Annual Report
2016
2017

IOM-NCGM
Research Collaboration OfficeFebruary 2018
Kathmandu, Nepal
Tokyo, Japan
ISBN 978-909675-44-6

2

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Preface

The Medical Education Project, which was implemented from 1980 to 1996, contributed
to the establishment and enhancement of basic and the clinical medicine of the Institute ofMedicine (IOM) at Tribhuvan University along with its attached teaching hospital (TUTH). During
the project period experts dispatched from NCGM worked together with IOM staff and good
relationship was built between the two institutions.
After the completion of the project IOM achieved further development and currently
it is greatly contributing to medical education, medical care, researches and human resource
development in Nepal. Besides, reliable relationship between NCGM and IOM has been
maintained until today. This is one of the valuable outcomes and legacies of the project.
In January 2013, Memorandum of Understanding (MOU) was concluded between NCGM
and IOM to start a unique cooperation focusing on research and related human resource
development. Five years have passed since the start of the new cooperation between NCGM
and IOM. Collaborations initiated based on the MOU are on track, and a number of fruitful
outcomes have been obtained.
It is a great pleasure for us to summarize the report of our new collaboration as Annual
Report 2016-17 following the publications of Annual Report 2013, 2014 and 2015. We also
would like to thank all those who worked hard for the success of the project and toward
the realization of new collaborations between NCGM and IOM. I sincerely hope that therelationship of mutual trust between the two institutions will be further strengthened.

						
					

Hiroshi Ohara, M.D.,Ph.D.

						Director, Division of Global Networking,						Bureau of International Health Cooperation,						
National Center for Global Health and Medicine						Tokyo, Japan

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

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IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Abbreviations

AMR		

Antimicrobial Resistance

ESBL		

Extended Stratum beta Lactamase

GFATM		

Global Fund against AIDS, Tuberculosis and Malaria

IOM		

Institute of Medicine

JICA		

Japan International Cooperation Agency

MBBS		

Medicine Bachelor Bachelor Surgery

MOHP		

Ministry of Health and Population

MOU		

Memorandum of UnderstandingNCD		

Non Communicable Diseases

NCGM		

National Center for Global Health and Medicine

NDM		

New Delhi Methalo-β-Lactamase

ODA		

Official Development Assistance

TUTH		

Tribuban University Teaching Hospital

WHO		

World Health Organization

WPRO		

Western Pacific Regional Office

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

5

Contents

Preface
1. NCGM............................................................................................................................. 03
2. IOM................................................................................................................................. 04
Abbreviations...................................................................................................................... 05
Contents.............................................................................................................................. 06
I.

Outline of Collaboration between NCGM and IOM
1. Background....................................................................................................................

07

2. Start of the new collaboration........................................................................................ 08
3. Purpose of the collaboration between NCGM and IOM, and expected outcomes.......

09

II. Research activities
1. Overview of researches................................................................................................... 10
2. Visit to Nepal for a collaborative research on a subnational health impact ofthe 2015 Nepal earthquake............................................................................................ 12
Researches No.1- No.5.......................................................................................................... 13
List of papers published on international journals from 2012 to 2015:
Achievement in NCGM-IOM Collaboration Office................................................................. 18
Attachment: Scientific papers.............................................................................................. 21

6

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

I. Outline of Collaboration between NCGM and IOM

1.

Background
The Institute of Medicine (IOM) at Tribhuvan University was established as the first medical school

in Nepal with the support of Japan’s Grant Aid in 1980 and then a technical cooperation project (Medical
Education Project) was implemented from 1980 to 1996 supported by Japan International Cooperation Agency(JICA). During this period, the National Center for Global Health and Medicine (NCGM) dispatched project teamleaders, doctors and other medical professionals.
The Medical Education Project was implemented as a technical cooperation project in two phases.
In the first phase (1980-1989), strengthening education, clinical practice, and the research infrastructure forbasic medicine and clinical medicine were mainly implemented. In the second phase (1989-1996), a technicalguidance was conducted, focusing mainly on improving medical education to have international accreditation,enhancing the function of basic medicine, clinical medicine, and research, as well as improving hospitalmanagement function.
During the 16-year technical cooperation period almost all the basic and advanced techniques
regarding diagnosis, examination, treatment and clinical record, which were indispensable to the daily medicalcare and medical education in IOM and its attached teaching hospital (Tribhuvan University Teaching Hospital;
TUTH), were guided (however, nosocomial infection control was not included as awareness on it was still low
in those days even in advanced countries). IOM/TUTH had grown to be a medical institution that carried out akernel function of Nepalese medical care, and had gained extremely high reliance from Nepalese people.
After the completion of the project, IOM has been functioning as a core in medical education, medicalcare and medical human resource development in Nepal, and TUTH has gained the trust and popularity of thepeople in Nepal.
Nepal suffered political instability from1996 to 2006 followed by a transition from theKingdom of Nepal to the Federal Democratic
Republic of Nepal in 2008. The politicalinstability affected economic growth andcompromised the delivery of social and publichealth interventions in the country. During thisperiod, cooperative relations between IOM and
NCGM were temporarily suspended, but withthe stabilizing of political conditions, the good
relationship resumed.

Medical Education Project in Nepal (JICA)

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

7

In September 2009, a joint symposium on nosocomial infection control was held at IOM organizedby NCGM and IOM, resulting in a greater awareness of the importance of infection control and researchcollaboration. At the conference, successful technical cooperation in nosocomial infection control in Vietnam
was introduced inviting director of Bach Mai Hospital in Hanoi.
2.

Start of the new collaboration
On January 18, 2013, NCGM concluded an agreement of cooperation with IOM in Nepal for researchand related human resource development, and IOM became the fifth overseas platform of NCGM. The
Memorandum of Understanding (MOU) was signed by the President of NCGM in Tokyo firstly, and then, wassigned by the Dean of IOM at IOM in the capital Kathmandu. Based on this MOU, unique activities of NCGM,
such as collaborative research on infectious diseases, nosocomial infection, dual burden of infectious diseases
and non-communicable diseases, and development of human resources related to these researches cameto be actively promoted, seeking to improve healthcare in Nepal. Collaboration between NCGM and IOM
including related institutions Kathmandu University School of Medicine, Clinics in Kathmandu, etc. mainly on
research field has continued thereafter. Conclusion of MOU is considered to be highly effective for the smoothand efficient implementation of collaborations including related institutions.
In September 2013, the IOM-NCGM Research Collaboration Office was established in the academic
building of IOM, and the necessary equipment (desk,
chair, computer, projector and scanner) was installed.
One assistant was employed and given an orientation.
Initially, grants from the International Health Cooperation
Research (24-5, 25-7), a grant from the Ministry of Health,
Labor and Welfare of Japan, were used and thereaftersome other grants have also been utilized to conductvarious activities. IOM’s Department of Microbiology
and NCGM’s Bureau of International Health Cooperation
manage the office operation.
Following the ceremony of the MOU signing “the1st Joint Conference on Infectious Diseases with Growing
Concern in Recent Years in Nepal” was held at IOM on
January 18, 2013 (the second conference was held in
December 2014).
8

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Signing of MOU at NCGM and IOM (January 2013)

3.

Purpose of the collaboration between NCGM and IOM, and expected outcomes
The purpose of this collaboration is to contribute to the healthcare in both countries by collaborative

research and related human resource development, and to strengthen a reliable relationship.
Expected Outcomes
1. To strengthen research activities by the
publication and presentation at academic
conferences, etc. of the collaborative
researches between NCGM and IOM.
2. To obtain research results effectively
through collaboration with IOM that is themain medical care and medical education

IOM-NCGM Research Collaboration Officeinstitution in Nepal.
3. To provide benefits to younger staff members of NCGM by learning about the actual conditions of
medical care in developing countries and how international collaborations are conducted.
4. To promote a relationship by utilizing a good relationship built on the results of the technicalcooperation project.
5. To contribute to the quality improvement of medical care in Nepal by conducting researches focusedon high-priority infectious diseases and other emerging health priorities in Nepal and providing related
technical assistance. These results are also beneficial for medical care in Japan.
6. To enable wider range of cooperation in the future, because the inclusion of medical institutions otherthan IOM allows providing further possible collaboration fields.
7. To contribute to expansion of the benefit of the Japan’s ODA projects.

IOM-NCGM Research Collaboration Office Annual Report 2016-20179II. Research activities

1.

Overview of researches
The main theme of the ongoing researches is “Infectious diseases with growing concern in recent

years in Nepal.” In other words, “Emerging health priorities in infectious diseases in Nepal”. Infectious
diseases change over time, as can be seen by the appearance of emerging or re-emerging infectious diseases,
Antimicrobial Resistance (AMR), spread of drug-resistant malaria, etc. These changes are often associated withsocial and environmental factors including development, population movement, climate change, nutritional
status, condition of health system, inappropriate use of antibiotics. Control strategy and assistance of foreigncountries also affect infectious diseases.
Infectious diseases, which have growing issues in recent years in Nepal but appropriate studies alongwith control measures have not been done, are target of these researches. These researches are aiming toanalyze the latest situation of the target infectious diseases and the causative factors of growing issues. Suchanalysis is crucial to implement effective control.
ØØ As a result of preliminary survey and a series of discussion in our research team (both Japanese and
Nepalese), we recognized that the following topics were suitable in consideration of the concept of themain theme and feasibility.
① AMR② Healthcare associated infections (Nosocomial infection)③ Dual burden of infectious diseases and non-communicable diseases④ Viral hepatitis⑤ Malaria control and health system⑥ Diarrhea caused by emerging pathogens
Among them researches on ①,②,③,④ have been conducted up to 2017. Researches ⑤,⑥were
conducted in 2013-15 and these researches have already concluded by the end of 2015. (Cf. Annual
reports 2013, 2014, 2015)
http://www.ncgm.go.jp/kyokuhp/library/annual/index.html
ØØ Outline of each research which was conducted in 2016-17 is shown in on pages 13-17.
ØØ List of scientific papers which were published on international journals as a result of collaborative studies
during the period from 2012 to 2017 are shown on pages 18-20.
•
10

As the published papers show, we have already made some valuable achievements. Among them it is
IOM-NCGM Research Collaboration Office Annual Report 2016-2017

noteworthy that 3 new NDM variants were discovered from the nosocomial infection cases and named
NDM-8, 12 and 13. Including these discoveries it was revealed that in medical settings in Nepal, AMR
particularly in gram negative bacilli were spreading and it was suggested that immediate countermeasures
are needed.
ØØ To implement collaborative activities and strengthen the IOM-NCGM Research Collaboration Office, thefollowing grants were utilized in 2016-2017.
•

Grants from the International Health Cooperation Research, grants from the Ministry of Health, Laborand Welfare of Japan (25-5, 25-7, 27-4, 29-5).

•

Clinical Epidemiology Research, St. Luke’s International University, Tokyo, Japan (2016)•

Grant-in-Aid for Scientific Research (B) (Overseas Academic Research) from Japan Society for the
Promotion of Science to NM

ØØ NCGM planned a new research proposal on thehealth impact of the 2015 Nepal earthquake (Cf.
page 10). This topic is one of the emerging healthpriorities in Nepal. Hereafter the main theme
will be widened from “Emerging health priorities
in infectious diseases in Nepal” to “Emerging
health priorities in Nepal”.
Training course on nosocomial infection control
for newly recruited nurses (TUTH)

Internal Medicine Ward (TUTH)

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

11

2.

Visit to Nepal for a collaborative research on a subnational health impact of the 2015 Nepal
earthquake

Date:		Visitors:

September, 20–24th, 2017
Hidechika Akashi1, Yoko Iwaki2, Kazuki Miyazaki1, Shuhei Nomura2, Hiroshi Ohara1,

		Manami Uechi2
1. Department of Global Network and Partnership, Bureau of International Health
Cooperation, National Center for Global Health and Medicine
2. Institute for Global Health Policy Research, Bureau of International Health Cooperation
A 7.8 magnitude earthquake struck
Nepal on April 2015, causing massive damage on
Kathmandu and other nearby areas. Institute for
Global Health Policy Research (iGHP) in NCGM
will launch the collaborative research with Nepal’s
Institute of Medicine (IOM), Tribhuvan University
Teaching Hospital, with the aim to understand thepotential med to long-term health impact of theearthquake at subnational, district level in order
to inform the creation of disaster resilient regional health system after a major disaster, including earthquake.
With logistics and administrative support from the Bureau of International Health Cooperation, we
visited Nepal (in particular the IOM) in September 20–24th, 2017. The purposes of the visit were:
1. to coordinate the research governance by gaining agreement on the research proposal with theprinciple researchers from the IOM
2. to discuss the arrangement of data access (the Annual Report of the Department of Health Service,
Ministry of Health and Population, Nepal), identifying administrative division/persons of the data
3. to confirm the approval process and necessity materials for research ethics in the country
4. to conduct an inspection of the most affected area (Bhaktapur), where the damage of the earthquake
was the greatest
During the 4-day visit in Nepal, we have confirmed the concrete direction of the research activities
with the IOM, including data access and ethics approval. In addition, we shared the experience of Japan’s 2011
Great East Earthquake with the 15 researchers from IOM (photo above).

12

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Research No.1
1.

Title(in English)

Study on effective use of the surveillance results for drug resistant pathogens in nosocomial
infection control

2.

Title(in Japanese)

院内感染対策における耐性菌サーベイランスの活用

3.

Main researcher

Hiroshi Ohara (Bureau of International Health Cooperation, National Center for Global Healthand Medicine)4.

Co-Researcher(s)Pokhrel BM, Shrestha RK, Dahal RK, Mishra SK, Kattel HP, Rijal BP (Dept. of Microbiology,
Institute of Medicine, Tribhuvan University, Nepal)
Jeevan B. Sherchand (Dept. of Public Health, Institute of Medicine, Tribhuvan University,
Nepal)
Jatan B. Sherchan (Dept. of Clinical Microbiology, Kathmandu University, School of Medicine,
Nepal)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (25-7, 27-4)

6.

Affiliation(s) in Nepal

Department of Microbiology, Institute of Medicine, Tribhuban University,
Dept. of Medical Microbiology, Kathmandu University, School of Medicine

7.

Period of the research

September 2014- March 20178.

Publications in
FY 2016 - 2017

Sherchan JB, Gurung P, Karkee P, Ranabhat N, Shrestha N, Ohara H. Microbiological andclinical profile of uropathogenic Escherichia coli isolates in Kathmandu University Hospital. J
Nepal Health Res Counc 2016; 14(32): 33-38.
Ohara H, Sherchan JB, Pokhrel BM, Sherchand JB. Review of collaboration between Tribhuvan
University Institute of Medicine in Nepal and National Center for Global Health and Medicine
in Japan on Nosocomial infection control and proposal for improvement. J Inst Med 2017;39(2): 101-108.

IOM-NCGM Research Collaboration Office Annual Report 2016-201713

Research No.2
1.

Title(in English)

Molecular and Clinical Epidemiology of Salmonella Paratyphi A Isolated from Patients with
Bacteremia in Nepal

2.

Title(in Japanese)

ネパールにおけるパラチフス菌血症患者の分子微生物学的および臨床疫学的検討

3.

Main researcher

Kayoko Hayakawa (Disease Control and Prevention Center, National Center for Global Healthand Medicine)
Jatan Bahadur Sherchan (Department of Clinical Microbiology, Kathmandu University Schoolof Medical Sciences, Dhulikhel, Nepal)

4.

Co-Researcher(s)Masatomo Morita, Takashi Matono, Hidemasa Izumiya, Makoto Ohnishi(Department ofBacteriology I, National Institute of Infectious Diseases, Tokyo, Japan), Jeevan B. Sherchand,Sarmila Tandukar, Ujjwal Laghu (Public Health Research Laboratory, Institute of Medicine,
Tribhuvan University Teaching Hospital, Kathmandu, Nepal), Maki Nagamatsu, Yasuyuki Kato,
Norio Ohmagari (Disease Control and Prevention Center, National Center for Global Healthand Medicine, Tokyo, Japan)

5.

Resource of fund

Clinical Epidemiology Research, St. Luke’s International University, Tokyo, Japan (2016).

6.

Affiliation(s) in Nepal

Department of Medical Microbiology, Kathmandu University, School of Medical Sciences

7.

Period of the research

December 2014-March, 20178.

Publications in
FY 2016 - 2017

Miyoshi-Akiyama T, Sherchan JB, Doi Y, Nagamatsu M, Sherchand JB, Tandukar S, Ohmagari
N, Kirikae T, Ohara H, Hayakawa K. Comparative gene anakysis of extended –spectrum –β -lactamase producing Escherichia coli sequence type 131 Strains from Nepal and Japan.
Therapeutics Prevention 2016 e00289-16; 1(5): 1-13.
Sherchan JB, Morita M, Matono T, Izumiya H, Ohnishi M, Sherchand JB, Tandukar S, Laghu
U, Nagamatsu M, Kato Y, Ohmagari N, Hayakawa K. Molecular and clinical epidemiology of
Salmonella paratyphi A isolated from patients with bacteremia in Nepal. Am J Trop Med Hyg
2017; 97(6): 1706-1709.

14

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Research No.3
1.

Title(in English)

Molecular epidemiology of multidrug-resistant pathogens and development of diagnosis
systems in Nepal

2.

Title(in Japanese)

ネパールの医療施設で分離される多剤耐菌の分子疫学解析および診断法の開発

3.

Main researcher

Tatsuya Tada (Department of Infectious Diseases, Research Institute, National Center for Global
Health and Medicine)
Shinichiro Morioka (Disease Control and Prevention Center, National Center for Global Health andMedicine)

4.

Co-Researcher(s)Jeevan B. Sherchand (Dept. of Medical Microbiology, Tribhuvan University Teaching Hospital, Nepal)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (29-5)

6.

Affiliation(s) in Nepal

Department of Microbiology, Institute of Medicine, Tribhuban University

7.

Period of the research

April 2016- March 20178.

Publications in
FY 2016 - 2017

Shrestha S, Tada T, Shrestha B, Kirikae T, Ohara H, Rijal BP, Pokhrel BM, Sherchand JB. Emergence ofaminoglycoside resistance due to armA methylase in multi-drug resistant Acinetobacter baumanii
isolates in a University Hospital in Nepal. J Nepal Health Res Counc 2016; 14(33): 72-76.
Tada T, Shrestha S, Shimada K, Ohara H, Sherchand JB, Pokhrel BM, Kirikae T. PER-8 a novel
extended-sprctrum β -lactamase PER variant, from an Acinetobacter baumannii clinical isolate in
Napal. Antimicrob Agents Chemother 2017; 61(3) e00694-17: 1-5.
Tada T, Shimada K, Satou K, Hirano T, Pokhrel BM, Sherchand JB, Kirikae T. Pseudomonas aeruginosa
clinical isolates in Nepal coproducing metallo-β -lactamase and 16S rRNA methyltransferases.
Antimicrob Agents Chemother 2017; 61(9) e00694-17: 1-6.
Shrestha B, Tada T, Shimada K, Shrestha S, Ohara H, Pokhrel BM, Sherchand B, Kirikae T. Emergenceof Various NDM-type-metallo-β -lactamase-producing Escherichia coli clinical isolates in Nepal.
Antimicrob Agents Chemother 2017; 61(12) e01425-17: 1-6.

IOM-NCGM Research Collaboration Office Annual Report 2016-201715

Research No.4
1.

Title(in English)

Study on double burden tuberculosis (TB) and non-communicable diseases (NCD) in Nepal

2.

Title(in Japanese)

ネパールにおける結核と非感染性疾患の二重負荷に関する研究

3.

Main researcher

Takanori Hirayama, Shinsaku Sakurada (Bureau of International Health Cooperation, NCGM)

4.

Co-Researcher(s)Yuko Tsuda (Health Bureau of Osaka City), Kiyohiko Izaumi (JATA/RIT), Shyam K Shrestha (FSB Clinic,
Kathmandu), Jatan B Sherchan (Kathmandu University School of Medicine, Dhulikhel), Kishor S
Manandhar (CF Clinic, Kathmandu), Jeevan B Sherchand (Tribhuvan University, Kathmandu)

16

5.

Resource of fund

Grants of National Center for Global Health and Medicine (24-5, 27-4)

6.

Affiliation(s) in Nepal

Department of Public Health, Institute of Medicine, Tribhuvan University, Kathmandu, Nepal

7.

Period of the research

September, 2012-March, 20188.

Publications in
FY 2016 - 2017

None

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Research No.5
1.

Title(in English)

The survey of the present status of viral hepatitis treatment in Nepal

2.

Title(in Japanese)

ネパールにおけるウイルス肝炎治療に関する実態調査

3.

Main researcher

Naohiko Masaki, MD, PhD (Medical Director, Laboratory Testing Department, National Center for
Global Health and Medicine)4.

Co-Researcher(s)Prof. Pradeep K. Shrestha (Department. of Internal Medicine, Teaching Hospital, Tribhuvan
University), Prof. Yasuhito Tanaka (Departments of Virology & Liver Unit, Nagoya City
University Graduate School of Medical Sciences), Prof. Hiroshi Ichimura (Department ofViral infection and International Health, Graduate School of Medical Sciences, Kanazawa
University)

5.

Resource of fund

Grant-in-Aid for Scientific Research (B) (Overseas Academic Research) from Japan Society for the
Promotion of Science to NM

6.

Affiliation(s) in Nepal

Department of Internal Medicine, Teaching Hospital, Tribhuvan University

7.

Period of the research

April 2015 - March 20188.

Publications in
FY 2016 - 2017

None in 2016, 2017
(Abstract of the following paper published in 2015 was put in this annual report 2016- 2017
for reference)
Masaki N, Shrestha PK, Nishimura S, Ito K, Sugiyama M, Mizokami M. Use of nucleosideanalogs in patients with chronic hepatitis B in Nepal: a prospective cohort study in a single
hospital. Hepatology Research 2015; 45: 1163-1169.

IOM-NCGM Research Collaboration Office Annual Report 2016-201717

List of papers published on international journals from 2012 to 2015:
Achievement in NCGM-IOM Collaboration Office
Scientific papers which were published on international journals as results of collaborative studies
during the period from 2012 to 2015 are shown below.
		◎ Full papers are put in this annual report 2016-2017 (Cf. Attachment)
		○ Full papers are put in the annual report 2014 or 2015
These reports are available at: http://www.ncgm.go.jp/kyokuhp/library/annual/index.html
		△ Abstract is put in the annual report 2016-2017

Papers18

Sherchan JB, Ohara H, Sakurada S, Basnet A, Tandukar S, Sherchand JB, Bam DS. Enteric
1 opportunistic parasitic infections among HIV-seropositive patients in Kathmandu, Nepal.
Kathmandu Univ Med J 2012; 38(2):14-17.

○

Sherchan JB, Ohara H, Sherchand JB, Tandukar S, Sakurada S, Gurung B, Ansari S, Rijal
2 BP, Pokhrel BM. Molecular evidence based hospital acquired rotavirus gastroenteritis in
Nepal. Prime J Mirobobiol Res 2011; 1(2): 16-21.

○

Shrestha S, Chaudhari R, Karmacharya S, Kattel HP, Mishra SK, Dahal RK, Bam N, Banjade
N, Rijal BP, Sherchand JB, Ohara H, Koirala J, Pokhrel BM. Prevalence of nosocomial lower3
respiratory tract infections caused by multi drug resistant pathogens. J Inst Med 2012;33(2): 7-14.

○

Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Shimada K, Kirikae T, Pokhrel BM.
4 NDM-8 metallo-β -lactamase in a multidrug-resistant Escherichia coli strain isolated in
Nepal. Antimicrob Agents Chemother 2013; 57(5): 2394-2396.

○

Shrestha RK, Dahal RK, Mishra SK, Parajuli K, Rijal BP, Sherchand JB, Kirikae T, Ohara H,
5 Pokhrel BM. Ventilator associaed pneumonia in tertiary care hospital, Maharajgunj,
Kathmandu, Nepal. J Inst Med 2013; 35(3): 21-28.

○

Ohara H, PokhrelBM, DahalRK, Mishra SK, Kattel HP, Shrestha DL, Haneishi Y, Sherchand
6 JB. Fact-finding survey of nosocomial infection control in hospitals in Kathmandu, Nepal
and trial to improvement. Tropical Med Health 2013; 41:113-119.

○

Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Shimada K, Kirikae T, Pokhrel
7 BM. NDM-1 metalloβ-lactamase and ArmA 16S rRNA methylase producing Providencia
rettgeri clinical isolates in Nepal. BMC Infect Dis 2014; 14:56-60○

Tada T, Miyoshi-Akiyama T, Dahal RK, Mishra SK, Ohara H, Shimada K, Kirikae T, Pokhrel
BM. Dissemination of multidrug-resistant Klebsiella pneumoniae clinical isolates with
8various combinations of carbapenemases (NDM-1 and OXA-72) and 16S rRNA methylases(ArmA, RmtC and RmtF) in Nepal. Int J Antimicrob Agents 2014; 42(4):372-374.

○

IOM-NCGM Research Collaboration Office Annual Report 2016-2017

Papers

Tada T, Shrestha B, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T, Pokhrel BM.
NDN-12, a Novel New Delhi Metallo-β -Lactamase Variant from a Carbapenem9
Resistant Escherichia coli Clinical Isolate in Nepal. Antimicrob Agents Chemother 2014;
58(10):6302-6305.
Tada T, Miyoshi-Akiyama T, Dahal RK, Shyam MK, Shimada K, Ohara H, Kirikae T, Pokhrel
BM. Identification of a Novel 6’-N-Aminoglycoside Aetyltransferase, AAC(6’)-Iak from a
10
Multidrug-resistant Clinical Isolates of Stenotrophomonas maltophilia. Antimicrob Agents
Chemother 2014; 58(10):6324-6327.
Sha MK, Mishra SK, Ohara H, Kirikae T, Sherchand JB, Rijal BP, Pokhrel BM. Nosocomial
11 bacterial infection and Antimicrobial resistant Pattern in a tertiary Care hospital in Nepal.
J Inst Med 2014; 36(3): 38-48.
Sherchan JB, Hayakawa K, Miyoshi-Akiyama T, Ohmagari N, Kirikae T, Nagamatsu M, Tojo
M, Ohara H, Sherchand JB, Tandukar S. Clinical epidemiology and molecular analysis of
12
extended-spectrum β -lactamase (ESBL)-producing Escherichia coli in Nepal: Characteristics
of sequence types 131 and 648. Antimicrob Agents Chemother 2015; 59(6): 3424-3432.
Ohara H, Sherchand JB, Pokhrel BM, Hirayama T, Nam VH, Sherchan JB. Assessment ofhealth systems in relation to interface between malaria control programs and health13
system strengthening: Comparative study between Nepal and Viet Nam. J Inst Med 2015;
37(1): 11-20.

○

○

○

○

○

Shrestha B, Tada T, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T, Pokhrel BM.
14 Identification of a novel NDM variant, NDM-13, from a multidrug-resistant Escherichia
coli clinical isolate in Nepal. Antimicrob Agents Chemother 2015; 59(9): 5847-5850.

○

Tada T, Miyoshi-Akiyama T, Shimada K, Dahal RK, Mishra SK, Ohara H, Kirikae T, Pokhrel
15 BM. A novel 6’-N aminoglycoside acetyltransferase, AA(6’)-lai, from a clinical isolate ofSerratia marcescens. Microbial Drug Resist 2015.

○

Masaki N, Shrestha PK, Nishimura S, Ito K, Sugiyama M, Mizokami M. Use of nucleoside
16 analogs in patients with chronic hepatitis B in Nepal: a prospective cohort study in asingle hospital. Hepatology Research 2015; 45: 1163-1169.

○
△
P21

Sherchand JB. Earthquake disaster-associated health effects and the need for improved
preventive measures. J Inst Med 2015; 37(1): 1-3.

○

Sherchan JB, Gurung P, Karkee P, Ranabhat N, Shrestha N, Ohara H. Microbiological
18 and clinical profile of uropathogenic Escherichia coli isolates in Kathmandu University
Hospital. J Nepal Health Res Counc 2016; 14(32): 33-38.

◎
P22

Shrestha S, Tada T, Shrestha B, Kirikae T, Ohara H, Rijal BP, Pokhrel BM, Sherchand JB.
Emergence of aminoglycoside resistance due to armA methylase in multi-drug resistant19
Acinetobacter baumanii isolates in a University Hospital in Nepal. J Nepal Health Res
Counc 2016; 14(33): 72-76.

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Miyoshi-Akiyama T, Sherchan JB, Doi Y, Nagamatsu M, Sherchand JB, Tandukar S,
Ohmagari N, Kirikae T, Ohara H, Hayakawa K. Comparative gene anakysis of extended
20-spectrum -β -lactamase producing Escherichia coli sequence type 131 Strains from Nepal
and Japan. Therapeutics Prevention 2016 e00289-16; 1(5): 1-13.
Tada T, Shrestha S, Shimada K, Ohara H, Sherchand JB, Pokhrel BM, Kirikae T. PER-8 a
21 novel extended-sprctrum β -lactamase PER variant, from an Acinetobacter baumannii
clinical isolate in Napal. Antimicrob Agents Chemother 2017; 61(3) e00694-17: 1-5.
Sherchan JB, Morita M, Matono T, Izumiya H, Ohnishi M, Sherchand JB, Tandukar S, Laghu
U, Nagamatsu M, Kato Y, Ohmagari N, Hayakawa K. Molecular and clinical epidemiology
22of Salmonella paratyphi A isolated from patients with bacteremia in Nepal. Am J Trop
Med Hyg 2017; 97(6): 1706-1709.
Ohara H, Sherchan JB, Pokhrel BM, Sherchand JB. Review of collaboration between
Tribhuvan University Institute of Medicine in Nepal and National Center for Global Health23
and Medicine in Japan on Nosocomial infection control and proposal for improvement. J
Inst Med 2017; 39(2): 101-108.
Tada T, Shimada K, Satou K, Hirano T, Pokhrel BM, Sherchand JB, Kirikae T. Pseudomonas
24 aeruginosa clinical isolates in Nepal coproducing metallo-β -lactamase and 16S rRNA
methyltransferases. Antimicrob Agents Chemother 2017; 61(9) e00694-17: 1-6.
Shrestha B, Tada T, Shimada K, Shrestha S, Ohara H, Pokhrel BM, Sherchand B, Kirikae T.
25 Emergence of Various NDM-type-metallo-β -lactamase-producing Escherichia coli clinicalisolates in Nepal. Antimicrob Agents Chemother 2017; 61(12) e01425-17: 1-6

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Hepatology Research 2015; 45: 1163–1169

doi: 10.1111/hepr.12482

Original Article

Use of nucleoside analogs in patients with chronic hepatitis B
in Nepal: A prospective cohort study in a single hospital
Naohiko Masaki,1* Pradeep Krishna Shrestha,3 So Nishimura,2* Kiyoaki Ito,1*
Masaya Sugiyama1 and Masashi Mizokami1
1

The Research Center for Hepatitis and Immunology, 2Kohnodai Hospital, National Center for Global Health andMedicine, Chiba, Japan, and 3Department of Medicine, Tribhuvan University, Kathmandu, Nepal

Aim: There still remain many concerns about the present sta-

Results: The younger, especially female patients of reproduc-tus of antiviral therapy for chronic hepatitis B in developing
countries in Asia, where the monitoring systems of virologicalmarkers have not been well established, despite the high prevalence of hepatitis B virus (HBV) infection. To investigate it in Nepal, this prospective cohort study was conducted at the
Teaching Hospital of Tribhuvan University in Kathmandu.

tive age were more frequently prescribed with these NA, and anincreased preference for the use of TDF was observed over time.
However, there was insufﬁcient follow up of the NA-treated patients in this cohort, and not a few patients developed emergenceof NA-resistant HBV: known resistance to LMV in 3 patients andincidental resistance to non-administrated NA in four patients.

Methods: From 2007 to 2012, 65 patients were consecutively

Conclusion: The results of the present study indicate that edu-

enrolled, 44 of whom received nucleoside analogs (NA), suchas lamivudine (LMV), adefovir or tenofovir (TDF), on the decisionof the local hepatologist. Virological determinations were performed in Japan, by using the serially collected serum samples
at the Teaching Hospital. Statistical analysis was performed,
using Mann–Whitney U-test or Fisher’s exact test.

cation for physicians as well as for infected patients regarding
the proper use of NA, together with establishment of appropriate monitoring systems for virological markers, is warranted toprevent an increase in NA-resistant HBV infections in Nepal.

INTRODUCTION

deaths related to liver cirrhosis or hepatocellular carcinoma caused by chronic HBV infection occur annually.1
HBV infection is endemic, particularly in Asian and Africancountries, and the prevalence is reported to be as high as
20% of the general population in some countries.2 In therural areas of Nepal, one of the poorest countries in theworld, the prevalence of HBV and hepatitis C virus(HCV) among blood donors was reported to be 0.86%
and 0.52%, respectively, in 2001–2002.3 Our epidemiological study conducted between 1997 and 2002 also demonstrated that HBV and HCV carrier rates among 540
blood donors in the urban area of Nepal were 2.4% and1.1%, respectively (e.g. N. Masaki, unpubl. data, 2003).
The recent introduction of nucleoside analogs (NA)such as lamivudine (LMV), adefovir (ADF), entecavir(ETV) and tenofovir (TDF) into clinical settings has significantly attenuated the progression of liver ﬁbrosis andhepatocarcinogenesis,4 leading to improvements in the

A

CCORDING TO ESTIMATES by the World Health Organization, almost 240 million people are infected byhepatitis B virus (HBV) worldwide, and up to 780 000

Correspondence: Dr Naohiko Masaki, The Research Center for Hepatitis
and Immunology, National Center for Global Health and Medicine,
Kohnodai 1-7-1 Ichikawa, Chiba 272-8516, Japan. Email:nmasaki0808@gmail.com*
Author contribution: N. M. designed the research protocol, obtained
funding and wrote the ﬁrst draft of the manuscript. P. K. S. was thecounterpart of this research in Nepal and contributed to the collection ofserum samples as well as clinical information. S. N. and K. I. contributed tothe acquisition of data and the analysis. M. S. carried out the molecularanalyses of hepatitis B virus. M. M. supervised the entire research project. All
authors read and approved the ﬁnal manuscript.
*
Present addresses: S. N. and K. I. are now afﬁliated with Saiseikai Fukuoka
General Hospital, Fukuoka, Japan, and Department of Gastroenterology,
Aichi Medical University School of Medicine, Aichi, Japan, respectively.
Received 14 August 2014; revision 23 December 2014; accepted 5 January
2055.

Key words: antiviral therapy, chronic hepatitis B, hepatitis B
virus, nucleoside analogs, resistance© 2015 The Authors.
1163
Hepatology Research published by Wiley Publishing Asia Pty Ltd on behalf of The Japan Society of Hepatology
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made.

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J Nepal Health Res Counc 2016 Jan - Apr;14(32):33-8

Microbiological and Clinical Profile of Uropathogenic
Escherichia coli Isolates in Kathmandu University
Hospital
Sherchan JB,1 Gurung P,2 Karkee P,1 Ranabhat N,1 Shrestha N,1 Ohara H3
1
Department of Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel, 2Kathmandu
Medical College, Kathmandu, Nepal, 3National Center for Global Health and Medicine, Japan.

ABSTRACT
Background: Treatment of patients infected by multidrug resistant bacteria is a major challenge. Immunocompromised status,
prolonged hospital stay, malignancy, diabetes are some of the risk factors for emergence of multidrug resistance. Our study focusedon microbiological and clinical profile of multidrug resistant uropathogenic Escherichia coli.
Methods: This was a cross-sectional study conducted between June 2014-May 2015 in Kathmandu University Hospital. Urine
sample from outpatients and inpatients from which Escherichia coli isolated was included. Specimen collection, culture, identification
tests were done following guidelines given by American Society for Microbiology.
Results: Total number of urine samples received during the study were 3,554. Escherichia coli isolates were 645(18.14%) and245(37.98%) were Extended Spectrum Beta-Lactamase producer. Extended Spectrum Beta-Lactamase producers were foundmore among inpatients 148(60.41%) [p<0.001], patients with underlying urological abnormalities 38 (15.51%) [p=0.0039],
pregnant ladies 46(18.77%) [p=0.0028], diabetic patients 27 (11.02%) [p=0.0084], patients who received prior antibiotic
therapy 155 (63.26%) [p=0.0043] than Extended Spectrum Beta-Lactamase non-producer. Malignancy was seen more among
Extended Spectrum Beta-Lactamase producer having patients 5 (2.04%) [p=0.031] and all these isolates were more resistant tofluoroquinolones 168(68.57%), Trimethoprim-sulfamethoxazole 239 (97.55%) [p=0.0633], aminoglycosides [p=0.0001] but only2(0.80%) were resistant to carbapenems.

Conclusions: Diabetes, pregnancy, malignancy, prior antibiotic therapy, underlying urological abnormalities were foundassociated with emergence of Extended Spectrum Beta-Lactamase producer in urine samples. Proper antibiotic usage may help toovercome the problem of emergence of antibiotic resistance.

Keywords: Extended Spectrum Beta-Lactamase, Escherichia coli, multidrug resistant.
INTRODUCTION
Antimicrobials remain the mainstay of empiricaltherapy; however, indiscriminate use of antibiotics inmany developing countries including Nepal has resulted
in the outbreak of drug resistant microorganisms.1 It
has been found and mentioned that, the importance ofExtended Spectrum Beta-Lactamase producer (ESBL)mediated infections has been increasing.2-4 ESBLproducing infections have been found associated withboth negative clinical outcomes and increased cost.5,6
Failure to adhere to proper infection control technique,unrationale use of antibiotics, unhygienic practices,increased uses of antibiotics in animal and plants andmore so availability of antibiotics without prescription
and counterfeit products of dubious quality in developing
countries have resulted in spread of antimicrobial
resistance and selection of multidrug resistant bacterial
pathogens.1,7,8 Unless we gather the information about
the existing multidrug resistant (MDR) strains, the rateof emergence and spread of antimicrobial resistancecannot be reduced.9
With resistance to each additional class of antibiotics,
ESBL-EK (Escherichia, Klebsiella) infections become aCorrespondence: Jatan B. Sherchan, Department of Microbiology, Kathmandu University
School of Medical Sciences, Dhulikhel, Nepal. Email: jatansherchan@gmail.com.

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Microbiological and Clinical Profile of Uropathogenic Escherichia Coli Isolates
greater therapeutic challenge. Reliance on carbapenems
has increased; because they are the only class of agentsto which ESBL-EK remain almost uniformly susceptible.
However, empirical treatment of suspected ESBLEK infections with carbapenems has been associatedwith significant increases in carbapenem resistance inother organisms (e.g., Pseudomonas aeruginosa andAcinetobacter calcoaceticus).10-12
In 1983, the first outbreak involving extended spectrum
beta-lactamase (ESBL) producing organisms was reported
in Germany.13 These ESBL-producing pathogens are now
recognized globally as major causes of nosocomial andcommunity-acquired infections.14
Several cases of multidrug resistant bacterial outbreaksof significant clinical concern have been frequently
reported.15-17
Risk factors for colonization or infection with multidrugresistant bacterial species include prolonged length of
hospital stay, exposure to an ICU, receipt of mechanicalventilation, colonization pressure, and exposure to
broad-spectrum antimicrobial agents, recent surgery,invasive procedures, and underlying severity ofillness.18,19Patients who are infected with multidrug
resistant bacteria have to stay in the hospital or have to
be treated longer. Treating such patient has become a
big challenge to the clinicians and patients for economic
burden. Research on such situation may become a useful
tool to overcome such situation in future. This study
might be one of a good source to identify the burdenof multidrug resistance pathogen, which might help toprepare guidelines for infection control and antibiotic
policy.

METHODSThis was a cross-sectional study carried out at Kathmandu
University Hospital, Dhulikhel, Nepal. Urine sample fromoutpatients and inpatients collected between June 2014
to May 2015 from which Escherichia coli was isolated(with special reference to ESBL) was included in the
study. Clinical and epidemiological information wascollected from the patient after taking informed consent
from the patient.
Ethical clearance was taken from Institutional Review
Committee of Kathmandu University Hospital before thestudy was conducted.
Specimen collection, culture, identification tests weredone according to the guidelines given by American
Society for Microbiology. 20 The antibiotic susceptibility

test of the pathogens isolated from the clinicalspecimen against different antibiotics was done using
Mueller Hinton agar (MHA) (Oxoid, United Kingdom)by the standard disk diffusion technique of modified
Kirby-Bauer method as recommended by Clinical andLaboratory Standards Institute (CLSI). 21
Definition of ESBL: They are capable of hydrolyzing
penicillins,
broad-spectrum
cephalosporins
andmonobactams, but they do not affect the cephamycins or
carbapenems and their activity is inhibited by clavulanic
acid. 22
The initial screen test for the production of ESBL
was performed by using ceftriaxone (CRO) (30 μg),
ceftazidime (CAZ) (30 μg) and cefotaxime (CTX) (30 μg)
disks (Oxoid, UK). If the zone of inhibition (ZOI) was ≤ 25
mm for CRO, ≤ 22 mm for CAZ and/or ≤ 27 mm for CTX,
the isolate was considered a potential ESBL- producer asrecommended by CLSI.21
Combination disk (CD) method was used for thephenotypic confirmation of ESBL-producing strains inwhich CTX and CAZ (30 μg), alone and in combination
with clavulanic acid (CA) (10 μg) was used (Becton
Dickinson, USA). An increased ZOI of ≥ 5 mm for either
antimicrobial agent tested in combination with CA versus
its zone when tested alone confirmed ESBL.21
Data were analyzed by (SPSS) version 11.5 software andP value less than 0.05 was considered to be significant.
RESULTS
Total number of urine sample received during the study
period was 3,554 out of which significant bacterial
growth was observed in 835 (23.49%) samples and outof this total number Escherichia coli isolates was 645(18.14%). Of total 645 E. coli 245(37.98%) were ESBL
producer and 400(62.01%) were non-ESBL producer. Of
total 245 ESBL E. coli 148 (60.41%) isolates were foundamong inpatients and 97 (39.59%) were found among
outpatients. Of total 400 non-ESBL E. coli 142 (35.50%)
isolates were found among inpatients and 258 (64.50%)
were found among outpatients. ESBL E. coli isolates
were found more among inpatients (60.41%) comparedto Non-ESBL E. coli (35.5%), which was statisticallysignificant. (p<0.001)
38 (15.51%) patients from whom ESBL E. coli was isolated
had underlying urological abnormalities whereas only
32 (8.00%) of patients from whom non-ESBL E. coli
was isolated had underlying urological abnormalities.
(p=0.0039)

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Microbiological and Clinical Profile of Uropathogenic Escherichia Coli Isolates
46 (18.77%) patients from whom ESBL E. coli was isolated
were pregnant where as only 41 (10.25%) patients fromwhom non-ESBL E. coli was isolated were pregnant. (pvalue=0.0028)
91 (37.14%) patients from whom ESBL E. coli was
isolated had complicated UTI whereas only 55 (13.75%)
patients from whom non-ESBL E. coli was isolated hadcomplicated UTI. (p<0.001)
27 (11.02%) patients from whom ESBL E. coli was

isolated were diabetic whereas only 21(5.25%) patientsfrom whom non-ESBL E. coli was isolated were diabetic.
(p=0.0084)
5 (2.04%) patients from whom ESBL E. coli was isolated
had malignancy whereas only 1 (0.25%) patients fromwhom non-ESBL E. coli was isolated had malignancy. (pvalue = 0.031)
155 (63.26%) patients from whom ESBL E. coli was
isolated received prior antibiotic therapy whereas only

Table 1. Clinico-epidemiological characteristics of ESBL vs. non-ESBL Escherichia coli from urine samples
ESBL isolates

Non-ESBL isolates

ESBL vs. Non-ESBL(n = 245, 38 %)(n = 400, 62 %)P value

Male patients

74 (30.20%)

124 (31.00%)

0.86

Female patients

171 (69.79%)

276 (69.00%)

Outpatients97 (39.59%)

258 (64.50%)

0.0001

Patients with underlying
urological condition

38 (15.51%)

32 (8.00%)

0.0039Pregnant patients46 (18.77%)

41 (10.25%)

0.0028Complicated UTI

91 (37.14%)

55 (13.75%)

0.0001

Uncomplicated UTI

154 (62.85%)

345 (86.25%)

Patients with DM

27 (11.02%)

21 (5.25%)

0.0084

Patients with cancer

5 (2.04%)

1 (0.25%)

0. 031

Prior antibiotic therapy

155 (63.26%)

207 (51.75%)

0.0043

Table 2. Susceptibility profile of ESBL- E. coli and non-ESBL E. coli for various antibiotics.
Antimicrobial agents
Ampicillin/Amoxicillin (10µg)

ESBL-E. coli resistant(n=245)

Non-ESBL-E. coli
resistant (n=400)

245 (100%)

267 (66.75%)

p-value

Cefuroxime (30µg)

245 (100%)

233 (58.25%)

Norfloxacin (10µg)

168 (68.57%)

245 (61.25%)

0.0633

Trimethoprim-sulfamethoxazole(1.25/23.75 µg)

239 (97.55%)

373 (93.25%)

0.0163

Gentamicin (10µg)

166 (67.75%)

102 (25.50%)

0.0001

Amikacin (30µg)

25 (10.20%)

5 (1.25%)0.0001

Amoxycillin-clavulanic acid (20/10µg)

245 (100%)

15 (3.75%)

Cefotaxime (30µg)

245 (100%)

12 (3.00%)

Netilmicin (30µg)

148 (60.40%)

17 (4.25%)

Aztreonam (30µg)

245 (100%)

7 (1.75%)

Cefepime/Cefpirome (30µg)

211 (86.12%)

4 (1.00%)

Cefoperazone-salbactum (75/30µg)

245 (100%)

2 (0.50%)

Piperacillin-tazobactum (100/10µg)

245 (100%)6 (1.5%)

Ticarcillin-clavulanic acid (75/10µg)

245 (100%)8 (2.00%)

Imipenem (10µg)

2 (0.80%)0 (0.00%)

Meropenem (10µg)

2 (0.80%)0 (0.00%)

0.0001

Includes intermediate and resistant isolates, based on CLSI criteria, 2013
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Microbiological and Clinical Profile of Uropathogenic Escherichia Coli Isolates
207 (51.75%) from whom non-ESBL E. coli was isolated
received prior antibiotic therapy. (p value=0.0043).
Fluoroquinolone resistance was observed more among
ESBL E. coli 168 (68.57%) than non-ESBL E. coli 245(61.25%). [p=0.0633]
Trimethoprim-sulfamethoxazole resistance was observed
more among ESBL E. coli 239 (97.55%) than non-ESBL E.
coli 373 (93.25%). [p=0.0163]
Aminoglycoside resistance was also observed moreamong ESBL E. coli [p<0.001]
Of total 245 ESBL E. coli only 2 (0.80%) were resistant toCarbapenems.

DISCUSSION
Certain strains of Escherichia coli can cause a wide
variety of intestinal and extra-intestinal diseases suchas urinary tract infection, diarrhea, septicemia andneonatal meningitis.23 Nosocomial infections caused
by Extended Spectrum Beta-Lactamase producing
pathogens are associated with risk factors such as elderly
age, prolonged hospitalization, previous antibiotic
use, and presence of invasive devices.14,24 In our study
duration total urine sample received was 3,554 out of
which 835 had significant bacterial growth and out of
this 645 (77.24%) were Escherichia coli. Among total
645 E. coli, which were screened for ESBL production,
245 (37.98%) were ESBL producer and 400 (62.02%)
were non-ESBL producer. This finding was more than
the finding done by Datta et.al in which out of total
140 strains of E. coli, which were screened for ESBL
production, only 30 (21.4%) isolates were positive.25 This
may reflect that their antibiotic policy and antibiotic
usage is better. ESBL E. coli isolates were found moreamong inpatients 148 (60.41%) compared to Non-ESBL
E. coli 142 (35.50%), which was statistically significant(p<0.001). This means ESBL E. coli isolated were moreencountered among hospital isolates indicating it might
be nosocomial infection. This finding is similar to thefinding observed by Husam. et.al in which ESBL producers
among urinary E. coli isolates was significantly higher
among in-patients. 26 Mean age of the patients with ESBL
E. coli was 48.8 ± 22.3 and mean age of patients with
non-ESBL E. coli was 40.8 ± 23.2. This means ESBL E. coli
was detect more among patients of higher age group andold age remains one of the risk factor, which is similar toobservation by Husam. et.al. 26 In our study of total 645
patients from whom E. coli was isolated 447 (69.30%)
were female and 198 (30.70%) were male. It is true andit has been mention in many studies that females are
more prone to urinary tract infection compared to themales. 27-29 Of total 245 patients from whom ESBL E. coli
was isolated 171 (69.79%) were female and 74 (30.21%)
were male.
In the past few years, the number of complicated UTI
due to resistant gram-negative bacteria has risen,
mainly due to spread of extended spectrum β-lactamase(ESBL) bacteria, which pose a significant therapeuticchallenge. Although a broad range of pathogens cancause complicated UTI, Escherichia coli remains themost common.30
Our study found similar observation as mentioned abovein which 91 (37.14%) patients from whom ESBL E. coli was
isolated had complicated UTI whereas only 55 (13.75%)
patients from whom non-ESBL E. coli was isolated
had complicated UTI (p<0.001) This means underlying
urological abnormalities such as nephrolithiasis, benign
prostatic hyperplasia, prostatic cancer, presence ofinvasive devices etc may be risk factor for acquiringurinary tract infection by ESBL E. coli.
In our study, 46 (18.77%) patients from whom ESBL E. coli
was isolated were pregnant where as only 41 (10.25%)
patients from whom non-ESBL E. coli was isolated werepregnant. (p value=0.0028) This means pregnancy might
be risk factor for acquiring UTI by ESBL E. coli. In study
conducted by Dutta et. al out of total 30 patients fromwhom ESBL E. coli isolates were detected, 11 (37%) werepregnant, which seems much more than our findings. 25 It
may be because our maternity and birth center follows
guidelines more strictly than their settings. In a study
conducted by Aswani Srinivas et.al ESBL producing E.
coli was significantly higher in diabetics (p value= 0.001)
compared to non-diabetics. 31 In our study though we did
not compare prevalence of ESBL E. coli among diabetics
and non-diabetics we found higher number 27(11.02%)
diabetic patients with ESBL E. coli in urine compared toless number 21(5.25%) diabetic patients with non-ESBL
E. coli. (p value=0.0084) This result shows that diabetes
may be another risk factor for acquiring multidrug
resistant bacteria.
In our study 5 (2.04%) patients from whom ESBL E. coli
was isolated had malignancy whereas only 1(0.25%)
patients from whom non-ESBL E. coli was isolated has
malignancy. (p value = 0.031) indicating underlying
comorbid illness like malignancy might be a risk factor
for acquiring ESBL E. coli.
In our study Fluoroquinolone resistance was observed
more among ESBL E. coli 168 (68.57%) than non-ESBL
E. coli 245 (61.25%). [p value=0.0633] which was quite

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Microbiological and Clinical Profile of Uropathogenic Escherichia Coli Isolates
similar to finding observed by Husam S. et. al in whichhigh levels of ciprofloxacin resistance was found among
ESBL isolates. 26 This means fluoroquinolones should be
used cautiously if ESBL E. coli is suspected from urine
sample. In our study Trimethoprim-sulfamethoxazole
resistance was observed more among ESBL E. coli 239(97.55%) than non-ESBL E. coli 373 (93.25%). [p=0.0163]
and aminoglycoside resistance was also observed moreamong ESBL E. coli [p<0.001]
In our study 155 (63.26%) patients from whom ESBL
E. coli was isolated received prior antibiotic therapy
whereas only 207 (51.75%) from whom non-ESBL E.
coli was isolated received prior antibiotic therapy.
(p value=0.0043) This finding might suggest that priorantibiotic therapy might be a risk factor for acquiring
ESBL E. coli.
This study do has some limitations. Since, it was carriedout in a single center the results may not be applicable
to settings with a different epidemiology. In addition,
only urine sample was screened for ESBL E. coli notincluding other clinical sample like blood, pus, wound
swab, sputum etc.

negative bacilli in the United States. The National
Nosocomial Infections Surveillance System. J Infect
Dis 1994; 170:1622–5.

3.

Itokazu GS, Quinn JP, Bell-Dixon C, Kahan FM,
Weinstein RA. Antimicrobial resistance rates among
aerobic gram-negative bacilli re- covered frompatients in intensive care units: evaluation of anational postmarketing surveillance program. Clin
Infect Dis 1996; 23:779–84.

4.

Nijssen S, Florijn A, Bonten MJM, Schmitz FJ,
Verhoef J, Fluit AC. b- Lactam susceptibilities andprevalence of ESBL-producing isolates among morethan 5000 European Enterobacteriaceae isolates.
Int J Antimicrob Agents 2004; 24:585–91.

5. Lautenbach E, Patel JB, Bilker WB, Edelstein PH,
Fishman NO. Ex- tended-spectrum b-lactamase–producing
Escherichia
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andKlebsiellapneumoniae: risk factors for infection and impactof resistance on outcomes. Clin Infect Dis 2001;
32:1162–71.

6. Paterson DL, Ko WC, Von Gottberg A, Mohapatra
S, Casellas JM, Goossens H, et al. International
prospective study of Klebsiella pneumoniae
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beta-lactamase production in nosocomial Infections.
Ann Intern Med 2004; 140:26–32.

CONCLUSIONS
Several factors were found associated with emergenceof Extended Spectrum Beta-Lactamase producing
Escherichia coli from urine sample. Extended Spectrum
Beta-lactamase producing Escherichia coli was foundmore resistant to other group of drugs as well. But lessthan one percent of them were resistant to carbapenem.
Hospital-acquired Extended Spectrum Beta-Lactamase
producers are emerging challenge and proper antibiotic
usage following antibiotic policy may help to someextent to over come this.

ACKNOWLEDMENTS
We
express
ourprofoundgratitude
toKathmandu
University
HospitalandNational
Center for Global Health and Medicine, Tokyo,
Japan for their valuable support in this study.

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Microbiological and Clinical Profile of Uropathogenic Escherichia Coli Isolates
pneumoniae.

24. Panhotra BR, Saxena AK, Al-Ghamdi AM. Extended-

13. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi

spectrum beta-lactamase-producing Klebsiellapneumoniae hospital acquired bacteremia. Risk
factors and clinical outcome. Saudi Med J. 2004;
25: 1871-1876.

Escherichia coli and KlebsiellaPharmacotherapy 2001; 21:583–92.

S. Transferable resistance to cefotaxime, cefoxitin,
cefamandole and cefuroxime in clinical isolates ofKlebsiella pneumoniae and Serratia marcescens.
Infection 11: 315-317.

14. Bradford PA. Extended-spectrum beta-lactamasesin the 21st century: characterization, epidemiology,
and detection of this important resistance threat.
Clin Microbiol Rev. 2001;14: 933-951.

15. Rai SK, Pokhrel BM, Tuladhar NR, Khadka JB,
Upadhyaya MP. Methicillin resistant coagulase
negative Staphylococci. J Inst Med (Nepal) 1987; 9:
23-8.

16.

Rai SK, Tuladhar NR, Shrestha HG. Methicillin
resistant Staphylococcus aureus in a tertiary
medical care center, Nepal. Indian J Med Microbiol
1990; 8:108-9.

17. Pokharel BM, Koirala J, Dahal RK, Mishra SK, Khadga
PK, Tuladhar NR. Multidrug-resistant and extendedspectrum
beta-lactamase(ESBL)-producing
Salmonella enterica (serotypes Typhi and ParatyphiA) from blood isolates in Nepal: surveillance ofresistance and a search for newer alternative. Int’l
J Infect Dis 2006; 6: 434-38.

18. Fournier PE, Richet H. The epidemiology and controlof Acinetobacter baumannii in health care facilities.
Clin Infect Dis. 2006; 42(5): 692-9.

19. Playford EG, Craig JC, Iredell JR. Carbapenemresistant Acinetobacter baumannii in intensive
care unit patients: risk factors for acquisition,
infection and their consequences. J Hosp Infect.
2007;65(3):204-11.

25. Datta P, Gupta V, Sidhu S. Extended spectrum
beta lactamase positive uropathogenic E. coli
– Epidemiological Factors and Resistance. BJMP
2014;7(2):a718

26. Husam S. Khanfar, Khalid M. Bindayna, Abiola C.
Senok, and Giuseppe A. Botta Extended spectrum
beta-lactamases (ESBL) in Escherichia coli andKlebsiella pneumoniae: trends in the hospital andcommunity settings. J Infect Dev Ctries 2009; 3(4):295-9.

27. Komaroff AL. Urinalysis and urine culture in womenwith dysuria. Ann Intern Med. 1986;104:212-8.

28. Stamm WE, Hooton TH. Management of urinary tract
infections in adults. N Engl J Med. 1993;329:132834.

29. Wong ES, Fennell Cl, Stamm WE. Urinary tract
infection among women attending a clinic forsexually transmitted diseases. Sex Transm Dis. 1984;
11:18-23.

30. Nicolle LE. A practical guide to the management ofcomplicated urinary tract infection. Drugs. 1997;
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31. Aswani Srinivas M, Chandrashekar UK, Shivashankara
KN, Pruthvi BC. Clinical profile of urinary tract
infections in diabetics and non-diabetics. Australas
Med J. 2014; 7 (1): 29-34.

20. Isenberg, HD. Clinical Microbiology Procedures
Handbook. 2nd Ed. Washington D.C., ASM press,
2004.

21. Clinicaland Laboratory Standards Institute.
Performancestandards
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22. Paterson DL, Bonomo RA. Extended-spectrum betalactamases: a clinical update. Clin Microbiol Rev.
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and disease in man and animals. Can J Microbiol.
1992;38:699-704.

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J Nepal Health Res Counc 2016 May - Aug;14(33):72-6

Emergence of Aminoglycoside Resistance Due to armA
methylase in Multi-drug Resistant Acinetobacter
Baumannii Isolates in a University Hospital in Nepal
Shrestha S,1 Tada T,2 Shrestha B,1 Kirikae T,2 Ohara H,2 Rijal BP,1 Pokhrel BM,1 Sherchand JB1
1
Department of Microbiology, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal,
2

National Center for Global Health and Medicine, Tokyo, Japan.

ABSTRACT
Background: The emergence of multidrug-resistant Acinetobacter baumannii associated with hospital-acquired
infections has been increasingly reported worldwide. 16S rRNA methylase producing Gram-negative bacteria arehighly resistant to all clinically important aminoglycosides. We analyzed A. baumannii clinical isolates resistant toaminoglycosides from hospitalized patients. The objective of this study was to investigate the emergence of armA in
A.baumannii species associated with nosocomial infection in a university hospital in Nepal.
Methods: This was a cross-sectional study conducted at the department of Clinical Microbiology,Tribhuvan University
Teaching Hospital (TUTH), from December 2013 to December 2014. A total of 246 Acinetobacter species were isolated
from different patients were screened for MDR A. baumannii. Identification at the species level was confirmed by 16S
rRNA sequencing. Drug susceptibility testing was performed by Kirby- Bauer disc diffusion method and minimum
inhibitory concentrations (MICs) were determined using the guidelines of the Clinical and Laboratory Standards
Institute (CLSI). Screening for 16S rRNA methylase-production was done for the isolates resistant to gentamicin andamikacin. Detection of 16S rRNA methylase gene was done by PCR.
Results: All 122 multidrug-resistant A. baumanniiisolates were resistant to majority of the antibiotics used except
polymyxin and tigecycline. Ninty-six MDR A. baumannii isolates had MICs of > 512 mg/L to amikacin and arbekacin
indicating their high resistance to aminoglycosides.Of the 96 pan-aminoglycoside resistant isolates, 75 isolates had
16SrRNAmethylasewith all isolates harboring armA gene.
Conclusions: This is the first report describing multidrug-resistant A. baumannii strains harboring armA from
hospitalized patients in Nepal. A methylase gene (armA), conferring high level of resistance to aminoglycosides, wasdetected in majority of our isolates.
Keywords: Acinetobacterbaumannii; aminoglycosideresistance; 16S rRNAmethylase; multidrug-resistant.
INTRODUCTIONEnterobacteriaceae and glucose–non-fermenting organisms

Acinetobacterbaumannii is a gram-negative, non-lactosefermenting

organism

recognized

as

a

major

pathogenincluding Pseudomonas aeruginosa and Acinetobacter species.3
The armA gene encoding a 16S rRNA methylase, was initially

responsible for nosocomial infections.1 It plays a significant

identified in Citrobacterfreundii in 2002 in Poland4 causingrole in the colonization and infection of hospitalized patients

global dissemination of hazardous multiple aminoglycosideimplicating variety of nosocomial infections, particularly inresistance genes. Although data on the prevalence of

patients admitted to intensive care units.2

aminoglycoside resistance mediated by 16S rRNA methylation

Methylation of 16S ribosomal RNA (rRNA) has emerged asa new mechanism of resistance to clinically importantaminoglycosides among gram-negative pathogens of the family

among gram-negative bacilli is still scarce, the presence of 16SrRNA methylases has already been reported worldwide

METHODS

Correspondence: Shovita Shrestha, Department of Microbiology, Institute ofMedicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal.
Email: shovitashrestha@gmail.com, Phone: +977-9851098729.

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Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii IsolatesThis study was approved by Institutional Review Board,
Institute of Medicine, Research Department, Kathmandu,
Nepal. It is a cross-sectional study, conducted at
the Department of Clinical Microbiology, Tribhuvan
University Teaching Hospital (TUTH).

5′-TATCAAGTATATAAGTTCTGTTCCG-3′(reverse),
which amplify a 741-bp DNA fragment;and for
rmtD, 5′-CGGCACGCGATTGGGAAGC-3′ (forward) and5′-CGGAAACGATGCGACGAT-3′ (reverse), which amplify a
401-bp DNA fragment.4

From December 2013 to September 2014, two hundredand forty six Acinetobacter spp.were isolated from nonduplicate, non-consecutive samples of blood, wound,urine, sputum and respiratory tract of patients fromdifferent wards of TU Teaching hospital in Nepal.

RESULTS

Specimen collection, culture, identification tests were
performed according to the guidelines given by American
Society of Microbiology (ASM).5
Phenotypic identification was performed by conventional
biochemical methods.6 Species identification was
confirmed by 16SrRNA sequencing7 also confirmed for
blaOXA-51 genes by PCR.8
Drug susceptibility testing was performed by KirbyBauer disc diffusion method as recommended by Clinicaland Laboratory Standards Institute (CLSI) guidelines
2015.9Escherichia coli ATCC 25922 was used as thequality control strain.
Minimum inhibitory concentration (MICs) were performedby broth micro-dilution method and interpreted
according to guidelines of the CLSI 2015.9
MDR A. baumannii strains are defined as isolates not
susceptible to at least one agent in three or more
antimicrobial categories, including aminoglycosides,
antipseudomonal
carbapenems,
antipseudomonal
fluroquinolones, antipseudomonal penicillins/β-lactam
inhibitors, extended-spectrum cephalosporins, folate
pathway inhibitors, penicillins/β-lactamase inhibitors,
polymyxins and tetracyclines.10
A. baumannii isolates showing resistance to gentamicin
and amikacin in disk diffusion were subjected to screen
for producing 16 SrRNA methylase.3
PCR analysis was done using 16S rRNAmethylase
gene specific primers to detect the armA, rmtA,
rmtB, rmtD genes. The PCR primers used were,
for armA, 5′-ATTCTGCCTATCCTAATTGG-3′ (forward)
and5′-ACCTATACTTTATCGTCGTC-3′(reverse),
which amplify a 315-bp DNA fragment; forrmtA,
5-CTAGCGTCCATCCTTTCCTC-3′(forward)
and5′-TTGCTTCCATGCCCTTGCC-3′(reverse),
which amplify a 635-bp DNA fragment for rmtB,5′-ATGAACATCAACGATGCCCTCACC-3′(forward)andOf the 246 Acinetobacter spp. isolates tested, 129(52.43%) were multidrug-resistant (MDR). Of the
129 MDR isolates, 122 were A. baumannii, 6 were A.
calcoaceticusand 1 was A. berezinaie. The majority of
MDR A. baumannii isolates were resistant to at least one
agent in 7 or more antimicrobial categories.
One-hundred-nine (89.34%) and 97(79.50%) isolates
showed MICs of ≥512 mg/L to amikacin and arbekacin
respectively. All isolates were resistant to ceftazidime
and 119(97.54%) were resistant to meropenem. All weresensitive to colistin with MICs of ≤ 2 mg/L. Ninety isolates(73.77%) had MICs of ≤1 mg/L to tigecyline, whereas 4
isolates (3.27%) had MICs of 8 mg/L.
Among the total bacterial isolates (n=122), majority wasfrom respiratory tract (n=60, 49.18%) followed by pus/
wounds (n=30, 24.59%) and urine (n=13, 10.65%). (Table
1)
Table 1. Distribution of MDR A. baumannii in various
samples (n=122).
SpecimenNo.
%
Respiratory tract
60
49.18
Pus
30
24.59
Urine
13
10.65
Blood
9
7.37
CSF
7
5.73
Others
3
2.45
Total
122
100
Majority of the isolates were resistant to all the antibiotics
tested, which is shown in the table below (Table 2).
All the isolates weresensitive to polymyxin,colistin
andtigecycline
Table 2. Antimicrobial susceptibility profile of 122
MDR A.baumannii isolates.
Resistant,Sensitive,
Antibiotic
number (%)
number (%)
Ciprofloxacin
122 (100)0Ceftriaxone
122 (100)0Ceftazidime
122 (100)
0Gentamicin
120 (98.36)
2 (1.63)
Cefepime
120 (98.36)
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Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii Isolates

Co-trimoxazole
Meropenem
Ampicillin/
sulbactam
Amikacin
Piperacillin/tazobactam
Imipenem
Levofloxacin
Doxycycline
Colistin
Polymyxin
Tigecycline

119 (97.54)
119 (97.54)

3 (2.45)
3 (2.45)

118 (96.72)

4 (3.27)

117 (95.90)

5 (4.09)

115 (94.26)

7 (5.73)

97 (79.50)
92 (75.40)
72 (59.01)00025(20.49)
30 (24.59)
50 (40.98)
122 (100)
122 (100)
122 (100)

Majority of the isolataes were from Intensive care unit
(ICU) followed by Surgical ward and Medical ward.
(Figure 1)

Fig 1. Wardwise distribution of MDR A. baumannii(n=122).
Ninety-six of the 122 MDR A. baumannii isolates had MICsof > 512 mg/L to amikacin and arbekacin which indicates
their high resistance to aminoglycosides. 9 isolates had
MICs of >512mg/L to amikacin but <64mg/L to arbekacin.
3 isolates had MICs of 512mg/L to amikacin but <16mg/L
to arbekacin. The remaining 14 isolates had MICs of<512mg/L to amikacin and< 64mg/L to arbekacin.
Of the 96 pan-aminoglycoside resistant isolates, 75
isolates had 16SrRNAmethylasewith all isolates harboringarmA gene. None of the isolates harbored genes otherthan armA. (Figure 2)

Fig 2. Electrophoresis profile of the PCR product ofarmA genes: laneM- marker, lane1- positive control,lane 2- negative control, lane 3, 5, 7 and 9 - armA
positive.

DISCUSSION
Multi-drug resistantA. baumannii has been the cause of
an increasing threat in hospitals and a global challenge
nowadays. A. baumannii is an important nosocomial
pathogen associated with a wide variety of illnesses
in hospitalized patients especially in the intensive
care units imposing greater challenge to the patientsmanagement and infection control. In our study also
majority of the isolates were recovered from patientsfrom ICU (60, 49.18%) followed by surgical ward (22,18.03%). Antimicrobial resistance among A. baumannii
has substantially increased in the past decade creatinga major public health dilemma. Carbapenems are themost potent antibiotic currently available but resistantstrains have emerged.11
In our study, A. baumannii was frequently isolated from
respiratory tract (49.18%) followed by pus (24.59%),
urinary tract (10.65%), blood (7.37%), CSF (5.73%) andother sources (2.45%). In another study from India, 59.8%
A. baumannii isolates were reported from respiratory
tract followed by 18.6% from blood.12
We have studied the antimicrobial resistance patternof 122 A. baumannii isolates. In our study, A. baumannii
isolates showed resistance to most of the antibiotic
tested. All the isolates were completely sensitive topolymyxin, colistin and tigecycline only.
Aminoglycosides continue to play an important rolein the management of serious infections causedby gram-negative pathogens, often in combination
with broad-spectrum beta-lactams but the activityof aminoglycosides is lower for MDR isolates of A.
baumannii compared with non-multiresistant ones. To
survive in this niche, A. baumannii developed a large
variety of resistance traits including production of 16SrRNA methylases with the major representative armA.
Incidence of infection by A. baumannii with armA 16S
rRNAmethylase has increased, leading to reports ofhigh-level resistance to most aminoglycosides.13In ourstudy, the prevalence of armA among MDR A. baumannii
was 61.47% and the isolates with armA in our study
were highly resistant to all available aminoglycosides.
According to a study done in Korea, the prevalence ofarmA among MDR A. baumannii was 97.8%13 which is

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Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii Isolates

quite higher than our study. Another study done in Egypt
found 94% prevalence of armA in clinical isolates of A.
baumannii.14

settings in Nepal. Multidrug resistantA. baumannii werecommon in our hospital and they usually harbored 16S
rRNA methylase gene, predominantly armA.

Antibiotics are among the most commonly prescribeddrugs in hospitals. In developed countries around 30% of
the hospitalized patients are treated with these drugs.15
Among the hospitalized patients in a medical settingsin Vietnam, 67.4% received antibiotics including 18.9%
receiving aminoglycosides with 30.8% inappropriately
prescribed.16A study done in Nepal documents 29.5% of
the patients were prescribed antibiotics. This is very
similar to the reports from developed countries.15A study
done in outpatients of Nepal concludes antibiotics werethe most frequently prescribed therapeutic class with
16.54% of the total prescribed drugs.17

The high prevalence of armA can threaten the existing
therapeutic qualities for such infections.Strict
surveillance and more rapid detection are essential toreduce the spread of MDRA. baumannii including 16SrRNA methylase.

Nosocomial infections caused by multidrug-resistant,gram-negative bacteria have become a serious problem
in clinical facilities. P. aeruginosa and Acinetobacter
spp. have been especially efficient at developing
multidrugresistanceagainstbroad-spectrum
β-lactams, fluoroquinolones, and aminoglycosides. The
identification of armA and rmtB genes in Europe andEast Asia suggests that we must pay consistent attention
to prevent further global proliferation. If 16S rRNA
methylase positive bacterial isolates disseminate widelyand extensively, the high level of pan-aminoglycosideresistance will undoubtedly have an impact on illness,
deaths, and costs of care.18
To our knowledge, this is the first report of 16S rRNA
methylase in A. baumannii isolates causing high level ofresistant to aminoglycosides in medical settings in Nepal.
Some of the A. baumannii strains were simultaneously
resistant to other classes of antimicrobials including
carbapenems. The high prevalence of 16S rRNA
methylase producing MDR A. baumannii in our hospitalwould have resulted from the high rate of the use ofaminoglycosides.
Our study strongly suggests that A. baumannii isolates
producing a 16S rRNA methylase, armA, have emerged anddisseminated in Nepal. A methylase gene (armA), which
confers high levels of resistance to aminoglycosides, wasdetected in the majority of our isolates. This suggeststhat aminoglycosides may no longer be recommended
as a first-line treatment for multidrug resistant A.
baumannii infections in our settings.

ACKNOWLEDGEMENTS
Authors would like to offer their sincere gratitude
to all the staffs of the Department of Microbiology
and different wards as well as all the lab members of
National Center for Global Health and Medicine, NCGM,
Tokyo. We are grateful to National Academy of Scienceand Technology (NAST) for providing fellowship grant forthis study. Also we would like to thank all the patients of
TUTH for their generous support throughout the study.

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Shahcheraghi F, Abbasalipour M, Feizabadi MM,
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Clarridge JE, Alerts C. Impact of 16S rRNA genesequence analysis for identification of bacteria on clinical

CONCLUSIONSThis is the first report describing the presence of
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Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii Isolates

microbiology and infectious diseases. Clin Microbiol Rev.
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Turton JF, Woodford N, Glover J, Yarde S, Kaufmann
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2007

10. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y,
Falagas ME, Monnet DL. Multidrug-resistant, extensively
drug-resistant and pandrug-resistant bacteria: Aninternational expert proposal for interim standard
definitions for acquired resistance. Clin Microbiol
Infect. 2012;18(3):268-281. doi:10.1111/j.14690691.2011.03570.x.
11. Shrestha S, Tada T, Shrestha B, Kirikae T, Pokhrel BM,
Sherchand JB. Phenotypic characterization of multidrugresistant Acinetobacter baumannii with special reference
to metallo-β-lactamase production from the hospitalized
patients in a tertiary care hospital in Nepal . J Inst Med(Nepal) 2015;37 (3):3-10
12. Jaggi N, Sissodia P, Sharma L. Acinetobacter baumannii
isolates: epidemiology, antibiogram and nosocomial
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doi:10.1186/1753-6561-5-S6-P291.

13. Lim J, Cho HH, Kim S, et al. The genetic characteristics ofmultidrug-resistant Acinetobacter baumannii coproducing
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jbv.2013.43.1.27.
14. Ahmed ES, Amin MA, Tawakol WM, Loucif L, Bakour S,
Rolain JM. High prevalence of blaNDM-1carbapenemaseencoding gene and 16S rRNA armA methyltransferase
gene among Acinetobacter baumannii clinical isolates in
Egypt. Antimicrob Agents Chemother. 2015;59(6):36023605. doi:10.1128/AAC.04412-14.
15. Shankar RP, Partha P, Shenoy NK, Easow JM,
Brahmadathan KN. Prescribing patterns of antibiotics
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Antimicrob. 2003;2:7. doi:10.1186/1476-0711-2-7.
16. Tada T, Akiyama TM, Kato Y, Ohmagari V, Takeshita N,
Kirikae T. Emergence of 16S rRNA methylase-producing
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2013;13(1):1-6. doi:10.1186/1471-2334-13-251.
17. Kumar J, Shaik M, Kathi M, Deka A, Gambhir S. Prescribing
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18. Yamane K, Wachino J, Suzuki S, Shibata N, Kato H,
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doi:10.3201/eid1304.060501.

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RESEARCH ARTICLE
Therapeutics and Prevention

crossmarkTohru Miyoshi-Akiyama,a Jatan Bahadur Sherchan,b Yohei Doi,cMaki Nagamatsu,d,e Jeevan B. Sherchand,f Sarmila Tandukar,f Norio Ohmagari,dTeruo Kirikae,e Hiroshi Ohara,g Kayoko Hayakawad
Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japana; Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel,
Nepalb; Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania,
USAc; Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japand;Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japane; Public Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital,
Kathmandu, Nepalf; Department of International Medical Cooperation, National Center for Global Health andMedicine, Tokyo, Japang

ABSTRACT The global spread of extended-spectrum--lactamase (ESBL)-producing

Escherichia coli (ESBL-E. coli) has largely been driven by the pandemic sequence type
131 (ST131). This study aimed to determine the molecular epidemiology of theirspread in two Asian countries with contrasting prevalence. We conducted wholegenome sequencing (WGS) of ESBL-E. coli ST131 strains collected prospectively from
Nepal and Japan, two countries in Asia with a high and low prevalence of ESBL-E.
coli, respectively. We also systematically compared these genomes with those reported from other regions using publicly available WGS data for E. coli ST131 strains.
Further, we conducted phylogenetic analysis of these isolates and all genome sequence data for ST131 strains to determine sequence diversity. One hundred ﬁve
unique ESBL-E. coli isolates from Nepal (February 2013 to July 2013) and 76 isolates
from Japan (October 2013 to September 2014) were included. Of these isolates, 54(51%) isolates from Nepal and 11 (14%) isolates from Japan were identiﬁed as ST131
by WGS. Phylogenetic analysis based on WGS suggested that the majority of ESBL-E.
coli ST131 isolates from Nepal clustered together, whereas those from Japan weremore diverse. Half of the ESBL-E. coli ST131 isolates from Japan belonged to virotype
C, whereas half of the isolates from Nepal belonged to a virotype other than virotype A, B, C, D, or E (A/B/C/D/E). The dominant sublineage of E. coli ST131 was
H30Rx, which was most prominent in ESBL-E. coli ST131 isolates from Nepal. Our results revealed distinct phylogenetic characteristics of ESBL-E. coli ST131 spread in thetwo geographical areas of Asia, indicating the involvement of multiple factors in its
local spread in each region.

Received 28 September 2016 Accepted 29
September 2016 Published 26 October 2016
Citation Miyoshi-Akiyama T, Sherchan JB, Doi
Y, Nagamatsu M, Sherchand JB, Tandukar S,
Ohmagari N, Kirikae T, Ohara H, Hayakawa K.
2016. Comparative genome analysis ofextended-spectrum-β-lactamase-producing
Escherichia coli sequence type 131 strains from
Nepal and Japan. mSphere 1(5):e00289-16.
doi:10.1128/mSphere.00289-16.
Editor Patricia A. Bradford, Antimicrobial
Development Specialists, LLC
Copyright © 2016 Miyoshi-Akiyama et al. Thisis an open-access article distributed under theterms of the Creative Commons Attribution 4.0
International license.
Address correspondence to Kayoko Hayakawa,kayokohayakawa@gmail.com.

Downloaded from http://msphere.asm.org/ on December 26, 2017 by guest

Comparative Genome Analysis ofExtended-Spectrum--LactamaseProducing Escherichia coli SequenceType 131 Strains from Nepal and Japan

IMPORTANCE The global spread of ESBL-E. coli has been driven in large part bypandemic sequence type 131 (ST131). A recent study suggested that, within E. coli
ST131, certain sublineages have disseminated worldwide with little association with
their geographical origin, highlighting the complexity of the epidemiology of thispandemic clone. ST131 bacteria have also been classiﬁed into four virotypes basedon the distribution of certain virulence genes. Information on virotype distribution in
Asian ST131 strains is limited. We conducted whole-genome sequencing of ESBL-E.
coli ST131 strains collected in Nepal and Japan, two Asian countries with a high andlow prevalence of ESBL-E. coli, respectively. We systematically compared these ST131
genomes with those reported from other regions to gain insights into the molecular
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Miyoshi-Akiyama et al.

epidemiology of their spread and found the distinct phylogenetic characteristics ofthe spread of ESBL-E. coli ST131 in these two geographical areas of Asia.
KEYWORDS: CTX-M, Escherichia coli, ST131, antimicrobial resistance, extended-

spectrum beta-lactamase, whole-genome sequence

T

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he emergence of extended-spectrum--lactamase (ESBL)-producing Escherichia coli
(ESBL-E. coli) is a global problem. However, its prevalence and epidemiology differ
signiﬁcantly depending on its geographical location. According to a recent report fromthe World Health Organization (WHO) (1), the prevalences of E. coli resistance tothird-generation cephalosporins were 14% in Europe, 27% in the Americas (15% in the
United States), and 30% in Southeast Asia. The prevalence of E. coli resistance to thesecephalosporins was the highest in developing countries in Asia (38% in Nepal, 68% in
Myanmar, and 16% to 95% in India) while relatively low in more-developed Asian
countries, including Japan (17%) (1).
The global spread of ESBL-E. coli has been driven in large part by pandemic
sequence type 131 (ST131); E. coli ST131 bacteria are often resistant to multiple drugs(2). A recent study suggested that, within E. coli ST131, certain sublineages havedisseminated worldwide with little association with their geographical origin, highlighting the complexity of the epidemiology of this pandemic clone (3). ST131 has also beenclassiﬁed into four virotypes based on the distribution of certain virulence genes, andvirotype C has been reported to be globally disseminated (4). However, information
pertaining to virotype distribution among Asian ST131 strains is limited.
In this study, we conducted whole-genome sequencing (WGS) of ESBL-E. coli ST131
strains collected prospectively in Nepal (5) and Japan. Although both countries arelocated in Asia, the socioeconomic statuses of Japan and Nepal are different and theyalso have distinct prevalences of ESBL-E. coli (Japan has a low prevalence, and Nepal has
a high prevalence). We systematically compared these ST131 genomes with thosereported from other regions to gain insights into the epidemiology of their spread.
RESULTS AND DISCUSSION
Characteristics of ESBL-E. coli ST131 isolates and E. coli ST131 genomes. During thestudy period, 105 and 76 unique ESBL-E. coli isolates were identiﬁed from Nepal andJapan, respectively. Of these isolates, 54 (51%) isolates from Nepal and 11 (14%) isolates
from Japan were identiﬁed as ST131 by WGS based on sequence typing and includedin further analyses. Forty (38.1%) of 105 ESBL-E. coli isolates from Nepal and 19 (25%)
of 76 ESBL-E. coli isolates from Japan were isolated from outpatients. Nineteen (35.2%)
of 54 ESBL-E. coli ST131 isolates from Nepal and 2 (18.2%) of 11 of ESBL-E. coli ST131
isolates from Japan were from outpatients. This suggests that ESBL-E. coli ST131
bacteria were isolated from outpatients almost twice as often in Nepal as in Japan.
The median age of patients with ESBL-E. coli ST131 was signiﬁcantly lower in Nepal(39 years [interquartile range {IQR}, 25 to 61 years]) than in Japan (83 years [IQR, 73 to
86 years]) (P  0.001). The numbers of male patients with ESBL-E. coli ST131 weresimilar in the two countries (19 [35%] in Nepal and 4 in Japan [36%]). The majority of
isolates were collected from urine samples (54 [100%] in Nepal and 8 [73%] in Japan).
Pregnant female patients with ESBL-E. coli ST131 were more frequent in Nepal (n  9[26%]) than in Japan (n  0) (P  0.001). Comorbid conditions, such as malignancy (n 
2 [3.7%] in Nepal; n  3 [27.3%] in Japan) and underlying urological conditions (e.g.,benign prostatic hyperplasia, urolithiasis, obstructive urinary diseases) (n  10 [18.5%]
in Nepal; n  6 [54.5%] in Japan) were more common in patients with ESBL-E. coli inJapan than in patients in Nepal (P  0.031 and P  0.02, respectively). ESBL-E. coli
isolates from Nepal and Japan had distinct clinical characteristics.
In a study that identiﬁed ESBL-E. coli from travelers who returned from India, 47% of
17 ESBL-E. coli isolates collected from 2004 to 2006 were ST131 (6). The prevalence of
ST131 among ESBL-E. coli bacteria isolated in Nepal in our study is in line with theprevious data, considering the geographical proximity and trafﬁc of visitors between
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Genome Analysis of E. coli ST131 in Asia

TABLE 1 Comparison of ﬁmH alleles and H30Rx and H30R sublineages in Escherichia coli ST131
No. of isolates (%) from:ﬁmH allele or
sublineage
H30
H30Rx
H30R (non-Rx)
H22
H27
H41

Total no. of
isolates(n  132)
110
88
19
9
2
11

Nepal
(n  54)54 (100)
54 (100)
0 (0)
0 (0)
0 (0)
0 (0)

Japan
(n  12)a11 (91.7)
8 (66.7)
3 (25)0 (0)0 (0)1 (8.3)

Other Asian
countries(n  3)b3 (100)2 (66.7)
1 (33.3)
0 (0)
0 (0)
0 (0)

USA
(n  29)b
27 (93.1)
16 (55.2)
11 (37.9)
0 (0)
0 (0)
2 (6.9)Europe(n  29)b,c
12 (41.4)
6 (20.7)
3 (10.3)
9 (31)0 (0)8 (27.6)

Africa(n  5)b3 (60)
2 (40)
1 (20)0 (0)2 (40)0 (0)P valued<0.001<0.0010.006<0.001<0.0010.001

aIncludes one publicly available sequence of E. coli ST131.
cOne isolate from Austria belonged to H31.
dThe P values are comparing the prevalence of each antibiotic resistance gene among geographical regions. P values in boldface type represent statistically signiﬁcantresults.

Nepal and India. The ST131 prevalence of 14% found among ESBL-E. coli isolates fromJapan is also similar to that reported in a previous study where 21% of 130 ESBL-E. coli
isolates collected between 2002 and 2003 from Japan were ST131 (7). However, theprevalence was lower than that reported in a more recent study conducted in Japan,
in which 37% of 581 ESBL-E. coli isolates collected from 2001 to 2010 were identiﬁed as
ST131 (8). This discrepancy might be attributed to the differences in the year ofcollection, geographical locations in the country, and methods used to identify ST131(PCR-based screening versus WGS).
ﬁmH alleles and H30Rx sublineages in E. coli ST131. ﬁmH alleles and H30Rx
sublineages in E. coli ST131 were determined and compared among various geographical regions (Table 1). ﬁmH30, especially ﬁmH30Rx comprised the majority of the ﬁmHalleles in various geographical regions, followed by ﬁmH41 and ﬁmH22. ﬁmH27 wasobserved only in E. coli ST131 from Africa. We found that all 54 ESBL-E. coli isolates from
Nepal belonged to the H30Rx sublineage. There is little data on the prevalence of the
H30Rx sublineage in South Asia. A previous study that included only a few ST131 strainsfrom India revealed that all of them belonged to clade C (i.e., H30) and produced
CTX-M-15 (3). Our result is in accordance with the high prevalence of CTX-M-15producing ESBL-E. coli reported among travelers returning from the Indian subcontinent (6). However, the limited isolate collection period in one facility might haveaffected the clonal distribution of ESBL-E. coli ST131 isolates from Nepal in our study.
H30R (non-Rx) were more prevalent among ESBL-E. coli ST131 isolates from Japan
compared to isolates from Nepal, although more than 60% of ESBL-E. coli ST131 isolates
from Japan still belonged to H30Rx. A previous report from Japan suggested a higher
prevalence of H30R (non-Rx) and H22 among ESBL-E. coli ST131 isolates collected from2001 to 2012 in 10 Japanese acute-care hospitals located in Kyoto, Shiga area, which ismore than 460 km (286 miles) away from the hospital in this study (9). The relatively
high prevalence of H30R (non-Rx) and H22 observed in this study in the United Statesand Europe, respectively, were consistent with previous reports (3, 10).
Antimicrobial susceptibility and antibiotic resistance genes. Antimicrobial
susceptibility data were available for 54 and 11 ESBL-E. coli ST131 isolates from Nepal
and Japan, respectively (Fig. 1). The susceptibilities of the 105 ESBL-E. coli isolates(including 54 ESBL-E. coli ST131 isolates described in this study) from Nepal were
described elsewhere (5). The susceptibilities to trimethoprim-sulfamethoxazole, gentamicin, and levoﬂoxacin were not signiﬁcantly different in isolates from Nepal and Japan.
All ESBL-E. coli ST131 isolates from Nepal and Japan were susceptible to cefmetazole
and fosfomycin.
The frequency of resistance genes among the E. coli ST131 isolates is summarized inTable 2. More than 94% (n  51) and 66% (n  8) of ESBL-E. coli ST131 isolates werepositive for blaCTX-M-15 in Nepal and Japan, respectively. Overall, 50 (70%) of the 71
blaCTX-M-15-positive isolates in the entire cohort were also positive for blaOXA-1 andVolume 1 Issue 5 e00289-16

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bPublicly available sequence data for E. coli ST131.

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FIGURE 1 Phylogenetic trees of ST131 strains. ML phylogenetic trees were estimated using PHYML 3.0 and SH statistics for branch support. Antibiotics
are abbreviated as follows: A/C, amoxicillin and clavulanate; CMZ, cefmetazole; ST, trimethoprim-sulfamethoxazole; FOM, fosfomycin; LVX, levofloxacin;(Continued)
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aac(6=)-Ib-cr (encoding an aminoglycoside/ﬂuoroquinolone acetyltransferase); of these
71 isolates, 38 isolates were obtained from Nepal, 5 isolates from Japan, and 6 isolates
from Europe.
The aforementioned study from Japan (9) revealed higher prevalence of blaCTX-M-14
and blaCTX-M-27 in addition to blaCTX-M-15 among ESBL-E. coli isolates collected fromanother area in Japan. A study from Korea on E. coli isolates collected from 2012 to 2013
suggested that the majority of H30Rx isolates harbored blaCTX-M-15, whereas about halfof the H30 non-Rx isolates harbored blaCTX-M-14 or blaCTX-M-27 (11). Geographicaldifferences and different times of these studies may account for these discrepancies.
Virotypes and virulence-associated genes. The distribution of virotypes andvirulence-associated genes among the E. coli ST131 strains is summarized in Table 3.
The predominant virotypes differed across the geographical regions. The majority ofE. coli ST131 isolates from the United States and half of those from Japan belonged to
virotype C, whereas almost half of the isolates from Nepal belonged to virotypes otherthan A, B, C, D, and E. Two-thirds of E. coli ST131 isolates from Europe belonged toeither virotype C or D. The virulence-associated genes such as iha, sat, fyuA, traT, ompT,and malT were highly prevalent among isolates from most geographical regions. The
papGII gene was frequently observed only among isolates from Nepal and Tanzania,
and hlyA and cnf1 were common only in E. coli ST131 isolates obtained from Africa.
Most of the virotypes obtained in our study belonged to virotype C (Table 1). Ourﬁnding that virotype C is the most prevalent virotype among E. coli ST131 isolates is
consistent with previous reports on virotype distribution in ESBL-E. coli ST131 (4, 12). As
previously suggested (4, 12), our results also suggested the association between
virotype C and H30Rx sublineage. In our study, more isolates were identiﬁed as “other”
virotype than previous studies (4, 12), which might be due to the different methodology used (e.g., WGS versus PCR). The majority of the ESBL-E. coli ST131 isolates (n  28[52%]) from Nepal were positive for papGII and sat but were negative for hlyA, and thus,
were not categorized into any of the previously described virotypes (A to E) (13).
Plasmid replicon types. The distribution of plasmid replicon types was similar
across geographical regions except that FIA was prevalent in Nepal and the UnitedStates and considerably less prevalent in Europe; other geographical regions showedintermediate prevalence (Table 4). FII was commonly found among ESBL-E. coli ST131
isolates from Nepal, whereas it was considerably less prevalent in Japan. IncP was
detected in 40% of E. coli ST131 isolates from Africa, but it was very rarely found or notfound in isolates from other areas.
The association of blaCTX-M-15 and IncF replicon has been reported previously (14),and the H30Rx sublineage was created by introduction of IncF (15). We previously
found that a plasmid resembling pEC958 and harboring FIA and FII (16) was present inapproximately 80% of the ESBL-E. coli ST131 isolates from Nepal (5). The high prevalence of IncF (IncFIA, FIB, and FII) observed among ESBL-E. coli ST131 isolates from
Nepal is consistent with these previous reports and is reasonable considering the highprevalence of H30Rx among ESBL-E. coli ST131 isolates.
Phylogenetic analysis of E. coli ST131 in different geographical regions. In
the phylogenetic analysis based on WGS (Fig. 1), the majority of ESBL-E. coli ST131
isolates from Nepal clustered together, whereas those from Japan were more diverse.
Thus, ESBL-E. coli ST131 may have been introduced into Japan more sporadically overtime. Strict requirements for antibiotic prescription in Japan might have resulted inrelatively low selective pressure, along with more advanced medical and social infrastructure. Carriers of ESBL-E. coli are usually placed under contact precaution in health

Figure Legend Continued
GM, gentamicin. The countries are shown in parentheses after the strain name and abbreviated as follows: DNK, Denmark; NPL, Nepal; GBR, Great Britain;
JPN, Japan; TZA, Tanzania; KOR, South Korea; LBN, Lebanon; AUT, Austria; THA, Thailand. Susceptibility data are available only for the 11 ESBL-E. coli ST131
isolates from the National Center for Global Health and Medicine, Japan, and 54 ESBL-E. coli ST131 isolates from Tribhuvan University, Nepal. For isolates
from Japan, ampicillin-sulbactam was used instead of amoxicillin-clavulanate. For resistance genes, a filled square means positive for the resistance gene
and an open square means negative for the resistance gene.
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Miyoshi-Akiyama et al.

TABLE 2 Comparison of resistance genes in Escherichia coli ST131
No. of isolates (%) from:
Antibiotic(s) andantibiotic resistancegene
Aminoglycosideaac(3)-IIaaac(3)-IId

aadA2
aadA5

strA

strB

Aminoglycoside andﬂuoroquinolone
aac(6’)-Ib-cr

Beta-lactams
blaCTX-M-14
blaCTX-M-15
blaCTX-M-27
blaCTX-M-55
blaOXA-1
blaTEM-1B

blaTEM-1C

Total
H30Rx
H30R
Total
H30Rx
H30R
H27
H41
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H27
H41
Total
H30Rx
H30R
H22
H27
H41

Nepal
(n  54)

Japan(n  12)a

26
25
1
17
3
6
1
7
5
1
3
1
11
8
3
68
51
9
8
45
28
7
2
8
49
30
3
5
2
9

15 (27.8)
15 (27.8)

5 (41.7)
4 (33.3)
1 (8.3)2 (16.7)
2 (16.7)
0

Total
H30Rx
H30R

55
53
2

39 (72.2)
39 (72.2)

6 (50)
5 (41.7)
1 (8.3)

Total
H41
Total
H30Rx
H30R
Total
H30Rx
H30R
Total
H30Rx
H30R
Total
H30Rx
H30R
Total
H30Rx
H30R
H22
H27
H41
Total
H22

2
2
71
69
2
3
2
1
4
1
3
54
52
2
61
33
13
1
2
10
3
3

0
51 (94.4)
51 (94.4)

1 (1.9)1 (1.9)0

Other Asian
countries(n  3)bUSA(n  29)b

Europe(n  29)b,c

Africa(n  5)b

P valued0

3 (10.3)
3 (10.3)

3 (10.3)
3 (10.3)

0

0.05

1 (33.3)

5 (17.2)

3 (60)
1 (20)
1 (20)
1 (20)

0.003

1 (33.3)

5 (17.2)
1 (3.4)2 (6.9)02 (6.9)2 (6.9)5 (17.2)
3 (10.3)
1 (3.4)1 (3.4)1 (3.4)1 (3.4)

0

0.213

0

0.329

1 (20)
1 (20)

0.007

3 (60)
1 (20)

0.211

2 (6.9)8 (14.8)
8 (14.8)0031 (57.4)
31 (57.4)

6 (50)
4 (33.3)
2 (16.7)

020 (37)20 (37)2 (16.7)
1 (8.3)1 (8.3)

1 (33.3)
1 (33.3)

2 (6.9)2 (6.9)21 (72.4)
13 (44.8)
6 (20.7)
2 (6.9)13 (44.8)
6 (20.7)
5 (17.2)

1 (3.4)9 (31)
2 (6.9)1 (3.4)4 (13.8)
6 (20.7)

2 (6.9)15 (51.7)
8 (27.6)

6 (20.7)
7 (24.1)

2 (40)
20 (37)20 (37)2 (16.7)
1 (8.3)1 (8.3)

1 (33.3)

4 (80)
1 (20)
1 (20)

1 (33.3)

0.054

5 (17.2)
2 (40)

2 (3.7)2 (3.7)038 (70.4)
38 (70.4)
20 (37)20 (37)2 (6.9)7 (24.1)0

3 (10.3)
3 (10.3)

7 (24.1)
6 (20.7)
1 (3.4)0<0.001

0

0

0

0

0.205

8 (66.7)
7 (58.3)
1 (8.3)0

1 (33.3)
1 (33.3)

4 (13.8)
4 (13.8)

1 (20)
1 (20)<0.0010

1 (3.4)2 (6.9)2 (6.9)6 (20.7)
5 (17.2)
1 (3.4)
0

0

0.874

1 (33.3)

1 (3.4)00

0<0.0016 (20.7)
5 (17.2)
1 (3.4)16 (55.2)
4 (13.8)
1 (3.4)1 (3.4)0<0.0015 (100)2 (40)
1 (20)

0.036

3 (25)
1 (8.3)2 (16.7)
7 (58.3)
6 (50)
1 (8.3)3 (25)
1 (8.3)2 (16.7)

1 (33.3)
0
1 (33.3)

3 (10.3)
3 (10.3)

1 (33.3)

16 (55.2)
6 (20.7)
8 (27.6)02 (6.9)02 (40)
00

8 (27.6)
3 (10.3)
3 (10.3)

0

0.05(Continued on following page)

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aadA1

ﬁmH allele or
sublineage

Total no. of
isolates(n  132)

Attachment

Genome Analysis of E. coli ST131 in Asia

TABLE 2 (Continued)
No. of isolates (%) from:
Antibiotic(s) andantibiotic resistancegene
blaKPC-3
Macrolide
mphA

catB3

Sulfonamide
sul1

sul2

Trimethoprim
dfrA12
dfrA17

dfrA1

Tetracyclinetet(A)

Total
H30Rx
H30R
H41

Nepal
(n  54)0Japan(n  12)a
0

69
56
7
4

46 (85.2)
46 (85.2)

5 (41.7)
3 (25)
2 (16.7)

Total
H30Rx
H27
H41
Total
H30Rx
H30R

4
1
1
2
51
48
3

0

Total
H30Rx
H30R
H27
H41
Total
H30Rx
H30R
H27
H41

Other Asian
countries(n  3)b0

USA(n  29)b
6 (20.7)
6 (20.7)

Europe(n  29)b,c0Africa(n  5)b0

P valued<0.0010

10 (34.5)
5 (17.2)
4 (13.8)
1 (3.4)8 (27.6)2 (6.9)1 (3.4)3 (10.3)

0<0.001000

2 (6.9)2 (40)
1 (20)
1 (20)<0.00137 (68.5)
37 (68.5)

6 (50)
5 (41.7)
1 (8.3)0

2 (6.9)1 (3.4)1 (3.4)2 (6.9)6 (20.7)
5 (17.2)
1 (3.4)0<0.001

82
60
11
1
8
52
32
9
2
9

39 (72.2)
39 (72.2)

6 (50)
4 (33.3)
2 (16.7)

022 (75.9)
13 (44.8)
7 (24.1)

13 (44.8)
3 (10.3)
2 (6.9)2 (40)
1 (20)

0.01

2 (6.9)18 (62.1)
9 (31)
7 (24.1)

6 (20.7)
8 (27.6)
1 (3.4)2 (6.9)7 (24.1)

Total
H30Rx
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H22
H41

8
8
67
50
9
6
5
1
1
1
2

8 (14.8)
8 (14.8)
31 (57.4)
31 (57.4)

0

0

0

0

0

0.031

6 (50)
4 (33.3)
2 (16.7)

0

0.014

0

0

9 (31)
2 (6.9)1 (3.4)4 (13.8)
4 (13.8)
1 (3.4)

1 (20)
1 (20)0

20 (69)12 (41.3)
6 (20.7)
2 (6.9)1 (3.4)

0

0.054

Total
H30Rx
H30R
H22
H27
H41

86
61
10
4
2
9

40 (74.1)
40 (74.1)

4 (80)
1 (20)
1 (20)

0.292

1 (20)
20 (37)20 (37)2 (16.7)
1 (8.3)1 (8.3)

1 (33.3)
1 (33.3)

3 (60)
1 (20)

0.041

2 (40)

1 (3.4)1 (3.4)2 (6.9)8 (66.7)
7 (58.3)
1 (8.3)

1 (33.3)
1 (33.3)

18 (62.1)
11 (37.9)
5 (17.2)

15 (51.7)
2 (6.9)2 (6.9)4 (13.8)

2 (6.9)7 (24.1)

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Chloramphenicol
catA1

ﬁmH allele or
sublineage
Total
H30Rx

Total no. of
isolates(n  132)66

2 (40)

aIncludes one publicly available sequence of E. coli ST131.
bPublicly available sequence data for E. coli ST131.
cThree isolates from Europe were from Lebanon.
dThe P values are comparing the prevalence of each antibiotic resistance gene among geographical regions. P values in boldface type represent statistically signiﬁcantresults.

care facilities in Japan, including the hospital in this study, to minimize transmission.
These factors might explain at least in part the differences in the prevalence andclonality of ST131 between these two Asian countries. In contrast, prevalent clonal
spread of ESBL-E. coli appears to have occurred in Nepal, where poor infection control
practices and sanitation might facilitate dissemination of antimicrobial-resistant strains.
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TABLE 3 Comparison of virotypes and virulence-associated traits and genes in Escherichia coli ST131
No. of isolates (%) from:
Virotype orvirulence-associatedtrait and gene
Virotypes
A

B

D

e

E
Other

Virulence-associatedtraits and genes
Adhesin
papGII

iha

hra

Toxin
hlyA

cnf1

sat

Siderophore
iroN

Total no. of
isolates(n  132)

Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H41
Total
H22
Total
H30Rx
H30R
Total
H30Rx
H30R
H27
H41

12
9
1
2
4
1
1
2
54
33
14
4
9
9
18
17
1
35
28
2
2
3

Total
H30Rx
H30R
H27
Total
H30Rx
H30R
H22
H41
Total
H30Rx
H27

46
43
1
2
119
86
16
4
10
33
11
1

35 (64.8)
35 (64.8)

3 (25)
2 (16.7)
1 (8.3)

1 (33.3)
1 (33.3)

2 (6.9)2 (6.9)1 (3.4)1 (3.4)53 (98.1)
53 (98.1)

10 (83.3)
8 (66.7)
2 (16.7)

3 (100)2 (66.7)
1 (33.3)

27 (93.1)
16 (55.2)
9 (31)

11 (20.4)

3 (25)
3 (25)0

2 (6.9)8 (27.6)
8 (27.6)

24 (82.8)
6 (20.7)
3 (10.3)
4 (13.8)
8 (27.6)
0

Total
H30Rx
H30R
H27
Total
H30Rx
H30R
H27
Total
H30Rx
H30R
H22
H41

20
7
1
2
19
7
1

10 (18.5)

3 (25)
2 (16.7)
1 (8.3)0

2 (6.9)2 (6.9)1 (3.4)1 (3.4)4 (80)
2 (40)

9 (16.7)

3 (25)
2 (16.7)
1 (8.3)0

2 (6.9)2 (6.9)1 (3.4)1 (3.4)2 (40)
4 (80)
2 (40)

118
33
16
4
8

54 (100)

10 (83.3)
8 (66.7)
2 (16.7)

3 (100)2 (66.7)
1 (33.3)

26 (89.7)
15 (51.7)
9 (31)

22 (75.9)
6 (20.7)
3 (10.3)
4 (13.8)
6 (20.7)

Total
H30Rx
H30R
H22
H41

11
1
1
7
2

0

Nepal
(n  54)

Japan(n  12)a5 (9.3)5 (9.3)2 (16.7)
1 (8.3)1 (8.3)00
13 (24.1)
13 (24.1)

Other Asian
countries(n  3)bUSA(n  29)b

Europe(n  29)b,c

Africa(n  5)bPvalued0

1 (3.4)1 (3.4)4 (13.8)
2 (6.9)

0

0.619

0

0

0

0

0.012

6 (50)
5 (41.7)
1 (8.3)2 (66.7)
1 (33.3)
1 (33.3)

1 (20)<0.001000

23 (79.3)
12 (41.4)
9 (31)
2 (6.9)0

10 (18.5)
10 (18.5)

3 (25)
2 (16.7)
1 (8.3)1 (8.3)0

2 (6.9)2 (6.9)1 (33.3)
1 (33.3)

3 (10.3)
1 (3.4)2 (6.9)26 (48.1)
26 (48.1)

2 (6.9)4 (13.8)
1 (3.4)1 (3.4)2 (6.9)9 (31)
2 (6.9)2 (6.9)2 (6.9)9 (31)
9 (31)
1 (3.4)1 (3.4)

1 (20)

2 (6.9)0<0.001

2 (40)
2 (40)

0.09

2 (40)<0.001

2 (40)
1 (8.3)2 (6.9)4 (80)
2 (40)
2 (40)
2 (40)
1 (20)
1 (20)
1 (20)<0.001

0.001

0.085

1 (20)

2 (6.9)000

11 (37.9)
1 (3.4)1 (3.4)7 (24.1)
2 (6.9)2 (40)
3 (60)
2 (40)
1 (20)

0<0.001<0.001

0.004<0.001(Continued on following page)

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C

ﬁmH
allele orsublineage

Attachment

Genome Analysis of E. coli ST131 in Asia

TABLE 3 (Continued)
No. of isolates (%) from:
Virotype orvirulence-associatedtrait and gene
fyuA

ireA

iss

Invasin
ibeA
Miscellaneous
traT

ompT

malX

Total no. of
isolates(n  132)
130
87
19
9
2
11
3
3

Total
H30Rx
H30R
H22
H27
Total
H30Rx
H30R
H22
H41

43
28
8
4
1
9
1
1
5
2

13 (23.6)
13 (23.6)

4 (33.3)
2 (16.7)
2 (16.7)

0

0

Total
H22

9
9

0

0

0

0

Total
H30Rx
H30R
H22
H27
H41
Total
H30Rx
H30R
H22
H27
H41
Total
H30Rx
H30R
H22
H27
H41

107
68
16
9
1
10
129
87
18
9
2
11
131
88
19
9
2
11

47 (87)47 (87)

10 (83.3)
7 (58.3)
3 (25)

1 (33.3)

Nepal(n  54)53 (98.1)
53 (98.1)

Japan(n  12)a12 (100)
8 (66.7)
3 (25)

Other Asian
countries(n  3)b3 (100)2 (66.7)
1 (33.3)

USA(n  29)b
29 (100)
16 (55.2)
11 (37.9)

Europe(n  29)b,c
28 (96.6)
6 (20.7)
3 (10.3)
9 (31)

2 (6.9)0

8 (27.6)
00

13 (44.8)
9 (31)
4 (13.8)

9 (31)
2 (6.9)1 (3.4)4 (13.8)

1 (33.3)
1 (33.3)

08 (27.6)

Africa(n  5)b
5 (100)2 (40)
1 (20)

P
valued0.911

2 (40)
1 (8.3)0

2 (3.7)2 (3.7)

1 (33.3)
1 (33.3)00.009

4 (80)
2 (40)
1 (20)

0.066

1 (20)
0<0.0019 (31)
9 (31)

0<0.00129 (100)
6 (20.7)
3 (10.3)
9 (31)

3 (60)
2 (40)<0.001

1 (33.3)

17 (58.6)
6 (20.7)
9 (31)

2 (66.7)
1 (33.3)
1 (33.3)

2 (6.9)28 (96.6)
16 (55.2)
10 (34.5)

8 (27.6)
28 (96.6)
6 (20.7)
3 (10.3)
9 (31)

3 (100)2 (66.7)
1 (33.3)

2 (6.9)29 (100)
16 (55.2)
11 (37.9)

8 (27.6)
28 (96.6)
6 (20.7)
3 (10.3)
9 (31)

2

8 (27.6)

1 (3.4)5 (17.2)2 (6.9)1 (20)
54 (100)
54 (100)

12 (100)
8 (66.7)
3 (25)

5 (100)2 (40)
1 (20)

0.01

2 (40)
54 (100)
54 (100)

1 (8.3)12 (100)
8 (66.7)
3 (25)

5 (100)2 (40)
1 (20)

0.911

2 (40)
1 (8.3)aIncludes one publicly available sequence of E. coli ST131.
bPublicly available sequence data for E. coli ST131.
cThree isolates from Europe were from Lebanon.

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Protectins
kﬁA

ﬁmH
allele orsublineage
Total
H30Rx
H30R
H22
H27
H41
Total
H30Rx

dThe P values are comparing the prevalence of each antibiotic resistance gene among geographical regions. P values in boldface type represent statistically signiﬁcantresults.
eAll isolates of virotype D were identiﬁed as D3.

In Nepal, antibiotics can be purchased in the community at general retail stores andpharmacies. According to recent reports, 80% of the drugs are purchased outside thegovernment-supplied health system (17, 18). In addition, inappropriate prescription
occurs in up to 40% of patients (17, 18).
E. coli ST131 isolates from the United States are distributed across the phylogeny,
even those isolates that were collected in the same year. The wide diversity of
population in the United States might explain in part this phylogenetic characteristic.
E. coli ST131 isolates from Europe (including three E. coli ST131 isolates from Lebanon)
could be roughly divided into four clusters, with one major cluster consisting mainly of
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Miyoshi-Akiyama et al.

TABLE 4 Comparison of plasmid replicon types of Escherichia coli ST131
No. of isolates (%) from:
Replicontype
IncFIA

IncFIB

IncFII

IncI1

IncFrepB

IncB/O
IncY
IncN

IncP
IncA/C

Total no. of
isolates
84
62
17
2
89
48
18
9
2
10
3
1
2
95
60
17
8
8
9
5
2
2
40
32
4
4
1
1
2
1
1
8
6
1
1
3
1
2
2
2

Nepal
(n  54)38 (70.4)
38 (70.4)

Japan(n  12)a6 (50)
4 (33.3)
2 (16.7)

40 (74.1)
40 (74.1)

8 (66.7)
5 (41.7)
3 (25)

Other Asian
countries(n  3)b2 (66.7)
1 (33.3)
1 (33.3)
1 (33.3)

USA(n  29)b25 (86.2)
14 (48.3)
11 (37.9)

1 (33.3)

14 (48.3)
2 (6.9)10 (34.5)
2 (6.9)0Europe(n  29)b,c
10 (34.5)
3 (10.3)
2 (6.9)2 (6.9)23 (79.3)
1 (3.4)3 (10.3)
9 (31)

Africa(n  5)b3 (60)
2 (40)
1 (20)
3 (60)

P valued
0.002

0.09

1 (20)
2 (40)000

43 (79.6)
43 (79.6)

4 (33.3)
2 (16.7)
2 (16.7)

1 (33.3)

2 (16.7)
1 (8.3)1 (8.3)

1 (33.3)

13 (24.1)
13 (24.1)

3 (25)
2 (16.7)
1 (8.3)0

15 (51.7)
12 (41.4)
3 (10.3)

0

0

0

0

0

0

1 (3.4)1 (3.4)1 (3.4)0

2 (16.7)
1 (8.3)1 (8.3)0

1 (3.4)2 (6.9)2 (6.9)

0

0

0

0

0

0

0

2 (6.9)2 (6.9)3 (5.6)3 (5.6)

1 (33.3)

23 (79.3)
11 (37.9)
10 (34.5)
2 (6.9)1 (3.4)1 (3.4)8 (27.6)
3 (10.3)
1 (3.4)2 (6.9)21 (72.4)
2 (6.9)3 (10.3)
8 (27.6)
6 (20.7)
2 (6.9)00.053

3 (60)
2 (40)
1 (20)

0.017

0

0.29

2 (6.9)7 (24.1)
3 (10.3)

2 (40)
2 (40)

0.091

4 (13.8)
0

0

0.611

1 (3.4)1 (3.4)00.763

4 (13.8)
3 (10.3)00.092

2 (40)<0.001

2 (40)
00.205

1 (33.3)

1 (3.4)1 (3.4)1 (3.4)0

aIncludes one publicly available sequence of E. coli ST131.
bPublicly available sequence data for E. coli ST131.
cThree isolates from Europe were from Lebanon.
dThe P values are comparing the prevalence of each antibiotic resistance gene among geographical regions. P values in boldface type represent statistically signiﬁcantresults.

E. coli ST131 isolates from Denmark and one cluster consisting of two E. coli ST131
isolates from Lebanon. E. coli ST131 isolates from Denmark predominantly consisted ofE. coli ST131 from Europe (72%). Four E. coli ST131 isolates from Tanzania and one
isolate from South Africa were included in E. coli ST131 from Africa. E. coli ST131 isolates
from Africa were phylogenetically diverse. E. coli ST131 isolates from Asian countries
other than Nepal and Japan included one isolate from South Korea, one isolate from
India, and one isolate from Thailand. They did not belong to the same cluster.
There are several limitations to this study. Since ESBL-E. coli ST131 isolates from
Nepal and Japan were collected during a relatively short period of time, epidemiologically related isolates may have been included in the analysis, and the trends acrossyears could not be elucidated. However, as shown in Fig. 1, the distribution of ESBL-E.
coli ST131 isolates in each region suggests that the contribution of such clonal isolates
was unlikely to have been remarkable. Due to the small number of isolates from Japan,
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IncFICﬁmH allele or
sublineage
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H22
H27
H41
Total
H22
H41
Total
H30Rx
H30R
H22
H41
Total
H30Rx
H30R
H41
Total
H30Rx
H30R
H22
Total
H30R
Total
H30Rx
H30R
Total
H30Rx
H30R
H22
Total
H30R
H27
Total
H30R

Attachment

Genome Analysis of E. coli ST131 in Asia

the power of the statistical comparisons between isolates from Nepal and Japan islimited. Also, only small numbers of E. coli ST131 isolates from some geographicalregions such as Africa could be included due to limited publicly available data. Finally,
the difference in the molecular analyses, i.e., WGS and PCR, used might have caused thedifference in the identiﬁcation of the target genes.
In conclusion, comparative analysis of ESBL-E. coli ST131 genomes from Japan and
Nepal and those from other geographical regions revealed distinct phylogenetic characteristics of the spread of ESBL-E. coli ST131 in these two geographical areas of Asia.
Multiple yet distinct factors might contribute to the local spread of ESBL-E. coli ST131
in each region.

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MATERIALS AND METHODS
Isolates and susceptibility testing. All ESBL-E. coli isolates were serially collected from the Tribhuvan
University Teaching Hospital (444 beds), Kathmandu, Nepal, between 1 February 2013 and 31 July 2013
and from the National Center for Global Health and Medicine (NCGM) (781 beds), Tokyo, Japan, between
1 October 2013 and 30 September 2014; both centers are tertiary teaching hospitals. Some of the isolates
from Nepal were included in our previous study (5).
The E. coli strains from both Nepal and Japan were identiﬁed, and their susceptibility was tested inaccordance with the Clinical and Laboratory Standards Institute (CLSI) criteria (19) by using an automatedbroth microdilution system (MicroScan; Siemens AG, Germany) unless otherwise stated. The MIC of
fosfomycin was determined using an NC6.11J panel (Siemens AG, Germany) which contains glucose-6phosphate, and its susceptibility status was determined as previously reported (20). ESBL production was
conﬁrmed by disc diffusion tests in accordance with the 2009 CLSI criteria (19). If multiple ESBL-E. coli
isolates were identiﬁed from a patient during the study period, only the ﬁrst isolate was included.
Whole-genome sequencing and phylogenetic analysis. Molecular analysis was conducted at the
Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine,Tokyo, Japan. The strains were cultured overnight in lysogeny broth (LB) (Nakarai Tesque, Kyoto, Japan),and genomic DNA was puriﬁed using a DNeasy blood and tissue kit (Qiagen, Venlo, Netherlands). The
genomes of the isolates were then subjected to MiSeq sequencing by using Nextera XT library kits
(Illumina, Inc., San Diego, CA), according to the manufacturer’s instructions. Approximately 1 million
paired-end reads (301 bp  2) were obtained from each genome and analyzed using the CLC Genomics
Workbench software (CLC Bio, Aarhus, Denmark). The reads from each isolate were trimmed by screening
for base quality (quality score limit of 0.05; reads that contained greater than two ambiguous nucleotides
or that were less than 15 bp in length were removed) (21). The resulting sequencing data were registeredwith the DNA Data Bank of Japan (DDBJ) (DDBJ accession no. DRA003515). Velvet and Mummer (opensource MUMmer 3.0) described previously were used for de novo assembly to prepare contigs and single
nucleotide polymorphism (SNP) calling, respectively (21, 22).
All genome sequence data for ST131 strains available up to January 2015 were downloaded from the
NCBI database (http://www.ncbi.nlm.nih.gov/genome/genomes/167?genome_assembly_idgroup161531;
accessed 26 September 2016) and included for phylogenetic analysis to determine the sequence
diversity. SNP concatemers were prepared using a custom script and aligned using MAFFT (23). E. coli
SE15 (accession no. NC_013654.1) was used as the reference to call SNPs of the isolates after mobile
elements were removed from the chromosome. The appropriate evolutionary model (transversion modelplus gamma distribution) was determined using jModelTest2 (24). Maximum likelihood (ML) phylogenetic trees were estimated using PHYML 3.0 (25). Branch support for nodes was assessed using the
Shimodaira and Hasegawa (SH) test implemented in PHYML.
The contigs were subjected to further analyses by using the BLAST algorithm (26) and ResFinder (27)
to identify whether virulence marker genes and drug resistance genes were present in the genomes (28,29). An identiﬁcation rate of more than 97% was considered positive for each target gene. Phylotypes(30), virulence genotypes (31), ﬁmH alleles and H30R and H30Rx sublineages of ST131 (10), anddistribution of acquired drug resistance genes were determined. The nucleotide sequences corresponding to the ST131 multilocus sequence typing (MLST) allelic proﬁle (adk53, fumC40, gyrB47, icd13, mdh36,
purA28, and recA29) were downloaded from the University of Warwick (http://mlst.warwick.ac.uk/mlst/)
and compared by BLAST algorithm to the WGS data (3, 26). Virotypes were determined based on thevirulence gene scheme (13). Sequence typing of plasmid replicons was performed as described elsewhere (32, 33).
We added one E. coli ST131 isolate from Japan for which sequence data were available publicly(http://www.ncbi.nlm.nih.gov/genome/genomes/167?genome_assembly_idgroup161531;
accessed
26 September 2016). SE15 is a completely sequenced ST131 representative strain, which was used as areference in the current study and a previous study as well (3).
Statistical analysis. All statistical analyses were performed using IBM-SPSS statistics 20 (2012).
Bivariate analyses were performed using Fisher’s exact test or chi-square test for categorical variables andthe Mann-Whitney U test for continuous variables. All P values were two sided. The percentage values
included in this article are the “valid percentages,” which exclude the missing data.
Accession number(s). Sequence data were deposited in the DNA Data Bank of Japan (DDBJ) underaccession no. DRA003515.
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Miyoshi-Akiyama et al.

ACKNOWLEDGMENTS
We thank Y. Sakurai for help in the preparation of the ﬁgure.
This work was supported in part by a grant in Clinical Epidemiology Research, St.
Luke’s International University, Tokyo, Japan (2016).
FUNDING INFORMATIONThis work was funded in part by St. Luke’s International University, Tokyo, Japan.

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Clinical epidemiology and molecular analysis of extended-spectrumbeta-lactamase-producing Escherichia coli in Nepal: characteristics ofsequence types 131 and 648. Antimicrob Agents Chemother 59:
3424 –3432. http://dx.doi.org/10.1128/AAC.00270-15.
6. Pitout JD, Campbell L, Church DL, Gregson DB, Laupland KB. 2009.
Molecular characteristics of travel-related extended-spectrum-betalactamase-producing Escherichia coli isolates from the Calgary HealthRegion. Antimicrob Agents Chemother 53:2539 –2543. http://dx.doi.org/
10.1128/AAC.00061-09.
7. Suzuki S, Shibata N, Yamane K, Wachino J, Ito K, Arakawa Y. 2009.
Change in the prevalence of extended-spectrum-beta-lactamaseproducing Escherichia coli in Japan by clonal spread. J Antimicrob Chemother 63:72–79. http://dx.doi.org/10.1093/jac/dkn463.
8. Matsumura Y, Yamamoto M, Nagao M, Hotta G, Matsushima A, Ito
Y, Takakura S, Ichiyama S, Kyoto-Shiga Clinical Microbiology Study
Group. 2012. Emergence and spread of B2-ST131-O25b, B2-ST131-O16
and D-ST405 clonal groups among extended-spectrum-beta-lactamaseproducing Escherichia coli in Japan. J Antimicrob Chemother 67:
2612–2620. http://dx.doi.org/10.1093/jac/dks278.
9. Matsumura Y, Johnson JR, Yamamoto M, Nagao M, Tanaka M,
Takakura S, Ichiyama S, Kyoto-Shiga Clinical Microbiology Study
Group. 2015. CTX-M-27- and CTX-M-14-producing, ciproﬂoxacinresistant Escherichia coli of the H30 subclonal group within ST131 drive
a Japanese regional ESBL epidemic. J Antimicrob Chemother 70:
1639 –1649. http://dx.doi.org/10.1093/jac/dkv017.
10. Price LB, Johnson JR, Aziz M, Clabots C, Johnston B, Tchesnokova V,
Nordstrom L, Billig M, Chattopadhyay S, Stegger M, Andersen PS,
Pearson T, Riddell K, Rogers P, Scholes D, Kahl B, Keim P, Sokurenko
EV. 2013. The epidemic of extended-spectrum-beta-lactamaseproducing Escherichia coli ST131 is driven by a single highly pathogenic
subclone, H30-Rx. mBio 4:e00377-13. http://dx.doi.org/10.1128/
mBio.00377-13.
11. Kim SY, Park YJ, Johnson JR, Yu JK, Kim YK, Kim YS. 2016. Prevalence
and characteristics of Escherichia coli sequence type 131 and its H30 and
H30Rx subclones: a multicenter study from Korea. Diagn Microbiol Infect
Dis 84:97–101. http://dx.doi.org/10.1016/j.diagmicrobio.2015.10.016.
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Genome Analysis of E. coli ST131 in Asia

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30. Clermont O, Christenson JK, Denamur E, Gordon DM. 2013. The

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AUTHOR CORRECTION

crossmCorrection for Miyoshi-Akiyama et al.,“Comparative Genome Analysis ofExtended-Spectrum--LactamaseProducing Escherichia coli SequenceType 131 Strains from Nepal and Japan”
Tohru Miyoshi-Akiyama,a Jatan Bahadur Sherchan,b Yohei Doi,cMaki Nagamatsu,d,e Jeevan B. Sherchand,f Sarmila Tandukar,f Norio Ohmagari,dTeruo Kirikae,e Hiroshi Ohara,g Kayoko Hayakawad
Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japana; Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel,
Nepalb; Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania,
USAc; Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japand;Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japane; Public Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital,
Kathmandu, Nepalf; Department of International Medical Cooperation, National Center for Global Health andMedicine, Tokyo, Japang

Volume 1, no. 5, e00289-16, 2016, https://doi.org/10.1128/mSphere.00289-16. Due tothe classiﬁcation deﬁnitions that we used for our work, six isolates were misclassiﬁed forthe ﬁmH allele or sublineage in our paper. The revised ﬁmH alleles or sublineages are
as follows: HVH186 (H30Rx), J069 (H30Rx), N439/N1027/N1471 (H30R [non-Rx]), andN1449 (H41). A revised ﬁgure and revised tables are available from the authors uponrequest. (We are extremely grateful to Yasufumi Matsumura, Kyoto University Graduate
School of Medicine, Japan, for his tremendous help on molecular analysis.)

Published 24 May 2017
Citation Miyoshi-Akiyama T, Sherchan JB, Doi
Y, Nagamatsu M, Sherchand JB, Tandukar S,
Ohmagari N, Kirikae T, Ohara H, Hayakawa K.
2017. Correction for Miyoshi-Akiyama et al.,“Comparative genome analysis of extendedspectrum-β-lactamase-producing Escherichia
coli sequence type 131 strains from Nepal andJapan.” mSphere 2:e00136-17. https://doi.org/
10.1128/mSphere.00136-17.
Copyright © 2017 Miyoshi-Akiyama et al. Thisis an open-access article distributed under theterms of the Creative Commons Attribution 4.0
International license.
Address correspondence to Kayoko Hayakawa,kayokohayakawa@gmail.com.

46

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MECHANISMS OF RESISTANCE

PER-8, a Novel Extended-Spectrum
-Lactamase PER Variant, from an
Acinetobacter baumannii Clinical Isolate
in NepalTatsuya Tada,a Shovita Shrestha,b Kayo Shimada,a Hiroshi Ohara,cJeevan B. Sherchand,b Bharat M. Pokhrel,d Teruo Kirikaea
Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japana; Department of Medical Microbiology, Tribhuvan University, Maharajgunj, Kathmandu, Nepalb;
Department of International Medical-Cooperation, National Center for Global Health and Medicine, Tokyo,
Japanc; Department of Microbiology, Institute of Medicine, Tribhuvan University, Maharajgunj, Kathmandu,
Nepald

ABSTRACT A novel PER-type extended-spectrum -lactamase, PER-8, was identiﬁed
in an Acinetobacter baumannii clinical isolate obtained in Nepal. The amino acid sequence of PER-8 has a substitution at position 39 (Gly to Glu) compared with that of
PER-7. The kcat/Km ratio of PER-8 for aztreonam was lower than that of PER-7, while
the kcat/Km ratio of PER-8 for imipenem was higher than that of PER-7. The genomic
environment surrounding blaPER-8 was intI1 blaPSE-1 qacEDI sulI ISCR1-blaPER-8 gts sulI
orfX on a 100-kb plasmid.
KEYWORDS Acinetobacter baumannii, PER-type ESBLs, plasmid-mediated resistanceT

he class A extended-spectrum -lactamases (ESBLs) confer resistance to expandedspectrum cephalosporins and are inhibited in vitro by clavulanic acid and tazobactam (1). Resistance to broad-spectrum cephalosporins in Acinetobacter baumannii
mostly results from overexpression of the natural AmpC-type enzyme or from acquisition of ESBLs. To date, the following 5 types of ESBL genes have been reported in A.
baumannii: blaPER (2), blaGES (3), blaVEB (4), blaTEM (5), and blaCTX-M (6). The blaPER-1 gene
was ﬁrst found in a Pseudomonas aeruginosa isolate (7). Since then, it has been reported
worldwide in Enterobacteriaceae (8–11) and A. baumannii (2, 4). Until now, 7 types of
PER variants have been reported in clinical isolates of Enterobacteriaceae (12–16) and A.
baumannii (17) in various countries. The phylogenic tree based on amino acid sequences (Clustal W2) revealed two clusters in PER-type variants, one containing PER-1,
PER-3, PER-4, PER-5, and PER-7 and the other containing PER-2 and PER-6.
Multidrug-resistant A. baumannii IOMTU442 and IOMTU448 were isolated fromsamples of wound swabs from hospitalized patients at a university hospital in 2013 in
Nepal. The isolates were identiﬁed phenotypically, and species identiﬁcation was
conﬁrmed by comparing sequences of 16S rRNA, gyrB, and blaOXA-51-like genes. Escherichia coli DH5 (TaKaRa Bio, Shiga, Japan) and E. coli BL21-CodonPlus(DE3)-RIP (Agilent
Technologies, Santa Clara, CA) were used as hosts for recombinant plasmids andexpression of blaPER genes, respectively.
MICs were determined using the broth microdilution method as recommended by
CLSI (M100-S23). Whole genomes of IOMTU442 and IOMTU448 were extracted with
DNeasy blood and tissue kits (Qiagen, Tokyo, Japan) and sequenced by MiSeq (Illumina,
San Diego, CA). Multilocus sequence typing (MLST) was performed as described by the
protocols of the Institut Pasteur MLST (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db
pubmlst_abaumannii_pasteur_seqdef) databases.
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Received 1 November 2016 Returned for
modiﬁcation 20 November 2016 Accepted
17 December 2016
Accepted manuscript posted online 28
December 2016
Citation Tada T, Shrestha S, Shimada K, Ohara
H, Sherchand JB, Pokhrel BM, Kirikae T. 2017.
PER-8, a novel extended-spectrum β-lactamase
PER variant, from an Acinetobacter baumannii
clinical isolate in Nepal. Antimicrob Agents
Chemother 61:e02300-16. https://doi.org/
10.1128/AAC.02300-16.
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Teruo Kirikae,
tkirikae@ri.ncgm.go.jp.
T.T. and S.S. contributed equally to this article.

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Tada et al.

Antimicrobial Agents and Chemotherapy

MIC (mg/liter)
Antibiotic(s)Amikacin
Ampicillin
Ampicillin-sulbactam
Arbekacin
Aztreonam
Cefepime
Cefmetazole
Cefotaxime
Cefoxitin
Cefpirome
Ceftazidime
Cephradine
Ciproﬂoxacin
Colistin
Fosfomycin
Gentamicin
Imipenem
Kanamycin
Levoﬂoxacin
Meropenem
Moxalactam
Penicillin G
Piperacillin
Piperacillin-tazobactam
Tigecyclinea

IOMTU442
1,024
1,02464
1,024
1,024
512
256
512
512
256
1,024
1,02432
0.25
128
1,0242
1,02432
1128
1,024
1,02464
0.25

IOMTU4488
1,02464
2
1,024512
1,024
1,024
1,024256
512
1,02432
0.5
256
1,0248
1,0248
16
128
1,024512
256
0.5

pHSG398/PER-7
NDb
32
2
ND
8
0.125
1
16
4
0.063
32
128
ND
ND
ND
ND
0.125
ND
ND
0.063
0.06364
4
2
ND

pHSG398/PER-8
ND
16
2
ND
4
0.25
1
8
4
0.063
64
128
ND
ND
ND
ND
0.125
ND
ND
0.063
0.06364
4
2
ND

pHSG398
ND
2
2
ND0.063
0.0631
0.0634
0.063
0.5
16
ND
ND
ND
ND
0.063ND
ND
0.063
0.06332
2
2ND

aThe ratio of ampicillin to sulbactam was 2:1. The ratio of piperacillin to tazobactam was 4:1.
bND, not determined.

The blaPER-7 and blaPER-8 were cloned into the corresponding sites of the pHSG398
vector plasmid (TaKaRa, Shiga, Japan) using the primer set EcoRI-PER-F (5=-GGGAATTCATGGAATTGCCCAATATTATG-3=) and PstI-PER-R (5=-AACTGCAGTCAGCGCAGCTTGTCGGCCAT-3=). E. coli DH5 was transformed with pHSG398-PERs, and the transformants
were selected on chloramphenicol-containing plates (30 g/ml).
The open reading frames of PER-7 and PER-8, without signal peptide regions, werecloned into the pET28a expression vector (Novagen, Inc., Madison, WI, USA) using theprimer set BamHI-TEV-PER-F (5=-ATGGATCCGAAAACCTGTATTTCCAAGGCCAGCAAATGG
AAACTGGCGAC-3=) and XhoI-PER-R (5=-ATCTCGAGTCAGCGCAGCTTGTCGGCCATG-3=).
The plasmids were used to transform E. coli BL21-CodonPlus(DE3)-RIP (Agilent Technologies, Santa Clara, CA, USA). Recombinant PERs were puriﬁed, and initial hydrolysis
rates were determined as previously described (18).
To determine the size of the plasmid harboring blaPER-8, plasmid DNA in iOMTU442
was extracted and digested with S1 nuclease. Pulsed-ﬁeld gel electrophoresis (PFGE)
and Southern hybridization were performed. A probe for blaPER from IOMTU442 wasampliﬁed by PCR using the EcoRI-PER-F and PstI-PER-R primer set. A DNA plug of
IOMTU448, digested with I-CeuI, was prepared, separated by pulsed-ﬁeld gel electrophoresis, and subjected to Southern hybridization using 16S rRNA and blaPER-7 probes.
Signal was detected using digoxigenin (DIG) High Prime DNA labeling and detection
starter kit II (Roche Applied Science, Indianapolis, IN, USA).
IOMTU442 had blaOXA-70, blaPSE-1, and a novel blaPER variant, blaPER-8. IOMTU448 had
blaOXA-23, blaOXA-371, blaOXA-420 (blaOXA-58-like), and blaPER-7. The blaOXA-371 gene in
IOMTU 448 was grouped with the blaOXA-69-type genes, whereas the blaOXA-70 gene in
IOMTU442 was not grouped with the blaOXA-66-type genes, the blaOXA-69-type genes, orthe blaOXA-71-type genes. Neither blaOXA-70 nor blaOXA-371 was ﬂanked by ISAba1. The
MICs for A. baumannii IOMTU442 and IOMTU448 are shown in Table 1. IOMTU442 andIOMTU448 were found to belong to ST103 and ST623, respectively. A. baumannii
isolates belonging to ST103 have been found in Egypt (19) and Portugal (20), and ST623
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TABLE 1 MICs of various -lactams for A. baumannii strains IOMTU442 and IOMTU448 and E. coli DH5 transformed with PER-7- or PER8-encoding plasmids

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Antimicrobial Agents and Chemotherapy

PER-7

-Lactam
Ampicillin
Penicillin G
Piperacillin
Cefepime
Cefmetazole
Cefotaxime
Cefoxitin
Ceftazidime
Cephradine
Moxalactam
Aztreonam
Imipenem
Meropenem

Km (M)b13  413.7  2.812.6  3.681  7NHc
138  63
NH
258  2254  5
NH
17  3172  33
28  8PER-8kcat (s1)b52  216.0  0.20.60  0.02
10.2  0.3NH84  20
NH33  262  2NH
8.8  0.2
0.13  0.01
0.13  0.01

kcat/Km (M1 s1)
4.4
1.2
0.051
0.13
NH
0.65
NH
0.13
1.2
NH
0.54
0.00076
0.0051Km (M)b
19  313.7  3.18.6  1.8110  16
NH
114  17
NH
212  26
48  8
NH
14  2101  1032  3

kcat (s1)b54  1
16.4  0.30.65  0.03
12  1NH70  4NH
31  266  2NH
8.8  0.2
0.16  0.01
0.17  0.01

kcat/Km (M1 s1)
2.9
1.2
0.076
0.11
NH
0.62
NH
0.15
1.4
NH
0.64
0.00160.0053aThe proteins were initially modiﬁed by the use of a His tag, which was removed after puriﬁcation.
m and kcat values represent the means  standard deviations of results of 3 independent experiments.
cNH, no hydrolysis was detected under conditions with substrate concentrations up to 1 mM and enzyme concentrations up to 700 nM.
bK

belonged to CC1, which is known as international clone I, disseminated worldwide. The
sequence of blaPER-8 showed a nucleotide substitution compared with blaPER-7. Similarly, analysis of their predicted amino acid sequences revealed that PER-8 had asubstitution (Gly39Glu) compared with PER-7; therefore, PER-7 was used as a control for
PER-8. The nucleotide sequences of blaPER-8 and its ﬂanking region have been deposited in GenBank under accession number AB985401.
Compared with E. coli DH5 harboring a pHSG398 control vector, DH5 harboring
blaPER-7 or blaPER-8 showed signiﬁcantly increased MICs of all penicillins and cephalosporins tested, except cefmetazole, cefpirome, cefoxitin, and piperacillin, as well asslightly increased MICs of imipenem (Table 1). DH5 harboring blaPER-7 and blaPER-8 had
similar MICs of -lactams (Table 1). Recombinant PER-7 and PER-8 hydrolyzed all-lactams tested, except for cefmetazole, cefoxitin, and moxalactam (Table 2). PER-7
and PER-8 also hydrolyzed imipenem and meropenem, although their kcat/Km ratios
against these substrates were quite low. The kinetic proﬁles of PER-8 against the-lactams tested, except for imipenem, were similar to those of PER-7. The kcat/Km ratios
of PER-8 were 2-fold higher for imipenem than those of PER-7 (Table 2).
PFGE analysis showed that blaPER-7 in IOMTU448 was located on the chromosome,whereas blaPER-8 in IOMTU442 was located on a 100-kb plasmid whose replicon type
was classiﬁed into the GR12 type of Acinetobacter plasmids (21). The genomic environments surrounding blaPER-7 in IOMTU448 and blaPER-8 in IOMTU442 are shown in Fig. 1.
The genomic environments surrounding blaPER-7 (nucleotide [nt] 1162 to nt 8196;
GenBank accession no. LC020101) showed 99.4% nucleotide sequence identity with theregion from nt 2172 to nt 9204 of the plasmid of P. aeruginosa RJ248 producing PER-1
in China (GenBank accession no. KU133340). The genomic environment surrounding
blaPER-8 (from nt 1994 to nt 8195; GenBank accession no. AB985401) showed more than
99.9% nucleotide sequence identity with the region from nt 3083 to nt 9284 of theplasmid from A. baumannii A068 producing PER-7 in Sweden (GenBank accession no.
KT317086). blaPER-7 and blaPER-8 were both located downstream of ISCR1 and had
identical genetic structures for the sequence between qacEDI and orfX (ofrX is a geneencoding a putative ABC transporter ATP-binding protein) in their respective plasmids(Fig. 1). In our previous study, we reported PER-7-producing A. baumannii IOMTU433 in2015 in Nepal (22). The structures upstream of the 3= coding sequence (CS) in
IOMTU442 and IOMTU448 completely differed from the corresponding regions of aplasmid (pIOMTU433) in A. baumannii IOMTU433 (22) (Fig. 1).
A. baumannii harboring blaPER genes, including blaPER-7 and blaPER-8, mediated byplasmids or chromosomes may be spreading in medical settings in Nepal, because ourprevious study showed that 49.2% of A. baumannii clinical isolates in Nepal harbored
blaPER genes, including blaPER-7 and blaPER-8 (22). The blaPER-7 gene was ﬁrst identiﬁed
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TABLE 2 Kinetic parameters of the PER-7 and PER-8 enzymes in hydrolyzing -lactamsa

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FIG 1 Genetic environments surrounding blaPER genes in A. baumannii IOMTU442 (GenBank accession no.
AB985401), IOMTU448 (GenBank accession no. LC020101), IOMTU433 (GenBank accession no. AP014650) (22), andAP2 (GenBank accession no. HQ713678) (17). The blaPER-7 gene in A. baumannii AP2 was located on thechromosome, whereas the blaPER-7 gene in A. baumannii IOMTU433 and the blaPER-8 gene in A. baumannii
IOMTU442 were located on plasmids.

in A. baumannii AP2 (GenBank accession no. HQ713678) in France, and the gene was
located on the chromosome (17). As shown in Fig. 1, the genetic structures surrounding
blaPER genes in IOMTU442 and IOMTU448 differ from that in AP2 because bothIOMTU442 and IOMTU448 harbor intI1 in the region upstream of blaPER-7 and blaPER-8,
respectively, but AP2 does not. The upstream region of blaPER-7 in A. baumannii AP2,
arr-2 cmlA7 qacED1 sulI ISCR1, had a structure identical to that in pIOMTU433 in A.
baumannii IOMTU433 discovered in Nepal. The data from our present study suggestthat PER-producing A. baumannii in Nepal probably has at least two types of genetic
structures surrounding blaPER genes.
The insertion element ISCR1 in the upstream region of blaPER genes appears to be
involved in the acquisition of blaPER genes in A. baumannii in Nepal. The structure that
includes 3= CS-ISCR1 is commonly associated with the recent emergence of drugresistant pathogens, including E. coli, Klebsiella pneumoniae, A. baumannii, and P.
aeruginosa, which are linked to the drug resistance genes encoding not only metallo-lactamases but also 16S rRNA methylases (23). The ISCR1 may be associated with thegenetic diversity of a -lactamase-resistant factor in A. baumannii (24).
The blaOXA-70 gene in IOMTU442 was ﬁrst identiﬁed in A. baumannii clinical isolatesin Hong Kong (25), whereas the blaOXA-371 gene in IOMTU448 was ﬁrst identiﬁed in A.
baumannii clinical isolates in 2014 in Nepal (22). To date, blaOXA-70 harboring A.
baumannii was reported in 2014 in Canada (26). The blaOXA-70 gene had 11, 17, and 17
nucleotide substitutions compared with blaOXA-71, blaOXA-66, and blaOXA-69, respectively.
The blaOXA-371 gene had only one nucleotide substitution compared with blaOXA-69.
In conclusion, this is the ﬁrst report of A. baumannii isolates producing PER-7 andPER-8 in Nepal. The results of the present study indicate that plasmid- or chromosomemediated PER-producing A. baumannii strains will spread in medical settings in Nepal.
Accession number(s). The nucleotide sequences for blaPER-8 and its ﬂanking regionin A. baumannii IOMTU442 and for blaPER-7 and its ﬂanking region in A. baumannii
IOMTU448 have been deposited in the GenBank database under accession numbers
AB985401 and LC020101, respectively.
ACKNOWLEDGMENTS
This study was approved by the Institutional Review Board of the Institute ofMedicine, Tribhuvan University (reference 6-11-E), and the Biosafety Committee, National Center for Global Health and Medicine (approval no. 26-M-023 and 26-D-088).
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2. Poirel L, Karim A, Mercat A, Le Thomas I, Vahaboglu H, Richard C,
Nordmann P. 1999. Extended-spectrum beta-lactamase-producing strainof Acinetobacter baumannii isolated from a patient in France. J Antimicrob Chemother 43:157–158.
3. Bonnin RA, Nordmann P, Potron A, Lecuyer H, Zahar JR, Poirel L. 2011.
Carbapenem-hydrolyzing GES-type extended-spectrum beta-lactamasein Acinetobacter baumannii. Antimicrob Agents Chemother 55:349 –354.
https://doi.org/10.1128/AAC.00773-10.
4. Poirel L, Menuteau O, Agoli N, Cattoen C, Nordmann P. 2003. Outbreakof extended-spectrum beta-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J Clin Microbiol 41:3542–3547.
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hospital of a clonal Acinetobacter baumannii strain producing theTEM-92 extended-spectrum beta-lactamase. Antimicrob Agents Chemother 51:2211–2214. https://doi.org/10.1128/AAC.01139-06.
6. Nagano N, Nagano Y, Cordevant C, Shibata N, Arakawa Y. 2004. Nosocomial transmission of CTX-M-2 beta-lactamase-producing Acinetobacterbaumannii in a neurosurgery ward. J Clin Microbiol 42:3978 –3984.
https://doi.org/10.1128/JCM.42.9.3978-3984.2004.
7. Nordmann P, Ronco E, Naas T, Duport C, Michel-Briand Y, Labia R. 1993.
Characterization of a novel extended-spectrum beta-lactamase from
Pseudomonas aeruginosa. Antimicrob Agents Chemother 37:962–969.
8. Mantengoli E, Rossolini GM. 2005. Tn5393d, a complex Tn5393 derivative
carrying the PER-1 extended-spectrum beta-lactamase gene and other
resistance determinants. Antimicrob Agents Chemother 49:3289 –3296.
https://doi.org/10.1128/AAC.49.8.3289-3296.2005.
9. Poirel L, Cabanne L, Vahaboglu H, Nordmann P. 2005. Genetic environment and expression of the extended-spectrum beta-lactamase blaPER-1
gene in Gram-negative bacteria. Antimicrob Agents Chemother 49:
1708 –1713. https://doi.org/10.1128/AAC.49.5.1708-1713.2005.
10. Picão RC, Poirel L, Demarta A, Petrini O, Corvaglia AR, Nordmann P. 2008.
Expanded-spectrum beta-lactamase PER-1 in an environmental Aeromonas media isolate from Switzerland. Antimicrob Agents Chemother 52:
3461–3462. https://doi.org/10.1128/AAC.00770-08.
11. Xia R, Guo X, Zhang Y, Xu H. 2010. qnrVC-like gene located in a novel
complex class 1 integron harboring the ISCR1 element in an Aeromonas
punctata strain from an aquatic environment in Shandong Province,
China. Antimicrob Agents Chemother 54:3471–3474. https://doi.org/
10.1128/AAC.01668-09.
12. Bauernfeind A, Stemplinger I, Jungwirth R, Mangold P, Amann S, Akalin
E, Ang O, Bal C, Casellas JM. 1996. Characterization of beta-lactamasegene blaPER-2, which encodes an extended-spectrum class A betalactamase. Antimicrob Agents Chemother 40:616 – 620.
13. Orman BE, Pineiro SA, Arduino S, Galas M, Melano R, Caffer MI, Sordelli
DO, Centron D. 2002. Evolution of multiresistance in nontyphoid Salmonella serovars from 1984 to 1998 in Argentina. Antimicrob Agents
Chemother 46:3963–3970. https://doi.org/10.1128/AAC.46.12.3963-3970.2002.
14. Quinteros M, Radice M, Gardella N, Rodriguez MM, Costa N, Korbenfeld

March 2017 Volume 61 Issue 3 e02300-16

D, Couto E, Gutkind G; Microbiology Study Group. 2003. Extendedspectrum beta-lactamases in Enterobacteriaceae in Buenos Aires, Argentina, public hospitals. Antimicrob Agents Chemother 47:2864 –2867.
https://doi.org/10.1128/AAC.47.9.2864-2867.2003.
15. Vignoli R, Varela G, Mota MI, Cordeiro NF, Power P, Ingold E, Gadea P,
Sirok A, Schelotto F, Ayala JA, Gutkind G. 2005. Enteropathogenic Escherichia coli strains carrying genes encoding the PER-2 and TEM-116
extended-spectrum beta-lactamases isolated from children with diarrhea in Uruguay. J Clin Microbiol 43:2940 –2943. https://doi.org/10.1128/
JCM.43.6.2940-2943.2005.
16. Girlich D, Poirel L, Nordmann P. 2010. PER-6, an extended-spectrum
beta-lactamase from Aeromonas allosaccharophila. Antimicrob Agents
Chemother 54:1619 –1622. https://doi.org/10.1128/AAC.01585-09.
17. Bonnin RA, Potron A, Poirel L, Lecuyer H, Neri R, Nordmann P. 2011.
PER-7, an extended-spectrum beta-lactamase with increased activity
toward broad-spectrum cephalosporins in Acinetobacter baumannii. Antimicrob Agents Chemother 55:2424 –2427. https://doi.org/10.1128/
AAC.01795-10.
18. Tada T, Shrestha B, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T,
Pokhrel BM. 2014. NDM-12, a novel New Delhi metallo-beta-lactamasevariant from a carbapenem-resistant Escherichia coli clinical isolate in
Nepal. Antimicrob Agents Chemother 58:6302– 6305. https://doi.org/
10.1128/AAC.03355-14.
19. Kaase M, Nordmann P, Wichelhaus TA, Gatermann SG, Bonnin RA, Poirel
L. 2011. NDM-2 carbapenemase in Acinetobacter baumannii from Egypt.
J Antimicrob Chemother 66:1260 –1262. https://doi.org/10.1093/jac/dkr135.
20. Grosso F, Quinteira S, Peixe L. 2011. Understanding the dynamics ofimipenem-resistant Acinetobacter baumannii lineages within Portugal.
Clin Microbiol Infect 17:1275–1279. https://doi.org/10.1111/j.1469-0691.2011.03469.x.
21. Bertini A, Poirel L, Mugnier PD, Villa L, Nordmann P, Carattoli A. 2010.
Characterization and PCR-based replicon typing of resistance plasmids
in Acinetobacter baumannii. Antimicrob Agents Chemother 54:
4168 – 4177. https://doi.org/10.1128/AAC.00542-10.
22. Shrestha S, Tada T, Miyoshi-Akiyama T, Ohara H, Shimada K, Satou K,
Teruya K, Nakano K, Shiroma A, Sherchand JB, Rijal BP, Hirano T, Kirikae
T, Pokhrel BM. 2015. Molecular epidemiology of multidrug-resistantAcinetobacter baumannii isolates in a university hospital in Nepal reveals
the emergence of a novel epidemic clonal lineage. Int J Antimicrob
Agents 46:526 –531. https://doi.org/10.1016/j.ijantimicag.2015.07.012.
23. Toleman MA, Walsh TR. 2011. Combinatorial events of insertion sequences and ICE in Gram-negative bacteria. FEMS Microbiol Rev 35:
912–935. https://doi.org/10.1111/j.1574-6976.2011.00294.x.
24. Toleman MA, Bennett PM, Walsh TR. 2006. ISCR elements: novel genecapturing systems of the 21st century? Microbiol Mol Biol Rev 70:
296 –316. https://doi.org/10.1128/MMBR.00048-05.
25. Brown S, Amyes SG. 2005. The sequences of seven class D betalactamases isolated from carbapenem-resistant Acinetobacter baumannii
from four continents. Clin Microbiol Infect 11:326 –329. https://doi.org/
10.1111/j.1469-0691.2005.01096.x.
26. Loewen PC, Alsaadi Y, Fernando D, Kumar A. 2014. Genome sequence ofa tigecycline-resistant clinical isolate of Acinetobacter baumannii strain
AB031 obtained from a bloodstream infection. Genome Announc
2:e01036-14. https://doi.org/10.1128/genomeA.01036-14.

Downloaded from http://aac.asm.org/ on December 25, 2017 by Kohnodai Hospital, National Center for Global Health and Medicine

The research was supported by a grant of the Research Program on Emerging andRe-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED), a grant (27-A-1102) from International Health Cooperation Research,and JSPS KAKENHI grant number 16K19133.

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Am. J. Trop. Med. Hyg., 97(6), 2017, pp. 1706–1709
doi:10.4269/ajtmh.17-0227
Copyright © 2017 by The American Society of Tropical Medicine and Hygiene

Molecular and Clinical Epidemiology of Salmonella Paratyphi A Isolated from Patients with
Bacteremia in Nepal
Jatan Bahadur Sherchan,1 Masatomo Morita,2 Takashi Matono,2 Hidemasa Izumiya,2 Makoto Ohnishi,2 Jeevan B. Sherchand,3Sarmila Tandukar,3 Ujjwal Laghu,3 Maki Nagamatsu,4 Yasuyuki Kato,4 Norio Ohmagari,4 and Kayoko Hayakawa4*
1
Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel, Nepal; 2Department of Bacteriology I,
National Institute of Infectious Diseases, Tokyo, Japan; 3Public Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching
Hospital, Kathmandu, Nepal; 4Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japan

Abstract. Little is known about the epidemiology of typhoid and paratyphoid fever in Nepal. We aimed to elucidate themolecular and clinical epidemiology of Salmonella Paratyphi A in Nepal. Isolates were collected from 23 cases of bacteremia due to S. Paratyphi A between December 2014 and October 2015. Thirteen patients (57%) were male, and the
median age was 21 years. None of the patients had an underlying chronic disease. All S. Paratyphi A isolates weresensitive to ampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, and chloramphenicol. All isolates were resistant tonalidixic acid and were categorized as intermediately susceptible to levoﬂoxacin. Phylogenetic analysis revealed close
relatedness among the isolates, including several clonal groups, suggesting local spread. Patients with bacteremia due to
S. Paratyphi A in Kathmandu, Nepal, were relatively young and nondebilitated. Improving control of S. Paratyphi infections
should focus on effective infection control measures and selection of empirical therapy based on current resistancepatterns.

Minimum inhibitory concentrations (MICs) were determinedby the broth microdilution method using the Dry Plate Eiken(Eiken Chemical, Tokyo, Japan), in accordance with the Clinical and Laboratory Standard Institutions (CLSI) criteria(M100-S26) at the National Center for Global Health andMedicine. Whole-genome sequencing and phylogenetic
analysis were conducted at the National Institute of Infectious
Diseases. Genomic DNA was prepared using the DNeasy
Blood & Tissue kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. Genomic DNA libraries wereprepared using the Nextera XT DNA sample prep kit (Illumina,
San Diego, CA) and paired-end (300 × 2 bp) short reads foreach library were sequenced on a MiSeq instrument (Illumina).
Sufﬁcient DNA sequence reads were generated to cover the
genome at least 60 folds. Sequence reads were assembled
with the de novo genome assembly programCLC Genomics
Workbench v.8.5.1 (CLC Bio, Aarhus, Denmark) to generate amulticontig draft genome for each sample. All contigs werecompared with the reference genome for S. Paratyphi A strain
ATCC 9150 (CP000026.1) to detect mutations on gyrA, gyrB,
parC, and parE genes. To compare short-read mapping data
for all strains with the reference chromosomal sequence of S.
Paratyphi A strain ATCC 9150 (CP000026.1), bwasw4 andsamtools5 software were used with default parameters. Single
nucleotide polymorphisms (SNPs) were extracted with VarScan v.2.3.46 using default parameters. The SNPs in repetitiveand recombination regions were excluded for further analyses. Exact and inexact repeat regions were detected using the
MUMmer v.3.23.7 RecHMM was used to identify recombination regions.8 The remaining 254 SNPs were concatenated to generate a pseudosequence for phylogenetic
analysis; maximum likelihood phylogenetic analysis was
performed using RAxML v.8.2.09 with 1,000 bootstrap
iterations.

INTRODUCTION
Salmonella enterica serotype Typhi or S. enterica serotype
Paratyphi cause an estimated 22 million new cases of entericfever (typhoid or paratyphoid) annually, and 200,000 deaths.1
Nepal is located in South Asia where typhoid and paratyphoid
fever are most prevalent.2 Especially, Kathmandu is known tohave the signiﬁcant burden of enteric fever caused by S. Typhi
and S. Paratyphi A.3 The recent spread of multidrug resistant
S. Typhi and S. Paratyphi A is a serious threat to public health;however, little is known about the epidemiology of typhoid andparatyphoid fever in Nepal. Previous reports have suggested
an increase in the incidence of paratyphoid fever, against
which currently available typhoid vaccines provide little to noprotection.2,3 This study aimed to elucidate the molecular andclinical epidemiology including the prevalence of drugresistant strains and phylogenetic analyses of S. Paratyphi A
in Nepal.
METHODS
From December 2014 to October 2015, S. Paratyphi A
isolates were collected from patients with bacteremia at
Tribhuvan University, Kathmandu, Nepal. For patients fromwhom more than one S. Paratyphi A strain was isolated during
the study period, only the ﬁrst episode was analyzed (i.e.,
unique patient episodes). Institutional review boards at the
Tribhuvan University approved the study before its initiation.
Parameters retrieved from the patient records included demographics, background conditions and clinical symptoms,
duration of hospital stay (for patients who were hospitalized),
empiric antimicrobial treatment; information on occupationand exposure (e.g., animal contact, contact to similar cases),and clinical outcome.

RESULTS* Address correspondence to Kayoko Hayakawa, Disease Control andPrevention Center, National Center for Global Health and Medicine,
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail:kayokohayakawa@gmail.com

During the study period, 23 cases of bacteremia due to
S. Paratyphi A, and 86 cases of bacteremia due to S. Typhi

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MOLECULAR AND CLINICAL EPIDEMIOLOGY OF S. PARATYPHI A IN NEPALTABLE 1
Clinical characteristics of patients with bacteremia due to Salmonella
Paratyphi A (N = 23)
Number of patients (%)

Demographics
Age, median (IQR)
Male patients
Inpatients/out patientsOccupation
Student
House wife
Farmer/gardener
Driver
Nurse
Pastry shop worker
Clinical symptom
Fever
Abdominal pain
Diarrhea
Vomiting
Empiric antimicrobial treatment
Fluoroquinolone*
CeﬁximeCeftriaxone
Azithromycin
Duration of hospitalization, median (IQR), days

21 (17–23)
13 (56.5)
12 (52.2)/11 (47.8)
12 (52.2)
4 (17.4)
3 (13)
2 (8.7)1 (4.3)1 (4.3)23 (100)
10 (43.5)
3 (13)
2 (8.7)
12 (52.2)
4 (17.4)
2 (8.7)1 (4.3)5 (3–6)IQR = interquartile range. One patient received ceﬁxime, azithromycin, ceftriaxone, andanother patient received ceftriaxone, azithromycin, and levoﬂoxacin.
* Fluoroquinolones include ciproﬂoxacin (N = 10), oﬂoxacin (N = 1), and levoﬂoxacin (N = 1).

were identiﬁed. Among the 23 cases of bacteremia due to S.
Paratyphi A, 13 patients (56.5%) were male, the median age
was 21 years (interquartile range [IQR]: 17–23 years, range:
5–68 years). Among the 86 cases of bacteremia due to S.
Typhi, 51 patients (59.3%) were male, the median age was 21
years (IQR: 17–26 years, range: 4–70 years). Clinical characteristics are summarized in Table 1. Twelve (52.2%) patients were hospitalized, the other were outpatients. Noneof the patients had an underlying chronic disease or immunosuppressive status. Twelve patients (52.2%) were students,

four (17.4%) were housewives, three were farmers or gardeners, two (8.7%) were drivers, one (4.3%) was a nurse, andone (4.3%) worked at a pastry shop. The most common clinical symptom was fever (N = 23, 100%), followed by abdominal pain (N = 10, 43.5%), diarrhea (N = 3, 13%), and vomiting(N = 2, 8.7%). Most commonly prescribed empiric antimicrobial treatment was ﬂuoroquinolone (N = 12, 52.2%), followedby cephalosporins (ceﬁxime [N = 4, 17.4%], ceftriaxone [N = 2,
8.7%]), and azithromycin (N = 1, 4.3%). The median duration of
hospitalization was 5 (IQR: 3–6) days, and no patient hadcomplications or died.
Antibiotic susceptibility and MICs of the S. Paratyphi A
isolates are listed in Table 2. All isolates were sensitive toampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, andchloramphenicol, and had an azithromycin MIC of less than
16 μg/mL. All isolates were resistant to nalidixic acid. The
susceptibilities to ﬂuoroquinolones differed, i.e., all isolates
were resistant to oﬂoxacin based on CLSI criteria (M100-S26),
four (17.4%) were resistant to ciproﬂoxacin, and all isolates were categorized as intermediately susceptible to
levoﬂoxacin.
Phylogenetic analyses showed that except for one strain(151006PA), all isolates clustered together, with SNP distances of 0–9 SNPs (Figure 1). Clonal isolates (geneticallyclosest isolates with 0 SNPs) were detected in ﬁve groups,
whereas deﬁnitive outbreak from food handlers (the housewives and pastry shop worker) and family infection were notfound. Among the ﬁve groups of clonal isolates, each isolate
was predominantly detected 1 month or more apart, and only
three paired isolates had a near sampling dates: 151080PA(on July 1, 2015) and 151086PA (on July 16, 2015); 151029PA(on March 5, 2015) and 151032PA (on March 12, 2015); and151033PA (March 15, 2015) and 151031PA (March 8, 2015).
All S. Paratyphi A isolates had a same single mutation C248T
in gyrA encoding Ser83Phe; however, no mutation was foundin gyrB, parC, and parE.

TABLE 2
Antibiotic susceptibility and minimal inhibitory concentration (MIC) of Salmonella Paratyphi A (N = 23)
Antibiotic susceptibility, number of resistant* isolates (%)

MIC (μg/mL)

Antibiotics

S

I

R

MIC50

MIC90

Nalidixic acid
Ciproﬂoxacin
Levoﬂoxacin
Oﬂoxacin
Norﬂoxacin†
Gatiﬂoxacin†
Pruliﬂoxacin‡
Tosuﬂoxacin‡
Gentamicin
Kanamycin
Trimethoprim- sulfamethoxazole
Tetracycline
Minocycline
Azithromycin‡
Cefotaxime
Ceftriaxone
Ampicillin
Aztreonam‡
Imipenem
Panipenem‡
Biapenem‡
Chloramphenicol

–
–
–
–
–
–
–
–
23 (100)
23 (100)
23 (100)
23 (100)
23 (100)
23 (100)
23 (100)
23 (100)
23 (100)–
23 (100)––
23 (100)–
19 (82.6)
23 (100)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

23 (100)
4 (17.4)–
23 (100)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

> 128
0.5
1
2
4
0.5
0.5
0.5
0.12
0.5
0.12/2.38
2
4
8
0.12
0.12
4
0.12
0.25
0.12
0.25
8

> 128
1
1
2
4
1
0.5
0.5
0.25
1
0.25/4.75
4
4
8
0.25
0.25
4
0.12
0.25
0.12
0.25
8

* Based on Clinical and Laboratory Standard Institutions (CLSI) criteria 2016 (M100-S26) unless otherwise noted.
† CLSI criteria 2016 (M100-S26) did not specify Salmonella spp. as they did for other ﬂuoroquinolones, and thus, antibiotic susceptibility was left blank.
‡ No breakpoint is available for S. Paratyphi based on CLSI criteria 2016 (M100-S26).

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SHERCHAN AND OTHERSFIGURE 1. Maximum-likelihood tree generated from single nucleotide polymorphisms in the core genome. (A) Phylogenetic tree of Salmonella
Paratyphi A isolates from Tribhuvan University, Kathmandu, Nepal. (B) Phylogenetic sub-tree of major endemic S. Paratyphi A isolates.

DISCUSSION
In this study, we identiﬁed the molecular and clinicalepidemiology of S. Paratyphi A in Kathmandu, Nepal. As
we included sequential patients with bacteremia due to S.
Paratyphi A, our ﬁndings would reﬂect the epidemiologic
characteristics of this endemic region. It was revealed that
S. Paratyphi A mainly affects relatively young subjects. The
high prevalence of fever is consistent with recent reports
on S. Typhi and S. Paratyphi bacteremia. 10 In our cohort,the prevalence of abdominal pain was slightly higher,
whereas diarrhea and vomiting were less prevalent than inprevious reports. 10 This might be due to differences inbaseline characteristics, including age, of the tested
population.
In this study, the antibiotic susceptibility and MICs
revealed patterns of re-emergence of susceptibility to conventional antibiotics (e.g., chloramphenicol, trimethoprim/sulfamethoxazole, and ampicillin) similar to those described
previously.11 On the other hand, MICs for quinolones were
elevated. Importantly, S. Paratyphi A isolates of 10 patients
who received ciproﬂoxacin as empiric therapy showed
ciproﬂoxacin MICs ³ 0.5 μg/mL, and one patient who received oﬂoxacin carried S. Paratyphi A with an oﬂoxacin MIC
of 2 μg/mL. Along with recent trial results on the use ofgatiﬂoxacin,10 reconsideration is warranted in terms of empiric antimicrobial treatment against S. Paratyphi A bacteremia in Nepal.
Based on phylogenetic analyses, there seem to be two
distinct endemic strains of S. Paratyphi A in the study region.
The major endemic strain was genetically diverged as compared with outbreak-associated isolates in China.12 The relatively high prevalence of patients who handle food (e.g.,food-handling business, housewives) and patients who haveclose contact with other people (e.g., students) in this study
suggests a potential point of intervention to reduce the54transmission of S. Paratyphi, for which no vaccine is readily
available.
High relatedness, coupled with high prevalence of reduced
susceptibility to ﬂuoroquinolones, was observed in S. Paratyphi isolated from patients with bacteremia in Nepal, Kathmandu. Further studies are warranted in terms of appropriate
effective therapy and effective infection control approaches inthis region.
Received March 21, 2017. Accepted for publication July 26, 2017.
Published online October 9, 2017.
Financial support: This work was supported by a grant in Clinical
Epidemiology Research, St. Luke’s International University, Tokyo,
Japan (2016).
Authors’ addresses: Jatan Bahadur Sherchan, Department of ClinicalMicrobiology, Kathmandu University School of Medical Sciences,
Dhulikhel, Nepal, E-mail: jatansherchan@gmail.com. Masatomo
Morita, Takashi Matono, Hidemasa Izumiya, and Makoto Ohnishi,
Department of Bacteriology I, National Institute of Infectious Diseases,
Tokyo, Japan, E-mails: mmorita@niid.go.jp, tmatono@niid.go.jp,
izumiya@nih.go.jp, and ohnishi7@nih.go.jp. Jeevan B. Sherchand,Sarmila Tandukar, and Ujjwal Laghu, Department of Microbiology andParasitology, Tribhuvan University Teaching Hospital, Kathmandu,
Nepal, E-mails: jeevansherchand@hotmail.com, sar1234tan@gmail.
com, and ujjwal111@iom.edu.np. Maki Nagamatsu, Yasuyuki Kato,
Norio Ohmagari, and Kayoko Hayakawa, Disease Control and Prevention Center, National Center for Global Health and Medicine,Tokyo, Japan, E-mails: dfrmm217@yahoo.co.jp, ykato@hosp.ncgm.go.
jp, nohmagari@hosp.ncgm.go.jp, and kayokohayakawa@gmail.com.

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EPIDEMIOLOGY AND SURVEILLANCE

Pseudomonas aeruginosa Clinical Isolates
in Nepal Coproducing Metallo-Lactamases and 16S rRNA
Methyltransferases
Tatsuya Tada,a,b Kayo Shimada,a Kazuhito Satou,c Takashi Hirano,c,dBharat M. Pokhrel,e Jeevan B. Sherchand,e Teruo Kirikaea,bDepartment of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo,
Japana; Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japanb; Okinawa
Institute of Advanced Sciences, Okinawa, Japanc; SENTAN Pharma, Inc., Fukuoka, Japand; Tribhuvan University,
Institute of Medicine, Department of Microbiology, Maharajgunj, Kathmandu, Nepale

ABSTRACT A total of 11 multidrug-resistant Pseudomonas aeruginosa clinical isolates were obtained in Nepal. Four of these isolates harbored genes encoding one ormore carbapenemases (DIM-1, NDM-1, and/or VIM-2), and ﬁve harbored genes encoding a 16S rRNA methyltransferase (RmtB4 or RmtF2). A novel RmtF variant, RmtF2,
had a substitution (K65E) compared with the same gene in RmtF. To our knowledge,
this is the ﬁrst report describing carbapenemase- and 16S rRNA methyltransferasecoproducing P. aeruginosa clinical isolates in Nepal.
KEYWORDS 16S rRNA methylase, Pseudomonas aeruginosa, carbapenemase,multidrug resistanceM

etallo--lactamases (MBLs) confer resistance to all -lactams, except the monobactams, and are characterized by their efﬁcient hydrolysis of carbapenems (1).
The metallo--lactamase DIM-1 was ﬁrst identiﬁed in a Pseudomonas stutzeri strain
obtained from a Dutch patient in 2007 (2). DIM-1 hydrolyzes broad-spectrum cephalosporins and carbapenems but not monobactams. Since then, DIM-1 producers,
including P. stutzeri and Enterobacteriaceae spp., have been isolated in India (3) and
Sierra Leone (4), respectively.
Acquired 16S rRNA methyltransferase genes responsible for an extremely high levelof resistance against various aminoglycosides are widely distributed among Enterobacteriaceae and glucose-nonfermentative bacteria (5). To date, 10 different 16S rRNA
methyltransferases, including ArmA, RmtA, RmtB, RmtC, RmtD, RmtE, RmtF, RmtG,
RmtH, and NpmA, have been found in clinical isolates (6–9). One of these, RmtB, wasfound to have three variants, RmtB2 (accession no. JN968578), RmtB3 (accession no.
JN968579), and RmtB4 (accession no. KM999534). The 16S rRNA methyltransferase
RmtF was ﬁrst identiﬁed in a clinical isolate of Klebsiella pneumoniae on the island of
Réunion in 2011 (7). Since then, RmtF-producing Enterobacteriaceae have been isolated
in India, the United Kingdom, the United States, and Nepal (7, 10, 11).
Between 2012 and 2013, 11 multidrug-resistant Pseudomonas aeruginosa clinicalisolates were obtained from 11 inpatients treated at a university hospital in Nepal.
Multidrug-resistant Pseudomonas aeruginosa isolates are deﬁned as strains showing
resistance to carbapenem (MIC  16 g/ml), amikacin (MIC  32 g/ml), and ﬂuoroquinolone (MIC  4 g/ml), as previously described (12). Of these isolates, 7 were fromsputum, 3 from urine samples, and 1 from a pus sample. The MICs of various antibiotics
were determined using the microdilution method, according to the guidelines of theClinical and Laboratory Standards Institute (13). The entire genomes of these isolates
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Received 3 April 2017 Returned for
modiﬁcation 27 April 2017 Accepted 24 June2017
Accepted manuscript posted online 10 July
2017
Citation Tada T, Shimada K, Satou K, Hirano T,
Pokhrel BM, Sherchand JB, Kirikae T. 2017.
Pseudomonas aeruginosa clinical isolates in
Nepal coproducing metallo-β-lactamases and
16S rRNA methyltransferases. Antimicrob
Agents Chemother 61:e00694-17. https://doi.org/10.1128/AAC.00694-17.
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Tatsuya Tada,
ttada@ri.ncgm.go.jp.

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were sequenced by MiSeq (Illumina, San Diego, CA). Their genomes were searchedfor drug resistance genes, including genes encoding -lactamases (carbapenemases and extended-spectrum -lactamases), 16S rRNA methyltransferases, andaminoglycoside-acetyl/adenyltransferases, using ResFinder 2.1 (https://cge.cbs.dtu.dk/
services/ResFinder/). Point mutations associated with quinolone resistance weresearched in gyrA and parC. Multilocus sequence type (MLST) was deduced, as describedby the protocols of the PubMLST (http://pubmlst.org/paeruginosa/) databases. The
complete genome of P. aeruginosa IOMTU133 was determined using PacBio RS II(Menlo Park), as described previously (14). The genomic environments surrounding
genes encoding carbapenemases and/or 16S rRNA methyltransferases were conﬁrmed
by Sanger sequencing. DNA plugs of all isolates tested (digested with I-CeuI or S1
nuclease) were prepared and separated by pulsed-ﬁeld gel electrophoresis, and Southern hybridization was performed using probes of 16S rRNA, blaDIM-1, blaNDM-1, blaVIM-2,
rmtB4, and rmtF2 (15, 16).
All 11 isolates were resistant to meropenem, aztreonam, amikacin, and ciproﬂoxacin(Table 1), with MICs 16 g/ml. Three isolates showed higher MICs to imipenem ormeropenem, 64 g/ml, than the other isolates. Five of the 11 isolates were extremely
highly resistant to amikacin and arbekacin, with MICs 1,024 g/ml, and to ciproﬂoxacin, with MICs of 32 to 256 g/ml. All isolates were susceptible to colistin, with MICs
0.5 g/ml.
Of the 11 isolates, three had a novel rmtF variant, designated rmtF2 (accession no.
LC050387). Analysis of its predicted amino acid sequence revealed a substitution (K65E)
compared with the sequence of RmtF. Four isolates had genes encoding one or moremetallo--lactamases, i.e., blaDIM-1, blaNDM-1, and/or blaVIM-2; and four had genes encoding other -lactamases, i.e., blaPDCs, blaPSE-2, blaTEM-1, or blaVEB-1a (Table 1). In
addition, 5 isolates had a 16S rRNA methyltransferase encoding gene, rmtB4 or rmtF2;
and nine had genes encoding an aminoglycoside acetyl- and adenylyl-transferase,including AAC(6=)-Ib, AACA7, AACC5b, and AADB (Table 1). A novel rmtF2 gene was
located in the class 1 integron (Fig. 1). All isolates except for IOMTU3 had amino acidsubstitution point mutations S83I in GyrA and S80L in ParC; IOMTU3 had amino acidsubstitution point mutations S83L and D87E in GyrA and S80L in ParC.
A total of 6 isolates were classiﬁed as ST664, two as ST235, and one each as ST244,
ST654, and ST1047. The two ST235 isolates harbored blaNDM-1, blaVIM-2, and rmtB4; twoof the ST664 isolates harbored rmtF2; the ST654 isolate harbored blaVIM-2; and theST1047 isolate harbored blaDIM-1 and rmtF2.
Because IOMTU133 belonged to ST1047 and harbored several drug resistancegenes, its complete genome was sequenced and deposited in GenBank under accession no. AP017302. This isolate had no plasmids. The complete genome sequence of
IOMTU133 had 283-fold coverage for one chromosome, IOMTU133, which consisted ofa single circular chromosome of 6,897,018 bp with an average GC content of 65.98%.
The chromosome was found to contain 6,245 protein-encoding genes, including 63
tRNA genes and one transfer messenger RNA (tmRNA) gene for all amino acids.
IOMTU133 also harbored a carbapenemase-encoding gene, blaDIM-1; a 16S rRNA methyltransferase encoding gene, rmtF2; and an aminoglycoside acetyltransferase encoding
gene, aac(6=)-Ib. The blaDIM-1 gene and a novel rmtF2 gene were located within thesame integron on the chromosome (Fig. 1).
The genomic environments surrounding blaDIM-1, blaNDM-1, blaVIM-2, rmtB4, andrmtF2 are shown in Fig. 1. The genomic environments of blaDIM-1, blaNDM-1, and blaVIM-2
were IS6-intI1-blaDIM-1-dfr2e-aac(6=)-Ib-rmtF2-insE-cat-orf1 (gene encoding a hypothetical protein)-IS6 (accession no. AP017302), tnp-blaNDM-1-orf2 (gene encoding a hypothetical protein) (accession no. LC054839), and intI1-aadB-aacA7-blaVIM-2-dhfrB5-aacA5tniR-tniQ-tniB-tniA (accession no. LC054840), respectively. The genomic environments
surrounding these carbapenemase-encoding genes were unique to these isolates.
The genomic environment of rmtB4 was tnp-groEL-orf3 (gene encoding queuinetRNA-ribosyltransferase)-orf4 (gene encoding a hypothetical protein)-rmtB4-orf5 (geneencoding a putative Na/H antiporter) (accession no. LC052325), whereas theaac.asm.org 2

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MLST
654
244
235
1047
664
664
664
664
664
235
664

IPM
128
16
512
32
1
1
1
1
8
512
2

MEM
32
32
1024
64
4
4
4
4
32
1024
32

ATM
16
1024
32
16
16
32
16
32
128
32
128

CAZ
128
1024
1024
256
8
8
8
4
1024
1024
512

AMK
128
32
1024
1024
128
128
128
128
1024
1024
1024

ABK
1
16
1024
1024
1
1
1
1
1024
1024
1024

CIP
256
32
64
32
32
32
32
32
64
64
32

CST
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5

-Lactamase(s)VIM-2, PDC-58
VEB-Ia, PDC-61
NDM-1, VIM-2, PDC-35
DIM-1, PDC-32
PDC-98
PDC-98
PCD-98TEM-1, PDC-98
PSE-2, PDC-98
NDM-1, VIM-2, PDC-35
PSE-2, PDC-98

aIPM, imipenem; MEM, meropenem; ATM, aztreonam; CAZ, ceftazidime; AMK, amikacin; ABK, arbekacin; CIP, ciproﬂoxacin; CST, colistin.

StrainIOMTU 3IOMTU 7IOMTU 9IOMTU 133IOMTU 143IOMTU 155IOMTU 161IOMTU 179IOMTU 184
IOMTU 304IOMTU 487
RmtF2
RmtB4
RmtF2

RmtB4
RmtF2

16S rRNA
methylase

Aminoglycosideacetyl/adenylyltransferase(s)AADB
AAC(6=)-Ib
AACA7, AACC5b
AAC(6=)-Ib
AAC(6=)-Ib
AAC(6=)-Ib
AAC(6=)-Ib
AAC(6=)-Ib
AAC(6=)-Ib
AACA7, AACC5b
AADB
GyrA
S83L, D87E
S83I
S83I
S83I
S83I
S83I
S83I
S83I
S83I
S83I
S83I

ParC
S80L
S80L
S80L
S80L
S80L
S80L
S80L
S80L
S80L
S80L
S80L

Mutation(s) in DNA
gyraseEmergence of DIM-1 and RmtF2 Coproducing P. aeruginosa

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66

MICs (g/ml) fora:

TABLE 1 Summary of characteristics of the 11 Pseudomonas aeruginosa strains, including antimicrobial resistance proﬁles and resistant genes

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genomic environment of rmtF2 was tnpA-tnpR-intI1-blaOXA-10-aac(6=)-Ib-rmtF2-orf6(gene encoding a hypothetical protein)-blaPAC-1 (accession no. LC224309). The rmtF2 in
IOMTU133 was located in the same integron as blaDIM-1 (Fig. 1). Compared to the
genomic environment surrounding rmtF in K. pneumoniae UCLAOXA232KP plasmid
pUCLAOXA232-3.X (accession no. CP012569), both rmtF and rmtF2 were located in class
I integron, which contained aac(6=)-Ib in the upstream regions of rmtF and rmtF2;
however, the other allelic proﬁles in each integron were different (Fig. 1). The genomic
environments surrounding rmtB4 and rmtF2 were unique to these isolates.
Of all the isolates tested, only IOMTU487 had a 120-kbp plasmid, but the plasmid did
not harbor the blaDIM-1, blaNDM-1, blaVIM-2, rmtB4, or rmtF2 genes (see Fig. S1 in the
supplemental material). All of these genes were located in the chromosomes (see Fig.
S2 in the supplemental material).
The ﬁndings of this study indicate that ST664 P. aeruginosa clinical isolates spreadin a medical setting in Nepal, because the majority of P. aeruginosa isolates obtained in
Nepal were classiﬁed as ST664. To date, seven ST664 isolates (PubMLST no. 3401, 3707,
4018, 4033, 4052, 4060, and 4787) have been registered on the PubMLST website(https://pubmlst.org/paeruginosa/). Of these, PubMLST no. 4787 (Pseudomonas aeruginosa VRFPA06) was isolated from human blood in 2012 in India (17), although thedetails of others were not reported. Of our 11 isolates, only two were classiﬁed as ST235,
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FIG 1 Genomic environment surrounding blaDIM-1 in IOMTU133 (accession no. AP017302); blaNDM-1 in IOMTU9 and IOMTU304 (accession no. LC054839); blaVIM-2
in IOMTU3, IOMTU9 and IOMTU304 (accession no. LC054840); rmtB4 in IOMTU9 and IOMTU304 (accession no. LC052325); rmtF2 in IOMTU184 and IOMTU487(accession no. LC224309); and rmtF in Klebsiella pneumoniae UCLAOXA232KP (accession no. CP012569).

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SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00694-17.
SUPPLEMENTAL FILE 1, PDF ﬁle, 0.1 MB.
ACKNOWLEDGMENTS
This study was reviewed and approved by the Institutional Review Board of theInstitute of Medicine at Tribhuvan University (reference no. 6-11-E) and the Biosafety
Committee at the National Center for Global Health and Medicine (approval no.
28-M-053). The study was supported by grants from International Health Cooperation
Research (grant 29-S-5), the Okinawa Communicable Disease Research Hub Formation
Promotion Project, Okinawa Prefectural Government Commissioned Projects For Fiscal
Year 2016, the Research Program on Emerging and Re-emerging Infectious Diseases
from Japan Agency for Medical Research and Development (AMED), and JSPS KAKENHI(grant 16K19133).

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Kirikae T, Pokhrel BM. 2013. Dissemination of multidrug-resistant Klebsiella pneumoniae clinical isolates with various combinations of carbapenemases (NDM-1 and OXA-72) and 16S rRNA methylases (ArmA, RmtC
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which has been recognized as one of three high-risk clones, i.e., ST235, ST111, andST175 (18). A P. aeruginosa strain belonging to ST1047, which was originally obtainedin Norway and found to produce VIM-type MBLs, was ﬁrst registered on the PubMLST
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This is the ﬁrst report describing carbapenemase- and 16S rRNA methyltransferasecoproducing P. aeruginosa clinical isolates in Nepal. Carbapenemase- and 16S rRNA
methyltransferase-coproducing P. aeruginosa was reported in 2007 in Brazil (19) and in2015 in northeast India (20), which is bordered by Nepal. It is therefore necessary tosurvey multidrug-resistant P. aeruginosa in medical settings in Nepal.
Accession number(s). The sequences described were submitted to GenBank under
the accession numbers LC050387, LC052325, LC054839, LC054840, LC224309, andAP017302.

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18. Oliver A, Mulet X, Lopez-Causape C, Juan C. 2015. The increasing threatof Pseudomonas aeruginosa high-risk clones. Drug Resist Updat 21–22:
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integron, from a carbapenem-resistant Serratia marcescens clinical isolate. Antimicrob Agents Chemother 55:5143–5149. https://doi.org/10.1128/AAC.05045-11.
16. Tada T, Miyoshi-Akiyama T, Shimada K, Kirikae T. 2014. Biochemical
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3538 –3540. https://doi.org/10.1128/AAC.02793-13.
17. Murugan N, Malathi J, Umashankar V, Madhavan HN. 2014. Comparative
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2:e00140-14. https://doi.org/10.1128/genomeA.00140-14.

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EPIDEMIOLOGY AND SURVEILLANCE

Emergence of Various NDM-TypeMetallo--Lactamase-Producing
Escherichia coli Clinical Isolates in Nepal
Basudha Shrestha,a Tatsuya Tada,b,c Kayo Shimada,c Shovita Shrestha,aHiroshi Ohara,d Bharat Mani Pokhrel,a Jeevan B. Sherchand,a Teruo Kirikaeb,cDepartment of Microbiology, Institute of Medicine, Tribhuvan University, Kathmandu, Nepala; Department ofMicrobiology, Juntendo University School of Medicine, Tokyo, Japanb; Department of Infectious Diseases,
Research Institute, National Center for Global Health and Medicine, Tokyo, Japanc; Bureau of International
Medical Cooperation, National Center for Global Health and Medicine, Tokyo, Japand

ABSTRACT Of 250 clinical isolates of Escherichia coli obtained in Nepal, 38 werecarbapenem resistant, with MICs of imipenem or meropenem of 4 g/ml. All 38
isolates harbored the following blaNDMs: blaNDM-1, blaNDM-3, blaNDM-4, blaNDM-5,
blaNDM-7, blaNDM-12, and blaNDM-13. Most of these isolates also harbored the 16S
rRNA methylase gene(s) armA, rmtB, and/or rmtC.
KEYWORDS NDM-type metallo--lactamase, carbapenem-resistant Escherichia coli,

molecular epidemiology

M

etallo--lactamases (MBLs) can confer resistance to carbapenems, reducing theirsusceptibility to carbapenems, as well as to cephalosporins and penicillins but not
to monobactams (1). New Delhi metallo--lactamase-1 (NDM-1) was initially isolated in2008 from Klebsiella pneumoniae and Escherichia coli strains originating in India (2).
To date, 16 NDM variants have been identiﬁed (ftp://ftp.ncbi.nlm.nih.gov/pathogen/betalactamases/Allele.tab). Since the initial observation, NDM-producing Enterobacteriaceae strains have been isolated in various parts of the world, including Australia,
Bangladesh, Belgium, Canada, France, India, Kenya, Japan, Nepal, the Netherlands,
New Zealand, Pakistan, Singapore, Taiwan, and the United States (3–6). This study
analyzed the molecular epidemiology of carbapenem-resistant E. coli isolates obtained
from a university hospital in Nepal.
A total of 250 consecutive nonrepetitive clinical isolates of E. coli were obtained from250 patients hospitalized between December 2013 and December 2014 at a university
hospital in Kathmandu, Nepal. These bacterial isolates were collected from urine (n 92), pus (n  65), sputum (n  36), catheter specimens (n  35), blood (n  9), bile (n 5), body ﬂuid (n  4), and other specimens (n  4). Species identiﬁcation was conﬁrmed
by 16S rRNA sequencing (7). Antimicrobial susceptibility to amikacin, arbekacin, ceftazidime, ciproﬂoxacin, colistin, imipenem, meropenem, and tigecycline was tested by
the microdilution method according to the guidelines of the Clinical and Laboratory
Standards Institute (8). Carbapenem-resistant E. coli isolates were deﬁned as having
imipenem or meropenem MICs of 4 g/ml. The whole genomes of all carbapenemresistant E. coli isolates were extracted using DNeasy blood and tissue kits (Qiagen,Tokyo, Japan) and sequenced with a MiSeq sequencer (Illumina, San Diego, CA). The
raw reads were assembled using CLC Genomics Workbench version 8.0.2 (CLC bio,Tokyo, Japan). A summary of the assembly is shown in Table S1 in the supplemental
material. The whole-genome sequences of all 38 isolates were deposited in GenBank asaccession no. DRA005225. To analyze the relationships among these 38 E. coli isolates,
the complete genome of an E. coli sequence type 2747 (ST2747) strain (GenBank
accession no. CP007394) was used as a reference, because a BLAST search showed that
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Received 13 July 2017 Returned for
modiﬁcation 2 August 2017 Accepted 30
September 2017
Accepted manuscript posted online 9
October 2017
Citation Shrestha B, Tada T, Shimada K,
Shrestha S, Ohara H, Pokhrel BM, Sherchand
JB, Kirikae T. 2017. Emergence of various
NDM-type-metallo-β-lactamase-producing
Escherichia coli clinical isolates in Nepal.
Antimicrob Agents Chemother 61:e01425-17.
https://doi.org/10.1128/AAC.01425-17.
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Tatsuya Tada,
t-tada@juntendo.ac.jp.

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Shrestha et al.

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Antimicrobial
agent
AmikacinArbekacin
Ceftazidime
Ciproﬂoxacin
Colistin
Imipenem
Meropenem
Tigecycline

Breakpoint forresistance (g/ml)
64% of strains
resistant
87

16
4
4
4
4
4

100
100
0
100
100
5

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TABLE 1 MIC90 and MIC50 values and percentages of antimicrobial resistance among E.
coli clinical isolatesa
MIC (g/ml)
Range
2–512
1–512
512–5128–256
0.125–1
4–256
8–128
0.125–4

MIC50
512
512
512
128
0.125
16
32
2

MIC90
512
512
512
512
1
64
64
2

aThirty-eight isolates were tested. Breakpoints for antimicrobial resistance were determined according to

guidelines of the Clinical and Laboratory Standards Institute. The breakpoint for tigecycline resistance wasprovided for Enterobacteriaceae by EUCAST and the U.S. Food and Drug Administration (FDA).

this strain was genetically close to the isolates tested (https://blast.ncbi.nlm.nih.gov/
Blast.cgi?PROGRAMblastn&PAGE_TYPEBlastSearch&LINK_LOCblasthome). Concatenated single nucleotide polymorphism (SNP) sequences were aligned by MAFFT(http://mafft.cbrc.jp/alignment/server/). Models and parameters used for the phylogenetic analyses were computed using jModelTest 2.1.4. A maximum-likelihood phylogenetic tree was constructed from SNP alignment with PhyML 3.0 (9). The sequences ofdrug resistance genes, including -lactamase-encoding genes, aminoglycoside resistance genes, and quinolone resistance genes, were determined using ResFinder 2.1(https://cge.cbs.dtu.dk/services/ResFinder/). Multilocus sequence types (MLSTs) werededuced as described in the protocols of the University of Warwick MLST databases(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli), and clonal complexes (CC) were determinedby eBURST version 3 (http://eburst.mlst.net). To determine the sizes of the plasmids
harboring blaNDMs, plasmid DNAs in each isolate were extracted and digested with S1
nuclease. Pulsed-ﬁeld gel electrophoresis (PFGE) and Southern hybridization were
performed (10).
Of the 250 E. coli isolates, 38 were carbapenem resistant; 9 of these were from
respiratory tracts, 10 were from pus and wounds, 18 were from urinary tracts, and 1 wasfrom other sources. According to CLSI guidelines (11), 33 (87%) of the 38 isolates were
multidrug resistant (Table 1). All isolates were resistant to ceftazidime, ciproﬂoxacin,
and meropenem, but they were susceptible to colistin. Thirty-six isolates (95%) weresensitive to tigecycline (MICs of 1 g/ml), whereas for two isolates (5%), the MICs
were 4 g/ml. The 38 carbapenem-resistant E. coli isolates belonged to 11 different
MLSTs: ST38 (3 isolates), ST94 (1 isolate), ST101 (6 isolates), ST167 (5 isolates), ST361 (2isolates), ST405 (8 isolates), ST448 (3 isolates), ST648 (5 isolates), ST2083 (2 isolates),
ST2659 (1 isolate), and ST4108 (2 isolates) (Table 2). eBURST analysis revealed that the
ST38 and ST2659 isolates belonged to CC38 and that ST94, ST448, and ST2083 isolates
belonged to CC94. A maximum-likelihood phylogenetic tree constructed from the 38
carbapenem-resistant E. coli isolates revealed three clades (Fig. 1). Clade A consisted of
the isolates belonging to ST38, ST405, ST2659, and ST2747 (an E. coli ST2747 strain was
the reference strain). Clade B comprised the isolates belonging to ST94, ST101, ST167,
ST361, ST448, ST2083, and ST4108. Clade C had one isolate belonging to ST648. CladeA contained subclade CC38, which consisted of the isolates belonging to ST38 andST2659, and clade B contained subclade CC94, which consisted of the isolates belonging to ST94, ST448, and ST2083. All 38 carbapenem-resistant E. coli isolates harbored
blaNDMs, with 16, 11, 7, 1, 1, 1, and 1 harboring blaNDM-1, blaNDM-5, blaNDM-7, blaNDM-3,
blaNDM-4, blaNDM-12, and blaNDM-13, respectively (Table 2). In addition, 30 isolates, 29
isolates, 1 isolate, and 1 isolate harbored blaCTX-M-15, blaTEM-1, blaTEM-166, and blaOXA-181,
respectively (Table S2).
Of the 38 isolates, 33 (87%) harbored 16S rRNA methylase-encoding genes, including 14, 11, and 4 harboring armA, rmtB, and rmtC, respectively; 2 harboring both armAand rmtB; 2 harboring both armA and rmtC; and 30 harboring aac(6=)-Ib-cr (Table S2).
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Various NDM Variant-Producing E. coli Strains in Nepal

Antimicrobial Agents and Chemotherapy

MLST
ST38

No. ofisolates3

Clonalcomplex
CC38

ST94
ST101

1
6

CC94

ST167

5

ST361

2

ST405

8

ST448

3

ST648

5

ST2083
ST2659ST4108

2
1
2

CC94

CC94
CC38

Carbapenemase- and extendedspectrum -lactamase-encoding
gene(s)blaNDM-1 (2/3), blaNDM-3 (1/3),
blaCTX-M-15
blaNDM-1
blaNDM-1 (3/6), blaNDM-5 (1/6),
blaNDM-7 (1/6), blaNDM-13 (1/6),
blaCTX-M-15 (5/6), blaTEM-166 (1/6)
blaNDM-5, blaCTX-M-15 (4/5),
blaOXA-181 (1/5)
blaNDM-1 (1/2), blaNDM-5 (1/2),
blaCTX-M-15
blaNDM-1 (4/8), blaNDM-4 (1/8),
blaNDM-5 (2/8), blaNDM-7 (1/8),
blaCTX-M-15 (7/8)
blaNDM-1 (1/3), blaNDM-5 (1/3),
blaNDM-12 (1/3), blaCTX-M-15 (1/3)
blaNDM-1 (1/5), blaNDM-5, (1/5),
blaNDM-7 (3/5), blaCTX-M-15 (4/5)
blaNDM-1, blaCTX-M-15 (1/2)
blaNDM-1, blaCTX-M-15
blaNDM-7, blaCTX-M-15

16S rRNA methylase- and aminoglycosideacetyltransferase-encoding genes
rmtC, aac(6’)-1b-cr, aac(3)-IIa (1/3), aadA1

Mutation(s) in DNA gyrase
GyrA
S83L, D87N

ParC
S80I, E84G

armA, aac(6’)-1b-cr, aadA1, aadA2
armA (2/6), armA and rmtB (2/6), rmtB (1/6),
aac(6’)-1b-cr, aadA1 (3/6), aadA2 (2/6)

S83L, D87N
S83L, D87N

S80I
S80I

rmtB, aac(6’)-1b-cr (2/5), aadA1 (1/5), aadA2(3/5), aadA5 (1/5)
rmtB (1/2), rmtC (1/2), aac(6’)-1b (1/2),
aadA1, aadA2 (1/2), aphA6 (1/2)
armA (4/8), armA and rmtC (1/8), rmtB (2/8),
aac(6’)-1b-cr (6/8), aadA2, aphA6 (2/8),
aac(3)-IIa (1/8)
armA(1/3), rmtB (1/3), aac(6’)-1b-cr, aac(3)1Id (1/3), aadA1 (1/3), aadA2 (1/3)
armA (2/5), armA and rmtC (1/5), rmtB (1/5),
aac(6’)-1b-cr (4/5), aadA1 (1/5), aadA2(4/5)
armA, aac(6’)-1b-cr, aadA1, aadA2
rmtC, aac(6’)-1b-cr
armA, aac(6’)-1b-cr, aadA1, aadA2

S83L, D87N

S80I

S83L, D87N

S80I

S83L, D87N

S80I

S83L, D87N

S80I, E84G

S83L, D87N

S80I

S83L, D87N
S83L, D87N
S83L, D87N

S80I
S80I
S80I

ParE

E455D

aNumbers in parentheses are the number of isolates with the named gene/number tested.

Sequences derived from each contig datum after assembly of the raw read data
showed that 16 types of genetic structures surrounded blaNDMs (Fig. 2). Of the 16
isolates harboring blaNDM-1, 7 were type B, 5 were type A, and 1 each was type C, D, E,
or F. The genomic environment surrounding blaNDM-3 was tnpA blaNDM-3 bleMBL trpF

FIG 1 Molecular phylogeny of the 38 E. coli strains. A maximum-likelihood phylogenetic tree constructedfrom the 38 carbapenem-resistant isolates revealed three clades. The length of the concatemer was
244,995 bp. Clade A consisted of the isolates belonging to ST38, ST405, ST2659, and ST2747 (E. coli
ST2747 was the reference strain). Clade B consisted of the isolates belonging to ST94, ST101, ST167,
ST361, ST448, ST2083, and ST4108. Clade C consisted of only one isolate, and it belonged to ST648. CladeA contained subclade CC38, consisting of isolates belonging to ST38 and ST2659, and clade B contained
subclade CC94, consisting of isolates belonging to ST94, ST448, and ST2083.
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TABLE 2 Summary of the characteristics of the 38 carbapenem-resistant E. coli strains, including MLSTs and drug resistance genesa

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FIG 2 Structures of the genomic environments, including of blaNDM-1 (A to F), blaNDM-3 (G), blaNDM-4 (H),
blaNDM-5 (I to L), blaNDM-7 (M and N), blaNDM-12 (O), and blaNDM-13 (P), from sequences from each contig datum
after assembling. orfA and orfB, hypothetical protein-encoding genes.

blaDHA-1 ampR hybF qacEdelta1 sulI (type G in Fig. 2). This sequence showed greaterthan 99.9% identity with that in NDM-3-producing E. coli EC2 plasmid pEC2-NDM-3 inAustralia (12). The genomic environment surrounding blaNDM-4 was tnpA blaNDM-4
bleMBL trpF dsbC tnpA sul1 qacEdelta1 aadA2 orf111 dfrA12 intI1 tnpM (type H in Fig. 2).
This sequence had more than 99.9% identity with that in NDM-1-producing E. coli GUE
plasmid pGUE-NDM in India (13). The genomic environments surrounding blaNDM-5 had
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4 types of structures: types I, J, K, and L (Fig. 2). Of 11 NDM-5 producers, 6 were typeI, 3 were type J, and 2 each were type K or L. The genomic environment surrounding
blaNDM-7 had two types of structures, M and N (Fig. 2). The structure in type M wasunique, whereas the structure in type N was identical to that in the NDM-7producing E. coli ABC133 plasmid pABC133-NDM found in 2012 in the United ArabEmirates (GenBank accession no. KX214671). The genomic environment surrounding
blaNDM-12 was blaNDM-12 bleMBL trpF dsbC tnpA sulI qacEdeltaI (type O in Fig. 2). Thissequence showed greater than 99.9% identity with the pGUE-NDM plasmid (NCBI
accession no. JQ364967) from E. coli strain GUE, which was isolated in India, as well as
99.9% identity with the pEC77-NDM plasmid (NCBI accession no. AB898038) from E. coli
strain NCGM77, which emerged in Japan (14). The genomic environment surrounding
blaNDM-13 was tnpA IS30 blaNDM-13 bleMBL trpF dsbC cutA groES groL (type P in Fig. 2); thissequence has been deposited in GenBank (accession no. LC012596). This structure,
except for blaNDM-13, was identical to that of pPMK1 expressed by K. pneumoniae PMK1
(from Nepal) (NCBI accession no. CP008933), the Enterobacter hormaechei CCHB10892
plasmid (from Brazil) (accession no. KF727591), pKPX-1 from K. pneumoniae KPX (fromTaiwan) (accession no. AP012055), and pNDM-MAR isolated from K. pneumoniae in
Morocco (accession no. JN420336). PFGE and Southern hybridization analyses revealed
that blaNDMs in 19 isolates were found in the plasmids (Fig. S1).
There were no relationships among drug susceptibility proﬁles, drug resistancegenes, MLSTs, and plasmid sizes.
To our knowledge, this is the ﬁrst molecular epidemiological analysis of carbapenemresistant E. coli clinical isolates in Nepal indicating that the isolates have various typesof blaNDMs. Five isolates belonging to ST648 in clade C were found, despite the lack ofisolation of any carbapenem-resistant E. coli strain belonging to ST131 in medical
settings in Nepal. CTX-M-producing E. coli ST131 and ST648 isolates have been reported
to cause community-acquired infections in Nepal (15). The dissemination of ST648 E.
coli isolates in various communities of Nepal appears to be advancing into medical
settings, and ST648 E. coli isolates are acquiring carbapenem and aminoglycosideresistance.
In conclusion, carbapenem-resistant E. coli isolates producing NDMs and 16S rRNA
methylases have been spreading in medical settings in Nepal. Gram-negative pathogens producing NDMs and 16S rRNA methylases are resistant to clinically important
carbapenems (16) and aminoglycosides (17), respectively. Our previous studies revealed that Gram-negative pathogens, including Acinetobacter baumannii (18), E. coli(6), K. pneumoniae (19), and Providencia rettgeri (20), that produce NDMs and 16S rRNA
methylases have emerged in medical settings in Nepal. Although this study addressed
only carbapenem-resistant E. coli isolates, these results suggest the necessity of monitoring the dissemination of both aminoglycoside- and carbapenem-resistant E. coli
strains in medical settings in Nepal.
This study was reviewed and approved by the Institutional Review Board of theInstitute of Medicine at Tribhuvan University (reference no. 6-11-E). The study
protocol was carefully reviewed and approved by the ethics committee of the
National Center for Global Health and Medicine (reference no. 1268). Individual
informed consent was waived by the ethics committee listed above because thisstudy used currently existing samples collected during routine medical care and did
not pose any additional risks to the patients. Patient information was anonymized
and deidentiﬁed prior to the analysis. The study protocol was reviewed and approvedby the Biosafety Committee, National Center for Global Health and Medicine (approval
no. 28-M-053).
Nucleotide sequence accession number(s). The whole-genome sequences of all
38 isolates were deposited in GenBank as accession no. DRA005225 (for the experiment,
accession no. DRX069506 to DRX069543; for the run, accession no. DRR075609 to
DRR075646).
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SUPPLEMENTAL FILE 2, XLSX ﬁle, 0.1 MB.
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ACKNOWLEDGMENTS
The study was supported by grants from International Health Cooperation Research(29-S-5), JSPS KAKENHI (16K19133), and the Research Program on Emerging andReemerging Infectious Diseases from AMED.

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Contact details for further information
Hiroshi OHARA, M.D., Ph.D.
Bureau of International Health Cooperation, Japan
National Center for Global Health and Medicine, Japan
Email: oharah@it.ncgm.go.jp
Annual Report 2016-2017
IOM-NCGM Research Collaboration Office© National Center for Global Health and Medicine, JapanAll rights reserved.
National Center for Global Health and Medicine, Japan
Bureau of International Health Cooperation, Japan
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655
Tel: +81-3-3202-7181 / Fax: +81-3-3205-6780
info@it.ncgm.go.jp