# Molecular Characteristics and Antibiotic Resistance Profiles ofKlebsiella Isolates in Mthatha, Eastern Cape Province, South Africa

Authors: Sandeep Vasaikar; Larry Obi; Isaac Morobe; Mary Bisi-Johnson
Journal: International Journal of Microbiology
 (2017)

---

## Abstract

The increase in the incidence of extended-spectrumβ-lactamase- (ESBL-) producingKlebsiella species has become a serious problem worldwide, because of their incrimination in antibiotic resistance. The objective of this study is to investigate the resistance genes responsible for ESBL-producingKlebsiellaspecies and carbapenemase-producingKlebsiella (CRE) isolated in Mthatha and to study their epidemiology. A prospective, descriptive study of 202 nonrepetitive samples from patients was obtained from Nelson Mandela Academic Hospital. The culturedKlebsiella isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction of blaCTX-M, blaTEM, blaSHV, blaKPC, and blaNDM genes. OverallK. pneumoniaewere the majority with 169 (83.7%) species isolates, followed byK. oxytoca with 29 (14.4%), whileK. ozaenae andRaoultella ornithinolytica were 2 (0.9%) each. The prevalence of ESBL production in allKlebsiella species was 117 (57.9%). ESBL-genotypic resistance is driven in Mthatha by blaSHV 121 (77.1%) followed by blaTEM 105 (66.9%) and blaCTX-M at 89 (56.7%). The most common ESBL genotype combination among theKlebsiellawas blaTEM+blaSHV + blaCTX-M at 79 (50.3%). There is a steady increase in the rate of ESBL genes in the last five years.


---

## Body

## 1. Introduction

The genusKlebsiella belongs to the Enterobacteriaceae family and comprises Gram-negative opportunistic nonmotile pathogens with a mucoid aspect. The gastrointestinal tract serves as a reservoir and is often the latent source for infections [1]. The genusKlebsiellais classified into four species:Klebsiella pneumoniae (K. pneumoniae),Klebsiella oxytoca(K. oxytoca),Klebsiella terrigena (K. terrigena), andKlebsiella planticola(K. planticola), withK. pneumoniaeconsisting of three subspecies,K. pneumoniaesubsp.pneumoniae,K. pneumoniaesubsp.ozaenae, andK. pneumoniaesubsp.rhinoscleromatis[2]. K. pneumoniae is an opportunistic microorganism which causes serious diseases such as septicemia, pneumonia, urinary tract infections (UTIs), chronic lung disorders, and nosocomial infections in immunocompromised patients [3]. The emergence of extended-spectrum β-lactamase- (ESBL-) producing bacteria, particularly inK. pneumoniae, is now a critical concern for the development of therapies against bacterial infection [4]. These strains are resistant to extended-spectrum beta-lactam antibiotics, aminoglycosides, and fluoroquinolones [5]. The currently dominant ESBLs belonging to class A in the Ambler classification are blaTEM, blaSHV, and blaCTX-M types. The detection of the common ESBL genes such as blaTEM, blaSHV, and blaCTX-M by molecular methods in the ESBL-producing bacteria and their patterns of antimicrobial resistance can provide useful information about their epidemiology and can aid in developing rational antimicrobial therapy.K. pneumoniaecarbapenemase (KPC) are Ambler class A plasmid-encoded enzymes that are capable of hydrolyzing all beta-lactam antibiotics, including monobactams, extended-spectrum cephalosporins, and carbapenems. Originally described in 2001, pathogens harbouring these antibiotic resistance enzymes have been reported from the United States of America, France, China, Sweden, Norway, Colombia, and Brazil. Molecular detection of the blaKPC gene by polymerase chain reaction (PCR) assay provides laboratories with a means to quickly identify the presence of this important resistance determinant gene [6]. Though many studies have reported the drug resistance ofK. pneumoniae andK. oxytoca worldwide [4, 7–9]. Epidemiology studies on ESBL-producingK. pneumoniae in Republic of South Africa (RSA) from different provinces have been reported [10–13], but little is known in the Eastern Cape Province (ECP) about the epidemiology and molecular characteristics of ESBLs. The aim of this study was to investigate the resistance mechanisms to among ESBL-producing differentKlebsiellaspecies and carbapenemase-producingKlebsiella (CRE) isolated in Mthatha and surrounding areas and to study antimicrobial susceptibility to parenteral and oral antimicrobials.


## 2. Materials and Methods

### 2.1. Experimental Design

#### 2.1.1. Ethical Considerations

Ethical approval for the study was granted by the Health Research Ethics and Biosafety Committee of the Walter Sisulu University (WSU) certificate number 022/110 and the Nelson Mandela Academic Hospital Ethics Committee (NMAH), Mthatha, ECP.


#### 2.1.2. Study Design and Setting

A prospective, descriptive study based on laboratory investigations at the Microbiology Laboratory of the National Health Laboratory Services (NHLS) at NMAH and the Department of Medical Microbiology, Faculty of Health Sciences, WSU was undertaken. In this study 203 nonrepetitive (one per patient) samples from patients were randomly obtained from August 2011 to May 2014. Figure1 shows the specimen catchment area, that is, Mthatha and surrounding clinics. Mthatha (formerly Umtata) is the main town of the King Sabata Dalindyebo Local Municipality in the Oliver Reginald Tambo District of the ECP in South Africa. Study areas and health facilities in Mthatha and surrounding areas were primary health centres/clinics, secondary district hospitals, and a tertiary/academic hospital.Figure 1
Map of South Africa showing study area, Umtata (now Mthatha), in the province of Eastern Cape (by courtesy of Encyclopaedia Britannica, Inc., copyright 2009; used with permission) [14].


#### 2.1.3. Specimens

Nonduplicate, randomly selectedKlebsiella isolates were collected from Mthatha and surrounding-area clinics. Specimens included blood culture and catheter tips, swabs from abscesses, eye, ear, and vagina, sputum and throat swabs, urine, and sterile fluids (plural fluid, synovial fluid, etc.). Demographic data of the patients recorded were date of specimen collection, age, gender, specimen, tests ordered, and hospital/clinic and provisional diagnosis.


### 2.2. Microbiologic Methods

All samples were routinely cultured on MacConkey and blood agar plates. Blood and sputum were also cultured on chocolate agar. All suspected colonies were identified by gram staining, colony characteristics, motility, and so forth. Strains were identified to the species level with bioMérieux API20E and confirmed by Siemens MicroScan Negative ID panel Type 2. MICs were determined using MicroScan dehydrated broth microdilution panel negative MIC Type 37 (Siemens Medical Solutions Diagnostics, West Sacramento, CA), following the manufacturer’s guidelines and Clinical Laboratory Standards Institute (CLSI) [15]. MICs were interpreted following CLSI guidelines, including the new clinical breakpoints published in 2010 for carbapenems [16]. ESBL detection: phenotypic—the ESBL detection was done as was recommended by the CLSI confirmatory procedure, by using cefotaxime (30 μg) and ceftazidime (30 μg) discs alone and in combination with clavulanic acid discs.K. pneumoniae (ATCC-700603) were used as the controls throughout the study [17]. The ESBL production was confirmed by MicroScan MIC 37 panel using combination of cefotaxime/K clavulanate (Cft/CA) and ceftazidime/K clavulanate (Caz/CA) [18].


### 2.3. Molecular ESBL Detection by rPCR

#### 2.3.1. DNA Extraction

DNA extraction was done using Roche MagNA Pure Bacteria Lysis Buffer, MagNA Pure Compact Nucleic Acid Isolation Kit 1 in MagNA Pure Compact System (Roche Applied Science, Indianapolis).


#### 2.3.2. Real-Time PCR forblaCTX-M, blaTEM, blaSHV, blaNDM, and blaKPC

Real-time polymerase chain reaction (rPCR) was carried out in the LightCycler 2.0 instrument (Roche Applied Science, Germany) using LightCycler 480 Probes Master kit (Roche Diagnostics, USA). TheblaCTX-M, blaTEM, and blaSHV for ESBL and blaNDM and blaKPC genes for CRE were amplified by singleplex rPCR using the primers shown in Table 1. Primers were designed by TIB-Molbiol (Berlin, Germany) based on primers used by Turton et al. [19]. rPCR assay was performed in a 32 carousels using 20 μL capillaries with each capillary containing a total volume of 20 μL including 2 μL of LightCycler FastStart DNA Master HybProbe, 2 μL of primers (0.5 mM for each forward and reverse), 2.4 μL of MgCl2, 2 μL of extracted DNA, and water to make up the volume of 20 μL. DNA amplification was carried out using preincubation step at 95°C for 30 s, followed by 45 cycles of amplification with denaturation at 95°C for 30 s, annealing and extension at 60°C for 1 minute without the third step, and then a single cycle of cooling. Absolute quantification was carried out using the LightCycler software 4.05.Table 1
Primer sequences used forblaCTX-M, blaTEM, blaSHV, blaKPC, and blaNDM genes detection.
Target gene Primer sequences (5′-3′) Temp in °C Target size bp CTX-M forward primer ATG TGC AGY ACC AGT AAR GTK ATG GC 58.7 336 bp CTX-M reverse primer ATC ACK CGG RTC GCC NGG RAT 59.3 CTX-M1 probe FAM-CCC gAC AgC Tgg gAg ACg AAA CgT 70.2 CTX-M2 probe (gr 2) YAK-CAggTgCTTATCgCTCTCgCTCTgTT 66.3 CTX-M9 all/1 probe LC640-Cg AC AAT ACNgCC ATg AA 41.0 CTX-M9 probe LC610-CTggATCgCACTgAACCTACgCTgA 53.0 TEM forward primer AAG TTC TGC TAT GTG CGG TA 59.7 180 bp TEM reverse primer TGT TAT CAC TCA TGG TTA TGG CAG C 59.5 SHV forward primer CAG GAT CTG GTG GAC TAC T 57.2 195 bp SHV reverse primer GTC AAG GCG GGT GAC GTT 59.3 SHV-A primer AAG GCG GGT GAC GTT GTC 59.3 SHV-S primer CCG GTC AGC GAA AAA CAC 57.0 SHV probe Cy5-TCT GGC GCA AAA AGG CAG TCA-BBQ 64.5 NDM forward primer GAC CGC CCA GAT CCT CAA 57.5 51 bp NDM reverse primer CGC GAC CGG CAG GTT 60.2 NDM probe FAM-TGG ATC AAG CA+GGA+GAT-BBQ 48.8 KPC forward primer GGC CGC CGT GCA ATA C 58,6 61 bp KPC reverse primer GCC CAA CTC CTT CA 59,6 KPC probe Cy5-TGA TAA CGC GCG CAA TTT GT-BBQ 68.9 Relevant positive strains forKlebsiellaused as positive control in rPCR: CTX-M (group 1)—E. coli ATCC 25922, CTX-M (all groups except group 1)—CAP2006-D17∗, TEM—K. pneumoniaeATCC 51503, SHV—K. pneumoniae ATCC 700603, KPC—K. pneumoniaeATCC BAA 1705, NDM—K. pneumoniaeATCC BAA 21246. Obtained from NICD (National Institute of Communicable Diseases, Johannesburg, South Africa).


### 2.4. Statistical Analysis

Our data was entered into a database using SPSS 23 for Windows (SPSS Inc., Chicago, IL). The primary analysis involved using chi-square test in order to look for association between dependable variable (ESBL production) with covariable, for example, type of hospital/clinic, gender, and type of specimens. Significant variables were included in binary logistical regression to calculate the odds ratio and 95% confidence interval. All statistical testing was two tailed and statistical significance was defined as ≤0.05. Comparison of ESBL and non-ESBL groups was done using Epidat 3.1 software.


## 2.1. Experimental Design

### 2.1.1. Ethical Considerations

Ethical approval for the study was granted by the Health Research Ethics and Biosafety Committee of the Walter Sisulu University (WSU) certificate number 022/110 and the Nelson Mandela Academic Hospital Ethics Committee (NMAH), Mthatha, ECP.


### 2.1.2. Study Design and Setting

A prospective, descriptive study based on laboratory investigations at the Microbiology Laboratory of the National Health Laboratory Services (NHLS) at NMAH and the Department of Medical Microbiology, Faculty of Health Sciences, WSU was undertaken. In this study 203 nonrepetitive (one per patient) samples from patients were randomly obtained from August 2011 to May 2014. Figure1 shows the specimen catchment area, that is, Mthatha and surrounding clinics. Mthatha (formerly Umtata) is the main town of the King Sabata Dalindyebo Local Municipality in the Oliver Reginald Tambo District of the ECP in South Africa. Study areas and health facilities in Mthatha and surrounding areas were primary health centres/clinics, secondary district hospitals, and a tertiary/academic hospital.Figure 1
Map of South Africa showing study area, Umtata (now Mthatha), in the province of Eastern Cape (by courtesy of Encyclopaedia Britannica, Inc., copyright 2009; used with permission) [14].


### 2.1.3. Specimens

Nonduplicate, randomly selectedKlebsiella isolates were collected from Mthatha and surrounding-area clinics. Specimens included blood culture and catheter tips, swabs from abscesses, eye, ear, and vagina, sputum and throat swabs, urine, and sterile fluids (plural fluid, synovial fluid, etc.). Demographic data of the patients recorded were date of specimen collection, age, gender, specimen, tests ordered, and hospital/clinic and provisional diagnosis.


## 2.1.1. Ethical Considerations

Ethical approval for the study was granted by the Health Research Ethics and Biosafety Committee of the Walter Sisulu University (WSU) certificate number 022/110 and the Nelson Mandela Academic Hospital Ethics Committee (NMAH), Mthatha, ECP.


## 2.1.2. Study Design and Setting

A prospective, descriptive study based on laboratory investigations at the Microbiology Laboratory of the National Health Laboratory Services (NHLS) at NMAH and the Department of Medical Microbiology, Faculty of Health Sciences, WSU was undertaken. In this study 203 nonrepetitive (one per patient) samples from patients were randomly obtained from August 2011 to May 2014. Figure1 shows the specimen catchment area, that is, Mthatha and surrounding clinics. Mthatha (formerly Umtata) is the main town of the King Sabata Dalindyebo Local Municipality in the Oliver Reginald Tambo District of the ECP in South Africa. Study areas and health facilities in Mthatha and surrounding areas were primary health centres/clinics, secondary district hospitals, and a tertiary/academic hospital.Figure 1
Map of South Africa showing study area, Umtata (now Mthatha), in the province of Eastern Cape (by courtesy of Encyclopaedia Britannica, Inc., copyright 2009; used with permission) [14].


## 2.1.3. Specimens

Nonduplicate, randomly selectedKlebsiella isolates were collected from Mthatha and surrounding-area clinics. Specimens included blood culture and catheter tips, swabs from abscesses, eye, ear, and vagina, sputum and throat swabs, urine, and sterile fluids (plural fluid, synovial fluid, etc.). Demographic data of the patients recorded were date of specimen collection, age, gender, specimen, tests ordered, and hospital/clinic and provisional diagnosis.


## 2.2. Microbiologic Methods

All samples were routinely cultured on MacConkey and blood agar plates. Blood and sputum were also cultured on chocolate agar. All suspected colonies were identified by gram staining, colony characteristics, motility, and so forth. Strains were identified to the species level with bioMérieux API20E and confirmed by Siemens MicroScan Negative ID panel Type 2. MICs were determined using MicroScan dehydrated broth microdilution panel negative MIC Type 37 (Siemens Medical Solutions Diagnostics, West Sacramento, CA), following the manufacturer’s guidelines and Clinical Laboratory Standards Institute (CLSI) [15]. MICs were interpreted following CLSI guidelines, including the new clinical breakpoints published in 2010 for carbapenems [16]. ESBL detection: phenotypic—the ESBL detection was done as was recommended by the CLSI confirmatory procedure, by using cefotaxime (30 μg) and ceftazidime (30 μg) discs alone and in combination with clavulanic acid discs.K. pneumoniae (ATCC-700603) were used as the controls throughout the study [17]. The ESBL production was confirmed by MicroScan MIC 37 panel using combination of cefotaxime/K clavulanate (Cft/CA) and ceftazidime/K clavulanate (Caz/CA) [18].


## 2.3. Molecular ESBL Detection by rPCR

### 2.3.1. DNA Extraction

DNA extraction was done using Roche MagNA Pure Bacteria Lysis Buffer, MagNA Pure Compact Nucleic Acid Isolation Kit 1 in MagNA Pure Compact System (Roche Applied Science, Indianapolis).


### 2.3.2. Real-Time PCR forblaCTX-M, blaTEM, blaSHV, blaNDM, and blaKPC

Real-time polymerase chain reaction (rPCR) was carried out in the LightCycler 2.0 instrument (Roche Applied Science, Germany) using LightCycler 480 Probes Master kit (Roche Diagnostics, USA). TheblaCTX-M, blaTEM, and blaSHV for ESBL and blaNDM and blaKPC genes for CRE were amplified by singleplex rPCR using the primers shown in Table 1. Primers were designed by TIB-Molbiol (Berlin, Germany) based on primers used by Turton et al. [19]. rPCR assay was performed in a 32 carousels using 20 μL capillaries with each capillary containing a total volume of 20 μL including 2 μL of LightCycler FastStart DNA Master HybProbe, 2 μL of primers (0.5 mM for each forward and reverse), 2.4 μL of MgCl2, 2 μL of extracted DNA, and water to make up the volume of 20 μL. DNA amplification was carried out using preincubation step at 95°C for 30 s, followed by 45 cycles of amplification with denaturation at 95°C for 30 s, annealing and extension at 60°C for 1 minute without the third step, and then a single cycle of cooling. Absolute quantification was carried out using the LightCycler software 4.05.Table 1
Primer sequences used forblaCTX-M, blaTEM, blaSHV, blaKPC, and blaNDM genes detection.
Target gene Primer sequences (5′-3′) Temp in °C Target size bp CTX-M forward primer ATG TGC AGY ACC AGT AAR GTK ATG GC 58.7 336 bp CTX-M reverse primer ATC ACK CGG RTC GCC NGG RAT 59.3 CTX-M1 probe FAM-CCC gAC AgC Tgg gAg ACg AAA CgT 70.2 CTX-M2 probe (gr 2) YAK-CAggTgCTTATCgCTCTCgCTCTgTT 66.3 CTX-M9 all/1 probe LC640-Cg AC AAT ACNgCC ATg AA 41.0 CTX-M9 probe LC610-CTggATCgCACTgAACCTACgCTgA 53.0 TEM forward primer AAG TTC TGC TAT GTG CGG TA 59.7 180 bp TEM reverse primer TGT TAT CAC TCA TGG TTA TGG CAG C 59.5 SHV forward primer CAG GAT CTG GTG GAC TAC T 57.2 195 bp SHV reverse primer GTC AAG GCG GGT GAC GTT 59.3 SHV-A primer AAG GCG GGT GAC GTT GTC 59.3 SHV-S primer CCG GTC AGC GAA AAA CAC 57.0 SHV probe Cy5-TCT GGC GCA AAA AGG CAG TCA-BBQ 64.5 NDM forward primer GAC CGC CCA GAT CCT CAA 57.5 51 bp NDM reverse primer CGC GAC CGG CAG GTT 60.2 NDM probe FAM-TGG ATC AAG CA+GGA+GAT-BBQ 48.8 KPC forward primer GGC CGC CGT GCA ATA C 58,6 61 bp KPC reverse primer GCC CAA CTC CTT CA 59,6 KPC probe Cy5-TGA TAA CGC GCG CAA TTT GT-BBQ 68.9 Relevant positive strains forKlebsiellaused as positive control in rPCR: CTX-M (group 1)—E. coli ATCC 25922, CTX-M (all groups except group 1)—CAP2006-D17∗, TEM—K. pneumoniaeATCC 51503, SHV—K. pneumoniae ATCC 700603, KPC—K. pneumoniaeATCC BAA 1705, NDM—K. pneumoniaeATCC BAA 21246. Obtained from NICD (National Institute of Communicable Diseases, Johannesburg, South Africa).


## 2.3.1. DNA Extraction

DNA extraction was done using Roche MagNA Pure Bacteria Lysis Buffer, MagNA Pure Compact Nucleic Acid Isolation Kit 1 in MagNA Pure Compact System (Roche Applied Science, Indianapolis).


## 2.3.2. Real-Time PCR forblaCTX-M, blaTEM, blaSHV, blaNDM, and blaKPC

Real-time polymerase chain reaction (rPCR) was carried out in the LightCycler 2.0 instrument (Roche Applied Science, Germany) using LightCycler 480 Probes Master kit (Roche Diagnostics, USA). TheblaCTX-M, blaTEM, and blaSHV for ESBL and blaNDM and blaKPC genes for CRE were amplified by singleplex rPCR using the primers shown in Table 1. Primers were designed by TIB-Molbiol (Berlin, Germany) based on primers used by Turton et al. [19]. rPCR assay was performed in a 32 carousels using 20 μL capillaries with each capillary containing a total volume of 20 μL including 2 μL of LightCycler FastStart DNA Master HybProbe, 2 μL of primers (0.5 mM for each forward and reverse), 2.4 μL of MgCl2, 2 μL of extracted DNA, and water to make up the volume of 20 μL. DNA amplification was carried out using preincubation step at 95°C for 30 s, followed by 45 cycles of amplification with denaturation at 95°C for 30 s, annealing and extension at 60°C for 1 minute without the third step, and then a single cycle of cooling. Absolute quantification was carried out using the LightCycler software 4.05.Table 1
Primer sequences used forblaCTX-M, blaTEM, blaSHV, blaKPC, and blaNDM genes detection.
Target gene Primer sequences (5′-3′) Temp in °C Target size bp CTX-M forward primer ATG TGC AGY ACC AGT AAR GTK ATG GC 58.7 336 bp CTX-M reverse primer ATC ACK CGG RTC GCC NGG RAT 59.3 CTX-M1 probe FAM-CCC gAC AgC Tgg gAg ACg AAA CgT 70.2 CTX-M2 probe (gr 2) YAK-CAggTgCTTATCgCTCTCgCTCTgTT 66.3 CTX-M9 all/1 probe LC640-Cg AC AAT ACNgCC ATg AA 41.0 CTX-M9 probe LC610-CTggATCgCACTgAACCTACgCTgA 53.0 TEM forward primer AAG TTC TGC TAT GTG CGG TA 59.7 180 bp TEM reverse primer TGT TAT CAC TCA TGG TTA TGG CAG C 59.5 SHV forward primer CAG GAT CTG GTG GAC TAC T 57.2 195 bp SHV reverse primer GTC AAG GCG GGT GAC GTT 59.3 SHV-A primer AAG GCG GGT GAC GTT GTC 59.3 SHV-S primer CCG GTC AGC GAA AAA CAC 57.0 SHV probe Cy5-TCT GGC GCA AAA AGG CAG TCA-BBQ 64.5 NDM forward primer GAC CGC CCA GAT CCT CAA 57.5 51 bp NDM reverse primer CGC GAC CGG CAG GTT 60.2 NDM probe FAM-TGG ATC AAG CA+GGA+GAT-BBQ 48.8 KPC forward primer GGC CGC CGT GCA ATA C 58,6 61 bp KPC reverse primer GCC CAA CTC CTT CA 59,6 KPC probe Cy5-TGA TAA CGC GCG CAA TTT GT-BBQ 68.9 Relevant positive strains forKlebsiellaused as positive control in rPCR: CTX-M (group 1)—E. coli ATCC 25922, CTX-M (all groups except group 1)—CAP2006-D17∗, TEM—K. pneumoniaeATCC 51503, SHV—K. pneumoniae ATCC 700603, KPC—K. pneumoniaeATCC BAA 1705, NDM—K. pneumoniaeATCC BAA 21246. Obtained from NICD (National Institute of Communicable Diseases, Johannesburg, South Africa).


## 2.4. Statistical Analysis

Our data was entered into a database using SPSS 23 for Windows (SPSS Inc., Chicago, IL). The primary analysis involved using chi-square test in order to look for association between dependable variable (ESBL production) with covariable, for example, type of hospital/clinic, gender, and type of specimens. Significant variables were included in binary logistical regression to calculate the odds ratio and 95% confidence interval. All statistical testing was two tailed and statistical significance was defined as ≤0.05. Comparison of ESBL and non-ESBL groups was done using Epidat 3.1 software.


## 3. Results

During the study period, a total of 202Klebsiella species were isolated from a range of clinical specimens of patients hospitalized in various wards of NMAH in Mthatha and surrounding areas with secondary hospitals and clinics.K. pneumoniae were the majority 169 (83.7%) species isolated followed byK. oxytoca29 (14.4%) whileK. ozaenae andR. ornithinolytica were 2 (0.9%). The prevalence of ESBL production in allKlebsiella species was 122 (60.4%) while ESBL-producingK. pneumoniae were 117 (69.2%) followed byK. oxytoca5 (17.9%) and bothK. ozaenae andR. ornithinolytica were ESBL negative. Female population was slightly more 106 (52.5%) than males 96 (47.5%).In the multivariate analysis (backward logistic regression), using age group from day 1 to 5 years and tertiary level of hospitalization were found to be independent risk factors for infection due to ESBLKlebsiella species (Table 2).Table 2
Multivariate analysis of risk factors for ESBL-producing and nonproducingKlebsiella species in Mthatha and surrounding areas.
(a) Quantitative variable ESBL NIL OR (95% CI) P value Mean ± SD Mean ± SD Age (groups 0–5) 22.78 (22.77) 30.9 (22.7) 2.32 (1.20–4.52) 0.012  (b) Variable ESBL NIL OR (95% CI) P value Number (%) Number (%) Gender Male 59 (60.8) 38 (39.2) 1.18 (0.68–2.06) 0.551 Female 58 (55.2) 47 (44.8) Type of hospital Primary∗ 3 (21.4) 11 (78.6) — — Secondary 41 (47.1) 46 (52.9) 2.06 (0.76–5.57) 0.153 Tertiary 73 (72.3) 28 (27.7) 5.96 (2.21–16.03) 0.000 ∗Reported as resistant due to ESBL production.High antibiotic resistance in decreasing order was observed in amp/sulbactam, mezlocillin 167 (82.7%), piperacillin 160 (79.2%), trimeth/sulfa 143 (70.8%), cefazolin 139 (68.8%), cefepime 130 (64.4%), cefuroxime 129 (63.9%), cefpodoxime 127 (62.9%), aztreonam 126 (62.4%), ceftazidime 124 (61.4%), and tobramycin 108 (53.5%).Tables4 and 5 show rPCR data, blaCTX-M, blaTEM, blaSHV, blaNDM, and blaKPC rPCR, and LightCycler 2.0 results; real-time PCR was done on 157 specimens.rPCR was done on 157 specimens which were 103 (65.6%) of ESBL positiveK. pneumoniae andK. oxytoca and 54 (34.4%) ESBL negativeK. pneumoniae,K. oxytoca,K. ozaenae, andR. ornithinolytica. In rPCR the most common genotype was blaSHV 121 (77.1%) followed by blaTEM 105 (66.9%) and blaCTX-M at 89 (56.7%) as last.As seen in Table5 there is increase trend of ESBL genotypes over four years of study from 2011 to 2014 except in year 2013. The carbapenemase genes blaNDM and blaKPC were not detected.


## 4. Discussion

The emergence of plasmid mediated ESBLs among the members ofKlebsiellahas increased worldwide. The incidence of ESBLs in the different parts of South Africa has been reported from 36.1% to be as high as 68.3% (Gauteng, Western Cape, KwaZulu-Natal, Free State, and Limpopo) [11–13]. ESBL inK. pneumoniae showed rate of 62% from seven public sector hospitals in 2010 [20]. We reported 57.9% (117) ESBL producers, which is less than the above-mentioned South African provinces. In all of the above-mentioned studies ECP was not represented; this could be reason for slightly less rate of ESBLs in this area. In Africa ESBLs rate in different countries have increased from 11.7 to 77.8% amongK. pneumoniae (study periods: 1999–2005 and 2010) [7, 21]. Our ESBL rate of 57.9% is within this range, but it is on higher side. It was interesting to note that specific ESBLs appeared to be unique to a certain country or region. Though the prevalence of ESBLs has been recognized in various parts of the country, there is only limited data on its genotypes in this part of South Africa.High antibiotic resistance in decreasing order was observed in penicillins, cephalosporins, folate pathway inhibitors, monobactams, and aminoglycosides. Percentage of low resistance was seen in carbapenems, aminoglycosides (only amikacin), glycylcyclines (tigecycline), cephamycins (cefoxitin), quinolone (levofloxacin), phosphonic acids (fosfomycin), antipseudomonal penicillins +β-lactamase inhibitor (pip/tazo), and fluoroquinolones (ciprofloxacin) which can be considered for treatment ofKlebsiella species in Mthatha. Although resistance ofK. pneumoniae to ceftazidime is a useful marker of presence of ESBLs, some types of ESBL-producing organisms appear susceptible to ceftazidime according to standard methods, and ceftazidime resistance may be due to mechanisms other than ESBL production. We detected 61.4% resistance to ceftazidime by MIC method while 57.9% (117)Klebsiella were ESBL producers by the CLSI confirmatory test which correlates well. Antibiotic resistance to cefotaxime, ceftazidime, and cefepime was 125 (61.9%), 124 (61.4%), and 130 (64.4%), respectively, which is almost identical. This suggests that there are multiple copies of several ESBL genes in the sample, which is supported by the genotypic results in which 66.2% specimens had two or more ESBL genes (Table 3). We observed that nearly two-thirds of theKlebsiella ESBL isolates were also resistant to at least 2 useful non-β lactam antibiotics used to treat UTI, such as ciprofloxacin, trimethoprim-sulfamethoxazole, and gentamicin. Similar observations have been made by other investigators [22].Table 3
Percentage of resistance (included intermediate resistance for statistical analysis) in ESBL and non-ESBLKlebsiella(N=202).
Antibiotic Total (n=202) ESBL (n=117) NIL (n=85) OR (95% CI) P value a Number (%) Number (%) Number (%) Amikacin 10 (5.0) 5 (4.3) 5 (5.9) 0.71 (0.20–2.55) 0,604 Amox/K clav 111 (55.0) 98 (83.3) 13 (15.3) 28.6 (13.25–61.59) 0,000 Ampicillin 137 (67.8) 115 (98.3) 22 (25.9) 162.0 (36.88–711.93) 0,000 Amp/sulbactam∗ 201 (99.5) 117 (100.0) 84 (98.8) — 1,000 Aztreonam∗ 126 (62.4) 117 (100.0) 9 (10.6) — 0,996 Cefazolin∗ 139 (68.8) 117 (100.0) 22 (25.9) — 0,996 Cefepime∗ 130 (64.4) 117 (100.0) 13 (15.3) — 0,996 Cefotaxime 124 (61.4) 117 (100.0) 7 (8.2) 116.5 (136.9–9105.9) 0,000 Cefoxitin 18 (8.9) 10 (8.5) 8 (9.4) 0.90 (0.34–2.38) 0,831 Cefpodoxime∗ 127 (62.9) 117 (100.0) 10 (11.8) — 0,996 Ceftazidime∗ 124 (61.4) 117 (100.0) 7 (8.2) — 0,996 Cefuroxime 129 (63.9) 117 (99.1) 13 (15.3) 642.5 (82.29–5016.08) 0,000 Chloramphenicol 71 (35.1) 53 (45.3) 18 (21.2) 3.25 (1.71–6.19) 0,000 Ciprofloxacin 60 (29.7) 55 (47.0) 5 (5.9) 14.19 (5.36–37.58) 0,000 Ertapenem 7 (3.5) 4 (3.4) 3 (3.5) 0.97 (0.21–4.44) 0,966 Fosfomycin 26 (12.9) 15 (12.8) 11 (12.9) 0.97 (0.42–2.25) 0,954 Gentamicin 103 (51.0) 92 (78.6) 11 (12.9) 24.76 (11.44–53.60) 0,000 Imipenem 0 (0.0) 0 (0.0) 0 (0.0) — — Levofloxacin 23 (11.4) 19 (16.2) 4 (4.7) 3.88 (1.27–11.86) 0,018 Meropenem 0 (0.0) 0 (0.0) 0 (0.0) — — Mezlocillin∗ 167 (82.7) 117 (100.0) 50 (58.8) — 0,997 Moxifloxacin 77 (38.1) 65 (55.6) 12 (14.1) 8.43 (4.10–17.32) 0,000 NitrofurantoinΦ 1 (0.5) 0 (0.0) 1 (1.2) — 1,000 NorfloxacinΦ 3 (1.5) 3 (2.6) 0 (0.0) — 0,999 Pip/tazo 31 (15.3) 28 (23.9) 3 (3.5) 8.60 (2.52–29.36) 0,001 Piperacillin∗ 160 (79.2) 117 (100.0) 43 (50.6) — 0,997 Tetracycline 57 (28.2) 44 (37.6) 13 (15.3) 3.29 (1.64–6.63) 0,001 Tigecycline 17 (8.4) 9 (7.7) 8 (9.4) 0.80 (0.30–2.17) 0,664 Tobramycin 108 (53.5) 102 (87.2) 6 (7.1) 88.40 (32.79–238.28) 0,000 Trimeth/sulfa 143 (70.8) 115 (98.3) 28 (32.9) 117.05 (26.93–508.72) 0,000 aExact P values were determined by the χ2 test. For statistical analysis, the response of isolates to antibiotics was categorized as susceptible and nonsusceptible (consisting of intermediate and resistant groups). A P value < 0.05 was considered statistically significant. ∗Reported as resistance due to ESBL production according to confirmation by Autoscan MIC37 panel. Φ: used only in urinary tract infections.Table 4
Extended-spectrumβ-lactamase (ESBL) genotypes in Klebsiella strains.
Positive by PCR for ESBL genes Number amplified K. pneumoniae(n=139) K. oxytoca   (n=16) R. ornithinolyticaandK. ozaenae   (n=2) Total(N=157) (A) Single ESBL gene 28 2 2 32 (20.4%) blaTEM only 5 2 1 8 (5.1%) blaSHV only 22 0 1 23 (14.7%) blaCTX-M only 1 0 0 1 (0.6%) blaKPC only∗ 0 0 0 0 blaNDM only∗ 0 0 0 0 (B) Two or more ESBL genes 100 3 0 103 (65.6%) blaCTX-M+blaTEM 4 0 0 4 (2.6%) blaCTX-M+blaSHV 6 0 0 6 (3.8%) blaTEM+blaSHV 12 2 0 14 (8.9%) blaTEM+blaSHV+blaCTX-M 78 1 0 79 (50.3%) blaKPC+blaNDM∗ 0 0 0 0 ∗Done in 52 selective ESBL positive isolates.Table 5
Genotypes ofKlebsiellablaTEM, blaSHV, and blaCTX-M from 2011 to 2014.
Genotypes 2011 2012 2013 2014 Total (N=157) b l a T E M positive alone or in combination 23/34 (67.7%) 63/98 (67.3%) 6/8 (75%) 13/17 (76.5%) 105 (66.9%) b l a S H V positive alone or in combination 19/34 (55.9%) 79/98 (80.6%) 8/8 (100%) 15/17 (88.2%) 121 (77.1%) b l a CTX-M positive alone or in combination 17/34 (50%) 54/98 (55.1%) 6/8 (75%) 12/17 (70.6%) 89 (56.7%)In this study the most common genotype wasblaSHV 118 (75.2%) followed by blaTEM 99 (63.1%) and blaCTX-M 89 (56.7%) as last. These genotypes were found in majority of ESBL positiveKlebsiella species as compared to ESBL negativeKlebsiella. Our study finding is similar to the study done from Italy in which blaSHV was found to be the dominant ESBL genotype and also from Spain [22, 23]. ESBL genotype varies in different parts of the world; blaCTX-M is the most prevalent gene in high proportion of samples from different parts of the world and also Morocco [7, 9]. In Africa blaCTX-M15 is the most prevalent gene in a high proportion of the samples, disregarding country. It is usually combined with other types of blaCTX-M, blaTEM, and blaSHV genes [7]. In other parts of the world, for example, Canada [8] and Turkey [24], blaTEM was the most common ESBL genotype. The most common ESBL genotype combination among theKlebsiella species (especially inK. pneumoniae) was combination of blaTEM + blaSHV + blaCTX-M which is similar to a previous report from South Africa [12]. The second most common genotype combination was the combination of blaTEM+blaSHV which is similar to the study from Japan [4]. We found increased trend ofKlebsiella ESBL genotypes over 4 years; this trend is seen in studies from different parts of the world, Japan and Canada [4, 8].CRE has become an international health issue and poses a major threat to the viability of currently available antibiotics. First KPC was reported from South Africa in August 2011 from private hospital in Gauteng [25]. In our study we observed resistance to carbapenems by phenotypic test as ertapenem 3.5% while meropenem and imipenem were all susceptible. But all specimens were negative for blaKPC and blaNDM by genotypic test; this could be due to other resistance mechanisms such as newer CRE genes Verona Integron-Mediated Metallo-β-Lactamase (VIM), Imipenemase Metallo-Beta-Lactamase (IMP), or class D OXA-type enzymes [26].In multivariate analysis we found age group 1–5 years (OR 2.32 [CI 1.20–4.52]) and tertiary health centre (OR 5.96 [CI 2.21–16.03]) were risk factors for developing ESBL. In ESBLs we found statistically significant (P<0.005) antibiotic resistance in amox/clav, ampicillin, cefotaxime, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin, levofloxacin, moxifloxacin, pip/tazo, tetracycline, tobramycin, and trimeth/sulfa.


## 5. Conclusion

This is the first report of molecular characteristics and antibiotic resistance profiles ofKlebsiella isolates from clinical samples of patients in Mthatha, ECP, South Africa. The majority ofKlebsiella species in our area areK. pneumoniae followed byK. oxytoca and less than 1% ofK. ozaenae andR. ornithinolytica. This study reveals high ESBL rate inKlebsiella species especially inK. pneumoniaein this area. We also identified ESBL-producing three genes of blaTEM, blaSHV, and blaCTX-M by rPCR and our antibiotic resistance inKlebsiella species in Mthatha which is driven by combination of blaTEM+blaSHV+blaCTX-M and the most common genotype was blaSHV followed by blaTEM and blaCTX-M as last. For treatment of drug resistantKlebsiella choice of antibiotics in decreasing order is carbapenems, amikacin, tigecycline, cefoxitin, levofloxacin, pip/tazo, ciprofloxacin, and fosfomycin for UTI. Fortunately we did not detect CRE-formingKlebsiella in Mthatha. There is steady increase in rate of ESBL genes in the last five years; therefore continuous surveillance is essential to monitor the ESBL-producingKlebsiellain hospitals and community and also for CRE.



---

*Source: 8486742-2017-01-30.xml*