# Salmonella Agona harboring genomic island 1-A

B. Doublet, P. Butaye, H. Imberechts, J.M. Collard, E. Chaslus-Dancla, Axel Cloeckaert

► To cite this version:

B. Doublet, P. Butaye, H. Imberechts, J.M. Collard, E. Chaslus-Dancla, et al.. Salmonella Agona harboring genomic island 1-A. Emerging Infectious Diseases, 2004, 10 (4), pp.756-758. 10.3201/eid1004.030417. hal-02683157

HAL Id: hal-02683157

https://hal.inrae.fr/hal-02683157

Submitted on 1 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /><figcaption>Figure 1. Electron micro-
graph of O34 vibriophage.
Bar represents 100 nm.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Figure 2. Electron micro-
graph of O6 vibriophage.
Bar represents 100 nm.</figcaption></figure>

isolate more phages in Brazil and
neighboring countries.

To date, serotyping is the only
identification tool for the characteri-
zation of non-O1 strains of V. choler-
ae (8). However, serotyping is only
performed at a limited number of lab-
oratories. For this study, all isolates
from Brazil were sent to laboratories
outside the country for serotyping.
This step was expensive and time-
consuming and posed risks during
transit.

An alternative method is the use of
phages for identifying non-O1 strains.
This method offers an affordable
monitoring system in less-developed
countries such as Brazil. Phage O6
and O34 should at least be useful for
confirming the diagnosis of V. choler-
ae O6 and O34 infection and for dif-
ferentiating V. cholerae O1 and non-
O1 strains.

Acknowledgment

The authors are grateful to Eliseu
Alves Waldman for providing valuable
information.

B.L. Sarkar* Amar N. Ghosh,*
Anindito Sen,*
and D. P. Rodrigues†

*National Institute of Cholera & Enteric Diseases (ICMR) World Health Organization Collaborating Centre for Research and Training on Diarrheal Diseases, Kolkata, India; and †Instituto Oswaldo Cruz, Rio de Janerio, Brazil

**References**

1. Sarkar BL. A new phage-typing scheme for *Vibrio cholerae* biotype EITor developed at the National Institute of Cholera and Enteric Diseases, Calcutta, India. *Indian Journal of Medical Research Bulletin* 1993;23:82-3.

2. Sarkar BL, Roy MK, Chakraborti AK, Niyogi SK. Distribution of phage type of *Vibrio cholerae* O1 biotype in Indian

Scenario (1991–98). Indian J Med Res 1999;109:204–7.

3. Chakrabarti AK, Ghosh AN, Nair GB, Niyogi SK, Bhattacharya SK, Sarkar BL. Development and evolution of a phage typing scheme for *Vibrio cholerae* O139. J Clin Microbiol 2000;38:44–9.

4. Rodrigues DP, Seki LM, Sarkar BL. The use of phage typing scheme in *Vibrio cholerae* strains isolated in Brazil. Proc. 33rd US-Japan cholera and related diarrheal diseases joint panel meeting, Dec 3–5, 1997. Clearwater Beach (FL): U.S.-Japan Cooperative Medical Science Program; 1997. p. 188.

5. Chattopadhyay DJ, Sarkar BL, Ansari MQ, Chakrabarti BK, Roy MK, Ghosh AN, et al. New phage typing scheme for *Vibrio cholerae* O1 biotype El Tor strains. J Clin Microbiol 1993;31:1579–85.

6. Chakrabarti AK, Ghosh AN, Sarkar BL. Isolation of *Vibrio cholerae* O139 phages to develop a phage typing scheme. Indian J Med Res 1997;105:254–7.

7. Ghosh AN, Ansari MQ, Dutta GC. Isolation and morphological characterization of EIT or cholera phages. J Gen Virol 1989;70:2241–3.

8. Sarkar BL. Cholera bacteriophages revisited. ICMR Bull 2002;32:33–7.

Address for correspondence: B. L. Sarkar, National Institute of Cholera & Enteric Diseases (ICMR) WHO Collaborating Centre for Research and Training on Diarrheal Diseases, P-33, C.I.T. Road, Scheme-XM, Beleghata, Kolkata 700010, India; fax: +91-33-2350-506; email: bl_sarkar@hotmail.com

Salmonella Agona
Harboring Genomic
Island 1-A

**To the Editor:** Multidrug-resistant *Salmonella enterica* serovar Typhimurium definitive phage type 104 has emerged during the 1980s and 1990s as a world health problem because of its implications in animal and human disease (1–3). Epidemic serovar Typhimurium definitive phage type 104 isolates are commonly resistant to ampicillin (Ap), chlo-
ramphenicol (Cm)/florfenicol (Ff), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracyclines (Tc) (1,3). This multidrug-resistance phenotype is conferred by an antibiotic resistance gene cluster included in a 43kb genomic island named *Salmonella* genomic island 1 (4). *Salmonella* genomic island 1 has been recently characterized and located between the *thdF* and *int2* genes of the chromosome. The *int2* gene is part of a retron sequence found only in serovar Typhimurium. Downstream of the retron sequence is the *yidY* gene, which is also found in the chromosome of other *S. enterica* serovars. The antibiotic resistance gene cluster of approximately 13 kb is located at the 3' end of *Salmonella* genomic island 1 (4). All resistance genes are clustered and are bracketed by two integron structures (5,6). The first integron carries the *aadA2* gene, which confers resistance to Sm and Sp. The second integron contains the β-lactamase gene *pse-1*, conferring resistance to Ap. Flanked by these two integron structures are the *floR* gene, which confers cross-resistance to Cm and Ff, and the tetracycline-resistance genes *tetR* and *tet(G)* (5,6). Recently, *Salmonella* genomic island 1 has also been identified in other serovars of *S. enterica* namely Agona (4,7), Paratyphi B (8), and Albany (9), indicating the horizontal transfer potential of *Salmonella* genomic island 1. In these serovars, *Salmonella* genomic island 1 has the same chromosomal location as in serovar Typhimurium definitive phage type 104, except that they lack the retron sequence found downstream of *Salmonella* genomic island 1 (4,8,9). Moreover, six variant *Salmonella* genomic island 1 antibioticic resistance gene clusters (*Salmonella* genomic island 1-A to -F) have recently been reported for serovars Typhimurium DT104, Agona, and Albany to confer different multidrug resistance phenotypes (9,10). These clusters of genes were

probably generated after chromosomal recombinational events or by antibiotic resistance gene cassette replacement in the integron structures. In particular, the *dfrA10* gene coding for trimethoprim (Tm) resistance was found downstream of the *pse-1* integron in a third unusual integron structure involving *orf513* in the variant antibiotic resistance gene cluster called *Salmonella* genomic island 1-A (ApCmFfSmSpSuTcTm) (10).

Multidrug-resistant serovar Typhimurium definitive phage type 104 was disseminated globally with several outbreaks in humans and animals. At present, in contrast to the world health problem of multidrug-resistant serovar Typhimurium definitive phage type 104, human cases of infections with other *S. enterica* serovars harboring *Salmonella* genomic island 1 have not yet been reported. *Salmonella* genomic island 1-multidrug-resistant serovars Agona, Paratyphi B, and Albany were isolated from different animal species and countries (7-9). In this study, we analysed the first *Salmonella* genomic island 1 positive serovar Agona strain (02/01177) isolated from a human case in Belgium.

A Belgian patient, who had been infected by a multidrug-resistant serovar Agona strain was travelling to Turkey; subsequent to the multidrug-resistant serovar Agona strain, gastroenteritis developed. While in Turkey the patient sought medical care and was treated unsuccessfully with antimicrobial agents. Upon his return to Belgium, this serovar Agona strain was isolated from his stools, and he recovered after treatment with ciprofloxacin. The serovar Agona strain 02/01177 displayed the multidrug resistance profile ApCmFfSm SpSuTcTm, which suggested the possible occurrence of *Salmonella* genomic island 1-A (10). Moreover, the strain showed the same level of resistance to Ff as *Salmonella* genomic island 1 harboring *S. enterica*

serovars (MIC of 64 µg/mL) (7-9).

To assess the presence of *Salmonella* genomic island 1 and its location in the chromosome, polymerase chain reactions (PCRs) were performed using primers corresponding to the left and the right (with or without retron) *Salmonella* genomic island 1 junctions to the chromosome as described previously (4,8-10). PCR results were positive for the left junction between the *thdF* gene of the chromosome and the *int* gene of *Salmonella* genomic island 1 (4). For the right junction, PCR results were positive between open reading frame (ORF) S044 of *Salmonella* genomic island 1 and *yidY* gene of the chromosome. Thus, these data indicate that this serovar Agona human isolate contains *Salmonella* genomic island 1 at the same chromosomal location as in other *Salmonella* genomic island 1 positive serovars but lacks the retron sequence found to date only in serovar Typhimurium strains (4,8,9).

PCR mapping of the typical antibiotic resistance genes and integrons associated with *Salmonella* genomic island 1 was realized as described previously (4,8-10). PCR amplifications on genomic DNA extracted from serovar Agona strain 02/01177 yielded all specific fragments of the sizes expected from DNA of serovar Agona control strain 1169SA97 harboring *Salmonella* genomic island 1-A (data not shown) (10). These PCR mapping results indicated the presence of the typical *Salmonella* genomic island 1 resistance gene cluster with the insertion of the third unusual *orf513* integron structure carrying *dfrA10* (8-10). These data are in accordance with the multidrug resistance phenotype of serovar Agona strain 02/01177 and indicate the presence of the variant antibiotic resistance gene cluster *Salmonella* genomic island 1-A (10).

Macorestriction analysis by pulsed-field gel electrophoresis of DNA from serovar Agona strain 02/01177 cut by *Xba*I or *Bln*I, showed
that this human isolate is indistin-
guishable by its XbaI or BlnI macrore-
striction patterns from the other mul-
tidrug-resistant *Salmonella* genomic
island 1-carrying serovar Agona
strains isolated from poultry in
Belgium (data not shown) (7). Thus,
the human serovar Agona isolate
appears clonally related to those from
poultry.

To our knowledge, this is the first
report describing a human infected by
a serovar Agona strain harboring
*Salmonella* genomic island 1-A.
Moreover, it shows the first case
where another *S. enterica* serovar har-
boring *Salmonella* genomic island 1
than the epidemic serovar
Typhimurium definitive phage type
104 clone is implicated in human
infection. The patient could probably
have been infected before his travel to
Turkey by a *Salmonella* genomic
island 1-A carrying serovar Agona
strain in Belgium where this type of
strain is frequently isolated from
poultry (Doublet et al., pers. comm.).
This hypothesis is also supported by
macrorestriction analysis, which
showed that the strains from poultry
and the human case-patient had iden-
tical XbaI and BlnI pulsed-field gel
electrophoresis patterns and thus indi-
cate that they are clonally related.
Moreover, the patient was not in con-
tact with poultry during his stay in
Turkey and, to date, very little is
known about the epidemiology of
multidrug-resistant serovar Agona
strains in this country. Further investi-
gations on the epidemiology of mul-
tidrug-resistant serovar Agona strains
harboring *Salmonella* genomic island

1 are warranted to avoid such strains
becoming a worldwide epidemic, as
was the case for multidrug-resistant
serovar Typhimurium definitive
phage type 104.

Acknowledgments

We thank C. Mouline for expert tech-
nical assistance. We also thank D. Boyd
and M.R. Mulvey for their collaboration.

This work was supported by a grant
from the French Institut National de la
Recherche Agronomique (INRA, Action
Transversalité 2001-2003).

Benoît Doublet,* Patrick Butaye,†

Hein Imberechts,†

Jean-Marc Collard,‡

Elisabeth Chaslus-Dancla,*

and Axel Cloeckaert*

*Institut National de la Recherche
Agronomique, Nouzilly, France; †Centre
d'Etude et de Recherche Vétérinaires et
Agrochimiques, Brussels, Belgium; and
‡Scientific Institute of Public Health,
Brussels, Belgium

**References**

1. Cloeckaert A, Schwarz S. Molecular char-
acterization, spread and evolution of mul-
tidrug resistance in *Salmonella enterica*
serotype Typhimurium DT104. Vet Res
2001;32:301-10.

2. Glynn MK, Bopp C, Dewitt W, Dabney P, Mokhtar M, Angulo FJ. Emergence of multidrug-resistant *Salmonella enterica* serotype Typhimurium DT104 infections in the United States. N Engl J Med 1998;338:1333-8.

3. Ribot EM, Wierzba RK, Angulo FJ, Barrett TJ. *Salmonella enterica* serotype Typhimurium DT104 isolated from humans, United States, 1985, 1990, and 1995. Emerg Infect Dis 2002;8:387-91.

4. Boyd D, Peters GA, Cloeckaert A, Sidi Boumedine K, Chaslus-Dancla E,

Imberechts H, et al. Complete nucleotide
sequence of a 43-kilobase genomic island
associated with the multidrug resistance
region of *Salmonella enterica* serovar
Typhimurium DT104 and its identification
in phage type DT120 and serovar Agona. J
Bacteriol 2001;183:5725-32.

5. Arcangioli MA, Leroy-Sétrin S, Martel JL, Chaslus-Dancla E. A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in *Salmonella typhimurium* DT104. FEMS Microbiol Lett 1999;174:327-32.

6. Briggs CE, Fratamico PM. Molecular char-
acterization of an antibiotic resistance gene
cluster of *Salmonella typhimurium* DT104.
Antimicrob Agents Chemother
1999;43:846-9.

7. Cloeckaert A, Sidi Boumedine K, Flaujac G, Imberechts H, D'Hooghe I, Chaslus-Dancla E. Occurrence of a *Salmonella enterica* serovar Typhimurium DT104-like antibiotic resistance gene cluster including the *floR* gene in *S. enterica* serovar Agona. Antimicrob Agents Chemother 2000;44:1359-61.

8. Meunier D, Boyd D, Mulvey MR, Baucheron S, Mammina C, Nastasi A, et al. *Salmonella enterica* serotype Typhimurium DT 104 antibiotic resistance genomic island I in serotype Paratyphi B. Emerg Infect Dis 2002;8:430-3.

9. Doublet B, Lailler R, Meunier D, Brisabois A, Boyd D, Mulvey MR, et al. Variant *Salmonella* genomic island 1 antibiotic resistance gene cluster in *Salmonella enterica* serovar Albany. Emerg Infect Dis 2003;9:585-91.

10. Boyd D, Cloeckaert A, Chaslus-Dancla E, Mulvey MR. Characterization of variant *Salmonella* genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 2002;46:1714-22.

Address for correspondence: Axel Cloeckaert,
Unité 086, Bio-Agresseurs, Santé,
Environnement Institut National de la
Recherche Agronomique, 37380 Nouzilly,
France, email: cloeckae@tours.inra.fr

Search past issues of EID at www.cdc.gov/eid