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High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island
G Model
VETMIC-6508; No. of Pages 5 Veterinary Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island O. Belmonte a, H. Pailhorie`s b,c, M. Kempf b,c, M.P. Gaultier b, C. Lemarie´ b, C. Ramont b, M.L. Joly-Guillou b,c, M. Eveillard b,c,* a b c
Laboratoire de Bacte´riologie, CHU Saint-Denis, La Re´union, Saint-Denis, France Laboratoire de Bacte´riologie, CHU Angers, Angers, France Groupe d’e´tude des interactions hoˆtes pathoge`nes (GEIHP, EA 3142), UFR Me´decine, Universite´ Angers, Angers, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 November 2013 Received in revised form 28 January 2014 Accepted 31 January 2014
Our objective was to study the carriage of Acinetobacter baumannii (AB) in pets in Reunion Island (RI), a French territory in Indian Ocean. Overall, 138 pets were sampled (rectum, mouth, wounds if applicable) in 9 veterinary clinics (VC). The prevalence of AB carriage was 6.5% (95%CI; 2.4, 10.6) and 9 carriers were indentified from 4 VC. Hospitalization in a VC and antimicrobial treatment administered within the 15 preceding days were significantly associated with AB carriage (P < 0.01 and P < 0.05, respectively). Despite the VC in which animals have been sampled were located all around RI, most isolates (8/9) were closely-related (>90% similarity by pulsed-field gel electrophoresis). Additional studies are needed to improve the understanding about interactions between the different reservoirs of AB in RI. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Acinetobacter baumannii Animal reservoir Multicentre study
1. Introduction Acinetobacter baumannii (AB) has emerged over the last decade as a cause of healthcare-associated infections. Its clinical significance has been propelled by its remarkable ability to upregulate or acquire resistance determinant, making it one of the organisms threatening the current antibiotic era (Peleg et al., 2008; Higgins et al., 2010; Kempf and Rolain, 2012). The cross-transmission of this organism from patients to patients generating outbreaks, particularly in intensive care units and the possibilities of
* Corresponding author at: CHU Angers, Bacte´riologie-hygie`ne, 4 rue Larrey, 49000 Angers, France. Tel.: +33 2 41 35 33 15; fax: +33 2 41 35 41 64. E-mail address: [email protected] (M. Eveillard).
outbreak extension by patient transfers have been demonstrated (Naas et al., 2006; Giamarellou et al., 2008). In addition during the last decade, a lot of studies have reported the emergence of AB as an organism responsible for community-acquired infections, especially in tropical areas (Eveillard et al., 2013). Reunion Island (RI) is a French territory located in the western Indian Ocean. Hospital-based retrospective data have demonstrated that AB is regularly isolated from clinical or screening samples obtained from patients within the 48 h following hospitalization in RI. Those data tend to indicate the existence of a human extra-hospital AB reservoir in RI. This carriage can be due to AB isolates acquired during a preceding hospitalization, but some other hypotheses can be proposed. One of them is an AB acquisition from an animal reservoir. In several studies
http://dx.doi.org/10.1016/j.vetmic.2014.01.042 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
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VETMIC-6508; No. of Pages 5 O. Belmonte et al. / Veterinary Microbiology xxx (2014) xxx–xxx
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conducted previously in veterinary clinics (VC), AB have been isolated in pets from clinical samples (Francey et al., 2000; Endimiani et al., 2011; Hamouda et al., 2011; Zordan et al., 2011). Our objective was to study the AB reservoir in pets in RI. 2. Materials and methods A multicentre cross-sectional study was conducted in October 2012. All cats and dogs present on one day in 9 veterinary clinics (VC) (30% of all VC in RI) were sampled by swabbing rectum, mouth and wounds if applicable. All swabs were inoculated in 2 agar media: the UTI chromogenic agar medium (UTI, Oxoid, Basingstoke, UK) and the ESBL agar medium (AES, France), a selective medium for gram-negative bacilli resistant to thirdgeneration cephalosporins. Bacterial identifications were performed with the Vitek2 system (bioMe´rieux, France). For each isolate identified ‘‘AB complex’’ with the Vitek2 system, an identification of the blaOXA-51 like genes by PCR (Gundi et al., 2009) and the partial sequencing of the rpoB gene (Turton et al., 2006) were performed systematically to formally identify AB. Antimicrobial susceptibility was tested by the disk-diffusion method according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (http://www.sfm-microbiologie.org/UserFiles/file/CASFM/CASFM_2012.pdf). Strains were typed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested genomic DNA and by multi-locus sequence typing performed as previously described (Seifert et al., 2005; Diancourt et al., 2010). A strain of Acinetobacter sp. Isolated from a dog during the same study was analyzed in the same gel as a control of non relatedness. This strain is not completely identified yet because the partial sequencing of the rpoB gene showed a new sequence. A standardized questionnaire was completed for each animal sampled. The main items were the age of the animal, information about lifestyle (in contact with other animals or not), invasive or surgical procedures, the mode
of ‘‘hospitalization’’ (a full hospitalization, a one-day hospitalization or just a visit to the veterinarian), and about antimicrobial agents administered on the day of sampling or within the 15 preceding days. Associations between AB carriage and the characteristics of animals were studied by the Fisher exact test. The animal owners were informed about the study and gave their consent for those painless samples. 3. Results Overall, 138 animals were sampled and 9 carriers (2 cats and 7 dogs) were identified from 4 VC. According to our methods of screening, cultivating and identifying, the prevalence of AB carriage was 6.5% (CI95%; 2.4, 10.6). Eight isolates had the same susceptibility pattern to antibiotics: they were susceptible to ticarcillin, carbapenems, aminoglycosides, rifampicin, and resistant to ciprofloxacine and cotrimoxazole. The other isolate was susceptible to all those antibiotics (Table 1). The PFGE analysis showed that the 8 isolates with the same susceptibility pattern to antibiotics were closelyrelated (>90% similarity) (cluster A) whereas the other isolate was clearly different (Fig. 1). The 8 isolates could be differentiated into 2 subclusters (A1 including 4 isolates and A3 including 3 isolates) presenting 100% similarity and one lonely isolate representing the subcluster A2. The 8 isolates belonging to the PFGE cluster A were assigned to ST25 whereas the other isolate was assigned to ST239. In PFGE, the Acinetobacter sp. control was related to neither AB ST clonal lineages. The geographical distribution of the AB isolated from pets in RI is presented on Fig. 2. The proportion of pets sampled which have been identified AB carriers varied according to the VC. The same strain (A3) has been identified in Saint-Paul and Saint-Andre´, two cities distant from 40 km in a straight line and 55 km by the road. Similarly, the same strain (A1) was isolated in Saint-Leu and Saint-Pierre which are distant from 25 km in a straight line and 30 km by the road.
Table 1 Acinetobacter baumannii ST25 and ST239: susceptibility to antibiotics. Antimicrobial category
Aminoglycosides
Antipseudomonal carbapenems
Antipseudomonal fluoroquinolones Antipseudomonal penicillin + ß-lactamase inhibitor Folate pathway inhibitors Polymyxines Cyclines
Antimicrobial agent
Gentamicin Tobramycin Amikacin Netilmicin Imipenem Meropenem Doripenem Ciprofloxacin Levofloxacin Piperacillin + tazobactam Ticarcillin + clavulanic acid Trimethoprim + surfamethoxazole Colistin Doxycyclin Tigecyclin
Result of antimicrobial susceptibility testing (S or NS) ST25
ST239
S S S S S S S NS NS S S NS S S S
S S S S S S S S S S S S S S S
S: Susceptible. NS: not susceptible.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
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Fig. 1. Pulsed-field gel electrophoresis dendogram to illustrate the relatedness of Acinetobacter baumannii isolates collected from pets in Reunion Island.
There was a significant association between a length of hospitalization >1 day on the day of the survey and AB carriage (OR = 10.8, P < 0.01). However, none of the 3 carriers hospitalized for >1 day were sampled in the same VC. There was also a significant association
between the treatment by at least one antimicrobial agent within the 15 days preceding the day of sampling and AB carriage (OR = 4.44, P < 0.05). Co-amoxyclav, cefalexin and enrofloxacin had been administered to AB carriers.
Fig. 2. Geographical distribution in Reunion Island of veterinary clinics (VC) in which animals have been sampled: number of animals sampled (number of Acinetobacter baumannii carriers) – PFGE subcluster – Sequence type.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
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4. Discussion Today, increased attention is devoted to companion animal welfare, resulting in increased expenditure on veterinary care and prevention and therapy of infectious diseases. This situation is associated with a frequent use of antimicrobial agents in pet animals, may result in some laxity in antimicrobial prescription, and therefore can lead to inappropriate use of antimicrobial agents. It is well established that pet animals can be reservoirs of antimicrobial-resistant bacteria (Guardabassi et al., 2004). For instance, a study conducted in The Netherlands identified a high prevalence of fecal carriage of Enterobacteriaceae resistant to third-generation cephalosporins in cats and dogs (Hordijk et al., 2013). Most preceding studies concerning AB in pets have been conducted in VC. However, they included only AB isolated from clinical samples (Francey et al., 2000; Endimiani et al., 2011; Hamouda et al., 2011; Zordan et al., 2011). Therefore, to our knowledge, few studies estimated the prevalence of AB carriage among pets by systematic screening. As the study was conducted apart from any outbreak context, the prevalence identified can be considered high. Most AB isolates were assigned to ST25. This ST is now considered as an emergent clonal lineage (Zarrilli et al., 2013). Indeed, it has been responsible for outbreaks of clinical infections in human medicine in Italy, Greece and Turkey (Di Popolo et al., 2010). However, those latter strains were resistant to carbapenems, contrarily to our isolates. Several carbapenemases including oxacillinases (OXA-58 and OXA-72) and metallo-ß-lactamases (NDM-1) have been assigned to this clonal lineage ST25 (Zarrilli et al., 2013). In a recent study, Giannouli et al. (2013) demonstrated that AB ST25 strains had an elevated resistance to desiccation, a high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells (human alveolar epithelial cells). Those characteristics could suggest the existence of an environmental reservoir outside the hospital setting and an implication in human or animal infections (related to invasive procedures and pneumonia). The main result of our study is that most AB isolates were closely-related even though they have been isolated from animals living in different areas of RI. Several hypotheses could be proposed to explain this situation. First cross-transmissions could occur inside the VC. Indeed, outbreaks have been described among hospitalized animals (Francey et al., 2000). However in our study, most carriers were not inpatients and the 3 carriers hospitalized for >1 day were sampled in different VC. It can be argued that carriers could have been contaminated during a preceding hospitalization. Unfortunately, data concerning preceding hospitalizations were not recorded. Another hypothesis could be the existence of a reservoir in stray animals, particularly stray dogs which are frequently encountered in RI. A recent study conducted in South Korea reported that multidrug-resistant Escherichia coli producing extended-spectrum-ß-lactamases or plasmidmediated AmpC ß-lactamases have been isolated from stray dogs (Tamang et al., 2012). The transmission to domestic dogs could be explained by the lot of contacts and
fights occurring between them and stray dogs. The existence of an environmental reservoir of AB could also be envisaged. However, the presence of AB in the extrahospital environment is still controversial (Peleg et al., 2008; Towner, 2009). Actually, it could be limited to certain areas, particularly those in close relation with hospitals. For instance, a recent study reported that carbapenem-resistant AB had been isolated from the sewage of several hospitals in Beijing (Zhang et al., 2013). Another hypothesis could be the existence of other animal reservoirs like cattle (Hamouda et al., 2011). Indeed, antibiotics have been widely used in livestock production. This treatment has been linked to the emergence and dissemination of resistant bacteria which can be passed to people or other animals (Phillips et al., 2004). Lastly, AB transmissions could occur between humans and pets, as initially hypothesized. Additional studies are needed to understand those preliminary results. The first step could be to compare the isolates obtained from pets with the isolates collected from patients at the time of admission to hospital in RI. A study conducted in the nearby islands of RI could also be interesting by estimating the extent of the spread of the major AB clone we found in the present study. Acknowledgements We are very grateful to H. Lotteau, P. Legendre, G. Holzapfel, P. Melot, T. Lostfelt, L. Venturini, D. Plazanet, I. Lemercier, K. Teppe, and W. Severin for their participation to the study by sampling animals in their veterinary clinic. A part of the results of this study was presented at the 9th International Symposium on the Biology of Acinetobacter, Cologne 2013, oral presentation O5-4. References Di Popolo, A., Giannouli, M., Triassi, M., Brisse, S., Zarilli, R., 2010. Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme. Clin. Microbiol. Infect. 17, 190–203. Diancourt, L., Passet, V., Nemec, A., Dijkshoorn, L., Brisse, S., 2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One 5, e10034. Endimiani, A., Hujer, K.M., Hujer, A.M., Bertschy, I., Rossano, A., Koch, C., Gerber, V., Francey, T., Bonomo, R.A., Perreten, V., 2011. Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data. J. Antimicrob. Chemother. 66, 2248–2254. Eveillard, M., Kempf, M., Belmonte, O., Pailhorie`s, H., Joly-Guillou, M.L., 2013. Reservoirs of Acinetobacter baumannii outside the hospital and potential involvement in emerging human community-acquired infections. Int. J. Infect. Dis. 17, e802–e805. Francey, T., Gaschen, F., Nicolet, J., Burnens, A.P., 2000. The role of Acinetobacter baumannii as a nosocomial pathogen for dogs and cats in an intensive care unit. J. Vet. Intern. Med. 14, 177–183. Giamarellou, H., Antoniadou, A., Kanellakopoulou, K., 2008. Acinetobacter baumannii: a universal threat to public health? Int. J. Antimicrob. Agents. 32, 106–119. Giannouli, M., Antunes, L.C.S., Marchetti, V., Triassi, M., Visca, P., Zarrilli, R., 2013. Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineage I–II and to the emerging genotypes ST25 and ST78. BMC Infect. Dis. 13, 282. Guardabassi, L., Schwarz, S., Lloyd, D.H., 2004. Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 54, 321– 332.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
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VETMIC-6508; No. of Pages 5 O. Belmonte et al. / Veterinary Microbiology xxx (2014) xxx–xxx Gundi, V.A., Dijkshoorn, L., Burignat, S., Raoult, D., La Scola, B., 2009. Validation of partial rpoB gene sequence analysis for the identification of clinically important and emerging Acinetobacter species. Microbiology 155, 2333–2341. Hamouda, A., Findlay, J., Al Hassan, L., Amyes, S.G.B., 2011. Epidemiology of Acinetobacter baumannii of animal origin. Int. J. Antimicrob. Agents 3, 314–318. Higgins, P.G., Dammhayn, C., Hackel, M., Seifert, H., 2010. Global spread of carbapenem-resistant Acinetobacter baumannii. J. Antimicrob. Chemother. 65, 233–238. Hordijk, J., Schoormans, A., Kwakernaak, M., Duim, B., Broens, E., Dierikx, C., Mevius, D., Wagenaar, J.A., 2013. High prevalence of fecal carriage of extended-spectrum ß-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs. Front. Microbiol. 4, 242. Kempf, M., Rolain, J.M., 2012. Emergence of resistance to carbapenems in Acinetobacter baumannii in Europe: clinical impact and therapeutic options. Int. J. Antimicrob. Agents 39, 105–114. Naas, T., Coignard, B., Carbonne, A., Blanckaert, K., Bajolet, O., Bernet, C., Verdeil, X., Astagneau, P., Desenclos, J.C., Nordmann, P., 2006. French Nosocomial Infection Early Warning Investigation and Surveillance Network. VEB-1 extended-spectrum b-lactamase-producing Acinetobacter baumannii, France. Emerg. Infect. Dis. 12, 1214–1222. Peleg, A.Y., Seifert, H., Paterson, D.L., 2008. Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev. 21, 538–582. Phillips, I., Casewell, M., Cox, T., De Groot, B., Friis, C., Jones, R., Nightingale, C., Preston, R., Waddell, J., 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J. Antimicrob. Chemother. 53, 28–52.
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Seifert, H., Dolzani, L., Bressan, R., van der Reijden, T., van Strijen, B., Stefanik, D., Heersma, H., Dijkshoorn, L., 2005. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. J. Clin. Microbiol. 43, 4328–4335. Tamang, M.D., Nam, H.M., Jang, G.C., Kim, S.R., Chae, M.H., Jung, S.C., Byun, J.W., Park, Y.H., Lim, S.K., 2012. Molecular characterization of extended-spectrum-ß-lactamase-producing and plasmidmediated AmpC ß-lactamase-producing Escherichia coli isolated from stray dogs in South Korea. Antimicrob. Agents Chemother. 56, 2705–2712. Towner, K.J., 2009. Acinetobacter: an old friend, but a new enemy. J. Hosp. Infect. 73, 355–363. Turton, J.F., Woodford, N., Glover, J., Yarde, S., Kaufmann, M.E., Pitt, T.L., 2006. Identification of Acinetobacter baumannii by detection of blaOXA-51-like carbapenemase gene intrinsic to this species. J. Clin. Microbiol. 44, 2974–2976. Zarrilli, R., Pournaras, S., Giannouli, M., Tsakris, A., 2013. Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages. Int. J. Antimicrob. Agents. 41, 11–19. Zhang, C., Qiu, S., Wang, Y., Qi, L., Hao, R., Liu, X., Shi, Y., Hu, X., An, D., Li, Z., Li, P., Wang, L., Cui, J., Wang, P., Huang, L., Klena, J.D., Song, H., 2013. Higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals in Beijing. PLoS One 8, e64857. Zordan, S., Prenger-Berninghoff, E., Weiss, R., van der Reijden, T., van den Broek, P., Baljer, G., Dijkshoorn, L., 2011. Multidrug-resistant Acinetobacter baumannii in veterinary clinics, Germany. Emerg. Infect. Dis. 17, 1751–1754.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
VETMIC-6508; No. of Pages 5 Veterinary Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island O. Belmonte a, H. Pailhorie`s b,c, M. Kempf b,c, M.P. Gaultier b, C. Lemarie´ b, C. Ramont b, M.L. Joly-Guillou b,c, M. Eveillard b,c,* a b c
Laboratoire de Bacte´riologie, CHU Saint-Denis, La Re´union, Saint-Denis, France Laboratoire de Bacte´riologie, CHU Angers, Angers, France Groupe d’e´tude des interactions hoˆtes pathoge`nes (GEIHP, EA 3142), UFR Me´decine, Universite´ Angers, Angers, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 November 2013 Received in revised form 28 January 2014 Accepted 31 January 2014
Our objective was to study the carriage of Acinetobacter baumannii (AB) in pets in Reunion Island (RI), a French territory in Indian Ocean. Overall, 138 pets were sampled (rectum, mouth, wounds if applicable) in 9 veterinary clinics (VC). The prevalence of AB carriage was 6.5% (95%CI; 2.4, 10.6) and 9 carriers were indentified from 4 VC. Hospitalization in a VC and antimicrobial treatment administered within the 15 preceding days were significantly associated with AB carriage (P < 0.01 and P < 0.05, respectively). Despite the VC in which animals have been sampled were located all around RI, most isolates (8/9) were closely-related (>90% similarity by pulsed-field gel electrophoresis). Additional studies are needed to improve the understanding about interactions between the different reservoirs of AB in RI. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Acinetobacter baumannii Animal reservoir Multicentre study
1. Introduction Acinetobacter baumannii (AB) has emerged over the last decade as a cause of healthcare-associated infections. Its clinical significance has been propelled by its remarkable ability to upregulate or acquire resistance determinant, making it one of the organisms threatening the current antibiotic era (Peleg et al., 2008; Higgins et al., 2010; Kempf and Rolain, 2012). The cross-transmission of this organism from patients to patients generating outbreaks, particularly in intensive care units and the possibilities of
* Corresponding author at: CHU Angers, Bacte´riologie-hygie`ne, 4 rue Larrey, 49000 Angers, France. Tel.: +33 2 41 35 33 15; fax: +33 2 41 35 41 64. E-mail address: [email protected] (M. Eveillard).
outbreak extension by patient transfers have been demonstrated (Naas et al., 2006; Giamarellou et al., 2008). In addition during the last decade, a lot of studies have reported the emergence of AB as an organism responsible for community-acquired infections, especially in tropical areas (Eveillard et al., 2013). Reunion Island (RI) is a French territory located in the western Indian Ocean. Hospital-based retrospective data have demonstrated that AB is regularly isolated from clinical or screening samples obtained from patients within the 48 h following hospitalization in RI. Those data tend to indicate the existence of a human extra-hospital AB reservoir in RI. This carriage can be due to AB isolates acquired during a preceding hospitalization, but some other hypotheses can be proposed. One of them is an AB acquisition from an animal reservoir. In several studies
http://dx.doi.org/10.1016/j.vetmic.2014.01.042 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
G Model
VETMIC-6508; No. of Pages 5 O. Belmonte et al. / Veterinary Microbiology xxx (2014) xxx–xxx
2
conducted previously in veterinary clinics (VC), AB have been isolated in pets from clinical samples (Francey et al., 2000; Endimiani et al., 2011; Hamouda et al., 2011; Zordan et al., 2011). Our objective was to study the AB reservoir in pets in RI. 2. Materials and methods A multicentre cross-sectional study was conducted in October 2012. All cats and dogs present on one day in 9 veterinary clinics (VC) (30% of all VC in RI) were sampled by swabbing rectum, mouth and wounds if applicable. All swabs were inoculated in 2 agar media: the UTI chromogenic agar medium (UTI, Oxoid, Basingstoke, UK) and the ESBL agar medium (AES, France), a selective medium for gram-negative bacilli resistant to thirdgeneration cephalosporins. Bacterial identifications were performed with the Vitek2 system (bioMe´rieux, France). For each isolate identified ‘‘AB complex’’ with the Vitek2 system, an identification of the blaOXA-51 like genes by PCR (Gundi et al., 2009) and the partial sequencing of the rpoB gene (Turton et al., 2006) were performed systematically to formally identify AB. Antimicrobial susceptibility was tested by the disk-diffusion method according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (http://www.sfm-microbiologie.org/UserFiles/file/CASFM/CASFM_2012.pdf). Strains were typed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested genomic DNA and by multi-locus sequence typing performed as previously described (Seifert et al., 2005; Diancourt et al., 2010). A strain of Acinetobacter sp. Isolated from a dog during the same study was analyzed in the same gel as a control of non relatedness. This strain is not completely identified yet because the partial sequencing of the rpoB gene showed a new sequence. A standardized questionnaire was completed for each animal sampled. The main items were the age of the animal, information about lifestyle (in contact with other animals or not), invasive or surgical procedures, the mode
of ‘‘hospitalization’’ (a full hospitalization, a one-day hospitalization or just a visit to the veterinarian), and about antimicrobial agents administered on the day of sampling or within the 15 preceding days. Associations between AB carriage and the characteristics of animals were studied by the Fisher exact test. The animal owners were informed about the study and gave their consent for those painless samples. 3. Results Overall, 138 animals were sampled and 9 carriers (2 cats and 7 dogs) were identified from 4 VC. According to our methods of screening, cultivating and identifying, the prevalence of AB carriage was 6.5% (CI95%; 2.4, 10.6). Eight isolates had the same susceptibility pattern to antibiotics: they were susceptible to ticarcillin, carbapenems, aminoglycosides, rifampicin, and resistant to ciprofloxacine and cotrimoxazole. The other isolate was susceptible to all those antibiotics (Table 1). The PFGE analysis showed that the 8 isolates with the same susceptibility pattern to antibiotics were closelyrelated (>90% similarity) (cluster A) whereas the other isolate was clearly different (Fig. 1). The 8 isolates could be differentiated into 2 subclusters (A1 including 4 isolates and A3 including 3 isolates) presenting 100% similarity and one lonely isolate representing the subcluster A2. The 8 isolates belonging to the PFGE cluster A were assigned to ST25 whereas the other isolate was assigned to ST239. In PFGE, the Acinetobacter sp. control was related to neither AB ST clonal lineages. The geographical distribution of the AB isolated from pets in RI is presented on Fig. 2. The proportion of pets sampled which have been identified AB carriers varied according to the VC. The same strain (A3) has been identified in Saint-Paul and Saint-Andre´, two cities distant from 40 km in a straight line and 55 km by the road. Similarly, the same strain (A1) was isolated in Saint-Leu and Saint-Pierre which are distant from 25 km in a straight line and 30 km by the road.
Table 1 Acinetobacter baumannii ST25 and ST239: susceptibility to antibiotics. Antimicrobial category
Aminoglycosides
Antipseudomonal carbapenems
Antipseudomonal fluoroquinolones Antipseudomonal penicillin + ß-lactamase inhibitor Folate pathway inhibitors Polymyxines Cyclines
Antimicrobial agent
Gentamicin Tobramycin Amikacin Netilmicin Imipenem Meropenem Doripenem Ciprofloxacin Levofloxacin Piperacillin + tazobactam Ticarcillin + clavulanic acid Trimethoprim + surfamethoxazole Colistin Doxycyclin Tigecyclin
Result of antimicrobial susceptibility testing (S or NS) ST25
ST239
S S S S S S S NS NS S S NS S S S
S S S S S S S S S S S S S S S
S: Susceptible. NS: not susceptible.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
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Fig. 1. Pulsed-field gel electrophoresis dendogram to illustrate the relatedness of Acinetobacter baumannii isolates collected from pets in Reunion Island.
There was a significant association between a length of hospitalization >1 day on the day of the survey and AB carriage (OR = 10.8, P < 0.01). However, none of the 3 carriers hospitalized for >1 day were sampled in the same VC. There was also a significant association
between the treatment by at least one antimicrobial agent within the 15 days preceding the day of sampling and AB carriage (OR = 4.44, P < 0.05). Co-amoxyclav, cefalexin and enrofloxacin had been administered to AB carriers.
Fig. 2. Geographical distribution in Reunion Island of veterinary clinics (VC) in which animals have been sampled: number of animals sampled (number of Acinetobacter baumannii carriers) – PFGE subcluster – Sequence type.
Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042
G Model
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4. Discussion Today, increased attention is devoted to companion animal welfare, resulting in increased expenditure on veterinary care and prevention and therapy of infectious diseases. This situation is associated with a frequent use of antimicrobial agents in pet animals, may result in some laxity in antimicrobial prescription, and therefore can lead to inappropriate use of antimicrobial agents. It is well established that pet animals can be reservoirs of antimicrobial-resistant bacteria (Guardabassi et al., 2004). For instance, a study conducted in The Netherlands identified a high prevalence of fecal carriage of Enterobacteriaceae resistant to third-generation cephalosporins in cats and dogs (Hordijk et al., 2013). Most preceding studies concerning AB in pets have been conducted in VC. However, they included only AB isolated from clinical samples (Francey et al., 2000; Endimiani et al., 2011; Hamouda et al., 2011; Zordan et al., 2011). Therefore, to our knowledge, few studies estimated the prevalence of AB carriage among pets by systematic screening. As the study was conducted apart from any outbreak context, the prevalence identified can be considered high. Most AB isolates were assigned to ST25. This ST is now considered as an emergent clonal lineage (Zarrilli et al., 2013). Indeed, it has been responsible for outbreaks of clinical infections in human medicine in Italy, Greece and Turkey (Di Popolo et al., 2010). However, those latter strains were resistant to carbapenems, contrarily to our isolates. Several carbapenemases including oxacillinases (OXA-58 and OXA-72) and metallo-ß-lactamases (NDM-1) have been assigned to this clonal lineage ST25 (Zarrilli et al., 2013). In a recent study, Giannouli et al. (2013) demonstrated that AB ST25 strains had an elevated resistance to desiccation, a high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells (human alveolar epithelial cells). Those characteristics could suggest the existence of an environmental reservoir outside the hospital setting and an implication in human or animal infections (related to invasive procedures and pneumonia). The main result of our study is that most AB isolates were closely-related even though they have been isolated from animals living in different areas of RI. Several hypotheses could be proposed to explain this situation. First cross-transmissions could occur inside the VC. Indeed, outbreaks have been described among hospitalized animals (Francey et al., 2000). However in our study, most carriers were not inpatients and the 3 carriers hospitalized for >1 day were sampled in different VC. It can be argued that carriers could have been contaminated during a preceding hospitalization. Unfortunately, data concerning preceding hospitalizations were not recorded. Another hypothesis could be the existence of a reservoir in stray animals, particularly stray dogs which are frequently encountered in RI. A recent study conducted in South Korea reported that multidrug-resistant Escherichia coli producing extended-spectrum-ß-lactamases or plasmidmediated AmpC ß-lactamases have been isolated from stray dogs (Tamang et al., 2012). The transmission to domestic dogs could be explained by the lot of contacts and
fights occurring between them and stray dogs. The existence of an environmental reservoir of AB could also be envisaged. However, the presence of AB in the extrahospital environment is still controversial (Peleg et al., 2008; Towner, 2009). Actually, it could be limited to certain areas, particularly those in close relation with hospitals. For instance, a recent study reported that carbapenem-resistant AB had been isolated from the sewage of several hospitals in Beijing (Zhang et al., 2013). Another hypothesis could be the existence of other animal reservoirs like cattle (Hamouda et al., 2011). Indeed, antibiotics have been widely used in livestock production. This treatment has been linked to the emergence and dissemination of resistant bacteria which can be passed to people or other animals (Phillips et al., 2004). Lastly, AB transmissions could occur between humans and pets, as initially hypothesized. Additional studies are needed to understand those preliminary results. The first step could be to compare the isolates obtained from pets with the isolates collected from patients at the time of admission to hospital in RI. A study conducted in the nearby islands of RI could also be interesting by estimating the extent of the spread of the major AB clone we found in the present study. Acknowledgements We are very grateful to H. Lotteau, P. Legendre, G. Holzapfel, P. Melot, T. Lostfelt, L. Venturini, D. Plazanet, I. Lemercier, K. Teppe, and W. Severin for their participation to the study by sampling animals in their veterinary clinic. A part of the results of this study was presented at the 9th International Symposium on the Biology of Acinetobacter, Cologne 2013, oral presentation O5-4. References Di Popolo, A., Giannouli, M., Triassi, M., Brisse, S., Zarilli, R., 2010. Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme. Clin. Microbiol. Infect. 17, 190–203. Diancourt, L., Passet, V., Nemec, A., Dijkshoorn, L., Brisse, S., 2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One 5, e10034. Endimiani, A., Hujer, K.M., Hujer, A.M., Bertschy, I., Rossano, A., Koch, C., Gerber, V., Francey, T., Bonomo, R.A., Perreten, V., 2011. Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data. J. Antimicrob. Chemother. 66, 2248–2254. Eveillard, M., Kempf, M., Belmonte, O., Pailhorie`s, H., Joly-Guillou, M.L., 2013. Reservoirs of Acinetobacter baumannii outside the hospital and potential involvement in emerging human community-acquired infections. Int. J. Infect. Dis. 17, e802–e805. Francey, T., Gaschen, F., Nicolet, J., Burnens, A.P., 2000. The role of Acinetobacter baumannii as a nosocomial pathogen for dogs and cats in an intensive care unit. J. Vet. Intern. Med. 14, 177–183. Giamarellou, H., Antoniadou, A., Kanellakopoulou, K., 2008. Acinetobacter baumannii: a universal threat to public health? Int. J. Antimicrob. Agents. 32, 106–119. Giannouli, M., Antunes, L.C.S., Marchetti, V., Triassi, M., Visca, P., Zarrilli, R., 2013. Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineage I–II and to the emerging genotypes ST25 and ST78. BMC Infect. Dis. 13, 282. Guardabassi, L., Schwarz, S., Lloyd, D.H., 2004. Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 54, 321– 332.
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Please cite this article in press as: Belmonte, O., et al., High prevalence of closely-related Acinetobacter baumannii in pets according to a multicentre study in veterinary clinics, Reunion Island. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/ j.vetmic.2014.01.042