Screening for fecal presence of colistin-resistant Escherichia coli and mcr-1 and mcr-2 genes in camel-calves in southern Tunisia

Screening for fecal presence of colistin-resistant Escherichia coli and mcr-1 and mcr-2 genes in... Camels (Camelus dromedarius) are known to harbor multidrug resistant Gram-negative bacteria and to be involved in the transmission of various microorganisms to humans. Data on the occurrence of colistin resistant Escherichia coli as well as mobilized colistin resistance (mcr) genes in camels are lacking. We investigated the presence of colistin resist- ance and mcr (1–2) genes in E. coli from the feces of camels in Tunisia. Presumptive E. coli isolates from camel-calves in southern Tunisia were qualitatively screened for growth on Mueller–Hinton agar supplemented with 2 mg/L of colis- tin. The minimal inhibitory concentration of colistin was determined for isolates growing on this medium. All isolates were screened for the presence of the mcr-1 and mcr-2 genes by polymerase chain reaction without detecting any of these genes. However, one isolate was confirmed resistant to colistin and further testing of this isolate revealed it to be Enterobacter cloacae. Our study demonstrated absence of colistin resistance and of the mcr-1 and mcr-2 genes in E. coli isolated from camel feces in southern Tunisia. Thus, there is no evidence that camels represent a major source of mcr genes contamination for the local population or for tourists visiting southern Tunisia. Keywords: Camel, Colistin resistance, E. coli, Human, MCR Findings antimicrobials approved to treat bacterial infections in One-humped camels (Camelus dromedarius) are raised dromedaries poses a major challenge for veterinarians extensively in southern Tunisia for the production of working in this animal production [5]. The use of anti - meat and milk as well as for activities related to Sahara microbials approved for ruminants, horses or other ani- tourism. One-humped camels are considered part of the mal species in the treatment of sick camels has not been cultural heritage and represents a national wealth for associated with satisfactory results because of the physi- many countries [1]. Camel-calves have a high suscepti- ological particularities of this animal species [5]. Colistin bility to bacterial infections, particularly those caused sulfate (CS), a polypeptide antibiotic, is used off-label by Escherichia coli [1–3]. In fact, colibacillosis in young in Tunisia for the oral treatment of Enterobacteriaceae camelids results in considerable economic losses in camel infections in camel-calves at the dose of 25,000  IU/kg farms, being associated with mortality, growth retarda- twice a day for 3 consecutive days. Following the recent tion and medical treatment costs [1, 4]. The absence of identification of a stable plasmid mediated mobilized colistin resistance (mcr-1) gene encoding for resistance to colistin in Enterobacteriaceae, this “novel” mecha- *Correspondence: mohamed.rhouma@umontreal.ca nism of colistin resistance has been widely studied in Department of Pathology and Microbiology, Faculty of Veterinary bacteria isolated from several origins such as farm ani- Medicine, University of Montreal, 3200 Sicotte St, Saint-Hyacinthe, QC J2S mals, humans, food and the environment [6, 7]. In July 2M2, Canada Full list of author information is available at the end of the article and August 2017, we searched PubMed with the terms © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rhouma et al. Acta Vet Scand (2018) 60:35 Page 2 of 5 “colistin in dromedary or colistin in camels”, “colistin Medenine  (n = 10)  (Fig.  1) with an average flock size of resistance in dromedary or in camels”, “MCR-1 in drom- 75 animals. Rectal swabs from 120 camel-calves aged edary or in camels” and “mcr genes in dromedary or in between 1 and 3 months, with or without diarrhea, were camels”. However, no studies on fecal presence of plas- suspended in buffered peptone water solution. Selected −1 −2 −3 mid-mediated colistin resistance genes (mcr-1 to -5) or dilutions (10, 10 , and 10 ) were plated on MacCo- E. coli colistin resistance in camels were found. On the nkey agar. The plates were incubated aerobically for 24 h other hand, it was been recently reported that camels are at 37  °C. Colonies growing on MacConkey agar with a a potential source of human contamination with multid- morphology typical for E. coli (red or pink, non-mucoid rug resistant (MDR) bacteria such as extended-spectrum colonies) were tested by polymerase chain reaction beta-lactamase (ESBL) producing E. coli or Pseudomonas (PCR) for the β-glucuronidase (uidA) housekeeping gene, aeruginosa [5, 8]. Moreover, co-localisation of mcr-1 and and PCR-positive colonies obtained (n = 52) were consid- ESBL genes on a unique plasmid has been reported [9]. ered as presumptive E. coli isolates (Table  1) [10]. These The present study aimed (1) to determine the fecal prev - isolates were stored at − 80 °C until further analysis. alence of E. coli colistin resistance in camel-calves with After thawing, the 52 isolates were inoculated on Muel- and without diarrhea in Tunisia, and (2) to determine the ler–Hinton agar supplemented with CS at the concen- prevalence of mcr-1 and mcr-2 genes among both colistin tration of 2  mg/L. Isolates demonstrating growth were resistant and susceptible E. coli isolates. considered as possibly colistin-resistant and were exam- In a previous study, the distribution of virulence ined for minimum inhibitory concentration (MIC) of genes, pathotypes, serogroups, phylogenetic groups, colistin, using the broth microdilution method in techni- and antimicrobial resistance of presumptive E. coli iso- cal duplicate according to the guidelines of the Clinical lated from camel-calves with and without diarrhea in Laboratory Standards Institute (CLSI) [11]. Enterobac- Tunisia was investigated [2]. Fecal samples had been teriaceae with colistin MICs > 2  mg/L were defined as collected between January 2011 and April 2013 from resistant (R), and those with colistin MICs ≤ 2  mg/L 25 extensive camel farms located in  three  districts  in as susceptible (S) according to the European Commit- southern Tunisia: Kebili (n = 13), Gabes (n = 2), and tee on Antimicrobial Susceptibility Testing (EUCAST) Fig. 1 Geographic distribution of the sampled farms by district in southern Tunisia with the dromedary density and its involvement in tourist activities Rhouma et al. Acta Vet Scand (2018) 60:35 Page 3 of 5 Table 1 Distribution of  possibly colistin resistant isolates according to  their origin; camel- calves with  or  without diarrhea Health status of camel-calves Number of isolates Species (number) Colistin resistant isolates MIC (number) of colistin value (mg/L) No diarrhea 23 E. coli (n = 23) 0 – Diarrhea 29 E. coli (n = 28) 0 – E. cloacae (n = 1) E. cloacae (n = 1) 4 Escherichia coli, Enterobacter cloacae guidelines (http://www.eucas t.org/clini cal_break point s/). as possible colistin resistant (Fig. 2). However, only one Isolates with colistin MICs > 2 mg/L were further identi- of these isolates was confirmed to be resistant to colis - fied using the API 20E system (bioMérieux, Quebec City, tin with an MIC value of 4  mg/L (Table  1). This resist - Canada). ant isolate, recovered from a camel-calf with diarrhea, Bacterial DNA extraction was performed using the was identified as Enterobacter cloacae. Chelex method (Bio-Rad, ON, Canada). All isolates Neither the mcr-1 nor mcr-2 gene were detected in were screened by PCR for the presence of mcr-1 and any of the colistin resistant or susceptible isolates. mcr-2 genes, using primers and conditions as previously It is important to monitor colistin resistance in drom- described [12, 13]. DNA from E. coli strains harbouring edary herds in Tunisia as the mcr-1 gene is highly prev- either mcr-1 gene [14] or mcr-2 gene [13] were used as alent in food-producing animals (such as chickens) in positive control. this country [15]. In addition, Tunisia being an impor- More than half of the presumptive E. coli isolates tant tourist destination [16], close contact between (55%) (n = 28) in both groups of animals were identified Fig. 2 Distribution of minimum inhibitory concentrations (MICs) in E. coli isolates possibly resistant to colistin (n = 28) recovered from camel-calves with and without diarrhea Rhouma et al. Acta Vet Scand (2018) 60:35 Page 4 of 5 camels and tourists visiting southern Tunisia may result Nevertheless, it highlights the need to widen the scope in spread of resistant bacteria internationally. of monitoring colistin resistance to other Enterobacte- Our finding that colistin-resistance and the mcr genes riaceae species than those conventionally identified in were absent in E. coli isolated from camel-calves in south- veterinary diagnostic laboratories, such as E. cloacae. ern Tunisia suggest that dromedaries are not a major Moreover, with the global initiatives for the establish- source of contamination with these genes for inhabitants ment of a One Health antimicrobial resistance surveil- as well as for tourists visiting this region. It should be lance approach, camels should be also considered as an noted that colistin-susceptible isolates were included in important animal species in the tracking of resistant bac- the current study, as previous studies have reported the teria at the human, animal and environment interface. identification of mcr-1 gene among colistin-resistant and colistin-susceptible isolates [6, 17]. Abbreviations We report here for the first time the isolation of E. cloa - CLSI: Clinical and Laboratory Standards Institute; CS: colistin sulfate; ESBL: cae resistant to colistin from a diarrheic camel. However, extended-spectrum beta-lactamase; EUCAST: European Committee on Anti- microbial Susceptibility Testing; DNA: deoxyribonucleic acid; MDR: multidrug this isolate did not harbor mcr-1 or mcr-2 genes. Simi- resistant; MICs: minimal inhibitory concentrations; PCR: polymerase chain larly, colistin-resistant E. cloacae isolates with or with- reaction. out the mcr-1 gene have been found in healthy people or Authors’ contributions primary care patients [18–20]. Currently, the mechanism MR, SB, IS and PF planned and designed the study. MR and WT carried out of colistin resistance in E. cloacae strains in the absence the microbiological and molecular biological tests. MR, PF, and JMF analyzed of plasmid-borne colistin resistance genes (mcr-1 to -5) and interpreted the data. MR drafted the manuscript. All authors read and approved the final manuscript. remains poorly understood. Zhong et  al. [21] recently identified 17 genes in the chromosome and one gene in Author details pASM1 encoding efflux pump proteins in a highly colis - Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte St, Saint-Hyacinthe, QC J2S 2M2, Canada. tin resistant E. cloacae strain. Nevertheless, mechanisms Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of colistin resistance not associated with plasmids in of Gabès, Médenine, Tunisia. Enterobacter spp. require further investigation. Acknowledgements It should be emphasized that we had no information We would like to thank Dr. Pascal Sanders (ANSES, Laboratoire de Fougères, regarding the medical treatment history of the sampled France) and Pr. Dr. Surbhi Malhorta-Kumar (University of Antwerp, Wilrijk, camel-calves, specifically, whether or not colistin has Belgium) for providing DNA from Escherichia coli harboring respectively, mcr-1 and mcr-2 genes. We gratefully thank Pr. Mohamed Gharbi (Laboratoire de been used in these dromedary farms. Additional studies parasitologie, Ecole Nationale de Médecine Vétérinaire de Sidi Thabet-Tunisia) are needed in order to assess the spread of colistin resist- for critical reading of the manuscript prior to submission. ant bacteria and mcr genes among enteric bacteria of Competing interests camel origin worldwide. Future surveys should take into The authors declare that they have no competing interests. consideration other areas where dromedary density is very high, such as the Arabian Peninsula where the mcr-1 Availability of data and materials All data generated or analysed during this study are included in this published gene has been identified in E. coli isolates [22]. article. The colistin resistant bacterial strain is available from the authors upon In the present study, the number of isolates obtained reasonable request. was limited because of the difficulty of handling camel- Ethics approval and consent to participate calves in extensive farms. However, we believe that our Not applicable. sampling was representative of dromedary production in Tunisia, as fecal samples were obtained from districts Funding This work was supported by the Natural Sciences and Engineering Research containing the highest density of dromedary farms. Council of Canada (NSERC) (412247-10). As expected, the use of Mueller–Hinton agar supple- mented with 2  mg/L of CS overestimated the frequency Publisher’s Note of presumed colistin resistant bacteria. But the culturing Springer Nature remains neutral with regard to jurisdictional claims in pub- on this medium was only used as a screening method for lished maps and institutional affiliations. reducing the number of isolates to be tested for colistin Received: 7 November 2017 Accepted: 26 May 2018 MIC. In addition, because of the poor diffusion of colistin in agar, it was always recommended to confirm presumed colistin resistant isolates, growing on colistin containing agar, by colistin CMI determination [23, 24]. The results suggest that camel-calves do not represent References 1. Al-Ruwaili MA, Khalil OM, Selim SA. Viral and bacterial infections associ- a major source of mcr gene contamination for the local ated with camel (Camelus dromedarius) calf diarrhea in North Province, population or for tourists visiting southern Tunisia. Saudi Arabia. Saudi J Biol Sci. 2012;19:35–41. Rhouma et al. Acta Vet Scand (2018) 60:35 Page 5 of 5 2. Bessalah S, Fairbrother JM, Salhi I, Vanier G, Khorchani T, Seddik MM, et al. livestock, 2007 to 2014. Euro Surveill. 2016. https ://doi.org/10.2807/1560- Antimicrobial resistance and molecular characterization of virulence 7917.ES.2016.21.6.30135 . genes, phylogenetic groups of Escherichia coli isolated from diarrheic 15. Grami R, Mansour W, Mehri W, Bouallègue O, Boujaâfar N, Madec J, and healthy camel-calves in Tunisia. Comp Immunol Microbiol Infect Dis. et al. Impact of food animal trade on the spread of mcr-1-mediated 2016;49:1–7. colistin resistance, Tunisia, July 2015. Euro Surveill. 2016. https ://doi. 3. El Wathig M, Faye B. Camel calf diarrhoea in Riyadh region, Saudi Arabia. J org/10.2807/1560-7917.ES.2016.21.8.30144 . Camel Pract Res. 2016;23:283–5. 16. Halioui S, Schmidt M. Participatory decision-making for sustainable tour- 4. Wernery U, Kinne J, Schuster RK, editors. Camelid infectious disorders. ism development in Tunisia. In: Tourism, culture and heritage in a smart Colibacillosis. 2014, World Organisation for Animal Health (OIE). Paris, economy: Katsoni V, Upadhya A, Stratigea A, Editors. Third international France; 2014. P. 100–5. conference IACuDiT, Athens. Springer proceedings in Business and 5. Fadlelmula A, Al-Hamam NA, Al-Dughaym AM. A potential camel economics; 2017. P. 323–38. reservoir for extended-spectrum beta-lactamase-producing Escherichia 17. Fernandes M, Moura Q, Sartori L, Silva K, Cunha M, Esposito F, et al. Silent coli causing human infection in Saudi Arabia. Trop Anim Health Prod. dissemination of colistin-resistant Escherichia coli in South America could 2016;48:427–33. contribute to the global spread of the mcr-1 gene. Euro Surveill. 2016. 6. Rhouma M, Beaudry F, Theriault W, Letellier A. Colistin in pig produc-https ://doi.org/10.2807/1560-7917.ES.2016.21.17.30214 . tion: chemistry, mechanism of antibacterial action, microbial resistance 18. Baron S, Bardet L, Dubourg G, Fichaux M, Rolain JM. MCR-1 plasmid-medi- emergence, and one health perspectives. Front Microbiol. 2016;7:1–22. ated colistin resistance gene detection in an Enterobacter cloacae clinical 7. Rhouma M, Beaudry F, Letellier A. Resistance to colistin: what is the isolate in France. J Glob Antimicrob Resist. 2017;10:35–6. fate for this antibiotic in pig production? Int J Antimicrob Agents. 19. Zurfluh K, Stephan R, Widmer A, Poirel L, Nordmann P, Nuesch HJ, et al. 2016;48:119–26. Screening for fecal carriage of MCR-producing Enterobacteriaceae in 8. Elhariri M, Hamza D, Elhelw R, Dorgham SM. Extended-spectrum healthy humans and primary care patients. Antimicrob Resist Infect beta-lactamase-producing Pseudomonas aeruginosa in camel in Egypt: Control. 2017. https ://doi.org/10.1186/s1375 6-017-0186-z. potential human hazard. Ann Clin Microbiol Antimicrob. 2017;16:21. 20. Lin J, Zhao F, Feng Y, Zong Z. Draft genome sequence of a high-level 9. Rhouma M, Letellier A. Extended-spectrum beta-lactamases, carbapen- colistin-resistant clinical strain of the Enterobacter cloacae complex. emases and the mcr-1 gene: is there a historical link? Int J Antimicrob Genome Announc. 2017. https ://doi.org/10.1128/genom eA.00131 -17. Agents. 2017;49:269–71. 21. Zhong C, Zhang C, Fu J, Chen W, Jiang T, Cao G. Complete genome 10. Walk ST, Alm EW, Gordon DM, Ram JL, Toranzos GA, Tiedje JM, et al. sequence of Enterobacter cloacae R11 reveals multiple genes potentially Cryptic lineages of the genus Escherichia. Appl Environ Microbiol. associated with high-level polymyxin E resistance. Can J Microbiol. 2009;75:6534–44. 2018;64:87–90. 11. Rhouma M, Beaudry F, Thériault W, Bergeron N, Laurent-Lewandowski 22. Sonnevend A, Ghazawi A, Alqahtani M, Shibl A, Jamal W, Hashmey R, et al. S, Fairbrother JM, et al. Gastric stability and oral bioavailability of colistin Plasmid-mediated colistin resistance in Escherichia coli from the Arabian sulfate in pigs challenged or not with Escherichia coli O149: F4 (K88). Res Peninsula. Int J Infect Dis. 2016;50:85–90. Vet Sci. 2015;102:173–81. 23. Rhouma M, Beaudry F, Theriault W, Bergeron N, Beauchamp G, Laurent- 12. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of Lewandowski S, et al. In vivo therapeutic efficacy and pharmacokinetics plasmid-mediated colistin resistance mechanism MCR-1 in animals and of colistin sulfate in an experimental model of enterotoxigenic Escherichia human beings in China: a microbiological and molecular biological study. coli infection in weaned pigs. Vet Res. 2016;47:58. Lancet Infect Dis. 2016;16:161–8. 24. Turlej-Rogacka A, Xavier BB, Janssens L, Lammens C, Zarkotou O, Pour- 13. Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, naras S, et al. Evaluation of colistin stability in agar and comparison of et al. Identification of a novel plasmid-mediated colistin-resistance gene, four methods for MIC testing of colistin. Eur J Clin Microbiol Infect Dis. mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 2016. https :// 2018;37:345–53. doi.org/10.2807/1560-7917.ES.2016.21.27.30280 . 14. Perrin-Guyomard A, Bruneau M, Houée P, Deleurme K, Legrandois P, Poir- ier C, et al. Prevalence of mcr-1 in commensal Escherichia coli from French Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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Screening for fecal presence of colistin-resistant Escherichia coli and mcr-1 and mcr-2 genes in camel-calves in southern Tunisia

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Abstract

Camels (Camelus dromedarius) are known to harbor multidrug resistant Gram-negative bacteria and to be involved in the transmission of various microorganisms to humans. Data on the occurrence of colistin resistant Escherichia coli as well as mobilized colistin resistance (mcr) genes in camels are lacking. We investigated the presence of colistin resist- ance and mcr (1–2) genes in E. coli from the feces of camels in Tunisia. Presumptive E. coli isolates from camel-calves in southern Tunisia were qualitatively screened for growth on Mueller–Hinton agar supplemented with 2 mg/L of colis- tin. The minimal inhibitory concentration of colistin was determined for isolates growing on this medium. All isolates were screened for the presence of the mcr-1 and mcr-2 genes by polymerase chain reaction without detecting any of these genes. However, one isolate was confirmed resistant to colistin and further testing of this isolate revealed it to be Enterobacter cloacae. Our study demonstrated absence of colistin resistance and of the mcr-1 and mcr-2 genes in E. coli isolated from camel feces in southern Tunisia. Thus, there is no evidence that camels represent a major source of mcr genes contamination for the local population or for tourists visiting southern Tunisia. Keywords: Camel, Colistin resistance, E. coli, Human, MCR Findings antimicrobials approved to treat bacterial infections in One-humped camels (Camelus dromedarius) are raised dromedaries poses a major challenge for veterinarians extensively in southern Tunisia for the production of working in this animal production [5]. The use of anti - meat and milk as well as for activities related to Sahara microbials approved for ruminants, horses or other ani- tourism. One-humped camels are considered part of the mal species in the treatment of sick camels has not been cultural heritage and represents a national wealth for associated with satisfactory results because of the physi- many countries [1]. Camel-calves have a high suscepti- ological particularities of this animal species [5]. Colistin bility to bacterial infections, particularly those caused sulfate (CS), a polypeptide antibiotic, is used off-label by Escherichia coli [1–3]. In fact, colibacillosis in young in Tunisia for the oral treatment of Enterobacteriaceae camelids results in considerable economic losses in camel infections in camel-calves at the dose of 25,000  IU/kg farms, being associated with mortality, growth retarda- twice a day for 3 consecutive days. Following the recent tion and medical treatment costs [1, 4]. The absence of identification of a stable plasmid mediated mobilized colistin resistance (mcr-1) gene encoding for resistance to colistin in Enterobacteriaceae, this “novel” mecha- *Correspondence: mohamed.rhouma@umontreal.ca nism of colistin resistance has been widely studied in Department of Pathology and Microbiology, Faculty of Veterinary bacteria isolated from several origins such as farm ani- Medicine, University of Montreal, 3200 Sicotte St, Saint-Hyacinthe, QC J2S mals, humans, food and the environment [6, 7]. In July 2M2, Canada Full list of author information is available at the end of the article and August 2017, we searched PubMed with the terms © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rhouma et al. Acta Vet Scand (2018) 60:35 Page 2 of 5 “colistin in dromedary or colistin in camels”, “colistin Medenine  (n = 10)  (Fig.  1) with an average flock size of resistance in dromedary or in camels”, “MCR-1 in drom- 75 animals. Rectal swabs from 120 camel-calves aged edary or in camels” and “mcr genes in dromedary or in between 1 and 3 months, with or without diarrhea, were camels”. However, no studies on fecal presence of plas- suspended in buffered peptone water solution. Selected −1 −2 −3 mid-mediated colistin resistance genes (mcr-1 to -5) or dilutions (10, 10 , and 10 ) were plated on MacCo- E. coli colistin resistance in camels were found. On the nkey agar. The plates were incubated aerobically for 24 h other hand, it was been recently reported that camels are at 37  °C. Colonies growing on MacConkey agar with a a potential source of human contamination with multid- morphology typical for E. coli (red or pink, non-mucoid rug resistant (MDR) bacteria such as extended-spectrum colonies) were tested by polymerase chain reaction beta-lactamase (ESBL) producing E. coli or Pseudomonas (PCR) for the β-glucuronidase (uidA) housekeeping gene, aeruginosa [5, 8]. Moreover, co-localisation of mcr-1 and and PCR-positive colonies obtained (n = 52) were consid- ESBL genes on a unique plasmid has been reported [9]. ered as presumptive E. coli isolates (Table  1) [10]. These The present study aimed (1) to determine the fecal prev - isolates were stored at − 80 °C until further analysis. alence of E. coli colistin resistance in camel-calves with After thawing, the 52 isolates were inoculated on Muel- and without diarrhea in Tunisia, and (2) to determine the ler–Hinton agar supplemented with CS at the concen- prevalence of mcr-1 and mcr-2 genes among both colistin tration of 2  mg/L. Isolates demonstrating growth were resistant and susceptible E. coli isolates. considered as possibly colistin-resistant and were exam- In a previous study, the distribution of virulence ined for minimum inhibitory concentration (MIC) of genes, pathotypes, serogroups, phylogenetic groups, colistin, using the broth microdilution method in techni- and antimicrobial resistance of presumptive E. coli iso- cal duplicate according to the guidelines of the Clinical lated from camel-calves with and without diarrhea in Laboratory Standards Institute (CLSI) [11]. Enterobac- Tunisia was investigated [2]. Fecal samples had been teriaceae with colistin MICs > 2  mg/L were defined as collected between January 2011 and April 2013 from resistant (R), and those with colistin MICs ≤ 2  mg/L 25 extensive camel farms located in  three  districts  in as susceptible (S) according to the European Commit- southern Tunisia: Kebili (n = 13), Gabes (n = 2), and tee on Antimicrobial Susceptibility Testing (EUCAST) Fig. 1 Geographic distribution of the sampled farms by district in southern Tunisia with the dromedary density and its involvement in tourist activities Rhouma et al. Acta Vet Scand (2018) 60:35 Page 3 of 5 Table 1 Distribution of  possibly colistin resistant isolates according to  their origin; camel- calves with  or  without diarrhea Health status of camel-calves Number of isolates Species (number) Colistin resistant isolates MIC (number) of colistin value (mg/L) No diarrhea 23 E. coli (n = 23) 0 – Diarrhea 29 E. coli (n = 28) 0 – E. cloacae (n = 1) E. cloacae (n = 1) 4 Escherichia coli, Enterobacter cloacae guidelines (http://www.eucas t.org/clini cal_break point s/). as possible colistin resistant (Fig. 2). However, only one Isolates with colistin MICs > 2 mg/L were further identi- of these isolates was confirmed to be resistant to colis - fied using the API 20E system (bioMérieux, Quebec City, tin with an MIC value of 4  mg/L (Table  1). This resist - Canada). ant isolate, recovered from a camel-calf with diarrhea, Bacterial DNA extraction was performed using the was identified as Enterobacter cloacae. Chelex method (Bio-Rad, ON, Canada). All isolates Neither the mcr-1 nor mcr-2 gene were detected in were screened by PCR for the presence of mcr-1 and any of the colistin resistant or susceptible isolates. mcr-2 genes, using primers and conditions as previously It is important to monitor colistin resistance in drom- described [12, 13]. DNA from E. coli strains harbouring edary herds in Tunisia as the mcr-1 gene is highly prev- either mcr-1 gene [14] or mcr-2 gene [13] were used as alent in food-producing animals (such as chickens) in positive control. this country [15]. In addition, Tunisia being an impor- More than half of the presumptive E. coli isolates tant tourist destination [16], close contact between (55%) (n = 28) in both groups of animals were identified Fig. 2 Distribution of minimum inhibitory concentrations (MICs) in E. coli isolates possibly resistant to colistin (n = 28) recovered from camel-calves with and without diarrhea Rhouma et al. Acta Vet Scand (2018) 60:35 Page 4 of 5 camels and tourists visiting southern Tunisia may result Nevertheless, it highlights the need to widen the scope in spread of resistant bacteria internationally. of monitoring colistin resistance to other Enterobacte- Our finding that colistin-resistance and the mcr genes riaceae species than those conventionally identified in were absent in E. coli isolated from camel-calves in south- veterinary diagnostic laboratories, such as E. cloacae. ern Tunisia suggest that dromedaries are not a major Moreover, with the global initiatives for the establish- source of contamination with these genes for inhabitants ment of a One Health antimicrobial resistance surveil- as well as for tourists visiting this region. It should be lance approach, camels should be also considered as an noted that colistin-susceptible isolates were included in important animal species in the tracking of resistant bac- the current study, as previous studies have reported the teria at the human, animal and environment interface. identification of mcr-1 gene among colistin-resistant and colistin-susceptible isolates [6, 17]. Abbreviations We report here for the first time the isolation of E. cloa - CLSI: Clinical and Laboratory Standards Institute; CS: colistin sulfate; ESBL: cae resistant to colistin from a diarrheic camel. However, extended-spectrum beta-lactamase; EUCAST: European Committee on Anti- microbial Susceptibility Testing; DNA: deoxyribonucleic acid; MDR: multidrug this isolate did not harbor mcr-1 or mcr-2 genes. Simi- resistant; MICs: minimal inhibitory concentrations; PCR: polymerase chain larly, colistin-resistant E. cloacae isolates with or with- reaction. out the mcr-1 gene have been found in healthy people or Authors’ contributions primary care patients [18–20]. Currently, the mechanism MR, SB, IS and PF planned and designed the study. MR and WT carried out of colistin resistance in E. cloacae strains in the absence the microbiological and molecular biological tests. MR, PF, and JMF analyzed of plasmid-borne colistin resistance genes (mcr-1 to -5) and interpreted the data. MR drafted the manuscript. All authors read and approved the final manuscript. remains poorly understood. Zhong et  al. [21] recently identified 17 genes in the chromosome and one gene in Author details pASM1 encoding efflux pump proteins in a highly colis - Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte St, Saint-Hyacinthe, QC J2S 2M2, Canada. tin resistant E. cloacae strain. Nevertheless, mechanisms Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of colistin resistance not associated with plasmids in of Gabès, Médenine, Tunisia. Enterobacter spp. require further investigation. Acknowledgements It should be emphasized that we had no information We would like to thank Dr. Pascal Sanders (ANSES, Laboratoire de Fougères, regarding the medical treatment history of the sampled France) and Pr. Dr. Surbhi Malhorta-Kumar (University of Antwerp, Wilrijk, camel-calves, specifically, whether or not colistin has Belgium) for providing DNA from Escherichia coli harboring respectively, mcr-1 and mcr-2 genes. We gratefully thank Pr. Mohamed Gharbi (Laboratoire de been used in these dromedary farms. Additional studies parasitologie, Ecole Nationale de Médecine Vétérinaire de Sidi Thabet-Tunisia) are needed in order to assess the spread of colistin resist- for critical reading of the manuscript prior to submission. ant bacteria and mcr genes among enteric bacteria of Competing interests camel origin worldwide. Future surveys should take into The authors declare that they have no competing interests. consideration other areas where dromedary density is very high, such as the Arabian Peninsula where the mcr-1 Availability of data and materials All data generated or analysed during this study are included in this published gene has been identified in E. coli isolates [22]. article. The colistin resistant bacterial strain is available from the authors upon In the present study, the number of isolates obtained reasonable request. was limited because of the difficulty of handling camel- Ethics approval and consent to participate calves in extensive farms. However, we believe that our Not applicable. sampling was representative of dromedary production in Tunisia, as fecal samples were obtained from districts Funding This work was supported by the Natural Sciences and Engineering Research containing the highest density of dromedary farms. Council of Canada (NSERC) (412247-10). As expected, the use of Mueller–Hinton agar supple- mented with 2  mg/L of CS overestimated the frequency Publisher’s Note of presumed colistin resistant bacteria. But the culturing Springer Nature remains neutral with regard to jurisdictional claims in pub- on this medium was only used as a screening method for lished maps and institutional affiliations. reducing the number of isolates to be tested for colistin Received: 7 November 2017 Accepted: 26 May 2018 MIC. In addition, because of the poor diffusion of colistin in agar, it was always recommended to confirm presumed colistin resistant isolates, growing on colistin containing agar, by colistin CMI determination [23, 24]. The results suggest that camel-calves do not represent References 1. Al-Ruwaili MA, Khalil OM, Selim SA. Viral and bacterial infections associ- a major source of mcr gene contamination for the local ated with camel (Camelus dromedarius) calf diarrhea in North Province, population or for tourists visiting southern Tunisia. Saudi Arabia. Saudi J Biol Sci. 2012;19:35–41. Rhouma et al. Acta Vet Scand (2018) 60:35 Page 5 of 5 2. 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Acta Veterinaria ScandinavicaSpringer Journals

Published: Jun 5, 2018

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