Extended-spectrum β-Lactamase producing Enterobacteriaceae (ESBL-E) is a public and veterinary health burden worldwide and particularly in West Indian ocean countries [1
]. These multi-resistant bacteria have been identified as a priority in terms of epidemiological surveillance in humans and animals from the Indian Ocean Commission (IOC) state members (i.e., Comoros, Madagascar, Mauritius, Reunion and Seychelles) and Mayotte (French oversea territory) [1
ESBL-E are resistant to almost all beta-lactam antibiotic drugs including third generation cephalosporin (3GC), co-resistance is often observed with other classes of antibiotics such as fluoroquionolones, aminoglycosides, sulfonamides and tetracyclins, leading to the use of last-resort antibiotics (i.e., carbapenems) in ESBL-E infections in humans [2
The occurrence of ESBL-E has been identified in broiler and swine farms in Europe [3
] and the CTX-M β-lactamases is the most frequently detected enzyme in livestock, especially blaCTX-M-1
Selection pressure exerted by antibiotic drugs on microbiota favours carriage and persistence of ESBL-E in humans (hospital and community) [6
], livestock and pets [7
]; thus, all could act as potential reservoirs of ESBL-E.
The main known risk factor identified in ESBL-E occurrence in livestock was “use of 3GC or fourth generation cephalosporin (4GC) (ceftiofur, cefoperazone and cefquinome) in the last 12 months” in dairy and pig farms [10
Other risk factors such as storage of slurry in a pit, operating an open herd policy and infrequent cleaning of calf feeding equipment were also identified in dairy farms [4
] and fish ponds presence in poultry farms of Vietnam [12
In IOC, no estimate of ESBL-E prevalence in livestock was available. Thus, the aim of this study was first estimate the prevalence of ESBL-E on beef cattle, broiler and pig commercial farms in Reunion, Mayotte and Madagascar Islands and identify ESBL enzymes occurrence in each production type and territory. Secondly, potential risk factors of ESBL-E occurrence in poultry, beef cattle and pig farms were explored.
Our study pointed out high ESBL-E prevalence in Madagascar, Reunion and Mayotte livestock commercial farms. Overall ESBL genes diversity in E. coli
was reduced with blaCTX-M-1
mainly identified. In Madagascar, all genes identified in pig and beef cattle were blaCTX-M-15
, main enzyme observed in humans [20
]. It could confirm circulation of ESBL-E between human and livestock. Concrete factors associated with an increased risk of ESBL-E occurrence in farms were identified such as pet presence, farmer visits and recent antibiotic use. Finally, biosecurity and hygienic measures (e.g., disinfection, water quality control, detergent use) were globally reducing ESBL-E occurrence in IOC farms.
Our study clearly pointed a high ESBL-E prevalence in Madagascar, Reunion (except beef cattle) and Mayotte. Prevalence estimate was not accurate as obtained with a limited sample size; Madagascar ESBL-E prevalence calculated could neither estimate the overall prevalence in this large territory nor be the reflect of livestock farms diversity. If ESBL characterization allowed, for the first time, to identify a circulation of blaCTX-M-1 in all livestock types, the limited number of ESBL found in each livestock and IO territory (N = 10) cannot rule out patterns. Less diversity was expected by livestock type (e.g., in poultry in each territory, pigs from Reunion and beef cattle from Mayotte) and could highlight needs of further enzyme identification as diversity could not be captured as a whole.
No phenotypic resistance to ertapenem in ESBL-E isolates was identified, which is in accordance with the absence of carbapenemase producing Enterobacteriacae (CPE) detection in IO livestock in 2018 [1
]. However, use of CPE selective media would be more suitable for CPE detection. Resistance to fluoroquinolone could be low in Mayotte as no resistance to ofloxacin was observed but should be confirmed as few isolates were tested. Hypothesis about risk factors identification in our study was opportunistic and case control or cohort study designs to rule out ESBL-E control measures would be needed. Furthermore, antibiotic drug use recently was identified as increasing ESBL-E occurrence in IO farms but the farmers were not able to tell which antibiotic drug was used. Further studies should be undertaken to evaluate antibiotic drugs consumption and practices in IO farms.
In broiler production, the estimated prevalence in IO territories was higher than 50.0% reported in 2012 in Germany [22
] but similar to 70.0% reported in Japan in 2007 [23
]. In India, in 2014, among 87.0% of ESBL-E were detected in broiler and 42.0% in layer farms [24
]. In pig farms, the prevalence in IO was higher than 8.3% reported in pigs in Japan in 2007 [23
]. For Madagascar, it was similar to the 88.2% of ESBL-E positive farms observed in 2012 in Germany [22
]. ESBL-E occurrence of Mayotte and Madagascar beef cattle farms were similar to data reported from other studies in Germany with 73.3% of farms tested positive in 2011 to 2012 (Bavaria) [25
] and 54.4% in 2012 in Mecklenburg-Vorpommern [22
]. In Reunion, the prevalence of ESBL-E in beef cattle farms tends to be significantly lower than in other territories. It could reflect the effectiveness of the French governmental antibiotic reduction plan (Ecoantibio) in Reunion and better biosecurity. Mayotte is a French oversea territory, breeding practices are clearly different from Reunion with mixed livestock farms and could explain observed differences.
Finally, the high ESBL-E prevalence observed in IO territories could point to important antibiotic drug use and/or misuse, including cephalosporins. This is particularly true for pigs in Madagascar where high antibiotic residues were reported in pork products at abattoirs [26
Main ESBL-E co-resistance were observed in Madagascar (i.e., ofloxacin, tetracyclin, nalidixic acid and gentamicin) and Reunion (i.e., ofloxacin, nalidixic acid and trimethoprime/sulfamethoxazole). High ESBL-E co-resistance observed in Madagascar could point out a drug overuse, particularly for widely available oral agents [1
]. Nalidixic acid resistant isolates were resistant to ofloxacin in Reunion and Madagascar pig productions as observed in majority of cases [27
]. Fluoroquinolone resistance was high in ESBL producing E. coli
in pig production of both territories which could indicate past or present use/misuse of this critically important antimicrobial drug. Pig production was identified as the most important antibiotic consumer worldwide [28
]. French national data indicated that fluoroquinolones use was higher in cattle production than in pig and poultry production [29
]; trends, not estimated in IO French overseas territories, could differ from mainland France.
The most common ESBL gene identified in E. coli
isolates tested was blaCTX-M-1
(54.4%) as observed in food-producing animals in European countries [30
]. CTX-M β-lactamase is largely located on plasmids, which allows the horizontal transfer between Enterobacteriaceae [31
] and explains the current epidemic spread of this enzyme worldwide.
Overall ESBL gene diversity was reduced in our study with circulation of few genes by production type (e.g., blaCTX-M-1
in pig and poultry from Reunion and blaCTX-M-15
in pigs and beef cattle in Madagascar). It probably indicated a common past source of contamination with introduction of ESBL-E carriers and diffusion due to close contact in livestock as reported with blaCTX-M-14
in cattle from the United Kingdom [10
]. Thus, overall introduction/exchanges of ESBL-E between reservoirs and environment seems limited as observed by Dorado-Garcia in the Netherlands (2005–2015) [32
]. A more diverse ESBL genes pool was identified in IO poultry production with at least three different genes detected in each territory. Most of ESBL genes were blaCTX-M-1
but SHV-ESBL and TEM-ESBL genes were also identified as in Dutch broilers [33
]. This diversity of ESBL genes in poultry could be related to close contact with poultry house surrounding environment. Interestingly, blaCTX-M-15
was observed in pig production, beef cattle and poultry from Mayotte and Madagascar; It is the main enzyme observed in humans in IO [1
] and circulation of ESBL-E between human and livestock could be suspected.
In broiler farms, “Premises building constructed after 1999” and “change of shoes/boots before entering the building” were significantly increasing ESBL-E occurrence in Reunion. Both factors were difficult to explain as related to improved biosecurity measures. Antibiotic drug use could be higher in modern farms and “change of shoes/boots” was identified also as a risk factor ESBL-E occurrence in Vietnam poultry production [12
] confirming that further investigations are needed to identify a potential confounding explanatory factor. In Madagascar, “chick production in the farm” significantly reduced occurrence of ESBL-E. This is in accordance with a vertical ESBL-E transmission into the production chain through external introduction such as imported day-old grandparent chickens as in Dutch poultry farms [34
]. In all IO territories, “water quality control” was a protective factor of ESBL-E occurrence in commercial farms. It was in accordance with studies on Campylobacter
spp. that showed that electrolyzed water or chlorinated-water allowed reducing bacterial presence [35
]. Rural surface water may become a large reservoir of antibiotic residues and resistant bacteria [37
], thus, in order to minimize transmission of enteropathogens, drinking water should be of potable quality to ensure freedom from enteric pathogens [37
In pig farms, both “rodent control” and “two disinfections between two consecutive batches” were significantly reducing ESBL-E occurrence in Reunion. Both measures are related to biosecurity and hygiene helping to control disease and antibiotic resistance spread. In all IO territories, “recent antibiotic use”, “soak the floor” and “farmer visits” were associated with an increase of ESBL-E occurrence in pig production whereas “detergent use for cleaning” was associated with a decreased occurrence. ESBL-E occurrence could be more determined by the presence or absence of cephalosporin use at the farm as in Dutch pig production [38
]. “Others farmer visits” has never been identified as increasing ESBL-E occurrence and could be more related to the frequency of visits as observed with the veterinarian in cattle farms in Israel [39
]. Visitors could contribute to ESBL-E introduction and could carry/share material that favours transmission pathways. Detergent use for cleaning was associated with a decreased ESBL-E occurrence in IO pig production. Using effective detergent for cleaning was identified to decrease the risk of batch infection by Enterobacteriaceae such as Salmonella
]. However, “soak floor” practice in IO pig farm production could be explained by wrong biosecurity practices; for instance, let water for a too short period could not allow complete cleaning. For instance, a period of one-hour soak time may could be insufficient to demonstrate a significant difference in organic matter removal in pig pens [41
]. Thus, cleaning and disinfection processes are a cornerstone in ESBL-E eradication which was obtained in pig farms under specific disinfection procedures [42
In beef cattle production, “clearing space around the building “and “clean condition around the farm” reduced significantly ESBL-E occurrence in Madagascar. This explanatory variable could be related to a confounding factor; garbage presence in the farm probably attracting potential ESBL-E reservoirs such as dogs, cats or rodents. Accordingly, pet presence in the farm was identified as increasing ESBL-E occurrence in IO beef cattle farms. This finding was in accordance with Santman-Berends et al. 2017 [43
] which found cat presence as an explanatory factor of ESBL-E occurrence in organic herds in the Netherlands in 2011. It could be due to the fact that pets could be both given antibiotic drugs by owners and/or play a role of reservoir/vector of ESBL-E from the close environment. Furthermore, “recent antibiotic use” was associated with an increased ESBL-E occurrence in beef cattle farms. However, 3rd or 4th generation cephalosporin use in IO beef cattle farm was not studied while use was estimated to increase by nearly 4 times ESBL producing E. coli
in dairy farms if used in the last 12 months [10
Factors associated with a decrease of ESBL-E occurrence in IO beef cattle farms were “livestock size” and “disinfection”. IO big farms, herd size (>25 cattle), could apply stricter biosecurity measures. However, Adler et al. (2017) reported that an increased density was associated with more ESBL-E carriage in Israeli cattle farms [39
]. As discussed before, cleaning and disinfection seems to be cornerstones in ESBL-E management and hygiene paucity was identified as a risk factors of ESBL-E occurrence on dairy farms (e.g., storage of slurry in a pit, infrequent cleaning of feeding equipment) [10
In IO ESBL-E occurrence in 2016–2017 was high probably pointing out antibiotic drug overuses and/or misuses and particularly cephalosporins. The situation could be reversible if better practices were implemented regarding antibiotic use. For instance, in the Netherlands in 2010–2011, 20% of prevalence was observed if no cephalosporin was used (3CG and 4CG) within the preceding year in pig farms and 79% if those antibiotics were used [11
BlaCTX-M-15 gene, mainly identified in humans both in hospital and community, was observed in IO livestock and particularly Madagascar Further investigations, including complete genome sequencing, are needed to evaluate the hypothesis of ESBL-E transmission and diffusion between reservoirs in this territory. Finally, interesting factors related to biosecurity and hygiene measures in commercial farms were identified (e.g., controlled water, disinfection, rodent control) to control ESBL-E occurrence.