Study of the effect of administration of narasin or antibiotics on in vivo selection of a narasin- and multidrug-resistant Enterococcus cecorum strain.

Enterococcus cecorum is a member of the normal poultry gut microbiota and an emerging poultry pathogen. Some strains are resistant to key antibiotics and coccidiostats. We evaluated the impact on chicken excretion and persistence of a multidrug-resistant E. cecorum of administering narasin or antibiotics. E. cecorum CIRMBP-1294 (Ec1294) is non-wild-type to many antimicrobials, including narasin, levofloxacin, oxytetracycline and glycopeptides, it has a low susceptibility to amoxicillin, and carries a chromosomal vanA operon. Six groups of 15 chicks each were orally inoculated with Ec1294 and two groups were left untreated. Amoxicillin, oxytetracycline or narasin were administered orally to one group each, either at the recommended dose for five days (amoxicillin, oxytetracycline) or continuously (narasin). Faecal samples were collected weekly and caecal samples were obtained from sacrificed birds on day 28. Ec1294 titres were evaluated by culture on vancomycin- and levofloxacin-supplemented media in 5 % CO2. For inoculated birds given narasin, oxytetracycline or no antimicrobials, vancomycin-resistant enterococci were searched by culture on vancomycin-supplemented media incubated in air, and a PCR was used to detect the vanA gene. Ec1294 persisted in inoculated chicks up to day 28. Compared to the control group, the Ec1294 titre was significantly lower in the amoxicillin- and narasin-receiving groups on days 21 and 28, but was unexpectedly higher in the oxytetracycline-receiving group before and after oxytetracycline administration, preventing a conclusion for this group. No transfer of the vanA gene to other enterococci was detected. Other trials in various experimental conditions should now be conducted to confirm this apparent absence of co-selection of the multi-drug-resistant E. cecorum by narasin or amoxicillin administration.

Determination of Epidemiological Cutoff Values for Antimicrobial Resistance of Enterococcus cecorum.

Enterococcus cecorum, a commensal Gram-positive bacterium of the chicken gut, has emerged as a worldwide cause of lameness in poultry, particularly in fast-growing broilers. It is responsible for osteomyelitis, spondylitis, and femoral head necrosis, causing animal suffering, mortality, and antimicrobial use. Research on the antimicrobial resistance of E. cecorum clinical isolates in France is scarce, and epidemiological cutoff (ECOFF) values are unknown. To determine tentative ECOFF (COWT) values for E. cecorum and to investigate the antimicrobial resistance patterns of isolates from mainly French broilers, we tested the susceptibility of a collection of commensal and clinical isolates (n = 208) to 29 antimicrobials by the disc diffusion (DD) method. We also determined the MICs of 23 antimicrobials by the broth microdilution method. To detect chromosomal mutations conferring antimicrobial resistance, we investigated the genomes of 118 E. cecorum isolates obtained mainly from infectious sites and described previously in the literature. We determined the COWT values for more than 20 antimicrobials and identified two chromosomal mutations explaining fluoroquinolone resistance. The DD method appears better suited for detecting E. cecorum antimicrobial resistance. Although tetracycline and erythromycin resistances were persistent in clinical and nonclinical isolates, we found little or no resistance to medically important antimicrobials.

Impact of colistin and colistin-loaded on alginate nanoparticles on pigs infected with a colistin-resistant enterotoxigenic Escherichia coli strain.

Colistin is frequently used for the control of post-weaning diarrhoea in pigs. Colistin resistance caused by plasmidic genes is a public health issue. We evaluated, in experimental animal facilities, whether free colistin or colistin-loaded on alginate nanoparticles (colistin/Alg NPs) could select a colistin-resistant Enterotoxigenic Escherichia coli. The Alg NPs were produced by a simple top-down approach through ball milling of sodium alginate polymer precursor, and colistin loading was achieved through physical adsorption. Colistin loading on Alg NPs was confirmed using various tools such Fourier transform infrared spectroscopy and dynamic light scattering measurements. Thirty-four piglets were orally inoculated or not with a mcr-1-positive, rifampicin-resistant enterotoxigenic E. coli strain, and the inoculated pigs were either treated or not during five days with commercial colistin (100,000 IU/kg) or colistin/Alg NPs (40,415 IU/kg). Clinical signs were recorded. Fecal and post-mortem samples were analyzed by culture. The result clearly indicated that colistin/Alg NPs had a slightly better therapeutic effect. Both treatments led to a transitory decrease of the total E. coli fecal population with a majority of colistin-resistant E. coli isolates during treatment, but the dominant E. coli population was found susceptible at the end of the trial. Further studies are needed to evaluate, in diverse experimental or field conditions, the therapeutic efficacy of colistin/Alg NPs for post-weaning diarrhoea.

Impact of Escherichia coli probiotic strains ED1a and Nissle 1917 on the excretion and gut carriage of extended-spectrum beta-lactamase-producing E. coli in pigs.

We evaluated the impact of the administration of two Escherichia coli probiotic strains (ED1a and Nissle 1917) to pigs on the gut carriage or shedding of extended-spectrum beta-lactamase-producing E. coli. The probiotics were given to four sows from 12 days before farrowing to the weaning day, and to the 23 piglets (infected treated group (IPro)) from birth to the age of 49 days. Four other sows and their 24 piglets (infected non-treated group (INT)) did not receive the probiotics. IPro and INT piglets (n = 47) were orally inoculated with the strain E. coli 17-348F-RifR carrying the bla CTX-M-1 gene and resistant to rifampicin. Cefotaxime-resistant (CTXR) E. coli and rifampicin-resistant (RifR) E. coli were cultured and excretion of probiotics was studied using PCR on individual faecal and post-mortem samples, and from manure collected after the challenge with resistant E. coli. CTXR and RifR E . coli isolates were characterized to detect transfer of the bla CTX-M-1 to other strains.. Overall, there was no significant reduction in faecal excretion of CTXR and RifR E. coli in IPro pigs compared with INT pigs, although the CTXR and RifR E. coli titres were slightly, but significantly lower in the colon, caecum and rectum at post mortem. Excretion of the probiotics decreased with age, but Nissle 1917 was detected in most pigs at post-mortem. No transfer of the bla CTX-M-1 gene to probiotic and other E. coli strains was detected. In conclusion, in our experimental conditions, the used probiotics did not reduce shedding of the challenge strain.

Variations of the Escherichia coli population in the digestive tract of broilers.

We explored the between-group and temporal variations in the intestinal Escherichia coli populations of broilers under experimental conditions, taking both antimicrobial resistance and virulence into consideration. Four replicates of 45 commercial chicks were reared in four animal facilities. On their first day of life (Day 0), they were orally inoculated with two extended-spectrum-cephalosporin-resistant (ESCR) E. coli (2.72 log10 CFU of a bla CMY-2- and 2.55 log10 CFU of a bla CTX-M-carrying E. coli). Faecal samples were then collected weekly and caecal samples were obtained from birds sacrificed on Days 21 or 42. The total, ESC-, ciprofloxacin- and gentamicin-resistant E. coli populations were enumerated on MacConkey (MC) and MC-supplemented media, and eight virulence-associated genes (VAGs) (iroN, iutA, iss, ompT, hlyF, vat, frzorf4 , and fyuA) were sought by PCR on isolates obtained on MC agar. The results showed significant between-group differences in the size of the resistant sub-populations and the presence of VAGs. Contrary to bla CTX-M-positive strains, bla CMY-positive strains persisted up to Day 42, but represented only a minor fraction of the total E. coli population. The ESC-, gentamicin- and ciprofloxacin-resistant populations decreased over time. Isolates obtained during the first week contained a mean of 5.1 VAGs. The percentages of some VAG profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. The fluctuations or differences between E. coli isolates according to group, age, and faecal or caecal origin need to be considered when designing experimental protocols and seeking to improve colibacillosis control. RESEARCH HIGHLIGHTS Temporal variations in the intestinal E. coli populations of broilers was studied. The antibiotic-resistant populations decreased over time. Virulence profiles differed between faecal isolates on Day 41 and caecal isolates on Day 42. Strains with the highest numbers of virulence genes were present during the first days.

Decrease in fluoroquinolone use in French poultry and pig production and changes in resistance among E. coli and Campylobacter.

This paper presents the impact on antimicrobial resistance (AMR) in poultry and pig bacteria of the French EcoAntibio plan, a public policy to reduce antimicrobial use in animals. The analysis was performed using sales data of veterinary antimicrobials and AMR data from bacteria obtained at slaughterhouse and from diseased animals. From 2011-2018, fluoroquinolones exposure decreased by 71.5 % for poultry and 89.7 % for pigs. For Campylobacter jejuni isolated from broilers at slaughterhouses, ciprofloxacin resistance increased from 51 % in 2010 to 63 % in 2018, whereas for turkeys the percentages varied from 56 % in 2014 to 63 % in 2018. For commensal E. coli isolated from the caecal content of broilers at slaughterhouses, the resistance to ciprofloxacin - assessed using an epidemiological cut-off value - increased in broiler isolates from 30.7 % in 2010 to 38.1 % in 2018. In turkeys, the percentage of resistant E. coli isolates decreased from 21.3 % in 2014 to 15.2 % in 2018, whereas in pigs, it increased from 1.9 % in 2009 to 5.5 % in 2017. However, for E. coli isolated from diseased animals, when the breakpoints of 2018 were applied, resistance to fluoroquinolones significantly decreased between 2010 and 2018 from 9.0%-5.4% for broilers/hens, from 7.4 % to 3.4 % for turkeys and from 9.4 % to 3.6 % for pigs. These data show that the major, rapid decrease in the exposition to fluoroquinolones had contrasting effects on resistance in the diverse bacterial collections. Co-selection or fitness of resistant strains may explain why changes in AMR do not always closely mirror changes in use.

Impact of colistin administered before or after inoculation on the transmission of a mcr-1 colistin-resistant Escherichia coli strain between pigs.

Colistin resistance associated with plasmidic resistance genes is a serious public health issue. We aimed at studying the transmission of an mcr-1 colistin- and rifampicin-resistant Escherichia coli strain between inoculated pigs and sentinels in different controlled conditions. Three groups of four pigs were bred in separated animal rooms and inoculated on Day 0 (D0). In each inoculated group, six contact pigs were introduced on D2. The first inoculated-and-contact group was left untreated. The ten pigs in the second inoculated-and-contact group received colistin (100 000 IU/kg) before inoculation or contact (D-7 to D-5), simulating prophylactic administration. Pigs in the third inoculated-and-contact group were treated just after inoculation or before transfer (D0 to D2), simulating metaphylactic administration. Faecal samples were regularly collected and segments of intestinal tracts were obtained at necropsy, on D20-D22. Samples were cultured on rifampicin-supplemented media, and PCR was used to detect the mcr-1 gene. The kinetics of infection, based on culture results, were analysed using an SIR model. The inoculated strain was detected in all inoculated and contact pigs. The SIR model showed that one infected pig could transmit the resistant bacteria to one susceptible individual in less than 3 h on average. Prophylactic administration significantly enhanced the transmission rate and resulted in more samples containing the mcr-1 resistance gene at necropsy. No effect of metaphylactic administration could be detected on the transmission rate, nor on the carriage of the resistant strain. Our study confirms that colistin should not be used in a prophylactic manner.

Dissemination of the mcr-1 colistin resistance gene among pigs: An experimental study.

Colistin resistance in Enterobacteriaceae is a public health problem. The present study was designed to evaluate the dissemination of a colistin-resistant Escherichia coli strain and its resistance gene, mcr-1, between orally inoculated pigs and their contacts. A non-inoculated control group, one low-dose and one high-dose group-both including two pens of two inoculated and three contact pigs-were raised in separate rooms. After inoculation of a colistin- and rifampicin-resistant E. coli suspension (2.5 × 105 CFU/pig for the low-dose group and 2.5 × 108 CFU/pig for the high-dose group), feces from inoculated and non-inoculated contact pigs were collected and inoculated on colistin- and rifampicin-supplemented media directly or after enrichment in rifampicin-supplemented media, then the isolates were characterized. PCR was used to detect the mcr-1 gene in lysates from feces cultivated in colistin-supplemented broth and DNA prepared from feces. Results showed that the low-dose inoculum was probably insufficient to obtain durable colonization, but could lead to the temporary presence of mcr-1-positive E. coli strains. The high-dose inoculum resulted in durable colonization of both inoculated and contact animals. In all groups, the mcr-1 gene was also detected in rifampicin-susceptible strains, suggesting its transfer to several commensal strains. A comparison of detection methods showed that more positive samples were obtained with cultures in rifampicin-supplemented media and suggests that current methods to evaluate the prevalence of colistin resistance in fecal samples suffer from poor sensitivity.

Campylobacter coli in Organic and Conventional Pig Production in France and Sweden: Prevalence and Antimicrobial Resistance.

The purpose of the study was to evaluate and compare the prevalence and antimicrobial resistance of Campylobacter coli in conventional and organic pigs from France and Sweden. Fecal or colon samples were collected at farms or at slaughterhouses and cultured for Campylobacter. The minimum inhibitory concentrations of ciprofloxacin, nalidixic acid, streptomycin, tetracycline, erythromycin, and gentamicin were determined by microdilution for a total of 263 French strains from 114 pigs from 50 different farms and 82 Swedish strains from 144 pigs from 54 different farms. Erythromycin resistant isolates were examined for presence of the emerging rRNA methylase erm(B) gene. The study showed that within the colon samples obtained in each country there was no significant difference in prevalence of Campylobacter between pigs in organic and conventional productions [France: conventional: 43/58 (74%); organic: 43/56 (77%) and Sweden: conventional: 24/36 (67%); organic: 20/36 (56%)]. In France, but not in Sweden, significant differences of percentages of resistant isolates were associated with production type (tetracycline, erythromycin) and the number of resistances was significantly higher for isolates from conventional pigs. In Sweden, the number of resistances of fecal isolates was significantly higher compared to colon isolates. The erm(B) gene was not detected in the 87 erythromycin resistant strains tested.

Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota.

Resistance to extended-spectrum cephalosporins (ESCs) is a matter of considerable concern for public health. Here, we studied the spontaneous loss of an extended-spectrum beta-lactamase (ESBL)-encoding plasmid from a rifampin-resistant Escherichia coli isolate orally inoculated into pigs under controlled conditions. Fecal samples were collected and cultured on rifampin-supplemented medium, and the resistance of the E. coli isolates to ESCs was studied by phenotypic tests, PCR detection of plasmid genes, and complete sequencing. The results showed that only 3 out of 353 rifampin-resistant E. coli isolates were ESC susceptible, and PCR and bioinformatics analysis confirmed the loss of the plasmid. These in vivo experiments indicate that the loss of an ESBL-encoding plasmid seems a rare event in gut microbiota.

Impact of colistin sulfate treatment of broilers on the presence of resistant bacteria and resistance genes in stored or composted manure.

The application of manure may result in contamination of the environment with antimicrobials, antimicrobial-resistant bacteria, resistance genes and plasmids. The aim of this study was to investigate the impact of the administration of colistin and of manure management on (i) the presence of colistin-resistant Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa and (ii) the prevalence of various antimicrobial resistance genes in feces and in composted or stored manure. One flock of chickens was treated with colistin at the recommended dosage and a second flock was kept as an untreated control. Samples of feces, litter and stored or composted manure from both flocks were collected for isolation and determination of the colistin-susceptibility of E. coli, K. pneumoniae and P. aeruginosa and quantification of genes coding for resistance to different antimicrobials. The persistence of plasmids in stored or composted manure from colistin-treated broilers was also evaluated by plasmid capturing experiments. Results revealed that colistin administration to chickens had no apparent impact on the antimicrobial resistance of the dominant Enterobacteriaceae and P. aeruginosa populations in the chicken gut. Composting stimulated an apparently limited decrease in genes coding for resistance to different antimicrobial families. Importantly, it was shown that even after six weeks of composting or storage, plasmids carrying antimicrobial resistance genes could still be transferred to a recipient E. coli. In conclusion, composting is insufficient to completely eliminate the risk of spreading antimicrobial resistance through chicken manure.

Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets.

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates. A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS. The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin. Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance. This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.

Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens.

An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

Antimicrobial resistance selection in avian pathogenic E. coli during treatment.

An experiment was performed to compare the microbiological efficacy of four treatments (oxytetracycline, trimethoprim-sulphonamide, amoxicillin (AMX) or enrofloxacin (ENR)) to control experimental colibacillosis induced by an avian pathogenic Escherichia coli (APEC) with reduced susceptibility to fluoroquinolones. The protocol was also developed in order to study resistance gene transfer. Broilers were first orally inoculated with multiresistant E. coli bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-M or blaFOX), tetracycline or trimethoprim-sulphonamide. They were then inoculated in their air sacs with the APEC and treated as soon as symptoms appeared. Internal organs from dead or sacrificed birds were cultivated on non-supplemented or supplemented media. The inoculated O78 APEC was recovered significantly less frequently in ENR treated group (26%) compared to untreated group (47%). This was not true for other treated groups. Isolates obtained on non-supplemented media had the same susceptibility profile as the inoculated APEC. However, one isolate from the AMX-treated group obtained on AMX-supplemented media was resistant to AMX only, and one isolate from the same group obtained on ENR-supplemented media, showed a resistance profile suggesting acquisition of one of the multiresistance plasmids present in the intestinal microbiota. Molecular analysis performed on this multiresistant isolate confirmed the presence of a conjugative plasmid with qnr and blaCTX-M resistance genes. Thus, the experiment illustrated the emergence of resistant isolates in internal organs, probably via acquisition of a plasmid from the intestinal microbiota.

Effect of in-feed paromomycin supplementation on antimicrobial resistance of enteric bacteria in turkeys.

Histomoniasis in turkeys can be prevented by administering paromomycin sulfate, an aminoglycoside antimicrobial agent, in feed. The aim of this study was to evaluate the impact of in-feed paromomycin sulfate supplementation on the antimicrobial resistance of intestinal bacteria in turkeys. Twelve flocks of breeder turkeys were administered 100 ppm paromomycin sulfate from hatching to day 120; 12 flocks not supplemented with paromomycin were used as controls. Faecal samples were collected monthly from days 0 to 180. The resistance of Escherichia coli, Enterococcus faecium and Staphylococcus aureus to paramomycin and other antimicrobial agents was compared in paromomycin supplemented (PS) and unsupplemented (PNS) flocks. E. coli from PS birds had a significantly higher frequency of resistance to paromomycin, neomycin and kanamycin until 1 month after the end of supplementation compared to PNS birds. Resistance to amoxicillin or trimethoprim-sulfamethoxazole was also more frequent in PS turkeys. Resistance was mainly due to the presence of aph genes, which could be transmitted by conjugation, sometimes with streptomycin, tetracycline, amoxicillin, trimethoprim or sulfonamide resistance genes. Resistance to kanamycin and streptomycin in E. faecium was significantly different in PS and PNS breeders on days 60 and 90. Significantly higher frequencies of resistance to paromomycin, kanamycin, neomycin and tobramycin were observed in S. aureus isolates from PS birds. Paromomycin supplementation resulted in resistance to aminoglycosides in bacteria of PS turkeys. Co-selection for resistance to other antimicrobial agents was observed in E. coli isolates.

Resistance gene transfer during treatments for experimental avian colibacillosis.

An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments-oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)-on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (bla(CTX-M) or bla(FOX)), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, "old" antimicrobials may coselect antimicrobial resistance to recent and critical molecules.