Eric Jouy

Staphylococcus aureus CC398: Host Adaptation and Emergence of Methicillin Resistance in Livestock

ABSTRACT Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production. Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

What do we know about resistance to colistin in Enterobacteriaceae in avian and pig production in Europe

Colistin is a cyclic decapeptide bound to a fatty acid chain. It is active against many Gram-negative bacteria by destabilising the bacterial outer membrane. Bacteria can become resistant to colistin by modification of their lipopolysaccharide, thereby reducing the affinity of polymyxins. Colistin is often administered orally in poultry and pig production to control colibacillosis. Resistant isolates are sometimes recovered from pathological cases, particularly in piglets. However, in Europe the percentage of resistance to colistin in Escherichia coli strains isolated from the digestive tract microbiota of healthy animals remains <1%.

Synergistic Effect between Colistin and Bacteriocins in Controlling Gram-Negative Pathogens and Their Potential To Reduce Antibiotic Toxicity in Mammalian Epithelial Cells

ABSTRACT Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 μg/ml. When nisin A (1.70 μg/ml) or pediocin PA-1/AcH (1.56 μg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 μg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 μm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Colistin use and colistin resistance in bacteria from animals.

Colistin has been used in veterinary medicine for decades, mainly for the prevention and treatment of Enterobacteriaceae infections. However, data regarding colistin resistance in bacteria from animals and food of animal origin are relatively scarce, partly because there are methodological difficulties hampering the analysis of susceptibility to colistin. Most data regarding clinical isolates are related to enteropathogenic Escherichia coli and Salmonella. The resistance percentages are sometimes high for pathogenic strains, and the mcr-1 gene has been detected in pathogenic E. coli isolates from pigs, cattle and poultry in different countries. The prevalence of colistin resistance in Salmonella from healthy animals is usually low but depends on the proportion of intrinsically colistin-resistant serotypes. For indicator E. coli, the resistance levels are often very low, although higher levels have been observed in Asia. The mcr-1 gene has been detected in indicator E. coli from pigs, cattle, poultry and their products. Thus, there is an urgent need to re-assess the use of colistin in livestock throughout the world to ensure a global strategy for preserving this last-resort antimicrobial.

Plasmid-borne florfenicol and ceftiofur resistance encoded by the floR and blaCMY-2 genes in Escherichia coli isolates from diseased cattle in France

This study was designed to determine the genetic basis of florfenicol and ceftiofur resistance in Escherichia coli isolates recovered from French cattle. In these isolates, ceftiofur resistance was conferred by bla(CMY-2) located on three distinct conjugative plasmids on a specific DNA fragment, ISEcp1-bla(CMY-2)-blc- sugE. Two of the plasmids also carried the floR gene conferring resistance to florfenicol. The floR gene was shown to be associated with the insertion sequence ISCR2. Mobile elements appear to contribute to the mobilization of floR and bla(CMY-2) genes in E. coli. The presence of bla(CMY-2) and floR on the same plasmid highlights the potential risk for a co-selection of the bla(CMY-2) gene through the use of florfenicol in food animal production.

Impact of Third-Generation-Cephalosporin Administration in Hatcheries on Fecal Escherichia coli Antimicrobial Resistance in Broilers and Layers

ABSTRACT We investigated the impact of the hatchery practice of administering third-generation cephalosporin (3GC) on the selection and persistence of 3GC-resistant Escherichia coli in poultry. We studied 15 3GC-treated (TB) and 15 non-3GC-treated (NTB) broiler flocks and 12 3GC-treated (TL) and 10 non-3GC-treated (NTL) future layer flocks. Fecal samples from each flock were sampled before arrival on the farm (day 0), on day 2, on day 7, and then twice more. E. coli isolates were isolated on MacConkey agar without antibiotics and screened for 3GC resistance, and any 3GC-resistant E. coli isolates were further analyzed. 3GC-resistant E. coli isolates were found in all 3GC-treated flocks on at least one sampling date. The percentages of 3GC-resistant E. coli isolates were significantly higher in TB (41.5%) than in NTB (19.5%) flocks and in TL (49.5%) than in NTL (24.5%) flocks. In the day 2 samples, more than 80% of the E. coli strains isolated were 3GC resistant. 3GC-resistant E. coli strains were still detected at the end of the follow-up period in 6 out of 27 3GC-treated and 5 out of 25 non-3GC-treated flocks. Many 3GC-resistant E. coli strains were resistant to tetracycline, and there were significant differences in the percentages of resistance to sulfamethoxazole-trimethoprim, streptomycin, or gentamicin between treated and nontreated flocks. blaCTX-M and blaCMY-2 were the most frequently detected genes. These results clearly demonstrated that 3GC-resistant strains are introduced early in flocks and that the use of 3GC in hatcheries promotes the selection of 3GC-resistant E. coli. Measures must be implemented to avoid the spread and selection of 3GC-resistant strains.

Estimation of transmission parameters of a fluoroquinolone-resistant Escherichia coli strain between pigs in experimental conditions

Antimicrobial resistance is of primary importance regarding public and animal health issues. Persistence and spread of resistant strains within a population contribute to the maintenance of a reservoir and lead to treatment failure. An experimental trial was carried out to study the horizontal transmission of a fluoroquinolone-resistant Escherichia coli strain from inoculated to naïve pigs. All naïve contact pigs had positive counts of fluoroquinolone-resistant E. coli after only two days of contact. Moreover, re-infections of inoculated pigs caused by newly contaminated animals were suspected. A maximum likelihood method, based on a susceptible-infectious-susceptible (SIS) model, was used to determine the transmission parameters. Two transmission levels were identified depending on the quantity of bacteria shed by infected individuals: (i) low-shedders with bacterial counts of resistant E. coli in the faeces between 5*103 and 106 CFU/g (βL= 0.41 [0.27; 0.62]), (ii) high shedders with bacterial counts above 106 CFU/g (βH= 0.98 [0.59; 1.62]). Hence, transmission between animals could be pivotal in explaining the persistence of resistant bacteria within pig herds.

Resistance Gene Transfer during Treatments for Experimental Avian Colibacillosis

ABSTRACT 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 (blaCTX-M or blaFOX), 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.

Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs

ABSTRACT Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, blaCTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the blaCTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the blaCTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.