Validation of an antimicrobial susceptibility testing protocol for Brachyspira hyodysenteriae and Brachyspira pilosicoli in an international ring trial.

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

Antimicrobial resistance trends in Escherichia coli isolated from diseased food-producing animals in France: A 14-year period time-series study.

Antimicrobial resistance (AMR) among bacteria isolated from food-producing animals is a growing concern with implications for public health. AMR surveillance is essential to identify resistance trends and help in the design of effective and efficient control strategies. The aim of the study was to describe the antimicrobial susceptibility of pathogenic Escherichia coli isolated from three livestock productions in France (cattle, swine and poultry). The trend in resistance to the most commonly prescribed antibiotics in animal health was analysed as follows: amoxicillin (penicillin), spectinomycin or streptomycin (aminoglycoside), tetracycline and trimethoprim-sulfamethoxazole/Enrofloxacin and ceftiofur were also taken into account as members of critically important antimicrobial families in human and veterinary medicine, that is fluoroquinolones and third-generation cephalosporins, respectively. Data collected between 2002 and 2015 by the French national surveillance network of AMR referred to as RESAPATH were analysed. Resistance trends were investigated using non-linear analysis (generalized additive models) applied to time-series stratified by livestock production and antibiotic. Irrespective of the species and the antibiotic considered, resistance signals over time showed no significant annual cycle. Resistance to third-generation cephalosporins emerged during the period of the study, with a peak at 22% [20.5; 24.0] in poultry in 2010, decreasing afterwards, while it remained consistently below 10% for the other species. The proportion of resistance to fluoroquinolones was broadly similar between species and remained under 30%, with a slight decreasing trend after 2009. Resistances to tetracycline and amoxicillin remained high, between 90% and 40% over time in cattle and swine. After 2010, there was a decrease in resistance to these antibiotics for all species, especially to tetracycline for poultry with a drop from 84% in 2009 to 43% in 2015. These results contribute to risk assessment and constitute objective evidence on which to evaluate the efficacy of control measures implemented to limit AMR occurrence.

Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-ß-Lactamase-Producing E. coli.

Four trials were conducted to evaluate the impact of Escherichia coli probiotic strain ED1a administration to pigs on the gut carriage or survival in manure of extended-spectrum-ß-lactamase-producing E. coli Groups of pigs were orally inoculated with strain E. coli M63 carrying the blaCTX-M-1 gene (n = 84) or used as a control (n = 26). In the first two trials, 24 of 40 E. coli M63-inoculated pigs were given E. coli ED1a orally for 6 days starting 8 days after oral inoculation. In the third trial, 10 E. coli M63-inoculated pigs were given either E. coli ED1a or probiotic E. coli Nissle 1917 for 5 days. In the fourth trial, E. coli ED1a was given to a sow and its 12 piglets, and these 12 piglets plus 12 piglets that had not received E. coli ED1a were then inoculated with E. coli M63. Fecal shedding of cefotaxime-resistant Enterobacteriaceae (CTX-RE) was studied by culture, and blaCTX-M-1 genes were quantified by PCR. The persistence of CTX-RE in manure samples from inoculated pigs or manure samples inoculated in vitro with E. coli M63 with or without probiotics was studied. The results showed that E. coli M63 and ED1a were good gut colonizers. The reduction in the level of fecal excretion of CTX-RE in E. coli ED1a-treated pigs compared to that in nontreated pigs was usually less than 1 log10 CFU and was mainly observed during the probiotic administration period. The results obtained with E. coli Nissle 1917 did not differ significantly from those obtained with E. coli ED1a. CTX-RE survival did not differ significantly in manure samples with or without probiotic treatment. In conclusion, under our experimental conditions, E. coli ED1a and E. coli Nissle 1917 could not durably prevent CTX-RE colonization of the pig gut.

Impact of two different colistin dosing strategies on healthy piglet fecal microbiota.

Colistin is often used in piglets but underdosing and overdosing are frequent. The impact of such administrations on fecal microbiota was studied. Piglets were given either underdoses of colistin by oral gavage for five days or overdoses by in-feed medication for 14days. The composition of fecal microbiota was studied by quantitative PCR, 16S rRNA sequencing, culture of Enterobacteriaceae, and quantification of short-chain fatty acids (SCFAs). The mean colistin concentrations during the treatment for underdosed and overdosed groups were 14.4µg/g and 64.9µg/g of feces respectively. Whatever the piglet and the sampling day, the two main phyla were Firmicutes and Bacteroidetes, The main families were Lactobacillaceae, Clostridiales, Lachnospiraceae and Ruminococcaceae. The main perturbation was the significant but transitory decrease in the Escherichia coli population during treatment, yet all the E. coli isolates were susceptible to colistin. Moreover, colistin did not affect the production of SCFAs. These results show that under- or overdoses of colistin do not result in any major disturbance of piglet fecal microbiota and rarely select for chromosomal resistance in the dominant E. coli population.

Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

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, bla CTX-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 bla CTX-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 bla CTX-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.

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation.

AIMS: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. METHODS AND RESULTS: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10(4) colony-forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. CONCLUSION: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10(4) colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

[Antibiotics susceptibility of Streptococcus and Enterococcus: data of Onerba network]. / Sensibilité aux antibiotiques chez les streptocoques (hors pneumocoque) et les entérocoques: données Onerba.

This work was aimed to analyze trends in susceptibility to antibiotics among the main species of beta-hemolytic streptococci involved in community-acquired infections in human (Streptococcus pyogenes and Streptococcus agalactiae), or in animals (Streptococcus suis and Streptococcus uberis) and also among the main enterocci species, Enterococcus faecalis and Enterococcus faecium. Data were recorded since 1996 through the Onerba networks. S. pyogenes, as the other beta-hemolytic streptococci studied remained fully susceptible to beta-lactam antibiotics. However, susceptibility to macrolides is clearly decreasing in S. pyogenes. In 2002, only 62 to 65% of the strains according to the network considered, were susceptible to erythromycin. A similar trend was observed for S. agalactiae with only 75% of erythromycin susceptibility in 2002, and for both species isolated from animals S. suis and S. uberis, with respectively 35 and 76% of strains susceptible to erythromycin. In enterococci, susceptibility to beta-lactams remained stable between 2000 and 2004. Indeed, the susceptibility to aminopenicillins remained high in E. faecalis (about 98%), whereas the proportion of E. faecium isolates susceptible to these antibiotics were lower than 60%. From 1999 to 2004, various studies conducted in French hospitals showed that the vancomycin resistance among enterococci accounted for less than 2%. However, the recent emergence of glycopeptide resistant enterococci clusters in French hospitals is a matter of concern and emphasizes the need for an ongoing surveillance. Such trend in macrolide resistance among S. pyogenes or S. agalactiae should consequently lead to propose other alternatives in case of beta-lactam allergy, and for pharyngitis, to rethink the place of the culture for susceptibility testing.

Quality-control ranges for antimicrobial susceptibility testing by broth dilution of the Brachyspira hyodysenteriae type strain (ATCC 27164T).

There are no approved standards for antimicrobial susceptibility testing of the fastidious spirochete Brachyspira hyodysenteriae. An interlaboratory study was performed to establish MIC quality control ranges for six antimicrobial agents for the type strain of B. hyodysenteriae using broth dilution. The results showed that B. hyodysenteriae B78T ATCC 27164T is a suitable quality control strain. This is a first step toward standardization of methods regarding this anaerobe.

Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens.

In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.

Antimicrobial susceptibility of Streptococcus suis isolated from swine in France and from humans in different countries between 1996 and 2000.

The susceptibility of 135 Streptococcus suis strains isolated from pigs (n = 110) and from humans (n = 25) to 13 antimicrobial agents was studied by microdilution and disc diffusion methods using Mueller-Hinton Agar II (MH) supplemented with either defibrinated sheep blood (MHSB) or horse serum (MHHS). Results were similar for both methods used except for penicillin G whose zone diameters were reduced with MHSB compared with MHHS. When MH was supplemented with sheep blood, 39% of S. suis strains classified as penicillin susceptible by MHHS microdilution showed intermediate susceptibility. Nearly all strains were susceptible to penicillin G (except by disc diffusion in MHSB), amoxicillin, ceftiofur, florfenicol, gentamicin and bacitracin. The least active antimicrobial agents were doxycycline and macrolides/lincosamides. High-level resistance (MIC > 500 mg/L or zone diameters < 10 mm) to streptomycin and kanamycin was detected in only a few strains. The virulence of strains did not seem to be related to antimicrobial resistance because no statistical difference was reported between the proportion of resistant strains of S. suis isolated from pigs with meningitis, septicaemia and arthritis, and those from tonsils and nasal cavities. However, significant differences were found in the proportions of macrolide- or doxycycline-resistant strains between S. suis serotype 2 and other serotypes. The results of antibiotic susceptibility testing presented in this study indicate that beta-lactams can be used in empirical treatment of human and pig S. suis infections in France.