Isabelle Kempf

Extended-Spectrum β-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France

ABSTRACT Genes encoding extended-spectrum β-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a blaCTX-M-1 gene located on transferable plasmids of different sizes and structures.

Copper Resistance in Enterococcus faecium, Mediated by the tcrB Gene, Is Selected by Supplementation of Pig Feed with Copper Sulfate

ABSTRACT The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.

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%.

In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae

The in vitro emergence of resistance to enrofloxacin, erythromycin, tylosin, tiamulin, and oxytetracycline in three avian Mycoplasma species, Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae was studied. Mutants were selected stepwise and their MICs were determined after 10 passages in subinhibitory concentrations of antibiotic. High-level resistance to erythromycin and tylosin developed within 2-6 passages in the three Mycoplasma species. Resistance to enrofloxacin developed more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in M. gallisepticum or M. synoviae after 10 passages whereas, resistant mutants were obtained with M. iowae. Cross-sensitivity tests performed on mutants demonstrated that mycoplasmas made resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin. Some M. iowae tiamulin-resistant mutants were also resistant to both macrolide antibiotics. Enrofloxacin and oxytetracycline did not induce any cross-resistance to the other antibiotics tested. These results show that Mycoplasma resistance to macrolides can be quickly selected in vitro, and thus, providing that similar results could be obtained under field conditions, that development of resistance to these antibiotics in vivo might also be a relatively frequent event.

Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae.

Pulsed-field gel electrophoresis (PFGE) analysis was developed and compared with random amplified polymorphic DNA (RAPD) method to type 18 Mycoplasma synoviae (MS) strains. All analysed strains were typeable by RAPD but only 89% of MS strains were typeable by PFGE because of DNA degradation. The discriminatory power of RAPD was greater than that of PFGE but the two techniques had a discriminatory index superior to 0.95, the threshold value for interpreting typing results with confidence. The in vitro, in ovo and in vivo reproducibility of both typing techniques was 100%. However, the interpretation of RAPD patterns was complicated because of inconsistent band intensity. Thus, these molecular typing techniques should be helpful for epidemiological studies of avian mycoplasma infections.

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.

Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction

Successful detection of Mycoplasma synoviae (MS) by culture and PCR from samples collected in the environment of experimentally infected chickens and turkeys, or under field conditions, is described. Results showed that in the experimental infection, 10/96 and 46/96 samples of food, drinking water, feathers, droppings or dust were positive by culture and Mycoplasma-PCR. In field conditions, the number of positive results for environmental samples were respectively 7/28 and 17/28. These observations highlight the high disseminating capacities of this mycoplasma and show the usefulness of the PCR method for epidemiological studies.

A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples

In order to study horizontal transmission of Mycoplasma synoviae an avian pathogen, a reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect viable Mycoplasma in environment. The test was based on the RT-PCR of the 16S ribosomal RNA (rRNA) of Mycoplasma genus. Results showed that Mycoplasma 16S rRNA was stable up to 23 h after cell death. Therefore, the test allowed detection of viable or very recently (less than 23 h) dead mycoplasmas. M. synoviae survival in artificially contaminated water, food and soil and in the environment of M. synoviae experimentally infected turkeys was estimated by culture and RT-PCR. The RT-PCR method was then applied in a naturally infected laying hen farm showing problems of recurrent mycoplasmosis in the hens. Results confirmed the usefulness of RT-PCR in checking the efficiency of biosecurity measures and in improving cleaning and disinfection protocols.

Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of Mycoplasma gallisepticum Mutants Obtained In Vitro

ABSTRACT Mycoplasma gallisepticum enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.

Polymerase chain reaction for detection of Mycoplasma gallisepticum in environmental samples.

The polymerase chain reaction (PCR) was used to detect Mycoplasma gallisepticum in samples collected from the environment of experimentally or naturally infected poultry. Culture was also used in the experimental infections. Of 160 samples of food, drinking water, feathers, droppings or dust collected during experimental infection, 103 were positive using a M. gallisepticum -specific PCR (MG-PCR) and 68 were positive using a PCR (mycoplasma-PCR) that detects all species of the genera Mycoplasma , Spiroplasma, Acholeplasma and Ureaplasma . Six of these samples were also positive by culture. In environmental samples collected on a depopulated M. gallisepticum -positive turkey farm, three and two out of a total of 12 were positive by mycoplasma-PCR and MG-PCR, respectively. These results indicate the disseminating capacity of this mycoplasma and the possible use of PCR methods for epidemiological analyses and control of farm decontamination before the introduction of new birds.