Staphylococcus aureus in the Processing Environment of Cured Meat Products.

The presence of Staphylococcus aureus in six dry-cured meat-processing facilities was investigated. S. aureus was detected in 3.8% of surfaces from five facilities. The occurrence was clearly higher during processing (4.8%) than after cleaning and disinfection (1.4%). Thirty-eight isolates were typified by PFGE and MLST. Eleven sequence types (STs) were defined by MLST. ST30 (32%) and ST12 (24%) were the most abundant. Enterotoxin genes were detected in 53% of isolates. The enterotoxin A gene (sea) was present in all ST30 isolates, seb in one ST1 isolate, and sec in two ST45 isolates. Sixteen isolates harbored the enterotoxin gene cluster (egc) with four variations in the sequence. The toxic shock syndrome toxin gene (tst) was detected in 82% of isolates. Regarding antimicrobial resistance, twelve strains were susceptible to all the antibiotics tested (31.6%). However, 15.8% were resistant to three or more antimicrobials and, therefore, multidrug-resistant. Our results showed that in general, efficient cleaning and disinfection procedures were applied. Nonetheless, the presence of S. aureus with virulence determinants and resistance to antimicrobials, particularly multidrug-resistant MRSA ST398 strains, might represent a potential health hazard for consumers.

Genomic diversity and characterization of Listeria monocytogenes from dry-cured ham processing plants.

Genomic diversity of Listeria monocytogenes isolates from the deboning and slicing areas of three dry-cured ham processing plants was analysed. L. monocytogenes was detected in 58 out of 491 samples from the environment and equipment surfaces, all from the deboning area, with differences in prevalence among facilities. The most frequent PCR-serogroup was IIa (74.1%) followed by IIb and IIc, and only one isolate was serogroup IVb. Twenty different pulsotypes and 11 sequence types (STs) grouped into 10 clonal complexes (CCs) were determined. ST121 (CC121) and ST9 (CC9) were the most abundant. Premature stop codons (PMSC6 and PMSC19) associated with attenuated virulence were found in the inlA sequence in 7 out of 12 selected strains. CC121 strains were strong biofilm formers and some harboured the transposon Tn6188, related with increased tolerance to quaternary ammonium compounds. L. monocytogenes clones considered hypovirulent resulted predominant in the deboning areas. The clonal structure and potential virulence of the isolates could help to establish adequate control measures and cleaning protocols for the comprehensive elimination of the pathogen in dry-cured ham processing environment.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, ß-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, ß-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed ß-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

[Application of restriction fragment length polymorphism-polymerase chain reaction-flaA and resistotype to identify potential undiagnosed outbreaks of campylobacteriosis in Spain]. / Aplicación del análisis de los polimorfismos de los tamaños de los fragmentos de restricción del gen flaA amplificado por reacción en cadena de la polimerasa y resistotipo para identificar potenciales brotes de campilobacteriosis no diagnosticados en España.

INTRODUCTION: Outbreaks of campylobacteriosis are infrequent and usually involve a low number of patients, although it is estimated that many more remain undiagnosed. The most successful techniques for outbreak investigation in Campylobacter spp. (PFGE, MLST) have the drawback of being laborious and not available in many laboratories. METHODS: During the year 2008, 352 isolates of C. jejuni and C. coli from 16 hospitals were received in our laboratory. All strains were genotyped by RFLP-PCR-flaA (flaA type) and phenotyped with their resistotype. It was established that the strains of the same species from the same hospital, isolated over a period of up to 11 days, with MIC values of±1 dilution with the same flaA type could belong to an outbreak. Strains that met these criteria would be later subtyped by KpnI-PFGE and MLST. RESULTS: A total of 23 out of 352 isolates, distributed in 10 groups, met the criteria for being associated with putative undiagnosed outbreaks. The similarity of the PFGE-profiles in 8 groups was greater than 95% among the isolates from each group. In 7 of the groups, the sequence types (MLST) were coincident. CONCLUSIONS: The use of 2 easy markers (resistotype and RFLP-PCR-flaA) may detect isolates probably belonging to an undiagnosed outbreak of campylobacteriosis. Accurate diagnosis requires other molecular markers and epidemiological data of each isolate. The study suggests that, as in other countries, the number of outbreaks of campylobacteriosis in Spain is probably underestimated.

[Accuracy of Etest method to study Campylobacter spp. susceptibility to erythromycin, ciprofloxacin and tetracycline]. / Precisión del método E-test para el estudio de la sensibilidad de Campylobacter spp. a eritromicina, ciprofloxacino y tetraciclina.

INTRODUCTION: In industrialized countries Campylobacter jejuni is the enteropathogen most frequently isolated from the feces of patients with gastroenteritis. The Etest accuracy to categorize Campylobacter spp. susceptibility to erythromycin, ciprofloxacin and tetracycline was evaluated. METHODS: Ninety strains were studied. The Etest® was performed following the manufacturer's instructions on commercial plates of Mueller-Hinton blood. The breakpoints were those recommended by the Clinical Laboratory Standards Institute (CLSI) for broth microdilution. The gold standard was the broth microdilution method as recommended by CLSI. RESULTS: The Etest agreement with the reference method was 100%, 97% and 98% for erythromycin, ciprofloxacin and tetracycline, respectively. No major or very major errors were found. CONCLUSIONS: The Etest results are equivalent to those obtained using the gold standard. The Etest is a valid method to determine susceptibility to tetracycline. It is also a suitable method to categorize strains classified as non-resistant to erythromycin and ciprofloxacin by the diffusion method.

Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli strains isolated from two early stages of poultry production.

Our aim was to monitor the resistance of Campylobacter isolates from two initial stages of broiler production in 5 grandparent breeder broiler farms (GPBFs) and 12 parent breeder broiler farms (PBFs) in which no antimicrobials were used during the study. Susceptibility tests were carried out for 805 strains (697 Campylobacter jejuni and 108 Campylobacter coli) against nalidixic acid, ciprofloxacin, erythromycin, amoxicillin, amoxicillin plus clavulanic acid, tetracycline, gentamicin, and chloramphenicol using the disk-diffusion method. Quinolone resistance was the most abundant overall (74.9%) and at each stage of production. The second largest resistance was for tetracycline with 48.2%. The resistance against amoxicillin plus clavulanic acid, gentamicin, and chloramphenicol was not found. The percentages of resistance and multidrug-resistant (MDR) isolates were always higher in the PBFs than in the GPBFs. However, pan-susceptible populations (total 10.3%) were isolated in our survey. C. coli isolates were more resistant to tetracycline and erythromycin (96.3% and 23.1%, respectively) than for C. jejuni (40.7% and 0%, respectively) and were more MDR (33.3% vs. 11.9%). In conclusion, as other authors have shown, even in the absence of antibiotic pressure, relatively high rates of quinolone resistance are found in Campylobacter. However, a decrease in quinolone resistance has been observed compared to other studies in Spain [i.e., 99%; Saenz et al. Antimicrob. Agents Chemother. 2000;44(2):267-271]. MDR, fluoroquinolone-, macrolide-, and tetracycline-resistant Campylobacter populations are issues of concern in public health.

Drinking water as the source of Campylobacter coli infection in grandparent heavy breeders.

The aim of the present study was the molecular identification of a common source of infection of Campylobacter coli in two grandparent breeder farms. Campylobacter jejuni and C. coli were isolated from well water and cloacal swabs from grandparent chickens. Colonies were genotyped using restriction fragment length polymorphism-flaA gene, pulsed field gel electrophoresis and multi-locus sequence typing. The same genotype of C. coli was found in both farms and in the well from which drinking water was supplied to the farms. The well water was epidemiologically linked as the source of C. coli infection. The molecular identification for epidemiological source-tracking of C. coli in breeder farms could aid in combating the colonization of this pathogen and therefore to reduce their incidence in human campylobacteriosis.