RESUMO
The prevalence of Listeria monocytogenes in 30 samples of poultry was determined using culture-dependent (isolation on OCLA and confirmation by conventional polymerase chain reaction -PCR-, OCLA&PCR) and culture-independent (real-time polymerase chain reaction, q-PCR) methods. L. monocytogenes was detected in 15 samples (50.0%) by OCLA&PCR and in 20 (66.7%) by q-PCR. The concentrations (log10 cfu/g) of L. monocytogenes (q-PCR) ranged from 2.40 to 5.22 (total cells) and from <2.15 to 3.93 (viable cells). The two methods, q-PCR using a viability marker (v-PCR) and OCLA&PCR (gold standard), were compared for their capacity to detect viable cells of L. monocytogenes, with the potential to cause human disease. The values for sensitivity, specificity and efficiency of the v-PCR were 100%, 66.7% and 83.3%, respectively. The agreement between the two methods (kappa coefficient) was 0.67. The presence of nine virulence genes (hlyA, actA, inlB, inlA, inlC, inlJ, prfA, plcA and iap) was studied in 45 L. monocytogenes isolates (three from each positive sample) using PCR. All the strains harbored between six and nine virulence genes. Fifteen isolates (33.3% of the total) did not show the potential to form biofilm on a polystyrene surface, as determined by a crystal violet assay. The remaining strains were classified as weak (23 isolates, 51.1% of the total), moderate (one isolate, 2.2%) or strong (six isolates, 13.3%) biofilm producers. The strains were tested for susceptibility to a panel of 15 antibiotics. An average of 5.11 ± 1.30 resistances per isolate was observed. When the values for resistance and for reduced susceptibility were taken jointly, this figure rose to 6.91 ± 1.59. There was a prevalence of resistance or reduced susceptibility of more than 50.0% for oxacillin, cefoxitin, cefotaxime, cefepime ciprofloxacin, enrofloxacin and nitrofurantoin. For the remaining antibiotics tested, the corresponding values ranged from 0.0% for chloramphenicol to 48.9% for rifampicin. The high prevalence and level of L. monocytogenes with numerous virulence factors in poultry underline how crucial it is to follow correct hygiene procedures during the processing of this foodstuff in order to reduce the risk of human listeriosis.
RESUMO
Twenty samples of minced chicken meat procured from butcher's shops in León (Spain; 10 samples) and Vila Real (Portugal; 10 samples) were analyzed. Microbial concentrations (log10 cfu/g) of 7.53 ± 1.02 (viable aerobic microbiota), 7.13 ± 1.07 (psychrotrophic microorganisms), and 4.23 ± 0.88 (enterobacteria) were found. The detection method described in the UNE-EN ISO 11290-1 standard (based on isolation from the chromogenic medium OCLA) with confirmation by the polymerase chain reaction (PCR; lmo1030) (OCLA−PCR), revealed Listeria monocytogenes in 14 samples (70.0% of the total), nine of Spanish origin and five of Portuguese (p > 0.05). The levels of viable and inactivated L. monocytogenes in the samples were determined with a q-PCR using propidium monoazide (PMAxx) as a viability marker. Seven samples tested positive both with the OCLA−PCR and with the q-PCR, with estimated concentrations of viable cells varying between 2.15 log10 cfu/g (detection limit) and 2.94 log10 cfu/g. Three samples tested negative both with the OCLA−PCR and with the q-PCR. Seven samples were positive with the OCLA−PCR, but negative with the q-PCR, and three samples tested negative with the OCLA−PCR and positive with the q-PCR. The percentage of viable cells relative to the total ranged between 2.4% and 86.0%. Seventy isolates of L. monocytogenes (five from each positive sample) were classified in PCR serogroups with a multiplex PCR assay. L. monocytogenes isolates belonged to serogroups IIa (52 isolates; 74.3%), IIc (7; 10.0%), IVa (2; 2.9%), and IVb (9; 12.9%). The susceptibility of the 70 isolates to 15 antibiotics of clinical interest was tested. The strains presented resistance to between three and eight antibiotics. The average number of resistances was greater (p < 0.001) among strains isolated from Spanish samples (6.20 ± 1.08), than in those from Portugal (5.00 ± 1.08). In both groups of strains, a prevalence of resistance higher than 95% was observed for oxacillin, cefoxitin, cefotaxime, and cefepime. The need to handle minced chicken meat correctly, taking care to cook it sufficiently and to avoid cross-contamination, so as to reduce the danger of listeriosis, is emphasized. A combination of culture-dependent and culture-independent methods offers complementary routes for the detection in food of the cells of L. monocytogenes in various different physiological states.
