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This study was conducted to determine the prevalence and antimicrobial resistance of Listeria monocytogenes recovered from chicken carcasses in slaughterhouses in Northern Greece. A total of 100 poultry samples (300 carcasses) were examined for Listeria spp. The samples were neck skin taken from four different slaughterhouses in Northern Greece. Forty samples were also taken from the environment of the slaughterhouses. Identification of L. monocytogenes was carried out by PCR and fingerprinting of the isolates by random amplified polymorphic DNA. L. monocytogenes strains isolated from chicken carcasses and from the environment of the slaughterhouses were also examined for antibiotic resistance. Fifty-five isolates of L. monocytogenes were tested for susceptibility to 20 antibiotics using the disk diffusion method. Listeria spp. were present in 99 of the poultry samples tested (99%), and 38 yielded L monocytogenes (38%). L. monocytogenes was also isolated in 80% of samples from the environment of a certain slaughterhouse, while the other slaughterhouses were found to be contaminated only with Listeria spp. All isolates were resistant to nalidixic acid and oxolinic acid, the majority of them to clindamycin, and only a few to tetracycline and oxytetracycline, whereas they were found to be susceptible to all other antimicrobials. The results of this study demonstrate a high prevalence of L. monocytogenes contamination in chicken carcasses, and all isolates were found to be sensitive to the antimicrobials most commonly used to treat human listeriosis.

The presence, genetic diversity, and antimicrobial susceptibility profile of Campylobacter spp. in retail lamb and goat kid carcasses were assessed. A total of 200 samples consisting of 100 meat and 100 liver surface swabs were collected from 47 lamb and 53 goat kid carcasses at 23 retail markets in Northern Greece, and 125 Campylobacter isolates were recovered from 32 meat surfaces (32%) and 44 liver surfaces (44%). Multiplex polymerase chain reaction and restriction fragment length polymorphism analysis specified Campylobacter coli as the most frequently detected species (59.2%) followed by C. jejuni (40.8%). Pulsed-field gel electrophoresis (PFGE) was applied in order to typify a subset of randomly selected isolates (n=80). SmaI-PFGE successfully clustered the 80 isolates in 38 SmaI-PFGE types, indicating high heterogeneity among the analyzed Campylobacter isolates, and provided data regarding the dissemination of Camplobacter among carcasses stored in the same retail market. Antimicrobial susceptibility profiles of Campylobacter isolates, assessed by the disk-diffusion method, indicated that 31 isolates (24.8%) were multidrug resistant, and the most common profile was the concurrent resistance to tetracycline and streptomycin. Overall, 56.8% of isolates (n=71, multidrug-resistant isolates included) exhibited resistance to at least one antimicrobial (tetracycline 34.4%, quinolones 27.2%, and streptomycin 20.8%). However, all isolates were susceptible to erythromycin and gentamicin. The findings of this study verify the contamination of retail lamb and goat kid carcasses with a heterogeneous population of thermotolerant campylobacters. These data underscore the fact that retail meat and liver of small ruminants could serve as vehicles for consumer contamination with Campylobacter and that further investigation is necessary in order to evaluate the risk imposed by such products within the epidemiology of human campylobacteriosis cases.

This study was conducted to determine the prevalence and antimicrobial resistance of Salmonella isolates recovered from chicken carcasses in slaughterhouses in Northern Greece. A total of 150 broiler samples were examined for Salmonella spp. and the results were reported as presence or absence of Salmonella spp. The samples were neck‐skin taken from 450 poultry carcasses at four slaughterhouses in Northern Greece. Salmonella was present in 56 (37%) of the samples tested. A total of 142 isolates belonging to six serovars were detected. The most common serotype identified was Salmonella blockley (73.2%), followed by Salmonella paratyphi B (16.9%), Salmonella bredeney (6.3%), Salmonella neftenbach (1.4%), Salmonella hadar (1.4%) and Salmonella thompson (0.7%). Salmonella spp. isolated from chicken carcasses was also examined for antibiotic resistance. Fifty‐six isolates of Salmonella (one strain from each positive sample) were tested for susceptibility to 20 antibiotics using the disk diffusion method. All isolates were resistant to four antimicrobials (penicillin, erythromycin, vancomycin and clindamycin) and the mass majority of them (more than 90%) to another three antimicrobials (tetracycline, oxytetracycline and streptomycin). The results of this study demonstrate a high prevalence of Salmonella spp. contamination in chicken carcasses and a large percentage of antimicrobial resistant strains. PRACTICAL APPLICATIONS This study complements the limited and out‐of‐date research performed in Greece about the prevalence and the antimicrobial resistance of Salmonella isolated from chicken carcasses. There is a high prevalence of Salmonella contamination in chicken carcasses in Northern Greece. Poultry meat must be considered as a possible cause of Salmonella poisoning and therefore it should be handled with care during its preparation, especially from the consumers. The Salmonella strains isolated from chicken carcasses are resistant to a significant percentage of the most commonly used antibiotics which confirm that poultry is a major reservoir of multiresistant Salmonella.

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Human breast milk samples (n=87) collected between July 2004 and July 2005 from primipara and multipara mothers from Thessaloniki, Greece were analysed for six groups of persistent organic pollutants (POPs): polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), chlordane compounds (CHLs), hexachlorocyclohexane isomers (HCHs) and hexachlorobenzene (HCB). DDTs [median: 410ng/g lipid weight (lw)], PCBs (median: 90ng/g lw) and HCHs (median: 40ng/g lw) were the predominantly identified compounds in all the breast milk samples. Levels of PBDEs (median: 1.5ng/g lw) in human breast milk samples from Thessaloniki, Greece were lower compared to other countries. Maternal age had a positive correlation with most compounds, but not with PBDEs. Women with a higher occupational exposure to PBDEs (i.e., working in office environments) had higher PBDE concentrations than all others and showed strong correlations, especially for BDE 47 and BDE 153. None of the analysed compounds showed any correlation with parity. Based on these levels, the daily intake of each group of POPs via human milk was calculated and compared with the tolerable daily intakes (TDI) or the reference doses (RfD). For the majority of samples (85 out of 87) a higher daily intake of PCBs than the TDI was calculated, while 11 out of 87 samples had a higher HCB intake than the TDI. The TDI and the RfD were not exceeded for DDTs and PBDEs, respectively. This is the first report of brominated flame retardants in human breast milk from Greece.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml−1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria. IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.