Characterization of Extended Spectrum Cephalosporin-Resistant Escherichia coli Strains Isolated from Raw Poultry Carcasses in Catering Services in Northern Greece.

Antimicrobial resistance is considered a topic of utmost interest under the concept of "One Health", having severe implications in both human and veterinary medicine. Among the antibiotic-resistant bacteria, gram-negative bacteria, especially those belonging to the order of Enterobacterales (such as Escherichia coli), hold a prominent position in terms of both virulence and possessing/disseminating antimicrobial resistance (AMR) traits. The aim of this study was to examine the presence of extended-spectrum ß-lactamase producing E. coli isolates in raw poultry carcasses collected from a university club. Five hundred raw poultry skin samples were collected from the Aristotle University of Thessaloniki (AUTh) club in Thessaloniki, Greece. A total of 64% of the samples were positive for the presence of extended-spectrum ß-lactamase (ESBL)-producing E. coli. The isolates were further examined for their susceptibility to selected antibiotics by the disc diffusion method and were characterized as true ESBL, as producing class C cephalosporinases (AmpC) or "of unknown etiology" by the combination disc test. The 86 of the 120 isolates (71.67%) were classified as true ESBL, 24 (20.00%) as AmpC, and 10 (8.33%) as "of unknown etiology". The isolates were screened for the occurrence of ß-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaOXA). Thirty-six isolates (32 ESBL- and 4 AmpC-phenotype) harbored both blaTEM and blaCTX-M genes, twenty-two isolates (among which 19 ESBL-phenotype and 2 AmpC-phenotype) harbored blaCTX-M only, whereas twenty-six (14 ESBL- and 12 AmpC-phenotype) isolates harbored blaTEM alone. No isolate harboring blaSHV or blaOXA was detected. The results demonstrate the existence of E. coli isolates producing extended-spectrum ß-lactamases in poultry carcasses from Greece, pausing a risk for antibiotic resistance transfer to humans.

Detection of Cryptosporidium and Giardia in foods of plant origin in North-Western Greece.

Giardia and Cryptosporidium are recognized as leading causes of waterborne and foodborne diarrhoeal disease with worldwide distribution. The study aimed to determine the protozoan contamination of various foods of plant origin. A total of 72 samples from 27 different varieties of fresh vegetables and fruits were collected from supermarkets and open markets in North-Western Greece and were examined using conventional diagnostic methods. Two out of 72 (2.8%) samples were found positive for Cryptosporidium oocysts, while no sample was found to be positive for Giardia cysts. The results show the presence of protozoan contamination in foods of plant origin, which may constitute a potential health hazard.

In vitro antimicrobial activity of five essential oils on multidrug resistant Gram-negative clinical isolates.

AIM/BACKGROUND: The emergence of drug-resistant pathogens has drawn attention on medicinal plants for potential antimicrobial properties. The objective of the present study was the investigation of the antimicrobial activity of five plant essential oils on multidrug resistant Gram-negative bacteria. MATERIALS AND METHODS: Basil, chamomile blue, origanum, thyme, and tea tree oil were tested against clinical isolates of Acinetobacter baumannii (n = 6), Escherichia coli (n = 4), Klebsiella pneumoniae (n = 7), and Pseudomonas aeruginosa (n = 5) using the broth macrodilution method. RESULTS: The tested essential oils produced variable antibacterial effect, while Chamomile blue oil demonstrated no antibacterial activity. Origanum, Thyme, and Basil oils were ineffective on P. aeruginosa isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration values ranged from 0.12% to 1.50% (v/v) for tea tree oil, 0.25-4% (v/v) for origanum and thyme oil, 0.50% to >4% for basil oil and >4% for chamomile blue oil. Compared to literature data on reference strains, the reported MIC values were different by 2SD, denoting less successful antimicrobial activity against multidrug resistant isolates. CONCLUSIONS: The antimicrobial activities of the essential oils are influenced by the strain origin (wild, reference, drug sensitive, or resistant) and it should be taken into consideration whenever investigating the plants' potential for developing new antimicrobials.

Agriculture and food animals as a source of antimicrobial-resistant bacteria.

One of the major breakthroughs in the history of medicine is undoubtedly the discovery of antibiotics. Their use in animal husbandry and veterinary medicine has resulted in healthier and more productive farm animals, ensuring the welfare and health of both animals and humans. Unfortunately, from the first use of penicillin, the resistance countdown started to tick. Nowadays, the infections caused by antibiotic-resistant bacteria are increasing, and resistance to antibiotics is probably the major public health problem. Antibiotic use in farm animals has been criticized for contributing to the emergence of resistance. The use and misuse of antibiotics in farm animal settings as growth promoters or as nonspecific means of infection prevention and treatment has boosted antibiotic consumption and resistance among bacteria in the animal habitat. This reservoir of resistance can be transmitted directly or indirectly to humans through food consumption and direct or indirect contact. Resistant bacteria can cause serious health effects directly or via the transmission of the antibiotic resistance traits to pathogens, causing illnesses that are difficult to treat and that therefore have higher morbidity and mortality rates. In addition, the selection and proliferation of antibiotic-resistant strains can be disseminated to the environment via animal waste, enhancing the resistance reservoir that exists in the environmental microbiome. In this review, an effort is made to highlight the various factors that contribute to the emergence of antibiotic resistance in farm animals and to provide some insights into possible solutions to this major health issue.

Vancomycin-resistance phenotypes, vancomycin-resistance genes, and resistance to antibiotics of enterococci isolated from food of animal origin.

In the present study, 500 raw beef, pork, and chicken meat samples and 100 pooled egg samples were analyzed for the presence of vancomycin-resistant enterococci, vancomycin-resistance phenotypes, and resistance genes. Of 141 isolates of enterococci, 88 strains of Enterococcus faecium and 53 strains of E. faecalis were identified. The most prevalent species was E. faecium. Resistance to ampicillin (n = 93, 66%), ciprofloxacin (n = 74, 52.5%), erythromycin (n = 73, 51.8%), penicillin (n = 59, 41.8%) and tetracycline (n = 52, 36.9%) was observed, while 53.2% (n = 75) of the isolates were multiresistant and 15.6% (n = 22) were susceptible to all antibiotics. Resistance to vancomycin was exhibited in 34.1% (n = 30) of the E. faecium isolates (n = 88) and 1.9% (n = 1) of the E. faecalis isolates (n = 53) using the disc-diffusion test and the E-test. All isolates were tested for vanA and vanB using real-time polymerase chain reaction (PCR) and multiplex PCR, and for vanC, vanD, vanE, vanG genes using multiplex PCR only. Among E. faecalis isolates, no resistance genes were identified. Among the E. faecium isolates, 28 carried the vanA gene when tested by multiplex PCR and 29 when tested with real-time PCR. No isolate carrying the vanC, vanD, vanE, or vanG genes was identified. Melting-curve analysis of the positive real-time PCR E. faecium isolates showed that 22 isolates carried the vanA gene only, 2 isolates the vanB2,3 genes only, and seven isolates carried both the vanA and vanB2,3 genes. Enterococci should be considered a significant zoonotic pathogen and a possible reservoir of genes encoding resistance potentially transferred to other bacterial species.

Modeling microbial ethanol production by E. coli under aerobic/anaerobic conditions: applicability to real postmortem cases and to postmortem blood derived microbial cultures.

The mathematical modeling of the microbial ethanol production under strict anaerobic experimental conditions for some bacterial species has been proposed by our research group as the first approximation to the quantification of the microbial ethanol production in cases where other alcohols were produced simultaneously with ethanol. The present study aims to: (i) study the microbial ethanol production by Escherichia coli under controlled aerobic/anaerobic conditions; (ii) model the correlation between the microbial produced ethanol and the other higher alcohols; and (iii) test their applicability in: (a) real postmortem cases that had positive BACs (>0.10 g/L) and co-detection of higher alcohols and 1-butanol during the original ethanol analysis and (b) postmortem blood derived microbial cultures under aerobic/anaerobic controlled experimental conditions. The statistical evaluation of the results revealed that the formulated models were presumably correlated to 1-propanol and 1-butanol which were recognized as the most significant descriptors of the modeling process. The significance of 1-propanol and 1-butanol as descriptors was so powerful that they could be used as the only independent variables to create a simple and satisfactory model. The current models showed a potential for application to estimate microbial ethanol - within an acceptable standard error - in various tested cases where ethanol and other alcohols have been produced from different microbes.

Prevalence, antimicrobial resistance and relation to indicator and pathogenic microorganisms of Salmonella enterica isolated from surface waters within an agricultural landscape.

During a 12 month period (June 2007-May 2008), the prevalence and susceptibility of Salmonella serovars and their relation to specific pathogenic and indicator bacteria in river and coastal waters was investigated. A total of 240 water samples were collected from selected sites in Acheron and Kalamas Rivers and the Ionian Sea coast in north western Greece. The samples were analyzed for Salmonella spp., Listeria spp., Campylobacter spp., Escherichia coli O157, Staphylococci, Pseudomonas spp., Total Coliforms, Fecal Coliforms, Fecal Streptococci, Total Heterotrophic Flora at 20°C and at 37°C, fungi and protozoa (Cryptosporidium, Giardia). Susceptibility tests to nine antimicrobials (ampicillin, amikacin, amoxicillin/clavulavic acid, cefuroxime, ciprofloxacin, cefoxitin, tetracycline, ticarcillin/clavulanic acid, ampicillin/sulbactam) were performed using the disk diffusion method for Salmonella isolates. We isolated 28 serovars of Salmonella spp. identified as Salmonella enteritidis (23), Salmonella thompson (3) and Salmonella virchow (2). Multi-drug resistant Salmonella serovars were isolated from both river and marine waters, with 34.8% of S. enteritidis and 100% of S. virchow being resistant to more than 3 antibiotics. Also we isolated 42 strains of Listeria spp. identified as L. monocytogenes (20), L. innocua (9), L. seeligeri (2) and L. ivanovii (11). All the Listeria isolates were susceptible to the tested antibiotics. No Campylobacter spp., E. coli O157, Cryptosporidium and Giardia were detected. The overall ranges (and average counts) of the indicator bacteria were: Total Coliforms 0-4×10(4)cfu/100ml (3.7×10(3)cfu/100ml), Fecal Coliforms 0-9×10(3)cfu/100ml (9.2×10(2)cfu/100ml), Fecal Streptococci 0-3.5×10(4)cfu/100ml (1.4×10(3)cfu/100ml), Total Heterotrophic Flora at 20°C 0-6×10(3)cfu/ml (10(3)cfu/ml) and at 37°C 0-5×10(3)cfu/ml (4.9×10(2)cfu/ml). Weak or non significant positive Spearman correlations (p<0.05, rs range: 0.13-0.77) were obtained between Salmonella, Listeria, fungi and indicator bacteria. The results underline the complexity of the interrelations between pathogens and indicator bacteria, and the necessity to assess the presence of resistant bacteria in the aquatic environments.

Microbial ethanol production: experimental study and multivariate evaluation.

Ethanol can be produced from all the postmortem available substrates, though with higher rates and yields from carbohydrates, during the early stages of putrefaction. The so-called higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-2-butanol) and 1-butanol could be produced, from all the available postmortem substrates. However, a quantitative relationship between the produced ethanol and the potentially produced other alcohols is still missing. The objective of this study was the development of a simple, mathematical model which could be able to approximate the microbial produced ethanol in correlation with other produced alcohols. The selected bacterial species included two Gram+ spore-forming anaerobic bacteria and two (one Gram+ one Gram-) aerobic/facultative anaerobic bacteria, all being common commensals of the digestive tract and common colonizers of the corpse. The selected bacterial strains, Escherichia coli, Clostridium perfrigens, Clostridium sporogenes and Enterococcus faecalis, were cultured separately at 25 °C, for 30 days, under controlled anaerobic conditions. The produced ethanol and the previously referred alcohols were determined in the culture medium in 24h intervals. Using partial least squares (PLS) regression, the estimation of the relevance score for the available descriptors established the statistical model to assess the ethanol concentration produced by each studied microbe. E. coli, C. perfrigens, and C. sporogenes produced different patterns of ethanol and other alcohols, while E. faecalis produced negligible amounts of ethanol and higher alcohols. In constructing the mathematical models to predict the produced ethanol, 1-propanol, 1-butanol, and isobutanol were significant for C. perfrigens and C. sporogenes, while 1-butanol, 1-propanol, and methyl-butanol were significant for E. coli. The applicability of these models was tested in microbial, anaerobic cultures of normal human blood and plasma at 25 °C. The results indicate that factors such as the type of microbe species, the glucose content and the medium composition apparently affect the procedure of microbial ethanol, and other alcohols production. However, the models can be applied with acceptable accuracy and they show potential for application in real postmortem cases.

Antimicrobial resistance of major foodborne pathogens from major meat products.

The bacterial contamination of raw and processed meat products with resistant pathogens was studied. The raw samples included sheep (40), goat (40), pork (120), beef (80), and chicken (19) meat, and the processed samples included turkey filets (33), salami (8), readymade mincemeat (16), stuffing (22), and roast-beef (50). The samples were collected from retail shops in Northwestern Greece over a period of 3 years. The isolated pathogens were evaluated for susceptibilities to 19 antimicrobial agents used in humans. Out of 428 samples, 157 strains of Escherichia coli, 25 of Yersinia enterocolitica, 57 of Staphylococcus aureus, 57 of Enterococcus spp., 4 of Salmonella spp., and 3 of Campylobacter jejuni were isolated. Among the isolates 14.6% of the E. coli, 10.5% of S. aureus, 4% of Y. enterocolitica, 25% of Salmonella spp., and 42.1% of Enterococcus spp. were susceptible to antibiotics. E. coli from chicken exhibited high rates of resistance to ciprofloxacin (62.5%) followed by lamb/goat (10.9%), pork (15.7%), and beef (27.9%) meat. Resistance to nitrofurantoin dominated in the lamb/goat isolates (60%). Resistance to tetracycline predominated in pork (68.2%) and chicken (62.5%), and resistance to aminoglycosides dominated in lamb/goat meat isolates. S. aureus resistance to clindamycin predominated in lamb/goat isolates (50%), whereas resistance to ciprofloxacin predominated in the pork strains, but no resistance to methicillin was observed. Of the enterococci isolates 21.1% were resistant to vancomycin. High resistance to ampicillin (96%) was observed in Y. enterocolitica and all of the C. jejuni isolates were resistant to ampicillin, cephalothin, and cefuroxime. These results indicate that meat can be a source of resistant bacteria, which could potentially be spread to the community through the food chain.

Chemical investigation and antimicrobial properties of mastic water and its major constituents.

Mastic water is a commercial flavouring obtained during the steam distillation of mastic resin (the resin of Pistacia lentiscus var. chia) for the production of mastic oil. The mastic water extracts were analysed by GC-MS. The major compounds identified were verbenone, α-terpineol, linalool and trans-pinocarveol. Overall the composition was found to be very different from that of mastic oil. Additional GC-MS revealed the enantiomeric ratio of the chiral constituents of mastic water. The antimicrobial activity of mastic water extract, as well as that of its major constituents, was examined against Escherichia coli, Staphylococcus aureus and Candida spp. including ATCC wild clinical and food-borne strains. Linalool and α-terpineol were found to be the most potent antimicrobial constituents. Finally the stability of mastic water at different temperatures was studied, showing no change in the GC-MS profile of the organic extract for a period of 4months at storage temperatures up to 4°C.

Microbiological quality of indoor and outdoor swimming pools in Greece: investigation of the antibiotic resistance of the bacterial isolates.

During 1997-2005, the microbiological quality and susceptibility of bacterial isolates of swimming pool waters were investigated. A total of 462 water samples were collected from three indoor swimming pools (a teaching pool, a competition public pool, a hydrotherapy pool) and two outdoor swimming pools (a hotel semi-public and a residential private pool) in Northwestern Greece. All water samples were analyzed for the presence of bacteria, protozoa and fungi and susceptibility tests were performed for the bacterial isolates. Sixty-seven percent of the examined water samples conformed to the microbiological standards and 32.9% exceeded at least one of the indicated limits. Out of 107 bacterial isolates, 38 (35.5%) resistant strains were detected. Multi-resistant Pseudomonas alcaligenes, Leuconostoc, and Staphylococcus aureus (isolated from the teaching pool), Staphylococcus wernerii, Chryseobacterium indologenes and Ochrobactrum anthropi (isolated from the competition pool), Pseudomonas aeruginosa, P. fluorescens, Aeromonas hydrophila, Enterobacter cloacae, Klebsiella pneumoniae and S. aureus (isolated from the hydrotherapy pool) and A. hydrophila (isolated from the hotel pool) were detected. The swimming pool with the poorest microbiological quality (THC 500 cfu/ml in 12.1% of the samples, P. aeruginosa counts 1500 cfu/100 ml in 6% of the samples) and the highest prevalence of multi-resistant isolates (73.6%) was the hydrotherapy pool. No Cryptosporidium or Giardia cysts and no Legionella, Mycobacteria and Salmonella were detected, but there were isolations of Candida albicans, Aspergillus spp., Mucor spp., Alternaria spp., Rhizopus spp., Trichophyton spp., and Penicillium spp.