Investigation of Potential Gut Health Biomarkers in Broiler Chicks Challenged by Campylobacter jejuni and Submitted to a Continuous Water Disinfection Program.

The exploration of novel biomarkers to assess poultry health is of paramount importance, not only to enhance our understanding of the pathogenicity of zoonotic agents but also to evaluate the efficacy of novel treatments as alternatives to antibiotics. The present study aimed to investigate potential gut health biomarkers in broiler chicks challenged by Campylobacter jejuni and subjected to a continuous water disinfection program. A total of 144 one-day-old hatched broiler chicks were randomly allocated to four treatment groups with four replicates each, according to the following experimental design: Group A received untreated drinking water; Group B received drinking water treated with 0.01-0.05% v/v Cid 2000™ (hydrogen peroxide, acetic acid and paracetic acid); Group C was challenged by C. jejuni and received untreated drinking water; and Group D was challenged by C. jejuni and received drinking water treated with 0.01-0.05% v/v Cid 2000™. The use of Cid 2000™ started on day 1 and was applied in intervals until the end of the experiment at 36 days, while the C. jejuni challenge was applied on day 18. Potential biomarkers were investigated in serum, feces, intestinal tissue, intestinal content, and liver samples of broilers. Statistical analysis revealed significant increases (p < 0.001) in serum cortisol levels in C. jejuni-challenged broilers. Serum fluorescein isothiocyanate dextran (FITC-d) increased significantly (p = 0.004) in broilers challenged by C. jejuni and treated with drinking water disinfectant, while fecal ovotransferrin concentration also increased significantly (p < 0.001) in broilers that received the drinking water disinfectant alone. The gene expression levels of occludin (p = 0.003) and mucin-2 (p < 0.001) were significantly upregulated in broilers challenged by C. jejuni, while mucin-2 significantly increased in birds that were challenged and received the drinking water disinfectant (p < 0.001). TLR-4 expression levels were significantly (p = 0.013) decreased in both groups that received the drinking water disinfectant, compared to the negative control group. Finally, the C. jejuni challenge significantly increased (p = 0.032) the crypt depth and decreased (p = 0.021) the villus height-to-crypt-depth ratio in the ileum of birds, while the tested disinfectant product increased (p = 0.033) the villus height in the jejunum of birds. Furthermore, the counts of C. jejuni in the ceca of birds (p = 0.01), as well as its translocation rate to the liver of broilers (p = 0.001), were significantly reduced by the addition of the water disinfectant. This research contributes to novel insights into the intricate interplay of water disinfection and/or C. jejuni challenge with potential intestinal biomarkers. In addition, it emphasizes the need for continued research to unveil the underlying mechanisms, expands our understanding of broiler responses to these challenges and identifies breakpoints for further investigations.

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.

Investigation of the Effect of Three Commercial Water Acidifiers on the Performance, Gut Health, and Campylobacter jejuni Colonization in Experimentally Challenged Broiler Chicks.

This study investigated the effect of three commercial water acidifiers on the performance, gut health, and C. jejuni colonization in experimentally challenged broiler chicks. A total of 192 one-day-old broiler chicks (Ross 308®) were randomly allocated into 6 treatment groups with 4 replicates according to the following experimental design: group A, birds were not challenged and received tap water; group B, birds were challenged and received tap water; groups C, D, E, and F, birds were challenged and received tap water treated with 0.1% v/v SPECTRON®, with 0.1-0.2% v/v ProPhorce™ SA Exclusive, with 0.1-0.2% v/v Premium acid, and with 0.1-0.2% v/v Salgard® Liquid, respectively. The continuous water acidification evoked undesirable effects on broilers' performance and to an increased number of birds with ulcers and erosions in the oral cavity and the upper esophageal area. ProPhorce™ SA Exclusive and Premium acid significantly reduced the C. jejuni counts in the crop, whereas Salgard® Liquid significantly reduced the C. jejuni counts in the ceca of birds. At slaughter age, only Premium acid significantly reduced C. jejuni counts in the ceca of birds. All the tested products ameliorated the changes induced by C. jejuni infection in the pH in the ceca of birds. It can be concluded that besides the effectiveness of the tested products in controlling C. jejuni in broilers, their continuous application evoked undesirable effects on broilers' performance, leading to the need to modify the dosage scheme in future investigations.

In Vitro Investigation of the Antibacterial Activity of Nine Commercial Water Disinfectants, Acidifiers, and Glyceride Blends against the Most Important Poultry Zoonotic Bacteria.

Identifying and monitoring the efficiency of alternative biocides that are presently used in livestock is gaining vast attention. The objective of this study was to determine, in vitro, the antibacterial activity of nine commercial water disinfectants, acidifiers, and glyceride blends against clinical isolates or reference strains of zoonotic pathogens belonging to the genera Escherichia spp., Salmonella spp., Campylobacter spp., Listeria spp., and Staphylococcus spp. For each product, the antibacterial activity was tested in concentrations ranging from 0.002 to 1.136% v/v and expressed as the minimum concentration of the product that inhibits bacterial growth (MIC). Water disinfectants Cid 2000™ and Aqua-clean® recorded MICs ranging from 0.002 to 0.142% v/v, while the lowest MICs were recorded at two strains of Campylobacter (0.002-0.004% v/v). Virkon® S displayed various MICs (0.013-0.409% w/v) and was highly effective at suppressing the growth of Gram-positive bacteria such as S. aureus (0.013-0.026% w/v). The MICs of water acidifiers (Agrocid Super™Oligo, Premium acid, and Ultimate acid) and glyceride blends (CFC Floramix, FRA®LAC34, and FRA®Gut Balance) ranged from 0.036 to 1.136% v/v, and for most of these products, MICs were closely correlated by their ability to modify the pH of the culture medium close to 5. In conclusion, most of the tested products showed promising antibacterial activity; as a result, they would be good candidates for pathogen control in poultry farms and for reducing the emergence of antimicrobial resistance. However, further in vivo studies are recommended to provide relevant information for the underlying mechanisms, as well as for the establishment of the optimal dosage scheme for each product and their possible synergies.

Effect of Chitosan and Alginate-Based Edible Membranes with Oregano Essential Oil and Olive Oil in the Microbiological, Physicochemical and Organoleptic Characteristics of Mutton.

Edible chitosan or alginate coatings and their combinations with oregano essential oil or olive oil, have been examined for their effect on the microbiological, physicochemical and organoleptic characteristics of mutton. The results indicated that these edible coatings can contribute to maintaining good quality characteristics and extending mutton shelf-life. The total mesophilic counts in mutton ranged from 3.48 to 8.00 log10 CFU/g, the total psychrophilic counts from 4.00 to 9.50 log10 CFU/g, the B. thermosphacta counts from 2.30 to 7.77 log10 CFU/g and the lactic acid bacteria counts from 2.00 to 5.85 log10 CFU/g. Chitosan coatings significantly (p < 0.05) reduced the total mesophilic, the total psychrophilic (1-2 log10 cfu/g), the B. thermosphacta and the lactic acid bacteria counts in mutton. Alginate exhibited a lower L* value and a higher a* value and chroma compared with the control and chitosan lots. No significant differences were observed in the chemical composition of meat pieces among the experimental groups. Oregano oil positively affected the sensory attributes of meat. The most favourable combination, based on the microbiological counts, the organoleptic characteristics and the shelf-life extension of mutton, was that of chitosan with oregano essential oil.

A Comprehensive Study of the Quality of Fat-Tailed Sheep Carcasses in Greece.

Sheep farming in Greece is focused on milk production. Meat is considered a by-product and consists mainly light carcasses of undefined quality. The main challenge of the sector is to ensure sustainability, and hence efforts are towards efficient use of available resources, including undervalued carcasses of local fat-tailed sheep. The objective here was twofold: (i) to assess the carcass quality of fat-tailed sheep slaughtered at different live weights and (ii) to compare them with carcasses from thin-tailed sheep. In total, 146 fat-tailed and 97 thin-tailed dairy sheep were used. They belonged to five live-weight categories (LWC), representing 25%, 35%, 50%, 70% and 100% of mature body weight. Carcass length/weight/yield/pH and wither height were recorded. Muscle fiber minimum Feret's diameter and meat color/tenderness/moisture/lipid and protein content were determined. Sex and LWC differences in fat-tailed sheep were assessed. Parametric and non-parametric tests were used to compare with thin-tailed sheep, considering the effects of LWC, sex and their interactions with sheep population (fat-tailed/thin-tailed). Most traits were significantly different (p < 0.05) between groups of fat-tailed sheep. Carcass yield of fat-tailed sheep was significantly higher compared to thin-tailed (p < 0.01). Interactions of sheep population with LWC or sex affected wither height, carcass pH, meat color and tenderness (p < 0.05). Fat-tailed sheep meat quality is equal or higher compared to thin-tailed. Finishing weights corresponding to 50 and 70% LWC may improve capitalization of fat-tailed carcasses.

A Comparative Study on Microbiological and Chemical Characteristics of Small Ruminant Carcasses from Abattoirs in Greece.

Meat quality dictates consumer preferences with hygiene forming a key component, especially in meat types with declining popularity, such as sheep and goat meat. Aiming to increase the marketability of sheep and goat meat, we examined 370 sheep and goat carcasses from two abattoirs in Greece. Tests included enumeration of the total mesophilic viable count, total psychrophilic viable count and coliform count, and detection of Salmonella spp., Listeria monocytogenes and presumptive ESBL Escherichia coli. Moreover, designated samples of meat were used to measure pH, moisture, total fat and protein content. Goat carcasses had significantly higher microbial counts compared to sheep carcasses. Lamb and kid carcasses had larger TMVC, TPVC and coliform counts compared to carcasses from adult animals. One strain of L. monocytogenes (0.8%), typed as serovar 1/2a (3a), was isolated from one adult sheep carcass. Twelve strains of ESBL Escherichia coli (25%) were isolated; there were not any strains of Salmonella spp. The average values of pH, moisture, total fat and total protein were 5.83%, 67.76%, 7.21% and 21.31%, respectively, for sheep carcasses and 5.70%, 68.2%, 5.69% and 24.10%, respectively, for goat carcasses. The results showed a small deviation in assessed parameters, implying the uniformity of the conditions concerning rearing and slaughtering.

Electrospun Nanofiber Films Suppress Inflammation In Vitro and Eradicate Endodontic Bacterial Infection in an E. faecalis-Infected Ex Vivo Human Tooth Culture Model.

Treatment failure of endodontic infections and their concurrent inflammations is commonly associated with microbial persistence and reinfection, also stemming from the anatomical restrictions of the root canal system. Aiming to address the shortcomings of current treatment options, a fast-disintegrating nanofibrous film was developed for the intracanal coadministration of an antimicrobial (ZnO nanoparticles) and an anti-inflammatory (ketoprofen) agent. The electrospun films were fabricated based on polymers that dissolve rapidly to constitute the actives readily available at the site of action, aiming to eliminate both microbial infection and inflammation. The anti-inflammatory potency of the nanofiber films was assessed in an in vitro model of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells after confirming their biocompatibility in the same cell line. The nanofiber films were found effective against Enterococcus faecalis, one of the most prominent pathogens inside the root canal space, both in vitro and ex vivo using a human tooth model experimentally infected with E. faecalis. The physical properties and antibacterial and anti-inflammatory potency of the proposed electrospun nanofiber films constitute a promising therapeutic module in the endodontic therapy of nonvital infected teeth. All manuscripts must be accompanied by an abstract. The abstract should briefly state the problem or purpose of the research, indicate the theoretical or experimental plan used, summarize the principal findings, and point out major conclusions.

Phenotypic and Molecular Patterns of Resistance among Campylobacter coli and Campylobacter jejuni Isolates, from Pig Farms.

The purpose of this research was to characterize the antibiotic resistance patterns of Campylobacter spp. isolated from commercial farrow to finish farms in Greece, and analyze the relevant molecular resistance mechanisms among the resistant Campylobacter isolates. Susceptibility testing to five different classes of antibiotics was performed in 100 C. coli and 100 C. jejuni, previously isolated and identified. All isolates were found susceptible to meropenem. Very high rates of resistance were recorded for tetracyclines (84.5%), medium rates of resistance were recorded regarding quinolones (23%), and low and very low rates of resistance were identified for macrolides such as erythromycin and aminoglycosides (12% and 4%, respectively). Only 12.5% of the Campylobacter isolates displayed MDR. Regarding the molecular mechanisms of resistance, all ciprofloxacin resistant isolates hosted the mutant type Thr-86-Ile region of the quinolone resistance-determining region (QRDR) of the gyrA gene. In all erythromycin resistant isolates, the transitional mutations A2075G and A2074C in the 23S rRNA gene were only amplified. Molecular screening of tetracycline resistance genes indicated that the vast majority of Campylobacter isolates (92.3%) were positive for the tet(O) gene. In summary, these findings and especially the very high and medium rates of resistance for tetracyclines and fluroquinolones, respectively recommend that a continuous monitoring of Campylobacter isolates susceptibility in combination with the proper use of antimicrobials in livestock production is of great importance for public health.

First report of detection of microcystins in farmed mediterranean mussels Mytilus galloprovincialis in Thermaikos gulf in Greece.

BACKGROUND: Microcystins are emerging marine biotoxins, produced by potentially toxic cyanobacteria. Their presence has been reported in aquatic animals in Greek freshwater, while data are few in marine environments. Since the climate change induces eutrophication and harmful algal blooms in coastal marine ecosystems affecting the public health, further research on microcystins' presence in marine waters is required. The aim of this study was to examine the potential presence of microcystins in mussels Mytilus galloprovincialis in the largest farming areas in Thermaikos gulf, in Northern Greece, and to investigate their temporal and spatial distribution, adding to the knowledge of microcystins presence in Greek Mediterranean mussels. RESULTS: A 4-year microcystins' assessment was conducted from 2013 to 2016, in farmed Mediterranean mussels M. galloprovincialis, in five sampling areas in Thermaikos gulf, in northern Greece, where the 90% of the Greek mussels' farming activities is located. The isolation of potentially toxic cyanobacteria was confirmed by molecular methods. An initial screening was performed with a qualitative and quantitative direct monoclonal (DM) ELISA and results above 1 ng g-1 were confirmed for the occurrence of the most common microcystins-RR, -LR and -YR, by Ultra High Performance Liquid Chromatography (UHPLC) coupled with a high- resolution mass spectrometer (HRMS) (Orbitrap analyzer). Microcystin-RR and microcystin-LR were detected, while the intensity of microcystin-YR was below the method detection limit. Most samples that exhibited concentrations above 1 ng g-1 were detected during the warm seasons of the year and especially in spring. Results indicated an overestimation of the ELISA method, since concentrations ranged between 0.70 ± 0.15 ng g-1 and 53.90 ± 3.18 ng g-1, while the confirmation denoted that the levels of microcystins were 6 to 22 times lower. CONCLUSIONS: Microcystin-RR and microcystin-LR were detected for the first time in mussel M. galloprovincialis, harvested from farms in Thermaikos gulf, in Central Macedonia, Greece. Their presence was linked to potentially toxic cyanobacteria. Bioaccumulation was observed in digestive gland, while the concentrations in muscles were found extremely low. Samples with levels above 1 ng g-1 were observed mostly during spring, confirming the seasonal distribution of microcystins. The comparison of the results by the ELISA and the LC-Orbitrap MS method indicated an overestimation of concentration by the ELISA method.

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.

Occurrence and molecular characterization of betanodaviruses in fish and invertebrates of the Greek territorial waters.

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.

Zika virus infection: Past and present of another emerging vector-borne disease.

Zika virus infection is an emerging mosquito-borne disease, first identified in Uganda in 1947. It is caused by the Zika arbovirus, and transmitted by the bites of infected mosquitoes of the genus Aedes. For almost half a century, the Zika virus was reported as the causative agent of sporadic human infections. In 2007, the Zika virus emerged outside Asia and Africa causing an epidemic on the Island of Yap in Micronesia. The manifestation of the newly acquired human infection varies from asymptomatic to self-limiting acute febrile illness with symptoms and clinical features similar to those caused by the Dengue virus ('Dengue-like syndrome'). The real-time PCR and serological methods have been successfully applied for the diagnosis of the disease. The treatment is symptomatic, since there is no specific antiviral treatment or a vaccine. During the recent outbreaks in French Polynesia and Brazil, incidents of Guillain-Barrι syndrome and microcephaly were associated with Zika virus infection, giving rise to fears of further global spread of the virus. Prevention and vector control strategies have to be urgently implemented by national health authorities in order to contain future outbreaks in vulnerable populations. This review summarizes the existing information on Zika virus characteristics, pathogenesis and epidemiology, the available methods for the diagnosis of Zika virus infection and recent approaches for prevention and control.

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.