Listeria monocytogenes isolates from Cornu aspersum snails: Whole genome-based characterization and host-pathogen interactions in a snail infection model.

Even though Listeria monocytogenes is an extensive-studied foodborne pathogen, genome analysis of isolates from snails that may represent a reservoir of L. monocytogenes are still scarce. Here, we use whole-genome sequencing (WGS) to assess the genomic diversity of hypervirulent, virulent and non-virulent phenotypes of 15 L. monocytogenes isolated from snails to unveil their survival, virulence, and host-pathogen mechanisms of interactions in a snail infection model. Most of isolates (66.7%) were characterized as multidrug resistant (MDR) and belonged to clonal complexes (CCs) which are strongly associated with cases of human infection. All isolates contained intact genes associated with invasion and infection while hypervirulent isolates are adapted to host environment, possessing genes which are involved in teichoic acid biosynthesis, peptidoglycan modification and biofilm formation, correlating with their tolerance to haemolymph plasma phenotype and biofilm formation ability. A snail infection model showed that hypervirulent isolates triggered programmed host cell death pathway by increasing up to 30% the circulating apoptotic hemocytes in combination with induced nitrate production and reactive oxygen species (ROS) generation in snails' haemolymph. In contrast, the administration of the non-virulent strain which possesses a truncated mogR gene that regulates flagellar motility gene expression led only to an increase of necrotic non-apoptotic cells. Overall, this study provides significant insights into the genetic diversity of L. monocytogenes from snails, the genomic features of them linked to their hypervirulent/non-virulent phenotype, and the mechanisms of host-pathogen interactions.

Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry.

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use. In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1. MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis' monitoring systems contributing in the effective control of the disease.

Listeria monocytogenes induced dysbiosis in snails and rebiosis achieved by administration of the gut commensal Lactobacillus plantarum Sgs14 strain.

Listeria monocytogenes strains were isolated from Cornu aspersum maxima snails from farm units experiencing high mortalities and were characterized by phenotypic, molecular and biochemical criteria. A high heterogeneity was observed in the pulsed-field gel electrophoresis (PFGE) pulsotypes as well as in the virulence (13-100% mortality) among the fifteen L. monocytogenes strains. One strain was characterized as non-virulent while three strains exhibited hypervirulent phenotype. Hypervirulence activity was associated with cell surface properties such as hydrophobicity, autoaggregation and biofilm formation, with increased tolerance to snail's gut barriers such as pedal mucus, gastric mucus, gastric juices, and acidic pH as well as with increased capacity to resist the antibacterial activity of snail haemolymph and modulate immune cell populations and functions such as chemotaxis and phagocytoses. L. monocytogenes dysbiosis was characterized by a clinicopathological phenotype including immobilization of snails' headfoot outside the shell, increased mucus-secreting cells in the intestinal epithelium and feces, alteration of intestinal ridges morphology and excessive increase of haemolymph immune cells and cell death. Rebiosis in L. monocytogenes SN3 strain infected snails was achieved by dietary supplementation of the snail-gut commensal probiotic L. plantarum Sgs14 strain by exhibiting anti-Listeria activity, reducing mortality and clinicopathological manifestations as well as exhibiting immunomodulatory activity.

First Report of Isolation and Genome Sequence of L. petauri Strain from a Rainbow Trout Lactococcosis Outbreak.

Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in bulk tank milk, livestock and dairy-farm personnel in north-central and north-eastern Greece: Prevalence, characterization and genetic relatedness.

Recently, there has been an increased tendency towards raw-milk consumption, which may pose a consumer risk, due to the possible presence of human pathogenic microorganisms, such as Staphylococcus aureus and even methicillin-resistant S. aureus (MRSA). The prevalence of S. aureus and methicillin-resistant S. aureus (MRSA) was investigated in 40 dairy (cattle, sheep and goat) farms in northern Greece. S. aureus and MRSA were detected in 47.8% and 4.1% of the 387 samples (raw milk, farmers and animal samples) tested, respectively. Most (81.3%) of the MRSA isolates harbored the mecA gene, whereas the mecC or Panton-Valentine Leucocidin (PVL) genes were not detected. Seven spa types were identified, with t127 being the most prevalent. Spa type t034 (CC398) was isolated for the first time from livestock in Greece. Staphylococcal enterotoxin genes were detected in 93.8% of the MRSA isolates. The MRSA isolates were genetically diverse and were all capable of biofilm production. Our results confirm the lurking threat of MRSA in raw milk and dairy farms and suggest the need for surveillance programs starting at the farm level.

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus (MRSA) along the production chain of dairy products in north-western Greece.

The objective of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the production chain of dairy products. Of 367 tested samples (36 bulk tank milk (BTM), 19 dairy products, 72 human, 185 animal, 55 equipment), 212 (57.8%) were found positive for S. aureus. Almost all isolates (99.6%) were resistant to at least one antimicrobial and 13.3% were multi-drug resistant (MDR), exhibiting resistance to three or more antibiotic classes. Eleven samples (3%) were found contaminated by MRSA carrying the mecA gene. None of the MRSA isolates carried the mecC or the Pandon-Valentine leucocidin (PVL) genes. Four spa types were identified among the MRSA isolates: t127, t3586, t1773, t4038, with t127 being the most prevalent (7 out of 11). Two of them, t3586 and t1773, were isolated for the first time in Greece. Furthermore, Pulse-Field Gel Electrophoresis (PFGE) analysis indicated clonal circulation through the dairy production chain. The presence of MDR S. aureus, and especially MRSA, in animals and dairy products represents a potential threat for the spread of this pathogen in the community. The results indicated that human, animal and environmental sources could be involved in the contamination of dairy products along their production chain and therefore further investigation of contamination sources is needed to control the dispersion of MRSA in the community.

Differences in sheep and goats milk microbiological profile between conventional and organic farming systems in Greece.

The aim of this study was to examine differences in the microbiological profile and antimicrobial resistance of bacteria isolated from milk from organic and conventional sheep and goat farms. Twenty-five organic and 25 conventional sheep and goat farms in the region of Thessaly, Greece participated in this study. A standardised detailed questionnaire was used to describe farming practices. A total of 50 samples were collected and analysed for total viable count (TVC), total coliform count (TCC) and somatic cell count (SCC), while Staphylococcus aureus and Escherichia coli were isolated using standard methods. Isolates were identified at species level by Api-test and Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS). Susceptibility to a panel of 20 for E. coli and 16 for S. aureus antimicrobials was determined by the agar dilution method. Pulsed Field Gel Electrophoresis (PFGE) was performed for S. aureus and E. coli isolates to determine predominant clones. Lower counts of TVC, TCC and SCC were identified in milk from the organic farms, possibly due to differences in the hygienic farming practices found on those farms. API-tests and MALDI-TOF MS showed no significant differences in the S. aureus and E. coli isolates. Overall, antimicrobial resistance rates were low, while a statistically higher percentage was estimated among strains originating from conventional farms in comparison with organic farms, possibly due to the restriction of antibiotic use in organic farming. PFGE revealed diversity among S. aureus and E. coli populations in both organic and conventional farms indicating circulation of 2-3 main clones changing slightly during their evolution. Consequently, there is evidence that milk from the organic farms presents a better microbiological profile when compared with milk from conventional farms.

River waters in Greece: A reservoir for clinically relevant extended-spectrum-ß-lactamases-producing Escherichia coli.

In the current study, the genotypic characteristics such as antimicrobial resistance and virulence genes, and plasmid replicons and phenotypic characteristics such as biofilm formation and antimicrobial resistance of 87 extended-spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-Ec) isolated from 7 water bodies in northern Greece were investigated. Our data show a high prevalence (60.0 %) of ESBL-Ec in surface waters that exhibit high genetic diversity, suggesting multiple sources of their transmission into the aquatic environment. When evaluating the antimicrobial resistance of isolates, wide variation in their resistance profiles has been detected, with all isolates being multi-drug resistant (MDR). Regarding biofilm formation capacity and phylogenetic groups, the majority (54.0 %, 47/87) of ESBL-Ec were classified as no biofilm producers mainly assigned to phylogroup A (35.6 %; 31/87), followed by B2 (26.5 %; 23/87). PCR screening showed that a high proportion of the isolates tested positive for the blaCTX-M-1 group genes (69 %, 60/87), followed by blaTEM (55.2 %, 48/87), blaOXA (25.3 %, 22/87) and blaCTX-M-9 (17.2 %, 15/87). A subset of 28 ESBL-Ec strains was further investigated by applying whole genome sequencing (WGS), and among them, certain clinically significant sequence types were identified, such as ST131 and ST10. The corresponding in silico analysis predicted all these isolates as human pathogens, while a significant proportion of WGS-ESBL-Ec were assigned to extraintestinal pathogenic E. coli (ExPEC; 32.1 %), and urinary pathogenic E. coli (UPEC; 28.6 %) pathotypes. Comparative phylogenetic analysis, showed that the genomes of the ST131-O25:H4-H30 isolates are genetically linked to the human clinical strains. Here, we report for the first time the detection of a plasmid-mediated mobile colistin resistance gene in ESBL-Ec in Greece isolated from an environmental source. Overall, this study underlines the role of surface waters as a reservoir for antibiotic resistance genes and for presumptive pathogenic ESBL-Ec.

Colour Doppler study of blood flow in the portal vein in relation to blood flow in the milk vein, milk yield and body condition of dairy cows during dry period and lactation.

In dairy cows, the liver supports the increased nutrient demands of the udder for milk production. Blood flow is key for the transport of these nutrients. This study investigated portal vein blood flow volume (PVBFVol) in relation to daily milk yield (DMY), milk vein blood flow volume (MVBFVol) and body condition parameters of high-producing dairy cows, starting from late lactation, throughout dry period, and consecutive early lactation. Seventeen repeated examinations were performed on 19 Holstein cows and 313 measurement days were finally included. Vein morphology and blood flow were examined via B-mode and spectral Doppler (triplex) ultrasonography, respectively. Body condition parameters recorded were body condition score (BCS), backfat thickness (BFT) measurement with ultrasonography, heart girth circumference (HG) and withers height (WH). Longitudinal relationship of PVBFVol with MVBFVol, DMY, BCS, BFT, HG and WH was analyzed with linear mixed models, with random intercept effects, using restricted cubic splines. A significant increase of 8.28% (p < 0.01) in PVBFVol appeared for every 1 L/min increase in MVBFVol in the univariable model. PVBFVol presented a significant negative association with BCS (p < 0.01) and BFT (p = 0.02), while interaction with production stage was significant, too. PVBFVol significantly increased by 0.38% (p = 0.04) for every 1 kg increase in DMY in the multivariable model. In conclusion, the increased PVBFVol during lactation accompanies the escalation in metabolic activity of the liver and the increased blood circulation through the udder, coping with the udder's escalating nutrient demands for milk synthesis.

Prevalence, Serotypes, Antimicrobial Resistance and Biofilm-Forming Ability of Listeria monocytogenes Isolated from Bulk-Tank Bovine Milk in Northern Greece.

The prevalence of Listeria monocytogenes in bovine bulk-tank milk (BTM) in Greece has not been previously investigated. The aim of the study was to estimate the prevalence of L. monocytogenes in bovine BTM in Greece and to characterize the isolates in terms of carriage of genes encoding for pathogenic determinants, assess the isolates' biofilm-forming ability and determine their susceptibility against 12 antimicrobials. Samples (n = 138) of bovine BTM were obtained from farms located throughout Northern Greece and were analyzed qualitatively and quantitatively for L. monocytogenes. Five samples (3.6%) tested positive for L. monocytogenes. The pathogen's populations in these positive samples were below 5 CFU/mL. Most isolates belonged to the molecular serogroup "1/2a, 3a". All isolates carried the virulence genes inlA, inlC, inlJ, iap, plcA and hlyA, but actA was detected in only three isolates. The isolates displayed weak to moderate biofilm-forming ability and distinct antimicrobial resistance profiles. All isolates were characterized as multidrug resistant, with resistance to penicillin and clindamycin being a common feature. Considering that L. monocytogenes constitutes a serious public health threat, the key findings of the study, related to the carriage of virulence genes and multidrug resistance, highlight the importance of continued monitoring of the pathogen in farm animals.

Udder Ultrasonography of Dairy Cows: Investigating the Relationship between Echotexture, Blood Flow, Somatic Cell Count and Milk Yield during Dry Period and Lactation.

Udder health of dairy cows is related to their productivity and welfare. The period from dry-off to calving and early lactation is crucial. Ultrasonography is a useful and practical tool for the examination of the mammary parenchyma and blood flow. This observational study investigated the relationship between udder echotexture features, blood flow volume (BFVol) in the milk vein, milk somatic cell count (SCC) and daily milk yield (DMY) from late lactation, throughout the dry period and consecutive early lactation. Seventeen repeated measurements were performed on twenty-one Holstein cows. The udder parenchyma was examined with B-mode ultrasonography. Udder echotexture was studied using 15 features: Numerical Pixel Value (NPV), Pixel Standard Deviation (PSD), Skewness, Excess, Contrast, Homogeneity, Correlation, Entropy, Run Percentage, Long-Run Emphasis, Grey Value Distribution, Runlength Distribution, Gradient Mean Value, Gradient Variance and Percentage of Non-zero Gradients. Blood flow in the milk vein was examined with spectral Doppler. Linear mixed-effects models were employed to investigate relationships between BFVol, udder echotexture features, SCC and DMY throughout the study period. Our models showed that a 1 kg increase in DMY was associated with a significant increase of 0.25 L/min in the expected BFVol and that a 1,000,000-cells/mL increase in SCC was associated with a significant BFVol decrease of 0.49 L/min, keeping all other variables constant. Multivariable models showed significant associations between DMY and NPV, between PSD and Long-Run Emphasis, and between SCC and NPV, PSD, Gradient Mean Value, Homogeneity, Gradient Variance and Entropy. In conclusion, udder echotexture and BFVol in the milk vein are related to SCC and milk yield. Ultrasonography can be used for the comprehensive assessment of udder health in support of precision dairy farming.

Circulation of Pestiviruses in Small Ruminants from Greece: First Molecular Identification of Border Disease Virus.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.

Prevalence, Infectious Characteristics and Genetic Diversity of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece.

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers' nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton-Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products.

Removal of faecal bacteria from septage by treating it in a full-scale duckweed-covered pond system.

Performance of a full-scale duckweed-covered treatment system in removing faecal bacteria is presented. The system consisted of three ponds in series and received septage from holding tanks. Inflow averaged between 36 m(3) d(-1) in the cold season and 60 m(3) d(-1) in the warm season, resulting in a total hydraulic retention time of 88 and 58 days, respectively. Duckweed (Lemna minor) colonized the ponds in the summer and continued to grow in the cold season. Due to the difficult harvesting process of the duckweed biomass, the investigation of the treatment efficiency was carried out without plant harvesting. The system was monitored for temperature, pH, oxygen, chlorophyll-a, Escherichia coli and Enterococci. Duckweed growth resulted in chlorophyll-a concentration reduction from 924 to 13 µg L(-1), causing neutral and anoxic conditions in the pond water. A temperature effect was noticed on the E. coli decay coefficient with a decreasing trend along the treatment system. Enterococci always decayed less than E. coli. Differences on decay coefficients and removal efficiencies were not observed between the three ponds for both bacterial types. Effluent quality in terms of E. coli was 489 and 1377 cfu/100 mL, in the warm and the cold seasons, respectively, with average removals of 99.65 ± 1.46% and 99.33 ± 3.03%. Total Enterococci removal was 88.91 ± 23.1% in the warm season and 94.43 ± 24.45% in the cold season, resulting in mean effluent values of 1058 and 1404 cfu/100 mL, respectively. The seasonal differences in total removal efficiencies were insignificant for both bacterial types.

Staphylococcus aureus Isolated from Ruminants with Mastitis in Northern Greece Dairy Herds: Genetic Relatedness and Phenotypic and Genotypic Characterization.

Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S. aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.

Cryptosporidium and Giardia in surface water and drinking water: Animal sources and towards the use of a machine-learning approach as a tool for predicting contamination.

Cryptosporidium and Giardia are important parasites due to their zoonotic potential and impact on human health, often causing waterborne outbreaks of disease. Detection of (oo)cysts in water matrices is challenging and few countries have legislated water monitoring for their presence. The aim of this study was to investigate the presence and origin of these parasites in different water sources in Northern Greece and identify interactions between biotic/abiotic factors in order to develop risk-assessment models. During a 2-year period, using a longitudinal, repeated sampling approach, 12 locations in 4 rivers, irrigation canals, and a water production company, were monitored for Cryptosporidium and Giardia, using standard methods. Furthermore, 254 faecal samples from animals were collected from 15 cattle and 12 sheep farms located near the water sampling points and screened for both parasites, in order to estimate their potential contribution to water contamination. River water samples were frequently contaminated with Cryptosporidium (47.1%) and Giardia (66.2%), with higher contamination rates during winter and spring. During a 5-month period, (oo)cysts were detected in drinking-water (<1/litre). Animals on all farms were infected by both parasites, with 16.7% of calves and 17.2% of lambs excreting Cryptosporidium oocysts and 41.3% of calves and 43.1% of lambs excreting Giardia cysts. The most prevalent species identified in both water and animal samples were C. parvum and G. duodenalis assemblage AII. The presence of G. duodenalis assemblage AII in drinking water and C. parvum IIaA15G2R1 in surface water highlights the potential risk of waterborne infection. No correlation was found between (oo)cyst counts and faecal-indicator bacteria. Machine-learning models that can predict contamination intensity with Cryptosporidium (75% accuracy) and Giardia (69% accuracy), combining biological, physicochemical and meteorological factors, were developed. Although these prediction accuracies may be insufficient for public health purposes, they could be useful for augmenting and informing risk-based sampling plans.

Assessment of reclaimed municipal wastewater application on rice cultivation.

Field research was carried out to assess the effects of the application of reclaimed municipal wastewater on rice cultivation in Thessaloniki, Greece during a 2-year period (1999-2000). Effects on production cost, soil composition, and health risk were examined. A randomized complete block design was used for the paddy field with three treatments and four replicates. The treatments were (1) river irrigation water with N-P fertilization, (2) reclaimed wastewater irrigation with surface N fertilization, and (3) reclaimed wastewater irrigation without fertilization. The results showed that the total production cost decreased 8.8% and 11.9% by applying the second and third treatments, respectively, compared to the first treatment, without significant differences in the agronomic and rice quality traits. Soil composition showed discrepancies between the 2 years and the three treatments, whereas the pathogens of the reclaimed wastewater and rice tissues posed a low human risk when taking the needed precautions.

Use of clay minerals for sewage sludge stabilization and a preliminary assessment of the treated sludge's fertilization capacity.

Preserving sewage sludge's N is important for its agronomic use and this could possibly be achieved by treating sludge with certain clay minerals. Nine clay minerals and additionally Ca(OH)2 were added to dewatered sewage sludge at 0-30 % rates (wet weight basis) (treatments). After 70 days of equilibration, all mixtures were analyzed for certain properties and the mineral-sludge mixtures which showed the highest microbial load reduction were further assayed, along with the limed and untreated sludge. From all minerals' treatments, the fecal indicators of sludge treated with 30% of two bentonites, attapulgite, saponite-attapulgite, and zeolite decreased considerably compared to the control. These treatments were performed also well regarding sludge's retention capacity of available inorganic N, with the attapulgite and zeolite treatments containing the significantly highest amounts of NO3-N and NH4-N, respectively. For the water-soluble inorganic N, similar results were obtained for the zeolite treatment, whereas the treatments with the two bentonites had the significantly highest NO3-N content. Also, considerable amounts of water-soluble P were obtained in all cases of the treated sludge with minerals. Limed sludge had the lowest content of the water-soluble inorganic N and P. As far as the micronutrients are concerned, only Zn and B were detectable in the water-soluble fraction of all five minerals' treatments. The heavy metals, which regulate sludge's agronomic use, were far below the respective permissible limits and lower than the untreated sludge, except for Ni and Cr in the attapulgite and saponite-attapulgite treatments. In conclusion, certain clay minerals, i.e., bentonite, attapulgite, mixed clay of saponite and attapulgite, and zeolite, seem promising materials for the stabilization of sewage sludge in the perspective of using them as a fertilizer. In addition, they seem to have higher fertilizing value than limed sludge. However, environmental (in respect of Ni and Cr) and agricultural (in respect of Zn and B) impacts must be considered.

Prevalence, antimicrobial susceptibility and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from dairy industries in north-central and north-eastern Greece.

Staphylococcus aureus is an important cause of food intoxication, whereas methicillin-resistant S. aureus (MRSA) constitutes a serious public-health concern due to its ability to colonize and infect humans and animals. S. aureus and MRSA have often been isolated from milk and dairy products. The objectives of this study were to estimate the prevalence and the antimicrobial resistance of S. aureus and MRSA in four Greek dairy industries, to identify virulence factors of MRSA isolates and to describe their genetic diversity, in order to identify possible epidemiological links and evaluate the risk of MRSA dissemination to the community. S. aureus was isolated from 67 out of 305 (22.0%) dairy industry samples (bulk-tank milk, dairy products, employee nasal swabs and equipment/surface swabs). Almost all (99%) of the 227 corresponding S. aureus isolates (approximately 4 isolates per positive sample) were resistant to at least one antimicrobial and 22% were multi-drug resistant (MDR). MRSA were isolated from 11 different samples (3.6%) originating from three of the dairy plants. All MRSA isolates were capable of forming biofilms, while staphylococcal enterotoxin (SE) genes were detected in 91% of the MRSA isolates, with sec being the most frequent. All of the MRSA isolates harbored the mecA gene but the mecC and Pandon-Valentine leucocidin (PVL) genes were not detected. Pulse-Field Gel Electrophoresis (PFGE) analysis showed genetic diversity among the MRSA isolates and indicated clonal circulation in one of the dairy plants. Seven spa types were identified among the MRSA isolates with the most prevalent (t065) isolated only in one dairy plant. Certain spa types (t065, t337 and t3536) were isolated for the first time in Greece. The presence of MDR, biofilm-forming and enterotoxigenic MRSA strains in dairy plant facilities may lead to their dissemination to the community, but also to staphylococcal food poisoning, when conditions are favorable. The study's findings highlight the need for continuous monitoring of the dairy production chain, the need for re-evaluating the implemented cleaning and sanitizing processes and the adoption of preventive strategies in order to minimize public-health risks.

Multiple clones and low antimicrobial resistance rates for Salmonella enterica serovar Infantis populations in Greece.

All the Salmonella enterica ser. Infantis strains isolated under official control programs in Greece during a four year period were studied, 23 of human origin, 16 from food animals and one from food. Molecular analyses (PFGE) in combination with antimicrobial susceptibility testing were used to study whether the occurrence S. Infantis in Greece resulted from different biotypes or a successful spread of one clone. Low rates of antimicrobial resistance were observed, except for streptomycin among human isolates (48%), indicating that selective pressure due to consumption of antimicrobials has not resulted the spread of dominant clones. Pulsed Field Gel Electrophoresis revealed 31 XbaI distinct pulsotypes among the 40 strains with 60% overall similarity reflecting diversity. Four main clusters were constructed, using an 85% cut off value, clusters A, B, C and D consisting of 14, 6, 8 and 8 isolates respectively. Point source of transmission was not hypothesized as multiple reservoirs of the serovar seem to be present in Greece during the study period.