In Silico Molecular Analysis of Carbapenemase-Negative Carbapenem-Resistant Pseudomonas aeruginosa Strains in Greece.

To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD porin, and overexpression of the efflux pump MexAB-OprM. Twelve carbapenemase-negative carbapenem-resistant Pseudomonas aeruginosa strains, isolated from patients hospitalized at the University Hospital of Larissa, Central Greece, during 2023, which belonged to various sequence types (STs), were selected and were studied focusing on the characterization of their ß-lactamases, on changes to OprD and its regulator MexT proteins, and on alterations to the MexAB-OprM regulator proteins encoded by the mexR, nalC, and nalD genes. Whole genome sequencing analysis revealed the presence of ß-lactamase encoding genes, with blaPAO present in all isolates. Additionally, seven different genes of the oxacillinase family (blaOXA-35, blaOXA-50, blaOXA-395, blaOXA-396, blaOXA-486, blaOXA-488, blaOXA-494) were identified, with each strain harboring one to three of these. Regarding the OprD, five strains had truncated structures, at Loop 2, Loop 3, Loop 4, and Loop 9, while the remaining strains carried previously reported amino acid changes. Further, an additional strain had a truncated MexR; whereas, two other strains had totally modified NalC sequences. The active form of MexT, responsible for the downregulation of OprD production, as the intact sequence of the NalD protein, was found in all the strains studied. It is concluded that the truncated OprD, MexR, and NalC proteins, detected in eight strains, probably led to inactive proteins, contributing to carbapenem resistance. However, four strains carried known modifications in OprD, MexR, and NalC, as previously reported in both susceptible and resistant strains, a finding that indicates the complexity of carbapenem resistance in P. aeruginosa.

Longitudinal Study of Antibiotic Resistance of Staphylococci from Cases of Subclinical Mastitis in Sheep in Greece: Incidence and Risk Factors.

The present paper extends a previous publication on a field study of subclinical mastitis in sheep and focuses on the following laboratory characteristics of the staphylococcal isolates: antibiotic resistance and association with biofilm formation. The specific objectives of the present study were (a) to describe the incidence of isolation of antibiotic-resistant staphylococci from cases of mastitis throughout the milking period in dairy sheep flocks and (b) to identify relevant risk factors, which would contribute to the sustainable control of the infection. Staphylococcal isolates from subclinical mastitis were evaluated for antibiotic resistance to 18 antibiotics. Antibiotic resistance was detected in 57 of the 179 staphylococcal isolates from subclinical mastitis (31.8%). Resistance was recorded against 11 antibiotics, most often against ampicillin (63.2% of resistant isolates), penicillin (63.2%) and tetracycline (47.4%). Isolates resistant to ampicillin and penicillin were recovered in all 12 farms. Twenty-one multidrug-resistant isolates (11.7%) were also recovered. The incidence risk of isolation of staphylococci resistant to at least one (any) antibiotic throughout the study period was 23.8%. The incidence risk of isolation of staphylococci resistant to oxacillin was 5.0%; that of isolation of multidrug-resistant staphylococci was 8.8%. With regard to increased incidence risk of isolation of staphylococci resistant to at least one (any) antibiotic and increased incidence risk of isolation of staphylococci resistant to oxacillin, the omission of anti-staphylococcal mastitis vaccination of ewes emerged as a risk factor. With regard to increased incidence risk of isolation of multidrug-resistant staphylococci, the following variables emerged as risk factors: (a) higher number of antibiotics used on the farm for the treatment of mastitis and (b) younger age of lambs taken away from their dam. Most biofilm-forming antibiotic-resistant staphylococci were recovered from farms where anti-staphylococcal mastitis vaccination was not applied (55.9% versus 44.1% from farms where anti-staphylococcal mastitis vaccination was applied).

The Molecular Characterization of blaNDM-1-Positive Acinetobacter baumannii Isolated in Central Greece.

The objective of the present study is to report the detection and the molecular characterization of nine blaNDM-1-positive Acinetobacter baumannii isolates, which were isolated from patients in a tertiary care hospital in Central Greece from December 2022 to August 2023. The isolates were characterized by whole genome sequencing to obtain Pasteur multilocus sequencing typing (MLST) and to identify the blaNDM-1-environment, resistome, and virulence genes content. In silico MLST analysis showed that the isolates belonged to four different clones (STs 160, 2, 85, and 2493). All strains, apart from the blaNDM-1-gene, possessed at least eight different genes, encoding resistance to various antimicrobial agents. Whole genome sequencing revealed two different structures of the blaNDM-1 environment. The first, detected in ST160 strain, was identical with the Tn125, whereas the second, found in STs 2, 85, and 2493 was associated with Tn7382. To our knowledge, after a sole strain reported in 2016 and imported by a patient hospitalized in a Libyan hospital, this is the first report of the emergence of polyclonal blaNDM-1-positive Acinetobacter baumannii in Greece. Our findings re-emphasize the need to apply diligent surveillance protocols in order to limit the horizontal transfer of the blaNDM-1 gene to other A. baumannii clones or to other recipient strains.

Recovery of Staphylococci from Teatcups in Milking Parlours in Goat Herds in Greece: Prevalence, Identification, Biofilm Formation, Patterns of Antibiotic Susceptibility, Predictors for Isolation.

The objectives of this work are (a) to describe staphylococci on the teatcups of milking parlours in goat farms and identify predictors for the presence of staphylococcal isolates on the teatcups, (b) to evaluate relationships with total bacterial counts and somatic cell counts in bulk-tank milk, and (c) to establish patterns of susceptibility to antibiotics for the staphylococcal isolates and identify predictors for the recovery of resistant isolates. In a cross-sectional study of 66 goat farms across Greece, swab samples were collected from 303 teatcups (upper and lower part) for staphylococcal recovery, identification, and assessment of biofilm formation. Details regarding health management on the farms (including conditions in the milking parlour) and the socio-demographic characteristics of farmers were collected by means of a structured questionnaire. A total of 87 contaminated teatcups (28.7%) were found on 35 goat farms (53.0%). Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 73 versus 43 teatcups, respectively. After identification, 67 staphylococcal isolates (i.e., excluding similar isolates) were recovered from the teatcups; Staphylococcus aureus, Staphylococcus capitis, and Staphylococcus equorum predominated. Of these isolates, 82.1% were biofilm-forming. In multivariable analysis, the annual incidence of clinical mastitis in the herd emerged as the only significant factor associated with the isolation of staphylococci from the teatcups. Of the 67 isolates, 23 (34.3%) were resistant to at least one antibiotic, and 14 (22.4%) were multi-resistant. Resistance was found most commonly against penicillin and ampicillin (22.4% of isolates), fosfomycin (17.9%), clindamycin (14.9%), erythromycin, and tetracycline (13.4%). In multivariable analysis, the annual incidence of clinical mastitis in the herd and the use of detergent for parlour cleaning emerged as significant factors associated with the isolation of staphylococci resistant to antibiotics.

First Detection and Molecular Characterization of Pseudomonas aeruginosa blaNDM-1 ST308 in Greece.

The objective of the present study is to report the detection and the molecular characterization of nine blaNDM-1-positive Pseudomonas aeruginosa isolates, all of which belonged to the epidemic high-risk international clone ST308, and all were isolated from patients in a tertiary care hospital in Central Greece from May to July 2023.The isolates were characterized by whole genome sequencing to obtain multi-locus sequencing typing (MLST) and identify the blaNDM1-environment and resistome and virulence genes content. In silico MLST analysis showed that all isolates belonged to the high-risk ST308 international clone. All strains possessed 22 different genes, encoding resistance to various antimicrobial agents. Whole genome sequencing revealed that the blaNDM-1 was chromosomally located within the integrative and conjugative element ICETn43716385 and that it was part of one cassette along with two other resistance genes, floR and msrE. Two additional resistance cassettes were also found in the genome, which included the arrays of aph(6)-Id, aph(3″)-Ib, floR, sul2 and aadA10, qnrVC1, aac(3)-Id, dfrB5, aac(6')-II. Additionally, the strains possessed various virulence genes, e.g., aprA, exoU, lasA, lasB, toxA, and estA. All of the isolates shared identical genomes, which showed 98% similarity with the P. aeruginosa ST308 genome (acc. no CP020703), previously reported from Singapore. To our knowledge, this is the first report of ST308 blaNDM-1-positive P. aeruginosa isolation in Europe, which indicates the transmission dynamics of this high-risk clone.

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece.

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to ß-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.

Isolation of Listeria ivanovii from Bulk-Tank Milk of Sheep and Goat Farms-From Clinical Work to Bioinformatics Studies: Prevalence, Association with Milk Quality, Antibiotic Susceptibility, Predictors, Whole Genome Sequence and Phylogenetic Relationships.

A cross-sectional study was performed in 325 sheep and 119 goat dairy farms in Greece. Samples of bulk-tank milk were examined by standard microbiological techniques for Listeria spp. Listeria monocytogenes was isolated from one (0.3%) and Listeria ivanovii from three (0.9%) sheep farms. No associations between the isolation of L. monocytogenes or L. ivanovii and milk quality were found. No resistance to antibiotics was identified. Three variables emerged as significant predictors of isolation of the organism: the presence of pigs, low average relative humidity and a high number of ewes on the farm. The three L. ivanovii isolates were assessed in silico for identification of plasmids, prophages, antibiotic resistance genes, virulence factors, CRISPRs and CAS genes. Phylogenetic analysis using the core genome revealed that the three strains belonged to the L. ivanovii subsp. ivanovii branch and were especially close to the PAM 55 strain. All strains of the branch appeared to be very similar, with the distance between them being small.

Detection of Zoonotic Gastrointestinal Pathogens in Dairy Sheep and Goats by Using FilmArray® Multiplex-PCR Technology.

The objectives of this study were (a) to detect gastrointestinal pathogens in faecal samples of sheep and goats using the FilmArray® GI Panel and (b) to evaluate factors that were associated with their presence. Faecal samples from ewes or does in 70 sheep flocks and 24 goat herds in Greece were tested for the presence of 22 gastrointestinal pathogens by means of the BioFire® FilmArray® Gastrointestinal (GI) Panel. The most frequently detected pathogens were Shiga-like toxin-producing Escherichia coli stx1/stx2 (94.7% of farms), Giardia lamblia (59.6%), and Campylobacter spp. (50.0% of farms). Other pathogens detected were Cryptosporidium spp., Salmonella spp., enterotoxigenic E. coli lt/st, Yersinia enterocolitica, E. coli O157, Rotavirus A, Shigella/enteroinvasive E. coli, and Plesiomonasshigelloides. There was a difference in the prevalence of detection of pathogens between sheep and goat farms only for Salmonella spp.: 18.3% versus 0.0%, respectively. Mixed infections were detected in 76 farms (80.9% of farms), specifically 57 sheep flocks and 19 goat herds, with on average, 2.5 ± 0.1 pathogens detected per farm. The body condition score of ewes in farms, in which only one pathogen was detected in faecal samples, was significantly higher than that of ewes in farms, in which at least two pathogens were detected: 2.55 ± 0.11 versus 2.31 ± 0.04. In sheep flocks, the number of pathogens in faecal samples was significantly higher in farms with semi-extensive management. In goat herds, the number of pathogens in faecal samples was positively correlated with average precipitation and inversely correlated with temperature range in the respective locations.

Plethora of Resistance Genes in Carbapenem-Resistant Gram-Negative Bacteria in Greece: No End to a Continuous Genetic Evolution.

Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca. This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution.

Prevalence, Patterns, Association with Biofilm Formation, Effects on Milk Quality and Risk Factors for Antibiotic Resistance of Staphylococci from Bulk-Tank Milk of Goat Herds.

The objectives of this work were to study the prevalence and the patterns of antibiotic resistance of staphylococcal isolates from bulk-tank milk of goat herds across Greece, to assess possible associations of the presence of antibiotic resistance with the quality of milk in these herds and to evaluate herd-related factors potentially associated with the presence of antibiotic resistance among these staphylococcal isolates. A cross-sectional study was performed on 119 goat herds in Greece. Bulk-tank milk samples were collected for bacteriological examination; staphylococcal isolates were evaluated for resistance to 20 antibiotics. Oxacillin-resistant, resistant to at least one antibiotic, and multi-resistant staphylococcal isolates were recovered from 5.0%, 30.3%, and 16.0% of herds, respectively. Of 80 isolates, 7.5% were resistant to oxacillin, 50.0% were resistant to at least one antibiotic and 27.5% were multi-resistant. Resistance was seen more frequently among coagulase-negative staphylococci (59.3%) than among Staphylococcus aureus (23.8%). Resistance was more frequent against penicillin and ampicillin (41.3% of isolates) and fosfomycin (27.5%). No association was found with biofilm formation by staphylococci. For recovery of oxacillin-resistant isolates, the presence of working staff in the herds emerged as a significant factor; respective factors for the isolation of staphylococci resistant to at least one antibiotic were part-time farming and high (>10) number of systemic disinfections in the farm annually. The same three factors concurrently were also identified to be significant for the recovery of multi-resistant isolates.

Antibiotic Resistance of Staphylococci from Bulk-Tank Milk of Sheep Flocks: Prevalence, Patterns, Association with Biofilm Formation, Effects on Milk Quality, and Risk Factors.

The objectives of this work were to study prevalence and characteristics of resistance to antibiotics of staphylococcal isolates from the bulk-tank milk of sheep flocks across Greece, to assess possible associations of the presence of antibiotic resistance with the quality of milk in these flocks and to evaluate flock-related factors potentially associated with antibiotic resistance among these isolates. A cross-sectional study was performed in 325 sheep flocks in Greece. Bulk-tank milk samples were collected for bacteriological examination; staphylococcal isolates were evaluated for resistance to 20 antibiotics. Oxacillin-resistant staphylococcal isolates, isolates resistant to any antibiotic, and multi-resistant isolates were recovered from 8.0%, 30.5%, and 12.0% of flocks, respectively. Of 232 isolates, 11.6% were resistant to oxacillin, 46.1% were resistant to at least one antibiotic, and 16.4% were multi-resistant. Resistance was seen more frequently among coagulase-negative (50.6%) than among Staphylococcus aureus (31.5%) isolates. Resistance was more frequent against penicillin and ampicillin (34.1% of isolates), clindamycin (17.7%), and fosfomycin (14.2%). An association was found between biofilm formation by staphylococci and resistance to fosfomycin. For recovery of oxacillin-resistant isolates, the lack of experience by farmers emerged as a significant factor; respective factors for the isolation of staphylococci resistant to any antibiotic or multi-resistant isolates were the early stage of the lactation period (0th-1st month) and the intensive management system applied in the flocks, respectively.

Extensive countrywide field investigation of somatic cell counts and total bacterial counts in bulk tank raw milk in goat herds in Greece.

Dairy goat farming is an important sector of the agricultural industry in Greece, with an annual total milk production exceeding 450 000 l and accounting for over 25% of all goat milk produced in the European Union; this milk is used mainly for cheese production. Despite the importance of goat milk for the agricultural sector in Greece, no systematic countrywide investigations in the bulk-tank milk of goats in Greece have been reported. Objectives were to investigate somatic cell counts (SCC) and total bacterial counts (TBC) in raw bulk-tank milk of goat herds in Greece, study factors influencing SCC and TBC therein and evaluate their possible associations with milk content. Throughout Greece, 119 dairy goat herds were visited for milk sampling for somatic cell counting, microbiological examination and composition measurement. Geometric mean SCC and TBC were 0.838 × 106 cells ml-1 and 581 × 103 cfu ml-1, respectively. Multivariable analyses revealed annual frequency of check-ups of milking system and total milk quantity per goat (among 53 variables) to be significant for increased SCC; no factor emerged (among 58 variables) to be significant for increased TBC. Negative correlation of SCC with total protein was found; mean total protein content in the bulk-tank milk in herds with SCC >0.75 × 106 cells ml-1 was 5.1% lower and in herds with SCC >1.5 × 106 cells ml-1, it was 7.8% lower.

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience.

The objective of the present study was to evaluate the true positivity among people, whose results of initial testing of nasopharyngeal swabs (NPS) showed a very low viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Seventy-seven people detected with low viral loads of SARs-CoV-2 in nasopharyngeal samples (Ct ≥ 35) were enrolled in the study. For this purpose, a second NPS was collected for rRT-PCR (real-time reverse transcription polymerase chain reaction) combined with a pair of serum samples for detection of anti-nucleocapsid (anti-N) and anti-spike (anti-S) antibodies. In 8 people, subsequent examinations indicated an increase in viral loads, thereafter, followed by an increase of anti-N and anti-S antibodies, findings compatible with an early stage of COVID-19 infection. In 9 people, who already had increased anti-N antibodies, subsequent examination showed a decrease or absence of viral load and an increase in antibodies, indicative of a late stage of COVID-19 infection. In 60 people, subsequent examination showed absence of infection (as indicated by absence of viral load and antibodies). We propose that the combination of a second NPS and one serum-specimen, both taken three days after the first NPS, helps significantly to avoid false-positive results.

Clostridioides difficile as a Dynamic Vehicle for the Dissemination of Antimicrobial-Resistance Determinants: Review and In Silico Analysis.

The present paper is divided into two parts. The first part focuses on the role of Clostridioides difficile in the accumulation of genes associated with antimicrobial resistance and then the transmission of them to other pathogenic bacteria occupying the same human intestinal niche. The second part describes an in silico analysis of the genomes of C. difficile available in GenBank, with regard to the presence of mobile genetic elements and antimicrobial resistance genes. The diversity of the C. difficile genome is discussed, and the current status of resistance of the organisms to various antimicrobial agents is reviewed. The role of transposons associated with antimicrobial resistance is appraised; the importance of plasmids associated with antimicrobial resistance is discussed, and the significance of bacteriophages as a potential shuttle for antimicrobial resistance genes is presented. In the in silico study, 1101 C. difficile genomes were found to harbor mobile genetic elements; Tn6009, Tn6105, CTn7 and Tn6192, Tn6194 and IS256 were the ones more frequently identified. The genes most commonly harbored therein were: ermB, blaCDD, vanT, vanR, vanG and vanS. Tn6194 was likely associated with resistance to erythromycin, Tn6192 and CTn7 with resistance to the ß-lactams and vancomycin, IS256 with resistance to aminoglycoside and Tn6105 to vancomycin.

Emergence of a mupirocin-resistant, methicillin-susceptible Staphylococcus aureus clone associated with skin and soft tissue infections in Greece.

BACKGROUND: Staphylococcus aureus causes various infections, including skin and soft tissue infections (SSTIs). In this study, methicillin-susceptible S. aureus (MSSA) from SSTIs among patients in three tertiary-care hospitals in Greece were studied in terms of antimicrobial resistance, clonal distribution, toxin and adhesin genes carriage. RESULTS: During a five-year period (2014-2018), 6145 S. aureus were recovered from 13,244 patients with SSTIs and tested for antimicrobial susceptibility. MSSA were 4806 (78.21 %) including 1484 isolates with mupirocin minimum inhibitory concentration (MIC) > 64 mg/L (30.88 %). Two hundred and sixty representative mupirocin-resistant MSSA were analyzed for genes encoding Panton-Valentine leukocidin (PVL, lukS/lukF-PV), exfoliative toxins (eta, etb), adhesin FnbA (fnbA) and resistance genes mupA (high-level resistance to mupirocin), fusB (fusidic acid), aminoglycosides' modifying enzymes, ermA, ermC and msrA (macrolides/lincosamides) by PCRs. Strains were classified into clones by PFGE and MLST. All mupirocin-resistant MSSA were penicillin-resistant; 92.7 % expressed resistance to fusidic acid and 88.9 % to tobramycin. All 260 molecularly analyzed isolates were mupA-positive; all fusidic acid-resistant (241/260) carried fusB whereas, the tobramycin-resistant ones (230), ant(4')-Ia. The majority carried eta (93.85 %), etb (98.08 %) and fnbA (88.85 %). PFGE typing revealed a mostly unvarying population; 260 MSSA were grouped into three types. One major eta/etb-positive clone comprising of 258/260 strains (99.2 %), PFGE type 1, was classified as ST121, including nine strains co-carrying PVL. Another PVL-positive strain was identified as ST1, and one toxins-negative as ST21. CONCLUSIONS: A mupirocin-resistant MSSA clone, ST121, carrying resistance, exfoliative toxins and adhesin genes, was spread and predominated in SSTIs from patients in Greece during the five-year studied period.

Antimicrobial Resistance Genes in ESBL-Producing Escherichia coli Isolates from Animals in Greece.

The prevalence of multidrug resistant, extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide. The present study aimed to provide an overview of the multidrug resistance phenotype and genotype of ESBL-producing Escherichia coli (E. coli) isolates of livestock and wild bird origin in Greece. Nineteen phenotypically confirmed ESBL-producing E. coli strains isolated from fecal samples of cattle (n = 7), pigs (n = 11) and a Eurasian magpie that presented resistance to at least one class of non ß-lactam antibiotics, were selected and genotypically characterized. A DNA-microarray based assay was used, which allows the detection of various genes associated with antimicrobial resistance. All isolates harbored blaCTX-M-1/15, while blaTEM was co-detected in 13 of them. The AmpC gene blaMIR was additionally detected in one strain. Resistance genes were also reported for aminoglycosides in all 19 isolates, for quinolones in 6, for sulfonamides in 17, for trimethoprim in 14, and for macrolides in 8. The intI1 and/or tnpISEcp1 genes, associated with mobile genetic elements, were identified in all but two isolates. This report describes the first detection of multidrug resistance genes among ESBL-producing E. coli strains retrieved from feces of cattle, pigs, and a wild bird in Greece, underlining their dissemination in diverse ecosystems and emphasizing the need for a One-Health approach when addressing the issue of antimicrobial resistance.

Association of Staphylococcal Populations on Teatcups of Milking Parlours with Vaccination against Staphylococcal Mastitis in Sheep and Goat Farms.

There is a paucity of information regarding staphylococcal populations on teatcups of milking parlours in sheep and goat farms. The objectives were to describe the populations of staphylococci on teatcups in milking parlours in sheep or goat farms in two field investigations throughout Greece and to potentially associate the findings with the use of anti-staphylococcal mastitis vaccinations in the farms visited during the two investigations. In a cross-sectional (255 sheep and 66 goat farms across Greece) and a longitudinal (12 sheep farms, four samplings, throughout lactation) study, swab samples were collected from 1418 teatcups (upper and lower part) for staphylococcal recovery, identification and assessment of biofilm-formation. A total of 328 contaminated teatcups (23.1%) were found in 105 sheep (41.2%) and 35 goat (53.0%) farms. Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 269 versus 139 teatcups, respectively. After identification, 253 staphylococcal isolates were found: Staphylococcus aureus, Staphylococcus equorum, Staphylococcus lentus, and Staphylococcus capitis predominated. Of these isolates, 87.4% were biofilm-forming. The proportion of contaminated teatcups was smaller in farms where vaccination against anti-staphylococcal mastitis in general or vaccination specifically against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, 19.7% or 10.9%, respectively, versus 25.5% in farms without vaccination. In the longitudinal study, contaminated teatcups were identified in 28 (58.3%) sampling occasions, with staphylococci being recovered more frequently from their upper part. The same species as in the cross-sectional study predominated. Of these isolates, 61.9% were biofilm-forming. In farms where vaccination against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, the proportion of contaminated teatcups was smaller: 20.4% versus 48.3% in farms without vaccination. There were no differences in proportions of contaminated teatcups between sampling occasions. In conclusion, the great majority of staphylococci recovered from teatcups of milking parlours in sheep and goat farms included biofilm-forming isolates. Reduced staphylococcal isolation was noted in farms where anti-staphylococcal vaccination was performed; this was possibly the effect of reduced excretion of staphylococci in the milk of vaccinated animals.

Poultry and Wild Birds as a Reservoir of CMY-2 Producing Escherichia coli: The First Large-Scale Study in Greece.

Resistance mediated by ß-lactamases is a globally spread menace. The aim of the present study was to determine the occurrence of Escherichia coli producing plasmid-encoded AmpC ß-lactamases (pAmpC) in animals. Fecal samples from chickens (n = 159), cattle (n = 104), pigs (n = 214), and various wild bird species (n = 168), collected from different Greek regions during 2018-2020, were screened for the presence of pAmpC-encoding genes. Thirteen E. coli displaying resistance to third-generation cephalosporins and a positive AmpC confirmation test were detected. blaCMY-2 was the sole pAmpC gene identified in 12 chickens' and 1 wild bird (Eurasian magpie) isolates and was in all cases linked to an upstream ISEcp1-like element. The isolates were classified into five different sequence types: ST131, ST117, ST155, ST429, and ST1415. Four chickens' stains were assigned to ST131, while five chickens' strains and the one from the Eurasian magpie belonged to ST117. Seven pAmpC isolates co-harbored genes conferring resistance to tetracyclines (tetM, tetB, tetC, tetD), 3 carried sulfonamide resistance genes (sulI and sulII), and 10 displayed mutations in the quinolone resistance-determining regions of gyrA (S83L+D87N) and parC (S80I+E84V). This report provides evidence of pAmpC dissemination, describing for the first time the presence of CMY-2 in chickens and wild birds from Greece.

Extensive Countrywide Field Investigation of Somatic Cell Counts and Total Bacterial Counts in Bulk-Tank Raw Milk in Sheep Flocks in Greece.

Objectives were to investigate somatic cell counts (SCC) and total bacterial counts (TBC) in the raw bulk-tank milk of sheep flocks in Greece, to study factors potentially influencing increased SCC and TBC in the bulk-tank milk of sheep and to evaluate possible associations of SCC and TBC with milk content. Throughout Greece, 325 dairy sheep flocks were visited for collection of milk sampling for somatic cell counting, microbiological examination and composition measurement. Geometric mean SCC were 0.488 × 106 cells mL-1; geometric mean TBC were 398 × 103 cfu mL-1; 228 staphylococcal isolates were recovered form 206 flocks (63.4%). Multivariable analyses revealed annual incidence risk of clinical mastitis, age of the farmer and month into lactation period (among 53 variables) to be significant for SCC > 1.0 × 106 cells mL-1 and month into lactation period at sampling and availability of mechanical ventilators (among 58 variables) to be significant for TBC > 1500 × 103 cfu mL-1. Negative correlation of SCC with fat, total protein and lactose and positive correlation of SCC with added water were found. With SCC > 1.0 × 106 cells mL-1, significant reduction of protein content (2%) was observed, whilst in flocks with SCC > 1.5 × 106 cells mL-1, significantly lower annual milk production per ewe (42.9%) was recorded.