In vitro activities of omadacycline, eravacycline, cefiderocol, apramycin, and comparator antibiotics against Acinetobacter baumannii causing bloodstream infections in Greece, 2020-2021: a multicenter study.

Resistance of Acinetobacter baumannii to multiple clinically important antimicrobials has increased to very high rates in Greece, rendering most of them obsolete. The aim of this study was to determine the molecular epidemiology and susceptibilities of A. baumannii isolates collected from different hospitals across Greece. Single-patient A. baumannii strains isolated from blood cultures (n = 271), from 19 hospitals, in a 6-month period (November 2020-April 2021) were subjected to minimum inhibitory concentration determination and molecular testing for carbapenemase, 16S rRNA methyltransferase and mcr gene detection and epidemiological evaluation. 98.9% of all isolates produced carbapenemase OXA-23. The vast majority (91.8%) of OXA-23 producers harbored the armA and were assigned mainly (94.3%) to sequence group G1, corresponding to IC II. Apramycin (EBL-1003) was the most active agent inhibiting 100% of the isolates at ≤16 mg/L, followed by cefiderocol which was active against at least 86% of them. Minocycline, colistin and ampicillin-sulbactam exhibited only sparse activity (S <19%), while eravacycline was 8- and 2-fold more active than minocycline and tigecycline respectively, by comparison of their MIC50/90 values. OXA-23-ArmA producing A. baumannii of international clone II appears to be the prevailing epidemiological type of this organism in Greece. Cefiderocol could provide a useful alternative for difficult to treat Gram-negative infections, while apramycin (EBL-1003), the structurally unique aminoglycoside currently in clinical development, may represent a highly promising agent against multi-drug resistant A. baumanni infections, due to its high susceptibility rates and low toxicity.

Prevalence and analysis of CRISPR/Cas systems in Pseudomonas aeruginosa isolates from Greece.

The objective of this study was to investigate the prevalence of CRISPR/Cas systems in P. aeruginosa, isolated from a Greek hospital. Additionally, we aimed to determine the origin of the sequenced spacers. A collection of 100 nonrepetitive P. aeruginosa was analyzed. Isolates were typed by MLST. The presence of CRISPR/Cas systems, as well as amplification of CRISPR arrays, was examined by PCR using specific primers. CRISPR/Cas systems were detected in 36 isolates, of which 27 isolates exhibited resistance to carbapenems, with 10 of the later isolates producing a VIM-type MßL. The majority (n = 19) of CRISPR/Cas-positive isolates harbored a type I-F system, while I-C and I-E systems were found in 9 and 8 isolates, respectively. Based on MLST, isolates carrying I-E and I-F systems belonged to different STs and included CRISPR arrays with diverse number of spacers. Isolates with I-C systems belonged to clonal complex 235 and exhibited identical CRISPR arrays. Among 425 unique spacers, identified during this study, BLASTn search showed that they matched with P. aeruginosa chromosomal sequences (47.0%), phages (31.9%), plasmids, PAGIs, and an ICE. 16.3% of the spacers exhibited no significant similarity with sequences submitted to GenBank database. In conclusion, we observed the presence of type I-C, I-E and I-F CRISPR/Cas systems in P. aeruginosa of clinical origin. CRISPR/Cas were also observed among isolates carrying the carbapenemase-encoding blaVIM gene, which is usually associated with integrons, questioning the defense role against mobile elements. Therefore, further experimental characterization is needed to clarify their functional role.

Prevalence of human papillomavirus and subtype distribution in male partners of women with cervical intraepithelial neoplasia (CIN): a systematic review.

BACKGROUND: Human Papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection. The present systematic review summarizes data regarding the prevalence of HPV and the distribution of subtypes in heterosexual male partners of women, who were diagnosed with any grade of cervical intraepithelial neoplasia (CIN). METHODS: We conducted a systematic review of the literature by Medline and Google Scholar databases using the terms "Human Papillomavirus" or "HPV" plus "men" or "male partners" or "women with CIN". We included original published English-language articles published from 1/1/2000 until 1/1/2018 that had screened male partners of women with CIN using HPV DNA testing. We excluded studies that they overlapped with other included studies or were unrelated to the study subject. RESULTS: We included a total of 12 publications, which reported the prevalence of HPV in free-clinical signs male partners of women with CIN. The largest proportion of the studies were from South America (seven studies), and the rest from Europe. The mean age of participants was 35.18 + - 3.47 years. HPV prevalence ranged from 12.9 to 86%; the total HPV prevalence among the studies was 49.1%, while ten out twelve studies (83.3%) demonstrated prevalence > 20%. Between the studies, the distribution of HPV subtypes varied on the basis of the method used, on the population and on the geographic region. A great variety of subtypes were detected, including 6, 11, 16, 18, 31, 33, 40, 42, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 68, 81 and 83. In six studies the HPV 16 was the most frequent, while in two others the HPV 6 and HPV 83. CONCLUSIONS: Until now, there are not precise screening or surveillance guidelines for the management of partners of women with CIN. This population is frequently colonized by various HPV subtypes and therefore need to be screened in an effort to reduce the infection in both sexes. The screening test could include detection/identification of HPV subtypes by a molecular assay, followed by peniscopy only in the positive cases.

Association of subclinical mastitis prevalence with sheep breeds in Greece.

The objective of the research described in this Research Communication was to describe potential associations of subclinical mastitis with sheep breeds in Greece. A countrywide survey (2198 ewes in 111 farms) was performed. Prevalence of subclinical mastitis was 0·260. Results did not indicate any difference in the prevalence of subclinical mastitis between farms with pure-bred and farms with cross-bred animals, nor any difference in prevalence between farms with Greek pure-bred animals and farms with imported pure-bred animals. Results indicated that prevalence of subclinical mastitis was smaller in farms with Assaf-breed (0·100) and higher in farms with Frisarta-breed (0·625) (P < 0·02). Prevalence of mastitis was smaller in farms with Greek traditional indigenous breeds (0·221) (P = 0·007). In a model that included sheep breed and management system in farm, breed emerged as a significant factor for prevalence of subclinical mastitis (P = 0·003).

Successful control of a neonatal outbreak caused mainly by ST20 multidrug-resistant SHV-5-producing Klebsiella pneumoniae, Greece.

BACKGROUND: Extended spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) infection can cause significant morbidity and mortality in neonates. We investigated a nosocomial ESBL-Kp outbreak in a neonatal intensive care unit (NICU) of the University Hospital of Larissa (UHL), Central Greece. METHODS: A total of sixty-four ESBL-Kp were studied; twenty six isolates were recovered from the NICU and were compared with thirty-eight randomly selected isolates from different wards of the hospital during the period March- December 2012. All isolates were characterized by antimicrobial susceptibility testing, ESBL-production by double-disk synergy test, molecular typing using BOX-PCR, whereas selected isolates were further characterized by beta lactamase and virulence gene content, multilocus sequence typing and phylogenetic analysis. All neonates affected by ESBL-Kp were put under strict contact isolation, along with appropriate infection control measures. RESULTS: The outbreak strain of ST20 multidrug-resistant SHV-5-producing K. pneumoniae was identified in all infected (n = 13) and three colonized neonates. A novel ST (ST1114) was also identified among SHV-5 producers (n = 10) recovered from nine colonized infants, but it was not related with ST20. Both STs were identified only in the NICU and not in other wards of the hospital. No ESBL-Kp were isolated from the hands of the nursing staff and the environment. Although we were not able to identify the source of the outbreak, no ESBL-Kp were isolated in the NICU after this period and we assumed that the outbreak was successfully controlled. All neonates received parenteral nutrition and most of them were delivered by caesarean section and showed low gestational age (<32 weeks) and low birth weights (<1500 g). CONCLUSION: According to our knowledge, this is the first description of an outbreak of multidrug-resistant SHV-5 producing K. pneumoniae assigned to ST20.

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.

M1UKStreptococcus pyogenes causing community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome.

OBJECTIVES: Streptococcus pyogenes causes superficial infections but can also cause deep-seated infections and toxin-mediated diseases. In the present study, phylogenetic and in silico prediction analyses were performed on an antimicrobial resistant M1UKS. pyogenes strain causing severe clinical manifestations during the current surge of invasive group A Streptococcus (iGAS) disease. METHODS: A 40-year-old patient was admitted to the hospital with fever, chest pain and fatigue. Based on the clinical and laboratory findings, a diagnosis of sepsis with disseminated intravascular coagulation, community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome was made. Microbial identification was performed by multiplex PCR and conventional culturing. Furthermore, antimicrobial susceptibility testing, whole genome sequencing, phylogenomic analysis and in silico prediction analysis of antimicrobial resistance genes and virulence factors were performed. RESULTS: S. pyogenes isolates were detected in pleural fluid and sputum of the patient. Both isolates belonged to the M1UK lineage of the emm1/ST28 clone, being closely related with an M1UK GAS strain from Australia. They exhibited resistance to erythromycin and clindamycin and susceptibility-increased exposure to levofloxacin and carried genes encoding for protein homologues of antibiotic efflux pumps. Moreover, several virulence factors, and a previously described single-nucleotide polymorphism in the 5' transcriptional leader sequence of the ssrA gene, which enhances expression of SpeA, were detected. CONCLUSIONS: The present antimicrobial-resistant M1UKS. pyogenes strain represents the first report of this emerging lineage associated with such manifestations of iGAS disease.

The Roles of GDF-9, BMP-15, BMP-4 and EMMPRIN in Folliculogenesis and In Vitro Fertilization.

Growth differentiation factor 9 (GDF-9) contributes to early ovarian development and oocyte survival. Higher concentrations of GDF-9 in follicular fluid (FF) are associated with oocyte nuclear maturation and optimal embryo development. In in vitro fertilization (IVF), GDF-9 affects the ability of the oocyte to fertilize and subsequent embryonic development. Bone morphogenetic protein 15 (BMP-15) is involved in the regulation of ovarian function and affects oocyte development. During IVF, BMP-15 contributes to the formation of competent blastocysts. BMP-15 may play a role in embryo implantation by affecting endometrial receptivity. Bone morphogenetic protein 4 (BMP-4) is involved in the regulation of follicle growth and development and affects granulosa cell (GC) differentiation. In relation to IVF, BMP-4 is important for embryonic development, influences cell fate and differentiation, and plays a role in facilitating embryo-endometrial interactions during the implantation process. Extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with ovulation and follicle rupture, promotes the release of mature eggs, and affects the modification of the extracellular matrix of the follicular environment. In IVF, EMMPRIN is involved in embryo implantation by modulating the adhesive properties of endometrial cells and promotes trophoblastic invasion, which is essential for pregnancy to occur. The purpose of the current article is to review the studies and recent findings of GDF-9, BMP-15, BMP-4 and EMMPRIN as fundamental factors in normal follicular development and in vitro fertilization.

Carbapenemase-producing Pseudomonas aeruginosa from central Greece: molecular epidemiology and genetic analysis of class I integrons.

BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa is a serious challenge for antimicrobial therapy of nosocomial infections, as it possesses several mechanisms of antimicrobial resistance. In Central Greece, a sudden increase of infections caused by carbapenem-resistant P. aeruginosa was observed during 2011, indicating the need for further analysis. METHODS: Five-hundred and sixty-eight P. aeruginosa isolates were collected consecutively during an 8-month period in 2011 from inpatients treated in three hospitals in the Thessaly region (1,000,000 habitants) of Greece. Carbapenem-resistant P. aeruginosa (n = 284) were characterized by antimicrobial susceptibility testing and ß-lactamase content, and the genetic relatedness of carbapenemase-producing isolates was assessed by BOX-PCR, multilocus sequence typing, and eBURST analysis. Mapping of the class I integrons of Verona integron-encoded metallo-ß-lactamase (VIM)-carrying isolates was also performed, and clinical data of the VIM producers were reviewed. RESULTS: Eighty (14.1%) out of the 568 P. aeruginosa isolates recovered from clinical specimens were VIM producers. Multilocus sequence typing revealed high prevalence of the international clones ST111 and ST235 among blaVIM-2- and blaVIM-4-positive isolates, respectively. blaVIM-17 was identified in an isolate of a novel sequence type (ST1457). blaVIM gene cassettes were carried by five distinct class I integrons, including two novel ones. CONCLUSIONS: Since the first report of VIM-producing P. aeruginosa in 2000, this microorganism still remains among the most prevalent multidrug resistant pathogens in Greece. The spread of VIM-producers belonging to the most common international clones (ST111 and ST235), the spread of integrons of divergent structures, and the emergence of novel integrons underscore their ongoing evolution.

Associations of Ambient Environmental Conditions with Growth and Dissemination of Staphylococcus epidermidis on the Surface of Teatcups from Sheep Milking Parlours.

The growth of two isolates of Staphylococcus epidermidis (one that was forming biofilm and one that was not) on new or used teatcups made of silicone for use in milking parlours for sheep, was assessed for 24 h after the application by smearing on the surface of the teatcup. Staphylococci were applied by smearing on an area of 0.0003142 (3.142 × 10−4) m2 on material obtained from the teatcups and their growth and expansion further on were monitored for 24 h at varying ambient conditions: temperature 21 °C or 31 °C and humidity 60% or 80%. No differences were evident between the two isolates in the frequency of recoveries in any of the conditions tested (p > 0.75 for all comparisons). Recovery rates were higher in humidity 80% compared to humidity 60%: 1678/2016 (83.2%) versus 1282/2016 (63.6%) (p < 0.0001), and in temperature 31 °C compared to temperature 21 °C: 1525/2016 (75.6%) versus 1435/2016 (71.2%) (p = 0.001). Recovery rates were also higher from new teatcups compared to used ones only in humidity 60%: 744/1008 (73.8%) versus 538/1008 (53.4%) (p < 0.0001). Humidity 80% was associated with higher speed of linear dissemination of the isolates on teatcup surface compared to humidity 60%: 0.000000640 (6.40 × 10−7) m s−1 versus 0.000000322 (3.22 × 10−7) m s−1 (+98.8%) (p < 0.0001); no such association was seen with higher temperature: 0.000000509 (5.09 × 10−7) m s−1 versus 0.000000453 (4.53 × 10−7) m s−1 for temperature 31 °C and 21 °C (+12.4%) (p = 0.29). As part of precision livestock farming, differing approaches can be instituted in accord with varying climatic conditions in different farms, as well as within the same farm with the change of seasons.

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.

Irregular Findings on Teatcups in Milking Parlours in Sheep and Goat Farms and Potential Predictors.

The objectives of the present study were as follows: (a) to describe the prevalence of irregular findings on teatcups in milking parlours with dairy sheep and goats after the post-milking cleaning procedures had been completed, (b) to associate staphylococcal isolation from teatcups with the presence with irregular findings and (c) to identify predictors of the presence of irregular findings on teatcups. The teatcups in the milking parlour of 255 sheep and 66 goat farms were macroscopically evaluated for the presence of irregular findings immediately after the completion of cleaning of the parlour. In total, 1115 and 303 teatcups, respectively, were assessed. A detailed interview with the farmer served to record the characteristics of the milking parlour and obtain information about husbandry and health management variables in the farm. Teatcups with macroscopically evident irregular findings were observed in 150 milking parlours (46.7%). Overall, 593 teatcups (41.8%) were found with macroscopically evident irregular findings. Dirt, milk residues and cracks or tears were recorded in the teatcups of 90.0%, 36.0% and 12.7% of parlours with irregular findings. Staphylococci were more frequently isolated from teatcups with irregular findings than from undamaged ones: from 37.4% (222/593) versus 12.8% (106/825). They were more frequently isolated from teatcups with milk residues (39.4%) and teatcups with dirt (39.0%). Via multivariable analysis, the following three variables emerged as significant predictors of presence of teatcups with irregular findings: the daily number of milking sessions, month into the lactation period at sampling and number of available milking units per animal position. The study provides, for the first time internationally, an appraisal of the frequency of problematic teatcups in the milking parlours of small ruminant farms. The analysis of predictors has provided a focus for specific management aspects, where interventions need to be performed, in order to improve the situation in farms with problems. The consequent increased staphylococcal burden on teatcups with irregular findings points to the increased risk of intramammary infections in such cases.

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.

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.

TNFRSF13B/TACI Mutations in Patients with Chronic Rhinosinusitis with Nasal Polyps.

BACKGROUND: The pathogenesis of Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) remains still inconclusive. Recent studies identified an increased expression of BAFF (a B cell-activating factor) and its receptor TACI (Transmembrane Activator and cAML Interactor) in nasal polyp samples, while TNFRSF13B/TACI mutations have been found in patients with benign lymphoproliferative disorders and primary antibody deficiencies. OBJECTIVE: The aim of our study was to evaluate the possible contribution of TNFRSF13B/TACI mutations in CRSwNP pathogenesis. METHODS: Forty-four (44) patients with CRSwNP (male/female: 33/11, mean age: 52.5 years, range: 16-83) were analyzed for TNFRSF13B/TACI mutations by PCR-sequencing. RESULTS: No pathogenic TNFRSF13B/TACI mutations were identified in our cohort study of CRSwNP patients. We detected two common missense mutations (p.P251L and p.V220A), along with other common silent mutations and intronic polymorphisms in an identical prevalence to healthy control population. CONCLUSION: TNFRSF13B/TACI mutations might not play a role in the pathogenesis of CRSwNP.

Isolation of Biofilm-Forming Staphylococci from the Bulk-Tank Milk of Small Ruminant Farms in Greece.

The objectives of this study were (i) to describe staphylococcal isolates recovered from bulk-tank raw milk collected from sheep and goat farms during a countrywide study performed in Greece, (ii) to study management factors potentially associated with their presence in bulk-tank milk and (iii) to provide evidence regarding their association with the quality of the milk. In total, 312 staphylococcal isolates, recovered from samples of bulk-tank raw milk from 444 small ruminant farms in Greece, were evaluated in this work. The in vitro formation of biofilm by the isolates was tested by combining the findings of (a) culture appearance on Congo Red agar plates and (b) results of a microplate adhesion test. The most frequently identified species was Staphylococcus aureus (75 isolates); other frequently recovered species were S. simulans (44 isolates), S. equorum (34 isolates) and S. haemolyticus (26 isolates); in total, 23 species were identified. In total, 224 (71.8%) isolates were biofilm-forming and were recovered from the bulk-tank milk samples of 148 sheep flocks (45.5%) and 55 goat herds (46.2%). There was evidence of seasonality in the isolation of staphylococci: during spring, mostly biofilm-forming isolates were recovered, whilst during summer, mostly non-biofilm-forming isolates were recovered. Among farms applying machine-milking, the proportion of farms from which biofilm-forming isolates were recovered was higher where water with temperature < 50 °C or ≥90 °C was used to clean the milking parlour. In the multivariable analyses, for farms applying machine-milking, the temperature of the water emerged as the only significant variable (p = 0.024), whilst in farms applying hand-milking, the only tendency that emerged was for the frequency of collection of milk from the farm tank (p = 0.08). In sheep flocks, recovery of biofilm-forming staphylococci from the bulk-tank milk was associated with higher somatic cell counts and higher total bacterial counts in the milk. The study identified abiotic factors related to the presence and isolation of these bacteria, specifically the temperature of water used for the cleaning of the milking parlour (in farms where machine-milking is applied) and the frequency of milk collection from the farm tank. These factors apply after the production of milk, and they could thus be regulated appropriately in order to reduce bacterial load and improve the quality of milk delivered to dairy plants. In sheep farms, an association was also seen between recovery of biofilm-forming staphylococci and high somatic cell counts in milk.

Longitudinal Study of Subclinical Mastitis in Sheep in Greece: An Investigation into Incidence Risk, Associations with Milk Quality and Risk Factors of the Infection.

The objectives of this work were (a) to describe the incidence risk of subclinical mastitis in dairy flocks throughout the milking period, (b) to present potential associations of subclinical mastitis with the quality of milk and (c) to identify risk factors for high-incidence risk of the infection. A longitudinal study was performed in 12 dairy sheep flocks in Greece. Mammary secretion samples from 240 ewes and bulk-tank milk samples were collected in four repeated visits over a period of six months for bacteriological, chemical and cytological examinations. Overall, the incidence risk of subclinical mastitis throughout the study period was 51.7%, and it varied among farms from 25.0% to 75.0%. The respective figure for staphylococcal subclinical mastitis was 48.8%. The incidence risk of recurrence of subclinical mastitis among ewes in the flocks was 35.4%. The most frequently identified bacteria from cases of subclinical mastitis were S. aureus and S. simulans; of the mastitis-causing staphylococcal isolates, 65.4% were biofilm-forming. Somatic cell counts in bulk-tank milk progressively increased as the lactation period advanced, with significant increases seen on the third and fourth visits to the farms. Somatic cell counts in bulk-tank milk correlated well with prevalence of subclinical mastitis in flocks. A significant inverse correlation and a significant positive correlation were seen for prevalence of subclinical mastitis versus total protein content or added water in bulk-tank milk. During multivariable analysis, younger age of newborns when taken away from the dam and consequently delayed start of milking of ewes, omission of anti-mastitis vaccination of ewes and lack of employed staff on the farms emerged to be significantly associated (positively) with increased incidence risk of subclinical mastitis.

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).

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.