Slime-producing staphylococci as causal agents of subclinical mastitis in sheep.

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection.

Extensive countrywide field investigation of subclinical mastitis in sheep in Greece.

The objectives of this work were (1) to investigate prevalence of subclinical mastitis, (2) to identify etiological agents involved, and (3) to study factors potentially predisposing ewes to subclinical mastitis. Milk samples were collected from 2,198 ewes in 111 farms with a total population of 35,925 ewes, in all 13 administrative regions of Greece, for bacteriological and cytological examination. Prevalence of subclinical mastitis was 0.260. Main etiological agents were staphylococci (Staphylococcus aureus and coagulase-negative species), which accounted for 0.699 of all isolates recovered; prevalence of staphylococcal mastitis was 0.191. In a multivariable mixed-effects analysis, the primary factor found to be associated with increased prevalence of subclinical mastitis was the management system practiced in flocks (flocks under a semi-intensive system had the highest prevalence). Other factors that were included in the multivariable model were the stage of lactation period (ewes in the 2nd month postpartum showed the highest prevalence) and application of postmilking teat dipping. In contrast, measures taken at the end of a lactation period (e.g., intramammary administration of antimicrobial agents) were not found to have an effect on prevalence of subclinical mastitis. The results confirmed the significance of subclinical mastitis as a frequent problem of ewes, with staphylococci as the primary etiological agent. The findings confirm the multifactorial nature of subclinical mastitis and indicate that its control should rely on many approaches.

First description in Europe of the emergence of Enterococcus faecium ST117 carrying both vanA and vanB genes, isolated in Greece.

OBJECTIVES: An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. METHODS: Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. RESULTS: E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. CONCLUSIONS: This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.

Emergence of sequence type 11 Klebsiella pneumoniae coproducing NDM-1 and VIM-1 metallo-ß-lactamases in a Greek hospital.

Sequence type 11 Klebsiella pneumoniae, coproducing NDM-1 and VIM-1 metallo-ß-lactamases, were isolated in a Greek hospital. blaNDM-1 was part of a Tn125 derivative, located on an ~90-kb plasmid similar to the NDM-1-encoding plasmid pB-3002cz. blaVIM-1 was located in an In-e541-like integron, carried on a multireplicon (IncA/C and IncR) plasmid of ~180kb.

Pitfalls in the identification of Enterococcus species and the detection of vanA and vanB genes.

UNLABELLED: The aims were to assess the performance of Vitek 2 in identifying enterococcal species and the implementation of GeneXpert(®) vanA/vanB PCR for the detection of vancomycin-resistant enterococci (VRE). Gram-positive cocci from clinical and environmental specimens (n = 431) suspicious of being enterococci by conventional methods were evaluated by Vitek 2. This system identified 296 Enterococcus faecium, 87 Enterococcus faecalis, 10 Enterococcus villorum, 9 Enterococcus gallinarum, 9 Enterococcus durans, 5 Enterococcus casseliflavus, 1 Enterococcus spp. and 14 isolates as Non-Enterococcus. All strains were submitted to pulsed field gel electrophoresis (PFGE) analysis showing 64 banding patterns. Representative strains from each banding pattern were further characterized to species level by 16S rDNA sequencing. The misidentification rate by Vitek 2 to species level among 429 molecularly identified enterococci was 6% (26 isolates). Additionally, 372 rectal swabs were obtained from critically ill patients. They were evaluated for the presence of VRE by ChromID VRE combined with in-house PCR vs GeneXpert(®) . GeneXpert(®) showed high (>92%) sensitivity, specificity, accuracy for vanA-positive Enterococcus detection, as well as, sensitivity and specificity for vanB-positive strains. Positive predictive value for detection of vanB-positive enterococci by GeneXpert(®) vanA/vanB was low (30%). GeneXpert(®) showed the same efficacy as ChromID VRE in detecting vanA-positive enterococci, but lower for vanB-gene detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that even though the performance of Vitek 2 Advanced Expert System was good in identifying enterococci to species level, it is important to verify results by a molecular method when phenotypic findings are discordant with epidemiologic patterns. Furthermore, GeneXpert(®) vanA/vanB PCR and ChromID VRE combined with in-house PCR were applied in rectal samples for the detection of VRE colonization among critically ill patients. GeneXpert(®) showed an excellent performance in detecting vanA-positive enterococci, but false-positive results for vanB-gene detection render its application problematic in departments with high incidence of vanB-positive enterococci.

Dissemination of intestinal pathogens between lambs and puppies in sheep farms.

Objectives of the present work were (i) to confirm pathogens implicated in cases of diarrhoea in newborn and young lambs in sheep farms in Greece and (ii) to investigate a possible relation in dissemination of pathogens between lambs and dogs present in the farm. Work was carried out in 22 sheep farms, with (i) flock size over 150 animals, (ii) presence of clinical signs of diarrhoea in lambs in the flock and (iii) close and continuous contact and movement of shepherd dogs within the animal shed of each farm. Faecal sample collection from lambs was performed within 48 h of onset of clinical signs and prior to administration of any antimicrobial or antiparasitic medication to lambs. Faecal samples were also collected from puppies in the farm. In total, samples were collected from 126 lambs and 58 puppies. Samples were processed by using established techniques for isolation of bacteria, detection of viruses and observation of protozoan oocycts. Escherichia coli isolates obtained during the study, were tested for antimicrobial resistance against a variety of antimicrobial agents. In total, 236 bacterial isolates were recovered from faecal samples of lambs and 165 isolates from faecal samples of puppies. E. coli was the most frequently isolated microorganism: 104 isolates from lambs and 109 isolates from puppies were recovered. Other bacteria isolated were Enterobacter spp., Proteus spp., Klebsiella spp., (lambs and puppies), Clostridium perfringens, Citrobacter freundi, Salmonella enterica subsp. diarizonae (only lambs) and Streptococcus spp. (only puppies). Group A Rotavirus was detected in samples from lambs (2.5%) and Parvovirus in samples from puppies (5%). Cryptosporidium spp. oocysts were observed in samples from lambs and puppies. This is the first report of isolation of S. enterica subsp. diarizonae and of detection of Rotavirus from lambs in Greece. Rates of E. coli isolates from puppies resistant to antimicrobial agents were, in general, smaller than respective rates in isolates from lambs. Two pairs of isolates from the same farm (one from a lamb and one from a puppy) with identical patterns of resistance to antimicrobial agents were detected, which provides some evidence in support of a hypothesis that members of each pair might possibly have been spread from one animal species to the other.

Rapid dissemination of colistin and carbapenem resistant Acinetobacter baumannii in Central Greece: mechanisms of resistance, molecular identification and epidemiological data.

BACKGROUND: Colistin-resistant/carbapenem-resistant Acinetobacter baumannii is a significant challenge for antibiotic treatment and infection control policies. Since 2012, in Central Greece an increase of colistin/pan- resistant A. baumannii has occurred, indicating the need for further analysis. METHODS: A total of 86 colistin-resistant/carbapenem-resistant out of 1228 A. baumannii clinical isolates, consecutively collected between 2012 and 2014 in a tertiary Greek hospital of Central Greece, as well as one environmental isolate from surveillance cultures were studied. Molecular typing and mechanisms of resistance to colistin and to carbapenems were assessed, whereas, epidemiological and clinical data of the patients were reviewed. RESULTS: During the study period, the rate of colistin resistance gradually increased and reached 21.1 % in 2014. All colistin-resistant/carbapenem-resistant A. baumannii belonged to 3LST ST101 clone that corresponds to the international clonal lineage II. Carbapenem resistance was associated with the presence of bla oxa-23-like, while resistance to colistin probably correlated with G54E and R109H amino acid substitutions in PmrA and PmrC, respectively. CONCLUSIONS: Epidemiological data of the patients indicated that the first detection of colistin-resistant/carbapenem-resistant ST101 clone in the University Hospital of Larissa (UHL) was associated with a patient who previously had received colistin, while, the movement of the infected patients into the hospital probably resulted to its spread.