Igor Loncaric

Characterization of methicillin-resistant Staphylococcus spp. carrying the mecC gene, isolated from wildlife

OBJECTIVES A recently identified mecA homologue, mecC, in methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from humans and different animal hosts. The aim of this study was to determine antimicrobial resistance and provide molecular characterization of MRSA and methicillin-resistant non-Staphylococcus aureus staphylococci (MRnSA) isolated from wildlife that carried the gene mecC. METHODS Five S. aureus and one coagulase-negative Staphylococcus isolate displaying phenotypic oxacillin resistance, but not recognized with conventional PCR for mecA, were further characterized by a polyphasic approach. The presence of mecC in all isolates was determined using specific PCR. PCR targeting Panton-Valentine leucocidin (PVL) genes of MRSA was performed. MRSA isolates were genotyped by spa typing and multilocus sequence typing. All isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing. 16S rDNA sequence analysis for MRnSA identification was performed. Antimicrobial susceptibility testing was performed for all isolates. RESULTS All five MRSA isolates contained the mecC gene, were PVL negative, carried SCCmec type XI and belonged to ST130 (where ST stands for sequence type), with spa types t843, t10513 or t3256, or to ST2620, with spa type t4335. The MRnSA isolate, most closely related to Staphylococcus stepanovicii, carried mecA and blaZ genes related to SCCmec XI. MRSA isolates exhibited resistance to the β-lactams only. CONCLUSIONS The MRSA isolates described in this study represent the first detection of mecC-positive MRSA in a European otter (Lutra lutra) and a European brown hare (Lepus europaeus). The MRnSA isolate represents the first isolation of MRnSA from a Eurasian lynx (Lynx lynx).

Identification and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian companion animals and horses

The aim of this study was to investigate the antimicrobial resistance, resistance gene patterns and genetic relatedness of a collection of Austrian methicillin-resistant Staphylococcus aureus (MRSA) isolates from companion animals and horses. A total of 89 non-repetitive MRSA isolates collected during routine veterinary microbiological examinations from April 2004 to the end of 2012, and one isolate from 2013 were used for this study. The presence of mecA and other resistance genes was confirmed by PCR. Isolates were genotyped by spa typing, two multiple-locus variable-number tandem repeat analyses (MLVA) analyses, SCCmec typing and multilocus sequence typing (MLST). PCR targeting Panton-Valentine leukocidin (PVL) and detection of staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST) was performed using PCR assays. Antimicrobial susceptibility testing was performed. Five sequence types (STs-ST398, ST254, ST22, ST5 and ST1), SCCmec types II, IVa, V, and non-type-abele, 8 spa-types (t003, t011, t036, t127, t386, t1348, and t4450), and two isolates could not be assigned, 21 MLVA-14Orsay types Multiplex-PCR MLVA (mMLVA) displayed 17 different MLVA types. The present study is the most comprehensive dealing with MRSA from Austrian companion animals and horses. The results confirm that MRSA ST398 is present in a wide range of animal species and is predominant especially in horses. In other companion animals it is unclear whether the infections with the different MRSA isolates investigated in the present study truly represents a rare phenomenon or may be an emerging problem in companion animals.

Genetic diversity among isolates of Paenibacillus larvae from Austria.

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and alphab). Genotypes aB and alphab are new and have not been reported in other studies using the same techniques.

Comparison of ESBL--and AmpC producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) isolated from migratory and resident population of rooks (Corvus frugilegus) in Austria.

In order to test whether rooks (Corvus frugilegus) represent good indicators for the potential circulation of antibiotics in their native habitat, two populations with different migratory behavior were tested for the presence of beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). In all, 54 and 102 samples of fresh feces of a migratory and a resident population were investigated. A total of 24 and 3 cefotaxime-resistant enterobacterial isolates were obtained from the migratory and resident population, respectively. In these isolates CTX-M-1 (n = 15), CTX-M-3 (n = 3), and CTX-M-15 (n = 3) genes were detected. TEM-1 and OXA-1 were associated with CTX-M in 3 and 2 isolates, respectively. In two E. coli isolates CMY-2 could be detected, where from one isolate displayed an overexpression of chromosomal AmpC as well. Among E. coli isolates the most common phylogenetic group was A (n = 11) and ST1683 (n = 5). In one E. coli of B2-ST131 the rfbO25b locus was detected. Three Enterobacter isolates were stably derepressed AmpC-producers. In five samples of the migratory population, PVL positive MRSA could be isolated. Two isolates were typed SCCmec IVa, spa type t127, and ST1. Three isolates carried a SCCmec type IVc, with spa type t852 and ST22. The highly significant difference of the occurrence of antibiotic resistance between the migratory population from eastern Europe compared to resident population in our study indicates that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria, especially due to their ecology, foraging behavior and differing migratory behavior.

Typing of Pantoea agglomerans isolated from colonies of honey bees (Apis mellifera) and culturability of selected strains from honey

Pantoea agglomerans is a possible biocontrol agent against fire blight (Erwinia amylovora) and a facultative pathogen of humans. Isolates were gathered from flowers, pollen loads, honey sacs, and freshly stored nectar (FSN). Under artificial inoculation conditions, strains completely lost their culturability at 24 °C after 120 h of incubation in honey and 156 h in honey solution, respectively. None of tested strains could be cultivated from FSN, honey, or honey solution after 48 h at temperatures higher then 28 °C. At different time intervals, culturable population levels at 35 °C and 24 °C were significantly higher in blossom honey or its solution than in blossom and honeydew honey or its solution. Our results indicated that P. agglomerans is widely spread throughout honey bee’s environment. Strains lost culturability after short periods of incubation in honey or honey solution. In samples of honey and royal jelly from test colonies, no culturable P. agglomerans isolates could be detected.ZusammenfassungZiel dieser Studie war die Untersuchung der Diversität von Pantoea agglomerans und seiner Rückverfolgbarkeit von Trachtpflanzen zum Bienenvolk, sowie die Abschätzung der kultivierbaren Keimzahl (CPL) dieses Bakteriums in Honig, Honiglösung und frisch eingelagertem Nektar (FSN). P. agglomerans ist ein möglicher Kandidat zur biologischen Bekämpfung von Feuerbrand (Erwinia amylovora),wurde aber auch als fakultativ human-pathogener Keim beschrieben.Blüten verschiedener Pflanzen, Pollenhöschen, Honigblaseninhalt und frisch eingelagerter Nektar wurden gesammelt, aufbereitet und das gewonnene Probenmaterial auf Agarplatten inkubiert, um Isolate von P. agglomerans zu gewinnen. Zur Bewertung der Diversität wurden SDS-PAGE, RAPD- and ERIC-PCR eingesetzt. Die Abschätzung der Verwandtschaft der Isolate erfolgte mittels Cluster-Analyse.Zur Untersuchung der Kultivierbarkeit von P. agglomerans in Honig, Honiglösung und frisch eingelagertem Nektar wurden diese mit einer wässrigen Suspension von 5 ausgewählten Stämmen des Bakteriums künstlich inokuliert und sorgfältig durchmischt. In bestimmten Intervallen wurden Proben entnommen, auf Agarplatten ausplattiert und diese bebrütet. Für jeden Stamm wurde die Zahl der kultivierbaren koloniebildenden Einheiten (KBE) ermittelt.Von Blüten verschiedener Pflanzenarten, Pollenhöschen, Honigblaseninhalten und frisch eingelagertem Nektar konnten einzelne Isolate von P. agglomerans gewonnen werden, nicht aber aus Honig- und Weiselfuttersaftproben der Testvölker. Auf Basis der Koloniemorphologie, Pigmentierung, biochemischen Eigenschaften und dem Vergleich der Proteinmuster mit Referenzstämmen nach Natriumsulfat-Polyacrylamid Gel Elektrophorese (SDS-PAGE) wurden 301 Isolate ausgewählt. Von diesen zeigten 50 Stämme unterschiedliche Eiweißprofile. Die Analyse mittels RAPD-PCR erbrachte die gleiche Profilanzahl PCR (Abb. 1). Eine Identifizierung der isolierten Stämme erfolgte mit ERIC-PCR (Abb. 2).Aus den künstlich inokulierten Proben konnte auf PYE Agar bei 24 °C und einer Inkubationsdauer von mehr als 120 h (Honig) bzw. 156 h (Honiglösung) keiner der Teststämme reisoliert werden. Bei Temperaturen über 28 °C war bereits nach 48 h Bebrütungsdauer keine Rückisolation der Teststämme aus Honig, Honiglösung oder frisch eingelagertem Nektar mehr möglich.Sowohl bei 35 °C als auch bei 24 °C war die kultivierbare Keimzahl der Teststämme zu verschiedenen Zeitintervallen in „Blütenhonig“ (BH) signifikant höher (P < 0,05) als in „Blütenmit Waldhonig“, oder ihren Lösungen (Abb. 4).Gestützt auf diese Ergebnisse kann geschlossen werden, dass die Zeit, in der die getesteten P. agglomerans Stämme aus Honig kultiviert werden konnten, beträchtlich kürzer ist, als die Zeitspanne, die in der imkerlichen Praxis zwischen dem Sammeln des Nektars und der Ernte des Honigs üblicherweise eingehalten wird. Im Falle eines Einsatzes von P agglomerans als biologisches Mittel zur Feuerbrandbekämpfung in blühenden Obstanlagen kann dies ein wichtiger Punkt sein.

Suspected Goat to Human Transmission of Methicillin-Resistant Staphylococcus aureus Sequence Type 398

ABSTRACT Transmission of methicillin-resistant Staphylococcus aureus (MRSA) between animals and humans is widely recognized. In this study, we describe the first case of infection of a goat and suspected transmission of MRSA ST398 to a human, which resulted in colonization of animal owners by MRSA sequence type 398.

DIVERSITY OF Staphylococcus aureus ISOLATES IN EUROPEAN WILDLIFE

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation.

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

Characterization of selected Gram-negative non-fermenting bacteria isolated from honey bees (Apis mellifera carnica)

This study was conducted to improve the knowledge about bacteria associated with honey bees, Apis mellifera carnica. In this survey, the diversity of Gram-negative non-fermenting bacteria isolated and cultivated from pollen loads, honey sac, freshly stored nectar, and honey was investigated. Bacteria were characterized by a polyphasic approach. Based on morphological and physiological characteristics and comparison of isolates protein patterns after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 11 protein similarity groups were established and confirmed by enterobacterial repetitive intergenic consensus PCR. One isolate, representing a protein similarity group (representative strain), was characterized in more detail by analysis of respiratory quinones, amplified ribosomal DNA restriction analysis, and 16S rDNA sequence analysis. Based on the results of these examinations, seven representative strains were identified as members of the genus Pseudomonas. The remaining representative strains were allocated to the genera Acinetobacter, Chryseobacterium, Stenotrophomonas, and Commamonas.

European multicenter study on antimicrobial resistance in bacteria isolated from companion animal urinary tract infections

BackgroundThere is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012–2013 and temporal trends of bacteria resistance were established by logistic regression.ResultsThe aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium.ConclusionsThis work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.