Complete sequences of two Staphylococcus aureus plasmids carrying genes for resistance to antibiotics, heavy-metals, biocides and/or virulence

[P292]Aim: To gain detailed insight into the genetic organization of two multidrug resistance plasmids, pSM31 and pSM39, from clinical S. aureus isolates collected in a hospital in Lisbon, Portugal, and to identify factors that may explain their selection and persistence. Methods: Plasmid pSM31 was sequenced by Sanger sequencing using cloning and primer walking whereas pSM39 was sequenced by next generation sequencing. Sequence analysis was carried out using the BlastN and BlastP programs and the ORF Finder software from NCBI.Results: The two plasmids fully characterized in this work were non-conjugative multiresistance plas-mids. pSM31 (27,424 bp) is closely related to the family of globally dispersed plasmids from the pGSA23 group harbouring determinants for resistance to β-lactams and cadmium as well several enterotoxin genes. pSM39 (26,036 bp) is a newly described member of the pGSA11 group that carries genes con-ferring resistance to β-lactams, biocides, cadmium, zinc and mercury and comprises a region that may have been acquired by recombination with a S. epidermidis plasmid. Conclusions: The sequence analysis of these two plasmids confirmed their involvement in antimicrobial resistance among S. aureus strains isolated from a clinical setting. It also provided genetic evidence for the potential risk of co-selection of antibiotic resistance genes and/or virulence genes by selective pressure of antibiotics, biocides and/or heavy metals.otherpublishe

Nucleotide sequence and transfer properties of two novel types of Actinobacillus pleuropneumoniae plasmids carrying the tetracycline resistance gene tet(H)

17 p.The aim of this study is to analyze the sequence and transfer properties of two tetracycline resistance plasmids found in clinical isolates of Actinobacillus pleuropneumoniae in order to assert their role in the spread of tetracycline resistenceS

Clonal diversity, virulence patterns and antimicrobial and biocide susceptibility among human, animal and environmental MRSA in Portugal

Objectives: The objective of this study was to identify the Staphylococcus aureus clonal types currently circulating in animals, humans in contact with animals and the environment in Portugal based on genetic relatedness, virulence potential and antimicrobial/biocide susceptibility. Methods: Seventy-four S. aureus isolates from pets, livestock, the environment and humans in contact with animalswere characterized by SCCmec typing, spa typing, PFGE and CC398-specific PCR, by antimicrobial and biocide susceptibility testing and by detection of resistance genes and genes for efflux pumps. Representative strains were analysed by DNA microarray and MLST. Results: The S. aureus isolates represented 13 spa types and 3 SCCmec types and belonged to three clonal complexes (CC5, CC22 and CC398). Most of the isolates were multiresistant and harboured the resistance genes that explained the resistance phenotype. The qacG and qacJ genes for biocide resistance were detected in 14 isolates (all MRSA CC398), while 4 isolates (3 CC5 and 1 CC22) had insertions in the 210 motif of the norA promoter. Isolates of the clonal lineages associated with pets (CC5 and CC22) harboured specific sets of virulence genes and often a lower number of resistance genes than isolates of the clonal lineage associated with livestock animals (CC398). Conclusions:We found, for the first time in animals in Portugal, four strains belonging to CC5, including ST105-II, a lineage that has been previously reported as vancomycin-resistant S. aureus in Portugal. Moreover, for the first time the qacG and qacJ genes were detected in MRSA CC398 strains. Active surveillance programmes detecting MRSA not only in livestock animals but also in companion animals are urgently needed

Draft Genome Sequences of Three Porcine Streptococcus suis Isolates Which Differ in Their Susceptibility to Penicillin

The draft genome sequences of three Streptococcus suis isolates, IMT40343, IMT40201, and IMT40738, are presented here. These isolates were obtained from bronchoalveolar lavage fluid of healthy and diseased weaners from different German piglet-producing farms and differed in their susceptibility to penicillin

Unusual small plasmids carrying the novel resistance genes dfrK or apmA isolated from methicillin-resistant or -susceptible staphylococci

Objectives: The aims of this study were to identify small staphylococcal plasmids that carry either the trimethoprim resistance gene dfrK or the apramycin resistance gene apmA and analyse them for their structure and organization with regard to their potential role as precursors of large multiresistance plasmids that carry these genes. Methods: Trimethoprim- or apramycin-resistant staphylococci from the strain collections of the two participating institutions were investigated for the presence of plasmid-borne dfrK or apmA genes. The dfrK- or apmA-carrying plasmids were sequenced completely and compared with sequences deposited in the databases. Results: Two small plasmids, the 4957 bp dfrK-carrying plasmid pKKS966 from porcine Staphylococcus hyicus subsp. hyicus and the 4809 bp apmA-carrying plasmid pKKS49 from porcine methicillin-resistant Staphylococcus aureus were identified. Structural analysis revealed that both plasmids had a similar organization, comprising a single resistance gene (dfrK or apmA), a plasmid replication gene (rep) and three partly overlapping genes for mobilization proteins (mobA, mobB and mobC). Comparisons showed 71%-82% amino acid identity between the Rep and Mob proteins of these two plasmids; however, distinctly lesser percentages of identity to Rep and Mob proteins of staphylococci and other bacteria deposited in the databases were detected. Conclusions: Both plasmids, pKKS966 and pKKS49, appeared not to be typical staphylococcal plasmids. The homology to larger plasmids that harbour the genes apmA and/or dfrK was limited to these resistance genes and their immediate upstream and downstream regions and thus suggested that these small plasmids were not integrated into larger plasmids

Proposal of Epidemiological Cutoff Values for Apramycin 15 μg and Florfenicol 30 μg Disks Applicable to Staphylococcus aureus

Funding Information This study was supported by Project BIOSAFE funded by FEDER through the Programa Operacional Factores de Competitividade–COMPETE and by Fundação para a Ciência e a Tecnologia (FCT, Portugal)—Grant LISBOA-01-0145-FEDER-030713, PTDC/CAL-EST/30713/2017 and by FCT through funds to GHTM (UID/04413/2020), CIISA Project (UID/CVT/00276/2020), and Project PTDC/CVT-CVT/28469/2017. The contributions of Andrea T. Feßler and Stefan Schwarz were financially supported by the German Federal Ministry of Education and Research (BMBF) under project numbers 01KI1727D and 01KI2009D as part of the Research Network Zoonotic Infectious Diseases. Part of this research was supported by Cost Action CA18217: European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT).Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.publishersversionpublishe

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

Objectives The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. Methods A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. Results Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n = 2), IV (n = 6), V (n = 14) or VII-241 (n = 4) or were non-typeable (n = 2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated β-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6′)-Ie-aph(2′)-Ia] (88.3%), kanamycin [aph(3′)-III] (90.3%), streptomycin [ant(6′)-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (catpC221) (57.3%) and rifampicin (1.9%). Conclusions Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challeng

Comparative Analysis of ESBL-Positive Escherichia coli isolates from Animals and Humans from the UK, the Netherlands and Germany

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans