Natacha Couto

Application of next generation sequencing in clinical microbiology and infection prevention

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.

Small plasmids carrying vga(A) or vga(C) genes mediate resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-resistant Staphylococcus aureus ST398 from swine

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) isolates of the sequence type ST398 have been identified recently from cases of exudative epidermitis in swine, but also as colonizers of apparently healthy swine in Portugal. A considerable number of these isolates displayed an unusual resistance phenotype, namely resistance to the lincosamide clindamycin, but susceptibility to the macrolide erythromycin. Since the genetic basis of this resistance phenotype is unknown, we selected five representative isolates to identify the respective resistance genes, to determine whether they are transferable and to investigate their genetic environment. Of the five MRSA ST398 isolates, three (E32, E33 and E49) were obtained from dust samples in three different holdings of breeding pigs during the European Community baseline study, one (E8) was from the nasal swab of an apparently healthy sow and the remaining one (E18) was from a skin lesion of a piglet suffering from exudative epidermitis. PCR screening for the presence of the lincosamide resistance gene lnu(A) yielded negative results in repeated experiments using plasmid DNA or chromosomal DNA as targets. A full antibiogram of these isolates, as conducted by broth microdilution according to CLSI document M31-A3, revealed that besides the previously reported resistance to oxacillin, clindamycin and tetracycline, all five isolates displayed high MIC values of ≥128 mg/L for the pleuromutilin tiamulin and ≥32 mg/L for the streptogramin A antibiotic virginiamycin M1. Combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics has recently been described to be associated with the resistance genes vga(A) and vga(C), both of which are located on plasmids. Plasmid profiling revealed 1–3 plasmids in each of the five MRSA ST398 isolates. The plasmids were subjected to transformation into the plasmid-free recipient strain S. aureus RN4220, with subsequent selection of the transformants on medium containing clindamycin (2 mg/L). All five isolates yielded transformants that harboured a single plasmid of either 5.3 (E49) or 5.7 kb (E8, E18, E32 and E33). Susceptibility testing confirmed that all transformants displayed resistance or high MICs (in cases where no applicable breakpoints are available to classify an isolate as resistant) to lincosamides, pleuromutilins and streptogramin A antibiotics. Restriction analysis with the enzymes PvuII, DraI, PstI and BglII showed that the 5.3 kb plasmid, designated pCPS49, differed distinctly from the 5.7 kb plasmids, which were indistinguishable based on restriction analysis with the aforementioned endonucleases, but also with HindIII, PstI, ClaI, EcoRI, XhoI and EcoRV. The plasmid obtained from the E32 transformant, designated pCPS32, was chosen as representative of this group of plasmids for further analysis. HindIII fragments of plasmids pCPS32 and pCPS49 were cloned into pBluescript II SK+, and sequenced completely on both strands. Plasmid pCPS32 was 5718 bp in size (accession no. FN806791). It showed 99.9% sequence identity to the 5713 bp plasmid pVGA obtained from a human clinical S. aureus in Portugal. Sequence analysis revealed the presence of three open reading frames (Figure 1). The first reading frame coded for a Rep protein of 312 amino acids involved in replication of plasmid pCPS32. The second reading frame encoded a Vga(A) ABC transporter protein of 522 amino acids that is responsible for combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The third reading frame coded for a Mob protein of 325 amino acids involved in plasmid mobilization. While the Rep and Vga(A) proteins of pCPS32 were indistinguishable from those of pVGA, the Mob protein showed a single amino acid exchange, Thr50 (pCPS32) versus Ala50 (pVGA). Plasmid pCPS49 was 5292 bp in size (accession no. FN806792). It also comprised three reading frames for proteins involved in plasmid replication, antimicrobial resistance and mobilization. Plasmid pCPS49 harboured a vga(C) gene encoding a 522 amino acid ABC transporter identical to that described in the revised sequence of plasmid pKKS825 (accession no. FN377602). The Vga(C) protein also conferred combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The pre/mob gene for plasmid recombination and

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period

OBJECTIVES The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. METHODS Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. RESULTS The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. CONCLUSIONS The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the dissemination of highly successful human clones. Urgent measures, such as determination of clinical breakpoints and guidelines for antimicrobial use, are needed.

First Report of OXA-23-Mediated Carbapenem Resistance in Sequence Type 2 Multidrug-Resistant Acinetobacter baumannii Associated with Urinary Tract Infection in a Cat

Carbapenem resistance in multidrug-resistant Acinetobacter baumannii has been challenging human medicine ([1][1], [2][2]) and has also emerged in Acinetobacter spp. from animals; it is associated with the expression of OXA-23 in cattle and horses and NDM-1 in a porcine isolate ([3][3][–][4][5][5

Evaluation of antimicrobial resistance and virulence of enterococci from equipment surfaces, raw materials, and traditional cheeses.

Forty enterococci isolated along the production chains of three traditional cheeses (PDO Pecorino Siciliano, PDO Vastedda della Valle del Belìce, and Caciocavallo Palermitano) made in Sicily (southern Italy) were studied for the assessment of their antibiotic resistance and virulence by a combined phenotypic/genotypic approach. A total of 31 Enterococcus displayed resistance to at least one or more of the antimicrobials tested. The strains exhibited high percentages of resistance to erythromycin (52.5%), ciprofloxacin (35.0%), quinupristin-dalfopristin (20.0%), tetracycline (17.5%), and high-level streptomycin (5.0%). The presence of tet(M), cat(pC221), and aadE genes for resistance to tetracycline, chloramphenicol, and streptomycin, respectively, was registered in all strains with resistance phenotype. The erm(B) gene was not detected in any erythromycin-resistant strain. The Enterococcus strains were further tested by PCR for the presence of virulence genes, namely, gelE, asa1, efaA, ace, and esp. Twenty strains were positive for all virulence genes tested. Among the enterococci isolated from final cheeses, three strains (representing 33.3% of total cheese strains) were sensible to all antimicrobials tested and did not carry any virulence factor. Although this study confirmed that the majority of dairy enterococci are vectors for the dissemination of antimicrobial resistance and virulence genes, only two strains showed a high resistance to aminoglycosides, commonly administered to combat enterococci responsible for human infections. Furthermore, the presence of the strains E. casseliflavus FMAC163, E. durans FMAC134B, and E. faecium PON94 without risk determinants, found at dominating levels over the Enterococcus populations in the processed products, stimulates further investigations for their future applications in cheese making. All strains devoid of the undesired traits were isolated from stretched cheeses. Thus, this cheese typology represents an interesting environment to deepen the studies on the risk/benefit role of enterococci in fermented foods for their qualified presumption of safety (QPS) assessment.

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 animals were 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 -10 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.

Risk factors for faecal colonisation with Escherichia coli producing extended-spectrum and plasmid-mediated AmpC β-lactamases in dogs

The aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC β-lactamase (pAmpC) Escherichia coli producers in dogs. A three-month cross-sectional study was conducted and 151 rectal swabs were obtained from healthy dogs. ESBL and pAmpC genes were detected by PCR and were sequenced. Logistic regression models were used to investigate risk factors for the carriage of ESBL and pAmpC-producing E. coli. About 15 per cent of the isolates carried ESBL genes (blaCTX-M-32 n=8, blaCTX-M-15 n=5, blaCTX-M-1 n=3, blaCTX-M-9-like n=4) and 20 per cent carried pAmpC genes (blaCMY-2 n=23, blaCMY-2-like n=2). Thirteen dogs carried an E. coli isolate with both an ESBL and a pAmpC gene. One E. coli isolate harboured the human blaDHA-1 pAmpC gene, which has not been previously reported in companion animals in Europe. Dogs with a history of antimicrobial therapy in the past year had a higher risk of being carriers of ESBL-producing (P=0.003, OR =7.85) and pAmpC-producing (P=0.005, OR=6.28) E. coli. Dogs from shelter/breeders were approximately three times more likely to have an ESBL- or a pAmpC-producing E. coli than dogs from private owners. Males have a reduced risk of carrying a pAmpC-producing E. coli than females (P=0.017, OR =0.28). The knowledge of potential risk factors may help to limit the impact of resistance through implementation of effective control measures and judicious antimicrobial therapy.

Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals.

ABSTRACT Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)–4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB–1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections.

Identification of vaccine candidate antigens of Staphylococcus pseudintermedius by whole proteome characterization and serological proteomic analyses

The recent emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) has complicated considerably the treatment of infections caused by these bacteria. Therefore new treatment strategies are urgently needed, namely through the development of vaccines towards the control of bacterial infections. Our study describes an extensive characterization of the proteome of S. pseudintermedius through a 2-DE MALDI-TOF/TOF approach, followed by SERological Proteome Analysis (SERPA) to identify potential vaccine candidate antigens. We were able to identify 361 unique proteins, of which 39 are surface proteins. In order to assess the immunogenic potential of S. pseudintermedius proteins, a Western blot analysis of two-dimensional gels was carried out with serum from healthy dogs, dogs with atopic dermatitis infected and not infected with S. pseudintermedius. Only immunogenic areas detected by ≥ 50% of the dogs with atopic dermatitis infected with S. pseudintermedius sera and by <50% of the healthy dogs sera were excised and identified from Coomassie-colloidal stained gels. The areas identified by IgE were not considered as vaccine targets, because those proteins could induce hypersensitivity. We were able to identify 13 unique proteins after in-gel digestion of selected protein gel spots, with 4 antigenic proteins showing promising features for vaccine development. No specific antibodies were identified in the dogs with atopic dermatitis not infected with S. pseudintermedius sera that could contribute to prevention of infection. The SERPA approach employed in this study revealed novel candidate therapeutic targets for the control of S. pseudintermedius infections.

First description of fexA-positive meticillin-resistant Staphylococcus aureus ST398 from calves in Portugal

Meticillin-resistant Staphylococcus aureus (MRSA) primarily causes human diseases, and food-producing animals are known to be reservoirs of MRSA clonal complex (CC) 398 [1,2]. MRSA CC398 has become a rapidly emerging cause of human infections, most often associated with livestock exposure [2]. A direct association between animal and human MRSA carriage has been established [3]. Until now, no data were available regarding MRSA in calves in Portugal. In this study, nasal swabs were taken from randomly selected breeding calves at two closed-cycle farms from distinct regions of Portugal. In total, 247 nasal swabs from Farm A and 60 from Farm B were cultured as previously described [4]. Antimicrobial susceptibility testing was performed using the broth microdilution method [MicroScan PM21; Siemens, Erlangen, Germany; and VetMIC (Large Animals); National Veterinary Institute, Uppsala, Sweden] for the following antibiotics: chloramphenicol; ciprofloxacin; erythromycin; florfenicol; fosfomycin; fusidic acid; gatifloxacin; gentamicin; levofloxacin; linezolid; moxifloxacin; mupirocin; netilmicin; oxacillin; penicillin; quinupristin/dalfopristin; rifampicin; tetracycline; teicoplanin; trimethoprim/sulfamethoxazole; and vancomycin. Results of susceptibility testing were interpreted according to Clinical and Laboratory Standards Institute (CLSI) standards. The mecA and/or mecC genes were identified by PCR (http://www.crlar.eu), and MRSA isolates were subjected to staphylococcal protein A (spa) typing (http://www.seqnet.org/), staphylococcal cassette chromosome mec (SCCmec) typing [5] and pulsed-field gel electrophoresis (PFGE) using ApaI restriction [4]. The results of PFGE were interpreted using BioNumerics software v.4.6 (Applied Maths, Sint-Martens-Latem, Belgium). The presence of fexA, tet(K),