Jesper Boye Nielsen

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of blaTEM, blaSHV, and blaCTX-M, and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and blaCTX-M15, blaSHV-28, and blaTEM-1 positive by PCR. Ten out of 52 were ST16 and tested positive for blaCTX-M15, blaSHV-1, and blaTEM-1. Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for blaCTX-M15, blaSHV-28, and blaTEM-1, and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing blaCTX-M15 and blaSHV-28 constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype

OBJECTIVES To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization. Hyperproduction of AmpC beta-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated with hyperproduction and gene mutations associated with extended-spectrum AmpC (ESAC) beta-lactamase activity. RESULTS Twenty-four isolates exhibited a positive AmpC disc test. IEF confirmed AmpC expression in all isolates except one. Four isolates contained a bla(CMY-2) gene. These were not clonally related by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime. Sequencing of ampC showed that most isolates were not clonally related. CONCLUSIONS E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC.

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012–13, investigated by WGS, MLST and PFGE

OBJECTIVES In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm. METHODS A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE. RESULTS One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. CONCLUSIONS This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

ABSTRACT spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance

ABSTRACT The origin of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA β-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

Host-Specific Patterns of Genetic Diversity among IncI1-Iγ and IncK Plasmids Encoding CMY-2 β-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark

ABSTRACT CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected bla CMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. bla CMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. IMPORTANCE CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli. This β-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 β-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.

The New Macrolide-Lincosamide-Streptogramin B Resistance Gene erm(45) Is Located within a Genomic Island in Staphylococcus fleurettii

ABSTRACT Genome alignment of a macrolide, lincosamide, and streptogramin B (MLSB)-resistant Staphylococcus fleurettii strain with an MLSB-susceptible S. fleurettii strain revealed a novel 11,513-bp genomic island carrying the new erythromycin resistance methylase gene erm(45). This gene was shown to confer inducible MLSB resistance when cloned into Staphylococcus aureus. The erm(45)-containing island was integrated into the housekeeping gene guaA in S. fleurettii and was able to form a circular intermediate but was not transmissible to S. aureus.

A multiplex, internally controlled real-time PCR assay for detection of toxigenic Clostridium difficile and identification of hypervirulent strain 027/ST-1

The purpose of this study was to validate a multiplex real-time PCR assay capable of detecting toxigenic Clostridium difficile and simultaneously identifying C. difficile ribotype 027/ST-1 by targeting the toxin genes tcdA, tcdB and cdtA in one reaction and in a separate reaction identifying the Δ117 deletion in tcdC associated with ribotype 027/ST-1. PCR was done prospectively on 704 samples routinely submitted to our department and results were compared to results of toxigenic culture. Sequencing of tcdC, multi locus sequence typing (MLST) and PCR ribotyping were done on cultured isolates to confirm the correct identification of the Δ117 deletion in tcdC and C. difficile ribotype 027/ST-1, respectively. The PCR assay displayed a sensitivity, specificity, PPV and NPV of 99.0%, 97.4%, 87.4% and 99.8%, respectively, compared to toxigenic culture on 665 samples evaluable both by PCR and culture. Sequencing of tcdC, ribotyping and MLST of cultured isolates validated the genotyping assay and confirmed the ability of the assay to correctly identify C. difficile ribotype 027/ST-1 in our current epidemiological setting. We describe the use of a combination of two separate PCR assays for sensitive and specific detection of toxigenic C. difficile and presumptive identification of C. difficile 027/ST-1.

Using MALDI-TOF mass spectrometry as a rapid and accurate diagnostic tool in infective endocarditis: a case report of a patient with mitral valve infective endocarditis caused by Abiotrophia defectiva.

Abstract A case of infective endocarditis caused by Abiotrophia defectiva is presented. The use of MALDI-TOF mass spectrometry as a rapid and accurate diagnostic tool in infective endocarditis is discussed.

Evidence for the evolutionary steps leading to mecA-mediated β-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA–an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all β-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the β-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to β-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of β-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.