Investigation of the introduction and dissemination of vanB Enterococcus faecium in the Capital Region of Denmark and development of a rapid and accurate clone-specific vanB E. faecium PCR.

BACKGROUND: During 2018-19, an increase of vanB vancomycin-resistant Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. vanA/vanB PCR performed directly on rectal swabs is accurate in detection of vanA; however, the positive predictive value for vanB-positive samples is low because of the presence of vanB in non-enterococcal gut commensals. OBJECTIVES: We investigated the epidemiology and clonal relatedness of vanB VREfm from the period 2015-19 and describe the application of a clone-specific vanB VREfm PCR assay for rapid and accurate detection of vanB VREfm in rectal screening samples. METHODS: vanB VREfm were investigated using epidemiological data and WGS data. The SeqSphere+ software was used to analyse MLST and cgMLST, and de novo assemblies were annotated to determine insertion sites for the vanB transposon (Tn1549). A clone-specific vanB VREfm PCR assay was designed to detect the sequence bridging Tn1549 and the E. faecium chromosome (araA2) in the dominant cluster. RESULTS: Two hundred and seventy-five vanB VREfm isolates were identified, of which 76% were identified in 2019. A dominant cluster (Cluster 1, n = 204, 74%), six minor clusters and 15 singletons were identified. All Cluster 1 isolates and six non-Cluster 1 isolates had Tn1549 integrated into araA2. In 2019, the PCR assay would have detected 92% of all rectal screening samples containing vanB VREfm. CONCLUSIONS: vanB VREfm increased due to the introduction and nosocomial transmission of the successful Cluster 1. The clone-specific PCR assay detected vanB VREfm outbreak isolates in rectal screening samples rapidly and accurately.

Comparing whole-genome sequencing with Sanger sequencing for spa typing of methicillin-resistant Staphylococcus aureus.

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.

Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance.

OBJECTIVES: To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300. METHODS: We reconstructed the phylogeny of S. aureus CC8 by mutation discovery at 112 genetic housekeeping loci from each of 174 isolates, sampled on five continents between 1957 and 2008. The distribution of antimicrobial resistance traits and of diverse mobile genetic elements was investigated in relation to the isolates' phylogeny. RESULTS: Our analyses revealed the existence of nine phylogenetic clades within CC8. We identified at least eight independent events of methicillin resistance acquisition in CC8 and dated the origin of a methicillin-resistant progenitor of the notorious USA300 clone to the mid-1970s. Of the S. aureus isolates in our collection, 88% carried plasmidic rep gene sequences, with up to five different rep genes in individual isolates and a total of eight rep families. Mapping the plasmid content onto the isolates' phylogeny illustrated the stable carriage over decades of some plasmids and the more volatile nature of others. Strikingly, we observed trends of increasing antibiotic resistance during the evolution of several lineages, including USA300. CONCLUSIONS: We propose a model for the evolution of S. aureus CC8, involving a split into at least nine phylogenetic lineages and a subsequent series of acquisitions and losses of mobile genetic elements that carry diverse virulence and antimicrobial resistance traits. The evolution of MRSA USA300 towards resistance to additional antibiotic classes is of major concern.

Nosocomial transmission of community-associated methicillin-resistant Staphylococcus aureus in Danish Hospitals.

OBJECTIVES: The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has changed the epidemiology of MRSA infections worldwide. In contrast to hospital-associated MRSA (HA-MRSA), CA-MRSA more frequently affects healthy individuals, both with and without recent healthcare exposure. Despite obvious epidemiological differences, it is unknown whether differences in nosocomial transmissibility exist. We have, therefore, quantified the transmissibility, expressed by the single admission reproduction number (R(A)), of CA-MRSA and HA-MRSA in hospital settings in Denmark. METHODS: MRSA index cases and secondary cases were investigated in four hospitals in the Copenhagen area. Index cases were defined as non-isolated, non-screened patients with MRSA, and secondary cases were defined as persons carrying MRSA isolates-identical to that of the corresponding index-as identified through contact screening. CA-MRSA and HA-MRSA were categorized upon genotyping [CA-MRSA: t008-ST8, PVL+; t019-ST30, PVL+; t127-ST1, PVL+; t044-ST80, PVL+; and their related spa types; and HA-MRSA: all other (where ST stands for sequence type and PVL stands for Panton-Valentine leucocidin)]. A mathematical model was applied to determine the genotype-specific transmission rate (i.e. R(A)) of CA-MRSA and HA-MRSA strains. RESULTS: During the 7 year study period there were 117 MRSA index cases with subsequent post-contact screening (of 1108 patients and healthcare workers), revealing 22 outbreaks with a total of 52 secondary patients. R(A) values were 0.07 (95% CI 0.00-0.28) and 0.65 (95% CI 0.48-0.84) for CA-MRSA and HA-MRSA, respectively. CONCLUSIONS: In four Danish hospitals the nosocomial transmission rate of CA-MRSA was 9.3 times lower than that of HA-MRSA.

Structural variations of staphylococcal cassette chromosome mec type IVa in Staphylococcus aureus clonal complex 8 and unrelated lineages.

PCR mapping of staphylococcal cassette chromosome mec type IVa and adjacent mobile elements in 94 methicillin-resistant Staphylococcus aureus (MRSA) strains identified two primary structures (A and B) that could be further classified into two (A1 and A2) and five (B1 to B5) variants, primarily based on structural differences in the orfX-J3 region. While spa type t008 (USA300) invariably contained the A variants, other spa types belonging to clonal complex 8 and unrelated lineages generally contained B variants. These findings have important implications for the typing and identification of MRSA strains containing B variants.

Changing epidemiology of methicillin-resistant Staphylococcus aureus in Iceland from 2000 to 2008: a challenge to current guidelines.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continuously changing. Iceland has a low incidence of MRSA. A "search and destroy" policy (screening patients with defined risk factors and attempting eradication in carriers) has been implemented since 1991. Clinical and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (spa types t037 and t2802), which were successfully controlled with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49 spa types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific (SWP) clones were common. SCCmec type IV was most common (72%), and 38% of the isolates were PVL positive. The incidence of MRSA in Iceland has increased since 1999 but remains low and has been stable in the last years. The search and destroy policy was effective to control MRSA in the health care setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system.

Growth phase-dependent regulation of the global virulence regulator Rot in clinical isolates of Staphylococcus aureus.

Current models for global virulence regulation in Staphylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (beta-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.

A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay.

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants.

Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either to mutation or to a true deficiency of spa.

Two distinct clones of methicillin-resistant Staphylococcus aureus (MRSA) with the same USA300 pulsed-field gel electrophoresis profile: a potential pitfall for identification of USA300 community-associated MRSA.

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.

Rapid increase of genetically diverse methicillin-resistant Staphylococcus aureus, Copenhagen, Denmark.

In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for <15 isolates per year during 1980-2002. However, since 2003 an epidemic increase has been observed, with 33 MRSA cases in 2003 and 110 in 2004. We analyzed these 143 cases epidemiologically and characterized isolates by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had no identified risk factors. We identified 29 spa types (t) and 16 sequence types (STs) belonging to 8 clonal complexes and 3 ST singletons. The most common clonal types were t024/ST8-IV, t019/ST30-IV, t044/ST80-IV, and t008/ST8-IV (USA300). A total of 86% of isolates harbored SCCmec IV, and 44% had PVL. Skin and soft tissue infections dominated. CO-MRSA with diverse genetic backgrounds is rapidly emerging in a low MRSA prevalence area.

First isolation of Leptospira fainei serovar Hurstbridge from two human patients with Weil's syndrome.

Leptospira fainei serovar Hurstbridge is a recently discovered Leptospira species and so far it has only been cultured from animal sources. Based on positive serology and positive PCR for L. fainei among patients suspected of having leptospirosis, a role in human disease seems likely. This study describes two patients with Weil's disease from whom L. fainei was cultured. A local source of the infections was suspected, as these two patients resided in the same area of Denmark, were hospitalised approximately at the same time and had not been travelling recently. The Leptospira species was determined by serology, PCR and sequencing of bacterial DNA. One patient developed autoimmune hepatitis in the course of the L. fainei infection and was treated with both antibiotics and immunosuppression with good effect. The other patient had a self-limiting disease and did not receive any treatment.

Insights into alpha-hemolysin (Hla) evolution and expression among Staphylococcus aureus clones with hospital and community origin.

BACKGROUND: Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of Staphylococcus aureus infection, being active against a wide range of host cells. Although hla is ubiquitous in S. aureus, its genetic diversity and variation in expression in different genetic backgrounds is not known. We evaluated nucleotide sequence variation and gene expression profiles of hla among representatives of hospital (HA) and community-associated (CA) S. aureus clones. METHODS: 51 methicillin-resistant S. aureus and 22 methicillin-susceptible S. aureus were characterized by PFGE, spa typing, MLST and SCCmec typing. The internal regions of hla and the hla promoter were sequenced and gene expression was assessed by RT-PCR. RESULTS: Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that hla may have evolved together with the S. aureus genetic background, except for ST22, ST121, ST59 and ST93. Conversely, the promoter region showed lack of co-evolution with the genetic backgrounds. Four non-synonymous amino acid changes were identified close to important regions of hla activity. Amino acid changes in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones. CONCLUSION: We propose that the hla gene has evolved together with the genetic background. Overall, CA genetic backgrounds showed higher levels of hla expression than HA, and a high strain-to-strain variation of gene expression was detected in closely related strains.

Associations between dru Types and SCCmec cassettes.

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.

Complete Genome Sequence of Staphylococcus aureus Strain M1, a Unique t024-ST8-IVa Danish Methicillin-Resistant S. aureus Clone.

We report the genome sequence, in five contigs, of a methicillin-resistant Staphylococcus aureus isolate designated M1. This clinical isolate was from the index patient of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in Copenhagen, Denmark, that started in 2003. This strain is sequence type 8 (ST8), spa type t024, and staphylococcal cassette chromosome mec element (SCCmec) type IVa.

Phylogeographic variation in recombination rates within a global clone of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Next-generation sequencing (NGS) is a powerful tool for understanding both patterns of descent over time and space (phylogeography) and the molecular processes underpinning genome divergence in pathogenic bacteria. Here, we describe a synthesis between these perspectives by employing a recently developed Bayesian approach, BRATNextGen, for detecting recombination on an expanded NGS dataset of the globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clone ST239. RESULTS: The data confirm strong geographical clustering at continental, national and city scales and demonstrate that the rate of recombination varies significantly between phylogeographic sub-groups representing independent introductions from Europe. These differences are most striking when mobile non-core genes are included, but remain apparent even when only considering the stable core genome. The monophyletic ST239 sub-group corresponding to isolates from South America shows heightened recombination, the sub-group predominantly from Asia shows an intermediate level, and a very low level of recombination is noted in a third sub-group representing a large collection from Turkey. CONCLUSIONS: We show that the rapid global dissemination of a single pathogenic bacterial clone results in local variation in measured recombination rates. Possible explanatory variables include the size and time since emergence of each defined sub-population (as determined by the sampling frame), variation in transmission dynamics due to host movement, and changes in the bacterial genome affecting the propensity for recombination.

Variations in spa types found in consecutive MRSA isolates from the same patients.

Very little is known about how the spa gene mutates over time in methicillin-resistant Staphylococcus aureus (MRSA) from the same patient. Copenhagen is an area with low prevalence of MRSA but with high variability in spa types. We collected 1536 MRSA isolates from 319 patients during a 5-year period and found spa type alterations in 30 MRSA isolates (2%) from 13 patients (4%). The alteration most often seen was the deletion of repeats followed by repeat duplication and point mutation.

An unexpected location of the arginine catabolic mobile element (ACME) in a USA300-related MRSA strain.

In methicillin resistant Staphylococcus aureus (MRSA), the arginine catabolic mobile element (ACME) was initially described in USA300 (t008-ST8) where it is located downstream of the staphylococcal cassette chromosome mec (SCCmec). A common health-care associated MRSA in Copenhagen, Denmark (t024-ST8) is clonally related to USA300 and is frequently PCR positive for the ACME specific arcA-gene. This study is the first to describe an ACME element upstream of the SCCmec in MRSA. By traditional SCCmec typing schemes, the SCCmec of t024-ST8 strain M1 carries SCCmec IVa, but full sequencing of the cassette revealed that the entire J3 region had no homology to published SCCmec IVa. Within the J3 region of M1 was a 1705 bp sequence only similar to a sequence in S. haemolyticus strain JCSC1435 and 2941 bps with no homology found in GenBank. In addition to the usual direct repeats (DR) at each extremity of SCCmec, M1 had two new DR between the orfX gene and the J3 region of the SCCmec. The region between the orfX DR (DR1) and DR2 contained the ccrAB4 genes. An ACME II-like element was located between DR2 and DR3. The entire 26,468 bp sequence between DR1 and DR3 was highly similar to parts of the ACME composite island of S. epidermidis strain ATCC12228. Sequencing of an ACME negative t024-ST8 strain (M299) showed that DR1 and the sequence between DR1 and DR3 was missing. The finding of a mobile ACME II-like element inserted downstream of orfX and upstream of SCCmec indicates a novel recombination between staphylococcal species.

Management of recurrent pacemaker-related bacteraemia with small colony variant Staphylococcus aureus in a haemodialysis patient.

A patient with chronic haemodialysis with a cardiac pacemaker was admitted for five episodes of bacteraemia with Staphylococcus during an 8-month period. The species identification was complicated since the morphological characters and biochemical reactions were unusual and differing. Molecular biological identification and typing methods revealed that the pathogens for all the episodes were the same strain of Staphylococcus aureus that had small colony variant characteristics. Continuous suppressive antibiotic treatment initiated after the last infection episode has been able to keep the patient free of bacteraemia relapse during the past 24 months without removing the pacemaker.

[Epidemic increase in methicillin-resistant Staphylococcus aureus in Copenhagen]. / Epidemisk stigning af methicillinresistente Staphylococcus aureus i København.

INTRODUCTION: We have found an epidemic increase in methicillin-resistant Staphylococcus aureus (MRSA) in Copenhagen. The increase has a complex background and involves hospitals, nursing homes and persons nursed in their own home. MATERIAL AND METHODS: We found 33 MRSA patients in 2003 and 121 in 2004. All isolates have been spa-typed and epidemiologic information collected. RESULTS: The number of MRSA cases has a doubling time of about six months. The epidemic has been caused by many different MRSA types and 31 staphylococcus protein A genotypes (spa types). MRSA has caused several hospital outbreaks and is endemic in 10 nursing homes. Five staff members from nursing homes have been infected with MRSA. MRSA commonly causes skin and soft tissue infections (76%), but serious infections such as septicaemia and pneumonia are also found. CONCLUSION: Treatment of MRSA-infected patients is costly due to isolation regimes, increase in bed-days and treatment with special antibiotics. After treatment of the infection and in cases of MRSA carriage, MRSA is found on the skin and in the nose. Carriage of MRSA can be eradicated by washing with chlorhexidine and nasal administration of mupirocin. The necessary resources for handling the epidemic are not available. Without active intervention, this situation will have serious implications for the health care system.