Anders Rhod Larsen

Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study

Summary Background Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Methods Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. Findings A divergent mecA homologue (mecALGA251) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecALGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecALGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Interpretation Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecALGA251. Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

Epidemiology of Emerging Methicillin-Resistant Staphylococcus aureus (MRSA) in Denmark: a Nationwide Study in a Country with Low Prevalence of MRSA Infection

ABSTRACT Strict infection control measures introduced during the 1970s have kept the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections extremely low in Denmark. Nevertheless, similarly to other countries, MRSA infections began to appear in the community in the late 1990s. A nationwide surveillance program has collected and stored all MRSA isolates since 1988 and, since 1999, clinical information has been also recorded. We used this information and isolates in a detailed epidemiological and molecular analysis of the 81 MRSA infections identified in Denmark in 2001. MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing. Comparison of the 45 community-onset MRSA (CO-MRSA) infections with the 36 hospital-acquired MRSA (HA-MRSA) infections showed several striking contrasts. Most CO-MRSA were recovered from skin and soft tissue infections caused by isolates carrying the Panton-Valentine leucocidin toxin genes, and the majority (84%) of isolates belonged to a single clonal type, ST80-IV, which has been found in the community in other European countries. Clone ST80-IV could be traced in Denmark back to 1993. ST80-IV was rarely found in HA-MRSA infections, which belonged to a large number of clonal types, including some pandemic MRSA clones. The low number of HA-MRSA infections and the diversity of MRSA clones in Danish hospitals may be the result of successful infection control measures that prevent spread of clones in hospitals. The mechanism of spread of the ST80-IV clone in the Danish community is not known, and new control measures are needed to control further spread of this and other CA-MRSA clones.

Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates.

Whole genome sequencing identifies zoonotic transmission of MRSA isolates with the novel mecA homologue mecC

Several methicillin‐resistant Staphylococcus aureus (MRSA) lineages that carry a novel mecA homologue (mecC) have recently been described in livestock and humans. In Denmark, two independent human cases of mecC‐MRSA infection have been linked to a livestock reservoir. We investigated the molecular epidemiology of the associated MRSA isolates using whole genome sequencing (WGS). Single nucleotide polymorphisms (SNP) were defined and compared to a reference genome to place the isolates into a phylogenetic context. Phylogenetic analysis revealed two distinct farm‐specific clusters comprising isolates from the human case and their own livestock, whereas human and animal isolates from the same farm only differed by a small number of SNPs, which supports the likelihood of zoonotic transmission. Further analyses identified a number of genes and mutations that may be associated with host interaction and virulence. This study demonstrates that mecC‐MRSA ST130 isolates are capable of transmission between animals and humans, and underscores the potential of WGS in epidemiological investigations and source tracking of bacterial infections.

Novel Types of Staphylococcal Cassette Chromosome mec Elements Identified in Clonal Complex 398 Methicillin-Resistant Staphylococcus aureus Strains

ABSTRACT The structures of staphylococcal cassette chromosome mec (SCCmec) elements carried by 31 clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from the participants at a conference were analyzed. The SCCmecs were classified into novel types, namely, IX, X, V(5C2&5) subtype c, and IVa. Type V(5C2&5) subtype c, IX, and X SCCmecs carried genes conferring resistance to metals. The structures of SCCmecs from CC398 strains were distinct from those normally found in humans, adding to the evidence that humans are not the original host for CC398.

Epidemiology of methicillin‐resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated (HA), community-associated (CA) and livestock-associated (LA) infections. Recently, the discovery of human and bovine MRSA isolates carrying a new mecA gene homologue, mecA(LGA251) (now designated mecC), has caused concern because they are not detected by conventional, confirmatory tests for MRSA. Very little is known about their frequency, epidemiology and possible transmission between livestock and humans. In this study, the epidemiology of the mecC isolates in Denmark was investigated by screening the national collections of MRSA cases (from 1988 onwards) and S. aureus bacteraemia cases (from 1958 onwards). Isolates carrying mecC were only recovered infrequently before 2003 (n = 2) but now seem to be increasing, with 110 cases in 2003-2011. Clinical data on mecC-carrying MRSA demonstrated that mecC-MRSA were primarily community-acquired (CA-MRSA) and affected persons typically living in rural areas, being older than other CA-MRSA patients. Among 22 cases in Region Zealand, four reported contact with cattle and sheep. Two of these persons lived on farms with livestock positive for mecC-carrying MRSA, sharing spa type (t843), MLVA (MT429) and PFGE pattern with the human isolates. These observations indicate that mecC-carrying MRSA can be exchanged between humans and ruminants.

The newly described mecA homologue, mecALGA251, is present in methicillin-resistant Staphylococcus aureus isolates from a diverse range of host species

OBJECTIVES A previously unidentified mecA homologue, mecA(LGA251), has recently been described in methicillin-resistant Staphylococcus aureus (MRSA) from humans and dairy cattle. The origin and epidemiology of this novel homologue are unclear. The objective of this study was to provide basic descriptive information of MRSA isolates harbouring mecA(LGA251) from a range of host animal species. METHODS A number of S. aureus isolates from historical animal isolate collections were chosen for investigation based on their similarity to known mecA(LGA251) MRSA isolates. The presence of mecA(LGA251) was determined using a multiplex PCR and antimicrobial susceptibility testing performed by disc diffusion. RESULTS MRSA harbouring mecA(LGA251) were found in isolates from a domestic dog, brown rats, a rabbit, a common seal, sheep and a chaffinch. All of the isolates were phenotypically MRSA, although this depended on which test was used; some isolates would be considered susceptible with certain assays. All isolates were susceptible to linezolid, rifampicin, kanamycin, norfloxacin, erythromycin, clindamycin, fusidic acid, tetracycline, trimethoprim/sulfamethoxazole and mupirocin. Five multilocus sequence types were represented (2273, 130, 425, 1764 and 1245) and six spa types (t208, t6293, t742, t6594, t7914 and t843). CONCLUSIONS The discovery of MRSA isolates possessing mecA(LGA251) from a diverse range of host species, including different taxonomic classes, has important implications for the diagnosis of MRSA in these species and our understanding of the epidemiology of this novel mecA homologue.

Distinct determinants of human immunodeficiency virus type 1 RNA and DNA loads in vaginal and cervical secretions.

The relationship between human immunodeficiency virus type 1 (HIV-1) viral RNA and proviral DNA load in vagina and cervix and that found in the plasma and peripheral blood mononuclear cells (PBMC) was investigated in 28 HIV-1-infected women. Of the patients, 64% had > or = 1 HIV-1 RNA-positive genital sample, while 71% had > or = 1 DNA-positive sample. The higher the cervical HIV load, the more widespread was the virus in the genital tract. A strong correlation was found between viral RNA load in plasma and the genital tract, whereas the association between proviral DNA load in PBMC and the genital tract was less evident. Cervical HIV-1 DNA correlated with a viral RNA load > or = 50,000 copies/mL. Cervical HIV-1 RNA levels ranged from 10% to 100% of the plasma levels. Thus, a continuous transmission risk from untraumatized genital epithelium exists in the majority of HIV-1-infected women at all stages of infection.

Livestock Origin for a Human Pandemic Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

ABSTRACT The importance of livestock as a source of bacterial pathogens with the potential for epidemic spread in human populations is unclear. In recent years, there has been a global increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections of healthy humans, but an understanding of the different evolutionary origins of CA-MRSA clones and the basis for their recent expansion is lacking. Here, using a high-resolution phylogenetic approach, we report the discovery of two emergent clones of human epidemic CA-MRSA which resulted from independent livestock-to-human host jumps by the major bovine S. aureus complex, CC97. Of note, one of the new clones was isolated from human infections on four continents, demonstrating its global dissemination since the host jump occurred over 40 years ago. The emergence of both human S. aureus clones coincided with the independent acquisition of mobile genetic elements encoding antimicrobial resistance and human-specific mediators of immune evasion, consistent with an important role for these genetic events in the capacity to survive and transmit among human populations. In conclusion, we provide evidence that livestock represent a reservoir for the emergence of new human-pathogenic S. aureus clones with the capacity for pandemic spread. These findings have major public health implications highlighting the importance of surveillance for early identification of emergent clones and improved transmission control measures at the human-livestock interface. IMPORTANCE Animals are the major source of new pathogens affecting humans. However, the potential for pathogenic bacteria that originally were found in animals to switch hosts and become widely established in human populations is not clear. Here, we report the discovery of emergent clones of methicillin-resistant Staphylococcus aureus (MRSA) that originated in livestock and switched to humans, followed by host-adaptive evolution and epidemic spread in global human populations. Our findings demonstrate that livestock can act as a reservoir for the emergence of new human bacterial clones with potential for pandemic spread, highlighting the potential role of surveillance and biosecurity measures in the agricultural setting for preventing the emergence of new human pathogens. Animals are the major source of new pathogens affecting humans. However, the potential for pathogenic bacteria that originally were found in animals to switch hosts and become widely established in human populations is not clear. Here, we report the discovery of emergent clones of methicillin-resistant Staphylococcus aureus (MRSA) that originated in livestock and switched to humans, followed by host-adaptive evolution and epidemic spread in global human populations. Our findings demonstrate that livestock can act as a reservoir for the emergence of new human bacterial clones with potential for pandemic spread, highlighting the potential role of surveillance and biosecurity measures in the agricultural setting for preventing the emergence of new human pathogens.

Characterization of the Epidemic European Fusidic Acid-Resistant Impetigo Clone of Staphylococcus aureus

ABSTRACT Resistance to the antibiotic fusidic acid in European strains of Staphylococcus aureus causing impetigo has increased in recent years. This increase appears to have resulted from clonal expansion of a strain we have designated the epidemic European fusidic acid-resistant impetigo clone (EEFIC), which carries the fusidic acid resistance determinant fusB on its chromosome. To understand better the properties of the EEFIC responsible for its success, we have performed detailed phenotypic and genotypic characterization of this clone. Molecular typing revealed the EEFIC to be ST123, spa type t171, and agr type IV and therefore unrelated to earlier prevalent fusB+ strains found in the United Kingdom. EEFIC strains exhibited resistance to fusidic acid, penicillin, and, in some cases, erythromycin, which are all used in the treatment of impetigo. PCR analysis of the EEFIC and complete DNA sequencing of the 39.3 Kb plasmid it harbors identified genes encoding several toxins previously implicated in impetigo (exfoliative toxins A and B and EDIN-C). The location of fusB was mapped on the chromosome and found to be associated with a novel 16.6-kb genomic island integrated downstream of groEL. Although this element is related to classical staphylococcal pathogenicity islands, it does not encode any known virulence factors and consequently has been designated SaRIfusB (for “S. aureus resistance island carrying fusB”).