Andreas Petersen

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.

Meticillin-resistant Staphylococcus aureus CC398 is an increasing cause of disease in people with no livestock contact in Denmark, 1999 to 2011.

Livestock constitutes a potential reservoir of meticillin-resistant Staphylococcus aureus isolates belonging to a recently derived lineage within clonal complex 398 (MRSA CC398-IIa). Since its discovery in the early 2000s, this lineage has become a major cause of human disease in Europe, posing a serious public health challenge in countries with intensive livestock production. To retrace the history of human colonisation and infection with MRSA CC398-IIa in Denmark, we conducted a nationwide, retrospective study of MRSA isolates collected from 1999 to 2011. Among 7,429 MRSA isolates screened, we identified 416 MRSA CC398-IIa isolates. Of these, 148 were from people with infections, including 51 from patients reporting no livestock exposure. The first cases of MRSA CC398-IIa infection in Denmark occurred in 2004. Subsequently, the incidence of MRSA CC398-IIa infection showed a linear annual increase of 66% from 2004 to 2011 (from 0.09 to 1.1 per 100,000 person-years). There were clear temporal and spatial relationships between MRSA CC398-IIa-infected patients with and without livestock exposure. These findings suggest substantial dissemination of MRSA CC398-IIa from livestock or livestock workers into the Danish community and underscore the need for strategies to control its spread both on and off the farm.

Origin and Evolution of European Community-Acquired Methicillin-Resistant Staphylococcus aureus

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA. With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Rapid differentiation between livestock-associated and livestock-independent Staphylococcus aureus CC398 clades.

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.

A Staphylococcus xylosus Isolate with a new mecC allotype

ABSTRACT Recently, a novel variant of mecA known as mecC (mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

Impact of medicated feed on the development of antimicrobial resistance in bacteria at integrated pig-fish farms in Vietnam.

ABSTRACT Integrated livestock-fish aquaculture utilizes animal excreta, urine, and feed leftovers as pond fertilizers to enhance the growth of plankton and other microorganisms eaten by the fish. However, antimicrobial-resistant bacteria may be transferred and develop in the pond due to selective pressure from antimicrobials present in animal feed, urine, and feces. In an experimental pig-fish farm located in periurban Hanoi, Vietnam, nine piglets were provided feed containing 5 μg of tetracycline (TET)/kg pig weight/day and 0.45 μg of enrofloxacin (ENR)/kg pig weight/day during the second and fourth (last) months of the experiment. The aim of this study was to determine the association between the provision of pig feed with antimicrobials and the development of antimicrobial resistance, as measured in a total of 520 Escherichia coli and 634 Enterococcus strains isolated from pig manure and water-sediment pond samples. MIC values for nalidixic acid (NAL) and ENR showed that E. coli and Enterococcus spp. overall exhibited significant higher frequencies of resistance toward NAL and ENR during the 2 months when pigs were administered feed with antimicrobials, with frequencies reaching 60 to 80% in both water-sediment and manure samples. TET resistance for both indicators was high (>80%) throughout the study period, which indicates that TET-resistant E. coli and Enterococcus spp. were present in the piglets before the initiation of the experiment. PCR-based identification showed similar relative occurrences of Enterococcus faecium, Enterococcus faecalis, and other Enterococcus spp. in the water-sediment and manure samples, suggesting that Enterococcus spp. isolated in the ponds originated mainly from the pig manure. The development of antimicrobial resistance in integrated animal husbandry-fish farms and possible transfers and the impact of such resistance on food safety and human health should be further assessed.

Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to β-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to β-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant β-lactam antibiotics. Methods The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays. Results We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types. Conclusions This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for β-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Fitness cost: a bacteriological explanation for the demise of the first international methicillin-resistant Staphylococcus aureus epidemic

OBJECTIVES Denmark and several other countries experienced the first epidemic of methicillin-resistant Staphylococcus aureus (MRSA) during the period 1965-75, which was caused by multiresistant isolates of phage complex 83A. In Denmark these MRSA isolates disappeared almost completely, being replaced by other phage types, predominantly only penicillin resistant. We investigated whether isolates of this epidemic were associated with a fitness cost, and we employed a mathematical model to ask whether these fitness costs could have led to the observed reduction in frequency. METHODS Bacteraemia isolates of S. aureus from Denmark have been stored since 1957. We chose 40 S. aureus isolates belonging to phage complex 83A, clonal complex 8 based on spa type, ranging in time of isolation from 1957 to 1980 and with various antibiograms, including both methicillin-resistant and -susceptible isolates. The relative fitness of each isolate was determined in a growth competition assay with a reference isolate. RESULTS Significant fitness costs of 2%-15% were determined for the MRSA isolates studied. There was a significant negative correlation between number of antibiotic resistances and relative fitness. Multiple regression analysis found significantly independent negative correlations between fitness and the presence of mecA or streptomycin resistance. Mathematical modelling confirmed that fitness costs of the magnitude carried by these isolates could result in the disappearance of MRSA prevalence during a time span similar to that seen in Denmark. CONCLUSIONS We propose a significant fitness cost of resistance as the main bacteriological explanation for the disappearance of the multiresistant complex 83A MRSA in Denmark following a reduction in antibiotic usage.

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.