In the early 2000s, a particular MRSA clonal complex (CC398) was found mainly in pigs and pig farmers in Europe. Since then, CC398 has been detected among a wide variety of animal species worldwide. We investigated the population structure of CC398 through mutation discovery at 97 genetic housekeeping loci, which are distributed along the CC398 chromosome within 195 CC398 isolates, collected from various countries and host species, including humans. Most of the isolates in this collection were received from collaborating microbiologists, who had preserved them over years. We discovered 96 bi-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed ‘clade (C)’) has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses in veterinary-care settings (p = 2×10−7), it remained extremely rare among S. aureus isolates from human infections.
A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection.
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.
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.
The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays.
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.
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.
β-lactams; MRSA; mecA; mecC
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.
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.
The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.
Staphylococcus aureus clonal complex 398 (CC398) isolates colonize livestock and can spread to human contacts. Genetic analysis of isolates epidemiologically associated with human-to-human, but not livestock, transmission in multiple countries and continents identified a common clade that was negative for tet(M) and positive for bacteriophage ϕ3. Another group of human-to-human-transmitted isolates belonged to the common livestock-associated clade but had acquired a unique ϕ7 bacteriophage.
The aim of this study was to determine the long-term carriage rates and transmission dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in pig farmers and their household members. During a 6-month period in 2009–2010, 4 pig farms in Denmark, Belgium, and the Netherlands, respectively, were studied for the presence of MRSA. The proportion of persistent carriers was significantly higher among farmers than among household members (87% vs. 11%) and significantly higher in household members from Belgium compared to those from Denmark and the Netherlands (29% vs. 0% vs. 6%). Determinant analysis of MRSA carriage revealed that pig contact was the most important determinant for MRSA carriage among household members and that the increased MRSA carriage rate observed among household members from Belgium is linked to country-specific differences in pig exposure.
These findings demonstrated that even in pig farms with very high carriage rates of MRSA both in livestock and farmers, the risk for household members to acquire MRSA is limited and still depends strongly on pig exposure. By restricting access to the stables and exposure to pigs, MRSA acquisition by household members could be greatly reduced.
Staphylococcus aureus ST291 has been reported as a homologue recombinant double locus variant of the livestock associated S. aureus ST398. However, whole genome sequencing show that ST291 is a unique genetic lineage with highly variable content within its accessory genome compared to both human and livestock associated genome sequenced CC398s.
Since its introduction to the market in the 1970s, the synthetic biocide triclosan has had widespread use in household and medical products. Although decreased triclosan susceptibility has been observed for several bacterial species, when exposed under laboratory settings, no in vivo studies have associated triclosan use with decreased triclosan susceptibility or cross-resistance to antibiotics. One major challenge of such studies is the lack of strains that with certainty have not been exposed to triclosan. Here we have overcome this challenge by comparing current isolates of the human opportunistic pathogen Staphylococcus epidermidis with isolates collected in the 1960s prior to introduction of triclosan to the market. Of 64 current S. epidermidis isolates 12.5% were found to have tolerance towards triclosan defined as MIC≥0.25 mg/l compared to none of 34 isolates obtained in the 1960s. When passaged in the laboratory in the presence of triclosan, old and current susceptible isolates could be adapted to the same triclosan MIC level as found in current tolerant isolates. DNA sequence analysis revealed that laboratory-adapted strains carried mutations in fabI encoding the enoyl-acyl carrier protein reductase isoform, FabI, that is the target of triclosan, and the expression of fabI was also increased. However, the majority of the tolerant current isolates carried no mutations in fabI or the putative promoter region. Thus, this study indicates that the widespread use of triclosan has resulted in the occurrence of S. epidermidis with tolerance towards triclosan and that the adaptation involves FabI as well as other factors. We suggest increased caution in the general application of triclosan as triclosan has not shown efficacy in reducing infections and is toxic to aquatic organisms.
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.
cattle; mecC; MRSA; sheep; zoonosis
The aim of this study was to investigate the occurrence of bacterial cross-contamination between the nasal cavity and leg ulcers. Sixteen patients were included in the study. Staphylococcus aureus was isolated from the leg ulcer of 13 patients and 6 of these patients also harboured S. aureus in the nasal cavity. Klebsiella oxytoca was found in the ulcer and the nasal cavity of one patient. PFGE analysis revealed that patients harbouring S. aureus both in the nasal cavity and the leg ulcer had the same bacterial type at both sites. None of the S. aureus isolates were methicillin resistant.
Staphylococcus aureus; nasal carriage; chronic leg ulcers; cross-contamination; Klebsiella oxytoca.
The objective of this study was to assess temporal changes in incidence and short term mortality of Staphylococcus aureus bacteraemia (SAB) from 1995 through 2008.
The study was conducted as a nation-wide observational cohort study with matched population controls. The setting was hospitalized patients in Denmark 1995-2008. Uni- and multivariate analyses were used to analyze the hazard of death within 30 days from SAB.
A total of 16 330 cases of SAB were identified: 57% were hospital-associated (HA), 31% were community-acquired (CA) and 13% were of undetermined acquisition. The overall adjusted incidence rate remained stable at 23 per 100 000 population but the proportion of SAB cases older than 75 years increased significantly. Comorbidity in the cohort as measured by Charlson comorbidity index (CCI) score and alcohol-related diagnoses increased over the study period. In contrast, among the population controls the CCI remained stable and alcohol-related diagnoses increased slightly. For HA SAB crude 30-day mortality decreased from 27.8% to 21.8% (22% reduction) whereas the change for CA SAB was small (26.5% to 25.8%). By multivariate Cox regression, age, female sex, time period, CCI score and alcohol-related diagnoses were associated with increased mortality regardless of mode of acquisition.
Throughout a 14-year period the overall incidence of SAB remained stable while the overall short term prognosis continued to improve despite increased age and accumulation of comorbidity in the cohort. However, age and comorbidity were strong prognostic indicators for short term mortality.
Bacteraemia; Epidemiology; Incidence; Mortality; Comorbidity; Alcoholism; Staphylococcus aureus; Charlson comorbidity index
The European methicillin-resistant Staphylococcus aureus (MRSA) clone ST80-IV has historically dominated community-associated infections in major parts of Europe and is a lineage strongly linked to skin and soft tissue infections. Here, we report the genome sequence of an ST80-IV representative, 11819-97, isolated from a skin infection in Denmark in 1997.
Background. Nasal carriage is a major risk factor for Staphylococcus aureus infection. Approximately, one-quarter of adults carry S. aureus. However, the role of host genetics on S. aureus nasal carriage is unknown.
Methods. Nasal swabs were obtained from a national cohort of middle-aged and elderly Danish twins. Subjects colonized with S. aureus were identified by growth on selective plates and spa typing. A second sample was obtained from twins initially concordant for carriage. Twins found to again be colonized with S. aureus were defined as persistent carriers.
Results. The prevalence of S. aureus carriage among 617 twin pairs (monozygotic/dizygotic pairs: 112/505) was 26.3% (95% confidence interval [CI], 24.0%–28.9%). The concordance rate for carriage did not differ significantly between pairs of monozygotic (37.5%; 95% CI, 22.3%–53.8%) twins and same sex (24.2%; 95% CI, 15.4%–34.5%), and opposite sex (21.4%; 95% CI, 12.0%–33.4%) dizygotic twins. Despite shared childhoods, only 1 of 617 pairs was concordant with respect to lineage. Although heritability increased for S. aureus and lineage persistency, no significant heritability was detected.
Conclusion. In this study, host genetic factors exhibited only a modest influence on the S. aureus carrier state of middle-aged and elderly individuals.
Staphylococcus aureus infective endocarditis (IE) is a critical medical condition associated with a high morbidity and mortality. In the present study, we prospectively evaluated the importance of screening with echocardiography in an unselected S. aureus bacteraemia (SAB) population.
Methods and results
From 1 January 2009 to 31 August 2010, a total of 244 patients with SAB at six Danish hospitals underwent screening echocardiography. The inclusion rate was 73% of all eligible patients (n= 336), and 53 of the 244 included patients (22%; 95% CI: 17–27%) were diagnosed with definite IE. In patients with native heart valves the prevalence was 19% (95% CI: 14–25%) compared with 38% (95% CI: 20–55%) in patients with prosthetic heart valves and/or cardiac rhythm management devices (P= 0.02). No difference was found between Main Regional Hospitals and Tertiary Cardiac Hospitals, 20 vs. 23%, respectively (NS). The prevalence of IE in high-risk patients with one or more predisposing condition or clinical evidence of IE were significantly higher compared with low-risk patients with no additional risk factors (38 vs. 5%; P < 0.001). IE was associated with a higher 6 months mortality, 14(26%) vs. 28(15%) in SAB patients without IE, respectively (P < 0.05).
SAB patients carry a high risk for development of IE, which is associated with a worse prognosis compared with uncomplicated SAB. The presenting symptoms and clinical findings associated with IE are often non-specific and echocardiography should always be considered as part of the initial evaluation of SAB patients.
Infective endocarditis; Echocardiography; Staphylococcus aureus; Screening
Human beta-defensins are key components of human innate immunity to a variety of pathogens, including Staphylococcus aureus. The aim of the present study was to investigate a potential association between gene variations in DEFB1 and DEFB103/DEFB4 and the development of S. aureus bacteremia (SAB) employing a case-control design.
Cases were unique patients with documented SAB, identified with the National S. aureus Bacteremia Register, a comprehensive dataset of all episodes of community associated-SABs (CA-SAB) occurring in children (≤20 yrs) in Denmark from 1990 to 2006. Controls were age-matched healthy individuals with no history of SAB. DNA obtained from cases and controls using the Danish Newborn Screening Biobank were genotyped for functional polymorphisms of DEFB1 by Sanger sequencing and copy number variation of the DEFB103 and DEFB4 genes using Pyrosequencing-based Paralogue Ratio Test (P-PRT).
193 ethnic Danish SAB cases with 382 age-matched controls were used for this study. S. aureus isolates represented a variety of bacterial (i.e., different spa types) types similar to SAB isolates in general. DEFB1 minor allele frequencies of rs11362 (cases vs. controls 0.47/0.44), rs1800972 (0.21/0.24), and rs1799946 (0.32/0.33) were not significantly different in cases compared with controls. Also, DEFB4/DEFB103 gene copy numbers (means 4.83/4.92) were not significantly different in cases compared with controls.
Using a large, unique cohort of pediatric CA-SAB, we found no significant association between DEFB1 genetic variation or DEFB4/DEFB103 gene copy number and susceptibility for SAB.
Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production.
Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.
Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community-associated pathogen. Recently, livestock-associated MRSA (LA-MRSA) has emerged and disseminated in Europe and North America and now constitutes a considerable zoonotic burden in humans with risk factors of pig exposure, whereas the extent of the livestock reservoir is relatively unknown on other continents.
From March through April 2011, MRSA was identified in pigs from 3 out of 30 production holdings in Chang Mai Province, Thailand. Representative isolates were subjected to molecular characterization and antimicrobial susceptibility testing; all isolates had genotypic and phenotypic characteristics of LA-MRSA previously characterized in the region: they belonged to ST9, lacked the lukF-lukS genes encoding Panton-Valentine leukocidin, and were resistant to multiple non-β-lactam antimicrobials. However, unlike other Asian LA-MRSA-ST9 variants, they were spa type t337 and harbored a different staphylococcal cassette chromosome mec IX.
A novel MRSA-ST9 lineage has been established in the pig population of Thailand, which differs substantially from LA-MRSA lineages found in other areas of the continent. The emergence of novel LA-MRSA lineages in the animal agriculture setting is worrisome and poses a serious threat to global public health.
Staphylococcus aureus bacteremia (SAB) is an urgent medical problem due to its growing frequency and its poor associated outcome. As healthcare delivery increasingly involves invasive procedures and implantable devices, the number of patients at risk for SAB and its complications is likely to grow. Compounding this problem is the growing prevalence of methicillin resistant S. aureus (MRSA) and the dwindling efficacy of vancomycin, long the treatment of choice for this pathogen. Despite the recent availability of several new antibiotics for S. aureus, new strategies for treatment and prevention are required for this serious, common cause of human infection.
Staphylococcus aureus; bacteremia; MRSA; epidemiology; infective endocarditis; treatment
We report the first detection of methicillin-resistant Staphylococcus aureus isolates in pigs in Peru. The isolates belong to a livestock-associated lineage previously reported in North America and Europe, CC398, and a highly virulent USA300-like ST8-IV variant, which is the predominant community-associated lineage in Latin America.
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.