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.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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.

Aims

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).

Conclusion

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.

Introduction

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.

Methods

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).

Results

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.

Conclusions

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.

ABSTRACT

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.

IMPORTANCE

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.

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.

Summary

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.

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.

A PCR targeting sau1-hsdS1 was developed for rapid detection of Staphylococcus aureus clonal complex 398 (CC398). High sensitivity (100%) and specificity (100%) were shown by evaluating the test on a large strain collection (n = 1,307). We recommend this test for accurate, rapid, and inexpensive diagnosis of methicillin-resistant S. aureus (MRSA) CC398 in hospitals and on farms.

Mupirocin is widely used to decolonize patients carrying Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). The aim of this study was to determine the presence of high-level mupirocin resistance by a new commercially available mupA genotypic diagnostic product, mupA EVIGENE assay (AdvanDx).

To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.

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.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of ≤4 μg/ml for mecA-negative and ≥6 or 8 μg/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of ≤2 μg/ml for mecA-negative and ≥4 μg/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.

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.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log10 number of CFU/ml (Δlog10 CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog10 CFU/ml) > cefuroxime (3.56 Δlog10 CFU/ml) > rifampin (1.86 Δlog10 CFU/ml) > gentamicin (0.61 Δlog10 CFU/ml) > azithromycin (0.21 Δlog10 CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.

Staphylococcus lugdunensis, a rare cause of severe infections such as native valve endocarditis, often causes superficial skin infections similar to Staphylococcus aureus infections. We initiated a study to optimize the identification methods in the routine laboratory, followed by a population-based epidemiologic analysis of patients infected with S. lugdunensis in Viborg County, Denmark. Recognition of a characteristic Eikenella corrodens-like odor on Columbia sheep blood agar combined with colony pleomorphism and prominent β-hemolysis after 2 days of incubation, confirmed by API-ID-32 Staph, led to an 11-fold increase in the detection of S. lugdunensis. By these methods we found 491 S. lugdunensis infections in 4 years, corresponding to an incidence of 53 per 100,000 per year, an increase from 5 infections per 100,000 inhabitants in the preceding years. Seventy-five percent of the cases were found in general practice; these were dominated by skin abscesses (36%), wound infections (25%), and paronychias (13%). Fifty-six percent of the infections occurred below the waist, and toes were the most frequently infected site (21%). Only 3% of the patients suffered from severe invasive infections. The median age was 52 years, and the male/female ratio was 0.69. Our study shows that S. lugdunensis is a common cause of skin and soft-tissue infections (SSTI) and is probably underrated by many laboratories. S. lugdunensis should be accepted as a significant pathogen in SSTI and should be looked for in all routine bacteriological examinations, and clinicians should be acquainted with the name and the pathology of the bacterium.

Background

The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood.

Methodology/Principal Findings

We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively.

Conclusion

The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome.

Persons living or working on farms, particularly pig farms, are at increased risk for infection.

An emerging subtype of methicillin-resistant Staphylococcus aureus (MRSA), clonal complex (CC) 398, is associated with animals, particularly pigs. We conducted a matched case–control and a case–case study comparing 21 CC398 case-patients with 2 controls randomly selected from the Danish Civil Registry and 2 case-patients infected with MRSA other than CC398. On farms of case-patients, animals were examined for MRSA. Thirteen case-patients reported pig exposure. Living or working on farms with animals was an independent risk factor for CC398 in the case–control (matched odds ratio [MOR] 35.4, 95% confidence interval [CI] 2.7–469.8) and the case–case study (MOR 14.5, 95%CI 2.7–76.7). History of hospitalization was associated with an increased risk only in the case–control study (MOR 11.4, 95% CI 1.4–94.8). A total of 23 of 50 pigs on 4 of 5 farms were positive for CC398. Our results, corroborated by microbiologic testing, demonstrate that pigs are a source of CC398 in Denmark.

A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 μg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 μg/ml ERY and 0.5 μg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 μg/ml correlated well with the D-zone test.