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Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

Evaluating trends in antibiotic resistance and communicating the results to a broad audience are important for dealing with this global threat. The Drug Resistance Index (DRI), which combines use and resistance into a single measure, was developed as an easy-to-understand measure of the effectiveness of antibiotic therapy. We demonstrate its utility in communicating differences in the effectiveness of antibiotic therapy across countries.We calculated the DRI for countries with data on antibiotic use and resistance for the disease-causing organisms considered by the WHO as priority pathogens:41 countries had the requisite data and were included in the study. Resistance and use rates were highly variable across countries, butThe DRI is a useful indicator of the problem of resistance. By combining data on antibiotic use with resistance, it captures a snapshot of how the antibiotics a country typically uses match their resistance profiles. This single measure of the effectiveness of antibiotic therapy provides a means of benchmarking against other countries and can, over time, indicate changes in drug effectiveness that can be easily communicated.

Background Staphylococcus argenteus and Staphylococcus schweitzeri, previously known as divergent Staphylococcus aureus clonal lineages, have been recently established as novel, difficult-to-delimit, coagulase-positive species within the S. aureus complex. Methicillin-resistant strains of S. argenteus are known from Australia and the UK. Knowledge of their epidemiology, medical significance and transmission risk is limited and partly contradictory, hampering definitive recommendations. There is mounting evidence that the pathogenicity of S. argenteus is similar to that of ‘classical' S. aureus, while as yet no S. schweitzeri infections have been reported. Aim To provide decision support on whether and how to distinguish and report both species. Sources PubMed, searched for S. argenteus and S. schweitzeri. Content This position paper reviews the main characteristics of both species and draws conclusions for microbiological diagnostics and surveillance as well as infection prevention and control measures. Implications We propose not distinguishing within the S. aureus complex for routine reporting purposes until there is evidence that pathogenicity or clinical outcome differ markedly between the different species. Primarily for research purposes, suitably equipped laboratories are encouraged to differentiate between S. argenteus and S. schweitzeri. Caution is urged if these novel species are explicitly reported. In such cases, a specific comment should be added (i.e. ‘member of the S.aureus complex') to prevent confusion with less- or non-pathogenic staphylococci. Prioritizing aspects of patient safety, methicillin-resistant isolates should be handled as recommended for methicillin-resistant Staphylococcus aureus (MRSA). In these cases, the clinician responsible should be directly contacted and informed by the diagnosing microbiological laboratory, as they would be for MRSA. Research is warranted to clarify the epidemiology, clinical impact and implications for infection control of such isolates.

Livestock-associated methicillin-resistant Staphylococcus aureus clonal complex CC398 (LA-MRSA CC398) is resistant to nearly all β-lactams and several non-β-lactam antimicrobials. Over the last decade, it has become widespread in pig farms across Europe and is now an important cause of human infections in countries with previously low levels of MRSA, such as the Netherlands and Denmark. The hitherto uncontrolled spread of LA-MRSA CC398 underscores an urgent need to understand its epidemiology in order to develop evidence-based interventions. This study demonstrates that pig movements between farms in combination with increased bacterial resistance to specific antibiotics and heavy metals were important drivers of the rapid spread of LA-MRSA CC398 in the Danish pig production system. These findings should be taken into consideration when researchers and policy makers evaluate and decide on actions and policies to limit the spread of LA-MRSA CC398 and other pathogens in food animals., The spread of livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398) within the Danish pig production system has been linked to an increased number of human infections. Yet, the population structure and transmission dynamics of this important pathogen remain poorly understood. In this study, whole-genome sequences from 371 LA-MRSA CC398 isolates collected between 2004 and 2015 were subjected to bioinformatic analyses. The isolates originated from Danish pig farms (n = 209) and people having livestock contact (n = 79). In addition, whole-genome sequence data from 82 isolates representing an international reference collection and 83 isolates from Danish patients were included in the analysis. The results demonstrated that the increasing prevalence of LA-MRSA CC398 in Danish pigs and patients was caused by clonal expansion of three dominant lineages. The results also showed that these lineages were enriched for the tetracycline resistance gene tet(K) and other determinants conferring resistance to some of the most frequently used antimicrobials in Danish pigs. The association between pig movements and the spread of LA-MRSA CC398 was assessed in a Poisson regression analysis of 17,009 pig movements into 273 farms with known LA-MRSA CC398 status. The results demonstrated that animal movements have played a critical role in the dissemination of LA-MRSA CC398 within the Danish pig production system, although other transmission routes may also have contributed. Consistent with this scenario, the genetic relatedness of isolates from different farms was positively correlated with the number of animal movements between the farms.

INTRODUCTION: Staphylococcus aureus bacteremia (SAB) is a high-risk infection and feared complication related to hemodialysis. This study aimed to investigate incidence and risk factors for SAB depending on hemodialysis access type.METHODS: The Danish National Registry on Regular Dialysis and Transplantation was used to identify patients from January 1, 1996 to December 31, 2011 with end-stage kidney disease. Patients were followed until death, the first episode of SAB, or end of study (December 31, 2011). Independent risk factors were assessed by multivariable Poisson regression with time-updated exposure variables.FINDINGS: Total of 9997 patients were included. The initial modality of renal replacement therapy was hemodialysis in 6826 patients and peritoneal dialysis in 2882 patients; 289 patients had preemptive kidney transplantation. SAB occurred in 1278 patients (12.8%). The incidence rate of SAB declined after 90 days and leveled off after 270 days in hemodialysis, peritoneal dialysis, and kidney transplanted. As compared to peritoneal dialysis, the adjusted rate ratio (RR) for SAB was 7.42 (95% CI 5.63-9.79) in uncuffed central venous catheter (CVC), 5.68 (95% CI 4.39-7.36) in cuffed CVC, 4.43 (95% CI 2.10-9.53) in arteriovenous graft, and 3.40 (95% CI 2.79-4.15) in arteriovenous fistula. SAB risk did not differ between uncuffed and cuffed CVC. The risk of SAB was increased during the first three months of renal replacement therapy especially for CVC (RR 11.37 [95% CI7.09-18.22]) compared with peritoneal dialysis. Diabetes mellitus (RR 1.35 [95% CI 1.20-1.51]) and male sex (RR 1.15 [95% CI 1.03-1.29]) were also associated with SAB.DISCUSSION: Patients on hemodialysis had a high incidence rate of SAB, particularly those undergoing hemodialysis via CVC. SAB risk was comparable for cuffed and uncuffed CVC. Diabetes mellitus, male sex, and the first three months in renal replacement therapy were independently associated with SAB.

The role of host genetic variation in the development of complicated Staphylococcus aureus bacteremia (SAB) is poorly understood. We used whole exome sequencing (WES) to examine the cumulative effect of coding variants in each gene on risk of complicated SAB in a discovery sample of 168 SAB cases (84 complicated and 84 uncomplicated, frequency matched by age, sex, and bacterial clonal complex [CC]), and then evaluated the most significantly associated genes in a replication sample of 240 SAB cases (122 complicated and 118 uncomplicated, frequency matched for age, sex, and CC) using targeted sequence capture. In the discovery sample, gene-based analysis using the SKAT-O program identified 334 genes associated with complicated SAB at p, Author summary Complications from bloodstream infection with Staphylococcus aureus (S. aureus) are important causes of hospitalization, significant illness, and death. The causes of these complications are not well understood, but likely involve genetic factors rendering people more susceptible to such infections, differences in the bacteria that cause the infection, and the interactions between them. We examined the parts of the human genome that code for proteins to find variations that were more common in people with complicated S. aureus bacteremia (SAB), and identified one gene, called GLS2, in which variation is more common in complicated SAB cases than uncomplicated cases. Expression of GLS2 is lower in people with SAB than controls and in mouse white blood cells exposed to S. aureus. GLS2 encodes a protein that regulates the metabolism of glutamine, a regulatory process that activates white blood cells. These cells are very important in the immune response to S. aureus infection, and therefore genetic variants that might influence their growth are important potential genetic risk factors for complicated SAB.

Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius.We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller-Hinton agar from two manufacturers.Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6-20 mm and 19-30 mm. For cefoxitin 16% (95% CI 14.8-18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6-2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) 20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively.This investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci.

Background The recommended duration of antibiotic treatment for uncomplicated Staphylococcus aureus bloodstream infections is 14 days. We compared the outcomes of patients receiving short-course (6–10 days) vs. prolonged-course (11–16 days) antibiotic therapy for S. aureus bacteremia (SAB). Methods 30-day outcome of patients with penicillin (PSSAB, n = 202)) or methicillin-susceptible SAB (MSSAB, n = 203) treated with in vitro active therapy in the range of 6–16 days was analyzed using pooled data from two previously published, observational studies. Individuals were matched 1:1 by nearest neighbor propensity score matching without replacement. Regression analysis was performed to estimate the risk of all-cause mortality within 30 days after the end of antibiotic treatment. Eligible individuals had to have >1 day of follow-up after discontinuation of antimicrobials. Individuals with a diagnosis of endocarditis, bone infection, meningitis or pneumonia were excluded. Results There were 107 well-balanced matched pairs; 58 in the PSSAB and 39 in the MSSAB cohort. For PSSAB, the median duration of therapy was 8 (interquartile range [IQR], 7–10) in the short-course group and 12 days (IQR, 10–13) in the prolonged-course group. For the MSSAB cohort, these numbers were 9 days (IQR, 7–10) and 14 days (IQR, 13–16 days), respectively. No difference in mortality between short-course and prolonged-course treatment was observed (adjusted hazard ratio [aHR], 0.74; 95% confidence interval [CI], .23–2.41) and 1.14; 95% CI, 0.31–4.20), respectively for PSSAB and MSSAB. Conclusion Short courses of antibiotic therapy yielded similar clinical outcomes as prolonged courses of antibiotic therapy for S. aureus bacteremia. The findings warrant a randomized clinical trial to study the safety and efficacy of shortened antimicrobial therapy for the treatment of uncomplicated SAB. Disclosures All authors: No reported disclosures.

OBJECTIVES: An association between infection and arterial thromboembolic events (ATE) has been suggested. Here we examined the risk of myocardial infarction (MI), stroke and other ATE after Staphylococcus aureus bacteremia (SAB).METHODS: Danish register-based nation-wide observational cohort study between 1995 and 2008 with matched control subjects from the general population.RESULTS: Within a year, 278 of 15669 SAB patients and 2570 of 156690 controls developed MI, stroke or another ATE. The incidence rates among SAB patients were highest within the first 30 days and decreased over a year. The adjusted relative risk of MI, stroke and other ATE during the first 30 days after SAB in patients compared to controls were 2.2 (95% CI: 1.6-3.1), 5.5 (95% CI: 3.8-8.3) and 15.5 (95% CI: 6.9-35), respectively. Compared to controls, the increased adjusted relative risk persisted for 30 days for MI, 180 days for stroke and one year for other ATE. Increasing age, hypertension, atrial flutter/fibrillation, prior ATE and endocarditis in SAB patients were associated with an increased risk of ATE.CONCLUSIONS: SAB was associated with a short-term increased risk of ATE that persisted longer dependent on type of event. Studies are warranted to investigate treatment strategies to diminish ATE after SAB.

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation., IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone’s geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone’s spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone’s geographic spread.

Objectives Community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to clonal complex 80 (CC80) are recognized as the European CA-MRSA. The prevailing European CA-MRSA clone carries a type IVc staphylococcal cassette chromosome mec (SCC mec ) and expresses Panton-Valentine leukocidin (PVL). Recently, a significant increase of PVL-negative CC80 MRSA has been observed in Denmark. The aim of this study was to examine their genetics and epidemiology, and to compare them to the European CA-MRSA clone in order to understand the emergence of PVL-negative CC80 MRSA. Methods Phylogenetic analysis of the CC80 S. aureus lineage was conducted from whole-genome sequences of 217 isolates (23 methicillin-susceptible S. aureus and 194 MRSA) from 22 countries. All isolates were further genetically characterized in regard to resistance determinants and PVL carriage, and epidemiologic data were obtained for selected isolates. Results Phylogenetic analysis revealed the existence of three distinct clades of the CC80 lineage: (a) an methicillin-susceptible S. aureus clade encompassing Sub-Saharan African isolates ( n = 13); (b) a derived clade encompassing the European CA-MRSA SCC mec -IVc clone ( n = 185); and (c) a novel and genetically distinct clade encompassing MRSA SCC mec- IVa isolates ( n = 19). All isolates in the novel clade were PVL negative, but carried remnant parts (8–12 kb) of the PVL-encoding prophage ΦSa2 and were susceptible to fusidic acid and kanamycin/amikacin. Geospatial mapping could link these isolates to regions in the Middle East, Asia and South Pacific. Conclusions This study reports the emergence of a novel CC80 CA-MRSA sublineage, showing that the CC80 lineage is more diverse than previously assumed.

The authors regret that the author name Silvano Esposito was published incorrectly. The authors would like to apologise for any inconvenience caused.

We compared Etest and disk diffusion to broth microdilution for the detection of fluoroquinolone resistance in 135 typhoidal and nontyphoidal serovars of Salmonella . Categorical agreements for the ciprofloxacin and levofloxacin Etests were 89.6 and 83.7%, respectively. Disk diffusion categorical agreements were 88.2 and 93.3%, respectively. Only minor errors were observed.

Background: In recent years, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) multi locus sequence type CC398 has spread widely in the livestock production in Europe. The rates of LA-MRSA in hospitals have been found to be largely determined by contact to and density of livestock in the area.Methods: This is a cross sectional study of the prevalence of LA-MRSA among hospital staff in a Danish hospital situated in a livestock production region. We analysed nasal swabs, air and dust samples for the presence of MRSA using PCR and mass spectrometry.Results: Of 1745 employees, 545 (31%) contributed nasal swabs. MRSA was not detected in any participant, nor was it detected in air or dust at the hospital or in houses of employees living on farms. Four percent of the participants had contact to pigs either directly or through household members. LA-MRSA was detected in two of 26 samples from animal sheds, both of them from pig farms. The participation rate was relatively low, but participants were representative for the source population with regards to animal contact and job titles.Conclusions: The study suggests a low point prevalence of LA-MRSA carriage in Danish hospital staff even in regions where livestock production is dense. Should more studies confirm our findings we see no need for additional hospital precautions towards LA-MRSA in Denmark at the moment. We think that our data might reduce potential stigmatization of hospital workers with contact to LA-MRSA positive farms at their work places and in their communities.

Most Staphylococcus aureus isolates can cause invasive disease given the right circumstances, but it is unknown if some isolates are more likely to cause severe infections than others. S. aureus bloodstream isolates from 120 patients with definite infective endocarditis and 121 with S. aureus bacteraemia without infective endocarditis underwent whole-genome sequencing. Genome-wide association analysis was performed using a variety of bioinformatics approaches including SNP analysis, accessory genome analysis and k-mer based analysis. Core and accessory genome analyses found no association with either of the two clinical groups. In this study, the genome sequences of S. aureus bloodstream isolates did not discriminate between bacteraemia and infective endocarditis. Based on our study and the current literature, it is not convincing that a specific S. aureus genotype is clearly associated to infective endocarditis in patients with S. aureus bacteraemia.

Significance USA300 is a hypervirulent, community-acquired, multidrug-resistant Staphylococcus aureus clone that started to spread in the United States around 17 years ago. Many studies detected it also in South America, Europe, and the Asia-Pacific region. In this study, we show that USA300 is also circulating in sub-Saharan Africa. Locating the temporal and spatial origin of clonal lineages is important with respect to epidemiology and molecular evolution of pathogens. We show that USA300 evolved from a less virulent and less resistant ancestor circulating in Central Europe around 160 years ago. Constant surveillance of pathogen transmission routes is vital to prevent and control potential outbreaks. Whole genome sequencing proved to be a useful tool for epidemiological surveillance., USA300 is a pandemic clonal lineage of hypervirulent, community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) with specific molecular characteristics. Despite its high clinical relevance, the evolutionary origin of USA300 remained unclear. We used comparative genomics of 224 temporal and spatial diverse S. aureus isolates of multilocus sequence type (ST) 8 to reconstruct the molecular evolution and global dissemination of ST8, including USA300. Analyses of core SNP diversity and accessory genome variations showed that the ancestor of all ST8 S. aureus most likely emerged in Central Europe in the mid-19th century. From here, ST8 was exported to North America in the early 20th century and progressively acquired the USA300 characteristics Panton–Valentine leukocidin (PVL), SCCmec IVa, the arginine catabolic mobile element (ACME), and a specific mutation in capsular polysaccharide gene cap5E. Although the PVL-encoding phage ϕSa2USA was introduced into the ST8 background only once, various SCCmec types were introduced to ST8 at different times and places. Starting from North America, USA300 spread globally, including Africa. African USA300 isolates have aberrant spa-types (t112, t121) and form a monophyletic group within the clade of North American USA300. Large parts of ST8 methicillin-susceptible S. aureus (MSSA) isolated in Africa represent a symplesiomorphic group of ST8 (i.e., a group representing the characteristics of the ancestor), which are rarely found in other world regions. Isolates previously discussed as USA300 ancestors, including USA500 and a “historic” CA-MRSA from Western Australia, were shown to be only distantly related to recent USA300 clones.

Currently, surveillance of livestock-associated meticillin-resistant

Background Staphylococcus aureus bacteremia (SAB) is the leading cause of infective endocarditis in several countries. Since socioeconomic status (SES) is known to influence the risk of infectious diseases in general, we aimed to investigate the association between SES and SAB, and risk of subsequent endocarditis in a nationwide adult population. Methods All Danish residents were consecutively included at age ≥ 30 years during 1996–2010. We obtained information on SES (highest attained educational level), comorbidities, and microbiologically verified SAB by cross-linking nationwide registries. The incidence rate ratios (IRRs) of SAB and later endocarditis were investigated using Poisson regression models adjusted for sex, age and year (reference = highest SES). Results Our study population comprised 3,394,936 individuals (median age = 43.2 years). Over a median follow-up of 15.9 years, 13,181 individuals acquired SAB. SES was inversely associated with SAB acquisition, which declined with increasing age, e.g. in individuals with lowest SES, IRRs were 3.78 (95% confidence interval [CI] = 2.89–4.95) in age 30–50 years, 1.87 (CI = 1.60–2.18) in age > 50–70 years and 1.31 (CI = 1.11–1.54) in age > 70 years (interaction-p

International audience; In this study, the performances of four methicillin-resistant Staphylococcus aureus (MRSA) chromogenic screening media were compared for detecting mecC-positive MRSA. Using 111 clinical isolates representative of the various mecC-MRSA clones in Europe, chromID\textregistered MRSA (bioMérieux) and BrillianceTM MRSA 2 (Oxoid Ltd.) showed higher sensitivity (99.1% and 97.3%, respectively) than BBLTM CHROMagar\textregistered MRSA II (BD Diagnostics) and MRSASelectTM (Bio-Rad) (79.3% and 63.1%, respectively) (P \textless0.0001). These findings have important implications for effective public health diagnostics and epidemiological surveillance of MRSA.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) from animals to humans is of great concern due to the implications for human health and the health care system. The objective was to investigate the frequency and duration of MRSA carriage in human volunteers after a short-term exposure in a swine farm. The experimental study included 34 human volunteers staying 1 h in a MRSA-positive swine farm in four trials. In two of the trials, the influence of farm work involving pig contact was studied using a crossover design. The quantities of MRSA in nasal swabs, throat swabs, and air samples were measured at different time points and analyzed in relation to relevant covariates. This investigation showed that, overall, 94% of the volunteers acquired MRSA during the farm visit. Two hours after the volunteers left the stable, the nasal MRSA count had declined to unquantifiable levels in 95% of the samples. After 48 h, 94% of the volunteers were MRSA-negative. Nasal MRSA carriage was positively correlated to personal exposure to airborne MRSA and farm work involving pig contact and negatively correlated to smoking. No association was observed between MRSA carriage and face touching behavior, nasal methicillin-susceptible Staphylococcus aureus (MSSA) carriage, age, or gender. The increase in human MRSA carriage among the volunteers with pig contact seems to be dependent on the increased concentration of airborne MRSA of the surrounding air and not directly on physical contact with pigs. MRSA was not detected in any of the throat samples. IMPORTANCE The experimental approach made it possible to elucidate the contributions of airborne MRSA levels and farm work to nasal MRSA carriage in a swine farm. Short-term exposure to airborne MRSA poses a substantial risk for farm visitors to become nasal carriers, but the carriage is typically cleared within hours to a few days. The risk for short-term visitors to cause secondary transmissions of MRSA is most likely negligible due to the observed decline to unquantifiable levels in 95% of the nasal samples after only 2 h. The MRSA load in the nose was highly correlated to the amount of MRSA in the air and interventions to reduce the level of airborne MRSA or the use of face masks might consequently reduce nasal contamination.

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was

Summary Background Data on risk factors and rates of reinfection associated with Staphylococcus aureus bacteraemia (SAB) are sparse. Methods We conducted a nationwide cohort study of cases of SAB diagnosed between 1995 and 2008. Reinfection was defined as an episode of SAB more than 90 days after the initial episode of SAB. Comorbidity was evaluated by the Charlson Comorbidity Index (CCI). Cox proportional hazards modelling was used to estimate hazard rates (HR). Results Of 10,891 eligible patients, 774 (7.1%) experienced reinfection a median of 458 days (range 90–5021 days) after their primary SAB episode corresponding to a reinfection rate of 1459 (95% confidence interval (CI): 1357–1562) per 100,000 personyears. In multivariate analysis, sex, origin, a vascular or peritoneal device, endocarditis and comorbidity were associated with reinfection. The association was more than two-fold higher among patients in dialysis and for patients with severe comorbidity (CCI≥2). HIV infection (Hazard ratio (HR) 6.18, 95% CI: 4.17–9.16), renal disease (HR 3.92, 95% CI: 3.22–4.78), diabetes with complications (HR 2.11, 95% CI: 1.69–2.62), diabetes without complications (HR 1.61, 95% CI: 1.34–1.93), mild (HR: 1.94, 95% CI: 1.36–2.76) and severe liver disease (HR 2.08, 95% CI: 1.08–4.03), peptic ulcer (HR 1.33, 95% CI: 1.03–1.72), and paraplegia (HR 2.15, 95% CI: 1.02–4.54) were each associated with an increased risk of reinfection. Conclusions Patients with previous SAB have a 60-fold higher risk of SAB compared to the general population. Patients with HIV infection, renal disease, diabetes, liver disease, peptic ulcer and paraplegia had the highest rates of reinfection.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 is widespread in the Danish pig production with around 90% of herds being positive. Since 2009, cases of CC398 LA-MRSA infections in Danish mink farmers have been observed. The objective of the study was to examine the presence of LA-MRSA in farmed mink. The investigation comprised three different sample types 1) clinical samples from carcasses submitted to the laboratory for diagnostic examination, 2) paws and pharyngeal swabs from healthy animals collected at pelting, and 3) feed samples from mink feed producers. In clinical samples, LA-MRSA was found in 34% of submissions and was most prevalent in samples from paws (33%) and pharynx (17%), followed by nasal and intestinal samples (each 13%), whereas it was never detected in perineal samples. LA-MRSA was found in healthy animals on 40% of the investigated farms, including paw samples (29%) and pharyngeal samples (16%). Twenty out of the 108 feed samples from feed producers were positive for LA-MRSA. The dominant spa -types were t034 and t011 associated to CC398, corresponding to the dominant spa -types detected in the Danish pig production, from which slaughter offal is used for mink feed. The spa -types, the high prevalence of LA-MRSA on paws and in pharynx, and its detection in feed samples, suggest feed as a possible source of LA-MRSA in mink.

Selective reporting of antibiotic susceptibility test (AST) results is one possible laboratory-based antibiotic stewardship intervention. The primary aim of this study was to identify where and how selective reporting of AST results is implemented in Europe both in inpatient and in outpatient settings. An ESCMID cross-sectional, self-administered, internet-based survey was conducted among all EUCIC (European Committee on Infection Control) or EUCAST (European Committee on Antimicrobial Susceptibility Testing) national representatives in Europe and Israel. Of 38 countries, 36 chose to participate in the survey. Selective reporting of AST results was implemented in 11/36 countries (31%), was partially implemented in 4/36 (11%) and was limited to local initiatives or was not adopted in 21/36 (58%). It was endorsed as standard of care by health authorities in only three countries. The organisation of selective reporting was everywhere discretionally managed by each laboratory, with a pronounced intra-and inter-country variability. The most frequent application was in uncomplicated community-acquired infections, particularly urinary tract and skin and soft-tissue infections. The list of reported antibiotics ranged from a few first-line options, to longer reports where only last-resort antibiotics were hidden. Several barriers to implementation were reported, mainly lack of guidelines, poor system support, insufficient resources, and lack of professionals' capability. In conclusion, selective reporting of AST results is poorly implemented in Europe and is applied with a huge heterogeneity of practices. Development of an international framework, based on existing initiatives and identified barriers, could favour its dissemination as one important element of antibiotic stewardship programmes. KEYWORDS: Antibiotic prescription; Antibiotic resistance; Antibiotic stewardship; Antibiotic susceptibility testing; Questionnaire; Selective reporting PMID: 28093208 DOI: 10.1016/j.ijantimicag.2016.11.014, Selective reporting of antibiotic susceptibility test (AST) results is one possible laboratory-based antibiotic stewardship intervention. The primary aim of this study was to identify where and how selective reporting of AST results is implemented in Europe both in inpatient and in outpatient settings. An ESCMID cross-sectional, self-administered, internet-based survey was conducted among all EUCIC (European Committee on Infection Control) or EUCAST (European Committee on Antimicrobial Susceptibility Testing) national representatives in Europe and Israel. Of 38 countries, 36 chose to participate in the survey. Selective reporting of AST results was implemented in 11/36 countries (31%), was partially implemented in 4/36 (11%) and was limited to local initiatives or was not adopted in 21/36 (58%). It was endorsed as standard of care by health authorities in only three countries. The organisation of selective reporting was everywhere discretionally managed by each laboratory, with a pronounced intra- and inter-country variability. The most frequent application was in uncomplicated community-acquired infections, particularly urinary tract and skin and soft-tissue infections. The list of reported antibiotics ranged from a few first-line options, to longer reports where only last-resort antibiotics were hidden. Several barriers to implementation were reported, mainly lack of guidelines, poor system support, insufficient resources, and lack of professionals' capability. In conclusion, selective reporting of AST results is poorly implemented in Europe and is applied with a huge heterogeneity of practices. Development of an international framework, based on existing initiatives and identified barriers, could favour its dissemination as one important element of antibiotic stewardship programmes.

BACKGROUND: Point-of-care testing (POCT) in primary care may improve rational antibiotic prescribing. We examined use of POCT in Denmark, including patient- and general practitioner (GP)-related predictors.METHODS: We linked nationwide health care databases to assess POCT use (C-reactive protein (CRP), group A streptococcal (GAS) antigen swabs, bacteriological cultures, and urine test strips) per 1,000 overall GP consultations, 2004-2013. We computed odds ratios (OR) of POCT in patients prescribed antibiotics according to patient and GP age and sex, GP practice type, location, and workload.RESULTS: The overall use of POCT in Denmark increased by 45.8% during 2004-2013, from 147.2 per 1,000 overall consultations to 214.8. CRP tests increased by 132%, bacteriological cultures by 101.7% while GAS swabs decreased by 8.6%. POCT preceded 28% of antibiotic prescriptions in 2004 increasing to 44% in 2013. The use of POCT varied more than 5-fold among individual practices, from 54.9 to 394.7 per 1,000 consultations in 2013. POCT use varied substantially with patient age, and males were less likely to receive POCT than females (adjusted OR = 0.75, 95% CI 0.74-0.75) driven by usage of urine test strips among females (18% vs. 7%). Odds of POCT were higher among female GPs and decreased with higher GP age, with lowest usage among male GPs >60 years. GP urban/rural location and workload had little impact.CONCLUSION: GPs use POCT increasingly with the highest use among young female GPs. In 2013, 44% of all antibiotic prescriptions were preceded by POCT but testing rates vary greatly across individual GPs.

Denmark is a country with high prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 in pigs. Even though pig farming is regarded as the main source of human infection or colonization with MRSA CC398, 10-15% of the human cases appear not to be linked to pigs. Following the recent reports of MRSA CC398 in horses in other European countries and the lack of knowledge on S. aureus carriage in this animal species, we carried out a study to investigate whether horses constitute a reservoir of MRSA CC398 in Denmark, and to gain knowledge on the frequency and genetic diversity of S. aureus in horses, including bothmethicillin-resistant and -susceptible S. aureus (MSSA). Nasal swabs were collected from 401 horses originating from 74 farms, either at their farms or prior to admission to veterinary clinics. Following culture on selectivemedia, species identification by MALDI-TOF MS and MRSA confirmation by standard PCR-based methods, S. aureus and MRSA were detected in 54 (13%) and 17 (4%) horses originating from 30 (40%) and 7 (9%) farms, respectively. Based on spa typing, MSSA differed genetically from MRSA isolates. The spa type prevalent among MSSA isolates was t127 (CC1), which was detected in 12 horses from 11 farms and represents the most common S. aureus clone isolated from human bacteremia cases in Denmark. Among the 17 MRSA carriers, 10 horses from three farms carried CC398 t011 harboring the immune evasion cluster (IEC), four horses from two farms carried IEC-negative CC398 t034, and three horses from two farms carried a mecC-positive MRSA lineage previously associated with wildlife and domestic ruminants (CC130 t528). Based on whole-genome phylogenetic analysis of the 14 MRSA CC398, t011 isolates belonged to the recently identified horse-adapted clone in Europe and were closely related to human t011 isolates fromthree Danish equine veterinarians, whereas t034 isolates belonged to pig-adapted clones. Our study confirms that horses carry an equine-specific clone of MRSA CC398 that can be transmitted to veterinary personnel, and reveals that these animals are exposed to MRSA and MSSA clones that are likely to originate from livestock and humans, respectively.

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) isolates is pivotal for the control of their dissemination and for accurate management of infected patients. In 2011, a new mec variant, named mecC , that shows only 70% nucleotide sequence homology with the classical mecA

Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.

Clin Microbiol Infect ABSTRACT: 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.

Denmark is a country with high prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 in pigs. Even though pig farming is regarded as the main source of human infection or colonization with MRSA CC398, 10–15% of the human cases appear not to be linked to pigs. Following the recent reports of MRSA CC398 in horses in other European countries and the lack of knowledge on S. aureus carriage in this animal species, we carried out a study to investigate whether horses constitute a reservoir of MRSA CC398 in Denmark, and to gain knowledge on the frequency and genetic diversity of S. aureus in horses, including both methicillin-resistant and -susceptible S. aureus (MSSA). Nasal swabs were collected from 401 horses originating from 74 farms, either at their farms or prior to admission to veterinary clinics. Following culture on selective media, species identification by MALDI-TOF MS and MRSA confirmation by standard PCR-based methods, S. aureus and MRSA were detected in 54 (13%) and 17 (4%) horses originating from 30 (40%) and 7 (9%) farms, respectively. Based on spa typing, MSSA differed genetically from MRSA isolates. The spa type prevalent among MSSA isolates was t127 (CC1), which was detected in 12 horses from 11 farms and represents the most common S. aureus clone isolated from human bacteremia cases in Denmark. Among the 17 MRSA carriers, 10 horses from three farms carried CC398 t011 harboring the immune evasion cluster (IEC), four horses from two farms carried IEC-negative CC398 t034, and three horses from two farms carried a mecC-positive MRSA lineage previously associated with wildlife and domestic ruminants (CC130 t528). Based on whole-genome phylogenetic analysis of the 14 MRSA CC398, t011 isolates belonged to the recently identified horse-adapted clone in Europe and were closely related to human t011 isolates from three Danish equine veterinarians, whereas t034 isolates belonged to pig-adapted clones. Our study confirms that horses carry an equine-specific clone of MRSA CC398 that can be transmitted to veterinary personnel, and reveals that these animals are exposed to MRSA and MSSA clones that are likely to originate from livestock and humans, respectively.

International audience; The evolution of meticillin-resistant Staphylococcus aureus (MRSA) from meticillin-susceptible S. aureus has been a result of the accumulation of genetic elements under selection pressure from antibiotics. The traditional classification of MRSA into healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) is no longer relevant as there is significant overlap of identical clones between these groups, with an increasing recognition of human infection caused by livestock-associated MRSA (LA-MRSA). Genomic studies have enabled us to model the epidemiology of MRSA along these lines. In this review, we discuss the clinical relevance of genomic studies, particularly whole-genome sequencing, in the investigation of outbreaks. We also discuss the blurring of each of the three epidemiological groups (HA-MRSA, CA-MRSA and LA-MRSA), demonstrating the limited relevance of this classification. © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Background: A genetic predisposition to Staphylococcus aureus bacteremia has been demonstrated in animals, suggesting that genetic differences might influence susceptibility to S aureus in humans.Objective: To determine whether a history of S aureus bacteremia in first-degree relatives increases the rate of the disease, and whether this rate is affected by the type of family relationship (that is, parent or sibling) or by how the relative acquired the infection.Design: Register-based nationwide cohort study (1992 to 2011).Setting: Denmark.Participants: First-degree relatives (children or siblings) of patients previously hospitalized with S aureus bacteremia.Measurements: Poisson regression models were used to calculate standardized incidence ratios (SIRs) of S aureus bacteremia, with the incidence rate in the population as a reference.Results: 34 774 individuals (the exposed cohort) with a first-degree relative (index case patient) previously hospitalized with S aureus bacteremia were followed up for a median of 7.8 years (interquartile range, 3.6 to 13.0). A higher rate of S aureus bacteremia was observed among these first-degree relatives (SIR, 2.49 [95% CI, 1.95 to 3.19]) than in the background population. The estimate was significantly higher if the index case patient was a sibling (SIR, 5.01 [CI, 3.30 to 7.62]) than a parent (SIR, 1.96 [CI, 1.45 to 2.67]; interaction P < 0.0001). No interaction was observed regarding the sex of the first-degree relative (interaction P for parents = 0.85; interaction P for siblings = 0.92). Stratifying by disease acquisition revealed the highest rates in individuals exposed to index case patients with non-hospital-acquired infection. Few were infected with genetically identical bacteremia isolates.Limitation: The rarity of the outcome limited the number of variables in the multiple regression analysis, and whether nonsignificant interactions were true or caused by insufficient statistical power remains uncertain.Conclusion: A significant familial clustering of S aureus bacteremia was found, with the greatest relative rate of disease observed in individuals exposed to siblings with a history of the disease.Primary Funding Source: The Danish Heart Foundation and the Christian Larsen and Judge Ellen Larsen Foundation.

This study provides strong, novel evidence that humans may introduce methicillin-resistant Staphylococcus aureus CC398 into closed pig populations; it also demonstrates that stringent control and eradication measures were effective and prevented dissemination from pig farms to the general human population., Background. Emerging livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) persist in livestock populations and represent a reservoir for transmission to humans. Understanding the routes of introduction and further transmission is crucial to control this threat to human health. Methods. All reported cases of livestock-associated MRSA (CC398) in humans and pigs in Norway between 2008 and 2014 were included. Data were collected during an extensive outbreak investigation, including contact tracing and stringent surveillance. Whole-genome sequencing of isolates from all human cases and pig farms was performed to support and expand the epidemiological findings. The national strategy furthermore included a “search-and-destroy” policy at the pig farm level. Results. Three outbreak clusters were identified, including 26 pig farms, 2 slaughterhouses, and 36 humans. Primary introductions likely occurred by human transmission to 3 sow farms with secondary transmission to other pig farms, mainly through animal trade and to a lesser extent via humans or livestock trucks. All MRSA CC398 isolated from humans without an epidemiological link to the outbreaks were genetically distinct from isolates within the outbreak clusters indicating limited dissemination to the general population. Conclusions. This study identified preventable routes of MRSA CC398 introduction and transmission: human occupational exposure, trade of pigs and livestock transport vehicles. These findings are essential for keeping pig populations MRSA free and, from a “One Health” perspective, preventing pig farms from becoming reservoirs for MRSA transmission to humans.

Background. Panton-Valentine leucocidin is a Staphylococcus aureus virulence factor encoded by lukF-PV and lukS-PV that is infrequent in S aureus bacteremia (SAB), and, therefore, little is known about risk factors and outcome of lukF-PV/lukS-PV-positive SAB. Methods. This report is a register-based nationwide observational cohort study. lukF-PV was detected by polymerase chain reaction. Factors associated with the presence of lukF-PV were assessed by logistic regression analysis. Adjusted 30-day hazard ratios of mortality associated with lukF-PV status were computed by Cox proportional hazards regression analysis. Results. Of 9490 SAB cases, 129 were lukF-PV-positive (1.4%), representing 14 different clonal complexes. lukF-PV was associated with younger age, absence of comorbidity, and methicillin-resistant S aureus. In unadjusted analysis, mortality associated with lukF-PV-positive SAB was comparable to SAB. However, lukF-PV-positive SAB nonsurvivors were significantly older and had more comorbidity. Consequently, by adjusted analysis, the risk of 30-day mortality was increased by 70% for lukF-PV-positive SAB compared with SAB (hazard ratio, 1.70; 95% confidence interval, 1.20–2.42; P = .003). Conclusions. lukF-PV-positive SAB is rare in Denmark but associated with a significantly increased risk of mortality. Although the risk of lukF-PV-positive SAB was highest in the younger age groups, >80% of deaths associated with lukF-PV-positive SAB occurred in individuals older than 55 years.

Summary Objectives Data describing long-term mortality in patients with Staphylococcus aureus bacteremia (SAB) is scarce. This study investigated risk factors, causes of death and temporal trends in long-term mortality associated with SAB. Methods Nationwide population-based matched cohort study. Mortality rates and ratios for 25,855 cases and 258,547 controls were analyzed by Poisson regression. Hazard ratio of death was computed by Cox proportional hazards regression analysis. Results The majority of deaths occurred within the first year of SAB (44.6%) and a further 15% occurred within the following 2–5 years. The mortality rate was 14-fold higher in the first year after SAB and 4.5-fold higher overall for cases compared to controls. Increasing age, comorbidity and hospital contact within 90 days of SAB was associated with an increased risk of death. The overall relative risk of death decreased gradually by 38% from 1992–1995 to 2012–2014. Compared to controls, SAB patients were more likely to die from congenital malformation, musculoskeletal/skin disease, digestive system disease, genitourinary disease, infectious disease, endocrine disease, injury and cancer and less likely to die from respiratory disease, nervous system disease, unknown causes, psychiatric disorders, cardiovascular disease and senility. Over time, rates of death decreased or were stable for all disease categories except for musculoskeletal and skin disease where a trend towards an increase was seen. Conclusion Long-term mortality after SAB was high but decreased over time. SAB cases were more likely to die of eight specific causes of death and less likely to die of five other causes of death compared to controls. Causes of death decreased for most disease categories. Risk factors associated with long-term mortality were similar to those found for short-term mortality. To improve long-term survival after SAB, patients should be screened for comorbidity associated with SAB.

This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

Background The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing. Methods In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin]. Results and discussion The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

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.

The increasing prevalence of ESBL-, AmpC- and carbapenemase-producing Enterobacteriaceae necessitates reliable phenotypic tests for detection and categorization. The main objective of this study was to evaluate ROSCO Neo-Sensitabs with different β-lactam-β-lactam inhibitor combinations for phenotypic detection and categorization of β-lactamases in Enterobacteriaceae. Using standard CLSI/EUCAST methodology, differences in zones of inhibitions between a β-lactam alone compared with the combination with a β-lactamase inhibitor as well as subjective synergy observations were determined for 172 well characterized Enterobacteriaceae strains with defined resistance mechanisms. The results showed that for all ESBL-positive strains (n = 66), combinations of clavulanic acid synergy with cefotaxime, ceftazidime or cefepime, were observed. All acquired AmpC β-lactamases (n = 17) were detected using cloxacillin combined with cefotaxime and/or ceftazidime (both combinations were required). Carbapenemase producers (n = 59) with the exception of one KPC-producer were correctly grouped using the combination of meropenem ± aminophenylboronic acid (APBA) or dipicolinic acid (DPA). Ethylene diamine tetraacetic acid (EDTA) also inhibited all metallo-β-lactamases, but as with DPA, one false positive result was observed. Based upon these data, we propose a tablet layout for 14 cm agar plates, which could be used as a whole or in a targeted approach for detection and categorizing of relevant acquired β-lactamases in Enterobacteriaceae.

The rapid dissemination of community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) since the early 2000s and the appearance of new successful lineages is a matter of concern. The burden of these infections varies widely between different groups of individuals and in different regions of the world. Estimating the total burden of disease is therefore problematic. Skin and soft-tissue infections, often in otherwise healthy young individuals, are the most common clinical manifestation of these infections. The antibiotic susceptibilities of these strains also vary, although they are often more susceptible to 'traditional' antibiotics than related hospital-acquired strains. Preventing the dissemination of these organisms throughout the general population requires a multifaceted approach, including screening and decolonisation, general hygiene and cleaning measures, antibiotic stewardship programmes and, in the future, vaccination. The current evidence on the prevention and control of CA-MRSA is appraised and summarised in this review.

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.

SUMMARYThe prevalence and risk factors associated with livestock-associated MRSA (LA-MRSA) carriage was examined in Danish and Belgian veterinarians. The MRSA and LA-MRSA carriage rates were 9·5% (95% CI 5·3–15·6) and 7·5% (95% CI 3·8–13·1) for MRSA and LA-MRSA, respectively, in Belgium and 1·4% (95% CI: 0·17–5·05) in Denmark (all Danish MRSA isolates belonged to the LA-MRSA genotype). All LA-MRSA isolates were resistant to tetracycline and 53·4% (7/13) showed a multi-resistant phenotype. LA-MRSA was significantly associated with veterinarians in contact with livestock (P=0·046). In the multivariable analysis, working with small animals in a veterinary clinic seems to be negatively associated (OR 0·15, 95% CI 0–1·0,P=0·05) and a strong direct association was found for LA-MRSA acquisition and exposure to live pigs (OR 12·1, 95% CI 1·6–548·5,P=0·01). Since carriage of MRSA ST398 may increase the risk of complications during hospitalization, our results underline that preventive measures may need to be developed for veterinary professionals, particularly for livestock veterinarians.

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 ( mecA LGA251 ) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCC mec . The mecA LGA251 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 mecA LGA251 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 mecA LGA251 . Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

OBJECTIVES: To assess the association between comorbidities and Staphylococcus aureus bacteremia in children aged 5-18 years, thus, in children with a matured immune system. Further, we aimed to identify presumably healthy children acquiring bacteremia.STUDY DESIGN: By cross-linking nationwide registries, we consecutively included all children born from 1995 onward at their 5-year birthday or date of immigration during 2000-2015. We examined incidence rate ratios (IRR) between preselected exposures and microbiologically verified S aureus bacteremia (reference = children without exposure) using Poisson regression models.RESULTS: We followed 1 109 169 children in 2000-2015 during which 307 children (incidence rate: 3.7 per 100 000 person-years) acquired S aureus bacteremia (methicillin-resistant S aureus = 8; 2.6%). Children without known comorbidities or recent contact with the healthcare system comprised 37.1% of infected children. The highest IRRs were observed in children undergoing dialysis or plasmapheresis (IRR = 367.2 [95% CI) = 188.5-715.3]), children with organ transplantation (IRR = 149.5 [95% CI = 73.9-302.2]), and children with cancer (IRR = 102.9 [95% CI = 74.4-142.2]). Positive associations also were observed in children with chromosomal anomalies (IRR = 7.16 [95% CI = 2.96-17.34]), atopic dermatitis (IRR = 4.89 [95% CI = 3.11-7.69]), congenital heart disease (IRR = 3.14 [95% CI = 1.92-5.11]), and in children undergoing surgery (IRR = 3.34 [95% CI = 2.59-4.28]). Neither premature birth nor parental socioeconomic status was associated with increased disease rates.CONCLUSIONS: S aureus bacteremia is uncommon in children between 5 and 18 years of age. Risk factors known from the adult population, such as dialysis, plasmapheresis, organ transplantation, and cancer, were associated with the highest relative rates. However, prematurity and parental socioeconomic status were not associated with increased rates. Approximately one-third of infected children were presumably healthy.

We recently reported a phenotypic association between reduced susceptibility to zinc and methicillin resistance in Staphylococcus aureus CC398 isolates from Danish swine (F. M. Aarestrup, L. M. Cavaco, and H. Hasman, Vet. Microbiol. 142: 455-457, 2009). The aim of this study was to identify the genetic determinant causing zinc resistance in CC398 and examine its prevalence in isolates of animal and human origin. Based on the sequence of the staphylococcal cassette chromosome mec (SCC mec ) element from methicillin-resistant S. aureus (MRSA) CC398 strain SO385, a putative metal resistance gene was identified in strain 171 and cloned in S. aureus RN4220. Furthermore, 81 MRSA and 48 methicillin-susceptible S. aureus (MSSA) strains, isolated from pigs (31 and 28) and from humans (50 and 20) in Denmark, were tested for susceptibility to zinc chloride and for the presence of a putative resistance determinant, czrC , by PCR. The cloning of czrC confirmed that the zinc chloride and cadmium acetate MICs for isogenic constructs carrying this gene were increased compared to those for S. aureus RN4220. No difference in susceptibility to sodium arsenate, copper sulfate, or silver nitrate was observed. Seventy-four percent ( n = 23) of the animal isolates and 48% ( n = 24) of the human MRSA isolates of CC398 were resistant to zinc chloride and positive for czrC . All 48 MSSA strains from both human and pig origins were found to be susceptible to zinc chloride and negative for czrC . Our findings showed that czrC is encoding zinc and cadmium resistance in CC398 MRSA isolates, and that it is widespread both in humans and animals. Thus, resistance to heavy metals such as zinc and cadmium may play a role in the coselection of methicillin resistance in S. aureus .

The worldwide emergence of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) during the last decade represents a significant change in the biology of MRSA strains and is changing the epidemiology of MRSA infections. CA-MRSA infections are caused by strains belonging to lineages distinct from HA-MRSA. In the community, CA-MRSA strains typically cause skin and soft tissue infections in children and younger adults. However, CA-MRSA strains increasingly cause healthcare-acquired infections including surgical site infections, ventilator-associated pneumonia and bacteraemia. A mathematical model showing the influence of MRSA transmission in the community on the prevalence of MRSA in hospitals is presented. The increasing prevalence of MRSA in the community also results in an increase in community-onset MRSA (CO-MRSA) among S. aureus bacteraemia and other invasive infections. These patients do not have typical risk factors for MRSA. Such changes may have profound implications for the choice of empirical therapy for serious infections where S. aureus is a possible cause. The new and potentially very large reservoir of MRSA in production animals with subsequent transmission to humans represents an additional serious threat to the control of MRSA both in general and as a cause of healthcare-acquired infections. CA-MRSA is thus a matter of serious concern and should be suppressed.

Three latex agglutination kits for rapid identification of Staphylococcus aureus were compared by testing a selection of isolates, which included methicillin resistant S. aureus, methicillin sensitive S. aureus and a diverse selection of coagulase-negative staphylococci. The sensitivities of Monostaph Plus (Bionor Laboratories, Skien, Norway), Pastorex Plus (Bio-Rad Laboratories, Hercules, CA) and Staphaurex Plus (Oxoid, Cambridge, UK) were 98.5, 98.3 and 98.3% respectively, and the specificities were 97.5, 97.0 and 96.5%, respectively. None of the kits detected Staphylococcus lugdunensis correctly. This evaluation shows that the Monostaph Plus agglutination kit performs well and comparable to Pastorex Staph-Plus and Staphaurex Plus. PRACTICAL APPLICATIONS Latex agglutination kits are very useful in the routine identification of Staphylococcus aureus, but can occasionally give false-positive or false-negative results. Knowledge of how the various available kits are performing is, therefore, very important when selecting a kit for use. Correct identification of S. aureus is important, especially in relation to methicillin-resistant S. aureus, since other staphylococci species are known to carry the methicillin-resistance determinant mecA, but are of less clinical importance. This study is the first study comparing an improved version of the Monstaph-Plus kit with other latex agglutination kits, and provides new data about this kit.

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.

Objectives: To analyse the sequence diversity of the tetracycline resistance gene tet(M) in Staphylococcus aureus from humans and animals and to determine mobile elements associated with tet(M) in S. aureus. Methods: In total, 205 tetracycline-resistant isolates were screened for tet(M) by PCR. tet(M) genes were sequenced and compared with tet(M) deposited in GenBank. Based on phylogenetic analysis isolates were screened for Tn916- and Tn5801-like xislint genes, and transposons were confirmed by linking PCR. spa typing was performed and selected isolates were used as donors in a filter mating experiment. Results: Forty-one isolates (21.3%, 60.7%, 2.6% and 4.4% of the human, pig, poultry and cattle isolates, respectively) were tet(M) positive. tet(M) was located on Tn5801-like and Tn916-like transposons in humans and on a specific Tn916-like element in animals. Human isolates were of different spa types (t034, t008, t037, t051, t065, t078, t318 and t964) corresponding to different clonal complexes (CC398, CC8, CC25 and CC30). Animal isolates were of spa type t034, t011 or t0571 corresponding to CC398. tet(M) sequence types correlated with CC types. Tn916-like and Tn5801-like (Tn6014) transposons were able to transfer to S. aureus recipients. Conclusion: S. aureus of human origin contained diverse tet(M) located on Tn916- and Tn5801-like (Tn6014) transposons, and S. aureus of animal origin contained Tn916-like tet(M) genes. This suggests that conjugative transposition plays an important role in the evolution and horizontal spread of tet(M) in S. aureus. This is the first study showing horizontal transfer of Tn5801 (Tn6014).