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Introduction. Transmission of Enterobacterales in neonatal intensive care units (NICU) can cause outbreaks of colonization and invasive infections among neonates. Two clusters of nosocomial transmission of Klebsiella pneumoniae identified by MALDI-ToF mass-spectrometry were suspected at two NICUs in July and August 2016. Aim. To assess the potential transmission of K. pneumoniae among neonates. Methodology. Whole-genome sequencing (WGS) was performed of K. pneumoniae isolates obtained through targeted surveillance of patients and environmental sampling. Results. WGS data revealed that patient and environmental isolates represented two species, K. pneumoniae and K. variicola . Core-genome multi-locus sequence typing (cgMLST) of the isolates identified three separate transmission clusters, in Hospital A a cluster of K. pneumoniae isolates in 12 children and two environmental samples and a second cluster of K. variicola isolates in five children. In Hospital B a cluster of K. pneumoniae isolates from three children and five unrelated isolates of K. pneumoniae and two unrelated isolates of K. variicola were found. Conclusion. K. variicola can cause hospital outbreaks of colonization and infection similar to other Klebsiella spp. Preliminary results from this study were presented at the 27th European Congress of Clinical Microbiology and Infectious Diseases, April 22-25, 2018, Vienna, Austria.

1AbstractAimThe aim of this study was to estimate the household transmissibility of SARS-CoV-2 for lineage B.1.1.7 compared with other lineages, by age and viral load. Further-more, we wanted to estimate whether there is a multiplicative or additive effect of the increased transmissibility of B.1.1.7 compared with other lineages.BackgroundNew lineages of SARS-CoV-2 are of potential concern due to higher transmissibility, risk of severe outcomes, and/or escape from neutralizing antibodies. Lineage B.1.1.7 has been estimated to be more transmissible than other previously known lineages, but the association between transmissibility and risk factors, such as age of primary case and viral load is still unknown.MethodsWe used comprehensive administrative data from Denmark, comprising the full population, all SARS-CoV-2 RT-PCR tests, and all WGS lineage data (January 11 to February 7, 2021), to estimate household transmissibility stratified by lineage B.1.1.7 and other lineages.ResultsWe included 5,241 households with primary cases; 808 were infected with SARS-CoV-2 lineage B.1.1.7 and 4,433 were infected with other lineages. The attack rate was 38% in households with a primary case infected with B.1.1.7 and 27% in households with a primary case infected with other lineages. Primary cases infected with B.1.1.7 had an increased transmissibility of 1.5-1.7 times that of primary cases infected with other lineages. The increased transmissibility of B.1.1.7 was multiplicative across age and viral load.ConclusionsThe results found in this study add new knowledge that can be used to mitigate the further spread of SARS-CoV-2 lineage B.1.1.7, which is becoming increasingly widespread in numerous countries. Our results clarify that the transmissibility of B.1.1.7 should be included as a multiplicative effect in mathematical models used as a tool for decision makers. The results may have important public health implications, as household transmission may serve as a bridge between otherwise separate transmission domains, such as schools and physical workplaces, despite implemented non-pharmaceutical interventions.

BackgroundThe pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and also to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. Blood donors comprise approximately 4.7% of the similarly aged population of Denmark and blood is donated in all areas of the country. The objective of this study was to perform real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR.MethodsAll Danish blood donors aged 17-69 years giving blood April 6 to 17 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI).ResultsThe first 9,496 blood donors were tested and a combined adjusted seroprevalence of 1.7% (CI: 0.9-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 82 per 100,000 (CI: 59-154) infections.ConclusionsThe IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR, including only individuals with no comorbidity, is likely several fold lower than the current estimate. This may have implications for risk mitigation. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

These bacteria are capable of adapting to humans, leading to increased spread into the community and healthcare settings., Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) can acquire phage-encoded immune modulators, such as the immune evasion cluster (IEC), which protects bacteria from components of the human innate immune system, and the enzyme TarP, which protects against antibody-mediated immune recognition. We used whole-genome sequencing and epidemiologic investigations to study the effects of IEC- and tarP-harboring phages on household transmission of LA-MRSA in North Denmark Region during 2004–2011. We reviewed information about all patients throughout Denmark who experienced LA-MRSA infection during 2007–2018 to determine whether IEC is associated with increased spread into the general population. Horizontal acquisition of IEC in the human host was associated with increased household transmission of LA-MRSA and spillover into the community and healthcare settings, whereas we found no evidence to suggest that IEC-positive LA-MRSA isolates have become self-sustainable in the general population. By contrast, TarP did not seem to influence household transmission of LA-MRSA.

Staphylococcus aureus uses several strategies to evade the host immune reaction including expression of the genes on the immune evasion cluster (IEC), which target the innate immune response, and tarP, which alters the structure of the bacterial wall teichoic acids to avoid binding of host antibodies. IEC is carried on an ΦSa3 prophage, while the tarP gene is found located on diverse prophages. Livestock-associated methicillin-resistant S. aureus of clonal complex 398 (LA-MRSA CC398) typically lacks the IEC, but a number of Danish isolates have been found to carry the elements regardless. In this study, whole-genome sequences of 96 isolates from humans and 45 isolates from pigs from the North Denmark Region were used to establish a maximum-likelihood phylogeny from core-genome SNPs and to investigate the prevalence of IEC and tarP. Furthermore, epidemiological and national surveillance data were used to investigate household transmission and the prevalence of IEC in LA-MRSA CC398 isolates from the general population. The study documents several independent acquisitions of IEC on distinct ΦSa3 prophages in humans and an almost 3-fold higher human-to-human transmission rate of LA-MRSA CC398 in households with strains carrying IEC than in households with strains lacking it. No such effect was found for tarP, which is widespread among LA-MRSA CC398 isolates from both humans and pigs. Moreover, IEC also seems to promote spread of LA-MRSA CC398 in the general population. Thus, LA-MRSA CC398 is capable of re-adapting to the human host by acquisition of human-specific immune evasion factors encoded on mobile genetic elements.

Optimal transportation of bacteria is important for accurate clinical interpretation, quantitative assays, and microbiome studies. A transport medium should ideally keep the bacteria alive without supporting growth or altering the relative proportions of the constituent species. We investigated the effect of nasal mucus and mucin on the growth and survival of two Staphylococcus aureus strains in liquid Amies transport medium at room temperature and 4 °C for 14 days. The study showed that the presence of nasal mucus in microbiological samples stimulated undesired S. aureus growth at room temperature in a dose-dependent manner. These findings underscore that microbiological samples from humans and animals should be stored at 4 °C until analysis to avoid undesired S. aureus growth.

Background: Aerococcus urinae and Aerococcus sanguinicola are relatively newcomers and emerging organisms in clinical and microbiological practice. Both species have worldwide been associated with urinary tract infections. More rarely cases of bacteremia/septicemia and infective endocarditis have been reported. Treatment options are therefore important. Just recently, European recommendations on susceptibility testing and interpretive criteria have been released. Objective: In this investigation 120 A. urinae and A. sanguinicola isolates were tested for susceptibility to six antimicrobial agents: Penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin. Methods: Three susceptibility testing methods were used; disk diffusion according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) standardized disk diffusion methodology and MIC determination with Etest and broth microdilution (BMD). All testing was performed with EUCAST media for fastidious organisms. Results: Data obtained in this study were part of the background data for establishing EUCAST breakpoints. MIC values obtained by Etest and BMD were well correlated with disk diffusion results. Conclusion: All isolates were found susceptible to all six antimicrobial agents: penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is increasingly related to human infections. Farmers and veterinarians have the highest risk, but infections have also occurred in individuals without prior contact to livestock. Clonal complex (CC) 398 is the predominant LA-MRSA lineage causing human infections, and although pigs are the major source of CC398 worldwide, poultry and other animals are also reservoirs. This raises concern for transmission of MRSA via meat. In this study, the occurrence and characteristics of S. aureus isolated from Danish retail meat were examined with main focus on chicken meat. A total of 145 meat samples from Danish supermarkets were examined, including chicken (Danish, n=102), turkey (non-Danish origin; n=23), and pork (Danish, n=20). S. aureus was detected in 69% of the meat samples. MRSA was detected in 19 meat samples (13%), resulting in MRSA prevalence of 4% of chicken, 52% of turkey, and 15% of pork. Three MRSA positive samples were obtained by direct plating (Brilliance MRSA2), whereas 16 MRSA positive samples were detected only after enrichment (TSB+6.5% NaCl and Brilliance MRSA2). Based on spa typing, 68% of MRSA isolates belonged to CC398 (spa t034, t011, t2582, t108), and hereof one isolate derived from chicken (1%). Further findings were spa type t1430 (CC9) in turkey samples (16%) and the human-associated t008 (CC8) in chicken samples (16%). In conclusion, S. aureus was readily detected in Danish retail meat, but presence of MRSA in chicken meat is rare and it is unlikely to be an important transmission factor of MRSA to humans.

Over the last decade, an increasing number of infections with livestock-associated methicillin-resistant Staphylococcus aureus of clonal complex 398 (LA-MRSA CC398) in persons without contact to livestock has been registered in Denmark. These infections have been suggested to be the result of repeated spillover of random isolates from livestock into the community. However, other studies also found emerging sub-lineages spreading among humans. Based on genome-wide SNPs and genome-wide association studies (GWAS), we assessed the population structure and genomic content of Danish LA-MRSA CC398 isolates from healthcare-associated infections from 2014 to 2016 (n = 73) and compared these to isolates from pigs in Denmark from 2014 (n = 183). Phylogenetic analyses showed that most human isolates were closely related to and scattered among pig isolates showing that the majority of healthcare-associated infections are the result of repeated spillover from pig farms, even though cases of human-to-human transmission also were identified. GWAS revealed frequent loss of antimicrobial resistance genes and acquisition of human-specific virulence genes in the human isolates showing adaptation in response to changes in selective pressures in different host environments, which over time could lead to the emergence of LA-MRSA CC398 lineages more adapted to human colonization and transmission.

Background: Increasing attention has been given to the risk of infective endocarditis (IE) in patients with certain blood stream infections (BSI). Previous studies have been conducted on selected patient cohorts, yet unselected data are sparse. We aimed to investigate the prevalence of IE in BSIs with bacteria typically associated with IE. Methods: By crosslinking nationwide registries from 2010-2016, we identified patients with BSIs typically associated with IE: Enterococcus faecalis (E. faecalis), Staphylococcus aureus (S. aureus), Streptococcus spp., and coagulase negative staphylococci (CoNS) and examined the concurrent IE prevalence. A trend test was used to examine temporal changes in the prevalence of IE. Findings: In total 60,119, distributed with 15,407, 16,790, and 27,922 BSIs were identified in the periods of 2010-2011, 2012-2013, and 2014-2016, respectively. Patients with E. faecalis had the highest prevalence of IE (16.3%) followed by S. aureus (10.2%), Streptococcus spp. (7.3%), and CoNS (1.6%). Throughout the study period, the prevalence of IE among patients with E. faecalis increased significantly (p=0.003). Male patients had a higher prevalence of IE for all microorganisms investigated compared with females. A significant increase in the prevalence of IE was seen for E. faecalis, Streptococcus spp., and CoNS with increasing age. Interpretation: For E. faecalis BSI, 1 in 6 had IE, for S. aureus BSI 1 in 10 had IE, and for Streptococcus spp. 1 in 14 had IE. Our results suggest that screening for IE seems reasonable in patients with E. faecalis BSI, S. aureus BSI, or Streptococcus spp. BSI. Funding Statement: The authors state: "No funding." Declaration of Interests: Lauge Ostergaard: no competing interest. Niels Eske Bruun: no competing interest. Marianne Voldstedlund: no competing interest. Magnus Arpi: no competing interest. Christian Ostergaard Andersen: no competing interest. Henrik C Schonheyder: no competing interest. Lars Lemming: reports grants from Merck Sharp & Dohme (MSD) outside the submitted work. Flemming Rosenvinge: no competing interest. Nana Valeur: no competing interest. Peter Sogaard: no competing interest. Paal Skytt Andersen: no competing interest. Robert Skov: no competing interest. Ming Chen: no competing interest. Kasper Iversen: no competing interest. Sabine Gill: no competing interest. Trine Kiilerich Lauridsen: no competing interest. Anders Dahl: no competing interest. Louise Bruun Oestergaard: no competing interest. Jonas Agerlund Povlsen: no competing interest. Claus Moser: no competing interest. Henning Bundgaard: no competing interest. Lars Kober: no competing interest. Emil Loldrup Fosbol: no competing interest. Ethics Approval Statement: This study was approved by the Danish Data Protection Agency. Register-based studies do not require ethical approval in Denmark.

Background Increasing attention has been given to patients with blood stream infection (BSI) and the concurrent prevalence of infective endocarditis (IE). However, there exist a gap in knowledge on the prevalence of IE among patients with a left-sided heart valve prosthesis and BSI. Purpose To examine the prevalence between different BSI and IE in patients with a left-sided heart valve prosthesis. Methods By linking Danish nationwide registries, we were able to identify patients with a left-sided prosthetic heart valve and first-time BSI of interest (Enterococcus faecalis, Staphylococcsus aureus, streptococci species (spp.), and coagulase negative staphylococci [CoNS]) in the period of 2010–2016. Concurrent admission for IE was identified from the Danish National Patient Registry in a period of up to 14 days after the BSI. Results A total of 1,465 patients with BSI were included (1,319 patients with an aortic valve prosthesis [82.6% bioprosthetic] and 146 patients with a mitral valve prosthesis [48.6% bioprosthetic]). Among the included patients, 244 had E. faecalis (median age: 78.3 years, 77.0% male), 301 had S. aureus (median age: 76.9 years, 69.1% male), 401 had streptococci spp. (median age: 78.0 years, 69.1% male) and 519 had CoNS (median age: 76.4 years, 64.2% male). The percentage of patients with a diagnosis of IE in relation to the BSI was 44.3%, 33.6%, 34.4%, and 7.9% for E. faecalis, S. aureus, streptococci spp., and CoNS, respectively. No difference in the overall results was seen by type of valve prosthesis, sex, or age group. Percentage of patients with endocarditis Conclusion In patients with a left-sided heart valve prosthesis, IE was diagnosed in almost half of the patients with E. faecalis, in 1/3 with S. aureus or streptococci spp. and in 1 in 12 with CoNS BSI. Future studies may try to explain why some microbes are more likely to cause prosthetic heart valve endocarditis, than others are.

The impact of antibiotic prophylaxis on fecal carriage of ESBL-/AmpC-/carbapenemase-producing Enterobacteriaceae (CPE) was investigated. Patients admitted for elective surgery or diagnostic procedure in a Department of Surgical Gastroenterology (SG) (n= 450) and Orthopedic Surgery (OS) (n= 300) provided a fecal swab at admission and responded to a questionnaire on possible exposures. SG patients received gentamicin/penicillin G (±metronidazole); OS patients received cefuroxime. Two days after surgery a second swab was taken. From SG patients, 6% of first swabs and 9% of second swabs were positive for ESBL-/AmpC-producers. A similar carriage rate was observed in OS patients (6% and 8%, respectively). No CPE were detected. Escherichia coli was the predominant species and blaCTX-M-15 (29% and 22%) and blaCTX-M-14 (11% and 17%) were the most prevalent ESBL genotypes among SG and OS patients. Two different prophylactic antibiotic regimens had no impact on carriage rates. Previous hospitalization and antimicrobial treatment were associated with carriage for SG patients.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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 .

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).

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.

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 log 10 number of CFU/ml (Δlog 10 CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog 10 CFU/ml) > cefuroxime (3.56 Δlog 10 CFU/ml) > rifampin (1.86 Δlog 10 CFU/ml) > gentamicin (0.61 Δlog 10 CFU/ml) > azithromycin (0.21 Δlog 10 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.

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 (SCC mec ) 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 SCC mec 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 SCC mec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

In Europe, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been caused predominantly by isolates belonging to the “European CA-MRSA” clone (sequence type 80, staphylococcal cassette chromosome mec type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene ( spa ) types, and susceptibility patterns.

The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http:// www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) communityoriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine wholegenome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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

Staphylococcus aureus is a leading cause of bacteraemia. This study analysed temporal trends from 18 702 adult cases of S. aureus bacteraemia in Denmark between 1981 and 2000. After stratification for mode of acquisition, 57% of cases were hospital-acquired (HA), 28% were community-acquired (CA) and 15% were of undetermined acquisition (UA). Incidence rates increased from 18.2 to 30.5 cases/100 000 population. Annual rates increased by 6.4% for CA, by 2.2% for HA and by 3.6% for UA cases, respectively. Case-mortality associated with HA bacteraemia decreased from 36.2% to 20.7% (43% rate reduction, p 0.0001), compared with a decrease from 34.5% to 26.5% (23% rate reduction, p 0.0001) for CA bacteraemia. Following multivariate analysis, age, pneumonia, endocarditis and chronic illness were associated with increased mortality, regardless of the mode of acquisition. Overall, mortality associated with S. aureus bacteraemia declined significantly between 1981 and 2000, but incidence rates doubled, so that the total number of deaths increased. These data emphasise the public health importance of S. aureus bacteraemia and the need for further preventive measures and improved care in order to reduce incidence rates and improve outcomes.

The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2*, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.

Detection of fluoroquinolone resistance in Salmonella enterica has become increasingly difficult due to evolving resistance mechanisms to this antimicrobial class in this organism. We evaluated two quinolone disks and five fluoroquinolone disks for their ability to act as a surrogate agent for the detection of fluoroquinolone resistance in a collection of 136 S. enterica isolates, including 111 with intermediate or resistant ciprofloxacin MICs mediated by a variety of resistance mechanisms. Ciprofloxacin, ofloxacin, and pefloxacin disks detected all isolates resistant to ciprofloxacin (0% very major error) and yielded false resistance (major error) in 8, 4, and 12% of susceptible isolates, respectively. Ciprofloxacin and pefloxacin provided clearer differentiation of susceptible and resistant isolates.

Fluoroquinolones (FQs) are among the drugs of choice for treatment of Salmonella infections. However, fluoroquinolone resistance is increasing in Salmonella due to chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes gyrA , gyrB , parC , and parE and/or plasmid-mediated quinolone resistance (PMQR) mechanisms including qnr variants, aac ( 6 ′)- Ib-cr , qepA , and oqxAB . Some of these mutations cause only subtle increases in the MIC, i.e., MICs ranging from 0.12 to 0.25 mg/liter for ciprofloxacin (just above the wild-type MIC of ≤0.06 mg/liter). These isolates are difficult to detect with standard ciprofloxacin disk diffusion, and plasmid-mediated resistance, such as qnr , is often not detected by the nalidixic acid screen test. We evaluated 16 quinolone/fluoroquinolone disks for their ability to detect low-level-resistant Salmonella enterica isolates that are not serotype Typhi. A total of 153 Salmonella isolates characterized for the presence ( n = 104) or absence ( n = 49) of gyrA and/or parC topoisomerase mutations, qnrA , qnrB , qnrD , qnrS , aac ( 6 ′)- Ib-cr , or qepA genes were investigated. All isolates were MIC tested by broth microdilution against ciprofloxacin, levofloxacin, and ofloxacin and by disk diffusion using EUCAST or CLSI methodology. MIC determination correctly categorized all isolates as either wild-type isolates (MIC of ≤0.06 mg/liter and absence of resistance genes) or non-wild-type isolates (MIC of >0.06 mg/liter and presence of a resistance gene). Disk diffusion using these antibiotics and nalidixic acid failed to detect some low-level-resistant isolates, whereas the 5-μg pefloxacin disk correctly identified all resistant isolates. However, pefloxacin will not detect isolates having aac ( 6 ′)- Ib-cr as the only resistance determinant. The pefloxacin disk assay was approved and implemented by EUCAST (in 2014) and CLSI (in 2015).