Unearthing new ccr genes and SCC elements in staphylococci through genome mining.

The SCCmec typing is crucial for investigating methicillin-resistant S. aureus, relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB and 3 ccrC) through mining of the NCBI database, which forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of five groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

The association between Staphylococcus aureus colonization on cheek skin at 2†months and subsequent atopic dermatitis in a prospective birth cohort.

BACKGROUND: Staphylococcus aureus may worsen already established atopic dermatitis (AD), but its primary role in the aetiopathogenesis and severity of AD is unclear. OBJECTIVES: To compare the prevalence of S. aureus colonization in early infancy in children who developed AD during the first 2 years of life with children who did not. METHODS: In this prospective birth cohort study, which included 450 infants, we analysed bacterial swabs collected from cheek skin at 0 and 2 months of age. The development of AD, and its severity, was diagnosed by a physician and monitored prospectively for 2 years. Information on parental atopy, filaggrin gene mutation status and use of antibiotics and emollients was included in the analyses. RESULTS: At birth, the occurrence of S. aureus colonization was similar in infants who developed subsequent AD and those who did not. At 2 months of age, S. aureus colonization was more common in children who later developed AD (adjusted hazard ratio 1.97, 95% confidence interval 1.21-3.19; P = 0.006). No association was found between S. aureus colonization and AD severity or age at onset. CONCLUSIONS: It remains unknown whether colonization with S. aureus may directly increase the risk of AD, or whether it should be considered as secondary to skin barrier impairment or a skewed immune activity, but according to our findings, S. aureus colonization is more commonly increased at 2 months of age in children who later developed AD.

Pivmecillinam, the paradigm of an antibiotic with low resistance rates in Escherichia coli urine isolates despite high consumption.

OBJECTIVES: Pivmecillinam, the oral version of mecillinam, represents one of the major recommended and used antibiotics for empiric and targeted treatment of urinary tract infections in primary care in Denmark, Norway and Sweden. Mecillinam resistant mutants in Escherichia coli develop easily in vitro, but their fitness cost has been shown to be high. METHODS: We revisited the resistance and consumption data from the monitoring programmes in the three countries and compared pivmecillinam with ciprofloxacin from 2010 to 2020. RESULTS: Mecillinam resistance rates in Escherichia coli remained around 6% in Denmark and Norway relative to a constant consumption in Norway of 1.6-1.8 DID (defined daily doses per 1000 inhabitants per day), and even increasing in Denmark from 1.6 to 2.3 DID. In Sweden resistance was significantly lower at 4% related to the lower consumption of 0.5 DID. For ciprofloxacin, resistance rates fluctuated around 6%-12%, highest in Sweden with the highest consumption (0.8-0.6 DID) and lowest in Denmark (0.55-0.35 DID) and Norway (0.7-0.3 DID), although consumption declined significantly in all three countries. CONCLUSIONS: Pivmecillinam is an example of an antibiotic, which easily develops resistance in vitro, but apparently can be used broadly in primary care without increase in resistance rates.

Possible misinterpretation of penicillin susceptibility in Staphylococcus aureus blood isolate due to in vitro loss of the blaZ gene.

We describe a case of recurrent catheter-related blood stream infections (BSI) with Staphylococcus aureus, in which the first isolate tested susceptible to penicillin, while subsequent isolates were resistant. Phenotypic susceptibility correlated with the absence/presence of the blaZ gene. The in vitro stability of penicillin resistance was investigated by subculturing single colonies. In two out of five colonies, phenotypical resistance was lost after a single subculture, which correlated with loss of the blaZ gene. This in vitro phenomenon probably resulted in a very major error in the microbiology report of the first BSI, where penicillin had been recommended as treatment.

Heterogeneity of Staphylococcus epidermidis in prosthetic joint infections: time to reevaluate microbiological criteria?

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

COVID-19 preventive measures coincided with a marked decline in other infectious diseases in Denmark, spring 2020.

We aimed to descriptively analyse the possible impact of the national COVID-19 interventions on the incidence of common infectious diseases in Denmark during spring and summer 2020. This observational study focused on national register data on infections caused by 16 different bacterial and viral pathogens. We included new cases registered between 1 January 2016 and 31 July 2020. The weekly number of new cases were analysed with respect to the COVID-19-related interventions introduced during 2020. We found a marked decrease in infections associated with droplet transmission coinciding with the COVID-19 interventions in spring and summer 2020. These included decreases in both viral and bacterial airway infections and also decreases in invasive infections caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. There was also a reduction in cases associated with foodborne transmission during the COVID-19 lockdown period. We found no effect of the lockdown on infections by invasive beta-haemolytic streptococci group B, C and G, Staphylococcus aureus bacteraemia, Neisseria gonorrhoeae or Clostridioides difficile. In conclusion, we found that the widespread interventions such as physical distancing, less travel, hygiene measures and lockdown of schools, restaurants and workplaces together coincided with a marked decline in respiratory infections and, to a smaller extent, some foodborne-transmitted infections.

Staphylococcus aureus induces cell-surface expression of immune stimulatory NKG2D ligands on human monocytes.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Evaluation of methods for detection of ß-lactamase production in MSSA.

OBJECTIVES: Correct determination of penicillin susceptibility is pivotal for using penicillin in the treatment of Staphylococcus aureus infections. This study examines the performance of MIC determination, disc diffusion and a range of confirmatory tests for detection of penicillin susceptibility in S. aureus. METHODS: A total of 286 consecutive penicillin-susceptible S. aureus blood culture isolates as well as a challenge set of 62 MSSA isolates were investigated for the presence of the blaZ gene by PCR and subjected to penicillin-susceptibility testing using broth microdilution MIC determination, disc diffusion including reading of the zone edge, two nitrocefin tests and the cloverleaf test. RESULTS: Using PCR-based detection of blaZ as the gold standard, both broth microdilution MIC testing and disc diffusion testing resulted in a relatively low accuracy (82%-93%) with a sensitivity ranging from 49%-93%. Among the confirmatory tests, the cloverleaf test performed with 100% accuracy, while zone edge interpretation and nitrocefin-based tests increased the sensitivity of ß-lactamase detection to 96%-98% and 82%-96% when using MIC determination or disc diffusion as primary test, respectively. CONCLUSIONS: This investigation showed that reliable and accurate detection of ß-lactamase production in S. aureus can be obtained by MIC determination or penicillin disc diffusion followed by interpretation of the zone edge as a confirmatory test for apparently penicillin-susceptible isolates. The more cumbersome cloverleaf test can also be used. Nitrocefin-based tests should not be used as the only test for confirmation of a presumptive ß-lactamase-negative isolate.

Dynamics of the Human Nasal Microbiota and Staphylococcus aureus CC398 Carriage in Pig Truck Drivers across One Workweek.

Drivers of pig trucks constitute a potential route of human transmission of livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398). In this study, we determined MRSA prevalence in pig truck drivers (n = 47) and monitored the nasal microbiota of 9 drivers 3 times daily throughout 1 workweek (n = 113 samples) and compared it to that of their spouses (n = 25 samples from 6 spouses) and 89 nonexposed subjects. S. aureus isolates (n = 232) derived from a subset of nasal and truck samples were whole-genome sequenced. The nasal alpha diversity of drivers in the beginning of the workday was lower than that of nonexposed subjects. During the workday, it increased significantly. Similarly, the drivers' nasal composition shifted during the workday, becoming increasingly different from that of their spouses and nonexposed individuals. Clustering into community state types (CSTs) revealed frequent switches from either S. aureus- or Corynebacterium-dominated CSTs in the mornings to a Psychrobacter-dominated CST during the workday. Six intermittent MRSA carriers were mostly MRSA negative in the mornings, and their nasal microbiota resembled that of nonexposed subjects. When acquiring MRSA during the workday, they switched to the Psychrobacter-dominated CST. In contrast, the nasal microbiota of two persistent MRSA carriers was dominated by staphylococci. In conclusion, we show that the nasal microbiota of pig truck drivers is very dynamic, undergoes drastic changes during workdays, and differs from that of nonexposed subjects even before pig contact. MRSA-carrying drivers may eventually introduce MRSA into the community and health care facilities. Carriage dynamics, however, showed that for most drivers, CC398 MRSA is rapidly lost and only rarely causes transmission to spouses. IMPORTANCE In Denmark, the number of human methicillin-resistant Staphylococcus aureus (MRSA) cases has increased dramatically since the early 2000s, starting from imported cases and spreading in the community. However, today, approximately one-third of all new cases are attributed to livestock-associated MRSA clonal complex 398 (LA-MRSA CC398). This mirrors the increase in pig farms, of which 95% are now positive for LA-MRSA, and this has been caused mainly by three dominant lineages enriched for a number of key antimicrobial resistance genes. Although most human LA-MRSA CC398 infections in Denmark are linked to livestock contact, still up to one-third are not. Pig truck drivers constitute a previously understudied occupation group which may transmit LA-MRSA CC398 to household members, the community, and hospitals. In this study, we demonstrate dramatic work-related changes in the nasal microbiota of pig truck drivers, as well as in their carriage of LA-MRSA CC398. However, they likely do not constitute an important reservoir for LA-MRSA CC398 dissemination.

Human genetic variation in GLS2 is associated with development of complicated Staphylococcus aureus bacteremia.

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<3.5 x 10-3. These, along with eight biologically relevant candidate genes were examined in the replication sample. Gene-based analysis of the 342 genes in the replication sample using SKAT-O identified one gene, GLS2, significantly associated with complicated SAB (p = 1.2 x 10-4) after Bonferroni correction. In Firth-bias corrected logistic regression analysis of individual variants, the strongest association across all 10,931 variants in the replication sample was with rs2657878 in GLS2 (p = 5 x 10-4). This variant is strongly correlated with a missense variant (rs2657879, p = 4.4 x 10-3) in which the minor allele (associated here with complicated SAB) has been previously associated with lower plasma concentration of glutamine. In a microarray-based gene-expression analysis, individuals with SAB exhibited significantly lower expression levels of GLS2 than healthy controls. Similarly, Gls2 expression is lower in response to S. aureus exposure in mouse RAW 264.7 macrophage cells. Compared to wild-type cells, RAW 264.7 cells with Gls2 silenced by CRISPR-Cas9 genome editing have decreased IL1-ß transcription and increased nitric oxide production after S. aureus exposure. GLS2 is an interesting candidate gene for complicated SAB due to its role in regulating glutamine metabolism, a key factor in leukocyte activation.

Origin, evolution, and global transmission of community-acquired Staphylococcus aureus ST8.

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.

Age-Dependent Increase in Incidence of Staphylococcus aureus Bacteremia, Denmark, 2008-2015.

Staphylococcus aureus bacteremia (SAB) is a major cause of illness and death worldwide. We analyzed temporal trends of SAB incidence and death in Denmark during 2008-2015. SAB incidence increased 48%, from 20.76 to 30.37 per 100,000 person-years, during this period (p<0.001). The largest change in incidence was observed for persons >80 years of age: a 90% increase in the SAB rate (p<0.001). After adjusting for demographic changes, annual rates increased 4.0% (95% CI 3.0-5.0) for persons <80 years of age, 8.4% (95% CI 7.0-11.0) for persons 80-89 years of age, and 13.0% (95% CI 9.0-17.5) for persons >90 years of age. The 30-day case-fatality rate remained stable at 24%; crude population death rates increased by 53% during 2008-2015 (p<0.001). Specific causes and mechanisms for this rapid increase in SAB incidence among the elderly population remain to be clarified.

International travel as source of a hospital outbreak with an unusual meticillin-resistant Staphylococcus aureus clonal complex 398, Denmark, 2016.

In May 2016, an unusual outbreak with the Panton-Valentine leukocidin-positive human variant of meticillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 occurred among mothers and infants in the maternity unit of a Danish hospital. MRSA sharing genotypic and phenotypic characteristics was confirmed in 36 cases, including 26 patients, nine household members and a healthcare worker (HCW) who had contact with all the patients. The national MRSA database contained 37 seemingly unlinked MRSA cases whose isolates shared the same genotypic and phenotypic characteristics as the outbreak strain. Whole genome sequencing showed that three of these isolates clustered together with the 36 outbreak isolates, suggesting spread outside the hospital. The HCW and 21 of 37 cases from the national MRSA database had links to south-eastern Asia, where the outbreak strain is endemic. These findings suggest that the HCW acquired the outbreak strain while travelling in south-eastern Asia and then introduced it into the hospital; from there, it spread within the patients' households and into the community. Screening of travellers returning from countries with high levels of MRSA could be an important intervention to prevent spread of these bacteria into hospitals via patients or HCWs.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Antibiotic resistance and molecular characteristics of Staphylococcus aureus isolated from backyard-raised pigs and pig workers.

Staphylococcus aureus is a commensal and pathogenic bacterium with impact on public health and livestock industry. The study investigated nasal carriage, antibiotic resistance, and molecular characterization of S. aureus in pigs and pig workers. Nasal swabs from 300 backyard-raised pigs and 101 pig workers were used for the study. Resulting isolates were confirmed using MALDI-TOF MS, tested for antibiotic resistance, and three different multiplex PCRs were used to detect enterotoxin, mecA, spaA, scn, and pvl genes. spa typing was used to annotate the isolates into MLST clonal complexes (CC). Structured questionnaire was used to access possible risk factors for S. aureus carriage. The prevalence of S. aureus in pigs and pig workers were 5.3 and 12.9%, respectively. The isolates were resistant to beta-lactams (97%), tetracycline (62%), sulfonamide (52%), aminoglycoside (20.6%), fluoroquinolone (24%), and mupirocin (3.4%). Twenty seven (93%) of the isolates carried scn, 7(24%) pvl, and 12 (41%) enterotoxin genes, respectively. Questionnaire survey showed medical-related occupation of household members was associated (p < 0.5) with S. aureus carriage. This study suggests the presence of human multidrug resistant strains of S. aureus, high carriage of pvl, and enterotoxin genes, and CC5, CC15, and CC152 were the CC-groups shared among pigs and pig workers.

Transmission of Methicillin-Resistant Staphylococcus aureus to Human Volunteers Visiting a Swine Farm.

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.

Evaluation of a widely used culture-based method for detection of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA), Denmark and Norway, 2014 to 2016.

We evaluated a widely used culture-based method for detection of livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) in samples collected from pigs and the environment inside pig stables in Denmark and Norway. Selective enrichment in tryptic soy broth containing cefoxitin and aztreonam led to a high ratio of false-negative results (26%; 57/221). On this basis, we recommend reconsidering the use of selective enrichment for detection of LA-MRSA in animal and environmental samples.

Methicillin-resistant Staphylococcus aureus biofilm formation on dacryocystorhinostomy silicone tubes depends on the genetic lineage.

PURPOSE: The aim of this study was to investigate biofilm formation on silicone tubes by genetically diverse methicillin-resistant Staphylococcus aureus (MRSA) strains. METHODS: Capacity of biofilm formation on dacryocystorhinostomy silicone tubes was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on silicone tubes. RESULTS: Tested MRSA strains were classified into SCCmec type I (33.3%), II (3.3%), III (20.0%), IV (26.7%) and V (16.7%), agr type I (56.7%), II (36.7%) and III (6.6%), and eight spa clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on silicone tubes was as follows: 33.3% of strains belonged to the category of low biofilm producers, and 66.7% to moderate biofilm producers. There was statistically significant correlation between spa CC and the category of biofilm production on silicone tubes (p = 0.01): CC5 and CC45 with moderate amount of biofilm, and CC8 with low amount of biofilm. A moderate amount of biofilm formed on silicone tubes correlated with agr type II MRSA strains (p = 0.008). CONCLUSIONS: Biofilm formation by MRSA on silicone tubes is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related ocular device infections. Genotyping and biofilm quantification may be helpful in determining when decolonisation and cohort isolation are required to prevent device-related infections.

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

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

Inhibitory Effect of Newly-Synthesized Chalcones on Hemolytic Activity of Methicillin-Resistant Staphylococcus aureus.

Pathogenicity of methicillin-resistant Staphylococcus aureus (MRSA) is associated with a broad spectrum of virulence factors, amongst which is α-hemolysin. The aim of this study was to investigate the effect of three newly-synthesized chalcones (1,3- Bis-(2-hydroxy-phenyl)-propenone, 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone) on a-hemolysin production of clinical isolates of MRSA. Subinhibitory concentrations of the tested compounds reduced hemolytic activity of MRSA strains, with almost complete abolishment of hemolysis at concentrations in the range of 1/2-1/4 x MIC (25-12.5 µg/ml). In conclusion, newly-synthesized chalcones tested in this study showed potent inhibitory activity on α-hemolysin production of multiresistant and genetically diverse MRSA strains.