An emerging Panton-Valentine leukocidin-positive CC5-meticillin-resistant Staphylococcus aureus-IVc clone recovered from hospital and community settings over a 17-year period from 12 countries investigated by whole-genome sequencing.

BACKGROUND: A novel Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC)5-MRSA-IVc ('Sri Lankan' clone) was recently described from Sri Lanka. Similar isolates caused a recent Irish hospital outbreak. AIM: To investigate the international dissemination and diversity of PVL-positive CC5-MRSA-IVc isolates from hospital and community settings using whole-genome sequencing (WGS). METHODS: Core-genome single nucleotide polymorphism (cgSNP) analysis, core-genome multi-locus sequence typing (cgMLST) and microarray-based detection of antimicrobial-resistance and virulence genes were used to investigate PVL-positive CC5-MRSA-IVc (N = 214 including 46 'Sri Lankan' clone) from hospital and community settings in 12 countries over 17 years. Comparators included 29 PVL-positive and 23 PVL-negative CC5/ST5-MRSA-I/II/IVa/IVc/IVg/V. RESULTS: Maximum-likelihood cgSNP analysis grouped 209/214 (97.7%) CC5-MRSA-IVc into Clade I; average of 110 cgSNPs between isolates. Clade III contained the five remaining CC5-MRSA-IVc; average of 92 cgSNPs between isolates. Clade II contained seven PVL-positive CC5-MRSA-IVa comparators, whereas the remaining 45 comparators formed an outlier group. Minimum-spanning cgMLST analysis revealed a comparably low average of 57 allelic differences between all CC5/ST5-MRSA-IVc. All 214 CC5/ST5-MRSA-IVc were identified as 'Sri Lankan' clone, predominantly spa type t002 (186/214) with low population diversity and harboured a similar range of virulence genes and variable antimicrobial-resistance genes. All 214 Sri Lankan clone isolates and Clade II comparators harboured a 9616-bp chromosomal PVL-encoding phage remnant, suggesting both arose from a PVL-positive meticillin-susceptible ancestor. Over half of Sri Lankan clone isolates were from infections (142/214), and where detailed metadata were available (168/214), most were community associated (85/168). CONCLUSIONS: Stable chromosomal retention of pvl may facilitate Sri-Lankan clone dissemination.

Staphylococcus aureus colonization during military service: a prospective cohort study.

OBJECTIVES: Staphylococcus aureus colonization leading to skin and soft-tissue infections (SSTI) are known challenges in crowded settings such as the military. The aim of the study was to establish and compare the prevalence of S. aureus colonization in recruits at enrolment and discharge after the first year of military service, and to investigate the prevalence of S. aureus SSTI. METHODS: All recruits entering first year of military service in January 2013 to be stationed at three garrisons in the northern part of Norway were invited to join this prospective cohort study. Swabs were taken from nose, throat and perineum. Staphylococcus aureus was identified using standard culturing methods. Methicillin resistance was determined by a cefoxitin disc diffusion test. RESULTS: Of the 923 eligible recruits, 512 were included at enrolment; 265/512 (52%) were also screened at discharge. Staphylococcus aureus colonization was high, and increased significantly during military service (166/265 versus 224/265, p < 0.001) mainly caused by increase in throat colonization alone or in combination with nasal colonization. All S. aureus isolates were susceptible to methicillin. SSTI was self-reported in 7/265 (3%) recruits, of which only one was confirmed by a military physician. CONCLUSION: Staphylococcus aureus colonization increased during military service, but there were few confirmed reports of SSTI. Inclusion of throat swab provides important information as ∼20% of the recruits were only positive in their throat. Further analyses need to be performed to investigate if the increase in colonization is caused by specific S. aureus stains.

Emerging multidrug-resistant Bengal Bay clone ST772-MRSA-V in Norway: molecular epidemiology 2004-2014.

A multidrug-resistant, methicillin-resistant Staphylococcus aureus (MRSA) clone, PVL-positive ST772-MRSA-V, named the Bengal Bay clone, is emerging worldwide. In Norway, where MRSA prevalence is low, a sudden increase in ST772-MRSA-V initiated a nationwide molecular epidemiological study. Clinical data were obtained from the Norwegian Surveillance System for Communicable Diseases (MSIS). S. aureus isolates were characterised by antibiotic susceptibility profiles and comprehensive genotyping (spa typing, MLVA, DNA microarray). ST772-MRSA was detected in 145 individuals during 2004-2014, with 60% of cases occurring in 2013-2014. Median age was 31 years and male/female ratio 1.16. The majority had a family background from the Indian subcontinent (70%). MRSA acquisition was mainly reported as unknown (39%) or abroad (42%), the latter associated with a home-country visit (59%), tourism (16%), and immigration (13%). Clinical infection was present in 75%, predominantly by SSTI (83%), 18% were admitted to hospital and 42% were linked to small-scale outbreaks (n = 25). All isolates were multidrug-resistant. Most isolates were resistant to erythromycin, gentamicin and norfloxacin. Genotyping revealed a conserved clone predominated by spa type t657 (83%), MLVA-type 432 (67%) and the genes lukF/S, sea, sec/sel, egc, scn, cna, ccrAA/ccrC, agrII and cap5. A few untypical ccr gene combinations were detected. Bengal Bay isolates have likely been imported on several occasions and revision of infection control guidelines may prevent further spread.

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

BACKGROUND: The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. AIM: To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. METHODS: A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. FINDINGS: All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. CONCLUSION: MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates.

Comparison of two single-use scrub suits in terms of effect on air-borne bacteria in the operating room.

A low level of air-borne bacteria in the operating room air can be achieved if all staff wear clothes made of low-permeability material (i.e. clean air suits). This study investigated if there was a difference in protective efficacy between two single-use scrubs made of polypropylene by testing them during routinely performed orthopaedic surgical procedures. No significant difference in the colony-forming unit count/m3 air was found between the two scrubs, so the choice can be based on which scrub type is more comfortable for staff.

DNA microarray analysis of Staphylococcus aureus causing bloodstream infection: bacterial genes associated with mortality?

Providing evidence for microbial genetic determinants' impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe-host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n = 305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n = 38) were characterised by DNA microarray analysis and spa typing. Fisher's exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing.

Population-based epidemiology of Staphylococcus aureus bloodstream infection: clonal complex 30 genotype is associated with mortality.

Staphylococcus aureus bloodstream infections (SABSI) are associated with a high burden of morbidity and mortality. The impact of specific S. aureus genotypes on outcome is unclear. The aim of this study was to evaluate the epidemiology and outcome of SABSI, with a special emphasis on the impact of bacterial clonal lineage on mortality. We conducted a 3-year population-based prospective study between 2011 and 2014, including 303 consecutive adult patients. Clinical data were obtained from interviews and medical records. S. aureus isolates were genotyped using DNA microarrays. The incidence rate of SABSI was 27.6 per 100,000 inhabitants [95 % confidence interval (CI) 24.6-31.0]. The median age of the patients was 71 years (interquartile range 56-81 years) and 61.4 % were male. Most SABSI (70.6 %) occurred in hospitals or associated to healthcare, and 34.1 % of these were associated with intravascular catheters. Only five (1.6 %) SABSI were caused by methicillin-resistant S. aureus (MRSA). The 30-day case fatality rate was 20.8 % (95 % CI 16.6-25.7). S. aureus clonal complex 30 [hazard ratio (HR) 3.9; 95 % CI 1.8-8.5, p = 0.001], unknown focus of infection (HR 4.5; 95 % CI 1.9-10.8, p = 0.001) and respiratory tract infection (HR 12.7; 95 % CI 4.6-34.6, p < 0.001) were independent predictors of mortality in a Cox regression analysis after adjusting for age, sex and underlying conditions. A high proportion of potential preventable SABSI calls for effective infection control measures. S. aureus clonal complex 30 genotype was associated with mortality in patients with bloodstream infections. The genetic basis underlying this association remains to be demonstrated.

spa typing alone is not sufficient to demonstrate endemic establishment of meticillin-resistant Staphylococcus aureus in a low-prevalence country.

BACKGROUND: The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) in Norway is low but increasing. Over the last decade, numerous nursing homes have experienced MRSA outbreaks. One genetic lineage, spa type t304, has been identified at multiple nursing homes and has caused large outbreaks lasting for several years. AIM: To evaluate whether spa typing is sufficient for the detection of MRSA spread and endemic establishment in a low-prevalence area, using spa type t304 as the test organism. METHODS: All spa type t304 isolates detected in 1991-2010 in the most densely populated area of Norway were included. Time and place of bacterial sampling were recorded. The isolates were analysed using multi-locus sequence typing, staphylococcal cassette chromosome mec typing, detection of lukS/F-PV and pulsed-field gel electrophoresis (PFGE). FINDINGS: In total, 181 spa type t304 isolates were identified in three of 23 municipalities. Most (91%) of the isolates could be linked to 13 nursing homes, eight of which experienced outbreaks. PFGE analysis revealed three PFGE types, consisting of 19 PFGE patterns; 95% of the isolates were PFGE type 2. In total, PFGE types 2 and 3 accounted for 99% of all nursing home isolates, and included isolates from different nursing homes, different outbreaks and different time periods. Additional genetic analyses did not further differentiate between the spa type t304 isolates. CONCLUSION: MRSA spa type t304 appears to have established itself as an endemic genetic lineage in the study area. spa typing does not provide sufficient resolution when investigating the spread of an endemic-like genetic lineage in a low-prevalence area, and should be supplemented by additional typing techniques.

Molecular characterisation of methicillin-sensitive Staphylococcus aureus from deep surgical site infections in orthopaedic patients.

Staphylococcus aureus is a leading cause of surgical site infections (SSIs). The association between S. aureus genotypes and the severity of illness is, however, incompletely understood. The aim of the study was to genotype S. aureus isolates from deep SSI in orthopaedic patients to identify molecular markers associated with invasive S. aureus infections. DNA microarray analysis was performed on S. aureus isolates collected from 60 patients with deep SSI following major orthopaedic surgery, while 57 isolates from nasal carriers served as controls. Genes associated with antibiotic resistance, adhesion, immune evasion, tissue invasion and toxin production were detected. The bone sialoprotein-binding protein gene (bbp) was more frequent in isolates from SSI patients compared to nasal carriers (95.0% vs. 82.5%), suggesting a role in invasive disease. No major differences in other molecular virulence markers could be distinguished among isolates from the two clinical groups, suggesting that any S. aureus strain may cause invasive infection. Our study reveals important genotypic information on isolates obtained from deep SSI following orthopaedic procedures.

Patterns and dynamics of airway colonisation in mechanically-ventilated patients.

The aim of this study was to investigate the patterns and dynamics of the microbiota in the airways of ventilated patients. Seventy-four mechanically-ventilated patients were recruited consecutively, and oropharyngeal, tracheal and bronchoalveolar (BAL) fluid specimens were collected 48 h after intubation, and every 72 h thereafter until the patient was extubated or a total of five sample sets had been collected. Ventilator-associated pneumonia (VAP) pathogens were identified, quantified and genotyped. Microbial findings were highly correlated both between airway locations and over time when samples were taken no more than 72 h apart. If no VAP pathogen was present in the oral flora, it was unlikely to be found in a lower airway sample; i.e., the positive predictive value of the oropharyngeal sample was 0.73 (95% CI 0.67-0.80), and the negative predictive value was 0.95 (95% CI 0.92-0.99). Colonisation with Enterobacteriacae, non-fermentative bacteria and Staphylococcus aureus was monoclonal in the airways and over time, whereas colonisation with microbes normally found in the oropharynx, i.e., Haemophilus influenzae, Haemophilus parainfluenzae and Streptococcus pneumoniae, was polyclonal. When antibiotics were used, the chance of recovering VAP pathogens from all sampling sites was reduced three-fold. No correlation was observed between a bacterial count of > or =10(4) CFU/mL in BAL fluid and chest X-rays compatible with VAP.

Does Pseudomonas aeruginosa colonization influence morbidity and mortality in the intensive care unit patient? Experience from an outbreak caused by contaminated oral swabs.

BACKGROUND: Contaminated oral swabs caused a nationwide monoclonal Pseudomonas aeruginosa outbreak involving 27 Norwegian hospitals. The aim of the study was to study the consequences on mortality and morbidity of the introduction of this P. aeruginosa strain to intensive care unit (ICU) patients. METHODS: Forty-four out of 96 patients admitted to the general ICU of Akershus University Hospital during the outbreak, ventilated for more than 24 h and with at least one microbiological sample, were included and followed until death or hospital discharge. All isolated P. aeruginosa strains were genotyped. Demographic data, admission diagnosis, Simplified Acute Physiology Score II (SAPS II), Sequential Organ Failure Assessment (SOFA) score, comorbidities, and antibiotics used in the first week were recorded. RESULTS: The outbreak strain was found in 18 patients (41%) of whom seven became infected. Median time to the first positive culture was 4 days. These 18 patients spent a significantly longer time on mechanical ventilation (P =0.03) and had a significantly higher hospital mortality, 55.5% vs. 19.2% (P =0.03), than non-colonized patients. The number of patients with severe underlying disease was significantly higher (P =0.01) and the decline in SOFA score was significantly slower in the pseudomonas group (P =0.02). Irrespective of colonization status, patients with severe underlying disease had a significantly higher mortality (58%) than those without (16%) (P =0.009). CONCLUSION: Use of contaminated oral swabs led to a high rate of early airways colonization. Patients with severe underlying disease were more likely to become colonized, but whether colonization has any influence on hospital mortality requires further study.