Population Genomics of Legionella longbeachae and Hidden Complexities of Infection Source Attribution.

Legionella longbeachae is the primary cause of legionellosis in Australasia and Southeast Asia and an emerging pathogen in Europe and the United States; however, our understanding of the population diversity of L. longbeachae from patient and environmental sources is limited. We analyzed the genomes of 64 L. longbeachae isolates, of which 29 were from a cluster of legionellosis cases linked to commercial growing media in Scotland in 2013 and 35 were non-outbreak-associated isolates from Scotland and other countries. We identified extensive genetic diversity across the L. longbeachae species, associated with intraspecies and interspecies gene flow, and a wide geographic distribution of closely related genotypes. Of note, we observed a highly diverse pool of L. longbeachae genotypes within compost samples that precluded the genetic establishment of an infection source. These data represent a view of the genomic diversity of L. longbeachae that will inform strategies for investigating future outbreaks.

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus.

Hospital-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health burden dominated by a small number of bacterial clones. The pandemic EMRSA-16 clone (ST36-II) has been widespread in UK hospitals for 20 y, but its evolutionary origin and the molecular basis for its hospital association are unclear. We carried out a Bayesian phylogenetic reconstruction on the basis of the genome sequences of 87 S. aureus isolates including 60 EMRSA-16 and 27 additional clonal complex 30 (CC30) isolates, collected from patients in three continents over a 53-y period. The three major pandemic clones to originate from the CC30 lineage, including phage type 80/81, Southwest Pacific, and EMRSA-16, shared a most recent common ancestor that existed over 100 y ago, whereas the hospital-associated EMRSA-16 clone is estimated to have emerged about 35 y ago. Our CC30 genome-wide analysis revealed striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements and nonsynonymous mutations affecting antibiotic resistance and virulence. Importantly, phylogeographic analysis indicates that EMRSA-16 spread within the United Kingdom by transmission from hospitals in large population centers in London and Glasgow to regional health-care settings, implicating patient referrals as an important cause of nationwide transmission. Taken together, the high-resolution phylogenomic approach used resulted in a unique understanding of the emergence and transmission of a major MRSA clone and provided molecular correlates of its hospital adaptation. Similar approaches for hospital-associated clones of other bacterial pathogens may inform appropriate measures for controlling their intra- and interhospital spread.

Phenotypic detection of mecC-MRSA: cefoxitin is more reliable than oxacillin.

OBJECTIVES: To investigate the reliability of cefoxitin and oxacillin for the detection of mecC-positive Staphylococcus aureus. METHODS: The susceptibility to cefoxitin and oxacillin of 62 mecC-positive S. aureus isolates was investigated using broth microdilution, agar dilution, Etest and disc diffusion on different types of media. The data were interpreted for the utility of cefoxitin and oxacillin in conjunction with the stated methodologies for the detection of mecC-positive isolates. RESULTS: Cefoxitin with Mueller-Hinton media from Becton Dickinson and Oxoid detected all mecC-positive isolates when tested by broth microdilution, agar dilution and disc diffusion. By Etest, one isolate was falsely susceptible. Mueller-Hinton agar from bioMérieux was substantially less able to detect these isolates. One isolate was falsely susceptible by agar dilution when using Iso-Sensitest and Columbia agar. Disc diffusion using cefoxitin on Iso-Sensitest agar missed 29% of the isolates. For oxacillin, only agar dilution on Columbia agar + 2% NaCl was able to detect all mecC-positive isolates successfully. CONCLUSIONS: Cefoxitin used with EUCAST methodology and oxacillin used with agar dilution on Columbia agar + 2% NaCl detected all mecC-positive isolates. These methods with their concomitant agars should be preferred over Iso-Sensitest, which is recommended by the BSAC. It should be noted that for disc diffusion Mueller-Hinton media from bioMérieux performed poorly, with 26%-47% of mecC isolates being falsely susceptible.

Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to ß-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to ß-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant ß-lactam antibiotics. METHODS: The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays. RESULTS: We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types. CONCLUSIONS: This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for ß-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.

Use of Vitek 2 antimicrobial susceptibility profile to identify mecC in methicillin-resistant Staphylococcus aureus.

The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.

Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy.

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.

Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton-Valentine leucocidin (PVL), mecA and homologue mecALGA251.

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.

Re-emergence of early pandemic Staphylococcus aureus as a community-acquired meticillin-resistant clone.

During the 1950s, the notorious penicillin-resistant clone of Staphylococcus aureus known as phage type 80/81 emerged and caused serious hospital-acquired and community-acquired infections worldwide. This clone was largely eliminated in the 1960s, concurrent with the widespread use of penicillinase-resistant beta lactams. We investigated whether early 80/81 isolates had the genes for Panton-Valentine leucocidin, a toxin associated with virulence in healthy young people. Multilocus sequence analysis suggested that descendants of 80/81 have acquired meticillin resistance, are re-emerging as a community-acquired meticillin-resistant S aureus (MRSA) clone, and represent a sister lineage to pandemic hospital-acquired MRSA.

Pneumococci causing invasive disease in children prior to the introduction of pneumococcal conjugate vaccine in Scotland.

This study aimed to determine the serotypes and sequence types (STs) of pneumococci causing paediatric invasive disease in Scotland prior to the introduction of pneumococcal conjugate vaccines (PCVs). All invasive pneumococci isolated between 2000 and 2004 from children aged less than 5 years in Scotland were used. The isolates were characterized by serotyping and multi-locus sequence typing. Two hundred and seventeen pneumococci were characterized into 22 different serogroups/types, the most common, in rank order, being 14, 19F, 6B, 18C, 23F, 9V, 4, 1, 19A and 6A. They were further genotyped into 77 different STs, the three most common being 9, 162 and 176. Common serotypes possessed multiple STs, but pneumococci of a particular clone were mostly associated with a particular serotype. The seven most common serotypes are included in the 7-valent polysaccharide conjugate vaccine (PCV7). Serotype coverage for PCV7 was 76.5% in those aged less than 5 years but increased to 88.9% for those aged 1 year. The introduction of PCV7 into the childhood immunization schedule would reduce the burden of pneumococcal disease in children, although continued surveillance of invasive pneumococcal disease will be required before, during and after the introduction of PCVs.

Molecular diagnosis of Legionella infections--Clinical utility of front-line screening as part of a pneumonia diagnostic algorithm.

OBJECTIVES: Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. METHODS: L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. RESULTS: L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. CONCLUSIONS: Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response.

Identification of source and sink populations for the emergence and global spread of the East-Asia clone of community-associated MRSA.

BACKGROUND: Our understanding of the factors influencing the emergence, dissemination and global distribution of epidemic clones of bacteria is limited. ST59 is a major epidemic clone of community-associated MRSA in East Asia, responsible for extensive morbidity and mortality, but has a much lower prevalence in other parts of the world. The geographic origin of ST59 and its international routes of dissemination are unclear and disputed in the literature. RESULTS: To investigate the origin and spread of the ST59 clone, we obtained whole genome sequences of isolates from four continents, sampled over more than a decade, and carried out a time-scaled phylogeographic analysis. We discover that two distinct ST59 clades emerged concurrently, in East Asia and the USA, but underwent clonal expansion at different times. The East Asia clade was strongly enriched for gene determinants associated with antibiotic resistance, consistent with regional differences in antibiotic usage. Both clones spread independently to Australia and Europe, and we found evidence of the persistence of multi-drug resistance following export from East Asia. Direct transfer of strains between Taiwan and the USA was not observed in either direction, consistent with geographic niche exclusion. CONCLUSIONS: Our results resolve a longstanding controversy regarding the origin of the ST59 clone, revealing the major global source and sink populations and routes for the spread of multi-drug resistant clones. Additionally, our findings indicate that diversification of the accessory genome of epidemic clones partly reflects region-specific patterns of antibiotic usage, which may influence bacterial fitness after transmission to different geographic locations.

Turning the tide or riding the waves? Impacts of antibiotic stewardship and infection control on MRSA strain dynamics in a Scottish region over 16 years: non-linear time series analysis.

OBJECTIVES: To explore temporal associations between planned antibiotic stewardship and infection control interventions and the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). DESIGN: Retrospective ecological study and time-series analysis integrating typing data from the Scottish MRSA reference laboratory. SETTING: Regional hospital and primary care in a Scottish Health Board. PARTICIPANTS: General adult (N=1,051,993) or intensive care (18,235) admissions and primary care registrations (460,000 inhabitants) between January 1997 and December 2012. INTERVENTIONS: Hand-hygiene campaign; MRSA admission screening; antibiotic stewardship limiting use of macrolides and '4Cs' (cephalosporins, coamoxiclav, clindamycin and fluoroquinolones). OUTCOME MEASURES: Prevalence density of MRSA clonal complexes CC22, CC30 and CC5/Other in hospital (isolates/1000 occupied bed days, OBDs) and community (isolates/10,000 inhabitant-days). RESULTS: 67% of all clinical MRSA isolates (10,707/15,947) were typed. Regional MRSA population structure was dominated by hospital epidemic strains CC30, CC22 and CC45. Following declines in overall MRSA prevalence density, CC5 and other strains of community origin became increasingly important. Reductions in use of '4Cs' and macrolides anticipated declines in sublineages with higher levels of associated resistances. In multivariate time-series models (R(2)=0.63-0.94) introduction of the hand-hygiene campaign, reductions in mean length of stay (when >4 days) and bed occupancy (when >74 to 78%) predicted declines in CC22 and CC30, but not CC5/other strains. Lower importation pressures, expanded MRSA admission screening, and reductions in macrolide and third generation cephalosporin use (thresholds for association: 135-141, and 48-81 defined daily doses/1000 OBDs, respectively) were followed by declines in all clonal complexes. Strain-specific associations with fluoroquinolones and clindamycin reflected resistance phenotypes of clonal complexes. CONCLUSIONS: Infection control measures and changes in population antibiotic use were important predictors of MRSA strain dynamics in our region. Strategies to control MRSA should consider thresholds for effects and strain-specific impacts.

Laboratory diagnosis of legionnaires' disease due to Legionella pneumophila serogroup 1: comparison of phenotypic and genotypic methods.

Laboratory results of 67 cases of legionnaires' disease caused by Legionella pneumophila serogroup (Sg) 1 spanning a 6-year period were analysed by both phenotypic and genotypic methods. The methods compared were urinary antigen enzyme immunoassay (EIA), an immunofluorescent antibody (IFA) test, direct fluorescent antibody (DFA), culture and a 5S rRNA PCR with Southern blotting confirmation. Urine was available in 53 cases, of which 35 (66%) were positive, with an antigen peak observed at 5-10 days after onset of disease symptoms. The IFA test was positive in 62 (92.5%) cases, with 56 (90.3%) cases producing a greater than fourfold rise in titre and 6 (9.7%) giving presumptive high titres of > or =1:128. There were two antibody peaks, one at 10-15 days and another at >25 days after onset. In 23 cases where samples were available, DFA and culture were respectively positive in 5 (22%) and 10 (48%) cases. There was a peak in culture-positives 5-10 days after onset of disease. A Legionella-specific 5S rRNA PCR on patient serum was positive in 54 (80.5%) cases, with a peak in PCR positivity at 6-10 days after disease onset. In 22 of the 67 cases, the full panel of diagnostic methods was available for comparison. The relative sensitivity and specificity of the urinary antigen EIA and the serum PCR was 100%. The IFA gave relative sensitivity and specificity values of 93.8 and 95%. DFA and culture, although 100% specific, produced only low sensitivities, of 19 and 42.8%, respectively. This study has shown that urinary antigen and serum PCR are valuable tests in the acute phase of disease, with excellent sensitivity and specificity values. At present, the Legionella species causing infection requires to be verified by IFA serology and/or culture, but this could become unnecessary as new antigen and L. pneumophila Sg 1-specific PCR tests become available.

Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires' disease outbreak.

BACKGROUND: Legionnaires' disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires' disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates. RESULTS: Our high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila. CONCLUSIONS: Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires' disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

Trends in serotypes and sequence types among cases of invasive pneumococcal disease in Scotland, 1999-2010.

INTRODUCTION: The 7-valent pneumococcal conjugate vaccine (Prevenar(®), Wyeth; PCV7) was introduced to the UK paediatric immunisation schedule in 2006. This study investigates trends in serotypes and multi locus sequence types (STs) among cases of invasive pneumococcal disease (IPD) in Scotland prior to, and following, the introduction of PCV7. METHODS: Scottish Invasive Pneumococcal Disease Enhanced Surveillance has records of all cases of IPD in Scotland since 1999. Cases diagnosed from blood or cerebrospinal fluid isolates until 2010 were analysed. Logistic and poisson regression modelling was used to assess trends prior to and following the introduction of PCV7. RESULTS: Prior to PCV7 use, on average 650 cases of IPD were reported each year; 12% occurred in those aged <5 years and 35% affected those aged over 65 years. Serotypes in PCV7 represented 47% of cases (68% in <5 year olds). The serotype and ST distribution was relatively stable with only serotype 1 and associated ST 306 showing an increasing trend. PCV7 introduction was associated with a 69% (95% CI: 50%, 80%) reduction in the incidence of IPD among those aged <5 years, a 57% (95% CI: 47%, 66%) reduction among those aged 5-64 years but no significant change among those aged 65 years and over where increases in non-PCV7 serotypes were observed. Serotypes which became more prevalent post-PCV7 are those which were associated with STs related to the PCV7 serotypes. CONCLUSIONS: Routine serotyping and sequence typing in Scotland allowed the assessment of the relationship between the capsule and the clones in the post vaccination era. Changes in the distribution of serotypes post PCV7 introduction appear to be driven by associations between serotypes and STs prior to PCV7 introduction. This has implications for the possible effects of the introduction of higher valency vaccines and could aid in predicting replacement serotypes in IPD.

Phylogenetic analysis of Staphylococcus aureus CC398 reveals a sub-lineage epidemiologically associated with infections in horses.

In the early 2000s, a particular MRSA clonal complex (CC398) was found mainly in pigs and pig farmers in Europe. Since then, CC398 has been detected among a wide variety of animal species worldwide. We investigated the population structure of CC398 through mutation discovery at 97 genetic housekeeping loci, which are distributed along the CC398 chromosome within 195 CC398 isolates, collected from various countries and host species, including humans. Most of the isolates in this collection were received from collaborating microbiologists, who had preserved them over years. We discovered 96 bi-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed 'clade (C)') has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses in veterinary-care settings (p = 2 × 10(-7)), it remained extremely rare among S. aureus isolates from human infections.

Implications of differential age distribution of disease-associated meningococcal lineages for vaccine development.

New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.