Reduced In Vitro Susceptibility of Streptococcus pyogenes to ß-Lactam Antibiotics Associated with Mutations in the pbp2x Gene Is Geographically Widespread.

Recently, two related Streptococcus pyogenes strains with reduced susceptibility to ampicillin, amoxicillin, and cefotaxime, antibiotics commonly used to treat S. pyogenes infections, were reported. The two strains had the same nonsynonymous (amino acid-substituting) mutation in the pbp2x gene, encoding penicillin-binding protein 2X (PBP2X). This concerning report led us to investigate our library of 7,025 genome sequences of type emm1, emm28, and emm89S. pyogenes clinical strains recovered from intercontinental sources for mutations in pbp2x We identified 137 strains that, combined, had 37 nonsynonymous mutations in 36 codons in pbp2x Although to a lesser magnitude than the two previously published isolates, many of our strains had decreased susceptibility in vitro to multiple beta-lactam antibiotics. Many pbp2x mutations were found only in single strains, but 16 groups of two or more isolates of the same emm type had an identical amino acid replacement. Phylogenetic analysis showed that, with one exception, strains of the same emm type with the same amino acid replacement were clonally related by descent. This finding indicates that strains with some amino acid changes in PBP2X can successfully spread to new human hosts and cause invasive infections. Mapping of the amino acid changes onto a three-dimensional structure of the related Streptococcus pneumoniae PBP2X suggests that some substitutions are located in regions functionally important in related pathogenic bacterial species. Decreased beta-lactam susceptibility is geographically widespread in strains of numerically common emm gene subtypes. Enhanced surveillance and further epidemiological and molecular genetic study of this potential emergent antimicrobial problem are warranted.

Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing.

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.

Genome sequence analysis of emm89 Streptococcus pyogenes strains causing infections in Scotland, 2010-2016.

PURPOSE: Strains of type emm89 Streptococcus pyogenes have recently increased in frequency as a cause of human infections in several countries in Europe and North America. This increase has been molecular epidemiologically linked with the emergence of a new genetically distinct clone, designated clade 3. We sought to extend our understanding of this epidemic behavior by the genetic characterization of type emm89 strains responsible in recent years for an increased frequency of infections in Scotland. METHODOLOGY: We sequenced the genomes of a retrospective cohort of 122 emm89 strains recovered from patients with invasive and noninvasive infections throughout Scotland during 2010 to 2016. RESULTS: All but one of the 122 emm89 infection isolates are of the recently emerged epidemic clade 3 clonal lineage. The Scotland isolates are closely related to and not genetically distinct from recent emm89 strains from England, they constitute a single genetic population. CONCLUSIONS: The clade 3 clone causes virtually all-contemporary emm89 infections in Scotland. These findings add Scotland to a growing list of countries of Europe and North America where, by whole genome sequencing, emm89 clade 3 strains have been demonstrated to be the cause of an ongoing epidemic of invasive infections and to be genetically related due to descent from a recent common progenitor.

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.

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.