Seeding and Establishment of Legionella pneumophila in Hospitals: Implications for Genomic Investigations of Nosocomial Legionnaires' Disease.

BACKGROUND: Legionnaires' disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1. METHODS: WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires' disease. RESULTS: Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1. CONCLUSIONS: WGS can be used to support or refute suspected links between hospitals and Legionnaires' disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations

A community outbreak of Legionnaires’ disease caused by outdoor hot tubs for private use in a hotel

During the period October–November 2017, an outbreak of Legionnaires’ disease involving 27 cases occurred in the tourist area of Palmanova (Mallorca, Spain). The majority of cases were reported by the European Centre of Disease Prevention and Control (ECDC) as travel associated cases of Legionnaires’ disease (TALD). Most cases belonged to different hotel cluster alerts. No cases were reported among the local population residing in the area. All tourist establishments associated with one or more TALD cases were inspected and sampled by public health inspectors. All relevant sources of aerosol emission detected were investigated and sampled. The absence of active cooling towers in the affected area was verified, by documents and on-site. Samples from hot tubs for private use located on the terraces of the penthouse rooms of a hotel in the area were included in the study. Extremely high concentrations (> 106 CFU/l) of Legionella pneumophila, including the outbreak strain, were found in the hot tubs of vacant rooms of this hotel thus identifying the probable source of infection. Meteorological situation may have contributed to the geographical distribution pattern of this outbreak. In conclusion, hot tubs for private use located outdoors should be considered when investigating community outbreaks of Legionnaires’ disease of unclear origin

Mycoplasma pneumoniae detections before and during the COVID-19 pandemic: results of a global survey, 2017 to 2021

Background Mycoplasma pneumoniae respiratory infections are transmitted by aerosol and droplets in close contact. Aim We investigated global M. pneumoniae incidence after implementation of non-pharmaceutical interventions (NPIs) against COVID-19 in March 2020. Methods We surveyed M. pneumoniae detections from laboratories and surveillance systems (national or regional) across the world from 1 April 2020 to 31 March 2021 and compared them with cases from corresponding months between 2017 and 2020. Macrolide-resistant M. pneumoniae (MRMp) data were collected from 1 April 2017 to 31 March 2021. Results Thirty-seven sites from 21 countries in Europe, Asia, America and Oceania submitted valid datasets (631,104 tests). Among the 30,617 M. pneumoniae detections, 62.39% were based on direct test methods (predominantly PCR), 34.24% on a combination of PCR and serology (no distinction between methods) and 3.37% on serology alone (only IgM considered). In all countries, M. pneumoniae incidence by direct test methods declined significantly after implementation of NPIs with a mean of 1.69% (SD ± 3.30) compared with 8.61% (SD ± 10.62) in previous years (p < 0.01). Detection rates decreased with direct but not with indirect test methods (serology) (–93.51% vs + 18.08%; p < 0.01). Direct detections remained low worldwide throughout April 2020 to March 2021 despite widely differing lockdown or school closure periods. Seven sites (Europe, Asia and America) reported MRMp detections in one of 22 investigated cases in April 2020 to March 2021 and 176 of 762 (23.10%) in previous years (p = 0.04). Conclusions This comprehensive collection of M. pneumoniae detections worldwide shows correlation between COVID-19 NPIs and significantly reduced detection numbers

Group B streptococcal carriage, serotype distribution and antibiotic susceptibilities in pregnant women at the time of delivery in a refugee population on the Thai-Myanmar border

<p>Abstract</p> <p>Background</p> <p>Group B Streptococcus (GBS) is the leading cause of neonatal sepsis in the developed world. Little is known about its epidemiology in the developing world, where the majority of deaths from neonatal infections occur. Maternal carriage of GBS is a prerequisite for the development of early onset GBS neonatal sepsis but there is a paucity of carriage data published from the developing world, in particular South East Asia.</p> <p>Methods</p> <p>We undertook a cross sectional study over a 13 month period in a remote South East Asian setting on the Thai-Myanmar border. During labour, 549 mothers had a combined vaginal rectal swab taken for GBS culture. All swabs underwent both conventional culture as well as PCR for GBS detection. Cultured GBS isolates were serotyped by latex agglutination, those that were negative or had a weak positive reaction and those that were PCR positive but culture negative were additionally tested using multiplex PCR based on the detection of GBS capsular polysaccharide genes.</p> <p>Results</p> <p>The GBS carriage rate was 12.0% (95% CI: 9.4-15.0), with 8.6% positive by both culture and PCR and an additional 3.5% positive by PCR alone. Serotypes, Ia, Ib, II, III, IV, V, VI and VII were identified, with II the predominant serotype. All GBS isolates were susceptible to penicillin, ceftriaxone and vancomycin and 43/47 (91.5%) were susceptible to erythromycin and clindamycin.</p> <p>Conclusions</p> <p>GBS carriage is not uncommon in pregnant women living on the Thai-Myanmar border with a large range of serotypes represented.</p

Phenotypic and genotypic antimicrobial susceptibility patterns of the emerging human respiratory pathogen Mycoplasma amphoriforme isolated from the UK and Denmark

Methods Seven isolates of M. amphoriforme were examined for antimicrobial susceptibility to seven antibiotics using the microbroth dilution assay in line with the CLSI guidelines for mycoplasmas. Each isolate was additionally subjected to WGS to identify resistance-associated mutations. Based on the consensus sequences from the genomic data, PCR primers were designed, and tested, for the amplification of the QRDR within the parC gene. Results Of the seven isolates investigated, four (57%) were resistant to moxifloxacin (0.5–1 mg/L) and levofloxacin (1–2 mg/L), compared with those that were susceptible (0.03–0.06 and 0.006 mg/L, respectively). Isolate H29 was resistant to five of the seven antibiotics tested: moxifloxacin, 0.5 mg/L; levofloxacin, 2 mg/L; azithromycin, 64 mg/L; erythromycin, 128 mg/L; and clindamycin, 64 mg/L. All isolates were susceptible to tetracycline (0.06 mg/L) and lefamulin (0.001–0.004 mg/L). Mutations from genomic data confirmed the presence of an S89F mutation within the ParC protein among all fluoroquinolone-resistant isolates and an A2059G mutation in the 23S rRNA gene in the macrolide- and lincosamide-resistant isolate H29. Conclusions To the best of our knowledge, this is the first time where phenotypic and genotypic resistance data have been paired for M. amphoriforme confirming a correlation between the two. These data suggest the need for focused testing and resistance determination of isolates from high-risk patients given the backdrop of a high prevalence of antimicrobial resistance

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates. \ua9 2014 Macmillan Publishers Limited. All rights reserved

Surveillance of tuberculosis (TB) cases attributable to relapse or reinfection in London, 2002-2015.

Recurrence of TB in an individual can occur due to relapse of the same strain or reinfection by a different strain. The contribution of reinfection and relapse to TB incidence, and the factors associated with each are unknown. We aimed to quantify and describe cases attributable to relapse or reinfection, and identify associated risk factors in order to reduce recurrence. We categorised recurrent TB cases from notifications in London (2002-2015) as relapse or reinfection using molecular (MIRU VNTR strain type) and epidemiological information (hierarchical approach using time since notification, site of disease and method of case finding). Factors associated with each outcome were determined using logistic regression in Stata Version 13.1 (2009-2015 only). Of 43,465 TB cases, 1.4% (618) were classified as relapse and 3.8% (1,637) as reinfection. The proportion with relapse decreased from 2002 (2.3%) to 2015 (1.3%), while the proportion of reinfection remained around 4%. Relapse was more common among recent migrants (11 years, OR = 1.67, p = <0.001), those with a social risk factor (OR = 1.96, p = <0.001) and within specific areas in London. Patients with social risk factors were at increased risk of both relapse and reinfection. Characterising those with relapsed disease highlights patients at risk and factors associated with reinfection suggest groups where transmission is occurring. This will inform TB control programs to target appropriate treatment and interventions in order to reduce the risk of recurrence

External Quality Assessment of a DNA Sequence-Based Scheme for Epidemiological Typing of Legionella pneumophila by an International Network of Laboratories▿

We report the results of an international external quality assessment (EQA) program to assess the performance of laboratories in genotyping Legionella pneumophila isolates using the standard European Working Group for Legionella Infections sequence-based typing protocol. Three coded distributions of L. pneumophila isolates were sent to laboratories in 12, 14, and 20 countries, respectively. The data were returned by 11 of 16, 18 of 19, and 27 of 29 centers, respectively. Incomplete submission of data resulted in exclusion from certain aspects of the analyses. The number of centers achieving 100% score, for all loci tested, rose successively from 50% (5 of 10) for the first EQA distribution, to 56% (9 of 16) for the second EQA distribution, to 76% (19 of 25) for the third EQA distribution. A number of additional centers made only a few errors (one to three) in each distribution. Sequence data from the first two distributions were collected in flat text file format and using specially developed software, the sequence quality tool (SQT), in the third distribution. The SQT allows users to upload trace files in standard file formats, automates basecalling using phred and phrap software, contig assembly, trimming, and matching against a reference library. The program described here allow users an independent measure of sequence quality, and such schemes are vital in order to identify strengths and weakness in centers responsible for the generation of genotyping data in legionella outbreak investigation. The present study demonstrates that DNA sequence data can be highly reproducible but, when independently assessed, in practice frequently falls short of this goal. However, experience and training in the methodology results in increased performance

Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates of Legionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of >0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country

Molecular exploration for Mycoplasma amphoriforme, Mycoplasma fermentans and Ureaplasma spp. from patient samples previously investigated for Mycoplasma pneumoniae infection

Objectives: To determine the presence and genotypic macrolide susceptibility of Mycoplasma amphoriforme, and presence of Ureaplasma spp., and Mycoplasma fermentans among clinical samples from England previously investigated for Mycoplasma pneumoniae. Methods: Quantitative and conventional PCR were used to retrospectively screen a collection of 160 clinical samples, previously submitted to Public Health England (PHE) for detection of M. pneumoniae, between October 2016 and December 2017. Samples which were positive for M. amphoriforme DNA were further investigated for mutations associated with genotypic macrolide resistance by sequencing of domain V of the 23s rRNA. Results: Mycoplasma amphoriforme were detected in 10/160 (6.3%) samples, Ureaplasma parvum were detected in 4/160 samples (2.5%) with 0/160 M. fermentans detections. Of the nine individuals (two samples were from the same patient) in which M. amphoriforme were detected, eight were male (10 – 60 years age range) and one was female (30 – 40 years age range). One individual, with cystic fibrosis was positive for both M. amphoriforme and U. parvum. All M. amphoriforme DNA were genotypically susceptible for macrolides. Conclusions: Mycoplasma amphoriforme were found in clinical samples including the lower respiratory tract samples of patients with pneumonia. In the absence of other respiratory pathogens, these data suggest a potential role of this organism in human disease, with no evidence of acquired macrolide resistance. Ureaplasma parvum was detected in cerebrospinal fluid and respiratory tract samples. These data suggest the need to consider these atypical respiratory pathogens in future diagnostic investigations