Genetic and genomic variability of Legionella pneumophila: applications to molecular epidemiology and public health

Tesis por compendio[EN] Legionella pneumophila is a strictly environmental and opportunistic pathogen that can cause severe pneumonia after inhalation of aerosols with enough bacterial load. Outbreaks and sporadic cases are usually localized in temperate environments, and the reservoirs are often water-related sources where biofilms are created. The existence of non-cultivable forms of the bacteria increases the risk for public health, as culture-based methods may miss them, thus complicating the environmental investigations of the sources. Genetic classification through the Sequence-Based Typing (SBT) technique allowed an increased discrimination among L. pneumophila strains compared to previous methods. SBT data can also be used for genetic variability and population structure studies, but a more exhaustive analysis can be performed using high-throughput genome sequencing strategies. This thesis describes the use of both SBT and genomic sequencing to evaluate and provide solutions to different public health needs in L. pneumophila epidemiology. We have focused in the Comunidad Valenciana (CV), the second region in Spain with the highest incidence of Legionellosis, with special interest in the city of Alcoy, where recurrent outbreaks have occurred since 1998. Firstly, SBT data were used to gain a deeper insight into the genetic variability and distribution of the most abundant Sequence Types (ST) in the CV area. We have shown that the level of variability in this region is comparable to that from other countries, revealing the existence of both locally and broadly extended profiles. Approximately half of the observed genetic diversity was found to result from geographical and temporal structure. Secondly, L. pneumophila detection from environmental sources remains a challenge for public health. A comparison between water and biofilm samples using a sensitive touchdown PCR (TD-PCR) strategy revealed that the use of biofilms increased by ten-fold the detection rate. This method allowed evaluating the hidden uncultivable L. pneumophila diversity in the locality of Alcoy and the real-time investigation of a Legionellosis outbreak affecting a hotel in Calpe (Southeast of Spain) in 2012. Thirdly, genomic sequencing was applied to a set of 69 strains isolated during 13 outbreaks occurred in Alcoy in the period 1999-2010, mainly the recurrent ST578. Higher intra-outbreak variability than expected was observed, pointing to the potential existence of multiple sources in this endemic area or high environmental diversity. Interestingly, above 98% of the genomic variability in this ST was found as being incorporated through recombination processes rather than through point mutations. Finally, a metagenomic analysis of environmental biofilms from Alcoy revealed a microbial community dominated by Proteobacteria, Cyanobacteria, Actinobacteria and Bacteroidetes. Despite the known endemism of Legionella in this area, the genus was only found in a relative abundance ranging 0.01-0.07%, which explains the low recovery from environmental sources. In summary, the results from this thesis can benefit public health efforts to control this pathogen in the environment, as we provide new insight into its molecular epidemiology, with immediate applications to surveillance and outbreak investigations.[ES] Legionella pneumophila es un patógeno oportunista estrictamente ambiental capaz de causar neumonía debido a la inhalación de aerosoles con suficiente carga bacteriana. Los brotes y casos esporádicos suelen producirse en ambientes templados y los reservorios encontrarse en zonas con agua donde pueden crearse biopelículas microbianas. La existencia de formas no cultivables de la bacteria aumenta el riesgo para la salud pública, ya que los métodos estándar basados en cultivo microbiológico no pueden detectarlas, complicando las investigaciones ambientales. La clasificación genética basada en el método Sequence-Based Typing (SBT) permite un mayor poder de discriminación entre cepas de L. pneumophila en comparación con métodos previos. Los datos derivados del SBT pueden utilizarse para estudios de variabilidad genética y estructura poblacional. Sin embargo, puede llevarse a cabo un análisis más exhaustivo mediante técnicas de secuenciación genómica de alto rendimiento. Esta tesis describe la utilización tanto de SBT como de secuenciación genómica para evaluar e incluso proponer soluciones a diferentes necesidades en salud pública relacionadas con la epidemiología de L. pneumophila. Nos centramos en la Comunidad Valenciana (CV), la segunda región en España con mayor incidencia de Legionelosis, con especial interés en la localidad de Alcoy, donde ocurren brotes de forma recurrente. En primer lugar, utilizamos datos derivados de SBT para conocer mejor la variabilidad y la distribución de los perfiles genéticos (Sequence Types, ST) en el área de la CV. Mostramos que el nivel de variabilidad en sólo esta región es comparable a la de otros países, con perfiles extendidos local y globalmente. Aproximadamente la mitad de la diversidad genética observada se estima que procede de estructuración geográfica y temporal. En segundo lugar, la detección de L. pneumophila a partir de fuentes ambientales sigue suponiendo un reto para la salud pública. En esta tesis realizamos una comparación entre la detección mediante touchdown PCR (TD-PCR) a partir de muestras de agua y biopelículas microbianas y mostramos que estas últimas proporcionan un aumento de 10 veces en la tasa de detección de la bacteria. Este método permitió evaluar la diversidad no cultivable de L. pneumophila en la localidad de Alcoy y la investigación a tiempo real de un brote en un hotel en Calpe (Sudeste de España) en 2012. A continuación, aplicamos la secuenciación genómica a 69 cepas aisladas durante 13 brotes ocurridos en Alcoy en el período 1999-2010, principalmente el recurrente ST578. Se observó mayor variabilidad entre cepas de un mismo brote que la esperada, lo cual apunta a la existencia potencial de múltiples fuentes en este área, o alta diversidad ambiental. Además, se observó que más del 98% de la variabilidad genómica fue introducida por procesos de recombinación y no de mutación puntual. Finalmente, se realizó un análisis metagenómico de biopelículas ambientales recogidas en Alcoy. Se encontró que la comunidad está dominada por Proteobacteria, Cyanobacteria, Actinobacteria y Bacteroidetes. A pesar del conocido endemismo de Legionella en el área, este género sólo se encontró en una abundancia relativa entre 0.01-0.07%, lo cual explica su baja tasa de recuperación a partir de muestras ambientales. En resumen, los resultados de esta tesis pueden ser de utilidad para los programas de control de este patógeno llevados a cabo por las autoridades de salud pública, ya que proporcionan una nueva percepción de su epidemiología molecular, con aplicación inmediata a la vigilancia e investigación de brotes.[CA] Legionella pneumophila és un patogen oportunista estrictament ambiental capaç d'ocasionar pneumònia degut a la inhalació d'aerosols amb la suficient carga bacteriana. Els brots i casos esporàdics solen ocórrer en ambients temperats, i els reservoris solen trobar-se en zones amb aigua on poden crear-se biopel·lícules microbianes. La existència de formes no cultivables del bacteri augmenten el risc per a la salut pública, ja que els mètodes estàndard basats en el cultiu microbiològic no poden detectar-les, complicant les investigacions ambientals. La classificació genètica basada en el mètode Sequence-Based Typing (SBT) permet un major poder de discriminació entre soques de L. pneumophila en comparació amb previs mètodes. Les dades derivades del SBT poden utilitzar-se per a estudis de variabilitat genètica i estructura poblacional, però un anàlisis més exhaustiu pot dur-se a terme a través de tècniques de seqüenciació genòmica d'alt rendiment. Esta tesis descriu la utilització tant del SBT com de la seqüenciació genòmica per a avaluar i proposar solucions a diferents necessitats en salut pública relacionades amb l'epidemiologia de L. pneumophila. Ens centrem en la Comunitat Valenciana (CV), la segona regió d'Espanya amb la major incidència de Legionel·losi, amb especial interès en la localitat d'Alcoi, on els brots ocorren de forma recurrent des de 1998. Primer, hem utilitzat dades derivades del SBT per a conèixer millor la variabilitat i la distribució dels perfils genètics (Sequence Types, ST) en l'àrea de la CV. Mostrem que el nivell de variabilitat en només aquesta regió és comparable a la d'altres països, amb perfils estesos tant de forma local com més amplia. Aproximadament la meitat de la diversitat genètica observada s'estima que procedeix d'estructuració geogràfica i temporal. Segon, la detecció de L. pneumophila a partir de fonts ambientals continua suposant un repte per a la salut pública. En aquesta tesis realitzem una comparació entre la detecció mitjançant touchdown PCR (TD-PCR) a partir de mostres d'aigua i biopel·lícules microbianes i mostrem que aquestes últimes proporcionen un augment de deu vegades en la tassa de detecció. A més, aquest mètode ens va permetre avaluar la diversitat no cultivable de L. pneumophila a la localitat d'Alcoi i la investigació a temps real d'un brot de Legionelosis que va afectar a un hotel en Calp (Sud-est d'Espanya) a l'any 2012. Tercer, vam aplicar la seqüenciació genòmica a 69 soques aïllades durant 13 brots ocorreguts a Alcoi en el període 1999-2010, principalment el recurrent ST578. Es va observar una major variabilitat entre soques d'un mateix brot de l'esperada, apuntant a l'existència potencial de múltiples fonts en aquesta àrea, considerada endèmica, o alta diversitat ambiental. A més, es va observar que més del 98% de la variabilitat genòmica havia sigut introduïda a partir de processos de recombinació i no de mutació puntual. Finalment, es va realitzar una anàlisi metagenòmica de biopel·lícules ambientals recollides a Alcoi. Varem trobar que la comunitat està dominada per Proteobacteria, Cyanobacteria, Actinobacteria i Bacteroidetes. A pesar del conegut endemisme de Legionella en l'àrea, aquest gènere només es va trobar en una abundància relativa entre 0.01-0.07%, el qual explica la seua baixa tassa de recuperació a partir de mostres ambientals. En resum, els resultats d'aquesta tesi poden ser d'utilitat per als programes de control d'aquest patogen duts a terme per les autoritats de salut pública, ja que proporcionen una nova percepció de la seua epidemiologia molecular, amb aplicació immediata a la vigilància i la investigació de brots.Sánchez Busó, L. (2015). Genetic and genomic variability of Legionella pneumophila: applications to molecular epidemiology and public health [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52854TESISPremios Extraordinarios de tesis doctoralesCompendi

Genetic variation regulates the activation and specificity of Restriction-Modification systems in Neisseria gonorrhoeae

ABSTRACT Restriction-Modification systems (RMS) are one of the main mechanisms of defence against foreign DNA invasion and can have an important role in the regulation of gene expression. The obligate human pathogen Neisseria gonorrhoeae carries one of the highest loads of RMS in its genome; between 13 to 15 of the three main types. Previous work has described their organization in the reference genome FA1090 and has experimentally inferred the associated methylated motifs. Here, we studied the structure of RMS and target methylated motifs in 25 gonococcal strains sequenced with Single Molecule Real-Time (SMRT) technology, which provides data on DNA modification. The results showed a variable picture of active RMS in different strains, with phase variation switching the activity of Type III RMS, and both the activity and specificity of a Type I RMS. Interestingly, the Dam methylase was found in place of the NgoAXI endonuclease in two of the strains, despite being previously thought to be absent in the gonococcus. We also identified the real methylation target of NgoAX as 5’-GCAGA-3’, different from that previously described. Results from this work give further insights into the diversity and dynamics of RMS and methylation patterns in N. gonorrhoeae

ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads.

Antimicrobial resistance (AMR) is one of the major threats to human and animal health worldwide, yet few high-throughput tools exist to analyse and predict the resistance of a bacterial isolate from sequencing data. Here we present a new tool, ARIBA, that identifies AMR-associated genes and single nucleotide polymorphisms directly from short reads, and generates detailed and customizable output. The accuracy and advantages of ARIBA over other tools are demonstrated on three datasets from Gram-positive and Gram-negative bacteria, with ARIBA outperforming existing methods

Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination

Genomic and Phenotypic Variability in Neisseria gonorrhoeae Antimicrobial Susceptibility, England.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern. Phylogenetic analyses resolve uncertainties regarding genetic relatedness of isolates with identical phenotypes and inform whether AMR is due to new mutations and clonal expansion or separate introductions by importation. We sequenced 1,277 isolates with associated epidemiologic and antimicrobial susceptibility data collected during 2013-2016 to investigate N. gonorrhoeae genomic variability in England. Comparing genetic markers and phenotypes for AMR, we identified 2 N. gonorrhoeae lineages with different antimicrobial susceptibility profiles and 3 clusters with elevated MICs for ceftriaxone, varying mutations in the penA allele, and different epidemiologic characteristics. Our results indicate N. gonorrhoeae with reduced antimicrobial susceptibility emerged independently and multiple times in different sexual networks in England, through new mutation or recombination events and by importation. Monitoring and control for AMR in N. gonorrhoeae should cover the entire population affected, rather than focusing on specific risk groups or locations

Phylogenomic analysis of Neisseria gonorrhoeae transmission to assess sexual mixing and HIV transmission risk in England: a cross-sectional, observational, whole-genome sequencing study.

BACKGROUND: Characterising sexual networks with transmission of sexually transmitted infections might allow identification of individuals at increased risk of infection. We aimed to investigate sexual mixing in Neisseria gonorrhoeae transmission networks between women, heterosexual men, and men who report sex with men (MSM), and between people with and without HIV. METHODS: In this cross-sectional observational study, we whole-genome sequenced N gonorrhoeae isolates from the archive of the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP).w Isolates that varied by five single nucleotide polymorphisms or fewer were grouped into clusters that represented sexual networks with N gonorrhoeae transmission. Clusters were described by gender, sexual risk group, and HIV status. FINDINGS: We sequenced 1277 N gonorrhoeae isolates with linked clinical and sociodemographic data that were collected in five clinics in England during 2013-16 (July 1 to Sept 30 in 2013-15; July 1 to Sept 9 in 2016). The isolates grouped into 213 clusters. 30 (14%) clusters contained isolates from heterosexual men and MSM but no women and three (1%) clusters contained isolates from only women and MSM. 146 (69%) clusters comprised solely people with negative or unknown HIV status and seven (3%) comprised only HIV-positive people. 60 (28%) clusters comprised MSM with positive and negative or unknown HIV status. INTERPRETATION: N gonorrhoeae molecular data can provide information indicating risk of HIV or other sexually transmitted infections for some individuals for whom such risk might not be known from clinical history. These findings have implications for sexual health care, including offering testing, prevention advice, and preventive treatment, such as HIV pre-exposure prophylaxis. FUNDING: National Institute for Health Research Health Protection Research Unit; Wellcome; Public Health England

European Chlamydia abortus livestock isolate genomes reveal unusual stability and limited diversity, reflected in geographical signatures.

BACKGROUND: Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics. RESULTS: Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied. CONCLUSIONS: The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable

Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.

BACKGROUND: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen. METHODS: Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes. RESULTS: Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1-508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana. CONCLUSION: Our data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups

A global resource for genomic predictions of antimicrobial resistance and surveillance of Salmonella Typhi at pathogenwatch.

As whole-genome sequencing capacity becomes increasingly decentralized, there is a growing opportunity for collaboration and the sharing of surveillance data within and between countries to inform typhoid control policies. This vision requires free, community-driven tools that facilitate access to genomic data for public health on a global scale. Here we present the Pathogenwatch scheme for Salmonella enterica serovar Typhi (S. Typhi), a web application enabling the rapid identification of genomic markers of antimicrobial resistance (AMR) and contextualization with public genomic data. We show that the clustering of S. Typhi genomes in Pathogenwatch is comparable to established bioinformatics methods, and that genomic predictions of AMR are highly concordant with phenotypic susceptibility data. We demonstrate the public health utility of Pathogenwatch with examples selected from >4,300 public genomes available in the application. Pathogenwatch provides an intuitive entry point to monitor of the emergence and spread of S. Typhi high risk clones

Europe-wide expansion and eradication of multidrug-resistant Neisseria gonorrhoeae lineages: a genomic surveillance study

Background: Genomic surveillance using quality-assured whole-genome sequencing (WGS) together with epidemiological and antimicrobial resistance (AMR) data is essential to characterise the circulating Neisseria gonorrhoeae lineages and their association to patient groups (defined by demographic and epidemiological factors). In 2013, the European gonococcal population was characterised genomically for the first time. We describe the European gonococcal population in 2018 and identify emerging or vanishing lineages associated with AMR and epidemiological characteristics of patients, to elucidate recent changes in AMR and gonorrhoea epidemiology in Europe. Methods: We did WGS on 2375 gonococcal isolates from 2018 (mainly Sept 1-Nov 30) in 26 EU and EEA countries. Molecular typing and AMR determinants were extracted from quality-checked genomic data. Association analyses identified links between genomic lineages, AMR, and epidemiological data. Findings: Azithromycin-resistant N gonorrhoeae (8·0% [191/2375] in 2018) is rising in Europe due to the introduction or emergence and subsequent expansion of a novel N gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroup, G12302 (132 [5·6%] of 2375; N gonorrhoeae sequence typing for antimicrobial resistance [NG-STAR] clonal complex [CC]168/63), carrying a mosaic mtrR promoter and mtrD sequence and found in 24 countries in 2018. CC63 was associated with pharyngeal infections in men who have sex with men. Susceptibility to ceftriaxone and cefixime is increasing, as the resistance-associated lineage, NG-MAST G1407 (51 [2·1%] of 2375), is progressively vanishing since 2009-10. Interpretation: Enhanced gonococcal AMR surveillance is imperative worldwide. WGS, linked to epidemiological and AMR data, is essential to elucidate the dynamics in gonorrhoea epidemiology and gonococcal populations as well as to predict AMR. When feasible, WGS should supplement the national and international AMR surveillance programmes to elucidate AMR changes over time. In the EU and EEA, increasing low-level azithromycin resistance could threaten the recommended ceftriaxone-azithromycin dual therapy, and an evidence-based clinical azithromycin resistance breakpoint is needed. Nevertheless, increasing ceftriaxone susceptibility, declining cefixime resistance, and absence of known resistance mutations for new treatments (zoliflodacin, gepotidacin) are promising.This study was supported by the European Centre for Disease Prevention and Control, the Centre for Genomic Pathogen Surveillance, the Li Ka Shing Foundation (Big Data Institute, University of Oxford), the Wellcome Genome Campus, the Foundation for Medical Research at Örebro University Hospital, and grants from Wellcome (098051 and 099202). LSB was funded by Conselleria de Sanitat Universal i Salut Pública, Generalitat Valenciana (Plan GenT CDEI-06/20-B), Valencia, Spain, and Ministry of Science, Innovation and Universities (PID2020–120113RA-I00), Spain, at the time of analysing and writing this manuscript.S