RESUMEN
Abnormal vaginal discharge may be caused by bacterial vaginosis, vulvovaginal candidiasis, trichomoniasis and/or aerobic vaginitis. For the development of a diagnostic algorithm, tree-based classification analysis was performed on symptoms, signs and bedside test results of 56 patients, and laboratory tests (culture, Nugent score, qPCRs) were compared. Amplicon sequencing of the 16S rRNA gene was used as reference test for bacterial vaginosis and aerobic vaginitis, culture for vulvovaginal candidiasis and qPCR for trichomoniasis. For bacterial vaginosis, the best diagnostic algorithm was to screen at the bedside with a pH and odour test and if positive, to confirm by qPCR (sensitivity 94%; specificity 97%) rather than Nugent score (sensitivity of 59%; specificity 97%; P = 0.031). The analysis for the other infections was less conclusive due to the low number of patients with these infections. For bacterial vaginosis, the developed algorithm is sensitive, specific, and reduces the need for laboratory tests in 50% of the patients.
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Algoritmos , Excreción Vaginal/diagnóstico , Adolescente , Adulto , Técnicas de Laboratorio Clínico , Femenino , Humanos , Concentración de Iones de Hidrógeno , Persona de Mediana Edad , Países Bajos , Odorantes , Proyectos Piloto , Excreción Vaginal/microbiología , Excreción Vaginal/parasitología , Vaginosis Bacteriana/diagnóstico , Adulto JovenRESUMEN
Bacterial vaginosis (BV) is perceived as a condition of disrupted vaginal microbiota, but remains of unknown aetiology. In this study, vaginal microbiota composition was determined in twenty-one women with BV, before and after treatment with metronidazole or clindamycin. Microbiota composition varied greatly between women and defining a (un)healthy vaginal microbiota state remains elusive, challenging BV diagnosis and treatment. While relative abundance of Lactobacillus increased after antibiotic treatment in two-third of women, its abundance was not associated with treatment outcome. Instead, remaining complaints of abnormal vaginal discharge were more common after metronidazole treatment and associated with increased relative abundance of Ureaplasma.
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Antibacterianos/uso terapéutico , Microbiota/efectos de los fármacos , Vaginosis Bacteriana/tratamiento farmacológico , Vaginosis Bacteriana/microbiología , Adulto , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , Clindamicina/uso terapéutico , Femenino , Especificidad del Huésped , Humanos , Metronidazol/uso terapéutico , ARN Ribosómico 16S/genética , Vagina/microbiología , Excreción Vaginal/tratamiento farmacológico , Excreción Vaginal/microbiologíaRESUMEN
BACKGROUND: The elderly (≥65 years) are one of the populations most at risk for respiratory tract infections (RTIs). The aim of this study was to determine whether nasal and/or oropharyngeal microbiota profiles are associated with age and RTIs. METHODS: Nasal and oropharyngeal swabs of 152 controls and 152 patients with an RTI were included. The latter group consisted of 72 patients with an upper respiratory tract infection (URTI) and 80 with a lower respiratory tract infection (LRTI). Both nasal and oropharyngeal swabs were subjected to microbiota profiling using amplicon sequencing of the 16S rRNA gene. Moraxella species were determined using quantitative real-time PCR and culture. RESULTS: Based on the microbiota profiles of the controls and the patients with an RTI, eight nasal and nine oropharyngeal microbiota clusters were defined. Nasal microbiota dominated by either Moraxella catarrhalis or Moraxella nonliquefaciens was significantly more prevalent in elderly compared to mid-aged adults in the control group (p = 0.002). Dominance by M. catarrhalis/nonliquefaciens was significantly less prevalent in elderly with an LRTI (p = 0.001) compared to controls with similar age. CONCLUSIONS: Nasal microbiota dominated by M. catarrhalis/nonliquefaciens is associated with respiratory health in the elderly population.
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Moraxella catarrhalis/aislamiento & purificación , Moraxella/aislamiento & purificación , Nariz/microbiología , Orofaringe/microbiología , Infecciones del Sistema Respiratorio/microbiología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Moraxella/genética , Moraxella catarrhalis/genética , Infecciones del Sistema Respiratorio/diagnóstico , Ribotipificación , Medición de Riesgo , Factores de Riesgo , Adulto JovenRESUMEN
Bacterial vaginosis (BV) is a common gynaecological condition. Diagnosis of BV is typically based on Amsel criteria, Nugent score and/or bacterial culture. In this study, these conventional methods and two CE-IVD marked quantitative real-time (q)PCR assays were compared with microbiota analysis for the diagnosis of BV. Eighty women were evaluated for BV during two sequential hospital visits by Amsel criteria, Nugent score, culture, the AmpliSens® Florocenosis/Bacterial vaginosis-FRT PCR kit (InterLabService, Moscow, Russia), and the BD MAX™ Vaginal Panel (BD Diagnostics, MD, USA). Microbiota analysis based on amplicon sequencing of the 16S ribosomal RNA gene was used as reference test. The microbiota profile of 36/115 (31%) included cases was associated with BV. Based on microbiota analysis, the sensitivity of detecting BV was 38.9% for culture, 61.15% for Amsel criteria, 63.9% for Nugent score and the BD MAX assay, and 80.6% for the AmpliSens assay, while the specificity of all methods was ≥ 92.4%. Microbiota profiles of the cases with discrepant results between microbiota analysis and the diagnostic methods were variable. All five diagnostic methods missed BV positive cases with a relatively high abundance of the genus Alloscardovia, Bifidobacterium, or Dialister, which were categorised as unspecified dysbiosis by the AmpliSens assay. Compared to Amsel criteria, Nugent score, culture, and the BD MAX assay, the AmpliSens assay was most in agreement with microbiota analysis, indicating that currently, the AmpliSens assay may be the best diagnostic method available to diagnose BV in a routine clinical setting.
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Bacterias/aislamiento & purificación , Técnicas Microbiológicas/normas , Microbiota , Vaginosis Bacteriana/diagnóstico , Adolescente , Adulto , Bacterias/genética , ADN Bacteriano/genética , Pruebas Diagnósticas de Rutina , Femenino , Humanos , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Vagina/microbiología , Vaginosis Bacteriana/microbiología , Adulto JovenRESUMEN
Bloodstream infections and graft-versus-host disease are common complications after hematopoietic stem cell transplantation (HSCT) procedures, associated with the gut microbiota that acts as a reservoir for opportunistic pathogens. Selective gut decontamination (SGD) and total gut decontamination (TGD) during HSCT have been associated with a decreased risk of developing these complications after transplantation. However, because studies have shown conflicting results, the use of these treatments remains subject of debate. In addition, their impact on the gut microbiota is not well studied. The aim of this study was to elucidate the dynamics of the microbiota during and after TGD and to compare these with the dynamics of SGD. In this prospective, observational, single-center study fecal samples were longitudinally collected from 19 children eligible for allogenic HSCT (TGD, n=12; SGD, n=7), weekly during hospital admission and monthly after discharge. In addition, fecal samples were collected from 3 family stem cell donors. Fecal microbiota structure of patients and donors was determined by 16S rRNA gene amplicon sequencing. Microbiota richness and diversity markedly decreased during SGD and TGD and gradually increased after cessation of decontamination treatment. During SGD, gut microbiota composition was relatively stable and dominated by Bacteroides, whereas it showed high inter- and intraindividual variation and low Bacteroides abundance during TGD. In some children TGD allowed the genera Enterococcus and Streptococcus to thrive during treatment. A gut microbiota dominated by Bacteroides was associated with increased predicted activity of several metabolic processes. Comparing the microbiota of recipients and their donors indicated that receiving an SCT did not alter the patient's microbiota to become more similar to that of its donor. Overall, our findings indicate that SGD and TGD affect gut microbiota structure in a treatment-specific manner. Whether these treatments affect clinical outcomes via interference with the gut microbiota needs to be further elucidated.
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Microbioma Gastrointestinal/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas/métodos , Microbiota/efectos de los fármacos , Acondicionamiento Pretrasplante/métodos , Adolescente , Niño , Preescolar , Descontaminación , Femenino , Humanos , Masculino , Estudios ProspectivosRESUMEN
The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.
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Achromobacter denitrificans/efectos de los fármacos , Achromobacter denitrificans/metabolismo , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Meropenem/farmacología , Proteómica , beta-Lactamasas/metabolismo , Achromobacter denitrificans/genética , Secuencia de Aminoácidos , Humanos , beta-Lactamasas/química , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: It has been suggested that the high incidence of ribotype 078 Clostridium difficile infections (CDI) in the Netherlands is related to pig farming. METHODS: We used data of hospitalised CDI patients (>2yrs of age) diagnosed between May 2009 and May 2015 in 26 hospitals participating in a national sentinel surveillance. We compared clinical and geographical characteristics of 078 CDI to other CDI. We investigated the association between 078 CDI incidence and four indicators of pig farming (piglet, pig, piglet farm and pig farm density) by mixed-effects Poisson regression. We used a space-time permutation model to search for community-onset 078 CDI clusters (using SaTScan). RESULTS: A total of 4,691 CDI were identified. Ribotype 078 was isolated in 493 of 3,756 patients (13.1%) including a typing result. These patients had slightly higher community-onset disease and a 35% increase of 30-day mortality compared to non-078 CDI patients. The pooled overall and 078 incidence rates were 2.82 (95% CI, 2.42-3.29) and 0.26 (95% CI, 0.21-0.31) CDI per 10,000 patients-days respectively. Hospital 078 CDI incidence was not associated with provincial pig (IRR, 0.98; 95% CI, 0.89-1.08), piglet (IRR, 0.95; 95% CI, 0.75-1.19), pig farm (IRR, 1.08; 95% CI, 0.84-1.39), or piglet farm density (IRR, 1.00; 95% CI, 0.56-1.79). No clusters of community-onset ribotype 078 CDI were found. CONCLUSIONS: Our results do not indicate that the ribotype 078 CDI incidence in hospitals is related to pig (farm) or piglet (farm) density. However, transmission beyond provincial borders or in non-hospitalised patients cannot be excluded.
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Agricultura , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/epidemiología , Ribotipificación/métodos , Vigilancia de Guardia , Anciano , Animales , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/transmisión , Análisis por Conglomerados , Femenino , Humanos , Incidencia , Masculino , Países Bajos/epidemiología , Distribución de Poisson , PorcinosRESUMEN
The emergence of the plasmid-mediated mcr colistin resistance gene in the community poses a potential threat for treatment of patients, especially when hospitalized. The aim of this study was to determine the prevalence of all currently known mcr mediated colistin resistance gene in fecal samples of patients attending a tertiary care hospital. From November 2014 until July 2015, fecal samples of patients attending the Leiden University Medical Center were collected and screened for presence of mcr using real-time PCR. Two of 576 patients were positive for mcr-1, resulting in a prevalence of 0.35%, whereas no mcr-2 was found. One of these samples was culture negative, the second sample contained a blaCMY-2 and mcr-1 containing E.coli. This strain belonged to Sequence Type 359 and serotype O177:H21. The mcr-1 containing E.coli was phenotypically susceptible to colistin with a MIC of ≤ 0.25mg/l, due to a 1329bp transposon IS10R inserted into the mcr-1 gene as identified by WGS. This prevalence study shows that mcr-1 is present in low levels patients out of the community attending a hospital. Furthermore the study underlines the importance of phenotypical confirmation of molecular detection of a mcr-1 gene.
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Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/genética , Heces/microbiología , Genes Bacterianos , Enterobacteriaceae/efectos de los fármacos , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Combined fecal microbiota transfer and antibiotic treatment prevented recurrences of urinary tract infections with multidrug-resistant (MDR) Pseudomonas aeruginosa, but it failed to eradicate intestinal colonization with MDR Escherichia coli. Based on microbiota analysis, failure was not associated with distinct diminished microbiota diversity.
RESUMEN
Background: Contact precautions are recommended by health authorities in Europe and the United States for patients with Clostridium difficile infection (CDI). Recently, the significance of nosocomial transmission has been challenged by screening on admission studies and whole-genome sequencing, providing evidence for an endogenous source of C. difficile. We discontinued contact precautions for patients with CDI, except for patients infected with hypervirulent ribotypes or with stool incontinence, to determine the rate of transmission. Methods: From January 2004 to December 2013, contacts of each index case with CDI were screened for toxigenic C. difficile by culturing rectal swabs. Transmission was defined as possible if toxigenic C. difficile was detected in contacts, as probable if the identical polymerase chain reaction ribotype was identified in indexcontact pairs, and as confirmed if next-generation sequencing (NGS) revealed clonality of strains. Results: Four hundred fifty-one contacts were exposed to 279 index patients nursed in 2-to 4-bed rooms. Toxigenic C. difficile was detected in 6.0% (27/451) after a median contact time of 5 days. Identical ribotypes were identified in 6 indexcontact pairs, accounting for probable transmission in 1.3% (6/451). NGS was performed for 4 of 6 pairs with identical strains, and confirmed transmission in 2 contact patients. Conclusions: The rate of transmission of toxigenic, predominantly nonhypervirulent C. difficile, was low and no outbreaks were recorded over a 10-year period after discontinuing contact precautions for patients with CDI who were not severely incontinent and who used dedicated toilets. As contact precautions may lead to lower levels of care, their implementation needs to be balanced against the risk of nosocomial transmission.
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Clostridioides difficile , Enterocolitis Seudomembranosa/microbiología , Enterocolitis Seudomembranosa/transmisión , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/transmisión , Comorbilidad , Infección Hospitalaria , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterocolitis Seudomembranosa/epidemiología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Mortalidad , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Ribotipificación , Factores de RiesgoRESUMEN
BACKGROUND: Little is known about pediatric Clostridium difficile infection (CDI) epidemiology. We describe the clinical and microbiological characteristics of CDI among hospitalized children in the Netherlands. METHODS: Between May 2009 and May 2015, 26 hospitals registered characteristics of pediatric (aged 2-18 years) and adult (aged 18 years) CDI in a national sentinel surveillance study. Routine polymerase chain reaction (PCR) ribotyping and multiple-locus variable-number tandem-repeat analysis (MLVA) of selected strains was performed. Pediatric and adult results were compared using proportion and 95% confidence interval (CI). Time trend of pediatric CDI was evaluated using a mixed-effect Poisson model. RESULTS: Pediatric CDIs were reported in 17 of the 26 participating hospitals (n = 135; 3% of all CDIs); the monthly number was constant over time. The median age of pediatric cases was 10 years (interquartile range, 4.7-14.5 years). Fifty-five percent of the children had community onset and 31% had severe CDI. Compared with adults (n = 4,556), complication and mortality rates were lower. Clostridium difficile PCR ribotype 265 (toxin A negative, B positive) was most prevalent in children (15%; 95% CI, 8.8%-24.0%) but rarely found in adults (1%; 95% CI, 0.9%-1.6%). This strain was rarely found in other countries, except for Belgium. MLVA showed genetic relatedness between three-fourths of pediatric and adult ribotype 265 strains, without a clear epidemiological link. CONCLUSIONS: Pediatric CDI in hospitals has remained stable over the last 6 years and resulted in fewer complications than for adult CDI. Further studies are needed to elucidate the source and epidemiology of PCR ribotype 265, primarily found in children.
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Clostridioides difficile/clasificación , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/microbiología , Hospitalización , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/transmisión , Femenino , Humanos , Masculino , Países Bajos/epidemiología , Ribotipificación , Evaluación de SíntomasRESUMEN
BACKGROUND: An outbreak of Clostridium difficile ribotype 027 infection (CDI) occurred at an university hospital, involving 19 departments. To determine what hospital-associated factors drove the outbreak of this particular strain we performed a case-control study. METHODS: Cases (n = 79), diagnosed with CDI due to C. difficile ribotype 027 were matched for age and treating medical specialty to four control patients (n = 316). Patients diagnosed with CDI due to other ribotypes were included as a second control group. A random selection of C. difficile ribotype 027 strains (n = 10) was genotyped by Whole Genome Sequencing (WGS). FINDINGS: WGS showed the outbreak was likely caused by a single strain of C. difficile (two or less single-nucleotide variants between isolates). Ninety-five percent of cases had used antibiotics, compared to 56% of controls. Previous admission to the intensive care unit (ICU) (OR: 2.4, 95% CI 1.0-5.6), longer length of stay (LOS), and recent hospital admission were associated with CDI ribotype 027. Cases were less likely to have been admitted to a ward with a known isolated CDI patient (OR: 0.2, 95% CI 0.1-0.6). Analysis of patients who stayed at the ICU (35 cases; 51 controls), indicated that the use of selective decontamination of the digestive tract (SDD) and a longer LOS in the ICU were associated with CDI risk. INTERPRETATION: In this large outbreak, any antibiotic use, including SDD use, appeared as a prerequisite for acquisition of the outbreak strain. The role of use of SDD and prolonged stay on the ICU could not be disentangled, but both factors can play a biologically plausible role in C. difficile acquisition and infection.
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Clostridioides difficile/clasificación , Clostridioides difficile/genética , Infecciones por Clostridium/epidemiología , Brotes de Enfermedades , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/mortalidad , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Infección Hospitalaria/mortalidad , Descontaminación/métodos , Femenino , Tracto Gastrointestinal/microbiología , Hospitales Universitarios , Humanos , Incidencia , Unidades de Cuidados Intensivos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Ribotipificación , Factores de RiesgoRESUMEN
The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database. It is fast and reliable, and costs are low compared to previous diagnostic approaches. However, the low resolution and dynamic range of the MALDI-TOF profiles have shown limited applicability for the discrimination of different bacterial strains, as achieved with typing based on genetic markers. This is pivotal in cases where certain strains are associated with, e.g., virulence or antibiotic resistance. Ultrahigh resolution MALDI-FTICR MS allows the measurement of small proteins at isotopic resolution and can be used to analyze complex mixtures with increased dynamic range and higher precision than MALDI-TOF MS, while still generating results in a similar time frame. Here, we propose to use ultrahigh resolution 15T MALDI-Fourier transform ion cyclotron resonance (FTICR) MS to discriminate clinically relevant bacterial strains after species identification performed by MALDI-TOF MS. We used a collection of well characterized Pseudomonas aeruginosa strains, featuring distinct antibiotic resistance profiles, and isolates obtained during hospital outbreaks. Following cluster analysis based on amplification fragment length polymorphism (AFLP), these strains were grouped into three different clusters. The same clusters were obtained using protein profiles generated by MALDI-FTICR MS. Subsequent intact protein analysis by electrospray ionization (ESI)-collision-induced dissociation (CID)-FTICR MS was applied to identify protein isoforms that contribute to the separation of the different clusters, illustrating the additional advantage of this analytical platform.
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Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Análisis por Conglomerados , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genéticaRESUMEN
Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged as a leading cause of antibiotic associated diarrheal disease in humans. The genetic basis of the pathogenicity of C. difficile remains poorly understood. In this study we aimed at characterizing the genetic diversity of C. difficile strains by three different methods (PCR ribotyping, multilocus sequence typing and genetic markers) to improve the typing of C. difficile. Our study was performed on a reference collection (Leeds-Leiden/ECDC) of C. difficile PCR ribotype (RT) strains (n=70) expanded with six PCR RT strains highly related to the emerging PCR RTs 027 and 078. Besides PCR ribotyping we used multilocus sequence typing (MLST) using seven housekeeping genes (MLST 7HG) that has recently been developed for characterizing C. difficile isolates as well as analysis of unique genetic markers. Evolutionary relatedness of the sequences determined by MLST 7HG was analyzed in phylogenetic analysis. In total 56 MLST 7HG sequence types (STs) were identified, nine of which were new. Phylogeny reconstruction of the reference set of strains supplemented with the online available C. difficile MLST reference database, revealed six monophyletic lineages of closely related STs. ST-122 (PCR RT131) formed a well-separated branch in the tree and was thus designated as a novel lineage. Furthermore, we confirmed that several PCR RTs are highly related to the emerging PCR RTs 027 and 078 since these types display the same STs (ST-1 and ST-11, respectively). Based on the observed results, we conclude that MLST 7HG is a valuable method to study C. difficile phylogeny.
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Clostridioides difficile/genética , Evolución Molecular , Genes Bacterianos , Genes Esenciales , Polimorfismo de Nucleótido Simple , Teorema de Bayes , Clostridioides difficile/clasificación , Variación Genética , Funciones de Verosimilitud , Modelos Genéticos , Tipificación de Secuencias Multilocus/normas , Filogenia , Reacción en Cadena de la Polimerasa , Estándares de ReferenciaRESUMEN
Rapid identification of hypervirulent Clostridium difficile strains is essential for preventing their spread. Recent completion of several full-length C. difficile genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between C. difficile strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase (thyX) gene and replaces it with an equivalent, catalytically more efficient, thyA gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within C. difficile that are highly related to hypervirulent strains.