The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Estudios de tipificación con MALDI-TOF / Studies of bacterial typing with MALDI-TOF

La espectrometría de masas MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) se ha convertido rápidamente en una herramienta de indiscutible utilidad para la identificación y caracterización de microorganismos en numerosos laboratorios de microbiología clínica. La técnica es rápida, fiable y económica y ofrece múltiples posibilidades en distintas áreas de la microbiología. En esta revisión se ofrece una visión general de su utilidad para los estudios de tipificación y epidemiología bacteriana detallando las aproximaciones metodológicas que pueden emplearse tanto para su realización como para el análisis de resultados que se obtienen. Por último, se resumen los estudios existentes sobre su uso en la caracterización de distintas especies bacterianas

MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry has emerged as a potential tool for microbial characterization and identification in many microbiology departments. The technology is rapid, sensitive, and relatively inexpensive in terms of both the labour and costs involved. This review provides an overview on its utility for strain typing and epidemiological studies and explains the methodological approaches that can be used both for the performance of the technique and for the analysis of results. Finally, the review summarizes studies on the characterization of distinct bacterial species

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species.

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole-genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype-host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.

Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture.

AIMS: The aim of this study was to determine the uropathogenic potential of Escherichia coli isolated from retail meats. METHODS AND RESULTS: Two hundred E. coli isolates recovered from retail meats, which were previously identified molecularly as extraintestinal pathogenic E. coli, were investigated for the presence of 21 uropathogenic E. coli (UPEC) virulence-associated genes. Twenty-three E. coli isolates were selected based on their serogroups and the number of virulence genes they contained, and further characterized using multilocus sequence typing, and by tissue culture assays for adherence to and invasion of T-24 human bladder cells and for their induction of interleukin (IL)-6 secretion. All virulence genes tested, except afa/dra and hlyD, were detected among the E. coli isolates. Multilocus sequence typing analysis of 23 selected isolates revealed that 17 isolates belonged to STs associated with human UPEC. Nearly all 23 isolates exhibited lower level of adherence and invasion compared to a clinical strain, UPEC CFT073. CONCLUSIONS: These observations suggested that a small proportion of E. coli isolates from retail meats carry uropathogenic associated virulence genes and thus may serve as a reservoir of these genes to UPEC in the human intestine. Their virulence potential seemed limited as they were only weakly invasive in human bladder cell culture. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the hypothesis that retail meat E. coli may play a role in relation to urinary tract infection (UTI) and may be considered in development of a UTI prevention strategy.

Development of a new nomenclature for Salmonella typhimurium multilocus variable number of tandem repeats analysis (MLVA).

Multilocus variable number of tandem repeats analysis (MLVA) has recently become a widely used highly discriminatory molecular method for typing of the foodborne pathogen Salmonella Typhimurium. This method is based on amplification and fragment size analysis of five repeat loci. To be able to easily compare MLVA results between laboratories there is a need for a simple and definitive nomenclature for MLVA profiles. Based on MLVA results for all human S. Typhimurium isolates in Denmark from the last five years and sequence analysis of a selection of these isolates, we propose a MLVA nomenclature that indicates the actual number of repeat units in each locus. This nomenclature is independent of the equipment used for fragment analysis and, in principle, independent of the primers used. A set of reference strains is developed that can be used for easy normalisation of fragment sizes in each laboratory.

[Amplification and restriction fragment length polymorphism analysis on the complete sequence of Sta56 gene of Orientia tsutsugamushi isolated from Shandong area].

OBJECTIVE: To analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank. METHODS: PCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software. RESULTS: The complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%. CONCLUSION: Data from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

Rapid diagnosis in pediatric infectious diseases: the past, the present and the future.

The focus of rapid diagnosis of infectious diseases of children in the last decade has shifted from variations of the conventional laboratory techniques of antigen detection, microscopy and culture to that of molecular diagnosis of infectious agents. Pediatricians will need to be able to interpret the use, limitations and results of molecular diagnostic techniques as they are increasingly integrated into routine clinical microbiology laboratory protocols. PCR is the best known and most successfully implemented diagnostic molecular technology to date. It can detect specific infectious agents and determine their virulence and antimicrobial genotypes with greater speed, sensitivity and specificity than conventional microbiology methods. Inherent technical limitations of PCR are present, although they are reduced in laboratories that follow suitable validation and quality control procedures. Variations of PCR together with advances in nucleic acid amplification technology have broadened its diagnostic capabilities in clinical infectious disease to now rival and even surpass traditional methods in some situations. Automation of all components of PCR is now possible. The completion of the genome sequencing projects for significant microbial pathogens, in combination with PCR and DNA chip technology, will revolutionize the diagnosis and management of infectious diseases.

Usefulness of sputum culture for diagnosis of bacterial pneumonia in HIV-infected patients.

The use of sputum culture in immunocompetent patients with community-acquired pneumonia is controversial. The usefulness of this technique in HIV-infected patients has not been evaluated. A prospective, observational, multicenter, hospital-based study of bacterial community-acquired pneumonia was carried out to analyze the value of sputum culture in HIV-infected patients. Only good-quality sputum samples were cultured. Altogether, 355 cases of bacterial community-acquired pneumonia were included. An etiological diagnosis was obtained in 190 (53.5%) cases. Sputum was cultured in 313 (88.1%) cases, being diagnostic in 108 (34.5%). The microorganism identified in sputum culture was the same as that identified in sterile samples in 26 of 27 (96.3%) cases in which both cultures were diagnostic. The microbiologic findings in sputum and bronchoscopic cultures were concordant in seven of eight (87.5%) cases in which both were positive. These results suggest that sputum culture is a useful technique, given its availability and ease of performance and its good correlation with culture of sterile samples.

Comparative and library epidemiological typing systems: outbreak investigations versus surveillance systems.

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.

[The use of molecular biology in the modeling of Pseudomonas aeruginosa strains recovered from nosocomial infections]. / Zastosowanie metod biologii molekularnej do typowania szczepów Pseudomonas aeruginosa izolowanych z zakazen szpitalnych.

Different methods of molecular typing (ribotyping, genomic DNA RFLP and RAPD) were tested on Pseudomonas aeruginosa strains isolated in Polish hospitals in order to elaborate a reliable typing scheme for epidemiological investigations. The combined RAPD analysis with the use of two different primers, RAPD-4 and RAPD-7, was found to have the highest discriminatory power which considering also the easiness and low time-consumption has suggested its high usefulness in studies of outbreaks caused by P. aeruginosa. Ribotyping was shown to be the least discriminatory, however, especially with the use of the PvuII restriction enzyme, this method can be very useful in revealing the genetic structure of P. aeruginosa populations persisting in hospital environments over longer periods. Clonal relations within populations of strains isolated in four different hospitals were revealed. In two of the hospitals P. aeruginosa populations demonstrated a very high diversity which suggested that infections had been caused by strains of different origins, probably introduced from other environments. P. aeruginosa strains from two remaining hospitals were found to form some clonally related clusters what revealed that in these hospitals epidemic strains of this microorganism have been circulating for prolonged periods and infecting predisposed patients.

Acinetobacter species in clinical specimens: biotypes and serotypes of acinetobacter baumannii strains isolated in Säo Paulo, Brazil

Foram estudadas 190 cepas de Acinetobcter (76 isoladas de líquido cefalorraquidiano, 30 de sangue e 84 de outros materiais clínicos), isoladas no período 1986-92, em Säo Paulo, Brasil. As espécies foram identificadas segundo Bouvet and Grimont e Vouvet and Jeanjean. Os sorotipos e biotipos foram determinados para A. baumannii seguindo os métodos descritos por Traub e Bouvet and Grimont, respectivamente. Entre as 10 espécies identificadas, o A.baumannii (60 por cento) e A.genospecies 3 (11.6 por cento) foram as mais frequentes. Verificou-se que os biotipos 2,6, e 9 e os sorotipos 04, 015 e 029 foram predominantes entre as cepas de A.baumannii

Bacterias identicadas por el sistema clásico y el sistema API-20E / Bacterias identificated by classic system and API-20E system

Se efectuaron 146 aislamientos bacterianos mediante el uso de hemocultivos. Dichos aislamientos se identificaron usando el método clásico y el sistema API-20E. Con el método clásico identificaron 84 cepas: 34 cepas de Estafilococo coagulasa negativa; 16 cepas de Estafilococo aureus patógeno; 9 cepas de Estreptococo no hemolítico; 13 cepas de Bacteroides; 4 cepas de citrobactter y una cepa de Shiguella flexneri. Con el sistena API-20E se identificaron 62 cepas; 7 cepas de Echerichia coli; 13 cepas de Salmonella tiphy; 22 cepas de Serratia marcescens; 8 cepas de Klebsiella neumoneae; 5 cepas de Enterobacter cloacae; 4 cepas de Acinetobacter calcoacético y 3 cepas de Pseudomona fluorences.

Klebsiella pneumoniae infection on a rehabilitation unit: comparison of epidemiologic typing methods.

OBJECTIVE: To identify factors associated with an increased occurrence of Klebsiella pneumoniae isolation in urine cultures and infected wounds on a rehabilitation unit and to compare typing methods for K pneumoniae isolates. DESIGN: Retrospective review of laboratory reports and patient records with case-control study. Analysis of K pneumoniae isolates using capsular serotyping, enzyme electrophoretic typing, ribotyping, and DNA typing. SETTING: 48-bed rehabilitation unit in an 1,100-bed tertiary care teaching hospital in Winnipeg, Manitoba. RESULTS: In 1988, 20 (19%) of 106 patients admitted to the rehabilitation unit had K pneumoniae isolated from urine or wound, and in 1989 31 (28%) of 111 patients had Klebsiella isolated. Review of ward practices revealed appropriate written policies but evidence of failure in execution leading to multiple opportunities for transmission among patients. Substantial environmental contamination was not identified, although a common urine graduate may have contributed to some transmission. Individuals with K pneumoniae isolated had a significantly longer duration of stay. Many of these were spinal cord-injured patients and were maintained on intermittent catheterization. One outbreak strain was identified in epidemiologic typing. Other strains were generally identified in individuals with non-nosocomial acquisition of infection. Comparison of epidemiologic typing methods suggests ribotyping may be the optimal method for typing K pneumoniae strains. CONCLUSIONS: K pneumoniae was acquired frequently by spinal cord-injured patients with extended admissions, re-emphasizing the importance of both patients and staff following appropriate infection control practices on rehabilitation wards. Ribotyping was the optimal method for typing K pneumoniae isolates.

Quién es quién en Bacteriología: Capnocytophaga spp / Who is who in Bacteriology: Capnocytophaga spp

Dentro del género Capnocytophaga existen dos grupos de bacilos Gram-negativos de dificil crecimiento previamente conocidos como grupos DF-1 y DF-2 del CDC. En ellos se incluye a las especies C. ochracea, C. gingivalis, C. sputigena (ex DF-1), C. canimorsus y C. cynodegmi (ex DF-2 y DF-2 like). Probablemente, en el futuro, se agregue a este género otro grupo de bacterias actualmente conocidas como grupo DF-3 del CDC. Las tres primeras especies se asocian a sepsis en pacientes neutropénicos que reciben quimioterapia, aunque también son capaces de producir infecciones en huéspedes normales. C. canimorsus ha sido reconocida como agente causal de infecciones severas secundarias a contacto previo con animales en pacientes alcohólicos, esplenectomizados o tratados con corticoides. C. cynodegmi ha sido aislada de mordeduras de animales en pacientes normales. Parecería que las bacterias del grupo DF-3 podrían tener que ver con cuadros de diarrea prolongada. Es destacable además el hallazgo de C. cynodegmi en un caso de endoftalmitis en un paciente con transplante de córnea y el informe de algunos casos de infecciones perinatales producidas por bacterias del antiguo grupo DF-1. La sensibilidad de estos microorganismos es bastante amplia. La mayor experiencia clínica es la registrada con penicilina, que parece ser el antibiótico de elección. Sin embargo, es necesario destacar que se han aislado cepas de Capnocytophaga productoras de beta-lactamasas. El aislamiento de estas bacterias a partir de muestras clínicas constituye un desafío para los microbiólogos. Su desarrollo puede lograrse utilizando placas de agar tripteína de soya adicionadas de sangre al 5 por ciento e incubándolas en aero o anaerobiosis en presencia de CO2 al 5 por ciento por espacio de al menos 5 días. Su diferenciación a nivel de especie puede resultar engorrosa por la superposición de resultados en varias pruebas. De todos modos, las pruebas de oxidasa, catalasa, movilidad y arginina separan bien a los antiguos grupos DF-1 y DF-2 entre sí y de otras bacterias similares

Quién es quién en Bacteriología: Capnocytophaga spp / Who is who in Bacteriology: Capnocytophaga spp

Dentro del género Capnocytophaga existen dos grupos de bacilos Gram-negativos de dificil crecimiento previamente conocidos como grupos DF-1 y DF-2 del CDC. En ellos se incluye a las especies C. ochracea, C. gingivalis, C. sputigena (ex DF-1), C. canimorsus y C. cynodegmi (ex DF-2 y DF-2 like). Probablemente, en el futuro, se agregue a este género otro grupo de bacterias actualmente conocidas como grupo DF-3 del CDC. Las tres primeras especies se asocian a sepsis en pacientes neutropénicos que reciben quimioterapia, aunque también son capaces de producir infecciones en huéspedes normales. C. canimorsus ha sido reconocida como agente causal de infecciones severas secundarias a contacto previo con animales en pacientes alcohólicos, esplenectomizados o tratados con corticoides. C. cynodegmi ha sido aislada de mordeduras de animales en pacientes normales. Parecería que las bacterias del grupo DF-3 podrían tener que ver con cuadros de diarrea prolongada. Es destacable además el hallazgo de C. cynodegmi en un caso de endoftalmitis en un paciente con transplante de córnea y el informe de algunos casos de infecciones perinatales producidas por bacterias del antiguo grupo DF-1. La sensibilidad de estos microorganismos es bastante amplia. La mayor experiencia clínica es la registrada con penicilina, que parece ser el antibiótico de elección. Sin embargo, es necesario destacar que se han aislado cepas de Capnocytophaga productoras de beta-lactamasas. El aislamiento de estas bacterias a partir de muestras clínicas constituye un desafío para los microbiólogos. Su desarrollo puede lograrse utilizando placas de agar tripteína de soya adicionadas de sangre al 5 por ciento e incubándolas en aero o anaerobiosis en presencia de CO2 al 5 por ciento por espacio de al menos 5 días. Su diferenciación a nivel de especie puede resultar engorrosa por la superposición de resultados en varias pruebas. De todos modos, las pruebas de oxidasa, catalasa, movilidad y arginina separan bien a los antiguos grupos DF-1 y DF-2 entre sí y de otras bacterias similares (AU)

Epidemiologic and clinical utility of typing systems for differentiating among strains of methicillin-resistant Staphylococcus aureus.

Typing systems for differentiating among strains of methicillin-resistant Staphylococcus aureus (MRSA) can be valuable tools for the epidemiologist and the clinician. Specific criteria for evaluating such systems are typeability, reproducibility, and discriminatory power. An ideal typing system also would be rapid, inexpensive, technically simple, and readily available. Systems based on the detection of phenotypic variations include antimicrobial susceptibility testing, bacteriophage typing, multilocus enzyme electrophoresis, and electrophoretic methods such as protein electrophoresis and immunoblotting. Systems that directly detect genotypic variations include plasmid profile analysis, restriction enzyme analysis of plasmid DNA, restriction enzyme analysis of chromosomal DNA, Southern blot analysis of specific restriction fragment length polymorphisms, and pulse field gel electrophoresis. In general, the more widely available typing systems based on phenotypic assays and plasmid analysis have limitations in typeability and/or discriminatory power. The chromosomal DNA-based techniques, although promising, are unproven approaches still under active investigation.