Next-generation sequencing technologies and their application to the study and control of bacterial infections.

BACKGROUND: With the efficiency and the decreasing cost of next-generation sequencing, the technology is being rapidly introduced into clinical and public health laboratory practice. AIMS: The historical background and principles of first-, second- and third-generation sequencing are described, as are the characteristics of the most commonly used sequencing instruments. SOURCES: Peer-reviewed literature, white papers and meeting reports. CONTENT AND IMPLICATIONS: Next-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics), could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.

Development and application of MLVA methods as a tool for inter-laboratory surveillance.

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

PulseNet USA standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio parahaemolyticus.

PulseNet is a national molecular subtyping network for foodborne disease surveillance composed of public health and food regulatory agencies. Participants employ molecular subtyping of foodborne pathogens using a standardized method of pulsed-field gel electrophoresis (PFGE) for conducting laboratory-based surveillance of foodborne pathogens. The PulseNet standardized PFGE protocols are developed through a comprehensive testing process. The reproducibility of the protocol undergoes an internal evaluation at the Centers for Disease Control and Prevention and an external evaluation in multiple PulseNet laboratories. Here we describe the development and evaluation of a rapid PFGE protocol for subtyping Vibrio parahaemolyticus for use in PulseNet activities. The protocol was derived from the existing standardized PulseNet protocols for Escherichia coli O157:H7 and Vibrio cholerae. An external evaluation of this protocol was undertaken in collaboration with three PulseNet USA participating public health laboratories. Comparative analysis of the PFGE fingerprints generated by each of these laboratories demonstrated that the protocol is both reliable and reproducible in the hands of multiple users.

The significance of the number of submitted samples and patient-related factors for faecal bacterial diagnostics.

The sensitivity of bacteriological testing of faecal samples from patients with diarrhoea has not been properly determined. The present study analysed the association between the results of stool sample examinations and the number of samples examined per patient and other patient-related factors. Data concerning faecal specimens referred for culture for enteric bacterial pathogens (Campylobacter, Salmonella, Shigella and Yersinia) to the central microbiological laboratory in Denmark between 1995 and 2003 were analysed. In total, 620 000 samples were sorted into 277 000 sample-series, i.e., samples submitted from the same individual on the same day. Data were analysed by multivariate logistic regression, with the outcome being a positive sample-series, i.e., one or more positive samples per series. Overall, 11.9% of the sample-series were positive. For adults (aged > or =18 years), the OR for a positive diagnosis was 1.20 (95% CI 1.18-1.21) for each additional sample. Positive diagnoses were also more likely during summer, if the patient was male, or if the patient was neither very young nor very old. The added diagnostic effect of additional samples was more pronounced for the group of patients with persistent (>2 weeks) diarrhoea. Overall, the probability of finding common pathogenic bacteria in faecal samples was found to vary according to the number of samples, the season and the patient's age and gender. Analysis of more than one sample improves the sensitivity of faecal culture by at least 20% for each additional sample.

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics.

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory.

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.

Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study.

Salmonella is one of the most common causes of foodborne infection in Europe with Salmonella enterica serovar Enteritidis (S. Enteritidis) being the most commonly identified serovar. The predominant phage type for S. Enteritidis is phage type (PT) 4, although PT 8 has increased in incidence. Within these phage types, pulsed-field gel electrophoresis (PFGE) provides a method of further subdivision. The international project, Salm-gene, was established in 2001 to develop a database of PFGE profiles within nine European countries and to establish criteria for real-time pattern recognition. It uses DNA fingerprints of salmonellas to investigate outbreaks and to evaluate trends and emerging issues of foodborne infection within Europe. The Salm-gene database contains details of about 11 700 S. Enteritidis isolates, demonstrating more than 65 unique PFGE profiles. The clonal nature of S. Enteritidis is evidenced by the high similarity and distribution of PFGE profiles. Over 56% (6603/11 716) of the submitted isolates of several different phage types were profile SENTXB.0001, although this profile is most closely associated with PT 4. The next most common profiles, SENTXB.0002 and SENTXB.0005, were closely associated with PT 8 and PT 21 respectively. Studies to investigate the relationship of profile types with outbreaks and possible vehicles of infection suggest that the incidence of PFGE profile SENTXB.0002, and thus PT 8, in some countries may be due to importation of foods or food production animals from Eastern Europe, where PT 8 is amongst the most frequently identified phage types. Collation of subtyping data, especially in the commonly recognized phage types, is necessary in order to evaluate trends and emerging issues in salmonella infection.

PulseNet USA: a five-year update.

PulseNet USA is the molecular surveillance network for foodborne infections in the United States. Since its inception in 1996, it has been instrumental in detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:[H7] (STEC O157), Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. This paper describes the current status of the network, including the methodologies used and its future possibilities. The currently preferred subtyping method in the network is pulsed-field gel electrophoresis (PFGE), a proven highly discriminatory molecular subtyping method. New simpler sequencebased subtyping methods are under development and validation to complement and eventually replace PFGE. PulseNet is essentially a cluster detection network, but the data in the system will now also be used in attribution analyses of sporadic infections. The PulseNet platform will also be used as a primary tool in preparedness and response to acts of food bioterrorism.

Standardized pulsed-field gel electrophoresis of Shiga toxin-producing Escherichia coli: the PulseNet Europe Feasibility Study.

PulseNet USA, the American molecular subtyping network for foodborne infections, has since 1996 been highly successful combating infections caused by Shiga toxin-producing Escherichia coli (STEC) O157, Salmonella, Listeria, and Shigella spp. The PulseNet Europe feasibility study was initiated to ascertain the interest of public health and veterinary reference laboratories to establish a similar network, and to determine if it was possible to perform standardized pulsed-field gel electrophoresis (PFGE) typing of Salmonella, Listeria, and STEC on a large scale in Europe. The results of the STEC part of that study are presented here. Twenty-seven veterinary and public health laboratories participated in the study. The participants subtyped eight E. coli strains by PFGE using the restriction enzyme XbaI according to the PulseNet or a similar protocol, with strict adherence to the electrophoretic conditions stated in the former and submitted an image of their gel for centralized anonymous analysis. The quality of the gels was first graded visually as "good," "intermediate," and "unsatisfactory." The number of gels graded this way was 11, 14, and 2, respectively. All "good" and "intermediate" gels were also analysed and compared by computerized analysis to a reference gel. For gels graded "good," on average 5.6, 7.4, and 8 patterns out of 8 per gel were identified with a similarity of 100%, >95%, and >90%, respectively. The corresponding numbers for gels graded "intermediate" were 1.7, 4.9, and 7.4, respectively. The problems causing the grading to be "intermediate" was overloaded lanes, overexposed images, not optimally focused images and incomplete digestion, all problems that led to misinterpretation of the number of restriction fragments in the gel. These problems may be corrected by simple adjustments to the subtyping procedure. Thus, there seems to be little need for training of the participants in PulseNet Europe.

Distribution of molecular subtypes within Salmonella enterica serotype Enteritidis phage type 4 and S. Typhimurium definitive phage type 104 in nine European countries, 2000-2004: results of an international multi-centre study.

This study investigates the distribution of pulsed-field gel electrophoresis (PFGE) profiles within Salmonella enterica serotype Enteritidis phage type (PT) 4 and S. Typhimurium definitive phage type (DT) 104, from cases of human infection in nine European countries from 2000 to 2004. Isolates were subtyped using standardized methods and gel images submitted by each participating country to the coordinating centre (Health Protection Agency Centre for Infections, London, UK), where they were entered into a central database, developed within BioNumerics software, and designated using an agreed nomenclature. S. Enteritidis PT4 (n=3637) was differentiated into 38 different profiles. Simpson's index of diversity (D) of profiles ranged from 0.2 to 0.4. Profile SENTXB.0001 represented at least 80% of all profiles in each country. S. Typhimurium DT104 (n=1202) was differentiated into 28 different profile types. Simpson's D was at least 0.6 in all countries except in Austria and Italy. In both these countries over 74% of S. Typhimurium DT104 profiles were STYMXB.0013. Profile STYMXB.0061, was predominant in Denmark, Spain, Finland and England and Wales where it represented between 36% and 45% of profiles. Profile STYMXB.0001 represented nearly half of all profiles in Scotland and 23% in England and Wales. PFGE is proving useful for further discrimination within S. Enteritidis PT4 and S. Typhimurium DT104. Ascertainment of international outbreaks involving common serotypes and phage types may be increased by the timely pooling of PFGE profiles within a central database readily accessible to all participating countries.

Invasive listeriosis in Denmark 1994-2003: a review of 299 cases with special emphasis on risk factors for mortality.

Listeriosis is a rare, but serious, foodborne infection which, in the invasive form, presents as bloodstream (BS) infection, an infection of the central nervous system (CNS), a maternofetal infection or a focal infection. The disease is notifiable in Denmark. This paper reviews the results of the Danish surveillance of invasive listeriosis from 1994 to 2003, excluding maternofetal cases. In total, 299 invasive cases of listeriosis were reported. Two-thirds of the cases were caused by isolates of serogroup 1/2, and one-third by serogroup 4. Most (70%) cases had conditions known to predispose to listeriosis. More patients with BS infection were predisposed because of concurrent underlying illness than were patients with CNS infection. Half of the patients were aged > 70 years, and 21% died of the disease. There was no change in the case fatality rate (CFR) during the 10-year period. The CFR was identical for men and women. BS and CNS infection caused the same incidence of mortality, but no mortality was observed in patients with focal infections at normally sterile body sites. In a multivariate analysis, isolates belonging to serogroup 4 were associated with a higher CFR than were isolates of serogroup 1/2. In patients aged < 70 years, underlying conditions predisposing to disease were related strongly to mortality, which was not the case in patients aged > 70 years. The underlying conditions associated most strongly with mortality in the younger age group were non-haematological malignancies.

Campylobacter concisus: an evaluation of certain phenotypic and genotypic characteristics.

The clinical relevance of Campylobacter concisus in gastrointestinal disease has not been determined definitively. This study investigated the phenotypic and genotypic characteristics of 39 C. concisus isolates from Danish patients with diarrhoea, three isolates from healthy individuals and the type strain. A cytolethal distending toxin (CDT)-like effect on Vero cells was observed in 35 (90%) isolates from patients with diarrhoea, in all three isolates from healthy individuals and in the type strain. Analysis of SDS-PAGE protein profiles and PCR amplification of 23S rDNA assigned the isolates into two distinct, but discordant groups. Automated ribotyping (RiboPrinting) identified 34 distinct patterns among the 43 isolates, but cluster analysis did not separate isolates from patients with diarrhoea from isolates from healthy patients. Random amplified polymorphic DNA (RAPD) analysis with three primers identified 37 unique profiles, but requires further evaluation. The isolates obtained from healthy carriers were distinguished by cluster analysis from the isolates obtained from patients with diarrhoea. All the isolates were susceptible to 11 antimicrobial agents tested. Overall, there was considerable variability between the C. concisus isolates, but there were no clear phenotypic or genotypic differences between isolates from patients with diarrhoea and isolates from healthy carriers. Further evidence is needed to support the possible role of C. concisus as a human enteric pathogen.

Trends in antimicrobial drug resistance in Salmonella enterica serotypes Typhi and Paratyphi A isolated in Europe, 1999-2001.

Results of antimicrobial sensitivity tests for strains of Salmonella enterica serotypes Typhi and Paratyphi A isolated from patients in ten European countries between 1999 and 2001 have been transferred electronically to the Enter-net surveillance hub. For Typhi between 22 and 29% of isolates were multiresistant (to four drugs or more) with decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) increasing from 20% in 1999 to 26% in 2001. Nineteen of 169 (11%) strains with decreased ciprofloxacin susceptibility were sensitive to nalidixic acid. For Paratyphi A multiple resistance increased from 9% in 1999 to 25% in 2001 and decreased ciprofloxacin susceptibility from 6 to 17%. Clinicians should be aware of the possibility of treatment failures when fluoroquinolones are used as the first-line drug for infections with Typhi and Paratyphi A, particularly for patients recently returning from areas where drug-resistant strains are endemic.

Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance.

The Enter-net surveillance system received results of antimicrobial sensitivity tests for isolates from over 27 000 cases of human salmonellosis in 2000 in 10 European countries. Almost 40% of isolates were resistant to at least one antimicrobial, with 18% multiresistant. Resistance to ampicillin, streptomycin, sulphonamides and tetracyclines was common, with over 20% of isolates resistant to at least one of these antimicrobials. Clinical resistance to ciprofloxacin was rare, with only 0.5% of isolates exhibiting such resistance (MIC >1.0 mg/l). Resistance to nalidixic acid coupled with a decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) was more common, with 14% of isolates showing these properties. Resistance to third-generation cephalosporins was rare with only 0.6% of isolates resistant to cefotaxime. In all countries multiple resistance was most common in Salmonella enterica serotype Typhimurium, with 51% of isolates multiresistant in total. In England and Wales multiple resistance was also prevalent in S. Virchow and S. Hadar, whereas in other countries multiple resistance was common in serotypes such as S. Blockley.

Absence of clonality of Campylobacter jejuni in serotypes other than HS:19 associated with Guillain-Barré syndrome and gastroenteritis.

Guillain-Barré syndrome (GBS) is recognized as a complication that occurs after Campylobacter infection. Certain Penner serotypes, such as HS:19, are linked particularly to GBS in some parts of the world, and there is good evidence for restricted genetic diversity in these isolates. However, GBS also occurs after Campylobacter infection due to other serotypes. Therefore, we asked whether Campylobacter jejuni non-HS:19 serotypes associated with GBS have a clonal structure and differ from strains isolated from patients with Campylobacter gastroenteritis. A worldwide selected population of C. jejuni non-HS:19 strains associated with GBS and gastroenteritis was analyzed by use of multilocus enzyme electrophoresis, automated ribotyping, pulsed-field gel electrophoresis, and flagellin gene typing. The results show that these isolates represent a heterogenic population and do not constitute a unique population across serotypes. No epidemiologic marker for GBS-associated strains was identified.

Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates.

The incidence of human Campylobacter jejuni and C. coli infections has increased markedly in many parts of the world in the last decade as has the number of quinolone-resistant and, to a lesser extent, macrolide-resistant Campylobacter strains causing infections. We review macrolide and quinolone resistance in Campylobacter and track resistance trends in human clinical isolates in relation to use of these agents in food animals. Susceptibility data suggest that erythromycin and other macrolides should remain the drugs of choice in most regions, with systematic surveillance and control measures maintained, but fluoroquinolones may now be of limited use in the empiric treatment of Campylobacter infections in many regions.

Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark.

Enterococcus faecalis and E. faecium isolated from humans in the community (98 and 65 isolates), broilers (126 and 122), and pigs (102 and 88) during 1998 were tested for susceptibility to 12 different antimicrobial agents and for the presence of selected genes encoding resistance using PCR. Furthermore, the presence of vancomycin resistant enterococci was examined in 38 human stool samples using selective enrichment. Widespread resistance to chloramphenicol, macrolides, kanamycin, streptomycin, and tetracycline was found among isolates from all three sources. All E. faecium isolates from humans and pigs were susceptible to avilamycin, whereas 35% of isolates from broilers were resistant. All E. faecium isolates from humans were susceptible to vancomycin, whereas 10% and 17% of isolates from broilers and pigs, respectively, were resistant. A vancomycin resistant E. faecium isolate was found in one of the 38 human fecal samples examined using selective enrichment. All vancomycin resistant isolates contained the vanA gene, all chloramphenicol resistant isolates the cat(pIP501) gene, and all five gentamicin resistant isolates the aac6-aph2 gene. Sixty-one (85%) of 72 erythromycin resistant E. faecalis examined and 57 (90%) of 63 erythromycin resistant E. faecium isolates examined contained ermB. Forty (91%) of the kanamycin resistant E. faecalis and 18 (72%) of the kanamycin resistant E. faecium isolates contained aphA3. The tet(M) gene was found in 95% of the tetracycline resistant E. faecalis and E. faecium isolates of human and animal origin, examined. tet(K) was not observed, whereas tet(L) was detected in 17% of tetracycline resistant E. faecalis isolates and in 16% of the E. faecium isolates. tet(O) was not detected in any of the isolates from pigs, but was observed in 38% of E. faecalis isolates from broilers, in two E. faecalis isolates from humans and in three E. faecium isolates from broilers. tet(S) was not detected among isolates from animals, but was observed in 31% of E. faecalis and one E. faecium isolate from humans. This study showed a frequent occurrence of antimicrobial resistance and the presence of selected resistance genes in E. faecalis and E. faecium isolated from humans, broilers and pigs. Differences in the occurrence of resistance and tetracycline resistance genes were observed among isolates from the different sources. However, similar resistance patterns and resistance genes were detected frequently indicating that transmission of resistant enterococci or resistance genes takes place between humans, broilers, and pigs.

Homogeneity of Danish environmental and clinical isolates of Shewanella algae.

Danish isolates of Shewanella algae constituted by whole-cell protein profiling a very homogeneous group, and no clear distinction was seen between strains from the marine environment and strains of clinical origin. Although variation between all strains was observed by ribotyping and random amplified polymorphic DNA analysis, no clonal relationship between infective strains was found. From several patients, clonally identical strains of S. algae were reisolated up to 8 months after the primary isolation, indicating that the same strain may be able to maintain the infection.