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.

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.

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.

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.

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.

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.

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.

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.

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level.

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.

Acinetobacter in Denmark: I. Taxonomy, antibiotic susceptibility, and pathogenicity of 112 clinical strains.

One hundred and twelve clinical strains of Acinetobacter were collected during a 7-month period in 1990-1991 from Danish clinical microbiology departments. According to the old notation, 37 strains were biovar anitratus and 75 b.lwoffii. Using the newest terminology, 35 strains were unambiguously identified as the species A. haemolyticus, A. johnsonii, A. radioresistens, and as the unnamed phenons 6, 10, and 14, by a numerical approach using a simplified phenotypical identification scheme. Most of the remaining strains were identified to the genotypically heterogeneous A. calcoaceticus-A. baumannii complex and the A. lwoffii phenotype. Sixteen strains (14%) were left unidentified. Eight A. lwoffii strains were glucose-oxidizing and were thus classified as b. anitratus using the old terminology. The strains were tested for susceptibility to ampicillin, piperacillin, ticarcillin, cefotaxime, ceftazidime, imipenem, gentamicin, tetracycline, sulphonamide, and nalidixic acid. All strains were susceptible to imipenem. The susceptibility to the remaining beta-lactams was variable, the A. lwoffii isolates being the most and the A. calcoaceticus-A. baumannii complex strains the least susceptible. All non-A. calcoaceticus-A. baumannii complex strains were susceptible to all other antibiotics tested, except for one A. lwoffii strain that was sulphonamide resistant. Twenty-two percent of the strains in the A. calcoaceticus-A. baumannii complex showed reduced susceptibility or resistance to gentamicin, but only sporadic resistance to sulphonamides, tetracyclines, and nalidixic acid. Eight infections were recorded: three cases of septicaemia, three cases of peritonitis related to continuous ambulatory peritoneal dialysis, and two cases of recurrent urinary tract infection. No epidemics were detected.

Acinetobacter in Denmark: II. Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex consists of four closely related "genospecies" or DNA groups: DNA group 1 (A. calcoaceticus), DNA group 2 (A. baumannii), DNA 3, and Tjernberg & Ursing's DNA group 13. Strains in this complex are so similar phenotypically that it is often impossible to identify them to the DNA group level by the use of biochemical tests. Twenty-three Danish clinical strains from 23 patients phenotypically identified to the A. calcoaceticus-A. baumannii complex were studied by ribotyping, plasmid profiling, and DNA/DNA hybridization. Multiple isolates were recovered from four patients. These were identical in each patient as judged by phenotype, ribotype and plasmic profile. Seventeen different ribotypes were observed among the 23 strains, and by using this method 19 out of the 23 strains could be identified to the DNA group level. Five strains were allocated to DNA group 2 (A. baumannii), eight to DNA group 3, and six to DNA group 13. These findings were confirmed by DNA/DNA hybridization. Two of the four unidentified strains were genotypically most closely related to but different from DNA groups 1 and 3. The last two strains were most closely related to DNA group 13. These four strains represent two new DNA groups within the A. calcoaceticus-A. baumannii-complex. One to four plasmids in the size range 2.1 kb- > kb were detected in 13 of the strains. Nine plasmid profiles were seen, indicating the usefulness of this typing method if the strains contain plasmids. The study also indicates that ribotyping is useful both for typing and for identification purposes, and that the genetic relationship in this area are more diverse than hitherto perceived. Taxonomic reconsiderations are warranted.

A new Danish Listeria monocytogenes phage typing system.

A phage typing system was developed for Listeria monocytogenes. Phages were released from clinical and from food strains of L. monocytogenes by mitomycin C induction. The system consists of two subsystems, one of which is used to type L. monocytogenes serotype 1 strains, containing 12 phages, and a second which is used to type serotype 4 strains, containing 14 other phages. For the serotype 1 subsystem, the reproducibility was > or = 90%, the typability 92%, and the discriminatory power, as judged by the discriminatory index, 80%. The corresponding figures for the serotype 4 subsystem were: reproducibility > or = 94%, typability 87%, and discriminatory index 87%. The performance of the whole typing system is sufficient for it to be used for screening purposes.

Epidemiological study of Acinetobacter species isolated from an intensive care unit.

An epidemiological survey of the increase in Acinetobacter species isolates occurring in the intensive care unit of a Spanish teaching hospital during 1993 and 1994 was carried out. Different laboratory methods were used to find out, whether there was a genetic linkage. The isolates were divided into three main groups according to the resistance patterns to 11 drugs. Using API 20NE biotyping, eight different types were found. The two most common contained 20 and 11 isolates, respectively. Five different plasmid profile types were observed, although plasmids were only demonstrated in 40% of the isolates. Ribotyping with EcoRI, SalI and ClaI enzymes revealed 10, 9, and 8 different patterns, respectively. In total, 15 different ribotypes were identified using these three enzymes. Twenty-one isolates belonged to exactly the same ribotype, and 13 were associated with two highly related ribotypes. In the first ribotype, only five isolates harboured plasmids. The ribotyping method produced 100% typability and ribotypes were easy to compare; it also had taxonomic value. Ribotyping allowed us to determine the genetic linkage between Acinetobacter isolates recovered from ICU patients.

Harmonization of antibiotic susceptibility testing for Salmonella: results of a study by 18 national reference laboratories within the European Union-funded Enter-net group.

For the effective surveillance of antimicrobial drug resistance within Salmonella organisms from humans, harmonization of methods used for sensitivity testing by laboratories responsible for the typing of such organisms is essential. A study of resistance or sensitivity to a panel of 11 antimicrobials by the Enter-net international surveillance network was therefore undertaken. There are 18 national Salmonella reference laboratories within this European Union-funded network. Forty-eight strains of Salmonella enterica were distributed to each laboratory for testing for resistance or sensitivity to ampicillin, cefotaxime, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfonamides, tetracyclines, trimethoprim, nalidixic acid, and ciprofloxacin. Over 8,500 tests were assessed involving disk diffusion (DD), agar breakpoint (BP), or full minimum inhibitory concentration (MIC). Results indicated that whichever method was used, there was a high degree of concordance for the detection of resistance to most antimicrobials; only for decreased sensitivity to ciprofloxacin was there substantial nonconcordance. Because all isolates with decreased sensitivity to ciprofloxacin were resistant to nalidixic acid, it is suggested that, if required, MICs to ciprofloxacin could be determined for isolates resistant to nalidixic acid. For the detection of sensitivity, the main area of nonconcordance was in the detection of sensitivity to streptomycin. With the exception of decreased sensitivity to ciprofloxacin, we are confident that a database of antimicrobial susceptibilities can now be established and harmonized antibiogram data for Salmonella can now be exchanged for national Salmonella reference laboratories within the European Union.

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.

Moraxella (Branhamella) catarrhalis: restriction enzyme analysis typing with HinfI, HaeIII and PstI.

Restriction enzyme analysis typing with HinfI, HaeIII and PstI was performed on Moraxella (Branhamella) catarrhalis strains consecutively collected from children suspected of respiratory tract infection and the type strain. Use of HinfI gave the most distinct patterns. Great polymorphism was seen among strains.

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.

Endemic occurrence of Acinetobacter calcoaceticus biovar anitratus in an intensive care unit.

One strain of Acinetobacter calcoaceticus biovar anitratus caused colonization of 111 patients admitted to an intensive care unit (ICU) during a 2-year period. All patients were intubated and had received antibiotic therapy prior to colonization. Morbidity due to the organism was about 1%. The colonization rate showed a decreasing trend during the study period, but no seasonal variation. The strain was found in the air in a low concentration and on the hands of 8-13% of the members of the staff. No chronic carriers were found.

Frequency of plasmids in strains of Acinetobacter calcoaceticus.

Plasmids were found in 75 out of 93 clinical isolates of Acinetobacter calcoaceticus. The strains harboured up to 20 plasmids each, with two-thirds containing two or more. Most plasmids were smaller than 15 Md. Strains of A. Iwoffi contained more plasmids than A. anitratus. Plasmid analysis may be an easy and valuable tool in the epidemiology of A. calcoaceticus, especially in conjunction with other typing methods.

Radiation resistance of clinical Acinetobacter spp.: a need for concern?

As part of an epidemiological investigation of hospital infections caused by Acinetobacter spp. the radiation resistance of 15 clinical isolates and four reference strains was assessed. The radiation resistance (in D-6 values, viz. the dose necessary for reducing the initial number of colony forming units by a factor of 10(6)) was, in general, higher in the isolates of A. radioresistens than in the isolates of the A. calcoaceticus-A. baumannii complex and of A. lwoffi. However, the least resistant isolates of A. radioresistens had a D-6 value equal to or lower than the most resistant isolates of the other groups. The lowest D-6 values found were for two of the reference strains. The highest D-6 value was 35 kGy. Three isolates of A. johnsonii could not survive long enough in a dried preparation to make an assessment of the D-6 values possible. The radiation resistance of the 15 clinical isolates in the present study was higher than the resistance found in a study of similar isolates in 1970.