Showing papers on "Typing published in 2003"

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management

Abstract: The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA) We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis One of two predominant spa types was replaced by a clonally related variant in the second study period Ten unique spa types, which were equally distributed in both study periods, were recovered The data show a rapid dynamics of clone circulation in a university hospital setting spa typing was valuable for tracking of epidemic isolates The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA

Multilocus Sequence Typing System for Group B Streptococcus

Abstract: A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.

Comparative Genotyping of Campylobacter jejuni by Amplified Fragment Length Polymorphism, Multilocus Sequence Typing, and Short Repeat Sequencing: Strain Diversity, Host Range, and Recombination

Abstract: Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.

New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates.

Abstract: The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant Staphylococcus aureus. Five staphylococcal VNTR loci (sdr, clfA, clfB, ssp, and spa) were subjected to analysis, and it was shown that the method allows typing of S. aureus strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.

Development of a Multilocus Sequence Typing Method for Analysis of Listeria monocytogenes Clones

Abstract: This study is a first step in the development of multilocus sequence typing (MLST) method for Listeria monocytogenes. Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain. These strains were previously characterized by pulsed-field gel electrophoresis (PFGE). Because of low diversity, two loci were discarded from the study. The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain. The results of this sequence analysis were generally consistent with those of PFGE. Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of L. monocytogenes clones in the environment.

Characterization of Waterborne Outbreak–associated Campylobacter jejuni, Walkerton, Ontario

Abstract: The Walkerton, Canada, waterborne outbreak of 2000 resulted from entry of Escherichia coli O157:H7 and Campylobacter spp. from neighboring farms into the town water supply. Isolates of Campylobacter jejuni and Campylobacter coli obtained from outbreak investigations were characterized by phenotypic and genotypic methods, including heat-stable and heat-labile serotyping, phage typing, biotyping, fla–restriction fragment length polymorphism (RFLP) typing, and pulsed-field gel electrophoresis. Two main outbreak strains were identified on the basis of heat-stable serotyping and fla-RFLP typing. These strains produced a limited number of types when tested by other methods. Isolates with types indistinguishable from, or similar to, the outbreak types were found only on one farm near the town of Walkerton, whereas cattle from other farms carried a variety of Campylobacter strains with different type characteristics. Results of these analyses confirmed results from epidemiologic studies and the utility of using several different typing and subtyping methods for completely characterizing bacterial populations.

Optimization and Validation of Multilocus Sequence Typing for Candida albicans

Abstract: Multilocus sequence typing (MLST) was applied to 75 Candida albicans isolates, including 2 that were expected to be identical, 48 that came from diverse geographical and clinical sources, and 15 that were sequential isolates from two patients. DNA fragments (≈500 bp) of eight genes encoding housekeeping functions were sequenced, including four that have been described before for C. albicans MLST, and four new gene fragments, AAT1a, AAT1b, MPI, and ZWF1. In total, 87 polymorphic sites were found among 50 notionally different isolates, giving 46 unique sequence types, underlining the power of MLST to differentiate isolates for epidemiological studies. Additional typing information was obtained by detecting variations in size at the transcribed spacer region of the 25S rRNA gene and tests for homozygosity at the mating type-like (MTL) locus. The stability of MLST was confirmed in two sets of consecutive isolates from two patients. In each set the isolates were identical or varied by a single nucleotide. Reference strain SC5314 and a derived mutant, CAF2, gave identical MLST types. Heterozygous polymorphisms were found in at least one isolate for all but 16 (18.4%) of the variable nucleotides, and 35 (41%) of the 87 individual sequence changes generated nonsynonymous amino acids. Cloning and restriction digestion of a gene fragment containing heterozygous polymorphisms indicated that the heterozygosity was genuine and not the result of sequencing errors. Our data validate and extend previous MLST results for C. albicans, and we propose an optimized system based on sequencing eight gene fragments for routine MLST with this species.

Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni.

Abstract: Multilocus sequence typing (MLST) has been proven useful for the study of the global population structure of Campylobacter jejuni; however, its usefulness for the investigation of outbreaks of disease caused by C. jejuni has not been proven. In this study, MLST plus sequencing of the flaA short variable region (SVR) were applied to 47 isolates from 12 outbreaks of C. jejuni infection whose relatedness has been determined previously, and the results were compared to those of serotyping and pulsed-field gel electrophoresis (PFGE). Isolates implicated in an outbreak were indistinguishable by all four subtyping methods, with sporadic isolates being distinguished from outbreak isolates. Two sporadic isolates from one outbreak were resistant to SmaI digestion and therefore nontypeable by PFGE but were differentiated from the outbreak strain by the other methods. PFGE and flaA SVR typing were the most discriminatory methods, with discriminatory indices (DI) of 0.930 and 0.923, respectively. However, an epidemic strain from one outbreak was distinguished from the other outbreak isolates by flaA SVR typing; its flaA allele was different at five nucleotides, suggesting that this change was possibly mediated by recombination. MLST was less discriminatory than PFGE and flaA SVR typing (DI = 0.859), and many of the epidemic strains possessed common sequence types (STs) including ST-8, -21, -22, and -42. However, further discrimination within STs was achieved by flaA SVR typing or PFGE. The results from this study demonstrate that a combined approach of MLST plus flaA SVR typing provides a level of discrimination equivalent to PFGE for outbreak investigations.

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Abstract: Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.

Multilocus Sequence Typing of Staphylococcus aureus with DNA Array Technology

Abstract: A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

Molecular Typing of Methicillin-Resistant Staphylococcus aureus: Can PCR Replace Pulsed-Field Gel Electrophoresis?

Abstract: Pulsed-field gel electrophoresis (PFGE) is considered the “gold standard” for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA). However, the method is time-consuming and expensive, and its discriminatory power may not be necessary in outbreak situations. We used a rapid multiplex PCR-based method with published primers and compared the results with those obtained by PFGE. A total of 75 clinical isolates were typed: 59 strains originated from our prospectively collected clinical strains and were epidemiologically unrelated; 16 strains came from an outbreak that was epidemiologically well defined in time and space. A primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to mecA gene was used for multiplex PCR. Both PFGE and PCR clustered the 75 strains into 41 different genotypes. Concordance of the results was 100% for strains originating from the outbreak. Overall, both methods produced concordant results in 72% of cases. A total of 16% were clustered together by PFGE, but not by PCR and 12% were clustered together by PCR but not by PFGE, respectively. The turnaround time was only 8 h for PCR but 5 days for PFGE. This PCR-based method is excellent for rapid and inexpensive typing of MRSA in an outbreak setting, but the discriminatory power and reproducibility are still insufficient to replace PFGE in longitudinal studies in the endemic setting.

Reduced keyboard system that emulates qwerty-type mapping and typing

Abstract: A reduced keyboard system (100), with a reduced number of keys (13, 15, 23, 25, 102, 104, 106, 108, 110, 112), but retaining the typing map of the conventional QWERTY or QWERTY-type keyboard by employing an intuitive and learning database engine. A plurality of letters, numerals, symbols and functions are assigned to a set of data keys, buttons or data inputs (milti-character keys). The arrangement of the multi-character keys together with the character assignments to the individual multi-character keys allows a user to use the same typing map, as when typing on a conventional QWERTY or QWERTY-type keyboard, be it visual mapping (i.e. physical location of keys) or finger mapping (i.e. touch typing for typist). This enhances the typing experience on the reduced keyboard system to have the same feeling, typing rhythm and speed as typing on a conventional QWERTY or QWERTY-type keyboard.

Sequence-based typing of Legionella pneumophila serogroup 1 offers the potential for true portability in legionellosis outbreak investigation.

Abstract: Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.

Mono-allelic amplification of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and -C genes: preparation and validation of ready-to-use pre-SBT mini-kits.

Abstract: Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.

Molecular techniques for MRSA typing: current issues and perspectives

Abstract: Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially as to therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Multilocus Sequence Typing Has Better Discriminatory Ability for Typing Vibrio cholerae than Does Pulsed-Field Gel Electrophoresis and Provides a Measure of Phylogenetic Relatedness

Abstract: Twenty-two Vibrio cholerae isolates, including some from “epidemic” (O1 and O139) and “nonepidemic” serogroups, were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) by using three housekeeping genes, gyrB, pgm, and recA; sequence data were also obtained for the virulence-associated genes tcpA, ctxA, and ctxB. Even with the small number of loci used, MLST had better discriminatory ability than did PFGE. On MLST analysis, there was clear clustering of epidemic serogroups; much greater diversity was seen among tcpA- and ctxAB-positive V. cholerae strains from other, nonepidemic serogroups, with a number of tcpA and ctxAB alleles identified.

Evaluation of the Polymorphisms Associated with Tandem Repeats for Pseudomonas aeruginosa Strain Typing

Abstract: We report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosa based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa. A free, online strain identification service based on the genotyping data produced herein has been developed.

Evaluation of Genotyping Large Numbers of Escherichia coli Isolates by Enterobacterial Repetitive Intergenic Consensus-PCR

Abstract: We analyzed a large collection of Escherichia coli isolates typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR with BioNumerics gel analysis software. However, the interexperimental variation of ERIC-PCR caused the computer software to classify repeated isolates as different. ERIC-PCR should not be used as the sole determinant of genetic similarity when typing large numbers of bacterial isolates via computer software.

Mycobacterial Interspersed Repetitive Unit Typing of Mycobacterium tuberculosis Compared to IS6110-Based Restriction Fragment Length Polymorphism Analysis for Investigation of Apparently Clustered Cases of Tuberculosis

Abstract: An evaluation of the utility of IS6110-based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method The number of copies of tandem repeats of the five loci ETR(A) to ETR(E) and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433) The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A All of these isolates had identical MIRU and VNTR types A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in "real time," influencing choices in contact tracing The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals

Typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the S1 gene

Abstract: A universal primer set was developed that amplifies a region covering hypervariable region (HVR) 1 and HVR 2 in the S1 gene of the infectious bronchitis virus (IBV). The universality of this primer set was confirmed by testing the reference strains of different serotypes or variants of the IBV present in the United States. An approximately 450-bp region containing HVR 1 and HVR 2 of 7 untyped field isolates obtained in 1999 and 2000 was amplified. Direct sequencing followed by phylogenetic analysis on that region allowed us to type those field isolates that were not typable by reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). Furthermore, it was found that typing by phylogenetic analysis of that region correlates with virus neutralization results. Together with RT-PCR and RFLP, this method will serve as a fast typing method for IBV diagnosis.

Patterns of Resistance Associated with Integrons, the Extended-Spectrum β-Lactamase SHV-5 Gene, and a Multidrug Efflux Pump of Klebsiella pneumoniae Causing a Nosocomial Outbreak

Abstract: Multiresistant Klebsiella pneumoniae caused a nosocomial outbreak. Resistance patterns of the presumed outbreak isolates varied among and within patients. In order to control the outbreak, screening for extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae was commenced. A number of susceptible K. pneumoniae strains were stored to serve as controls in genetic strain typing. Typing by pulsed-field gel electrophoresis proved the clonality of the strains in the recognized outbreak patients. Typing of the control strains by pulsed-field gel electrophoresis showed that at least one patient had been missed by the ESBL screening procedure. Further genetic typing confirmed the presence of the SHV-5 ESBL gene in all but one of the outbreak strains. Variable presence of integrons that carried the aminoglycoside resistance genes aadB and aadA2 was found. A gyrA mutation in codon 83 was present in all outbreak strains tested, despite considerable differences in ciprofloxacin MICs. The MICs of ciprofloxacin and the chemically unrelated drug cefoxitin were correlated (r = 0.86, P < 0.01) and were compatible with the overexpression of an efflux pump in a subset of the outbreak strains. We conclude that outbreak strains that express an ESBL gene only at a low level may pass unnoticed in a screening procedure, when the laboratory is unaware of variable ESBL expression. In this particular outbreak, screening for strains for which ciprofloxacin MICs were ≥0.25 μg/ml would in retrospect have been the most sensitive method for detection of the K. pneumoniae outbreak strain.

Phage-Based Typing Scheme for Salmonella enterica Serovar Heidelberg, a Causative Agent of Food Poisonings in Canada

Abstract: Salmonella enterica serovar Heidelberg is perhaps the second most frequent Salmonella serovar isolated from humans and the most common isolated from animals in Canada. This pathogen has shown increasing resistance to antimicrobial agents and mimics the multidrug resistance observed in S. enterica serovar Typhimurium strain DT 104. However, unlike for serovar Typhimurium, a rapid and inexpensive subtyping method has not been available for large-scale surveillance efforts. We developed a phage typing scheme and subtyped 2,523 strains of serovar Heidelberg from outbreaks, sporadic infections, and environmental sources in Canada between January 1991 and December 2000. All strains were sensitive to one or more phages and could be subdivided into 49 phage types. A total of 196 isolates from 13 major outbreaks could be subtyped into six phage types, while 86 strains from family outbreaks were assigned to seven phage types. All strains were typeable, and epidemiologically related strains isolated from patients and implicated foods had identical phage types, antibiograms, and pulsed-field gel electrophoresis (PFGE) patterns. Combining PFGE with phage typing increased the discriminatory power of the analysis beyond that of either method alone. We concluded that this phage typing scheme, in conjunction with PFGE, enhances subtyping of serovar Heidelberg strains. Furthermore, this phage typing scheme is a rapid, economical, stable, and reliable epidemiologic tool for tracing the origin of food-borne disease and for the surveillance of sporadic infections.

Prospective Value of PCR Amplification and Sequencing for Diagnosis and Typing of Old World Leishmania Infections in an Area of Nonendemicity

Abstract: We assessed the prospective value of PCR amplification of a repetitive sequence from Leishmania nuclear DNA and sequencing for the diagnosis and typing of Old World Leishmania infection in an area of nonendemicity. During this 42-month study, 29 of 168 consecutive samples were examined and classified as positive for Leishmania by direct examination and/or in vitro culture. This molecular approach showed excellent sensitivity (97%) and specificity (100%) compared to direct examination (86 and 100%, respectively) and in vitro culture (72 and 100%, respectively). Isoenzymatic and molecular typing allowed similar identification for 12 samples. Besides, PCR and subsequent sequencing of DNA products permitted the species identification of 14 samples for which parasite culture remained negative or did not allow isoenzymatic characterization, indicating the complementarity of parasitological and molecular tools.

Genetic Diversity of Neisseria gonorrhoeae Housekeeping Genes

Abstract: Molecular typing of Neisseria gonorrhoeae strains is an important tool for epidemiological studies of gonococcal infection and transmission. The recently developed multilocus sequence typing (MLST) method is based on the genetic variation among housekeeping genes. As a preliminary investigation for the development of such a method, we characterized the genetic diversity at 18 gonococcal housekeeping gene loci. Approximately 17,500 nucleotides, spanning 18 loci, were sequenced from 24 isolates. Including strain FA 1090, which has been fully sequenced, and three unique glnA sequences from GenBank, the number of alleles identified for the 18 loci ranged from 2 to 18, with a mean of 8.3 alleles per locus. The majority of polymorphic sites were distributed randomly along the genes, consistent with evolution of DNA sequences by point mutation. In addition, several examples of clustered mutations and insertions or deletions were detected, which most likely occurred by recombinational events. While purifying selection is the dominant force driving the evolution of these housekeeping genes, positive selection also appeared to operate on the abcZ and gpdh loci. The 25 completely characterized strains each had a unique allelic profile with as few as three loci (pilA, abcZ, and pip or pgi2). Molecular typing based on the allelic profile of housekeeping genes resolved the isolates better than either porB nucleotide sequencing or typing of the opa gene. The allelic profiles for the pilA, abcZ, and serC loci of paired strains from 16 sexual contacts were identical. A potential MLST for N. gonorrhoeae, based on ∼500- to 600-bp gene fragments of seven housekeeping gene loci, would include the pilA, abcZ, serC, glnA, gdh, gnd, and pip loci.

Clonal Characterization of Staphylococcus aureus by Multilocus Restriction Fragment Typing, a Rapid Screening Approach for Molecular Epidemiology

Abstract: We have developed a rapid and simplified approach for the strain characterization of Staphylococcus aureus on the basis of multilocus sequence typing (MLST) in which sequence variations in the MLST housekeeping gene loci are detected by restriction fragment pattern analysis rather than sequencing; we refer to this approach as multilocus restriction fragment typing (MLRFT). Briefly, MLRFT for S. aureus involves the PCR amplification of each of the seven MLST housekeeping gene loci by using the same primer pairs used in MLST. The amplicons are then digested directly with one or two restriction enzymes and the restriction fragments are resolved by agarose gel electrophoresis. Projection from published MLST data shows that MLRFT captures about 95% of the genetic diversity detected by MLST. The MLRFT approach was validated with a set of 59 methicillin-susceptible and 44 methicillin-resistant S. aureus isolates from community-acquired and nosocomial sources which had previously been characterized by pulsed-field gel electrophoresis (PFGE). MLRFT resolved the 103 isolates into 15 restriction fragment types, giving a discrimination index of 89.0%. Clonal groupings established by MLRFT correlated well with those established by PFGE. In short, MLRFT provides a convenient alternative to MLST and PFGE because it requires minimal laboratory facilities and is relatively simple and inexpensive to perform.

Stability of Repetitive-Sequence PCR Patterns with Respect to Culture Age and Subculture Frequency

Abstract: Methods for molecular typing of bacteria are powerful tools that can be used to determine whether isolates recovered from different patients or the environment are related and, in so doing, provide evidence for a common source of transmission of infection. Molecular typing can also be used to establish colonization of an individual by the same organism over time or to determine the relatedness of strains of bacteria possessing different antimicrobial resistance profiles. Epidemiological analysis of bacterial isolates has gained popularity over the past three decades, and many distinct approaches have been used for this purpose, including phenotypic and genotypic typing schemes. The limitations of phenotypic analysis have led to the progressive development of genotypic strategies, including analysis of plasmid content and plasmid restriction patterns (6), random amplified polymorphic DNA analysis (8), repetitive-sequence (rep) PCR (2, 13, 14), restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) (4), and multilocus sequence analysis of bacterial DNA (9). While RFLP/PFGE is considered the current “gold standard” for typing bacteria, the process is somewhat time consuming and technically demanding, limiting the laboratory’s ability to process large numbers of organisms simultaneously. Repetitive DNA sequences of approximately 30 to 400 bp have been found interspersed throughout a variety of prokaryotic (and eukaryotic) genomes (10). In bacteria, the most common repeat sequences are highly conserved across species. Some of these sequences, such as the repetitive extragenic palindrome sequence, are thought to play a role in gene regulation (7). Unlike the repetitive sequence analysis used in forensic medicine, the method developed for assessment of bacterial relatedness (termed rep PCR) examines the distance between repeat sequences by using primers directed outwardly from these sequences. If repetitive sequences are within a distance that can be spanned by Taq polymerase extension, products of various molecular sizes (depending upon the distance between repeats) are obtained. The amplified products are separated by gel electrophoresis; the gel image is then scanned and the banding patterns are analyzed visually or by using gel comparison software. Because repetitive sequences are interspersed throughout the genome, rep PCR is potentially capable of surveying the entire genome and has been shown to have similar or better strain differentiation power compared to other methods (12). rep PCR is rapid by comparison to RFLP/PFGE, has been shown to be reproducible, and requires few additional resources other than those already available in most molecular laboratories. Although molecular typing using rep PCR has become relatively popular, some assumptions regarding the performance of this method have been made that have not yet been substantiated. For example, molecular typing analyses have generally relied on DNA extracted from overnight or 24-h subcultures of the strains being examined. Presumably, this practice was adopted to exclude confounding factors that might alter rep PCR banding patterns due to increasing culture age and/or clonal expansion during growth of multiple subcultures. However, the practice of using overnight cultures for analysis might be unnecessary. To challenge this ritual, we evaluated six representative organisms for changes in rep PCR patterns with

Genotyping of Entamoeba Species in South Africa: Diversity, Stability, and Transmission Patterns within Families

Abstract: Using a recently described polymerase chain reaction-based DNA typing method for Entamoeba histolytica and E. dispar, we investigated the genetic diversity of these species in a geographically restricted region of South Africa. Patterns were stable over time in the same infection, and, with few exceptions, infected family members carried the same strain. However, both species exhibited remarkable variation, with no 2 family groups being infected with the same strain of E. histolytica. Mixed infections were rare. The results indicate that this typing method will be useful in identifying epidemiological linkage between infections.

Evaluation of Repetitive Element Sequence-Based PCR as a Molecular Typing Method for Clostridium difficile

Abstract: Repetitive element sequence-based PCR (rep-PCR) is a typing method that enables the generation of DNA fingerprinting that discriminates bacterial strains. In this study, we evaluated the applicability of rep-PCR in typing Clostridium difficile clinical isolates. The results obtained by rep-PCR were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. A high correspondence between pattern differentiations produced by rep-PCR and PFGE was observed, whereas PCR ribotyping showed a lower level of discriminatory power.

Comparison of three molecular methods for typing Aeromonas popoffii isolates

Abstract: Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP.

Characterization of Isolates of Methicillin-Resistant Staphylococcus aureus from Hong Kong by Phage Typing, Pulsed-Field Gel Electrophoresis, and Fluorescent Amplified-Fragment Length Polymorphism Analysis

Abstract: The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major “clones” at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.