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Population-associated differences between the phase variable LPS biosynthetic genes of Helicobacter pylori.

BACKGROUND: Population structures are normally determined using genes under minimal functional selection. In this study we have assessed genes that are not always essential, show differences in alleles between strains, and are involved in the directly host-selectable phenotype of LPS biosynthesis. RESULTS: Eight complete LPS biosynthesis genes, seven of which are associated with phase variation in some or all strains of Helicobacter pylori, have been sequenced and their divergence analyzed. The differences observed indicate that recombination within these genes largely reflects exchange between strains within the population lineages previously determined on the basis of MLST using housekeeping genes. This indicates that the differences that are used for MLST are likely to broadly associate with genes under functional selection, and differences in strain behaviour. However, instances of exchange between the subpopulations were identified, including the hpAfrica2 subpopulation. Further, there were other differences in gene complements and the chromosomal location of genes indicative of greater diversity within the population than is revealed by the available genome sequences and comparative genome hybridization studies. CONCLUSION: These results indicate that the described population structure based upon MLST is broadly a good basis for studying the biology of H. pylori, but that individual alleles may not follow these associations. As a consequence, when working in unsequenced strains, it is necessary to carefully check the presence, sequence, and distribution of any individual gene of interest.

Alleles↗

A test to approach maximal lactate steady-state in 12-year old boys and girls.

The purpose of this study was to measure the running velocity corresponding to the individual maximal lactate steady-state of a group of 12-year old boys and girls on a treadmill. This running velocity (v MLST) was compared with the maximal aerobic running velocity (v a max) at which maximal oxygen uptake (VO2 max) occurs. Thirteen pupils of the same school whose puberal maturation corresponded to the end of stage 2 and the beginning of stage 3 of Tanner: 6 boys (12.2 years old +/- 0.5, 38.4 +/- 2 kg, 150 +/- 4.8 cm: group 1) and 7 girls (12.3 years old +/- 0.5, 37.6 +/- 6 kg, 151.4 +/- 5.6 cm: group 2) carried out two tests at one week interval. The first test was a maximal incremental test for the determination of VO2 max with Douglas's bag method and v a max. The purpose of the second test was the determination of maximal lactate steadystate velocity (v MLST) With two stages of ten minutes at 60 +/- 5% and 74 +/- 4.5% v a max separated by 40 minutes of complete rest (Billat, 1992); VO2max and v a max were significantly different, equal to 49.4 +/- 7 ml.min-1.kg-1, 40.4 +/- 4.7 ml.min-1.kg-1 and 12.6 +/- 0.2 km.h-1, 11.2 +/- 1.2 km.h-1 for group 1 and 2 respectively (P < 0.05). Moreover, maximal lactate steady state velocity (v MLST) was respectively equal to 64.8 +/- 12.5% and 64.6% +/- 12.5% VO2 max respectively, representing 67.8 +/- 6.2% and 68.8% +/- 8.3% v a max and was not significantly different for group 1 and 2. In conclusion, this study shows that maximal lactate steady-state velocity is not significantly different between young boys and girls of 12 years old, when expressed in fraction of VO2 max or v a max. However, VO2 max and v a max were significantly higher in boys: +27.2 and +11.6% higher respectively.

Child↗

Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation.

A total of 814 isolates of the foodborne pathogen Campylobacter jejuni were characterized by multilocus sequence typing (MLST) and analysis of the variation of two cell-surface components: the heat-stable (HS) serotyping antigen and the flagella protein FlaA short variable region. We identified 379 combinations of the MLST loci (sequence types) and 215 combinations of the cell-surface components among these isolates, which had been obtained from human disease, animals, food, and the environment. Despite this diversity, 748 (92%) of the isolates belonged to one of 17 clonal complexes, 6 of which contained many (318, 63%) of the human disease isolates. Several clonal complexes exhibited associations with isolation source or particular cell-surface components; however, the latter were poorly predictive of clonal complex. These data demonstrate that the clonal complex, as defined by MLST, is an epidemiologically relevant unit for both long and short-term investigations of C. jejuni epidemiology.

Animals↗

Campylobacter jejuni multilocus sequence types in humans, northwest England, 2003-2004.

Detailed understanding of the epidemiology of Campylobacter is increasingly facilitated through use of universal and reproducible techniques for accurate strain differentiation and subtyping. Multilocus sequence typing (MLST) enables discriminatory subtyping and grouping of isolate types into genetically related clonal complexes; it also has the advantage of ease of application and repeatability. Recent studies suggest that a measure of host association may be distinguishable with this system. We describe the first continuous population-based survey to investigate the potential of MLST to resolve questions of campylobacteriosis epidemiology. We demonstrate the ability of MLST to identify variations in the epidemiology of campylobacteriosis between distinct populations and describe the distribution of key subtypes of interest.

Adolescent↗

Evaluation of molecular typing methods for Escherichia coli O157:H7 isolates from cattle, food, and humans.

Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

Animals↗

[Multilocus sequencing--a new method of genotyping bacteria and first results of its use].

Comparative characterization (molecular typing) of isolates within a bacterial species is one of the major problems in microbiology and epidemiology. However, it is rather difficult to correlate data obtained in various laboratories, because traditional, including molecular, methods employed in typing pathogenic microorganisms cannot be standardized. In 1998, Maiden et al. proposed multilocus sequence typing (MLST); through which alleles of several housekeeping genes are directly assessed by nucleotide sequencing, each unique allele combination determining a sequence type of a strain. The advantages of this approach are that the culturing of pathogenic microorganisms is avoided, as their gene fragments are amplified directly from biological samples, and that the sequencing data are unambiguous, easy to standardize, and electronically portable. The latter makes it possible to generate an expandable global database for each species at an Internet site, in order to use it for the purposes of genotyping pathogenic bacteria (and other infectious agents). MLST protocols have been elaborated for Neisseria meningitidis, Streptococcus pneumoniae, and Helicobacter pylori; those for Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae are now being developed. Basic principles and the first results of MLST have been reviewed, including data on the distribution and microevolution of N. meningitidis clones causing epidemic meningococcal infection, the relative recombination and mutation rates in the N. meningitidis genome, the identification of antibiotic-resistant S. pneumoniae clones causing severe generalized infection, the grouping of H. pylori isolates from various geographic regions, etc.

Bacteria↗

[Multilocus sequence typing of group A streptococci isolated from invasive diseases in the Czech Republic in 2003].

First results of multilocus sequence typing (MLST) for characterization of 29 invasive Streptococcus pyogenes strains isolated in the Czech Republic in the first half of 2003 are presented. None of 16 emm types detected among the study strains showed sequence type (ST) variability. The MLST results are indicative of differences between the strains causing serious diseases in the Czech Republic and those isolated in other countries. In seven strains, four new STs with known alleles in new combinations, ST134, ST308, ST336, ST340, and one new ST with three as yet undescribed alleles (gki 91, murI 65 and yqiL 60), ST341, were described. These newly described STs were submitted to the web-based reference MLST database for S. pyogenes.

Bacterial Typing Techniques↗

Whole-Genome Analysis Reveals Antimicrobial Resistance and Population Structure of Environmental and Veterinary Acinetobacter baumannii.

Acinetobacter (A.) baumannii is an important multidrug-resistant pathogen increasingly recognized across animal and environmental settings, and carbapenem-resistant A. baumannii (CRAB) is classified as a critical-priority pathogen by the World Health Organization. This study investigated the antimicrobial resistance (AMR) and genomic characteristics of 122 A. baumannii isolates comprising 72 veterinary and 50 environmental isolates collected in Andhra Pradesh, India. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), resistance and virulence gene profiling, multilocus sequence typing (MLST), core-genome analysis, single nucleotide polymorphism (SNP) phylogeny, and pan-genome analysis were performed. Overall, 58.2% of isolates were multidrug-resistant (MDR), and 41.8% were extensively drug-resistant (XDR). Sequence type (ST) 52 predominated among veterinary isolates, whereas ST2 was more frequent among environmental isolates. The presence of carbapenem-resistant isolates along with the ST2 lineage enhances the similarity to clinical A. baumannii. Several intrinsic resistance genes, including blaOXA-23, armA, aph(3&#x2033;)-Ib, aph(6)-Id, tet(B), mph(E), and msr(E), were more prevalent in the ST2-associated population. Virulence-associated determinants were widely conserved. Core-genome MLST (cgMLST) and core-genome SNP (cgSNP) analyses identified highly related isolates within both lineages, while pairwise SNP differences were 0-7. Pan-genome analysis identified 4204 gene clusters and distinct accessory gene patterns between ST2 and ST52. These findings indicate that resistance gene distribution was closely associated with lineage structure and support integrated genomic surveillance of A. baumannii across animal and environmental reservoirs.

Acinetobacter baumannii↗

Population structure and evolutionary dynamics of pathogenic bacteria.

Evidence concerning the significance of recombination within natural bacterial populations has historically come from two main sources: multilocus enzyme electrophoresis (MLEE) and nucleotide sequence data. Here we discuss evidence from a third method, multilocus sequence typing (MLST), which is a development of MLEE based on nucleotide sequencing that combines the advantages of both approaches. MLST has confirmed both the existence of clones and the high rates of recombination for several bacterial pathogens. The data are consistent with "epidemic" population structures, where clones are superimposed upon a backdrop of frequent recombination, thus, in the short term, resisting the homogenising effect of recombination. The nature of the selective advantage of clones, however, and how this advantage relates to virulence are unclear. The current evidence also has broader implications concerning bacterial species definition, the management of antibiotic-resistant bacteria and the assessment of the dangers of releasing genetically modified organisms into the environment.

Bacteria↗

Characterization of methicillin-resistant Staphylococcus aureus from Ulaanbaatar, Mongolia.

In order to expand current knowledge of the types of methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in central Asia, six MRSA strains collected from hospitals in Ulaanbaatar, Mongolia during 2000-2002 were examined. Three strains possessed a staphylococcal cassette chromosome mec (SCCmec) element of type IV c, were sequence type (ST) 154 according to multilocus sequence typing (MLST), and contained lukS-lukF (Panton-Valentine leukocidin). Another three strains contained a SCCmec element of type III and were MLST type ST 239. Using automated ribotyping, the six MRSA strains were divided into four different EcoRI ribotypes, and two groups of isolates were distinguished by means of SmaI-macrorestriction patterns. In comparison to other countries, the incidence of MRSA in Mongolia is low.

Anti-Bacterial Agents↗

Clonal variability of group B Streptococcus among different groups of carriers in southern Israel.

A high prevalence of maternal group B Streptococcus (GBS) carriage and an extremely low incidence of invasive neonatal disease have been reported from southern Israel. In order to obtain insight into this discrepancy, this study was performed to determine the population structure of GBS from asymptomatic pregnant women living in this area. Seventy-two strains from maternal GBS carriers were characterized using multilocus sequence typing (MLST). Epidemiologic characteristics of the carriers and their newborns, including demographic variables, obstetric status, and general health parameters, were collected by means of a postpartum interview and a review of the relevant medical records. The MLST analysis grouped the bacteria into six different lineages (clonal complexes). Lineage ST-2 was prevalent among Bedouin-Arabs (p=0.01) and lineage ST-22 among Jews (p=0.001). Lineage ST-17 was prevalent among carriers who emigrated after 1997 from western nations of the former USSR (p<0.001). Lineage ST-22 was associated with carriage of surface-protein C (p=0.01) and lineage ST-17 with surface-protein R (p<0.01). Lineage ST-2 was prevalent among consumers of antibiotics (p=0.02) and was associated with erythromycin-resistant strains (p<0.001). Each subgroup of the southern Israeli maternal population has a different distribution of GBS clones. The clones prevalent among the Bedouin-Arabs and the Jews are known to be of low virulence. Lineage ST-17, which is associated with invasive disease, is prevalent among women who emigrated from western Soviet nations. Therefore, a different policy of GBS prophylaxis, resembling the one executed in endemic areas, should be considered in this population.

Arabs↗

Mycoplasma and Bartonella in cats from the tropical tourist Gili Islands, Indonesia.

Bartonella spp. and haemotropic Mycoplasma spp. are important vector-borne bacteria of veterinary and zoonotic relevance, yet information on their circulation in Indonesian island ecosystems remains limited. We investigated their occurrence and molecular diversity in 117 domestic and free-roaming cats from the Gili Islands, Indonesia, using full-length 16S rRNA nanopore metagenomics followed by targeted PCR, sequencing, phylogenetic analysis and multilocus sequence typing (MLST). Bartonella DNA was detected in 18/117 (15.4%) cats and haemotropic Mycoplasma DNA in 40/117 (34.2%). Sequence analysis identified Bartonella henselae as the predominant species together with Bartonella clarridgeiae. MLST of B. henselae revealed three sequence types (ST1, ST16 and ST42), with ST1, a lineage reported in both feline and human isolates, predominating. Comparison with the PubMLST database showed significant geographical differences in the distribution of ST1 and ST42, supporting regional variation in the circulation of B. henselae lineages. Haemoplasma characterization identified Candidatus Mycoplasma haemominutum, Mycoplasma haemofelis, Candidatus Mycoplasma turicensis and a Mycoplasma feliminutum-like organism, comprising ten distinct sequence variants. Haemoplasma positivity was significantly associated with age, with adults showing higher positivity than younger animals (P < 0.001), whereas Bartonella infection was not associated with age, sex or island of origin. The detection of zoonotically relevant B. henselae lineages and the genetic diversity of feline haemoplasmas provide evidence of the circulation of vector-borne bacteria among cats in this tropical island ecosystem. These findings provide the first molecular epidemiological baseline for this region and contribute to understanding the circulation and genetic diversity of feline vector-borne pathogens in Southeast Asia.

Animals↗

High frequency of vancomycin-resistant Enterococcus faecium isolates with VanB phenotype and vanA genotype in Korean hospitals.

A total of 59 vancomycin-resistant Enterococcus faecium (VREF) clinical isolates were collected from 8 Korean hospitals for 2 months in 2004. They were investigated by genotyping for glycopeptide resistance, multilocus sequence typing (MLST), esp repeat profiling, and structural analysis of Tn1546-like element. Nine of 59 VREF isolates (15.3%) from 5 hospitals in Korea showed VanB phenotype, but they contained vanA gene. MLST and esp repeat profiling indicated that E. faecium isolates with VanB phenotype and vanA genotype occurred from independent genetic background except 3 isolates from 1 hospital. Structural analysis of Tn1546 also showed that these isolates were not clonally related. Data showed a relatively high frequency of VREF isolates with incongruence between phenotype and genotype for glycopeptide resistance in Korean hospitals.

Bacterial Proteins↗

Genetic evidence for recombination in Candida albicans based on haplotype analysis.

The possibility of sexual reproduction in the human pathogenic fungus Candida albicans is a question of great interest in medical mycology. Not only is it a fundamental biological issue, but it is also a potential mechanism for contributing to the phenotypic plasticity (and hence the virulence) of the organism. Molecular genotyping methods such as multi-locus sequence typing (MLST) are generating data that can shed light on this question. In the present study we have used MLST information to generate haplotypes that identify many different homologues of a chromosome within a collection of strains. Particular combinations of these haplotypes provide evidence for chromosomal segregation and intra-chromosome recombination. All of our observations of haplotype diversity could also be explained by other mechanisms, such as gene conversion or mitotic recombination, and the resolution of these issues will require a denser map of accurately localised markers. A common event observed in strain evolution is loss of heterozygosity at a particular marker. Our results contribute to the emerging picture of C. albicans as an organism whose primary means of reproduction is clonal, but with a small but important contribution from sexual reproduction, occurring in nature but not under commonly used laboratory conditions.

Alleles↗

Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation.

The presence and numbers of campylobacters on chicken carcasses from 26 slaughter groups, originating from 22 single-house flocks and processed in four UK plants, were studied in relation to the level of flock colonisation determined by examining the caecal contents of at least ten birds per group. The prevalence of campylobacters on carcasses from five campylobacter-negative flocks processed just after other negative flocks was low (</=30%). Campylobacters were isolated from 90 to 100% of carcasses from three flocks which were partly colonised, with 5, 5 and 30% of caecal contents positive, and which were processed after fully colonised flocks. All carcasses from the remaining fully colonised flocks were contaminated with campylobacters, and they had significantly (P<0.001) higher numbers per carcass (average of 5.3 log(10) cfu; range: 1.3 to >8.0 log(10) cfu) than carcasses originating from low prevalence flocks (average of 2.3 log(10) cfu; range: <1.1 to 4.1 log(10) cfu). There was a reduction in the numbers of campylobacters on carcasses between plucking and chilling in eight of ten fully colonised flocks. In another eight flocks, a significant (P<0.001) decrease (0.8 log(10) cfu) in the number of campylobacters on carcasses from just before to after chilling was detected. Campylobacter spp. could be isolated from aerosols, particles and droplets in considerable numbers in the hanging-on, defeathering and evisceration areas but not in the chillers. This was the case even when campylobacters were not isolated from the target flock. Campylobacters on carcasses from two partly colonised flocks were either the same subtype, as determined by speciation, Multi-Locus Sequence Typing (MLST) and flaA Restricted Fragment Length Polymorphism (RFLP) typing, as those in the fully colonised flocks processed previously, although not necessarily the most prevalent ones; or were the same subtypes as those found in the caeca of the flock itself. The prevalences of the different campylobacter subtypes found on carcasses from two fully colonised flocks did not closely reflect those found in the caeca. MLST combined with flaA RFLP provided a good method for ascertaining the relatedness of strains isolated from carcasses and caecal contents. This study showed that carcass contamination is related to the within-flock prevalence of campylobacter colonisation, but that contamination from previously processed flocks was also significant, especially on carcasses from low prevalence flocks. Forced dry air cooling of carcasses reduced contamination levels.

Animals↗

Outbreak of methicillin-resistant Staphylococcus aureus in two nursing homes in Central Norway.

Until recently, infections with methicillin-resistant Staphylococcus aureus (MRSA) have mainly been associated with hospital outbreaks in Norway. However, increasingly cases are contracted outside hospitals. This paper reports the first two outbreaks of MRSA in two nursing homes in central Norway, affecting 23 residents and five staff members. Pulsed-field gel electrophoresis analysis showed that all strains from nursing home A were identical and that the strains from nursing home B were genotypically similar with one or two band differences. Multi-locus sequence typing (MLST) showed that the strains from the two nursing homes belong to clonal complex 45, with each strain being a single-locus variant of sequence type 45 (ST 45), a well-known European epidemic strain. No evident source of the two outbreaks was found, and there was no obvious connection between the two outbreaks. The latter is also supported by the minor differences observed by MLST, suggesting a connection at some time in the past. The outbreaks led to a heavier workload and economic strain on both nursing homes. The outbreak in nursing home A was brought to an end, whereas two residents remained colonized in nursing home B despite several eradication attempts. These outbreaks show the potential for MRSA spread in a nursing home. If the prevalence of MRSA in Norway continues to increase, nursing home staff and residents may have to be included in the groups to be screened for MRSA upon hospital admission.

Aged↗

Changes in the epidemiology of meticillin-resistant Staphylococcus aureus associated with the emergence of EMRSA-16 at a university hospital.

This study investigated the molecular epidemiology of meticillin-resistant Staphylococcus aureus (MRSA) in the University Hospital of the Canary Islands (HUC) in order to evaluate epidemiological changes over a six-year period. Clinical and epidemiological data were collected between May 2000 and December 2003, and isolates were subjected to pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), SCCmec typing and spa typing. Since 2000, the rate of MRSA infections has increased at the HUC, coinciding with the emergence and spread of the EMRSA-16 clone (ST36-MRSA-II) and replacement of the Iberian clone (ST247-MRSA-I). Genotypic changes were associated with changes in the epidemiological profile. The mean age and proportion of patients over 60 years old (P=0.01) and the proportion of respiratory infections (P=0.001) increased significantly. Gentamicin and tetracycline susceptibility of MRSA isolates increased (P<0.001) following the emergence of EMRSA-16. Combining PFGE, SCCmec and MLST has been instrumental in understanding these changes and defining the clones circulating in the HUC patient population.

Age Factors↗

New Delhi metallo-&#x3b2;-lactamase-producing Acinetobacter baumannii in the USA from October, 2013, to March, 2022: a retrospective molecular epidemiological analysis.

BACKGROUND: Most US carbapenem-resistant Acinetobacter baumannii (CRAB) isolates harbour carbapenem-hydrolysing class D &#x3b2;-lactamases. Other carbapenemases, such as New Delhi metallo-&#x3b2;-lactamase (NDM), are uncommon but emerging. We describe the epidemiology of NDM-producing CRAB reported to the US Centers for Disease Control and Prevention (CDC). METHODS: We defined cases as A baumannii with blaNDM confirmed by molecular testing and isolated from any specimen source from a patient in the USA between Oct 1, 2013, and March 31, 2022, and passively reported to the CDC from regional, state, local public health, and CDC laboratories. Epidemiologically linked cases had epidemiological linkage (eg, overlapping health-care facility stay) with one or more other cases. We assessed case relatedness through analysis of whole-genome sequence data using traditional multilocus sequence typing (MLST; Oxford scheme [sequence typeOX]) and core genome MLST. To understand the potential origins of NDM-CRAB in the USA, we compared sequences of cases to US CRAB without NDM and to NDM-CRAB from non-US locations. FINDINGS: We identified 327 NDM-CRAB cases from 264 patients in 21 US states. Among patients with available epidemiological information, 192 (90%) of 214 had epidemiological linkage to at least one additional case and 13 (7%) of 193 were hospitalised outside the USA 12 months or less before index specimen collection. Five regionally distinct sequence type clusters were identified among the 264 case patients; three (sequence type OX218, sequence type OX281, and sequence type OX1697) were closely related to international NDM-CRAB isolates. INTERPRETATION: We identified regionally distinct NDM-CRAB strains, suggesting localised transmission in the USA. Some NDM-CRAB strains in the USA are closely related to strains identified outside the USA, suggesting that spread followed importation. FUNDING: None.

Acinetobacter baumannii↗