The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNA<Superscript>Asn</Superscript>

Background  

Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression.     

Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates

Background  

Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR. However, we have identified 23 Bacillus spp. isolates that gave discrepant results when assayed by standard microbiological methods and PCR. We used multiple-locus variable-number tandem repeat analysis (MLVA), multiple-locus sequence typing (MLST), and phenotypic analysis to characterize these isolates, determine if they cluster phylogenetically and establish whether standard microbiological identification or PCR were associated with false positive/negative results.     

Toxin production in a rare and genetically remote cluster of strains of the <Emphasis Type="Italic">Bacillus cereus</Emphasis> group

Background  

Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2.     

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

Background  

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis.     

Multi locus sequence typing of <Emphasis Type="Italic">Chlamydiales</Emphasis>: clonal groupings within the obligate intracellular bacteria <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

Background  

The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Performance of a 70-mer oligonucleotide microarray for genotyping of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.     

Molecular epidemiology of Neisseria meningitidis serogroup B in Brazil

Background

Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988–2006) for study (n = 372).

Methods

We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA.

Results

In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1.

Conclusions

A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.     

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis,reactive arthritis and Guillain–Barré syndrome

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA). Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002–2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST‐21, ST‐45 and ST‐22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST‐22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST‐22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA‐associated genes may vary, by recombination or expression differences, independent of MLST loci. Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.     

Proposal to create subspecies of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> based on multi-locus sequence typing and an emended description of <Emphasis Type="Italic">Rickettsia conorii</Emphasis>

Background  

Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Sporulation is dispensable for the vegetable-associated life cycle of the human pathogen Bacillus cereus

Bacillus cereus is a common food-borne pathogen that is responsible for important outbreaks of food poisoning in humans. Diseases caused by B. cereus usually exhibit two major symptoms, emetic or diarrheic, depending on the toxins produced. It is assumed that after the ingestion of contaminated vegetables or processed food, spores of enterotoxigenic B. cereus reach the intestine, where they germinate and produce the enterotoxins that are responsible for food poisoning. In our study, we observed that sporulation is required for the survival of B. cereus in leaves but is dispensable in ready-to-eat vegetables, such as endives. We demonstrate that vegetative cells of B. cereus that are originally impaired in sporulation but not biofilm formation are able to reach the intestine and cause severe disorders in a murine model. Furthermore, our findings emphasise that the number of food poisoning cases associated with B. cereus is underestimated and suggest the need to revise the detection protocols, which are based primarily on spores and toxins.     

Carvacrol and <Emphasis Type="Italic">p</Emphasis>-cymene inactivate <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in apple juice

Background  

Outbreaks of food poisoning associated with drinking un-pasteurised apple juice contaminated with enterohaemorrhagic Escherichia coli O157:H7 are a cause of serious illness and occasionally death. Whilst a well-established heat process (pasteurisation) will readily eliminate the pathogen, some consumers are demanding more fresh-like foods that have not been subjected to processing methods that are perceived as severe and may lead to loss of flavour and vitamins. Therefore, alternative methods are being investigated to replace pasteurisation and improve the safety of minimally-processed juices. The addition of natural antimicrobial substances such as the phenolic substances carvacrol and p-cymene (derived from the essential oils of herbs and spices) provides a potential new route to assure safety and extend the shelf-life of raw fruit juices.     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

Origins of asexuality in <Emphasis Type="Italic">Bryobia</Emphasis>mites (Acari: Tetranychidae)

Background  

Obligate asexual reproduction is rare in the animal kingdom. Generally, asexuals are considered evolutionary dead ends that are unable to radiate. The phytophagous mite genus Bryobia contains a large number of asexual species. In this study, we investigate the origin and evolution of asexuality using samples from 111 populations in Europe, South Africa and the United States, belonging to eleven Bryobia species. We also examine intraspecific clonal diversity for one species, B. kissophila, by genotyping individuals from 61 different populations. Knowledge on the origin of asexuality and on clonal diversity can contribute to our understanding of the paradox of sex.     

Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach

Background  

Multilocus Sequence Typing (MLST) is a frequently used typing method for the analysis of the clonal relationships among strains of several clinically relevant microbial species. MLST is based on the sequence of housekeeping genes that result in each strain having a distinct numerical allelic profile, which is abbreviated to a unique identifier: the sequence type (ST). The relatedness between two strains can then be inferred by the differences between allelic profiles. For a more comprehensive analysis of the possible patterns of evolutionary descent, a set of rules were proposed and implemented in the eBURST algorithm. These rules allow the division of a data set into several clusters of related strains, dubbed clonal complexes, by implementing a simple model of clonal expansion and diversification. Within each clonal complex, the rules identify which links between STs correspond to the most probable pattern of descent. However, the eBURST algorithm is not globally optimized, which can result in links, within the clonal complexes, that violate the rules proposed.