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.     

Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

Background

Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.

Methodology/Principal Findings

In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.

Conclusions/Significance

Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.     

Bayesian modeling of recombination events in bacterial populations

Background  

We consider the discovery of recombinant segments jointly with their origins within multilocus DNA sequences from bacteria representing heterogeneous populations of fairly closely related species. The currently available methods for recombination detection capable of probabilistic characterization of uncertainty have a limited applicability in practice as the number of strains in a data set increases.     

Differential effects of age on circulating and splenic leukocyte populations in C57BL/6 and BALB/c male mice

Background  

Despite several reports on age-related phenotypic changes of the immune system's cells, studies that use a multipoint age comparison between the specific and innate immune cell populations of prototypical Th1- and Th2-type polarized mouse strains are still lacking.     

Comparative genomics of Helicobacter pylori isolates recovered from ulcer disease patients in England

Background  

Genomic diversity of H. pylori from many different human populations is largely unknown. We compared genomes of 65 H. pylori strains from Nottingham, England. Molecular analysis was carried out to identify rearrangements within and outside the cag-pathogenicity-island (cag PAI) and DNA sequence divergence in candidate genes. Phylogenetic analysis was carried out based on various high-resolution genotyping techniques.     

Cheating does not explain selective differences at high and low relatedness in a social amoeba

Background  

Altruism can be favored by high relatedness among interactants. We tested the effect of relatedness in experimental populations of the social amoeba Dictyostelium discoideum, where altruism occurs in a starvation-induced social stage when some amoebae die to form a stalk that lifts the fertile spores above the soil facilitating dispersal. The single cells that aggregate during the social stage can be genetically diverse, which can lead to conflict over spore and stalk allocation. We mixed eight genetically distinct wild isolates and maintained twelve replicated populations at a high and a low relatedness treatment. After one and ten social generations we assessed the strain composition of the populations. We expected that some strains would be out-competed in both treatments. In addition, we expected that low relatedness might allow the persistence of social cheaters as it provides opportunity to exploit other strains.     

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia

Background

Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H. pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance.

Methods

Strains were isolated from the gastric mucosa of patients infected with H. pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby–Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed.

Results

We isolated 1955 H. pylori strains. The resistance rates of H. pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement.

Conclusion

H. pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H. pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H. pylori infection in Ningxia.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Background  

The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).     

Variation in tissue-specific gene expression among natural populations

Background  

Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.     

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli

Background  

To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate.     

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Aims

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP‐PCR) techniques.

Methods and Results

A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP‐PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP‐PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

Conclusions

Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

Significance and Impact of the Study

Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.     

Screening of white‐rot fungi for bioprocessing of wheat straw into ruminant feed

Aim

In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal‐treated wheat straw.

Methods and Results

Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0·05) IVGP compared to the control after 7 weeks (227·7 ml g^?1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g^?1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g^?1 OM, respectively.

Conclusion

Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains.

Significance and Impact of the Study

Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.     

Variation in tissue-specific gene expression among natural populations

Background

Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.

Results

Half of the genes (48%) were differentially expressed among individuals within a population-tissue group and 76% were differentially expressed among tissues. Differences among tissues reflected well established tissue-specific metabolic requirements, suggesting that these measures of gene expression accurately reflect changes in proteins and their phenotypic effects. Remarkably, only a small subset (31%) of tissue-specific differences was consistent in all three populations.

Conclusions

These data indicate that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types. We suggest that those subsets of treatment-specific gene expression patterns that are conserved between taxa are most likely to be functionally related to the physiological state in question.     

The effect of biotic and physical factors on the competitive ability of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>

Background  

Rhizobium leguminosarum bv. viciae (Rlv) is a soil bacterium which can form nitrogen-fixing symbiotic relationships with leguminous plants. Numerous rhizobial strains found in soils compete with each other. Competition can occur both during the saprophytic growth phase in the rhizosphere and inside plant tissues, during the symbiotic phase. Competition is important as it may affect the composition of rhizobial populations present in the soil and in the root nodules of plants.     

Hereditary profiles of disorderly transcription?

Background  

Microscopic examination of living cells often reveals that cells from some cell strains appear to be in a permanent state of disarray without obvious reason. In all probability such a disorderly state affects cell functioning.     

Genetic diversity and colony structure of Tapinoma melanocephalum on the islands and mainland of South China

Aim

Tapinoma melanocephalum is listed as one of the most important invasive pest species in China. Information regarding the patterns of invasion and effects of geographic isolation on the population genetics of this species is largely lacking.

Location

South China.

Methods

To address this problem, we genotyped 39 colonies (two colonies were collapsed due to genetic similarity) using microsatellite markers and mitochondrial DNA sequencing to compare colony genetic structure of T. melanocephalum on the mainland and islands of South China.

Results

An analysis of the colony genotypes showed that the genetic diversity of the mainland population was slightly higher than that of the island populations but not significantly so. However, the observed heterozygosity on Shangchuan Island (SCD) was significantly lower than that of the other colonies. We also found six haplotypes in 111 mitochondrial DNA COI sequences. The relatedness (r) value between colonies of SCD was 0.410, higher than that of the other populations. The genetic clusters among colonies were not related to geographic locations and exhibited admixture likely due to frequent human‐mediated dispersal associated with trade between the mainland population and the islands. Pairwise F_STs between populations showed differentiation among mainland populations, while SCD displayed high levels of divergence (F_ST > 0.15) from most mainland populations. There was no significant isolation by distance among colonies. Most populations showed signs of a bottleneck effect.

Main conclusions

Our study suggests that there was no significant difference in the genetic diversity among the islands and the mainland; however, the lower genetic diversity, the higher degree of genetic divergence from other colonies, and the higher relatedness among nestmates made the SCD population stand out from all the others.     

Differentiation and gene flow among European populations of Leishmania infantum MON-1

Background

Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1.

Methodology/Principal Findings

We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups—MON-1 and non-MON-1—could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands–Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles.

Conclusion

In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.     

Antibiotics resistance of Helicobacter pylori in children with upper gastrointestinal symptoms in Hangzhou,China

Background

The decreasing eradication rate of Helicobacter pylori is mainly because of the progressive increase in its resistance to antibiotics. Studies on antimicrobial susceptibility of H. pylori in children are limited. This study aimed to investigate the resistance rates and patterns of H. pylori strains isolated from children.

Materials and Methods

Gastric mucosa biopsy samples obtained from children who had undergone upper gastrointestinal endoscopy were cultured for H. pylori, and susceptibility to six antibiotics (clarithromycin, amoxicillin, gentamicin, furazolidone, metronidazole, and levofloxacin) was tested from 2012‐2014.

Results

A total of 545 H. pylori strains were isolated from 1390 children recruited. The total resistance rates of H. pylori to clarithromycin, metronidazole, and levofloxacin were 20.6%, 68.8%, and 9.0%, respectively. No resistance to amoxicillin, gentamicin, and furazolidone was detected. 56.1% strains were single resistance, 19.6% were resistant to more than one antibiotic, 16.7% for double resistance, and 2.9% for triple resistance in 413 strains against any antibiotic. And the H. pylori resistance rate increased significantly from 2012‐2014. There was no significant difference in the resistance rates to clarithromycin, metronidazole, and levofloxacin between different gender, age groups, and patients with peptic ulcer diseases or nonulcer diseases.

Conclusions

Antibiotic resistance was indicated in H. pylori strains isolated from children in Hangzhou, and it increased significantly during the 3 years. Our data strongly support current guidelines, which recommend antibiotic susceptibility tests prior to eradication therapy.     

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.