Single Reference Strain Research Articles

Symbiosis genes show a unique pattern of introgression and selection within a Rhizobium leguminosarum species complex.

Rhizobia supply legumes with fixed nitrogen using a set of symbiosis genes. These can cross rhizobium species boundaries, but it is unclear how many other genes show similar mobility. Here, we investigate inter-species introgression using de novo assembly of 196 Rhizobium leguminosarum sv. trifolii genomes. The 196 strains constituted a five-species complex, and we calculated introgression scores based on gene-tree traversal to identify 171 genes that frequently cross species boundaries. Rather than relying on the gene order of a single reference strain, we clustered the introgressing genes into four blocks based on population structure-corrected linkage disequilibrium patterns. The two largest blocks comprised 125 genes and included the symbiosis genes, a smaller block contained 43 mainly chromosomal genes, and the last block consisted of three genes with variable genomic location. All introgression events were likely mediated by conjugation, but only the genes in the symbiosis linkage blocks displayed overrepresentation of distinct, high-frequency haplotypes. The three genes in the last block were core genes essential for symbiosis that had, in some cases, been mobilized on symbiosis plasmids. Inter-species introgression is thus not limited to symbiosis genes and plasmids, but other cases are infrequent and show distinct selection signatures.

Landscape of gene expression variation of natural isolates of Cryptococcus neoformans in response to biologically relevant stresses.

Cryptococcus neoformans is an opportunistic fungal pathogen that at its peak epidemic levels caused an estimated million cases of cryptococcal meningitis per year worldwide. This species can grow in diverse environmental (trees, soil and bird excreta) and host niches (intracellular microenvironments of phagocytes and free-living in host tissues). The genetic basic for adaptation to these different conditions is not well characterized, as most experimental work has relied on a single reference strain of C. neoformans. To identify genes important for yeast infection and disease progression, we profiled the gene expression of seven C. neoformans isolates grown in five representative in vitro environmental and in vivo conditions. We characterized gene expression differences using RNA-Seq (RNA sequencing), comparing clinical and environmental isolates from two of the major lineages of this species, VNI and VNBI. These comparisons highlighted genes showing lineage-specific expression that are enriched in subtelomeric regions and in lineage-specific gene clusters. By contrast, we find few expression differences between clinical and environmental isolates from the same lineage. Gene expression specific to in vivo stages reflects available nutrients and stresses, with an increase in fungal metabolism within macrophages, and an induction of ribosomal and heat-shock gene expression within the subarachnoid space. This study provides the widest view to date of the transcriptome variation of C. neoformans across natural isolates, and provides insights into genes important for in vitro and in vivo growth stages.

Intra- and inter-laboratory agreement of the disc diffusion assay for assessing antimicrobial susceptibility of porcine Escherichia coli

Reliable assessment of the susceptibility of animal bacterial pathogens to antimicrobials is of paramount importance in the fight against antimicrobial resistance. This work aims to estimate the repeatability (intra-laboratory agreement) and reproducibility (inter-laboratory agreement) of the disc diffusion assay in veterinary laboratories to understand further if the assay has a role in the surveillance of antimicrobial resistance in animals. Seven major veterinary laboratories from all States in Australia participated, and each tested the same panel of isolates five times at three to four-week intervals, against six antimicrobial agents using Clinical and Laboratory Standards Institute protocols. The panel consisted of twenty different isolates from porcine Escherichia coli from clinical cases and a single reference strain (ATCC 25922). Laboratories were blinded to the identity of the isolates, replicates, and to each other. In total, 4200 inhibition zone diameters (mm) were collected, and analysed descriptively, graphically, and with linear mixed models. Regardless of the laboratories and isolate/antimicrobial combinations, the overall very major error rate (proportion of isolates classified as susceptible when actual status is resistant) was 1.6%; the major error rate (proportion of isolates classified as resistant when actual status is susceptible) was 1.6%; and the ‘minor error’ rate (proportion of isolates with intermediate susceptibility that measure fully susceptible or resistant or vice versa) was 2.4%. The variation between repeated measurements ranged between 4.4–7.2 mm depending on the antimicrobial agent assessed. The reproducibility was always more variable than the repeatability, which suggested some laboratory effects. The repeatability coefficient of disc diffusion was lowest for tetracycline (4.4 mm, 95% CI: 3.8–5.0 mm) and ampicillin (4.6 mm, 95% CI: 4.2–5.2 mm) and highest for trimethoprim-sulfamethoxazole (6.6 mm, 95% CI: 5.9–7.4 mm). The reproducibility coefficient of disc diffusion was lowest for gentamicin (5.4, 95% CI: 4.0–7.2) and highest for trimethoprim-sulfamethoxazole (7.2 mm, 95%CI: 4.5–11.7 mm). The precision of the disc diffusion assay was deemed satisfactory for use in a national surveillance program for clinical porcine E. coli isolates. However, measurement variation of the disc diffusion assay is of concern for isolates with marginal susceptibility or resistance due to increased risk of misclassification.

Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence

BackgroundStreptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains.ResultsPan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection.ConclusionThe gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first “whole-genome” comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1512-6) contains supplementary material, which is available to authorized users.

A reference pan-genome approach to comparative bacterial genomics: identification of novel epidemiological markers in pathogenic Campylobacter.

The increasing availability of hundreds of whole bacterial genomes provides opportunities for enhanced understanding of the genes and alleles responsible for clinically important phenotypes and how they evolved. However, it is a significant challenge to develop easy-to-use and scalable methods for characterizing these large and complex data and relating it to disease epidemiology. Existing approaches typically focus on either homologous sequence variation in genes that are shared by all isolates, or non-homologous sequence variation - focusing on genes that are differentially present in the population. Here we present a comparative genomics approach that simultaneously approximates core and accessory genome variation in pathogen populations and apply it to pathogenic species in the genus Campylobacter. A total of 7 published Campylobacter jejuni and Campylobacter coli genomes were selected to represent diversity across these species, and a list of all loci that were present at least once was compiled. After filtering duplicates a 7-isolate reference pan-genome, of 3,933 loci, was defined. A core genome of 1,035 genes was ubiquitous in the sample accounting for 59% of the genes in each isolate (average genome size of 1.68 Mb). The accessory genome contained 2,792 genes. A Campylobacter population sample of 192 genomes was screened for the presence of reference pan-genome loci with gene presence defined as a BLAST match of ≥70% identity over ≥50% of the locus length - aligned using MUSCLE on a gene-by-gene basis. A total of 21 genes were present only in C. coli and 27 only in C. jejuni, providing information about functional differences associated with species and novel epidemiological markers for population genomic analyses. Homologs of these genes were found in several of the genomes used to define the pan-genome and, therefore, would not have been identified using a single reference strain approach.

Molecular Epidemiologic Investigation of Polish Avian Pasteurella multocida Strains Isolated from Fowl Cholera Outbreaks Showing Restricted Geographical and Host-Specific Distribution

Molecular epidemiologic analyses of the 42 clinical isolates of Pasteurella multocida from various avian hosts (geese, ducks, turkeys, and laying hens) in Poland from 2001 to 2011, including a single reference strain, were performed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, single primer PCR, and repetitive extragenic palindromic (REP)-PCR. Forty-two isolates were identified as P. multocida (serotype A). The majority of P. multocida strains were obtained from waterfowl clustered within one genotype, and they were not consistent with the genotypes obtained from the turkey strains. Pasteurella multocida showed genetic homogeneity between the host species, especially when isolated on the same farm. Some of the clones also were characteristic to the particular farm. The strains obtained in different regions represent distinct molecular patterns. The present findings demonstrate that some clones of P. multocida are restricted in geographical and host distribution. In addition, this study suggests that ERIC-PCR, single primer PCR, and REP-PCR are suitable techniques for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

DNA Gyrase and Topoisomerase IV Mutations in Quinolone-ResistantFlavobacterium psychrophilumIsolated from Diseased Salmonids in Norway

Flavobacterium psychrophilum is the causative agent of the recognized diseases 'bacterial coldwater disease' and 'rainbow trout fry syndrome' and is found in many farmed freshwater and marine fish species. In Norway, the bacterium has mainly been isolated from Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.). In the present study, 26 isolates from Norwegian farmed salmonids were examined. All isolates were tested for susceptibility towards various antibacterial drugs by the disk diffusion method, and minimum inhibitory concentration values for oxolinic acid and flumequine were established for selected isolates. All isolates from rainbow trout displayed reduced susceptibility towards quinolones, while brown trout and Atlantic salmon isolates were susceptible. The quinolone resistance determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes were sequenced. Sequence analysis of the QRDR of gyrA in quinolone resistant isolates revealed a threonine:arginine amino acid substitution at position 82 in all 16 isolates from Norwegian rainbow trout and a single reference strain isolated from rainbow trout in Sweden. No evidence for plasmid-mediated quinolone resistance was found in any of the isolates. Pulsed-field gel electrophoresis and phylogenetic analysis of parC and gyrB sequences indicate a clonal relationship between rainbow trout isolates.

Phylogenetic Studies on Corynebacterium bovis Isolated from Bovine Mammary Glands

Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.

Serological analysis of feline calicivirus isolates from the United States and United Kingdom

The antigenic relationship of 181 feline calicivirus (FCV) isolates from the United States (USA) and United Kingdom (UK) to 5 reference viruses, representing 2 vaccine strains (F9 VacNor, 2280) and 3 field strains (H, J, TN) was determined by a neutralization assay using two standardized dilutions of reference sera. A comparison between USA and UK isolates indicated that significantly fewer UK isolates could be neutralized by sera specific for each of the reference strains. Isolates from the USA were also analyzed based on the time period and geographical location from which they were derived. Analysis of the cross-neutralization results for isolates derived from the USA indicated that no single reference strain was predominant in all the geographical locations, however, antisera specific to strains 2280, H and J neutralized the highest number of isolates overall. Analysis of the neutralization patterns based on the time period from which the isolates were derived indicated that F9 VacNor was antigenically similar to 86% of feline caliciviruses isolated in 1958–1979. This number decreased to 43% for isolates derived in 1980–1995. By contrast, the J strain maintained its antigenic relatedness to approximately 75% of representative FCV isolates circulating during the last 40 years.

Analysis of a single reference strain for determination of gentamicin regression line constants and inhibition zone diameter breakpoints in quality control of disk diffusion antibiotic susceptibility testing.

An equation was derived from known formulas to express the size of the inhibition zone diameter in the disk diffusion antibiotic susceptibility test as a function of the disk content of antibiotic. The equation permitted a calculation of regression line constants for the correlation between zone diameter and the minimum inhibitory concentration (MIC) with a single reference strain. Analysis of reference strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, as well as 12 clinical isolates belonging to these species, showed a linearity between zone size squared and the logarithm of disk content in tests with 10-, 30-, and 100-micrograms gentamicin disks. All three species, however, gave regression line constants which were characteristic for the individual bacterial species. Calculations of zone diameter breakpoints corresponding to recommended MIC limits with E. coli and P. aeruginosa reference strains gave an accurate prediction of gentamicin susceptibility. Histogram analysis of 48 strains of Streptococcus faecalis from clinical specimens showed a distribution of zone diameter values which would result in false classification of susceptibility with breakpoints calculated for the other bacterial species studied. Single reference strain analysis of S. faecalis ATCC 29212 (gentamicin MIC, 8 micrograms/ml) permitted the calculation of breakpoints which accurately assigned the strains tested to the intermediate category of susceptibility. Single reference strain analysis offers a quality control method for individual laboratories that allows the determination of inhibition zone diameter breakpoints corresponding to recommended MIC limits with no MIC determinations required.