Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin

Abstract 80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with ΔT_m-values ranging from 0.0–2.3°C. In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for β-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a ΔT_m-value of 5.7°C and produced γ-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate^TM, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.     

Calibration of the change in thermal stability of DNA duplexes and degree of base pair mismatch

Summary One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (T_m) to base pair mismatch have led to conflicting results. We have studied the T_m between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of T_m and percent base pair mismatch is remarkably linear over this range with a correlation coefficient >0.98. A T_m of 1°C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.     

Comparative genomic analysis of clinical strains of Campylobacter jejuni from South Africa

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the post-infectious neuropathies, Guillain-Barré and Miller Fisher syndromes. In the Cape Town area of South Africa, C. jejuni strains with Penner heat-stable (HS) serotype HS∶41 have been observed to be overrepresented among cases of Guillain-Barré syndrome. The present study examined the genetic content of a collection of 32 South African C. jejuni strains with different serotypes, including 13 HS∶41 strains, that were recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes. The sequence-based typing methods, multilocus sequence typing and DNA microarrays, were employed to potentially identify distinguishing features within the genomes of these C. jejuni strains with various disease outcomes.

Methodology/Principal Findings

Comparative genomic analyses demonstrated that the HS∶41 South African strains were clearly distinct from the other South African strains. Further DNA microarray analysis demonstrated that the HS∶41 strains from South African patients with the Guillain-Barré syndrome or enteritis were highly similar in gene content. Interestingly, the South African HS∶41 strains were distinct in gene content when compared to HS∶41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements (CJIEs). Only the integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS∶41 strains whereas this element was absent in two closely-related HS∶41 strains from Mexico. A more distantly-related HS∶41 strain from Canada possessed both integrated elements CJIE1 and CJIE2.

Conclusion/Significance

These findings demonstrate that CJIEs may contribute to the differentiation of closely-related C. jejuni strains. In addition, the presence of bacteriophage-related genes in CJIE1 may contribute to the genomic diversity of C. jejuni strains. This comparative genomic analysis of C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks.     

DNA base composition and biochemical characters of Campylobacter strains

We studied the relationship between ten authentic Campylobacter fetus and two C. bubulus strains, and seventeen named vibrios of animal and human origin. All organisms fall within the genus Campylobacter, as defined. Their DNA base composition ranges from 29.8 to 35.9% (G+C). On the basis of similarity of differential biochemical tests and % (G+C), they can be divided into four closely similar groups.

1. C. fetus var. intestinalis and var. venerialis with 34.7 to 35.9% (G+C).
2. Vibrio fecalis with 32.0 to 32.8% (G+C).
3. C. bubulus with 30.1 and 30.6% (G+C).
4. The cluster V. jejuni, V. coli, Vibrio sp. from fowl, and related vibrios from man and sheep with 29.8 to 34.0% (G+C).

We propose that all the vibrios used here be included in the genus Campylobacter.     

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains

Abstract Microcystis aeruginosa (Synechocystis ) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

PCR-RFLP and total DNA homology revealed three related genomic species among broad-host-range Frankia strains

Abstract: Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified nif D-K intergenic spacer (IGS) region was used to cluster 22 Frankia strains of the Elaeagnus host specificity group into seven genomic groups and to measure the degree of genetic similarity among them. This PCR-RFLP analysis could assign freshly isolated strains to described genomic species and revealed genomic groups not yet described among Frankia strains of the Elaeagnus specificity group. Six broad-host-range Frankia strains, infective on both Alnus and Elaeagnus , fell into three closely related PCR-RFLP clusters. DNA-DNA hybridization was then used to establish the correlations between PCR-RFLP clusters and total DNA relatedness groups. The three PCR-RFLP clusters agreed with two new and one reference genomic species, indicating that Frankia ability to nodulate with Alnus and Elaeagnus is a monophyletic trait shared by three genomic species.     

Genomic analysis of the diversity,antimicrobial resistance and virulence potential of clinical Campylobacter jejuni and Campylobacter coli strains from Chile

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n = 81), collected in 2017–2019 in Santiago, Chile. This culture collection accounts for more than one third of the available genome sequences from South American clinical strains. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen.     

Detection of DNA base sequence homology between entomopoxviruses isolated from lepidoptera and orthoptera

The extent of DNA base sequence homology between entomopoxviruses (EPVs) from Lepidoptera, Orthoptera, and the vertebrate poxvirus Vaccinia was investigated by DNA-DNA hybridization. α-³²P-Labeled DNA from Amsacta moorei EPV, Melanoplus sanguinipes EPV, and Vaccinia virus strain WR was hybridized with the DNA from six different entomopoxvirus isolates. Based on the thermal denaturation temperature of hybrid DNA molecules, approximately 54% base sequence homology was detected between Amsacta moorei and Euxoa auxiliaris EPV DNAs. Extensive DNA hybridization was detected between α-³²P-labeled Melanoplus sanguinipes EPV DNA and DNA from Arphia conspirsa and Phoetaliotes nebrascensis entomopoxviruses. No base sequence homology was detected between vaccinia DNA and DNA from any of the entomopoxvirus isolates used in this study.     

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

Background

Waterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.

Results

One of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.

Conclusions

Our results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-768) contains supplementary material, which is available to authorized users.     

DNA base compositions of halophilic and nonhalophilicBacillus firmus strains of marine origin

The mineral salt requirements of 27 strains ofBacillus firmus were determined. Twenty-six of these strains were of marine origin and one terrestrial strain was used as a reference. Three strains demonstrated strictly halophilic behavior, i.e., they showed no growth in media prepared without sodium chloride. Seven strains were nonhalophilic. The growth of 17 strains was stimulated by the addition of sodium chloride, but the cells were able to grow without it. These results were compared with the DNA base compositions of the strains. In contrast to literature data, relationships between the DNA base ratios and the halophilic or nonhalophilic behavior of the cells could not be detected. But strains with guanine plus cytosine values above 41 mol% did grow well at 44°C, and those strains showing poor growth at this temperature had lower guanine plus cytosine percentages.     

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non‐livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1–C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7‐C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.     

Estimates of relatedness and inbreeding in goose strains from DNA fingerprints

The purpose of this study was to determine if DNA fingerprints (DFPs) could be used to estimate relatedness and inbreeding of strains of geese and to compare three methods of calculating relatedness indices. Strains included a control and selected strain from each of the Chinese and Synthetic (Chinese, Hungarian and Pilgrim) breeds. DFP patterns for each strain were based on individual DNA samples from six females, or on pooled DNA from 15 females different from those used for individual samples. Three relatedness indices were used, namely, genetic distance, modified Rogers distance and band sharing. All relatedness indices showed a closer relationship of strains within than between breeds. Correlation coefficients among relatedness indices were higher based on pooled DNA (r ≥|0·97|) than those based on individual DNA (r ≥|0·741). Inbreeding estimates were higher for selected compared with control strains. It appears that the use of DFPs to estimate relatedness, regardless of index used, and inbreeding can be valuable for studying geese where there is a limited breeding history.