Outer Membrane Protein Gene Research Articles

한국에서 분리된 Vibrio harveyi 외막단백질의 유전적 차이와 넙치(Paralichthys olivaceus)에 대한 OmpW의 면역원성 분석

Vibrio harveyi is a pathogenic marine bacterium causing systemic symptoms resulting in mass mortalities in fishes and shrimps in aquaculture. Outer membrane proteins(OMPs) are related to the pathogenicity and thus good targets for diagnosis and vaccination for Gram negative bacteria. Recently vaccination strategies using the OMPs have been suggested to control vibriosis in several fish species. In this study, we have isolated V. harveyi from diseased marine fishes from different regions of Korea and investigated genetic variations of four OMP genes including OmpK, OmpU, OmpV and OmpW. Consequently, OmpK and U genes could be divided into 3 subgroups of type I, II, III and type A, B, C, respectively, without any correlation with geographical regions and species while OmpV and W were highly homologous. OmpW gene of V. harveyi FP4138 was fully sequenced and predicted the deduced amino acid sequence to form <TEX>${\beta}-barrel$</TEX> with hydrophobic channel. Indeed, the immunogenicity of recombinant OmpW produced in Escherichia coli was assessed by vaccinating flounder. As a result, the high antibody response with antibody titer of <TEX>$4.2{\pm}0.7$</TEX> and protection with relative percent survival of 60% against artificial infection of V. harveyi were demonstrated. This result indicates that OmpW is a virulence related factor and it can be a vaccine candidate to prevent a high mortality caused by V. harveyi infection in olive flounder, Paralichthys olivaceus.

DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases.

This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.

Vibrio cholerae pathogen from the freshwater-cultured whiteleg shrimp Penaeus vannamei and control with Bdellovibrio bacteriovorus

Vibriosis has become a major global economic problem in freshwater-farmed whiteleg shrimp (Penaeus vannamei). The prevention and control of vibriosis are now priority research topics. In this study, a pathogenic strain (QH) was isolated from vibriosis-infected freshwater-farmed P. vannamei that resulted in leg yellowing and was identified as a Vibrio cholerae isolate through phylogenetic analysis and the API 32GN system. A phylogenetic tree that was constructed using the neighbor-joining method further confirmed the QH isolate as a V. cholerae strain. A virulent outer membrane protein (ompU) gene was found to be present in the QH isolate, which further confirmed its pathogenicity. In addition, Bdellovibrio bacteriovorus conferred significant protection against V. cholerae: B. bacteriovorus exhibited significant bacteriolytic effects on the V. cholerae pathogen, possessed a wide prey range that included Vibrio pathogens, and displayed a positive protective efficacy against experimental V. cholerae infection in P. vannamei. To the best of our knowledge, this is the first report of the control of shrimp pathogen V. cholerae with B. bacteriovorus.

Sequence Type 4821 Clonal Complex Serogroup B Neisseria meningitidis in China, 1978-2013.

Serogroup B Neisseria meningitidis strains belonging to sequence type 4821 clonal complex (CC4821), a hyperinvasive lineage first identified for serogroup C in 2003, have been increasingly isolated in China. We characterized the outer membrane protein genes of 48 serogroup B and 214 serogroup C strains belonging to CC4821 and analyzed the genomic sequences of 22 strains. Four serogroup B strains had porin A (i.e., PorA), PorB, and ferric enterobactin transport (i.e., FetA) genotypes identical to those for serogroup C. Phylogenetic analysis of the genomic sequences showed that the 22 CC4821 strains from patients and healthy carriers were unevenly clustered into 2 closely related groups; each group contained serogroup B and C strains. Serogroup B strains appeared variable at the capsule locus, and several recombination events had occurred at uncertain breakpoints. These findings suggest that CC4821 serogroup C N. meningitidis is the probable origin of highly pathogenic CC4821 serogroup B strains.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

The Role of OmpK35, OmpK36 Porins, and Production of β-Lactamases on Imipenem Susceptibility in Klebsiella pneumoniae Clinical Isolates, Cairo, Egypt.

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. We aimed to study the effect of combined porin loss and production of extended-spectrum β-lactamases (ESBLs) on imipenem susceptibility among K. pneumoniae clinical isolates. This study included 91 suspected ESBL-producing K. pneumoniae clinical isolates, isolated from different patient specimens at the Cairo University hospital from January to June 2010. All isolates were subjected to genotypic analysis of the outer membrane protein gene expression using reverse transcription-PCR (RT-PCR) and analysis of OmpK35/36 of 38 isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By RT-PCR, loss of Omp35 was detected in 78 (85.7%) isolates, loss of Omp36 was detected in 64 (70.32%), and loss of both porins was detected in 62 (68.1%). Out of 91 isolates, 45 (49.5%) were resistant to cefoxitin, and 17 (18.7%) were confirmed as derepressed AmpC producers. Omp35 was lost in all FOX-resistant isolates, whereas Omp36 was lost in 42 (93.3%) (p-value 0.002). The mean of ceftazidime inhibition zone diameter was significantly decreased among ESBL-producing isolates with loss of Omp35/36 (p-value 0.041 and 0.006), respectively. The mean of imipenem minimal inhibitory concentration (MIC) was markedly increased to 8.55 μg/ml among AmpC-producing isolates with Omp35/36 loss, while the mean of imipenem MIC among the 66 confirmed ESBL producers was 0.32 μg/ml. Imipenem MIC was markedly increased among K. pneumoniae isolates showing AmpC production with loss of both porins OmpK35/36. Meanwhile, the association of porin OmpK35/36 loss with ESBL production was not a direct cause of resistance to imipenem.

Toxigenic Vibrio cholerae identified in estuaries of Tanzania using PCR techniques.

The current study assessed the occurrence of the Vibrio cholerae serogroups O1 and O139 in environmental samples along salinity gradients in three selected estuaries of Tanzania both through culture independent methods and by cultured bacteria. Occurrence of V. cholerae was determined by PCR targeting the V. cholerae outer membrane protein gene ompW. Furthermore, the presence of toxigenic strains and serogroups O1 and O139 was determined using multiplex PCR with specific primers targeting the cholera toxin gene subunit A, ctxA, and serotype specific primers, O1-rfb and O139-rfb, respectively. Results showed that V. cholerae occurred in approximately 10% (n = 185) of both the environmental samples and isolated bacteria. Eight of the bacteria isolates (n = 43) were confirmed as serogroup O1 while one belonged to serogroup O139, the first reported identification of this epidemic strain in East African coastal waters. All samples identified as serogroup O1 or O139 and a number of non-O1/O139 strains were ctxA positive. This study provides in situ evidence of the presence of pathogenic V. cholerae O1 and O139 and a number of V. cholerae non-O1/O139 that carry the cholera toxin gene in estuaries along the coast of Tanzania.

Comparative Study on Bacterial Outer Membrane Protein (BOMP) and Bacterial Outer Membrane Gene (BOMPG) Vaccination to Goldfish Carassius Auratus against Aeromonas Hydrophila

Aeromonas hydrophila is one of the serious bacterial pathogen in fresh water aquaculture industry. Recently, some attention has been given to bacterial outer membrane protein (BOMP) as potentially important vaccine components. Ornamental gold fish Carassius auratus immunized with the bacterial outer membrane protein encoding gene clone, BOMPG-pTZ57R/T that was named as BOMPG and the bacterial outer membrane proteins (BOMP) which were developed from a virulent strain of A. hydrophila and used as a vaccine candidate. In order to study the comparative effects of the vaccines, BOMPG and BOMP were delivered to the experimental fishes (16.4 ± 1 g mean weight) by intra peritoneal injection once in every 15 days interval. At the end of the experiment, the immunized and control fishes were challenged with virulent strain of A. hydrophila and assessed the immunological parameters. C. auratus succumbed to death 100% at five days when no vaccination was given whereas the vaccinated groups survived significantly (F=34.64; P ≤ 0.001) of 70 and 80% respectively in BOMPG and BOMP after 10 days of challenge. Surprisingly the vaccinated groups help to decrease the Aeromonas sp. load in blood and muscle of treated fishes and improve the serum biochemical parameters significantly (P ≤ 0.001) than the control group. The haematological and immunological parameters were also improve significantly (P ≤ 0.001) in the BOMP vaccinated fishes due to the immune enhancement by the vaccines. Among the two different BOMP deliveries, the BOMP highly influenced to improve the immune system against A. hydrophila challenge than BOMPG. It may be the reason the BOMPG vaccine may easily degraded in vivo conditions.

Cloning and sequence analysis of hsf, an outer membrane protein gene of Pasteurella multocida serotype B:2

Aim: The present study was undertaken to clone, sequence and analyze the hsf, an outer membrane protein gene of Pasteurella multocida serotype B:2 Materials and Methods: hsf gene was amplified from genomic DNA of P. multocida. Polymerase chain reaction (PCR) product was cloned in pET-32a vector and was characterized. hsf gene was sequenced, analyzed and phylogenetic tree was constructed taking sequences of other strains. Results: Amplicon size was found to be 785 bp. Recombinant got characterized through colony PCR and restriction enzyme analysis. Conclusion: hsf gene of P. multocida serotype B is similar to serotype A, but different from serotype D. Further work is needed to evaluate role of Hsf protein in protection studies and to study the antigenic properties of this recombinant protein as a candidate for vaccine.

First Report of Natural Infection by 'Candidatus Liberibacter solanacearum' in Bittersweet Nightshade (Solanum dulcamara) in the Columbia Basin of Eastern Oregon.

Potatoes are a major crop in the Columbia Basin of Oregon and Washington, representing an annual farm gate value of almost $750 million. Zebra chip disease (ZC), a new and economically important disease of potato, was first reported in Oregon and Washington in 2011 (1). The disease is caused by the bacterium 'Candidatus Liberibacter solanacearum' (Lso, also referred to as 'Ca. L. psyllaurous'), which is vectored by the potato psyllid (Bactericera cockerelli Sulc) (1,2). Identifying alternative hosts for Lso may facilitate management of ZC disease, which has increased potato production costs in the region. The perennial weed, bittersweet nightshade (Solanum dulcamara L.), is a year-round host of the potato psyllid (3) and is also a suspected host of Lso. However, little is known about the role of this weed in ZC epidemiology. Naturally occurring bittersweet nightshade plants (n = 21) were sampled at six different locations near Hermiston, Oregon, between May and October in 2012. These plants exhibited several symptoms associated with Lso, ranging from asymptomatic to slight purpling, chlorosis, or scorching of the foliage. However, S. dulcamara exhibits similar symptoms under a variety of environmental conditions (drought stress, etc.); therefore, it was difficult to identify potentially infected plants based solely on symptomology. Leaf and stem tissue (n = 21) was analyzed with high-fidelity PCR using species-specific primers for the 16S rDNA gene, CLipoF, and OI2c (2,4). Approximately 27.3% of the plants tested positive for Lso using these primers, including plants from the following locations on 16 April, 16 May, and 24 May, respectively: Hat Rock, OR (45°55.033' N, 119°10.495' W), Irrigon, OR (45°54.560' N, 119°24.857' W), and Stanfield, OR (45°46.971' N, 119°13.203' W). Three plants were selected for further PCR analysis with primers for the outer membrane protein gene, 1482f and 2086r (1). Amplicons obtained with both sets of PCR primers were directly sequenced. A BLAST analysis showed that the 16S rDNA gene sequence (993 to 1,000 bp) shared 99 to 100% identity with several Lso accessions, including JN848751.1 (from Washington) and JN848753.1 (from Oregon). Likewise, the outer membrane protein gene sequence (600 to 601 bp) shared 99 to 100% identity with 'Ca. L. solanacearum' accession KC768330.1 (from Honduras). All six sequences were deposited in GenBank (Accession Nos. KJ854199 to KJ854204). According to these findings, bittersweet nightshade may be an important annual source of Lso in the region, particularly since it serves as a host for the potato psyllid. Potato psyllids were also detected at two of the locations with infected S. dulcamara: Irrigon, OR, and Stanfield, OR. A subsample of the psyllids collected in 2012 were analyzed with PCR and Lso was detected in a sample from Stanfield, OR (5). Identifying perennial hosts of Lso promotes a better understanding of both ZC disease epidemiology and management. To our knowledge, this is the first report of Lso causing natural infections in S. dulcamara in the United States.

Mutations in the major outer membrane protein gene from Histophilus somni by an allelic exchange method

Functional roles of the major outer membrane protein (MOMP) gene from the bovine pathogen Histophilus somni have remained to be elucidated due to lack of mutagenesis methods easily applicable to this gene. In this study, the direct use of PCR-amplified mutated DNA flanking by an antibiotic selection marker for transformation of H. somni was applied to accomplish the site-directed mutagenesis via homologous recombination in H. somni non-pathogenic strain 129Pt and pathogenic strain 2336. A protocol for unmarking the antibiotic resistance gene from the created MOMP mutant by using a temperature-sensitive plasmid vector was also established. In both strains, no significant phenotypic difference was observed between the wild-type strain and its isogenic mutant expressing the exchanged MOMP in growth in broth medium and antibiotic susceptibility. However, the mutant 129Pt expressing MOMP from strain 2336 was significantly more susceptible to bacterial killing by fresh normal bovine serum than its wild-type parent strain. Serum susceptibility of the mutant 2336 expressing MOMP from strain 129Pt was significantly lower than its wild-type parent strain although the susceptibility difference was considerably less than that found between the 129Pt wild-type and MOMP mutant strains. From further assays using MOMP mutants that express chimeric MOMP consisting of the amino-terminal part that contains loops L1–L3 from one strain and the carboxyl-terminal part that contains loops L4–L8 from another strain, the C-terminal part of MOMP was found to affect serum susceptibility of H. somni.

Molecular identification of chlamydial cause of abortion in small ruminants in Jordan.

Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.

Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.

A mutation burst during the acute phase of Helicobacter pylori infection in humans and rhesus macaques.

The evolution rate and genetic changes that occur during chronic infection with Helicobacter pylori have been analysed, but little is known about the genomic changes during the initial, acute bacterial infection phase. Here we analyse the rate and pattern of genome evolution in H. pylori from the genomes of two input strains isolated from human volunteers with asymptomatic infection, and the genomes of two output strains collected 20 and 44 days after re-infection. Similarly, we analyse genome evolution in bacteria from the genome sequences of input and output strains sequentially taken after experimental infection of a rhesus macaque. The estimated mutation rate reveals a mutation burst during the acute infection phase that is over 10 times faster than the mutation rate during chronic infection, and orders of magnitude faster than mutation rates in any other bacteria. The elevated frequency of mutations in outer membrane protein genes suggests that the mutation burst facilitates rapid host adaptation of the bacteria.

Molecular characterization of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and domestic dogs, Buenos Aires (Argentina)

Rickettsioses, ehrlichioses and anaplasmoses are emerging diseases that are mainly transmitted by arthropods and that affect humans and animals. The aim of the present study was to use molecular techniques to detect and characterize those pathogens in dogs and ticks from Buenos Aires city. We studied 207 Rhipicephalus sanguineus ticks and 52 canine blood samples from poor neighborhoods of Buenos Aires city. The samples were molecularly screened for the genera Rickettsia, Ehrlichia, and Anaplasma by PCR and sequencing. DNA of Rickettsia massiliae (3.4%) and Anaplasma platys (13.5%) was detected in ticks and blood samples, respectively. For characterization, the positive samples were subjected to amplification of a fragment of the 190-kDa outer membrane protein gene (spotted fever group rickettsiae) and a fragment of the groESL gene (specific for A. platys). A phylogenetic tree was constructed using the neighbor-joining method, revealing that the sequences were closely related to those of strains from other geographic regions. The results indicate that human and animal pathogens are abundant in dogs and their ticks in Buenos Aires city and portray the potentially high risk of human exposure to infection with these agents, especially in poor neighborhoods, where there is close contact with animals in an environment of poor health conditions.

Detection and Identification of Pectinatus Brewery Contaminants Based on the Gene for the Major Outer Membrane Protein

Considering the recurrence of Pectinatus in breweries, detection and differentiation of contaminating isolates is important for quality control to mitigate recurring or newly developed spoilage problems. As a potential new PCR target, the gene for a major outer membrane protein (OMP) was sequenced for three species of Pectinatus, including one Pectinatus haikarae, two P. cerevisiiphilus, and three P. frisingensis isolates. Conserved regions in the OMP gene enabled the design of a multiplex PCR that concurrently targets all three species. The multiplex PCR demonstrates specificity for brewery Pectinatus isolates, and does not amplify DNA from various Megasphaera, Selenomonas, or Zymophilus species. Additionally, the OMP gene contains variable regions that allow for differentiation of Pectinatus strains. As such, by sequencing individual PCR amplicons, it is possible to determine if the Pectinatus contaminant is new or recurring. Furthermore, by targeting the OMP gene, strain-specific Pectinatus detection methods can be designed to enable rapid monitoring and quality control of recurring Pectinatus contaminants within a given brewery.

Is the role of Chlamydia trachomatis underestimated in reactive arthritis patients in India

Background: Although Chlamydia trachomatis-induced Reactive Arthritis (ReA) constitutes a significant disease burden in various countries, however it remains largely unrecognized and probably underestimated in India. Due to availability of multiple serovars, biphasic lifecycle and asymptomatic infection, diagnosis of this pathogen is difficult, thereby leading to chronic, persistent chlamydial infection in the synovium. The aim of the present prospective study was to find the presence of intra-articular C. trachomatis infection by nucleic acid amplification tests and secretory IgA determination in patients with ReA and in forme fruste of ReA, viz.: Undifferentiated Spondyloarthropathy (uSpA) attending a major tertiary Army hospital inNew Delhi (India). Methods & Materials: Synovial fluid was aspirated from the knee joints of age and sex-matched 30 arthritic patients, viz.: ReA/uSpA (n=15) and Osteoarthritis controls (OA) (n =15) following the European Spondyloarthropathy Study Group criteria for ReA while clinical and radiological diagnosis was done for OA patients. The clinical details were recorded in all patients. Both C. trachomatis-specific surface Major Outer Membrane Protein (MOMP) and endogenous plasmid genes were targeted using semi-nested Polymerase Chain Reaction (snPCR) and nested PCR (nPCR), while anti-C. trachomatis IgA antibodies were estimated by commercial enzyme-linked immunosorbent assay kit (Savyon Diagnostics, Israel). Statistical evaluation of data was done by using trial version of Graph Prism Pad software (version 5.0; GraphPad Software, Inc., San Diego, California, USA). Results: Overall, 33.3% (5/15) ReA/uSpA patients were found to be C. trachomatis-positive for either MOMP/plasmid gene in the joint fluid. 20% (3/15) were positive for plasmid gene while MOMPgenewas present in 13.3% (2/15) patients in the study group (ReA/uSpA). Intra-articular anti-C. trachomatis IgA antibodies were detected in 26.6% (4/15) patients in the synovial fluid. Significant correlation (‘p’ value <0.05) was found between PCR and serologic findings. None of OA controls was found positive for either chlamydial antibody/MOMP gene/plasmid gene. Conclusion: The observations in the present study indicate that intra-articular C. trachomatis infection in ReA/uSpA patients is both underestimated as well as underdiagnosed inIndia. It also suggests the usefulness of serology for better clinical management of such patients.

Development of a rapid detection system for opportunistic pathogenic Cronobacter spp. in powdered milk products

Certain species of the genus Cronobacter are considered opportunistic pathogens, but their detection in milk products according to ISO/TS 22964 may take up to six days. The aim of this study was to develop a fast and sensitive PCR-based detection system for these species including enrichment, DNA-isolation and detection by real-time PCR, using the outer membrane protein gene ompA as a target. The assay was successfully validated using type strains of the genus Cronobacter, as well as 18 strains of closely related genera as controls. A total of 40 Cronobacter spp. food isolates yielded positive results, while the food matrix itself did not influence the PCR reaction. An equal detection limit as achieved with the ISO/TS 22964 method was established in this study, when 0.01 CFU Cronobacter sakazakii DSM 4485T per gram powdered infant formula were successfully detected after 28 days of storage at ambient temperature. In comparison to the ISO/TS 22964 method, the method described here has an equal detection limit, but offers a specific detection at the genus level in an analysis time of 24 h.

Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups

To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V. cholerae) serogroups non-O1 and non-O139. The outer membrane protein gene (ompW) specific for V. cholerae, as well as O antigen rfb genes specific for both O1 and O139, were used for the design of the PCR primers. Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139. Specific rfb genes and ompW were developed to evaluate their specificity, limit of detection, reproducibility and consistency. We established multiple PCR and real-time SYBR green PCR methods. According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR), both methods could specifically detect the non-O1, non-O139 V. cholerae, and to differentiate them from O1,O139 V. cholerae, other five Vibrios and 3 intestinal bacteria. The detection limits were 7 × 10(4) cfu/ml (multiple PCR) and 7 × 10² cfu/ml(real-time SYBR green PCR), with statistically significant difference seen (P < 0.05). For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92% while the internal coefficient variation ranging from 0.27% to 1.41%. 370 strains of non-O1, non-O139 V. cholerae, were detected, with both consistency rates as 100% . Both multiple PCR and real-time SYBR green PCR could detect non-O1, non-O139 V. cholerae, rapidly, specifically, and reproducibly, that could all be used for the detection and identification of non-O1, non-O139 under different conditions.

Molecular Structural Analysis of Major Outer Membrane Protein (MOMP) Gene Clusters in Campylobacter Lari

Southern hybridisation shows that urease-negative (UN) Campylobacter lari JCM2530T carries two putative major outer membrane protein (MOMP) genes. Sequences of approximately 2.1 kbp, encoding non-coding (NC) regions, with possible open reading frames (ORFs) for MOMP (porAl or porA2) of approximately 1.2 kbp, NC regions and partial and putative Cla_0435 or Cla_1109 ORFs were identified in all five UN C. lari isolates examined, following polymerase chain reaction (PCR) cloning and sequencing. Each putative MOMP structural gene carried start and stop codons and ribosome binding sites of 1236–1278 bp in length. The putative σ70 transcriptional promoter and the hypothetical ρ-independent transcription terminator structures were also seen. Using Northern hybridisation, there was in vivo monocistronic MOMP gene transcription. In addition, in a Japanese urease-positive thermophilic Campylobacter (UPTC) CF89–12 strain, the porA1 gene locus, including an extra gene (approximately 2000 bp in length) was identified. The extra gene may occur within the porA1 gene locus in the eight UPTC isolates of the 23 C. lari isolates examined. Thus, a genetic heterogeneity occurred within the porA1 gene locus from some of the C. lari organisms including the UPTC CF89–12.