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PubMed2005

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis.

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.

Adolescent↗
PubMed2024

An integrated in-silico approach for drug target identification in human pathogen Shigella dysenteriae.

Shigella dysenteriae, is a Gram-negative bacterium that emerged as the second most significant cause of bacillary dysentery. Antibiotic treatment is vital in lowering Shigella infection rates, yet the growing global resistance to broad-spectrum antibiotics poses a significant challenge. The persistent multidrug resistance of S. dysenteriae complicates its management and control. Hence, there is an urgent requirement to discover novel therapeutic targets and potent medications to prevent and treat this disease. Therefore, the integration of bioinformatics methods such as subtractive and comparative analysis provides a pathway to compute the pan-genome of S. dysenteriae. In our study, we analysed a dataset comprising 27 whole genomes. The S. dysenteriae strain SD197 was used as the reference for determining the core genome. Initially, our focus was directed towards the identification of the proteome of the core genome. Moreover, several filters were applied to the core genome, including assessments for non-host homology, protein essentiality, and virulence, in order to prioritize potential drug targets. Among these targets were Integration host factor subunit alpha and Tyrosine recombinase XerC. Furthermore, four drug-like compounds showing potential inhibitory effects against both target proteins were identified. Subsequently, molecular docking analysis was conducted involving these targets and the compounds. This initial study provides the list of novel targets against S. dysenteriae. Conclusively, future in vitro investigations could validate our in-silico findings and uncover potential therapeutic drugs for combating bacillary dysentery infection.

Shigella dysenteriae↗
PubMed1999

Identification of bacterial pathogens in patients with endophthalmitis by 16S ribosomal DNA typing.

PURPOSE: To determine whether sequence analysis of 16S ribosomal DNA (rDNA) can be used to detect bacterial pathogens in patients with postoperative endophthalmitis. METHODS: In 10 eyes of 10 patients, vitreous specimens were collected for culture and rDNA typing. Variable segments of each ribosomal DNA specimen were amplified by polymerase chain reaction (PCR), sequenced, and aligned by BLAST, a computer alignment program, against sequences in GenBank at the National Institutes of Health. RESULTS: Specimens were available from five eyes with bacterial endophthalmitis diagnosed by Gram stain or culture. Amplified 16s rDNA sequences from the eyes of three patients were identical to microbiologic results. Polymerase chain reaction results were negative in two cases in which unusual organisms were detected. All five control specimens from patients with nonbacterial endophthalmitis or uveitis were PCR negative. Approximately 48 to 72 hours are required under ideal conditions for final species identification with this ribosomal typing technique. CONCLUSIONS: 16S rDNA typing shows potential as a relatively rapid technique for identifying bacteria in vitreous samples.

Bacteria↗
PubMed2003

Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures.

Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.

Bacteria↗
PubMed2004

Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes.

We developed real-time polymerase chain reaction (PCR) assays for rapid detection of the most common and clinically relevant bacteria in positive blood culture bottles, including Staphylococcus spp., S. epidermidis, S. aureus, Enterococcus spp. (including differentiation of E. faecalis and E. faecium), Streptococcus spp., Streptococcus agalactiae, Enterobacteriaceae, E. coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp., Bacteroides spp., Haemophilus influenzae, and Neisseria meningitidis. A total of 507 positive blood cultures were investigated according to a specific PCR algorithm based on the microscopic result of the blood culture, and the PCR results were compared to the culture results. Apart from-cross reactions between E. coli and Chryseomonas luteola and Enterococcus faecium and E. durans, the PCR assay correctly identified all bacteria in the blood cultures and did not show any false-positive results. Regarding blood cultures positive with a single species of bacteria (n = 474), 98.3% of all bacteria were correctly detected by the PCR algorithm within a few hours. However, in mixed infections, the sensitivity was lower. The PCR algorithm is well suited for rapid identification of the most common bacteria in positive blood cultures.

Algorithms↗
PubMed2001

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections.

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.

DNA, Fungal↗
PubMed2005

[Identification and phylogenetic study of pathogenic bacteria causing ulcer disease of cultured Turbot (Scophthalmus maximus)].

An outbreak of ulcer disease of the cultured turbot Scophthalmus maximus L. occurred in an indoor farm in Haiyang, Shandong Province in the sweltering August of 2004 and caused significant mortality. At the beginning, infected turbots displayed sluggish swimming and anorexia. Several days later, the turbot eyes swelled, the fins and tail turned red, and the back gradually ulcerated. Dissection of the moribund fish showed that the ulcerative gills paled, the liver becamed bloodshot, the kidney and gallbladder swelled, the intestinal wall became filmy and bloodshot. The time course from appearance of disease signs to death lasted about a week. A dominant strain of bacteria, which was Gram-negative and short rod with single polar flagellum under electron microscope, was isolated from the diseased turbot and designated as H1. In artificial infection test, all fish of the experimental groups died in a week after intramuscularly injected with bacterial suspension, while the control group showed no signs in 10 d post-challenge. The moribund experimental fish had similar gross signs as the natural infected fish. The bacteria re-isolated from the challenged fish also had the same characteristics as H1, which proved that the isolate H1 was the pathogenic bacteria that triggered this ulcer disease. Different methods were used to identify the pathogenic bacteria. The identification result by API 20NE and API 20E system indicated that H1 was Aeromonas hydrophila, with 99% reliability and 61.5% respectively. While traditional biochemical identification revealed that H1 exhibited relatedness to Vibrio harveyi. In order to confirm the different result, a 1424 bp sequence of H1's 16S rDNA was amplified and compared with other Vibrio spp. in GenBank, homology analysis and phylogenetic study showed that H1 has the highest similarity to V. harveyi, with 99% identity. According to morphological features, physiological and biological characteristics and 16S rDNA homology comparison of the bacteria, the pathogenic bacteria were V. harveyi. Drugs sensitivity test showed that the pathogenic bacteria were highly sensitive to nitrofurantoinum and ceftriaxone sodium etc. This is the first report that V. harveyi was found as the pathogenic bacteria of cultured turbot in China. The research suggests that V. harveyi should be regarded as an important pathogen of turbot and can causes ulcer disease under conditions of high temperatures. Therefore, it is necessary to prevent against ulcer disease in culture turbot in summer.

Animals↗
PubMed2000

Proteomics in medical microbiology.

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

Bacterial Proteins↗
PubMed1989

An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.

We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1990

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria.

Mutants of a tomato strain of Xanthomonas campestris pv. vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kb EcoRI fragment of p6AD4.

Cloning, Molecular↗
PubMed1995

An approach to the identification of the pathogens of bacterial meningitis by the polymerase chain reaction.

A combination of universal and species-specific primers was used to detect and differentiate by nested polymerase chain reaction (PCR) the four species most commonly causing bacterial meningitis. Primers recognising conserved sequences in the 16S and 23S ribosomal RNA genes were employed to amplify the 16S-23S spacer region from Neisseria meningitidis, Haemophilus influenzae (type b), Streptococcus pneumoniae, and Streptococcus agalactiae (group B streptococcus). The sequence of the most abundant spacer product was determined in each case and used to deduce species-specific primers. A nested PCR using universal primers in the first round and a species-specific primer in the second were able to detect and distinguish between the four common pathogens, in the presence of human DNA. Streptococcus pneumoniae DNA was detected in the cerebrospinal fluid of a meningitis patient with negative culture and Gram-stain results.

Base Sequence↗
PubMed2005

Identification of pathogen-responsive regions in the promoter of a pepper lipid transfer protein gene (CALTPI) and the enhanced resistance of the CALTPI transgenic Arabidopsis against pathogen and environmental stresses.

The 5' flanking region of the CALTPI gene, which encodes a basic lipid transfer protein, was isolated and characterized from the genomic DNA of Capsicum annuum. Four different regions of the promoter sequence of the CALTPI gene were fused to the beta-glucuronidase (GUS) coding region. In an Agrobacterium-mediated transient expression assay, the transcriptional activations of the promoter deletions were examined in tobacco leaves after infection with Pseudomonas syringae pv. tabaci, and treatment with ethylene and salicylic acid. The -808 bp region of the CALTPI gene promoter sequence exhibited full promoter activity. The W-box and ERE-box elements, which are essential for induction by all signals, were localized in the region between -555 bp and -391 bp upstream of the translation initiation site. A CALTPI transgene was then introduced under the control of the 35S promoter into the Arabidopsis ecotype Col-0. Transgenic Arabidopsis lines expressing the CALTPI gene developed rapidly compared to the wild-type plants, indicating that CALTPI may be involved in plant development. Overexpression of the CALTPI gene enhanced the resistance against infection by P. syringae pv. tomato and Botrytis cinerea. The transgenic plants expressing the CALTPI gene also showed high levels of tolerance to NaCl and drought stresses at various vegetative growth stages. No transcription of the PR-1, PR-2, PR-5, thionin, and RD29A genes was observed in untreated leaf tissues of the transgenic plants. The enhanced resistance to pathogen and environmental stresses in transgenic Arabidopsis correlated with the enhanced expression of the CALTPI gene.

Adaptation, Physiological↗
PubMed1991

Identification of foodborne pathogens by nucleic acid hybridization.

Nucleic acid hybridization methods have been developed and used to identify microorganisms in foods. Tests performed on mixed cultures save the time required to establish pure cultures. Enterotoxigenic or invasive strains of foodborne bacterial pathogens are detected with probes that identify genes responsible for virulence. Hybridization tests signal the presence or absence of a particular strain or an entire genus and are especially well suited for screening foods for specific pathogens. With the colony hybridization assay format, foodborne bacteria harboring a specific gene can be enumerated. However, hybridization tests require the presence of 10(5) to 10(6) cells to yield a positive result, thereby limiting sensitivity and necessitating a time-consuming growth step. In vitro DNA amplification techniques increase the amount of DNA segments 10(5)-10(6)-fold in 2 to 3 h, thus enhancing test sensitivity.

Bacteria↗
PubMed1986

Identification of enteric pathogens in the small and large intestine of children with diarrhea.

Enteric pathogens were identified in children with diarrhea from duodenal specimens obtained with a string capsule and from fecal specimens. Rotavirus was identified in stools of 43 of 100 children, and was recovered from the small intestine from nine (21%) children who were excreting this virus. Shigella was isolated from stools from 22, Salmonella from 17, enterotoxigenic Escherichia coli from eight, and Aeromonas hydrophila from one of 100 children with diarrhea. In contrast to rotavirus, Salmonella, Shigella, enterotoxigenic Escherichia coli, and A. hydrophila were not isolated from the small intestine. Nonenterotoxigenic Aeromonas species were recovered from the small intestine, but not the stool of five children. These children were also infected with Shigella or with rotavirus; this suggests that Aeromonas was not the cause of their diarrhea. None of 51 Escherichia coli isolated with the string capsules, or 67 isolated from stool that agglutinated in commercial enteropathogenic Escherichia coli antisera were of classical enteropathogenic Escherichia coli serotypes. One hundred and five of these 118 Escherichia coli did not hybridize with a deoxyribonucleic acid probe for plasmid mediated factors conferring adherence to HeLa cells. Examination of specimens collected with a string capsule from children with diarrhea did not identify any more enteric pathogens than examining stools. Furthermore testing Escherichia coli for agglutination in commercial enteropathogenic Escherichia coli antisera did not identify Escherichia coli of enteropathogenic serotypes.

Aeromonas↗
PubMed2005

Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain.

The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease.

Abortion, Veterinary↗