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PubMed2005

Threat of infection: microbes of high pathogenic potential--strategies for detection, control and eradication.

Infectious diseases due to microbes of high pathogenic potential remain a constant and variable threat for human and animal health. The emergence of new diseases or the re-emergence of diseases that were previously under control complicates the situation to date. Infectious disease research, which has undergone a dramatic progress in understanding disease mechanisms such as host-pathogen interactions, is now focusing increasingly on new strategies for prevention and therapy. Significant progress has been achieved in the development of delivery systems for protective heterologous protein antigens and in veterinary vaccinology. A landmark of infectious diseases research is the chemical synthesis of genomes, a major new field of research referred to as "synthetic biology", that to date has resulted in the chemical synthesis of the poliovirus and of phage phiX174 genomes and their expression as infectious viruses. On the molecular level the evolution of pathogens and mechanisms of genome flexibility, which account for several pathogenic properties of infectious agents, have received increased attention. Bacterial toxins are an additional threat to human health and their interference with host cells and cellular functions is receiving more attention.

Animals↗

PubMed2003

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.

Air Microbiology↗

PubMed1995

Explanation for false-positive urine cultures obtained by bag technique.

OBJECTIVE: To test whether a urine bag technique, previously shown in circumcised male infants 1 month to 1 year of age to yield no false-positive cultures, would give similar results in newborns (females and circumcised and uncircumcised males). DESIGN: Prospective study in which periurethral and urine specimens were obtained from healthy newborns. After the periurethral specimen was obtained, the perineum was washed and a urine bag applied. The urine bag was removed immediately after voiding and the urine was cultured. SETTING: Normal newborn nursery and pediatric hospital. SUBJECTS: Ninety-eight healthy full-term newborns (49 female and 49 male) admitted to the normal nursery during a 4-month period. MAIN RESULTS: Isolation of a pathogen from the bag urine reflected periurethral flora. In 20 (95%) of the 21 urine specimens from which a pathogen was isolated, the same pathogen was detected on the periurethra. Sixteen of the 21 urine cultures were falsely positive (> 10(4) colony-forming units of pathogen per milliliter). In 50 (98%) of the 52 urine samples that yielded no growth, the periurethral culture was also negative. In the remaining 25 urine samples in which nonpathogens were detected, the periurethra yielded nonpathogens or no growth. Thus, if a pathogen was isolated from a bag urine sample, the same pathogen was detected on the periurethra 95% of the time. Conversely, if the bag urine sample was negative for a pathogen, the periurethral culture was negative 100% of the time. The presence of a pathogen on the periurethra was more common in female than male neonates (16 of 49 vs four of 49; P = .004), and none of the 14 circumcised male neonates had a pathogen detected on their periurethra or in their urine. CONCLUSION: This study explains the finding of false-positive cultures with the bag technique. Pathogens detected in bag urine samples reflected pathogens on the periurethra. Until a bag collection technique that avoids contamination by periurethral flora can be developed, urethral catheterization and suprapubic aspiration remain the methods of choice for obtaining a urine specimen in female and uncircumcised male neonates.

Bacteria↗

PubMedDate not supplied

Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.

A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum.

Animals↗

PubMed2002

Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms.

Rainbow trout fry syndrome and cold-water disease are serious diseases in farmed salmonid fish. In the present study, three methods were compared, for the detection of the causative pathogen, Flavobacterium psychrophilum in water. The methods included traditional agar plate cultivation on tryptone yeast extract salts (TYES) agar, immunofluorescence antibody technique (IFAT) and nested PCR. The three methods were subsequently used for the detection of F. psychrophilum from fish farm environments. The nested PCR was the most sensitive method used for a detection of F. psychrophilum. As low as 3 CFU estimated by agar plate cultivation or 41 cells estimated by IFAT of F. psychrophilum per ml of non-sterile well water were needed for a detection using the nested PCR method. The obtained detection limits for the agar plate cultivation and the IFAT was 32 CFU/ml and 410 cells/ml, respectively. Using IFAT and nested PCR F. psychrophilum was detected most frequently in water samples from fish farms, but the pathogen was isolated from only a few samples using agar plate cultivation. In the present study IFAT and nested PCR proved to be rapid, specific and sensitive methods compared to traditional agar plate cultivation for the detection of F. psychrophilum from environmental samples. It is suggested that IFAT and nested PCR provide effective tools for the examination of F. psychrophilum in the environment.

Agar↗

PubMed2026

Metax enables accurate cross-domain taxonomic profiling of metagenomes.

Taxonomic profiling is fundamental to microbiome research, yet achieving high species-level accuracy remains challenging for complex communities that span bacteria, viruses, eukaryotes, and archaea, and these limitations are exacerbated in low-biomass, host-dominated samples. We introduce Metax, a cross-domain taxonomic profiler that integrates coverage-based probabilistic modeling with an expectation-maximization framework to distinguish true microbial signals from artifacts. Across >600 samples from host-associated, environmental, wastewater, and low-biomass clinical settings, including benchmarks with limited reference representation, Metax improved profiling accuracy, achieving on average 55% higher F1 scores and 45% lower Bray-Curtis dissimilarity than other methods. Moreover, this broad evaluation demonstrated that Metax resolved bacterial and viral signatures of peri-implantitis in oral microbiomes and revealed signals suggestive of reagent-borne contaminants and reference misassemblies in plasma-cell-free DNA. By leveraging genome-wide coverage evidence, Metax enables robust cross-domain profiling across diverse sample types and sequencing depths, including settings where reference databases are highly incomplete.

abundance estimation↗

PubMed1993

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Antibodies, Monoclonal↗

PubMed1997

Simultaneous multianalyte nucleic acid detection for gastrointestinal bacterial pathogens using GeneSTAR technology.

The use of Gene-based simultaneous target amplification and recognition (GeneSTAR) technologies allows for the rapid detection of five different bacterial gastrointestinal pathogens from stool sample. The process involves DNA isolation, multianalyte polymerase chain reaction (PCR) amplification and single base mismatch detection in a microtiter plate format. The analysis is instrument compatible and can be completed in less than five hours.

Bacteria↗

PubMed2005

[Detection of sexually transmitted pathogens in patients with hematospermia].

BACKGROUND: Although the current literature attributes most cases of hematospermia to an infectious agent, identification of the specific pathogens involved has been limited. OBJECTIVE: To determine the prevalence of different pathogens, in patients presenting with hematospermia to our sexually transmitted disease clinic. PATIENTS AND METHODS: We retrospectively examined the laboratory results of 30 patients who presented to our clinic with hematospermia after other non-infectious pathologies had been excluded by a referring physician. Firstly, void urine samples as well as genitourinary and serum specimens were tested for Herpes simplex, Chlamydia trachomatis and Ureaplasma urealyticum. Standard bacterial cultures were also performed. RESULTS: In 12 of the 18 patients a possible pathogenic cause for the hematospermia was found. Evidence for Herpes simplex virus (Type 2) was found in 5 of the patients (16.6%), for Chlamydia trachomatis in 3 patients (10%) and for Ureaplasma urealyticum in 2 of the patients. Enterococcus fecalis was detected in 2 (6.6%) of the patients and in 1 patient Herpes simplex virus (Type 1) antigen and antibodies were detected. In all cases in which evidence of a pathogen was identified, the appropriate antimicrobial agent was administered, with recurrence in two patients only. CONCLUSIONS: Recent advances in microbiologic diagnostic techniques have facilitated the detection of pathogens in patients with hematospermia, thereby enhancing the efficacy of treatment.

Chlamydia trachomatis↗

PubMed1980

Identification of pathogenic neisseriae by genetic transformation.

The detection of pathogenic neisseriae by genetic transformation of a naturally occurring proline auxotroph of Neisseria gonorrhoeae strain F62 is described. Of 169 clinical isolates of N. gonorrhoeae, approximately 90% gave a positive transformation assay. Twelve clinical isolates of N. meningitidis and stock cultures of the various meningococcal serogroups also gave a positive result. However, the sensitivity of the assay was found to be approximately 1000-fold lower with N. meningitidis as test organism. Eleven other members of the family Neisseriaceae failed to transform the recipient organism. Although proline requirement did not appear to limit the value of the assay greatly, it probably was the main reason for negative results. The sensitivity of the assay and its ability to detect non-viable gonococci suggests that this method merits further investigation as a possible aid to diagnosis of gonococcal infection in special circumstances.

Bacteriological Techniques↗

PubMed2000

Evaluation of batch and semicontinuous application of high hydrostatic pressure on foodborne pathogens in salsa.

The effects of high hydrostatic pressure (HPP; 545 MPa) on strains of Escherichia coli O157:H7, Listeria monocytogenes, enterotoxigenic Staphylococcus aureus, and nonpathogenic microorganisms were studied in tomato-based salsa. Products were evaluated for the survival of the inoculated pathogens following HPP treatment and after storage at 4 degrees C and 21 to 23 degrees C for up to 2 months. Inoculated samples without HPP treatment, stored under the same conditions, were also evaluated to determine the effects of the acid environment of salsa on the survival of inoculated strains. None of the inoculated pathogens were detected in the HPP-treated samples for all treatments throughout the storage period. Inoculated pathogens were detected in the non-HPP-treated samples stored at 4 degrees C after 1 month, with L. monocytogenes showing the highest level of survivors. In the non-HPP-treated samples stored at 21 to 23 degrees C, E. coli and S. aureus were not detected after 1 week, but L. monocytogenes was detected in low levels. Studies with nonpathogenic strains of the pathogens were conducted at Oregon State University using HPP treatments in a semicontinuous production system. The nonpathogenic microorganisms (E. coli, Listeria innocua, Listeria welshimeri, and nonenterotoxigenic S. aureus) were inoculated together into a feeder tank containing 100 liters of salsa. Microbiological results of samples collected before HPP treatment and from the aseptic filler were similar to those obtained for the pathogenic strains. No survivors were detected in any of the HPP-treated samples.

Bacteria↗

PubMed1995

Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplification with the polymerase chain reaction.

The utility of the 16S ribosomal RNA-based polymerase chain reaction (PCR) for detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola was examined and compared with that of anaerobic culture. Primer pairs consisting of 20-27 nucleotides amplified 404- to 688-bp regions of 16S ribosomal RNA genes of these organisms. This method had a lower detection limit of 50 target cells in a background of 10(7) cells. Its specificity for B. forsythus, P. gingivalis, and T. denticola seemed high. The primers for A. actinomycetemcomitans, C. rectus, and P. intermedia cross-reacted with some closely related species but did not reveal amplification products in tests with more distantly related organisms. The primers for E. corrodens did not seem to cross-react with oral organisms. This PCR technique was sensitive, reproducible, and easy to perform. PCR-based amplification may prove valuable for the detection of some periodontal pathogens in crude subgingival specimens.

Adult↗

PubMed1993

Digoxigenin-labelled inv- and ail-probes for the detection and identification of pathogenic Yersinia enterocolitica in clinical specimens and naturally contaminated pig samples.

A non-radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non-pathogenic strains. The method is based on, respectively, the presence of the inv-locus in all Yersinia spp. and the presence of the ail-gene in pathogenic Y. enterocolitica only. Hybridization results with ail-probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI+ strains and only two TCI- strains were positive by hybridization with ail. Hybridization results with inv- or ail-probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv- or ail-probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv- and/or ail-probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv- and/or ail-probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.

Abattoirs↗

PubMed2006

[Retrospective evaluation of the results of the microbiological meat examination (MFU) from calves and cows].

The microbiological meat examination (MFU), consisting of a bacteriological analysis and a testing for antibiotic residues, is one of several additional analyses used for an edibility rating of carcasses made during meat inspection. Reasons for performing a microbiological meat examination and procedures in the laboratory are defined in the Swiss ordinance for meat examination (FUV). The aim of this study was to analyze the data of 313 microbiological meat examinations from calves and 2882 microbiological meat examinations from cows carried out at the Institute for Food Safety and Hygiene during a period of 8 years. Reasons for microbiological meat examinations as reported by the meat inspectors were mainly classified to the category of "inflammation and necroses" (FUV, Annex 4, Pt 1.2; calves: 73%, cows: 48%). As declarations of the age of the pathological-anatomical changes (that influences directly the probability of detection of pathogens) were generally missing, it is not surprising that the compliance between a particular pathological-anatomical change and a specific detection of pathogens is poor (calves: 19%, cows: 18% of all MFU). About 18% (calves) and 45% (cows) of the reasons for microbiological meat examinations did not correspond to one of the reasons mentioned in the ordinance for meat examination. However, according to the data set, some reasons require a microbiological meat examination due to an often-found specific detection of pathogens. Otherwise, a remarkable number of reasons mentioned were missing the link to bacteriological etiology. Moreover, 14% (calf) and 7% (cow) of microbiological meat examinations with the declaration "no pretreatment" as well as 15% (calf) and 11% (cow) of microbiological meat examinations without declaration showed a positive result in the testing for antibiotic residues.

Animals↗

PubMed2000

[Detection of toxin-producing pathogenic bacterial strains by polymerase chain reaction].

Polymerase chain reaction (PCR) was used for detection of pathogenic Clostridium botulinum, Clostridium perfringens, Clostridium difficile, and Escherichia coli. With this aim in view, primers to botulinic toxins types A, B, C1, D, E, F, and G, perfringens enterotoxin, difficile toxin, and types 1 and 2 Shigella-like toxins were chosen and synthesized. Optimal amplification conditions were selected for each pair of primers, with DNA and the respective agent as the reaction mixture matrices. PCR was highly specific and sensitive in all cases. Its sensitivity was 10-100 cells/sample. Among the tested C. botulinum, C. perfringens, C. difficile, and E. coli strains, specific amplification products of expected size were observed only in the strains containing the respective toxin genes. These findings recommend the use of these methods in clinical microbiology. Strains containing type 2 Shigella-like toxin gene were detected among E. coli strains isolated from patients with the hemolytic uremic syndrome, which for the first time indicates that the problem with E. coli epidemic strain O157 is valid for Russia. As a result of our studies, test systems for detection of types A, B, C, D, E, F, and G C. botulinum strains, C. perfringens and C. difficile, and E. coli O157 strains are now available.

Bacterial Toxins↗

PubMed2000

Identification of enteric pathogens in HIV-positive patients with diarrhoea in northern India.

Enteric pathogens associated with chronic diarrhoea in HIV-positive patients were studied. The study was conducted during January 1995-December 1998. Stool specimens from all diarrhoea patients (n = 26) were examined microscopically for ova and parasites using wet preparations and stained smears. Stool samples from diarrhoea patients were also cultured on appropriate media to isolate enteric bacterial pathogens. Of the 59 patients, 26 (44%) had prolonged diarrhoea for more than 4 weeks. Enteric pathogens were detected in 19 (73%) of the 26 patients: 17 patients harboured a single pathogen, and 2 patients had mixed pathogens. The detection rate of emerging parasites, including Isospora, Cryptosporidium, Blastocystis hominis, and Strongyloides stercoralis as a single agent, was significantly higher than conventional pathogens (50% vs 19.2%; p < 0.05). Only one patient harboured both conventional and emerging pathogens (Entamoeba histolytica and Cryptosporidium). Isospora belli was detected in 8 (31%) of the 26 diarrhoea patients: in 7 (27%) patients as a single agent and in one patient with S. stercoralis. Cryptosporidium was identified in 3 (11%) diarrhoea patients: in 2 (8%) patients as a single agent and in one patient with E. histolytica, followed by B. hominis in 2 (8%) patients. E. histolytica was most commonly isolated (3/26; 11.5%), followed by Giardia lamblia, enteropathogenic Escherichia coli, and Campylobacter jejuni (one patient each). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhoea in northern India. I. belli was the most frequent parasite isolated, followed by Cryptosporidium. Stools of all HIV-positive patients with diarrhoea should thoroughly be investigated to identify aetiologic agents for proper management.

AIDS-Related Opportunistic Infections↗

PubMed2004

Isolation of pathogenic Listeria monocytogenes and detection of antibodies against phosphatidylinositol-specific phospholipase C in buffaloes.

The isolation of pathogenic Listeria spp. in bacteriological samples, and anti-phosphatidylinositol-specific phospholipase C (anti-PIPLC) antibodies in sera of buffaloes were studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mice incoulation test. Listeria spp. and L. monocytogenes were isolated from 8.8 and 2.4%, and 4.8 and 1.6% of 125 each meat and blood samples, respectively. Out of the 125 samples each of feacal, nasal and vaginal swabs from buffaloes 8 and 4%, 13.6 and 2.4%, and 6.4 and 2.4% were positive for Listeria spp. and L. monocytogenes, respectively. L. ivanovii was confirmed from 0.8% vaginal sample. A total of 125 serum samples were tested by phosphatidylinositol-specific phospholipase C (PIPLC) based indirect ELISA of which 4.0% turned out to be seropositive.

Animals↗

PubMed1999

A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni.

: A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.

Journal Article↗