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PubMed1987

Lectins for the identification of ocular bacterial pathogens.

In a preliminary in vitro investigation, fluorescein-conjugated lectins were used in the identification of bacteria commonly involved in ocular infections. Clinical isolates of Staphylococcus aureus, Staph. epidermidis, Streptococcus pneumoniae, Strep. pyogenes, Pseudomonas aeruginosa, Hemophilus influenzae, and Proteus mirabilis were incubated with each of eleven lectins using a slide technique. Bacterial fluorescence was readily observed with a fluorescence microscope. All clinical isolates bound wheat germ agglutinin. The Gram-positive isolates bound Concanavalin A, while the Gram-negative isolates did not, with rare exceptions. Streptococcal species isolates reacted with Dolichos biflorus agglutinin, while staphylococcal species isolates did not. Lectins may be useful in furthering the initial identification of causative organisms in bacterial ocular infections.

Bacterial Infections↗
PubMed2000

Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region.

In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.

Animals↗
PubMed1997

Lancefield grouping and smell of caramel for presumptive identification and assessment of pathogenicity in the Streptococcus milleri group.

AIM: To evaluate Lancefield grouping and caramel smell for presumptive identification of the Streptococcus milleri group, and to find whether Lancefield group, species, or protein profile correlated with virulence or infection site. METHODS: Prospective studies were made of 100 consecutive streptococcal isolates in blood cultures or pus from 100 patients in whom the severity of infection was categorised as serious, moderate, or not significant. The usefulness of Lancefield group and the caramel smell for presumptive identification was examined, and the relation of the S milleri species, Lancefield group, and SDS-PAGE protein analysis to severity of infection and infection site was investigated. Lower respiratory tract and genital tract specimens, strict anaerobes, group D streptococci, and strains identified as Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus agalactiae were excluded. RESULTS: Most streptococci occurring in pure or significant growth density were S milleri group (87/100; 87%, 95% confidence interval 0.81-0.93). Of these, 89.7% (78/87; 0.84-0.96) were associated with infection. Lancefield group F antigen predominated (41/87; 47.1%, 0.38-0.56). Lancefield group F alone or accompanied by the caramel smell had a specificity of 100%, but a sensitivity of only 47.3% for group F alone, and 19.5% for group F accompanied by the caramel smell. There was no significant association between species, Lancefield group, and severity of infection, site of infection, or pathogenicity. SDS-PAGE analysis failed to discriminate between strains. CONCLUSIONS: Neither species nor Lancefield antigen was related to the site of infection. The presence of Lancefield group F antigen alone or accompanied by a caramel smell was a useful indicator for the S milleri group when present, but was too insensitive to use as a screening test. Most streptococci occurring in pure culture or in significant growth density were of clinical importance. Such organisms should be identified to species level to detect the S milleri group.

Bacterial Proteins↗
PubMed1989

[Colicin genotype characteristics of pathogenic Escherichia circulating in the environment].

Colicin identification of pathogenic Escherichia, isolated from the environmental objects, showed that determination of colicinogenicity signs and sensitivity to standard colicins in pathogenic E. to a more considerable degree depended on strain sulfur-group membership than on the amanating objects. The analysis of colicin genotype characteristics of pathogenic E., circulating in environment, provided much information and could be used in identifying a source of infection and ways of escherichiosis pathogen transmission.

Bacteriocin Plasmids↗
PubMed2005

A fractal analysis of pathogen detection by biosensors.

A fractal analysis is presented for the detection of pathogens such as Franscisela tularensis, Yersinia pestis (the bacterium that causes plague), Bacillus anthracis, Venezuelan equine encephalitis (VEE) virus, Vavcinia virus, and Escherichia coli using a cellular analysis and notification of antigens risks and yields (CANARY) biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D. Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T. Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215. Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. In general, the binding and dissociation rate coefficients may be adequately described by either a single- or a dual-fractal analysis. An attempt is made to relate the binding rate coefficient to the degree of heterogeneity (fractal dimension value) present on the biosensor surface. Binding and dissociation rate coefficient values obtained are presented. Due to the dilute nature of the analyte(s) present, in some cases, a triple-fractal analysis is required to adequately describe the binding kinetics. It should be noted, and this is not entirely unexpected, that there is a lot of variation in the original experimental data when dilute concentrations of the analyte were analyzed by the CANARY biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. The data analyzed in this manuscript appears smoother since only discrete points at different time intervals were analyzed. The kinetics aspects along with the affinity values presented are of interest and should along with the rate coefficients presented for the binding and the dissociation phase be of significant interest in help designing better biosensors for an application area that is bound to gain increasing importance in the future.

B-Lymphocytes↗
PubMed2000

Rapid identification of the bacterial pathogens responsible for urinary tract infections using direct injection CE.

The use of high-performance capillary electrokinetic techniques for the separation, identification, and quantitation of intact microbes represents a new frontier for separation science. In this work, it is demonstrated that pathogens most responsible for urinary tract infections can be distinguished from one another after direct injection of untreated urine. High efficiencies (often exceeding 1000000 plates/m) and short analysis times (< 10 min) are characteristics of this approach. The concentration of the urine matrix appears to be able to cause a small, but definite, change in the electroosmotic flow velocity. This high-efficiency separation-based approach could prove to be invaluable for the diagnosis and tracking of certain diseases. It also could form the basis for a variety of rapid microbial assays.

Bacteriological Techniques↗
PubMed2000

Signature-tagged mutagenesis in the identification of virulence genes in pathogens.

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.

Candida↗
PubMed2002

Evaluation of the DipStreak, a new device with an original streaking mechanism for detection, counting, and presumptive identification of urinary tract pathogens.

DipStreak is a new urine culture device with two types of agar attached back-to-back on a plastic paddle. It combines dip-slide technology and an original streaking inoculation mechanism, allowing for bacterial counting and colony isolation. The performance of the DipStreak device with two different medium formulations, CHROMagar and MacConkey media in study A and UriSelect 3 and MacConkey media in study B, was evaluated and compared to that of the reference streak method by using plates of cystine-lactose-electrolyte-deficient (CLED) agar, tryptic soy agar with 5% sheep blood, and UriSelect 3 medium. In study A, 2,000 urine specimens were processed and 511 cultures were found positive. The DipStreak device and the UriSelect 3 and CLED medium plates gave the same detection rate, 99.7%. For the direct identification of Escherichia coli, Proteus mirabilis, and Enterococcus sp. isolates, the DipStreak device and the UriSelect 3 medium plate showed overall sensitivities of 97 and 93.4%, respectively. In study B, 3,000 urine specimens were processed and 714 cultures were found positive. The DipStreak device and the UriSelect 3 and CLED medium plates gave detection rates of 99.4, 99.9, and 99.2%, respectively. For the direct identification of E. coli, P. mirabilis, and Enterococcus sp. isolates, the DipStreak device and the UriSelect 3 medium plate showed overall sensitivities of 88 and 94.4%, respectively. In conclusion, the DipStreak device with both medium formulations represents an attractive and excellent screening method for the reliable detection, counting, and presumptive identification of urinary tract pathogens. It enables bedside urine inoculation and provides a valid means of transporting the sample back to the laboratory, decreasing drastically the rate of false-positive results due to bacterial overgrowth and reducing associated costs.

Bacteriological Techniques↗
PubMed1995

Evaluation of a new chromogenic agar medium for the identification of urinary tract pathogens.

This study evaluated the performance of CPS ID2 (bioMérieux) compared to that of conventionally used selective agar for the identification of bacteria responsible for urinary tract infections. This medium detects bacterial enzymes using chromogenic substrates. Two hundred and nineteen samples of urine were tested in order to evaluate new CPS ID2 in comparison to blood agar and MacConkey agar. According to our results, the CPS ID2 agar is an easy, rapid and sensitive method for the screening of colonies suspected of being Escherichia coli, reducing the number of biochemical tests needed. Also, enterococci and Proteae can be easily detected. Other micro-organisms require further identification. The greatest value of this medium is the accurate identification of polymicrobial cultures.

Agar↗
PubMed1977

[Pseudomonas putida: identification, antibiotic sensitivity and pathogenicity (author's transl)].

This work studies 51 strains of Pseudomonas putida, isolated from clinical specimens (17) and hospital environment (34). Identification is performed by study of 41 physiologica and biochemical characters and 78 nutritional characters. According to the two biotypes A and B, described by Stanier, Palleroni and Doudoroff, these 51 strains can be grouped as follows: 48 have typical characters of biotype A, widely predominant, 3 can be distinguished from biotype A only by their auxanogram and included in biotype B. Antibiogram pattern of P. putida shows two salient features: resistant to carbenicillin and sensitivity to kanamycin. Among 17 human isolates, only 4 have likely pathogenic significance. By intraperitoneal challenge in mice, one half of strains is avirulent, other strains have a very low virulence (LD50: from 2,2 to 5 X 10(8) viable cells). There is no relationship between experimental virulence and bacterial sources.

Animals↗
PubMed2002

Emerging foodborne pathogens.

The broad spectrum of foodborne infections has changed dramatically over time, as well-established pathogens have been controlled or eliminated, and new ones have emerged. The burden of foodborne disease remains substantial: one in four Americans is estimated to have a significant foodborne illness each year. The majority of these illnesses are not accounted for by known pathogens, so more must remain to be discovered. Among the known foodborne pathogens, those more recently identified predominate, suggesting that as more and more is learned about pathogens, they come under control. In addition to the emergence or recognition of new pathogens, other trends include global pandemics of some foodborne pathogens, the emergence of antimicrobial resistance, the identification of pathogens that are highly opportunistic, affecting only the most high-risk subpopulations, and the increasing identification of large and dispersed outbreaks. New pathogens can emerge because of changing ecology or changing technology that connects a potential pathogen with the food chain. They also can emerge de novo by transfer of mobile virulence factors, often through bacteriophage. Though this is rarely observed, it can be reconstructed. Better understanding of the ecology and dynamics of phage transmission among bacteria will help us to understand the appearance of new pathogens in the future. One may look for emerging foodborne pathogens among the silent zoonoses, and among the severe infections affecting the immunocompromised humans. We should expect the unexpected. In the past, separating human sewage and animal manure from human food and water supplies was critical to improving public health. Now, our health depends increasingly on the safety of the feed and water supplies for the animals themselves. The successes of the 20th century and the new challenges we face mean that public health vigilance, careful investigation of new problems, responsible attention to food safety from farm to table, and partnerships to bring about new foodborne disease control measures will be needed for the foreseeable future.

Animals↗
PubMed2000

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis.

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.

AIDS-Related Opportunistic Infections↗
PubMed2003

Species identification of medically important fungi by use of real-time LightCycler PCR.

Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients. Rapid identification of pathogenic fungi to species level is critical for disease treatment. A real-time LightCycler assay aiming at rapid detection and species identification of pathogenic fungi from clinical isolates was developed. Template DNAs of different species were amplified and detected in real time by employing SYBR Green fluorescent dye. The target sequences for species-level detection were located between the 18S and 28S rDNA. Seven fungal species encountered frequently in the clinical setting, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii and Cryptococcus neoformans, could be discriminated by species-specific primers and confirmed by melting-curve analyses. The range of linearity was from 1 ng to 1 pg (microl(-1) water) and the sensitivity was 1 pg fungal DNA microl(-1). Identification by this real-time PCR method matched biochemical identification for all 58 clinical strains. Therefore, the method is simple, rapid and sensitive enough for detection and identification of several fungal species.

Fungi↗
PubMed2000

Use of blood cultures in critically ill patients.

Infection, bacteremia, and sepsis are frequent complications in critically ill patients. Ideally, the infectious agent is readily identified to facilitate timely treatment to promote the patient's recovery. Use of blood cultures is one method of identifying the pathogen. Fever is the primary indicator for obtaining blood samples for culture, but other indicators may be considered, depending on the patient's medical history and condition. Use of appropriate techniques when collecting blood samples for culture will decrease contamination and improve the likelihood of identification of the infectious agent. One new technique being tested for the identification of pathogens that cause bacteremia involves genetic technology and the polymerase chain reaction. The polymerase chain reaction is used to identify the DNA of bacteria that are present in the blood. Blood cultures may not always result in identification of the pathogen because the organism may not grow once placed in culture medium. This new method that uses the polymerase chain reaction may be more sensitive than blood cultures because it requires only DNA from bacteria. Although early studies have not been conclusive in terms of the benefits of this new technology, additional research will improve methods for identification of pathogens in critically ill patients.

Blood Specimen Collection↗
PubMed2004

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring.

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.

Algorithms↗
PubMed1998

Presumptive identification of an emerging yeast pathogen: Candida dubliniensis (sp. nov.) reduces 2,3,5-triphenyltetrazolium chloride.

Developments in medical intervention and the increasing population of patients with immunodeficiencies and transient or long-term immunosuppression have increased the list of yeast species that can cause disease. Candida dubliniensis is a novel species with close genetic relatedness to C. albicans. The two species share many common physiological and biochemical properties thus making their distinction cumbrous. A rapid and inexpensive way to presumptively differentiate between the two species, having previously performed a germ tube test, is the ability of C. dubliniensis to reduce the tetrazolium salt and it is reported for the first time. Microbiological information about new and emerging yeast pathogens, including rapid means for their identification, equips medical microbiologists with the means to identify and physicians to treat effectively infections attributed to unusual yeasts.

Bacteriological Techniques↗
PubMed2003

Evaluation of a new chromogenic medium, Uriselect 4, for the isolation and identification of urinary tract pathogens.

AIMS: To compare the performance of a new chromogenic medium, Uriselect 4, with cystine lactose electrolyte deficient (CLED) agar and an established chromogenic agar, CPS ID 2 medium, for detection of urinary tract pathogens. METHODS: Using a semiquantitative culture method, 777 samples were inoculated on to the three test media in duplicate. All bacterial strains that yielded a potentially significant growth were observed for colony colour and identified using standard methods. RESULTS: Of the 777 samples tested, 589 urine samples yielded potentially significant growth of at least one strain. A total of 811 strains were isolated on at least one of the three media. A total of 168 urine samples yielded a mixture of at least two strains. Uriselect 4 medium showed the best sensitivity of the three media and only failed to recover 14 strains (1.7%). CPS ID 2 medium failed to recover 22 strains (2.7%). CLED medium showed the worst recovery and failed to recover 74 strains (9.1%). Both chromogenic media allowed for identification of Escherichia coli with a high degree of specificity (98% for Uriselect 4, 99.7% for CPS ID 2). Inclusion of a spot indole test increased the specificity of both chromogenic media to 100% for E coli. CONCLUSIONS: Uriselect 4 and CPS ID 2 were superior to CLED medium for the isolation of urinary tract pathogens mainly because of their ability to discriminate mixed cultures. Both chromogenic media were also useful for the preliminary identification of the most common urinary tract pathogens.

Agar↗