Identification Of Pathogenic Bacteria Research Articles

An epidemiological survey of neonatal sepsis in a hospital in western Nigeria

The study examined occurrence of bacterial pathogens associated with neonatal sepsis in a hospital setting. A prospective cross-sectional study was carried out on neonates with sepsis who were admitted to the neonatal ward of Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria over a six-month period. One hundred samples for microbiological evaluation were collected from the neonates excluding those on prior antibiotic therapy. Microbiological examination and analysis of a variety of samples, processed according to standard procedures for isolation and identification of pathogenic bacteria and antibiotic sensitivity testing, was carried out on bacterial isolates. Among the 100 samples collected and processed, Staphylococcus aureus accounted for 28%, Klebsiellaand Pseudomonas species 13% each, Proteus species 10%, otherEnterobacteriaciae 9%, Neisseria gonorrhoea 8%, beta-haemolytic Streptococcus5 and 14% showed no bacterial growth. Antibiotic sensitivity patterns of the bacterial isolates showed that most were sensitive to oxfloxacin. However, a significant number of them showed resistance to commonly used antibiotics such as ampicillin and penicillin. Bacterial pathogens in neonates with sepsis vary from Gram-positive bacteria, mostly S. aureus to Gram-negative bacilli, mainlyKlebsiella and Pseudomonas species. Improvement in hygienic practices in both the wards and nurseries is required to reduce mortality rate in the hospital. Key words: Neonatal sepsis, samples, infants, bacteria, antibiotic, Nigeria.

Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods

Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae were found to be the most prevalent bacteremia-causing bacteria in a survey in a medical center. A PCR method for identification of these five most common pathogens in blood cultures was developed. A unique sequence was chosen for each pathogen and used for primer design. Sixty-one blood samples (from hospitalized patients) in which bacterial growth was detected were processed in parallel by conventional microbiological methods and by the PCR method. The results obtained by PCR were identical to those obtained by conventional methods in 93.4% of the cases. PCR failed to identify bacteria which were found conventionally in only 6.6% of the cases (mostly bacteria not included in the PCR cassette). Another group of eighty-eight blood samples from patients were processed immediately upon their arrival at the laboratory by taking aliquots for the PCR method. The blood sample bottles were processed in parallel by conventional methods. In 78.4% of the cases the results of both methods were identical. In 12.5% of the cases, PCR afforded identification of bacteria but conventional methods showed no bacteria in the sample. On the other hand, PCR afforded 9.1% negative results while conventional methods identified bacteria not included in the PCR cassette. It is concluded that the molecular method appears to be a specific and precise method for identifying pathogenic bacteria in blood samples.

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.

Laser-Based Identification of Pathogenic Bacteria

Bacteria are ubiquitous in our world. From our homes, to our work environment, to our own bodies, bacteria are the omnipresent although often unobserved companions to human life. Physicists are typically untroubled professionally by the presence of these bacteria, as their study usually falls safely outside the realm of our typical domain. In the last 10 years, however, several events have occurred that demand the attention of the general populace — including the ranks of physicists among them.

Evaluation of methods for isolation of DNA for PCR based identification of pathogenic bacteria from pure cultures and water samples

Water Science and Technology 58(5), 995–999 Publisher's note. We regret that the version of this article used in production did not incorporate a number of corrections and clarifications. The corrected versions of the text are as given below.

Percutaneous Discectomy—Continuous Irrigation and Drainage for Tuberculous Lumbar Spondylitis: A Report of Two Cases

Percutaneous curettage and continuous irrigation were performed for definitive diagnosis and treatment of tuberculous (TB) lumbar spondylitis. Under local anaesthesia, affected lumbar discs were curetted using a procedure of percutaneous nucleotomy, and in-tube and the out-tube were placed for continuous irrigation. The period of continuous irrigation was 12–16 days. Mycobacterium tuberculosis was demonstrated in case 1 by culture and PCR, whereas histology showed tuberculous lesion with caseous necrosis in both cases. Postoperative MRI showed markedly reduced abscesses after 3 months in both cases. The signal intensity in vertebral bodies was improved. In Case 2, CT observations showed remodeling over time in the vertebral body cavities. This method is advantageous in that although minimally invasive, it achieves identification of pathogenic bacteria and treatment simultaneously. This surgical procedure is expected to prove effective for both TB and pyogenic spondylitis.

Pathogenic bacteria identification by diagnostic DNA microarray

Pathogenic bacteria identification by diagnostic DNA microarray

Evaluation of methods for isolation of DNA for polymerase chain reaction (PCR)-based identification of pathogenic bacteria from pure cultures and water samples

Polymerase chain reaction (PCR) provides a reliable detection of pathogenic bacteria in water samples. However, this method can be adversely influenced by the purity of the DNA template. This is a particularly important obstacle when the bacterial DNA is directly extracted from water samples. In this study we compared the suitability of 8 different methods for isolation of bacterial DNA from pure cultures and 10 different methods for isolation of DNA from water samples. The quality of extracted DNA was assessed by PCR amplification of target sequences derived from uid (E. coli and Shigella sp.), tuf (Enterococcus sp.) and hns (Salmonella sp.). Results indicated that there are differences among the methods tested and only a few of them gave satisfactory results. The method based on alkaline lysis of bacterial suspension, which was developed in our laboratory, seemed to be efficient enough for the detection of bacteria from pure cultures. Detection of bacteria directly from water samples was more difficult. The modified method developed by Slusarenko was found as the best of the tested methods.

Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens

The aim of this study was to develop a sensitive and reliable method for the molecular identification of pathogenic bacteria. A multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay was developed and evaluated for the rapid identification of 24 systemic and respiratory bacterial pathogens in routine diagnosis. All species-specific probes designed for the RLB hybridised with amplified DNA only from the corresponding species. Sensitivity limits of the mPCR/RLB assay varied among the 24 target organisms from 0.05 pg to 0.5 ng of genomic DNA. The sensitivity of the assay was 2 x 10(2) CFU/mL for Streptococcus pneumoniae and 6 x 10(2) CFU/mL for Escherichia coli. The specificity of each probe was tested against 24 species. There were no cross-reactions among any of the 43 probes. The mPCR/RLB assay appeared to be a useful alternative tool for the molecular identification of common pathogens.

An Innovative, Flow-Assisted, Noncompetitive Chemiluminescent Immunoassay for the Detection of Pathogenic Bacteria,

Up to 80 million cases of food-borne illness occur yearly in the US. Approximately 30% of these cases are caused by bacteria and their related toxic products (1)(2). The bacteria species that are most frequently responsible for food contaminations are Salmonella typhimurium (meat, milk, and eggs), Escherichia coli O157:H7 (meat), Staphylococcus aureus (milk, cream, and meat), Clostridium perfringens (sausages, preserved food), Campylobacter jejuni (poultry, eggs), Vibrio parahaemolyticus (shellfish), Yersinia enterocolitica (meat, milk), and Listeria monocytogenes (dairy products) (3). Conventional microbiological methods for the identification of pathogenic bacteria are labor-intensive (several enrichment steps) and time-consuming (2 to 3 days to obtain results) (4). Currently, new rapid procedures based on immunological, DNA hybridization, or biosensing methods have been proposed (5)(6). However, many of these rapid tests are expensive, laborious, and all of them are based on cultural enrichment to enhance sensitivity and selectivity before analysis. Simpler, faster and more sensitive diagnostic techniques are required to improve food safety (food production, processing, storage and distribution) and to screen for potential bacterial infections in humans. Several immuno-based detection methods for infectious agents have been developed recently (7)(8). Such methods are of great diagnostic importance because they offer unique detection specificity and sensitivity, with relatively simple and low-cost assay formats. Most commercially available immunological methods for the detection of bacteria are based on conventional sandwich immunoassays, which involve the formation of an immunocomplex with an immobilized antibody, the target bacteria, a second labeled antibody, and the separation of free and bound antibody fractions by washing steps. Unfortunately, these separation steps reduce the assay production, so alternative approaches are welcome. In general, flow-assisted immunoassays offer advantages over conventional microtiter solid-phase formats, mainly related to easier automation and faster kinetics of the immunological reaction (9)(10), which takes place …

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.

Identification of Pathogenic Bacteria in Mixed Cultures by FTIR Spectroscopy

Routine identification of pathogenic microorganisms predominantly based on nutritional and biochemical tests is a time-consuming process. In this study, a method based on FTIR spectroscopy was developed to detect and identify the presence of five possible pathogenic bacteria in ten different microorganism mixtures with each cocktail containing up to three different species at a concentration of 109 CFU/mL. A mathematical approach based on factoring out the common features in the spectral fingerprints was developed and implemented in conjunction with discriminant analysis. FTIR spectra of the mixtures were directly obtained and analyzed using canonical variate analysis based on the discriminant model for the presence of particular species in the pathogenic bacteria mixtures. In nine out of ten mixtures, the predictions were 100% accurate; in one mixture, only one false negative was reported, with no false positives. Results suggest that FTIR spectroscopy combined with a suitable analytical procedure has excellent potential as a fast, powerful, and reliable alternative for identifying a specific foodborne pathogen in a complex and/or competing system.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

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.

Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients.

Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

CP003 Rapid identification of pathogenic bacteria involved in nosocomial infections using DNA-microarrays

The functions of miR-9 in some cancers are recently implicated in regulating proliferation, epithelial–mesenchymal transition (EMT), invasion and metastasis, apoptosis, and tumor angiogenesis, etc. miR-9 is commonly down-regulated in nasopharyngeal carcinoma (NPC), but the exact roles of miR-9 dysregulation in the pathogenesis of NPC remains unclear. Therefore, we firstly used miR-9-expressing CNE2 cells to determine the effects of miR-9 overexpression on global gene expression profile by microarray analysis. Microarray-based gene expression data unexpectedly demonstrated a significant number of up- or down-regulated immune- and inflammation-related genes, including many well-known interferon (IFN)-induced genes (e.g., IFI44L, PSMB8, IRF5, PSMB10, IFI27, PSB9_HUMAN, IFIT2, TRAIL, IFIT1, PSB8_HUMAN, IRF1, B2M and GBP1), major histocompatibility complex (MHC) class I molecules (e.g., HLA-B, HLA-C, HLA-F and HLA-H) and interleukin (IL)-related genes (e.g., IL20RB, GALT, IL7, IL1B, IL11, IL1F8, IL1A, IL6 and IL7R), which was confirmed by qRT-PCR. Moreover, the overexpression of miR-9 with the miRNA mimics significantly up- or down-regulated the expression of above-mentioned IFN-inducible genes, MHC class I molecules and IL-related genes; on the contrary, miR-9 inhibition by anti-miR-9 inhibitor in CNE2 and 5–8F cells correspondingly decreased or increased the aforementioned immune- and inflammation-related genes. Taken together, these findings demonstrate, for the first time, that miR-9 can modulate the expression of IFN-induced genes and MHC class I molecules in human cancer cells, suggesting a novel role of miR-9 in linking inflammation and cancer, which remains to be fully characterized.

Rapid ribosequencing--an effective diagnostic tool for detecting microbial infection.

Rapid and reliable identification of microorganisms is a prerequisite for the diagnosis and subsequent treatment of infectious diseases. The identification of pathogenic bacteria is traditionally based on their isolation from clinical samples and propagation on culture medium in the routine laboratory. However, despite clinical signs of infection, culture of the pathogenic agent often fails. This may be due to a low number of microorganisms, prior antibiotic treatment, nonculturable microorganisms or specific culture requirements for presently unknown pathogens. Amplification and sequencing of the entire prokaryotic 16S-rRNA is time consuming, labor intensive and expensive. We describe here a procedure for the identification of a wide range of known and unknown clinically relevant microorganisms by sequencing a small, but highly informative region of the prokaryotic 16S-rRNA gene. This rapid ribosequencing method was evaluated with various reference strains and with clinical samples including eye anterior chamber fluid, cerebrospinal fluid (CSF) and blood cultures. All sequences obtained from the reference strains corresponded to the sequences in databases. We correlated severe eye infection with the isolation of Pseudomonas putida, neurological disorder with Tropheryma whippelii and disseminated visceral abscesses in a child with Blastobacter denitrificans. We consider the rapid ribosequencing method as a promising new tool for the analysis of infectious agents in primarily sterile body fluids where conventional culturing of microorganisms fails.

Rapid identification of pathogenic bacteria by single-enzyme amplified fragment length polymorphism analysis

Despite major progress in their treatment and prevention, bacterial infections remain a significant cause of morbidity and mortality worldwide. In responding to a disease outbreak, rapid and accurate identification of the bacterial species involved is of paramount importance. Strain level discrimination is desirable to allow selection of treatment modalities, and in the case of a deliberate release, for identification of the source. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of subgroup I Bacilli, Yersinia, Staphylococci and Escherichia coli. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level, even within the highly monomorphic species Bacillus anthracis. SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method. These features make SE-AFLP suitable for use in either field or laboratory applications.

Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes.

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureus and the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.