Identification Of Bacterial Strains Research Articles

Molecular diversity of peanut‐nodulating rhizobia in soils of Argentina

RSalpha sequencing is a valuable tool for identification of bacterial strains, and for evaluating the genetic structure of indigenous rhizobial populations. The purpose of this study was to evaluate, qualitatively, the presence or absence of RSalpha fragment in peanut-nodulating strains isolated from plants grown at four sites in central Argentina. RSalpha fragment was found in only three of 26 indigenous strains, and in one of three inoculant strains analyzed. In contrast to results from studies of other symbiotic nitrogen-fixing bacteria, such as soybean-nodulating strains, no correlation was found between generation time and presence of RSalpha sequence. Phylogenetic analysis of the 16S rRNA gene sequence grouped peanut-nodulating strains into two clusters, Bradyrhizobium japonicum vs. B. elkanii, and showed divergence among strains positive for RSalpha sequence. Our results confirm the genetic diversity previously reported for various peanut-nodulating rhizobial strains, and indicate that the RSalpha fragment is not applicable as a marker or tool for competition assays at the field or ecological level.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Isolation and Identifi cation of Bacterial Strains and Study of their Resistance to Heavy Metals and Antibiotics

The main objective of the work was to isolate and identify the bacterial strains and their resistance. A total of 9 strains could be isolated from the sample collected from the oxidation pond stage of waste water treatment of NIT Warangal. From all nine strains isolated, the maximum resistance was shown for chromium (490μg/ml for the strain1) and minimum for nickel (80μg/ml for strain6). The maximum resistance was shown for amoxilcillin (330 μg/ml for the strain8) and minimum for kanamycin (15μg/ml for the strain9). The metal resistant strains isolated from the waste water sample can be used for bioremediation process by construction of bioreactors where the strains can be immobilized for treating waste water effl uents from industrial or domestic sources.

Plant–rhizobacteria interactions alleviate abiotic stress conditions

Root-colonizing non-pathogenic bacteria can increase plant resistance to biotic and abiotic stress factors. Bacterial inoculates have been applied as biofertilizers and can increase the effectiveness of phytoremediation. Inoculating plants with non-pathogenic bacteria can provide 'bioprotection' against biotic stresses, and some root-colonizing bacteria increase tolerance against abiotic stresses such as drought, salinity and metal toxicity. Systematic identification of bacterial strains providing cross-protection against multiple stressors would be highly valuable for agricultural production in changing environmental conditions. For bacterial cross-protection to be an effective tool, a better understanding of the underlying morphological, physiological and molecular mechanisms of bacterially mediated stress tolerance, and the phenomenon of cross-protection is critical. Beneficial bacteria-mediated plant gene expression studies under non-stress conditions or during pathogenic rhizobacteria-plant interactions are plentiful, but only few molecular studies on beneficial interactions under abiotic stress situations have been reported. Thus, here we attempt an overview of current knowledge on physiological impacts and modes of action of bacterial mitigation of abiotic stress symptoms in plants. Where available, molecular data will be provided to support physiological or morphological observations. We indicate further research avenues to enable better use of cross-protection capacities of root-colonizing non-pathogenic bacteria in agricultural production systems affected by a changing climate.

Research in oral nursing frequency of patients undergoing orally mechanical ventilation

Objective To discuss oral nursing frequency of patients undergoing orally mechanical ventilation in order to ensure the oral cleanness of patients with the least oral nursing frequency and prevent the occurrence of ventilator associated pneumonia (VAP). Methods 36 patients undergoing orally me-chanical ventilation were randomly divided into groupA, B, C and D with 9 cases in each group, which all adopted 0.5% of pevidone iodine solution for oral nursing, time intervals of oral nursing were 4 hours, 6 hours, 8 hours and 12 hours. After the oral care and oral care before the next oropharyngeal samples for se-mi-quantitative bacterial colony counts, bacterial strain identification were obtained. The incidence rate of VAP was also compared and the results underwent χ2 test. Results Significant difference was seen in bacterial colony counts, especially during the time interval of 6 hours and 12 hours. No significant differ-ence was seen in incidence of VAP between the 4 groups. Conclusions The desirable frequency of oral nursing was every 6 hours on the premise of prevention of VAP. Key words: Orally mechanical ventilation; Patient; Frequency of oral care; 0.5% povidone iodine

Antibacterial activity of soaps against daily encountered bacteria

This study aims to check the antibacterial activity of various branded soaps against bacteria that are normally present in the environment. The proposed study includes selection of most common bacterial strains from the environment. Identification of bacterial strains was done by standard microbiological techniques, which include gram staining, biochemical testing and advanced identification by analytical profile index. Determination of minimal inhibitory concentration and minimum bactericidal activity of strains was performed by tube and microtitration method. Antibacterial soaps showed better MIC in comparison with beauty soaps. The most resistant bacterium to all the soaps is Klebsiella pneumoniae and Pseudomonas aeruginosa. It is obvious that antibacterial soaps have the antibacterial agents that can either kill or inhibit the bacterial cells. It might be possible that some bacterial strains become resistant which leads to their survival even at high concentrations of soaps.

Functional Nanoparticle-Based Proteomic Strategies for Characterization of Pathogenic Bacteria

Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) can be employed to rapidly characterize pathogenic bacteria, bacterial cultures are generally required to obtain sufficient quantities of the bacterial cells prior to MALDI MS analysis. If this time-consuming step could be eliminated, the length of time required for identification of bacterial strains would be greatly reduced. In this paper, we propose an effective means of rapidly identifying bacteria--one that does not require bacterial culturing--using functional nanoparticle-based proteomic strategies that are characterized by extremely short analysis time. In this approach, we used titania-coated magnetic iron oxide nanoparticles (Fe(3)O(4)@TiO(2) NPs) as affinity probes to concentrate the target bacteria. The magnetic properties of the Fe(3)O(4)@TiO(2) NPs allow the conjugated target species to be rapidly isolated from the sample solutions under a magnetic field. Taking advantage of the absorption of the magnetic Fe(3)O(4) NPs in the microwave region of the electromagnetic spectrum, we performed the tryptic digestion of the captured bacteria under microwave heating for only 1-1.5 min prior to MALDI MS analysis. We identified the resulting biomarker ions by combining their MS/MS analysis results with protein database searches. Using this technique, we identified potential biomarker ions representing five gram-negative bacteria: Escherichia coli O157:H7, uropathogenic E. coli, Shigella sonnei, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Finally, we demonstrated the practical feasibility of using this approach to rapidly characterize bacteria in clinical samples.

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.

Identification of bacterial strains by laboratories participating in the Deutsches Krebsforschungszentrum quality assurance programme

The quality assurance programme (QAP) of the Deutsches Krebsforschungszentrum (DKFZ) is a proficiency testing system developed to service the laboratory animal discipline. QAP comprises the quarterly distribution of two bacterial strains originating from various species of animals for identification to the species level and antibiotic susceptibility testing. We compared identification results reported by QAP participants over the years 1996-2004 with those obtained by the Dutch Bacterial Diagnostics reference laboratory on 68 samples comprising 71 bacterial strains and a fungus. Significant differences were found in the frequency of reported and correct identifications when bacteria were assigned to different groups based on morphology by Gram stain and on origin (animal versus environmental, rodent and rabbit versus other animal species, pathogen versus non-pathogens). Rodent and rabbit pathogens yielded 73% correct identifications, and with all bacterial strains only 60% of the identifications were correct. We assume that most QAP participants were from laboratory animal diagnostic laboratories. If this is true, the capabilities of laboratories in the laboratory animal discipline to correctly identify bacterial species are well below what are considered acceptable limits for human diagnostic laboratories. The distribution of cultured bacteria circumvents the most difficult step in the microbiological monitoring of animals, namely primary culture from clinical samples. We propose to set up a QAP that comprises the distribution of specimens mimicking clinical samples normally submitted to laboratory animal diagnostic laboratories.

Frequent Detection of Multidrug-Resistant Pneumonia-Causing Bacteria in the Pneumonia Lung Tissues of Patients with Hematological Malignancies

Pneumonia is a critical issue during the agonal phase, and often becomes lethal in the absence of pathogen detection. Autopsy is a powerful tool for analyzing the cause of a patient's death, progression of the disease, and the therapeutic response. However, it is frequently limited to the identification of bacterial strains. To elucidate the pathogenesis during the agonal phase of pneumonia, intrapulmonary sputum was harvested by directly inserting a swab into a resected lung, and the bacterial composition was analyzed using both pathological and microbiological techniques from 15 patients with hematological malignancies, and the results were compared with those from 25 patients with other medical and surgical diseases. Among the 54 bacteria strains isolated from the 40 patients, multidrug-resistant strains were significantly more prevalent in hematological group than in other diseases (16/21 versus 11/33, P = .002). Enterococcus faecium was preferentially isolated from the hematological patients, whereas the methicillin-resistant Staphylococcus aureus was predominantly found in the nonhematological group. Two coagulase-negative Staphylococcus epidermidis strains in hematological diseases may be diagnosed as causative bacteria of pneumonia by both bacterial and pathological techniques. Although the results of this study may not be directly applicable for clinical diagnosis, this approach has a potential to become not only a diagnostic method for bacterial pneumonia, but may be also useful for the analysis of multidrug-resistant pathogens.

Identification of Bacterial Strains Capable of Degrading Malaysian Petroleum Sludge

In an organic sludge bio degradation process, identification of bacterial strains with effective biodegradation capabilities indigenous to the sludge is an important step as isolation and subsequent usage of such strains can enhance the rate of the biodegradation process. In this study, the identification of bacterial strains capable of degrading Malaysian petroleum sludge was carried out using a biolog system kit subsequent to enrichment, isolation and degradation processes. A total of 53 purified strains was obtained from the isolation process. Three strains, namely, Clavibacter michiganesis ss insidiosus, Rhodococcus rhodochrus and Brevibacterium otitidis showed favorable degradation results for nearly all hydrocarbons especially for polycyclic aromatic hydrocarbon (PAHs) (anthracene, phenathrane and dibenzothiophene). Brevibacterium otitidis is especially interesting since it is the first time such a strain has been reported to have good PAHs degradation capabilities.

Microarray for serotyping of Bartonella species

BackgroundBacteria of the genus Bartonella are responsible for a large variety of human and animal diseases. Serological typing of Bartonella is a method that can be used for differentiation and identification of Bartonella subspecies.ResultsWe have developed a novel multiple antigenic microarray to serotype Bartonella strains and to select poly and monoclonal antibodies. It was validated using mouse polyclonal antibodies against 29 Bartonella strains. We then tested the microarray for serotyping of Bartonella strains and defining the profile of monoclonal antibodies.Bartonella strains gave a strong positive signal and all were correctly identified. Screening of monoclonal antibodies towards the Gro EL protein of B. clarridgeiae identified 3 groups of antibodies, which were observed with variable affinities against Bartonella strains.ConclusionWe demonstrated that microarray of spotted bacteria can be a practical tool for serotyping of unidentified strains or species (and also for affinity determination) by polyclonal and monoclonal antibodies. This could be used in research and for identification of bacterial strains.

Molecular Identification, Systematics, and Population Structure of Prokaryotes

Prokaryotic systematics is today perhaps one of the most fascinating and fast advancing fields in microbiology. It has become a widely respected scientific discipline, not least because of the application of new ideas and molecular techniques to bacterial identification and classification. Systematics describes the diversity of prokaryotes and classifies or arranges them into groups based on their common similarities. Although a major reason for establishing systematics has been to provide proper identification of bacterial strains, this discipline has developed into a global, multidisciplinary research field providing a phylogenetic framework for various areas within microbiology, including bacterial taxonomy, microbial ecology, clinical microbiology, population genetics, and environmental microbiology.

Identification of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with 16S ribosomal DNA-based oligonucleotide array hybridization

Rapid identification of the genus and species of bacteria in foods and clinical specimens is important. In this report, DNA sequences of bacterial 16S rDNA were used to develop the oligonucleotide array for the identification of bacterial strains of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. Most of these bacterial strains may cause food-borne outbreaks or sporadic cases. A rapid (< 4 h) detection method that used universal PCR primers to amplify the variable regions of bacterial 16S rDNA, followed by reverse hybridization of the PCR products, which were biotin labeled, to the oligonucleotides arrayed on the chip was developed. Fifteen oligonucleotide probes were selected and spotted on the nylon strip to determine the array hybridization patterns. It was successful in discriminating Bacillus spp., E. coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with identification, in general, to the genus level, not species level. As 182 randomly selected strains were assayed, the detection rate was found higher than 98%. Except for 3 strains, the remaining 179 strains were correctly identified and no cross reactions were observed. These 179 strains generated five hybridization patterns. Adding more oligonucleotide probes to the array may allow the detection of more bacterial genera and species without significantly increasing the complexity or cost.

Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius

Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae ( Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.

Interpretation of the results of antimicrobial susceptibility analysis of Escherichia coli isolates from bovine milk, meat and associated foodstuffs

Strains of indicator bacteria Escherichia coli ( E. coli) contaminating raw milk can, under specific conditions, become vectors of genes encoding resistance to antimicrobial drugs. The susceptibility of 1162 E. coli isolates originating from milk, milk products, meat, meat products and swabs from meat processing plant facilities to selected antimicrobial drugs (amikacin—AMI, ampicillin—AMP, cephalothin—CLT, cotrimoxazole—SXT, cefotaxime—CTX, gentamicin—GEN, neomycin—NEO, streptomycin—STR, tetracycline—TET, norfloxacin—NOR, chloramphenicol—CMP and erythromycin—ERY) was analysed. A standard microdilution method was used to detect minimal inhibition concentrations (MIC). The current criteria for interpretation (clinical breakpoints) do not allow identification of bacterial strains with acquired and potentially transmissible resistance to antimicrobial drugs. Microbiological breakpoints may be more appropriate because they take into account MIC distribution in a bacterial population of a certain species only. The aim of this study was to determine the microbiological breakpoints. Critical MIC R values (microbiological breakpoints) were derived, which enabled E. coli strains to be characterized by MIC values into two subgroups; the first one comprises susceptible strains (MIC<MIC R) and the second one comprises strains which are resistant to a given antimicrobial drug (MIC⩾MIC R). We obtained the following values of microbiological breakpoints: AMI-32, AMP-32, CLT-64, CTX-8, CMP-32, GEN-8, ERY-⩾512, NEO-16, NOR-1, STR-32, SXT-0.8/15.2 and TET-16 μg/ml.

Genetic relationships among strains of Xanthomonas campestrispv. campestrisrevealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA.

The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.

Electrochemical biosensor array for the identification of microorganisms based on lectin-lipopolysaccharide recognition.

Rapid identification of bacterial strains remains a well-known problem in applied medicine and, for viable pathogens, is an important diagnostic goal. We have investigated an electrochemical biosensor array, in which transduction is based on respiratory cycle activity measurements, where the microorganism's native respiratory chain is interrupted with non-native external oxidants. The selective biochemical recognition agents employed in this study are lectins that, once immobilized, recognize and bind to cell surface lipopolysaccharides. Porous membranes with different surface properties were examined as potential immobilization supports for these lectins. Optimizations performed using concanavalin A and E. coli JM105 show that immobilization methods involving pre-activated membranes significantly reduce the time required to create a functional lectin layer on the membrane surface. Overall, we found general agreement between agglutination test results and the electrochemical assessment of lectin-cell binding. Chronocoulometric measurements were made for cells captured on lectin-modified Immunodyne ABC membranes physically affixed to Pt working electrodes. This lectin-based sensor array was exposed to viable cells of gram-negative and gram-positive bacteria as well as yeast, and chronocoulometric measurements were used to generate a pattern of responses for each organism toward each lectin. Principal component analysis was used to classify the chronocoulometric results for the different microbial strains. With this new method, six microbial species (Baccilus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Enterobacter aerogenes, Saccharomyces cerevisiae) were readily distinguished.

Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy.

The use of Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques for the rapid detection and identification of bacterial contamination in liquids was evaluated. The complex biochemical composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification. Bacterial suspensions (Escherichia coli HB101, E. coli ATCC 43888, E. coli 1224, Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus cereus, and Listeria innocua) were filtered to harvest the cells and eliminate the matrix, which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 6000-4000 cm(-1). The method reproducibly distinguished between different E. coli isolates and conclusively identified the relationship between a new isolate and one of the test species. This methodology may allow for the rapid assessment of potential bacterial contamination in liquids with minimal sample preparation.