16S Ribosomal DNA Sequence Research Articles

Characterization of ancient DNA supports long-term survival of Haloarchaea.

Bacteria and archaea isolated from crystals of halite 10(4) to 10(8) years old suggest long-term survival of halophilic microorganisms, but the results are controversial. Independent verification of the authenticity of reputed living prokaryotes in ancient salt is required because of the high potential for environmental and laboratory contamination. Low success rates of prokaryote cultivation from ancient halite, however, hamper direct replication experiments. In such cases, culture-independent approaches that use the polymerase chain reaction (PCR) and sequencing of 16S ribosomal DNA are a robust alternative. Here, we use amplification, cloning, and sequencing of 16S ribosomal DNA to investigate the authenticity of halophilic archaea cultured from subsurface halite, Death Valley, California, 22,000 to 34,000 years old. We recovered 16S ribosomal DNA sequences that are identical, or nearly so (>99%), to two strains, Natronomonas DV462A and Halorubrum DV427, which were previously isolated from the same halite interval. These results provide the best independent support to date for the long-term survival of halophilic archaea in ancient halite. PCR-based approaches are sensitive to small amounts of DNA and could allow investigation of even older halites, 10(6) to 10(8) years old, from which microbial cultures have been reported. Such studies of microbial life in ancient salt are particularly important as we search for microbial signatures in similar deposits on Mars and elsewhere in the Solar System.

Vertical transmission of Theileria lestoquardi in sheep

This is the first report of an outbreak of Theileria lestoquardi abortion and stillbirth in a mob of 450 ewes in July 2012, during which, approximately 35 late-term ewes lost their fetuses over a 5-day period. A dead ewe and her aborted fetus were transported to the Ahvaz Veterinary Hospital for the diagnostic evaluation. The microbial cultures from the ewe vaginal discharges and fetal abomasal contents and the liver were negative. The blood films of the ewe and her fetus contained Theileria piroplasms and the impression smears from ewe liver and fetal spleen were positive for Theileria Koch blue bodies. The DNA was extracted from the liver and spleen of ewe and her fetus, respectively, and analyzed by polymerase chain reaction (PCR) using specific primers derived from the nucleotide sequences of 18S ribosomal DNA (rDNA) gene of T. lestoquardi. A single fragment of 428-bp fragment was amplified. The PCR product was directly sequenced and the alignment of the sequence with similar sequences in GenBank® showed 100% identities with 18S rDNA gene of T. lestoquardi. The present study is the first report of the T. lestoquardi vertical transmission that could be related to the abortion.

Detecting the Nonviable and Heat-Tolerant Bacteria in Activated Sludge by Minimizing DNA from Dead Cells

Propidium monoazide (PMA) has been used to determine viable microorganisms for clinical and environmental samples since selected naked DNA which was covalently cross-linked by this dye could not be PCR-amplified. In this study, we applied PMA to the activated sludge samples composed of complex bacterial populations to investigate the viability of human fecal bacteria and to determine the heat-tolerant bacteria by high-throughput sequencing of 16S ribosomal DNA (rDNA) V3 region. The methodological evaluation suggested the validity, and about 2-3 magnitude signals decreasing from the stained DNA were observed. However, the nest PCR, which was previously conducted to further minimize signals from dead cells, seemed not suitable perhaps due to the limitation of the primers. On one hand, for typical human fecal bacteria, less than half of them were viable, and most genera exhibited the similar viable percentages. It was interesting that many "unclassified bacteria" showed low viability, implying their sensitivity to environmental change. On the other hand, after heating at 60 °C for 4 h, the bacteria with high survival rate in activated sludge samples included those reported thermophiles or heat-tolerant lineages, such as Anoxybacillus and diverse species in Actinobacteria, and some novel ones, such as Gp16 subdivision in Acidobacteria. In summary, our results took a glance at the fate of fecal bacteria during sewage treatment and established an example for identifying tolerant species to lethal shocks in a complex community.

Alpine Scenedesmaceae from New Zealand: new taxonomy

Cultures from samples collected from the alpine zone in Canyon Creek, Canterbury, New Zealand, revealed three taxa belonging to Scenedesmaceae. Identifications were made based on morphological data (light microscopy and transmission and scanning electron microscopy) and sequences of 18S ribosomal DNA. Desmodesmus abundans is a commonly recorded species worldwide and in New Zealand, but Desmodesmus granulatus is a new record for this country. Coelastrella multistriata var. grandicosta is described as new to science. Cryptodesmus ellipsoideus, previously isolated from the alpine zone at Arthur's Pass, is transferred to Coelastrella ellipsoidea on the basis of new ultrastructural and molecular information. Analysis of 18S ribosomal DNA in Coelastrella indicates likely intragenomic recombination and possible horizontal transfer of introns between taxa in this genus.http://zoobank.org/urn:lsid:zoobank.org:pub:2D9CA9F7-72DE-462B-8201-2725F4F9C91F

Diversity of Lactic Acid Bacteria Associated with Fresh Coffee Cherries in Taiwan

A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.

Isolation, Identification and Characterization of Lactic Acid Bacteria From Shao-jiou-luo (Fermented Zoned Cerith), a Traditional Fermented Food in Taiwan

This study investigated the distribution of the native lactic acid bacteria (LAB) population in shao-jiou-luo (fermented zoned cerith), a popular traditional fermented food in Taiwan. A total of 129 cultures of LAB were isolated from shao-jiou-luo samples, and 14 cultures were isolated from its main raw material, zoned cerith. These isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. The identification results revealed 11 bacterial species belonging to five genera in shao-jiou-luo and three species belonging to two genera in zoned cerith. The most common bacterial genera in shao-jiou-luo were heterofermentative Weissella and Leuconostoc. On the other hand, Enterococcus aquimarinus was the most common species found in the raw zoned cerith sample. In conclusion, the results suggest that heterofermentative LAB species greatly contributed to the fermentation of shao-jiou-luo. This is the first report describing the distribution and varieties of LAB that exist in the shao-jiou-luo fermentation process and raw zoned cerith.

Isolation and characterization of lactic acid bacteria from Yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan

To isolate, characterize and identify lactic acid bacteria (LAB) in yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. A total of 226 LAB were isolated; 111 cultures were isolated from samples collected from seven different markets and 115 from six fresh broccoli samples. These isolates were characterized phenotypically and then initially divided into nine groups (r1 to r9) using restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Some isolates were further divided into four additional groups by other genetic analysis. The most common bacterial genera in yan-tsai-shin and fresh broccoli were Weissella, Lactococcus and Lactobacillus. Regional similarities in the LAB, with differences in diversity, were observed in this study. On the basis of phylogenetic analysis of 16S rRNA, rpoA, rpoB and pheS gene sequences, two strains were included in the genera Enterococcus and Lactococcus, respectively, and identified as potential novel species or subspecies. In addition, the novel enterococcal strain, and 33 L.lactis subsp. lactis and two Weissella cibaria strains were found to have bacteriocin-like inhibitory substance (BLIS) producing abilities. These results suggest that the LAB play important roles in the fermentation of yan-tsai-shin. This is the first report describing the distribution and varieties of LAB existing in yan-tsai-shin and fresh broccoli. In addition, two potential novel LAB species or subspecies and one potential novel BLIS were also found in this study.

Characterization and stability of lactobacilli and yeast microbiota in kefir grains

Characterization and stability of lactobacilli and yeasts from kefir grains using culture-dependent and culture-independent methods were investigated in this study. Culture-dependent analysis, followed by sequencing of 16S ribosomal DNA for bacteria and 26S rRNA gene for yeasts, revealed 3 different species of lactobacilli and yeasts, respectively. The most frequently isolated bacterial species were Lactobacillus kefiranofaciens ssp. kefirgranum, Lb. parakefiri, and Lb. kefiri, whereas yeasts belonged to Kluyveromyces marxianus, Kazachstania exigua, and Rhodosporidium kratochvilovae. This study is the first to report on the presence of R. kratochvilovae in kefir grains. On the other hand, PCR-denaturing gradient gel electrophoresis in the culture-independent method showed that the dominant microorganisms were Lb. kefiranofaciens ssp. kefirgranum, Kl. marxianus and Ka. exigua, but did not reveal bands corresponding to Lb. parakefiri, Lb. kefiri, or R. kratochvilovae. Our results support the necessity of combining more techniques for detailed and reliable study of microbial communities in kefir grains. Another interesting finding confirmed that the detected dominant microbiota of kefir grains is very stable and did not change over experimental time. This finding is important to ensure consistent product quality.

Isolation and characterization of lactic acid bacteria from pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan

Lactobacillus pobuzihii is a novel species which has been previously found in pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. However, the lactic acid bacteria (LAB) microflora in pobuzihi has not been studied in detail. In this study, LAB from pobuzihi were isolated, identified, and characterized. A total of 196 LAB were isolated; 79 cultures were isolated from the sample collected from a manufacturing factory, 38 from pobuzihi samples collected from 4 different markets, and 79 from 2 fresh cummingcordia samples. These isolates were characterized phenotypically and then divided into eight groups (A to H) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus plantarum was the most abundant LAB found in most samples during the fermentation of pobuzihi. On the other hand, Enterococcus casseliflavus and Weissella cibaria were, respectively, the major species found in the two fresh cummingcordia samples. A potential novel species or subspecies of lactococcal strain was found. In addition, seven L. plantarum and five W. cibaria strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157(T). This is the first report describing the distribution and varieties of LAB existing in the pobuzihi during its fermentation process and the final product on the market.

Comparison of sodA and 16S rDNA sequencing for accurate species-level identification of viridans group streptococci (VGS) isolated from patients with infective endocarditis

Background Viridans group streptococci (VGS) are commensal flora of the upper respiratory tract in humans and can cause serious infections, like infective endocarditis, septicemia, and meningitis. Accurate identification of VGS to the species level is difficult because they share many physiological characteristics. We report a study using sequencing of 16S ribosomal DNA (rDNA) gene and sodA gene to discriminate VGS up to species level.

The Melithaeidae (Cnidaria: Octocorallia) of the Ryukyu Archipelago: Molecular and morphological examinations

The family Melithaeidae (Octocorallia: Alcyonacea) is distributed in the West Pacific, Indian Ocean and the Red Sea. They are most abundant in warmer waters but can also be found in temperate waters. At present six genera are assigned to this family (Melithaea, Mopsella, Clathraria, Acabaria, Wrightella and Asperaxis), however overlapping characteristics make this group’s taxonomic identification difficult and their relationships unclear. There are only a few reports from the Ryukyu Archipelago in southern Japan of melithaeids and most other octocorals, despite the islands being an area of high octocoral diversity. To help resolve the taxonomic confusion in this family, samples from various Ryukyu Archipelago locations were collected and DNA sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome oxidase I (COI) were obtained. Additionally, SEM micrographs of the sclerites of specimens were taken to further confirm the molecular results. Three strongly supported clades were recovered from the COI and 28S rDNA analyses, corresponding to Melithaea, Acabaria, and Mopsella, and in most cases clades were clearly related with the sclerite shape reported for each genus. These results show clearly that molecular differences are present between the three genera, and also demonstrates the strong need of other molecular markers for resolving intra-generic phylogenies. Our results provide baseline data for future studies of this octocoral family, not only on taxonomy, but also with regards to their distribution in the Ryukyu Islands.

Isolation and characterisation of lactic acid bacteria from jiang‐gua (fermented cucumbers), a traditional fermented food in Taiwan

Jiang-gua (fermented cucumbers) is a popular traditional fermented food in Taiwan. The microflora of lactic acid bacteria (LAB) in jiang-gua have not been investigated in detail. In this study, LAB from jiang-gua were isolated, characterised and identified. A total of 103 LAB were isolated; 70 cultures were isolated from jiang-gua samples and 33 cultures were isolated from its raw substrate, cucumber. These isolates were mainly characterised phenotypically and then divided into seven groups (A-G) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. The isolates were identified as Enterococcus casseliflavus, Leuconostoc lactis, Leuconostoc mesenteroides, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactococcus lactis subsp. lactis, Weissella cibaria and Weissella hellenica. The antibacterial activities of the isolates were determined and 11 Lc. lactis subsp. lactis strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157(T) . Heterofermentative W. cibaria and Leu. lactis were the major LAB found in jiang-gua samples without soy sauce. In soy sauce-added samples, homofermentative L. pentosus and L. plantarum were the most abundant LAB. In addition, the results also suggested that HhaI and RsaI restriction enzymes could be applied to distinguish W. hellenica and Weissella paramesenteroides.

MALDI-TOF: Introduction in routine of a system for the rapid identification of microorganisms

Introduction. MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) is an recently introduced technique for the rapid identification of microorganisms by mass spectrometry (1,3) (Fig.1).The aim of this work was the evaluation and validation of the MALDI-TOF method (Bruker Daltonics) for inclusion in the routine workflow of a clinical microbiology laboratory. Methods.We analyzed strains isolated from different clinical specimens. For each species of microorganism we calculated the percentage of agreement by comparing the identification of MALDI-TOF with that obtained using the methods currently in use in the laboratory. In case of discordant results the conclusive identification was carried out by sequencing of 16S ribosomal DNA (Tab.3) using the MicroSeq identification system 500 (Applied Biosystems) (2). Alpha-hemolytic streptococci were instead identified by the ATB galleries (BioMerieux).ATCC strains were used as Quality Control. Results. 1340 strains were analyzed, including 479 enterobacteria, 188 gram-negative bacilli, 189 staphylococci, 140 enterococci, 84 Haemophilus spp, 114 streptococci, 40 yeasts and 106 less common microorganisms. Gramnegative bacteria, enterococci, Staphylococcus aureus, beta-hemolytic streptococci and yeasts showed full agreement (Tab.1 e Tab.2). In addition, MALDI-TOF has proved a reliable method for identification of fastidious germs such as Legionella, Branhamella, Neisseriae, Listeria monocytogenes, Corynebacteria, and anaerobes.Alpha-hemolytic streptococci were but in most cases identified as S. pneumoniae. Conclusions. The identification by mass spectrometry allows to obtain reliable results in minutes for most of the organisms isolated from the routine. Considerable importance for the performance of the system plays the quality of the database in the instrument. The high percentage of concordance between identification with the standard methods and MALDI-TOF allowed the introduction of this method in routine workflow with the exception of alpha hemolytic streptococci for which the current system of identification is still in use.

Pyrosequencing survey of the microbial diversity of ‘narezushi’, an archetype of modern Japanese sushi

This study aimed to analyse microbiota of the fermented food 'narezushi', an archetype of modern Japanese sushi. The pyrosequencing technique was used to analyse sequences of 16S ribosomal DNA contained in six narezushi products. The V1-V2 regions of the 16S ribosomal DNA were amplified from different narezushi products using PCR, and approximately 120,000 sequences were phylogenetically assigned at the genus level, using the Ribosomal Database Project classifier. In all samples, the microbial populations consisted of more than 90% Lactobacillales, mainly Lactobacillus or Pediococcus, reflecting their crucial role in narezushi fermentation. There were more than 700 operational taxonomy units in all samples, with Shannon-Wiener index varying from 1.69 to 2.60. The microbiota of all narezushi products were shown to consist largely of Lactobacillales populations. Interestingly, different species were found to be dominant in each product. This study provides an insight into the bacterial composition of fermented fish-based foods, which are consumed worldwide. Significant differences in the dominant species were observed between products, possibly because of the starter-free production process.

An unusual, flagellum-bearing hydrobiid snail (Gastropoda: Rissooidea: Hydrobiidae) from Greece, with descriptions of a new genus and a new species

A new genus and a new species of Hydrobiidae, Agrafia wiktori sp. nov., from the Agrafa Mountains, northern Greece, are described based on the shell, protoconch, radula and male and female reproductive organs. In the protoconch sculpture and female reproductive organs A. wiktori resembles a typical member of Hydrobiidae Sadlerianinae, but the penis with tubular penial gland terminating within the flagellum and extending into the cephalic haemocoele has not been found in any other representative of Hydrobiidae. Instead, it is comparable with the penes and tubular penial glands of some non-hydrobiid Rissooidea (Bithyniidae, Bythinellidae, Amnicolidae, Emmericidae). Analysis of partial sequences of 18S ribosomal DNA and cytochrome oxidase subunit I mitochondrial DNA in A. wiktori place it in Hydrobiidae Sadlerianinae. The evolution of the bifid penis and tubular penial gland is discussed.

Description of the two strains of turkey coccidia Eimeria adenoeides with remarkable morphological variability

Although oocyst morphology was always considered as a reliable parameter for coccidian species discrimination we describe strain variation of turkey coccidia, Eimeria adenoeides, which remarkably exceeds the variation observed in any other Eimeria species. Two strains have been isolated - the first strain maintains the typical oocyst morphology attributed to this species - large and ellipsoidal - while the second strain has small and ovoid oocysts, never described before for this species. Other biological parameters including pathogenicity were found to be similar. Cross-protection between these 2 strains in 2 immunization and challenge experiments was confirmed. Sequencing and analysis of 18S and ITS1 ribosomal DNA revealed a close relationship according to 18S and a relatively distant relationship according to ITS1. Analysis of 18S and ITS1 sequences from commercial turkey coccidiosis vaccines Immucox®-T and Coccivac®-T revealed that each vaccine contains a different strain of E. adenoeides and that these strains have 18S and ITS1 sequences homologous to the sequences of the strains we have isolated and described. These findings show that diagnostics of turkey coccidia according to oocyst morphology have to be carried out with caution or abolished entirely. Novel PCR-based molecular tools will be necessary for fast and reliable species discrimination.

Outbreak of a novel Enterobacter sp. carrying blaCTX-M-15 in a neonatal unit of a tertiary care hospital in Tanzania

Enterobacter hormaechei and Cronobacter sakazakii are amongst the most important causes of outbreaks of neonatal sepsis associated with powdered milk. In this study, we report for the first time an outbreak of a novel Enterobacter sp. harbouring bla CTX-M-15 in a neonatal unit in Tanzania. Seventeen Gram-negative enteric isolates from neonatal blood cultures were studied. Antibiotic susceptibility was assessed by disc diffusion testing, and the presence of the bla CTX-M-15 gene was established by polymerase chain reaction (PCR) and sequencing. Isolates were typed by pulsed-field gel electrophoresis (PFGE). Identification by biochemical profiling was followed by nucleotide sequencing of 16S ribosomal DNA (rDNA), rpoB and hsp60 alleles. Environmental sampling was done and control measures were established. Isolates were initially misidentified based on their fermentation characteristics and agglutination as Salmonella enterica serotype Paratyphi. All isolates were resistant to multiple antibiotics, except for ciprofloxacin and carbapenems, and were found to harbour bla CTX-M-15 on a 291-kb narrow-range plasmid. PFGE analysis indicated the clonal outbreak of a single strain, infecting 17 neonates with a case fatality rate of 35%. The same strain was isolated from a milk bucket. Phylogenetic analysis using 16S rDNA, rpoB and hsp60 sequences permitted no definitive identification, clustering the strains in the Enterobacter cloacae complex with similarities of 92–98.8%. The data describe an outbreak of a novel bla CTX-M-15-positive, multiresistant Enterobacter strain in an African neonatal unit that can easily be misidentified taxonomically. These data highlight the need for constant surveillance of bacteria harbouring extended-spectrum β-lactamases as well as improvements in hygiene measures in developing countries.

Isolation and characterization of novel sulphate-reducing Fusobacterium sp. and their effects on in vitro methane emission and digestion of wheat straw by rumen fluid from Indian riverine buffaloes

Seven isolates of sulphate-reducing bacteria were isolated from the rumen of buffalo and characterized morphologically, physiologically and phylogenically. A PCR assay and sequencing confirmed that all sulphate reducing isolates possessed the dissimilatory sulphite reductase gene. Sequencing of 16S ribosomal DNA indicated that the SRB isolates were highly homologous with Fusobacterium genus. The novel SRB isolates were evaluated for their suitability to reduce CH4 production and stimulate fibre digestion in vitro. When added to rumen fluid from buffalo, most isolates decreased (P<0.05) CH4 (mL/L) concentration, increased propionate production, but did not alter H2S production. The isolate SRBBR 5 had the highest decrease in CH4 emission compared to control, reducing it from 2.66 to 1.64mmol CH4/g DM truly digested after 72h of fermentation. Addition of SRBBR5 also increased (P<0.05) fibre digestion (15% in apparent digestibility and 40% in true digestibility), xylanase activity and numbers of cellulolytic bacteria in the incubation fluid with no change in the numbers of methanogens or fungi. Results show that SRBBR5 has potential as a microbial feed additive to reduce CH4 emissions and improve fibre digestibility in ruminants.This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Isolation and characterisation of lactic acid bacteria from yan-jiang (fermented ginger), a traditional fermented food in Taiwan.

Yan-jiang (fermented ginger) is a popular traditional fermented food in Taiwan. The lactic acid bacteria (LAB) microflora in yan-jiang has not been studied in detail. In this study, LAB from yan-jiang were isolated, characterised and identified. A total of 176 LAB were isolated; 160 cultures were isolated from yan-jiang samples and 16 cultures were isolated from raw ginger. These isolates were characterised phenotypically and then divided into nine groups (A to I) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus sakei and Lactococcus lactis subsp. lactis were the major LAB found in the initial 2 days of fermentation without pickled plums; these species were mostly replaced by Weissella cibaria and L. plantarum after 3 days of fermentation. In the fermentation bucket with added pickled plums, W. cibaria was the most abundant LAB found during fermentation. The antibacterial activities of the isolates were determined. Twenty-four Lc. lactis subsp. lactis and 19 W. cibaria strains showed inhibitory activity against the indicator strain L. sakei JCM 1157(T) . Results demonstrate that various LAB species were more numerous when fermentation was carried out without pickled plums. LAB also had effects on the aroma and flavour of yan-jiang.

Identification of Bacteria in Paraffin-Embedded Tissues Using 16S rDNA Sequencing from a Neonate with Necrotizing Enterocolitis

The conventional approach to bacterial identification in paraffin-embedded tissue relies mainly on morphology, with the aid of Gram stain. This approach is only able to provide some clues; it does not offer the capability of accurate identification. Bacterial identification based on sequencing of 16S ribosomal DNA (rDNA) is able to identify bacteria to the species level. Here we demonstrate the application of this technique in the postmortem examination, for which we had obtained different results from premortem and postmortem blood cultures. In the postmortem examination, abundant gram-negative rods were present in gastrointestinal tract lumen and lymphatic space. DNA was extracted from paraffin-embedded tissue of the above sections and subjected to 16S rDNA polymerase chain reaction for the first 500 base pairs, followed by sequencing. The results of sequencing correlated well with the postmortem culture result. In this review, the newly emerging field of bacterial identification with molecular techniques employing both broad-range and targeted approaches and their clinical applications and limitations are discussed.