16S RNA Gene Research Articles

Molecular identification of Babesia spp isolated from Polish cattle with asymptomatic protozoa infections

The aim of the paper was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine what species of protozoa infects Polish cattle. Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cows from various farms located in eastern Poland. The infection was detected in 10.4% of the samples. All animals were infected with Babesia occultans which sequence of the 18S RNA gene fragment showed a 93.1%, homology with the sequence of B. occultans EU 376017. This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland.

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression.

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome's molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.

Molecular typing of Polish strains of Babesia canis protozoa isolated from dogs in the years 2013-2016

The aim of the study was the molecular analysis of B. canis strains isolated from dogs from Poland based on 18S RNA and Bc28 genes. The study involved 140 protozoan strains derived from clinical disease cases. All DNA samples of parasites were analyzed in two ways (amplification and restriction digestion) which made it possible to demonstrate the polymorphism of the Bc28 gene, and to show 18S RNA gene polymorphism (amplification and restriction digestion). Amplification of the Bc28 gene fragment and digestion of the resulting PCR products allowed for the classification of 104 isolates of B. canis to the Bc28-A group, and 36 strains of protozoa to the Bc28-B group. Amplification of the Bc28 gene fragment and digestion of the resulting PCR products allowed for the classification of 33 isolates to 18S RNA-A group, while to 18S RNA-B – 107 parasite isolates. Comparison of both groups of protozoa among themselves allowed partial but not complete correlation of polymorphisms in Bc28 and 18S RNA genes..

The findings of escherichia coli in drinking water with reverse transcriptase polymerase chain reaction method at 16s RNA Gene

Drinking water is the fundamental needs of urban communities. Emerging drinking water refilled station (DWRS) causing decreased in water quality so required the existence of quality monitoring efforts. RT-PCR technique could detect the presence of Escherichia coli in drinking water. The RT-PCR method is superior in accuracy, efficiency, and specificity. This research aims to analyze the presence of Escherichia coli as an indicator of the quality of refilled drinking water with the technique of RT-PCR target 16s RNA. The sample in this study was ten drinking water refilled station with the total sample 30 samples measured in the inlet, outlet and output. The results of RT-PCR in Mariso district, obtained RNA Band in the gene 16S RNA at position 723-bp in the sample a. 13. While in Panakukkang district captured RNA Band in the gene 16S RNA at position 723-bp on sample B. 11; B. 12; B. 13. Conclusions, Genomic RNA template by RT-PCR can be used to detect bacteria Escherichia coli in drinking water more quickly and accurately than conventional methods.

Comparative Genome Analysis of 2 Mycobacterium Tuberculosis Strains from Pakistan: Insights Globally Into Drug Resistance, Virulence, and Niche Adaptation.

Multidrug-resistant Mycobacterium tuberculosis is a global threat particularly in developing countries like Pakistan. In this study, we identified 2 M tuberculosis strains, mnpk and swlpk, by 16S RNA genes, sequenced their draft genome, and compared the 2 genomes with reference strain H37Rv and gene expression analysis of selected virulent genes. Phylogenetic analysis of M tuberculosis strains, mnpk and swlpk, using 16S RNA genes revealed that the strains are closely related with reference strain H37Rv. The draft genome sequence of mnpk and swlpk contains 4305 and 4295 protein-coding genes, respectively, having 99.9% with high collinearity when compared with H37Rv. Although some important drug-resistant genes such as fabG, faDE24, and iniA were missing, genome mining also revealed key drug-resistant genes such as katG, inhA, rpoA, rpoB, and rpoC against first-line isoniazid and rifampicin drug. The strain mnpk and swlpk encodes 257 putative and 86 verified virulent genes including type 7 secretion system (T7SS) key genes. The variation in the expression profile of selected T7SS genes, particularly low expression level of EspK, raised concern that the mechanism of virulence of mnpk and swlpk might be different from H37Rv strains as espK is associated with ATPase EccC1a and EccC1b which showed high expression level. Briefly, this study shows that the strains mnpk and swlpk are linked with H37Rv having 99% similarity in genomes, but the absence of drug-resistant genes and variation in key genes’ expression profile espK, EccE1, PPE41, and espC provide a rationale for the future investigation of M tuberculosis mnpk and swlpk pathogenesis via RNA sequencing, single-nucleotide polymorphisms, as well as gene manipulation.

Clinical and symptom scores are significantly correlated with fecal microbiota features in patients with symptomatic uncomplicated diverticular disease: a pilot study.

There is growing consensus that symptomatic uncomplicated diverticular disease is a chronic inflammatory condition, and that alterations in the fecal microbiota may contribute to its pathogenesis. The aim of this study was to relate the fecal microbiota composition in symptomatic uncomplicated diverticular disease to measures of inflammation, symptoms, and history of previous acute diverticulitis. Fecal microbiota composition in 28 individuals with symptomatic uncomplicated diverticular disease was characterized by 16S RNA gene amplicon sequencing. Microbiota composition was related to clinical history, symptom and inflammation measures, and demographic variables. Previous acute diverticulitis was associated with higher relative abundance of Pseudobutyrivibrio, Bifidobacterium, Christensenellaceae family, and Mollicutes RF9 order (P=0.004, 0.006, 0.010, and 0.019, respectively), but not microbiota alpha or beta diversity. A higher bloating severity score was significantly correlated with a higher relative abundance of Ruminococcus (P=0.032), and significantly inversely correlated with the relative abundance of the Roseburia (P=0.002). Fecal calprotectin levels were positively correlated with alpha diversity (Shannon index, P=0.005) and the relative abundance of Lactobacillus (P=0.004). Pain score was positively correlated with the relative abundance of Cyanobacterium (adjusted P=0.032). Patient symptoms in symptomatic diverticular disease are significantly correlated with features of the fecal microbiota. Our findings suggest the potential utility of therapies that target intestinal microbiology, such as dietary prebiotic supplements.

Molecular discrimination of New Zealand sourced meat spoilage associated psychrotolerant Clostridium species by ARDRA and its comparison with 16s RNA gene sequencing

ARDRA analysis was carried out on 90 New Zealand psychrotolerant Clostridium isolates derived from three meat production animal types and their environments. The isolates included species associated with spoilage: C. gasigenes, C. algidicarnis, C. tagluense, C. frigidicarnis and C. estertheticum. The isolates fell into 14 distinct ARDRA Groups, with 13 previously characterised meat spoilage-associated isolates shared between 6 of the 14 groups. The accuracy of ARDRA profiling analysis was supported by sequencing the 16s rRNA gene from isolates, including the representative spoilage associated Clostridium species and was consistent with previous phylogenetic relationships and classical cultural characterisation. The ARDRA methodology described in this study successfully discriminated between the different spoilage-associated species of clostridia as well as other pyschrotolerant Clostridium species associated with meat production. This discriminatory molecular screen will aid future source attribution studies as well as enable meat processors to identify and validate control measures for clostridia contamination, thus gaining greater efficacy in controlling meat spoilage caused by psychrotolerant clostridia.

Evidence of multiple introductions and genetic admixture of the Asian brush-clawed shore crab Hemigrapsus takanoi (Decapoda: Brachyura: Varunidae) along the Northern European coast

The Asian brush-clawed shore crab Hemigrapsus takanoi is a non-indigenous species along the Northern European coast. Although the history of range expansion of European H. takanoi has been well-documented, little is known about the genetic compositions of either the introduced European populations or the native Asian ones. We therefore collected H. takanoi broadly from their native Asian sites and introduced European ranges, and genotyped them by sequencing the mitochondrial 16S RNA gene and by analyzing nuclear microsatellite loci. Our results revealed that the H. takanoi Bay of Seine (France) populations consisted of a genetic admixture between populations in Japan and those in the Yellow Sea region. These French populations should be carefully monitored in the future, since the genetic admixture of multiple source populations may accelerate range expansion in non-indigenous organisms. Our results also suggested that shipping lines from East Asia were more probable vectors than historical juvenile oyster transportations from Japan for the foundation of present European H. takanoi populations. Interestingly, gene flow between populations in Japan and those in the Yellow Sea region (i.e., domestic invasion) was not observed despite the higher potential for artificial translocations via shipping lines in the native Asian range compared with those from Asia to Europe. The lack of domestic invasions implied that intra-specific priority effects of the resident H. takanoi populations played an important role in preventing the successful colonization of artificially-transferred individuals.

Cryptosporidium baileyi in wild captive psittacines in Brazil.

Cryptosporidiosis in birds manifests as an acute or chronic disease of the respiratory or digestive tracts. The objective of our study was to perform the molecular characterization of Cryptosporidium spp. in wild psittacines kept in captivity at the Araçatuba Municipal Zoo, São Paulo, Brazil. A total of 47 fecal samples were collected from Amazona aestiva, Psittacara leucophthalma, and Ara ararauna. All samples were collected at the time the birds defecated. DNA extraction was performed using the ZR Fecal DNA MiniPrep™ kit (Zymo Research). Screening for Cryptosporidium spp. was accomplished using nested PCR targeting the 18S RNA gene and sequencing of amplified fragments. Positivity for Cryptosporidium spp. (10.64%; 5/47) was found in A. ararauna (4) and P. leucophthalma (1) samples. The amplified fragments were sequenced and showed 100% genetic similarity with Cryptosporidium baileyi.

Research of genetic bases of hereditary non-syndromic hearing loss.

Hearing loss is the most common sensory disorder that affects approximately one per 1000 live births. With this project, we aimed to identify gene variants that were common causes of hearing loss in Turkey to contribute to the planning of genetic screening programs for hearing loss, as well as to improve genetic counseling to affected families. Twenty-one families with at least two affected individuals and parental consanguinity who presented with non-syndromic severe-to-profound sensorineural hearing loss were included in this study. We first screened for mutations in GJB2 and mitochondrial DNA 12S RNA genes. Subsequently, we genotyped the TMIE c.250C>T and SNP markers flanking the SLC26A4, MYO7A, MYO15A, OTOF, CDH23, TMIE, TECTA, PCDH15, TMC1, TMPRSS3, TMHS genes in the remaining twelve families without mutations in GJB2. Screening for mutations in GJB2 gene showed c.[35delG];[35delG] mutation in four families, c.[35delG];[507C>A] mutation in two families, c.[35delG];[-23+1G>A] mutation in one family, and c.457G>A heterozygous mutation in one family. Genotyping SNP markers showed the c.[250C>T];[250C>T] mutation in TMIE in one family. A homozygous region with SNP genotypes was detected with the OTOF gene in one family, the TMPRSS3 gene in another family, and also a homozygous region was detected with TMHS, OTOF, and TMPRSS3 genes in another family. Further research will be required to determine the genetic bases of hearing loss in families with non-syndromic hearing loss.

Low occurrence of pathogenic Yersinia enterocolitica in pig tonsils at slaughter in Southern Brazil.

Yersinia enterocolitica is a foodborne pathogen and pigs are the main reservoir of it in their tonsils. As Brazil is a large producer and exporter of pork meat and information regarding this pathogen is still quite scarce, this study aimed at evaluating the direct detection of Y. enterocolitica followed by pathogenic Y. enterocolitica (PYE) determination in tonsils of slaughtered pigs. For this purpose, 400 pig tonsils were collected from 15 farms in four federally certified slaughterhouses in Southern Brazil. Initially, samples were screened using conventional PCR targeting of the 16sRNA gene, followed by multiplex PCR (mPCR) in order to detect three virulence genes (ail, yadA, and virF) and quantitative real-time PCR (qPCR) for the detection of the ail gene. One hundred and one (25.2%) of the samples tested positive for the 16sRNA gene. However, a PYE was detected in one out of the 101 Y. enterocolitica positive samples. The three virulence genes were determined by mPCR and confirmed by partial DNA sequencing. Thus, a significant occurrence of Y. enterocolitica was observed in pig tonsils from federally inspected slaughterhouses in Brazil, although the presence of pathogenic strains was quite low.

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting.

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect 2.5 × 103 CFU/ml of pLR colonies spiked in 106 CFU/ml of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Drug resistance and protoporphyrin ferrochelatase of Ralstonia mannitolilytica

To investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of Ralstonia mannitolilytica, and to explore its structure and pathogenic function. The strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene. A strain belonging to Ralstonia mannitolilytica was isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of Neisseria meningidis. The isolate of Ralstonia mannitolilytica has a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of Ralstonia mannitolilytica.

Sewers as potential reservoirs of antibiotic resistance

Wastewater transport along sewers favors the colonization of inner pipe surfaces by wastewater-derived microorganisms that grow forming biofilms. These biofilms are composed of rich and diverse microbial communities that are continuously exposed to antibiotic residues and antibiotic resistant bacteria (ARB) from urban wastewater. Sewer biofilms thus appear as an optimal habitat for the dispersal and accumulation of antibiotic resistance genes (ARGs). In this study, the concentration of antibiotics, integron (intI1) and antibiotic resistance genes (qnrS, sul1, sul2, blaTEM, blaKPC, ermB, tetM and tetW), and potential bacterial pathogens were analyzed in wastewater and biofilm samples collected at the inlet and outlet sections of a pressurized sewer pipe. The most abundant ARGs detected in both wastewater and biofilm samples were sul1 and sul2 with roughly 1 resistance gene for each 10 copies of 16s RNA gene. Significant differences in the relative abundance of gene intI1 and genes conferring resistance to fluoroquinolones (qnrS), sulfonamides (sul1 and sul2) and betalactams (blaTEM) were only measured between inlet and outlet biofilm samples. Composition of bacterial communities also showed spatial differences in biofilms and a higher prevalence of Operational Taxonomic Units (OTUs) with high sequence identity (>98%) to well-known human pathogens was observed in biofilms collected at the inlet pipe section. Our study highlights the role of sewer biofilms as source and sink of ARB and ARGs and supports the idea that community composition rather than antibiotic concentration is the main factor driving the diversity of the sewage resistome.

Interrelationships of Dictyocaulus spp. in Wild Ruminants with Morphological Description of Dictyocaulus cervi n. sp. (Nematoda: Trichostrongyloidea) from Red Deer, Cervus elaphus.

Lungworms from the genus Dictyocaulus cause parasitic bronchitis (dictyocaulosis) characterized by coughing and severe lung pathology in both domestic and wild ruminants. In this study we investigated the interrelationships of Dictyocaulus spp. from European bison (Bison bonasus L.), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) by nucleotide sequence analysis spanning the 18S RNA gene (small subunit [SSU]) and internal transcribed spacer 2 (ITS2) regions of the ribosomal gene array as well as the mitochondrial cytochrome c oxidase subunit 1 (cox1). Molecular analyses of sequence data obtained partly with novel primers from between 10 and 50 specimens from each host were carried out. Bayesian inference analysis revealed that each host species was infected with different genotypes. Analysis of cox1 sequence data showed a diverse genetic background and high evolutionary potential of Dictyocaulus taxa. Data from lungworms of European bison revealed a distinct genotype of Dictyocaulus viviparus, whereas Dictyocaulus capreolus was only found in roe deer. In contrast, red deer were infected with a taxon with unique SSU, ITS2, and cox1 sequences. These results indicate the occurrence of a novel genotype from red deer, which differs significantly from the National Center for Biotechnology Information reference sequence of Dictyocaulus eckerti. The molecular evidence was consistent with a morphological study with description and imaging of Dictyocaulus cervi n. sp. recovered from red deer. Dictyocaulus cervi n. sp. can be distinguished from D. eckerti on the basis of the absence of cervical papillae, the occurrence of a single ring of 4 symmetrical submedian cephalic papillae, length of the tail in females, morphometry of the female reproductive system, and measurements of gubernacula in males. In conclusion, our findings further strengthen the idea that the genetic complexity and diversity among Dictyocaulus lungworms infecting wildlife ruminants is larger than previously believed and warrants further investigation.

ContEst16S: an algorithm that identifies contaminated prokaryotic genomes using 16S RNA gene sequences.

Thanks to the recent advancement of DNA sequencing technology, the cost and time of prokaryotic genome sequencing have been dramatically decreased. It has repeatedly been reported that genome sequencing using high-throughput next-generation sequencing is prone to contaminations due to its high depth of sequencing coverage. Although a few bioinformatics tools are available to detect potential contaminations, these have inherited limitations as they only use protein-coding genes. Here we introduce a new algorithm, called ContEst16S, to detect potential contaminations using 16S rRNA genes from genome assemblies. We screened 69 745 prokaryotic genomes from the NCBI Assembly Database using ContEst16S and found that 594 were contaminated by bacteria, human and plants. Of the predicted contaminated genomes, 8 % were not predicted by the existing protein-coding gene-based tool, implying that both methods can be complementary in the detection of contaminations. A web-based service of the algorithm is available at www.ezbiocloud.net/tools/contest16s.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

A comparative in situ decomposition study using still born piglets and leaf litter from a deciduous forest

A cadaver and dead plant organic matter, or litter, are rich energy sources that undergo a complex decomposition process, which impact the surrounding environmental microbiota. Advances in molecular microbiology techniques, with study of the 16S RNA genes, in particular, have highlighted the application of forensic ecogenomics in addressing key knowledge gaps. To investigate subsurface microbiome shifts as a novel tool to establish “postmortem microbial clock” and augment postmortem interval (PMI) and time-since-burial estimations, an in situ study with triplicate underground burials of piglets as human taphonomic proxies and Quercus robur leaf litter was monitored for 270 days. Changes in microbial community structure and composition were related directly to changes in seasonal temperature, with microbial shifts more pronounced during the summer. For example, Methylococcaceae could be used as seasonal bacterial indicators, from winter to summer, in establishing postmortem microbial clock for this site. Furthermore, Methylophilaceae (Methylophilales order) and Anaerolineaceae would differentiate for the piglet and leaf litter soils, respectively, 180 days after internment.

Design of 2′-phenylethynylpyrene excimer forming DNA/RNA probes for homogeneous SNP detection: The attachment manner matters

1-Phenylethynylpyrene fluorophore (1-PEPy) has long-wavelength shifted emission and higher photostability compared to pyrene, retaining, however, pyrene's ability to form excimers. Here we report the synthesis of 2′-O-[3(and 4)-(pyren-1-ylethynyl)benzyl]-uridines and their tandem incorporation into deoxyribo- and 2′-O-Me-ribo-oligonucleotide probes. Excimer forming probes of type NN … NNXXNN … NN (X = 2′-O-[meta(or para)-(pyren-1-ylethynyl)-benzyl]uridine) containing two adjacent fluorescent nucleosides within an oligonucleotide are available in four types (meta-meta; para-meta; meta-para; para-para). Both DNA (N = deoxyribonucleotides) and 2′-O-Me-RNA (N = 2′-O-Me-ribo-nucleotides) probes were synthesized and their hybridization with complementary and singly mismatched DNA and RNA was studied. Several probes show a dramatic response of their excimer-to-monomer intensity ratio upon hybridization. Remarkably, most spectacular fluorescence changes were demonstrated for probes with para-meta and meta-para combination within 2′-O-Me-ribo-oligonucleotides. Using excimer forming probes, three natural SNP in Helicobacter pylori 23S RNA gene (A2144G, A2143G, A2143C) and the wild type gene can be distinguished.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization–time of flight mass spectrometry and cydB real-time quantitative PCR

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species.