Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Molecular epidemiological and clinical infection characteristics analysis of Ralstonia.

PURPOSE: This study was to clarify the molecular epidemiology and clinical infection characteristics of Ralstonia pickettii and establish sequence typing system. METHODS: 48 nonrepetitive Ralstonia pickettii strains were collected from January 2008 to December 2013 at the Chinese People's Liberation Army General Hospital (PLAGH) and were identified through a specific PCR experiment, 16 S rDNA experiment and VITEK 2 system to compare the identification accuracy. The sequence types of the strains were analyzed by multilocus sequence typing (MLST) method. The antibiotic sensitivity of these strains was determined with disc diffusion tests and broth microdilution method. The clinical data of Ralstonia pickettii infected patients were collected. RESULTS: All of the 48 strains were identified as Ralstonia pickettii by VITEK 2 system. 30 and 34 strains were identified as Ralstonia pickettii by PCR and 16 S rDNA experiment respectively. ST9 was the most sequence types (STs) in these 18 STs of 42 strains. 42 strains were divided into 2 groups (A and B) and 18 genotypes. Ralstonia pickettii was sensitive to some cephalosporins, ß-lactam/ß-lactamase inhibitor, levofloxacin and trimethoprim/sulfamethoxazole. Cough, sputum, shortness of breath and pulmonary rales were the common clinical symptoms of most Ralstonia pickettii infected patients. CONCLUSION: We established a sequence typing system with a relatively fine resolution and the PCR assay is a faster and more sensitive method for clinical identification of Ralstonia pickettii. ST9 is the most common sequence types of Ralstonia pickettii. The most common clinical characteristics of Ralstonia pickettii infected patients were cough, sputum, shortness of breath and pulmonary rales.

Vagococcus fluvialis isolation from the urine of a bladder cancer patient: a case report.

Vagococcus fluvialis infection is rare in humans, and there is limited research on the clinical manifestations and antimicrobial susceptibility testing of Vagococcus fluvialis infection. Here, We isolated Vagococcus fluvialis from the urine samples of bladder cancer patients at Hunan Provincial People's Hospital, and it is the first reported case of Vagococcus fluvialis isolated from the urine. The fully automated microbial identification system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified the bacterium as Vagococcus fluvialis with a confidence level of 99.9%. The VITEK-2Compact fully automated microbial susceptibility analysis system indicated that it was most sensitive to tigecycline, vancomycin, quinupristin/dalfopristin, linezolid, and showed moderate sensitivity to erythromycin, levofloxacin, ciprofloxacin, ampicillin/sulbactam, and tetracycline. Additionally, it exhibited synergy when combined with high-level gentamicin and vancomycin, showing sensitivity. However, it displayed poor activity against penicillin and furanth. According to our knowledge, this is the first study to isolate and identify Vagococcus fluvialis from the urine of bladder cancer patients and the systematically reviewed other reported Vagococcus infections on human, which provide an experimental basis for guiding the rational use of drugs in the clinical treatment and diagnose of Vagococcus fluvialis infection and related pathogenic mechanism research. Meanwhile, we have systematically reviewed other reported.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Trends in infectious spondylitis from 2000 to 2020.

PURPOSE: This study aimed to investigate the trends in infectious spondylitis over the past two decades. METHODS: We included 157 cases, from 2000 to 2020, of infectious spondylitis. The cases were divided into two groups: 00 (cases during 2000-2009; 82 cases:) and 10 (cases during 2010-2020; 75 cases) groups. Patients' age, sex, causative organism, and localization were examined and compared between the two groups. RESULTS: The proportions of women in the 00 and 10 groups were 30.5% and 38.7%, respectively, with no significant difference (P = 0.28). The average age was significantly higher in the 10 group (72.6 years) than in the 00 group (68.8 years; P < 0.01). A compromised host was the cause of infection in 52.4% and 36.0% of the patients in the 00 and 10 groups, respectively, showing a significant difference. The bacterial identification rates were 70.1% and 77.3% in the 00 and 10 groups, respectively (P < 0.01), and the genus Staphylococcus was the most common bacteria. The proportions of resistant bacteria such as methicillin-resistant Staphylococcus aureus in the 00 and 10 groups were 27.3% and 6.7%, respectively (P < 0.01). Conversely, infectious diseases caused by indigenous bacteria in the oral cavity and intestines were more common in the 10group (37.8%) than in the 00 group (13.0%), showing a significant difference (P < 0.01). CONCLUSION: Recently, infections caused by indigenous bacteria in the oral cavity and intestines have increased more than those caused by resistant bacteria over the past two decade.

A case of Vagococcus fluvialis isolated from the bile of a patient with calculous cholecystitis.

BACKGROUND: Chronic cholecystitis, characterized by persistent inflammation of the gallbladder, predominantly stems from the prolonged presence of gallstones. Calculous cholecystitis has demonstrated a consistent escalation in its incidence over time.Gallbladder stones have been recognized as a predisposing factor for the development of biliary tract infections.Concomitantly, there have been substantial shifts in the distribution and resistance profiles of pathogenic microorganisms responsible for biliary tract infections. The timely acquisition of bile samples for pathogen analysis is of paramount importance, given its critical role in guiding judicious clinical pharmacotherapy and enhancing patient prognosis. CASE PRESENTATION: We present a case involving a 66-year-old female patient who had previously undergone subtotal gastrectomy due to diffuse large B-cell lymphoma. The patient was admitted to our institution with complaints of abdominal pain. Subsequent diagnostic evaluation revealed concurrent choledocholithiasis and cholecystolithiasis. The patient underwent surgical cholecystectomy as the therapeutic approach. Histopathological examination of the excised gallbladder disclosed characteristic features indicative of chronic cholecystitis. Subsequent laboratory analysis of the patient's bile specimen yielded Gram-positive cocci, subsequently identified through biochemical assays, mass spectrometry, and 16 S rRNA analysis as Vagococcus fluvialis. Further in vitro antimicrobial susceptibility testing using disk diffusion and microfluidic dilution showed that this strain exhibited inhibition zone diameters ranging from 12.0 to 32.0 mm in response to 26 antibiotics, including ampicillin, cefazolin, cefuroxime, cefotaxime, ceftriaxone, cefepime, ampicillin/sulbactam, piperacillin, ciprofloxacin, cefoperazone/sulbactam, imipenem, meropenem, piperacillin/tazobarb, penicillin, erythromycin, chloramphenicol, vancomycin, methotrexate/sulfamethoxazole, teicoplanin, linezolid, tigecycline, cefoxitin, ceftazidime, levofloxacin, minocycline and tobramycin. However, the inhibition zone diameters were 6.0 mm for amikacin, oxacillin, clindamycin, and tetracycline. The patient received ceftazidime anti-infective therapy both preoperatively and within 24 h postoperatively and was discharged successfully one week after surgery. CONCLUSION: In this study, we present the inaugural isolation and identification of Vagococcus fluvialis from bile specimens of patients afflicted with calculous cholecystitis. This novel finding lays a substantial experimental groundwork for guiding clinically rational antimicrobial therapy and advancing the exploration of relevant pathogenic mechanisms pertaining to Vagococcus fluvialis infections.

Recent advances and perspectives of functionalized carbon dots in bacteria sensing.

Bacterial infectious diseases are severe threats to human health and increase substantial financial burdens. Nanomaterials have shown great potential in timely and accurate bacterial identification, detection, and monitoring to improve the cure rate and reduce mortality. Recently, carbon dots have been evidenced to be ideal candidates for bacterial identification and detection due to their superior physicochemical properties and biocompatibility. This review outlines the detailed recognition elements and recognition strategies with functionalized carbon dots (FCDs) for bacterial identification and detection. The advantages and limitations of different kinds of FCDs-based sensors will be critically discussed. Meanwhile, the ongoing challenges and perspectives of FCDs-based sensors for bacteria sensing are put forward.

Bacterial Colony Phenotyping with Hyperspectral Elastic Light Scattering Patterns.

The elastic light-scatter (ELS) technique, which detects and discriminates microbial organisms based on the light-scatter pattern of their colonies, has demonstrated excellent classification accuracy in pathogen screening tasks. The implementation of the multispectral approach has brought further advantages and motivated the design and validation of a hyperspectral elastic light-scatter phenotyping instrument (HESPI). The newly developed instrument consists of a supercontinuum (SC) laser and an acousto-optic tunable filter (AOTF). The use of these two components provided a broad spectrum of excitation light and a rapid selection of the wavelength of interest, which enables the collection of multiple spectral patterns for each colony instead of relying on single band analysis. The performance was validated by classifying microflora of green-leafed vegetables using the hyperspectral ELS patterns of the bacterial colonies. The accuracy ranged from 88.7% to 93.2% when the classification was performed with the scattering pattern created at a wavelength within the 473-709 nm region. When all of the hyperspectral ELS patterns were used, owing to the vastly increased size of the data, feature reduction and selection algorithms were utilized to enhance the robustness and ultimately lessen the complexity of the data collection. A new classification model with the feature reduction process improved the overall classification rate to 95.9%.

Shotgun Proteomics Analysis, Functional Networks, and Peptide Biomarkers for Seafood-Originating Biogenic-Amine-Producing Bacteria.

Biogenic amine-producing bacteria are responsible for the production of basic nitrogenous compounds (histamine, cadaverine, tyramine, and putrescine) following the spoilage of food due to microorganisms. In this study, we adopted a shotgun proteomics strategy to characterize 15 foodborne strains of biogenic-amine-producing bacteria. A total of 10,673 peptide spectrum matches belonging to 4081 peptides and corresponding to 1811 proteins were identified. Relevant functional pathways were determined, and strains were differentiated into hierarchical clusters. An expected protein-protein interaction network was created (260 nodes/1973 interactions). Most of the determined proteins were associated with networks/pathways of energy, putrescine metabolism, and host-virus interaction. Additionally, 556 peptides were identified as virulence factors. Moreover, 77 species-specific peptide biomarkers corresponding to 64 different proteins were proposed to identify 10 bacterial species. This represents a major proteomic dataset of biogenic-amine-producing strains. These results may also be suitable for new treatments for food intoxication and for tracking microbial sources in foodstuffs.

Species-specific identification of Pseudomonas based on 16S-23S rRNA gene internal transcribed spacer (ITS) and its combined application with next-generation sequencing.

BACKGROUND: Pseudomonas species are widely distributed in the human body, animals, plants, soil, fresh water, seawater, etc. Pseudomonas aeruginosa is one of the main pathogens involved in nosocomial infections. It can cause endocarditis, empyema, meningitis, septicaemia and even death. However, the Pseudomonas classification system is currently inadequate and not well established. RESULTS: In this study, the whole genomes of 103 Pseudomonas strains belonging to 62 species available in GenBank were collected and the specificity of the 16S-23S ribosomal RNA internal transcribed spacer (ITS) sequence was analysed. Secondary structures of ITS transcripts determining where the diversity bases were located were predicted. The alignment results using BLAST indicated that the ITS sequence is specific for most species in the genus. The remaining species were identified by additional frequency analyses based on BLAST results. A double-blind experiment where 200 ITS sequences were randomly selected indicated that this method could identify Pseudomonas species with 100% sensitivity and specificity. In addition, we applied a universal primer to amplify the Pseudomonas ITS of DNA extracts from fish samples with next-generation sequencing. The ITS analysis results were utilized to species-specifically identify the proportion of Pseudomonas species in the samples. CONCLUSIONS: The present study developed a species-specific method identification and classification of Pseudomonas based on ITS sequences combined NGS. The method showed its potential application in other genera.

First record of isolation and characterization of Vibrio sinaloensis from diseased orange-spotted grouper Epinephelus coioides.

A dominant bacterium, ZYL-12, isolated from the liver of a diseased orange-spotted grouper Epinephelus coioides, was identified as Vibrio sinaloensis, based on phenotypic and molecular analysis. The median lethal dosage of ZYL-12 was calculated as 1.6 × 105 CFU g-1 fish weight. The infection experiment indicated that ZYL-12 caused noticeable histological lesions to the liver, kidney and spleen of the fish. Growth characteristics showed that ZYL-12 possessed strong environmental adaptability. This note is the first report about the pathogenicity of V. sinaloensis isolated from diseased fish.

Bacteria in the cavity-restoration interface after varying periods of clinical service - SEM description of distribution and 16S rRNA gene sequence identification of isolates.

OBJECTIVES: To use extracted human teeth with amalgam (n = 26) or GIC (n = 3) restorations in service up to 20 years to evaluate microbiota at the cavity/restoration interface by SEM or culture. MATERIALS AND METHODS: Extracted teeth with intracoronal restorations (n = 20) of known history (2-20 years) were fixed, split, and prepared for SEM to ascertain the pattern and structure of bacterial aggregates on cavity and restoration surfaces. Another 9 teeth were anaerobically decontaminated, split and sampled (cavity/restorations), and cultured (anaerobically, aerobically); recovered isolates were identified by 16S rRNA gene sequencing. RESULTS: SEM showed rods, cocci, and filaments in 11/20 teeth (55%) on cavity and corresponding restoration surfaces; 4/20 (20%) on neither surface; 1/20 (5%) on just cavity; and 4/20 (20%) on just restoration. Microbial growth extended from marginal openings into the deeper interfacial microspace to varying extents but was not always evident. Restoration size or age did not predict bacterial presence. Bacteria-free surfaces (cavity/amalgam) showed possible calcification. Cultivation yielded 160 isolates, mainly Gram-positive (86%) and facultative (81%); and morphotypes of rods (43%), cocci (36%), and cocco-bacilli (18%) belonging to Actinobacteria (45%) and Firmicutes (50%). The most frequent genera were Staphylococcus, Streptococcus, Actinomyces, and Lactobacillus. Biofilms on cavity and restoration appeared independent of each other. CONCLUSIONS: Cavity and amalgam surfaces were independently colonised and some not. The penetration of microbiota into marginal gaps varied; resembled root caries and was dominated by Gram-positive species. CLINICAL RELEVANCE: Marginal gaps around restorations are unavoidable but are not always colonised by bacteria after long-term clinical service. Calcification of biofilms in the restorative interface may prevent further colonisation. The viable microbiota in the restorative interface resembled root caries and may be subject to ecological fluxes of activity and arrest and therefore preventative management.

Development of a Smartphone-Integrated Reflective Scatterometer for Bacterial Identification.

We present a smartphone-based bacterial colony phenotyping instrument using a reflective elastic light scattering (ELS) pattern and the resolving power of the new instrument. The reflectance-type device can acquire ELS patterns of colonies on highly opaque media as well as optically dense colonies. The novel instrument was built using a smartphone interface and a 532 nm diode laser, and these essential optical components made it a cost-effective and portable device. When a coherent and collimated light source illuminated a bacterial colony, a reflective ELS pattern was created on the screen and captured by the smartphone camera. The collected patterns whose shapes were determined by the colony morphology were then processed and analyzed to extract distinctive features for bacterial identification. For validation purposes, the reflective ELS patterns of five bacteria grown on opaque growth media were measured with the proposed instrument and utilized for the classification. Cross-validation was performed to evaluate the classification, and the result showed an accuracy above 94% for differentiating colonies of E. coli, K. pneumoniae, L. innocua, S. enteritidis, and S. aureus.

Evaluation of the Accelerate Pheno System for Rapid Identification and Antimicrobial Susceptibility Testing of Positive Blood Culture Bottles Inoculated with Primary Sterile Specimens from Patients with Suspected Severe Infections.

The Accelerate Pheno system is approved for rapid identification and phenotypic antimicrobial susceptibility testing (AST) of microorganisms grown from positive blood cultures inoculated with blood from septic patients. We evaluated the performance of the system for identification and AST from positive blood culture bottles inoculated with primary sterile nonblood specimens from patients with suspected severe infections. One hundred positive blood culture bottles with primary sterile specimens (63 cerebrospinal fluids, 16 ascites, 7 pleural fluids, 4 vitreous fluids, 5 joint aspirates, and 5 other aspirates) from 100 patients were included. Pathogen identification was in agreement with conventional methods for 72 of 100 cultures (72%) and for 81 of 112 (72%) pathogens when considering all pathogens and for 72 of 92 (78%) cultures and 81 of 104 (78%) pathogens when considering on-panel pathogens only. Eight of 31 isolates (26%) not identified by APS were pathogens not included in the APS panel. APS and conventional methods accordingly identified all pathogens from two of nine polymicrobial cultures (22%). APS generated antimicrobial resistance results for 57 pathogens of 57 cultures. The overall category agreement between APS and culture-based AST was 91.2%; and the rate for minor errors was 6.9%, for major was 1.7%, and for very major errors was 0.2%. APS may accelerate pathogen identification and phenotypic AST from positive blood culture bottles inoculated with primary sterile specimens from patients with serious infections, especially for hospitals without an on-site microbiology laboratory. However, the inclusion of nonblood specimens with a high likelihood of polymicrobial infections may result in an inferior performance.

Phenotypic and Genomic Profiling of Staphylococcus argenteus in Canada and the United States and Recommendations for Clinical Result Reporting.

Staphylococcus argenteus is a newly described species, formerly known as S. aureus clonal complex 75 (CC75). Here, we describe the largest collection of S. argenteus isolates in North America, highlighting identification challenges. We present phenotypic and genomic characteristics and provide recommendations for clinical reporting. Between 2017 and 2019, 22 isolates of S. argenteus were received at 2 large reference laboratories for identification. Identification with routine methods (biochemical, matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS], 16S rRNA gene analysis) proved challenging to confidently distinguish these isolates from S. aureus Whole-genome sequencing analysis was employed to confirm identifications. Using several different sequence-based analyses, all clinical isolates under investigation were confirmed to be S. argenteus with clear differentiation from S. aureus Seven of 22 isolates were recovered from sterile sites, 11 from nonsterile sites, and 4 from surveillance screens. While sequence types ST1223/coa type XV, ST2198/coa type XIV, and ST2793/coa type XId were identified among the Canadian isolates, the majority of isolates (73%) belonged to multilocus sequence types (MLST) ST2250/coa type XId and exhibited a high degree of homology at the genomic level. Despite this similarity, 5 spa types were identified among ST2250 isolates, demonstrating some diversity between strains. Several isolates carried mecA, as well as other resistance and virulence determinants (e.g., PVL, TSST-1) commonly associated with S. aureus Based on our findings, the growing body of literature on S. argenteus, the potential severity of infections, and possible confusion associated with reporting, including use of incorrect breakpoints for susceptibility results, we make recommendations for clinical laboratories regarding this organism.

Sputum sample positivity for Haemophilus influenzae or Moraxella catarrhalis in acute exacerbations of chronic obstructive pulmonary disease: evaluation of association with positivity at earlier stable disease timepoints.

BACKGROUND: Infection with Haemophilus influenzae (Hi) or Moraxella catarrhalis (Mcat) is a risk factor for exacerbation in chronic obstructive pulmonary disease (COPD). The ability to predict Hi- or Mcat-associated exacerbations may be useful for interventions developed to reduce exacerbation frequency. METHODS: In a COPD observational study, sputum samples were collected at monthly stable-state visits and at exacerbation during two years of follow-up. Bacterial species (Hi, Mcat) were identified by culture and quantitative PCR assay. Post-hoc analyses were conducted to assess: (1) first Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at the screening visit (stable-state timepoint); (2) first Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at stable timepoints within previous 90 days; (3) second Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at stable timepoints within previous 90 days. Percentages and risk ratios (RRs) with 95% confidence intervals were calculated. RESULTS: PCR results for analyses 1, 2 and 3 (samples from 84, 88 and 83 subjects, respectively) showed that the risk of an Hi- or Mcat-positive exacerbation is significantly higher if sputum sample was Hi- or Mcat-positive than if Hi- or Mcat-negative at previous stable timepoints (apart from Mcat in analysis 3); RRs ranged from 2.1 to 3.2 for Hi and 1.9 to 2.6 for Mcat.For all analyses, the percentage of Hi- or Mcat-positive exacerbations given previous Hi- or Mcat-positive stable timepoints was higher than the percentage of Hi- or Mcat-positive exacerbations if Hi- or Mcat-negative at previous stable timepoints. Percentage of Hi- or Mcat-positive exacerbations given previous Hi- or Mcat-negative stable timepoints was 26.3%-37.0% for Hi and 17.6%-19.7% for Mcat. CONCLUSIONS: Presence of Hi or Mcat at a stable timepoint was associated with a higher risk of a subsequent Hi- or Mcat-associated exacerbation compared with earlier absence. However, a large percentage of Hi- or Mcat-associated exacerbations was not associated with Hi/Mcat detection at an earlier timepoint. This suggests that administration of an intervention to reduce these exacerbations should be independent of bacterial presence at baseline. Trial Registration https://clinicaltrials.gov/ ; NCT01360398, registered May 25, 2011.

Direct detection of Corynebacterium striatum, Corynebacterium propinquum, and Corynebacterium simulans in sputum samples by high-resolution melt curve analysis.

BACKGROUND: Pulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus. METHODS: Three reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay's performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay. RESULTS: The melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5-99.9%) sensitivity and 100% (95% CI, 82.8-100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79-0.99]) was noted. CONCLUSIONS: The HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy combined with chemometric modelling for the classification of clinically relevant Enterococci.

AIMS: Attenuated Total Reflection Fourier Transform Infrared (ATR-FT-IR) Spectroscopy and chemometric modelling, including soft independent modelling by class analogy (SIMCA), partial least squares discriminant analysis (PLS-DA) and support vector machine (SVM), were applied to attempt to discriminate 60 clinical isolates of Enterococcus faecium and Enterococcus faecalis and hence evaluate the performance of the spectroscopic approach in identifying enterococci infections. METHODS AND RESULTS: The bacterial samples were identified by polymerize chain reaction (PCR) amplification and their ATR-FT-IR spectra acquired. Spectra were processed to the second derivative using the Savitzky-Golay algorithm and normalized using extended multiplicative signal correction employing the UnscramblerX (CAMO, Norway) software package. Multivariate classification models and their performance were evaluated using Cohen's Kappa coefficient. Principal component analysis (PCA) score plots showed separate clusters of spectra related to membership to E. faecium and E. faecalis, with this explained by bands assigned to PO2 (1230 cm-1 ), P-O-C (1114 cm-1 ), monosubstituted alkene (997, 987 cm-1 ) and C-O (1070, 1055, 1036 cm-1 ) corresponding to teichoic acids, polysaccharides and peptidoglycan from the cell wall in PCA and PLS-DA loading plots. The best classification model for E. faecium and E. faecalis is SVM, indicating via highest Kappa score. The classification coefficient between SIMCA, PLS-DA, SVM and PCR as reference method were 0·59, 0·9 and 1, respectively, shown as the Kappa scores. CONCLUSIONS: The main spectral differences observed between the two clinically relevant enterococci species were associated with changes in the teichoic acid content of cell walls. With regard to the binary classification method, SVM was found to be the best performing classification model, providing the highest correlation with the PCR results. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that ATR-FT-IR spectroscopy in combination with chemometric modelling can be applied for the phenotypic identification and discrimination of clinically relevant and similar enterococcal species.

Rapid identification of histamine-producing bacteria isolated from fish using MALDI-TOF MS.

Histamine-producing bacteria (HPB) produce histamine from histidine contained in food through the action of histidine decarboxylase. To identify HPB isolated from food, it is necessary to detect histamine produced by the bacteria. In this study, we concurrently identified HPB and detected histamine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After 24 h of incubation, 30 of 34 bacterial strains were correctly identified. Histamine was detected in all HPB cultured on Niven's medium, and 94% of HPB cultured in histidine broth, except for two strains with low histamine production. This method may greatly simplify the procedure and reduce the time required to identify HPB.

Discovery of thermophilic Bacillales using reduced-representation genotyping for identification.

BACKGROUND: This study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome. RESULTS: Based on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases. CONCLUSIONS: Previously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.