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Introduction: Veterinary clinical microbiology laboratories play a key role in antimicrobial stewardship, surveillance of antimicrobial resistance and prevention of healthcare associated-infections. However, there is a shortage of international harmonized guidelines covering all steps of veterinary bacterial culture from sample receipt to reporting. Methods: In order to gain insights, the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) designed an online survey focused on the practices and interpretive criteria used for bacterial culture and identification (C&ID), and antimicrobial susceptibility testing (AST) of animal bacterial pathogens. Results: A total of 241 microbiology laboratories in 34 European countries completed the survey, representing a mixture of academic (37.6%), governmental (27.4%), and private (26.5%) laboratories. The C&ID turnaround varied from 1 to 2 days (77.8%) to 3-5 days (20%), and 6-8 days (1.6%), with similar timeframes for AST. Individual biochemical tests and analytical profile index (API) biochemical test kits or similar were the most frequent tools used for bacterial identification (77% and 56.2%, respectively), followed by PCR (46.6%) and MALDI-TOF MS (43.3%). For AST, Kirby-Bauer disk diffusion (DD) and minimum inhibitory concentration (MIC) determination were conducted by 43.8% and 32.6% of laboratories, respectively, with a combination of EUCAST and CLSI clinical breakpoints (CBPs) preferred for interpretation of the DD (41.2%) and MIC (47.6%) results. In the absence of specific CBPs, laboratories used human CBPs (53.3%) or veterinary CBPs representing another body site, organism or animal species (51.5%). Importantly, most laboratories (47.9%) only report the qualitative interpretation of the result (S, R, and I). As regards testing for AMR mechanisms, 48.5% and 46.7% of laboratories routinely screened isolates for methicillin resistance and ESBL production, respectively. Notably, selective reporting of AST results (i.e. excluding highest priority critically important antimicrobials from AST reports) was adopted by 39.5% of laboratories despite a similar proportion not taking any approach (37.6%) to guide clinicians towards narrower-spectrum or first-line antibiotics. Discussion: In conclusion, we identified a broad variety of methodologies and interpretative criteria used for C&ID and AST in European veterinary microbiological diagnostic laboratories. The observed gaps in veterinary microbiology practices emphasize a need to improve and harmonize professional training, innovation, bacterial culture methods and interpretation, AMR surveillance and reporting strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Detecting pathogenic bacteria and their phenotypes including microbial resistance is crucial for preventing infection, ensuring food safety, and promoting environmental protection. Raman spectroscopy offers rapid, seamless, and label-free identification, rendering it superior to gold-standard detection techniques such as culture-based assays and polymerase chain reactions. However, its practical adoption is hindered by issues related to weak signals, complex spectra, limited datasets, and a lack of adaptability for detection and characterization of bacterial pathogens. This review focuses on addressing these issues with recent Raman spectroscopy breakthroughs enabled by machine learning (ML), particularly deep learning methods. Given the regulatory requirements, consumer demand for safe food products, and growing awareness of risks with environmental pathogens, this study emphasizes addressing pathogen detection in clinical, food safety, and environmental settings. Here, we highlight the use of convolutional neural networks for analyzing complex clinical data and surface enhanced Raman spectroscopy for sensitizing early and rapid detection of pathogens and analyzing food safety and potential environmental risks. Deep learning methods can tackle issues with the lack of adequate Raman datasets and adaptability across diverse bacterial samples. We highlight pending issues and future research directions needed for accelerating real-world impacts of ML-enabled Raman diagnostics for rapid and accurate diagnosis and surveillance of pathogens across critical fields. [ABSTRACT FROM AUTHOR]

IntroductionVeterinary clinical microbiology laboratories play a key role in antimicrobial stewardship, surveillance of antimicrobial resistance and prevention of healthcare associated-infections. However, there is a shortage of international harmonized guidelines covering all steps of veterinary bacterial culture from sample receipt to reporting.MethodsIn order to gain insights, the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) designed an online survey focused on the practices and interpretive criteria used for bacterial culture and identification (C&ID), and antimicrobial susceptibility testing (AST) of animal bacterial pathogens.ResultsA total of 241 microbiology laboratories in 34 European countries completed the survey, representing a mixture of academic (37.6%), governmental (27.4%), and private (26.5%) laboratories. The C&ID turnaround varied from 1 to 2 days (77.8%) to 3–5 days (20%), and 6– 8 days (1.6%), with similar timeframes for AST. Individual biochemical tests and analytical profile index (API) biochemical test kits or similar were the most frequent tools used for bacterial identification (77% and 56.2%, respectively), followed by PCR (46.6%) and MALDI-TOF MS (43.3%). For AST, Kirby-Bauer disk diffusion (DD) and minimum inhibitory concentration (MIC) determination were conducted by 43.8% and 32.6% of laboratories, respectively, with a combination of EUCAST and CLSI clinical breakpoints (CBPs) preferred for interpretation of the DD (41.2%) and MIC (47.6%) results. In the absence of specific CBPs, laboratories used human CBPs (53.3%) or veterinary CBPs representing another body site, organism or animal species (51.5%). Importantly, most laboratories (47.9%) only report the qualitative interpretation of the result (S, R, and I). As regards testing for AMR mechanisms, 48.5% and 46.7% of laboratories routinely screened isolates for methicillin resistance and ESBL production, respectively. Notably, selective reporting of AST results (i.e. excluding highest priority critically important antimicrobials from AST reports) was adopted by 39.5% of laboratories despite a similar proportion not taking any approach (37.6%) to guide clinicians towards narrower-spectrum or first-line antibiotics.DiscussionIn conclusion, we identified a broad variety of methodologies and interpretative criteria used for C&ID and AST in European veterinary microbiological diagnostic laboratories. The observed gaps in veterinary microbiology practices emphasize a need to improve and harmonize professional training, innovation, bacterial culture methods and interpretation, AMR surveillance and reporting strategies.

Abstract 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

The high demand for fish consumption has an impact on increasing aquaculture productivity and causes the vulnerability of increasing bacterial populations in aquaculture fields, so more rapid handling is needed. The use of phenotypic KIT methods (API 20E) has been applied as a targeted and efficient identification support in targeting better bacterial identification accuracy but often provides unequal results. Method validation is one of the general requirements for the competence of a laboratory evaluation: to provide coherent, interpretable, and accurate results with known uncertainties. The purpose of this study was to validate the API 20E KIT method for the identification of A. hydrophila. The conventional method used as a reference is SNI 7303.1:2015. The validation parameters consisted of determining the limit of detection, sensitivity, and specificity tests, as well as the positive predictive value and negative predictive value. The results showed that the limit of the detection value of the API 20E KIT was at a concentration of 100 CFU/mL with an ID of 99.00%. The sensitivity and specificity values in the positive and negative target samples were 100% with a positive predictive value and a negative predictive value of 0%, respectively. In conclusion, the API 20E KIT method as an alternative test method or rapid test was proven valid for identifying A. hydrophila by the test results using the reference method.

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, β-lactam/β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Infectious diseases caused by bacterial pathogens pose a significant public health threat, emphasizing the need for swift and accurate bacterial species detection methods. Hyperspectral microscopic imaging (HMI) offers nondestructive, rapid, and data‐rich advantages, making it a promising tool for microbial detection. In this research, we present a highly compatible and cost‐effective approach to extend a standard biomicroscope system into a hyperspectral biomicroscope using a prism‐grating‐prism configuration. Using this prototype, we generate 600 hyperspectral data cubes for Listeria, Bacillus typhi, Bacillus pestis, and Bacillus anthracis. Additionally, we propose a Transformer‐based classification network that achieves a 99.44% accuracy in classifying these infectious pathogens, outperforming traditional methods. Our results suggest that the successful combination of HMI and the optimized Transformer‐based classification network highlights the potential for rapid and precise detection of infectious disease pathogens. [ABSTRACT FROM AUTHOR]

One of the causes of disease attacks can be caused by bacteria, two of which are Edwardsiella ictaluri and Streptococcus agalactiae which often attack catfish. One of the causes of disease attacks can be caused by bacteria, two of which are Edwardsiella ictaluri and Streptococcus agalactiae which often attack catfish. Disease attacks can be prevented as early as possible by periodically identifying certain bacteria so that it can facilitate appropriate treatment to prevent them. This activity aimed to study bacterial identification techniques in catfish (Pangasius sp.) at the Fish Quarantine Station for Quality Control and Safety of Fishery Products in Palembang. The method used in this activity is quantitative by field observation and collecting primary and secondary data. Activities carried out include the preparation of tools and materials. Media creation, test sample preparation, bacterial isolation, bacterial purification, basic tests, biochemical tests, and instrument digestion. The approach was carried out through basic tests and biochemical tests. Data analysis was presented in tabular form and discussed descriptively. Results: There were no bacteria with the characteristics of Edwardsiella ictaluri and Streptococcus agalactiae found in catfish observed from December 2022 to January 2023. [ABSTRACT FROM AUTHOR]

The high demand for fish consumption has an impact on increasing aquaculture productivity and causes the vulnerability of increasing bacterial populations in aquaculture fields, so more rapid handling is needed. The use of phenotypic KIT methods (API 20E) has been applied as a targeted and efficient identification support in targeting better bacterial identification accuracy but often provides unequal results. Method validation is one of the general requirements for the competence of a laboratory evaluation: to provide coherent, interpretable, and accurate results with known uncertainties. The purpose of this study was to validate the API 20E KIT method for the identification of A. hydrophila. The conventional method used as a reference is SNI 7303.1:2015. The validation parameters consisted of determining the limit of detection, sensitivity, and specificity tests, as well as the positive predictive value and negative predictive value. The results showed that the limit of the detection value of the API 20E KIT was at a concentration of 100 CFU/mL with an ID of 99.00%. The sensitivity and specificity values in the positive and negative target samples were 100% with a positive predictive value and a negative predictive value of 0%, respectively. In conclusion, the API 20E KIT method as an alternative test method or rapid test was proven valid for identifying A. hydrophila by the test results using the reference method. [ABSTRACT FROM AUTHOR]

Gram staining can classify bacterial species into two large groups based on cell wall differences. Our study revealed that within the same gram group (gram‐positive or gram‐negative), subtle cell wall variations can alter staining outcomes, with the peptidoglycan layer and lipid content significantly influencing this effect. Thus, bacteria within the same group can also be differentiated by their spectra. Using hyperspectral microscopy, we identified six species of intestinal bacteria with 98.1% accuracy. Our study also demonstrated that selecting the right spectral band and background calibration can enhance the model's robustness and facilitate precise identification of varying sample batches. This method is suitable for analyzing bacterial community pathologies. [ABSTRACT FROM AUTHOR]

Bacterial cellulose (BC) stands out as a prominent biopolymer of global importance, distinguished by its unique advantages over plant-derived cellulose. Strains such as Acetobacter xylinum, renowned for their proficient BC production, draw considerable attention in both commercial and biomedical areas. This research aimed to selectively isolate cellulose-producing bacteria with enhanced efficiency from a variety of fruit samples utilizing a cost-effective methodology. A total of 60 fruit samples were selected, and the assessment focused on 17 strains derived from rotten banana, red apple, green apple, and pineapple samples. The evaluation encompassed an examination of bacteriological traits and cellulose synthesis, with subsequent identification of strains achieved through DNA extraction and 16S rRNA PCR analysis. The experimental findings reveal cellulose-producing strains, including model A. xylinum (KCCM 40407) obtained from the Pharmacy lab of COMSATS University Islamabad Abbottabad Campus, Pakistan, designated as number 2, serving as a control. Notably, strains isolated from deteriorated fruits (samples 1, 4, 8, 11, 12, and 15) demonstrated the capacity to produce soluble cellulose. A. xylinum (model strain 2) was cultured under static conditions in HS media, demonstrating remarkable efficacy for cellulose sheet production. Subsequent characterization employing scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) unveiled a nano-fiber mat featuring multi-layered fibers. This eco-friendly approach has the potential for large-scale, high-quality cellulose production, applicable in biomedical and industrial fields. The research highlights an environmentally sustainable and economically viable method for cellulose production, presenting potential applicability across biomedical and industrial arenas on a significant scale.

Abstract 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.

Bacterial cellulose (BC) stands out as a prominent biopolymer of global importance, distinguished by its unique advantages over plant-derived cellulose. Strains such as Acetobacter xylinum, renowned for their proficient BC production, draw considerable attention in both commercial and biomedical areas. This research aimed to selectively isolate cellulose-producing bacteria with enhanced efficiency from a variety of fruit samples utilizing a cost-effective methodology. A total of 60 fruit samples were selected, and the assessment focused on 17 strains derived from rotten banana, red apple, green apple, and pineapple samples. The evaluation encompassed an examination of bacteriological traits and cellulose synthesis, with subsequent identification of strains achieved through DNA extraction and 16S rRNA PCR analysis. The experimental findings reveal cellulose-producing strains, including model A. xylinum (KCCM 40407) obtained from the Pharmacy lab of COMSATS University Islamabad Abbottabad Campus, Pakistan, designated as number 2, serving as a control. Notably, strains isolated from deteriorated fruits (samples 1, 4, 8, 11, 12, and 15) demonstrated the capacity to produce soluble cellulose. A. xylinum (model strain 2) was cultured under static conditions in HS media, demonstrating remarkable efficacy for cellulose sheet production. Subsequent characterization employing scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) unveiled a nano-fiber mat featuring -layered fibers. This eco-friendly approach has the potential for large-scale, high-quality cellulose production, applicable in biomedical and industrial fields. The research highlights an environmentally sustainable and economically viable method for cellulose production, presenting potential applicability across biomedical and industrial arenas on a significant scale. [ABSTRACT FROM AUTHOR]

Objective This study aimed to evaluate the impact of short-term incubation (STI) protocol on clinical outcomes of bloodstream infection (BSI) patients. Methods A total of 1363 positive blood culture records from January 2019 to December 2021 were included. The main clinical outcomes included pathogen identification turnaround time (TAT), antimicrobial susceptibility testing (AST) TAT, and length of total hospital stay. Results The TAT of pathogen identification and AST significantly decreased after implementing the STI protocol (2.2 vs 1.4 days and 3.4 vs 2.5 days, respectively, with P <.001 for both). Moreover, for patients with Gram-negative bacteria (GNB)–infected BSIs, the length of total hospital stay decreased from 31.9 days to 27.1 days, indicating that these patients could be discharged 5 days earlier after implementing the STI protocol (P <.01). Conclusion The protocol led to a significant reduction in TAT and improved clinical outcomes, particularly for GNB organisms. The findings suggest that the STI protocol can improve patient outcomes and hospital resource utilization in the management of BSIs. [ABSTRACT FROM AUTHOR]

Phenotypic identification of bacteria by staining technique and biochemical tests. This practical was carried out to identify Proteus vulgaris using biochemical test, this test includes, citrate utilization, indole, carbohydrate fermentation, methyl red, voges Proskauer, motility, production of hydrogen sulphide, catalase, and oxidase tests. Results obtained shows that Proteus vulgaris was positive to indole, motility, methyl red and urease. Proteus vulgaris was unable to utilize citrate for carbon and energy, and does not synthesis the enzyme amylase to break down starch. The bacteria fermented glucose to produce acid and gas. [ABSTRACT FROM AUTHOR]

Antibiotics, metabolites of the antibiotics, and resistant bacteria reach the aquatic environment through treated and untreated sewage, hospital waste, aquaculture discharges and agricultural irrigation. Therefore, aquatic environments have a significant role in spreading the antibiotic-resistance genes. The current study demonstrates the prevalence of antibioticresistant bacteria in surface water and floating islands of Çat Dam Lake, Adiyaman, Turkey. A total of 79 E. coli colonies were isolated from Çat Dam Lake water samples and the floating islands, of which 36 were from the first period (August 2021), 28 were from the second period (November 2021), and 15 were from the third period (May 2022), which were also confirmed as E. coli by polymerase chain reaction (PCR). The confirmed isolates were tested for susceptibility using the EUCAST protocol. The results showed that the prevalence of resistance to erythromycin (E), ceftaroline (CPT) and cefazolin (CZ) was significantly higher than other tested antibiotics. In total, 96.2% of the isolated bacteria from all three periods were resistant to E, 77.21% to CPT and 48.1% to CZ, 12.65% to tetracycline (TE), 8.86% to cefuroxime (CXM), 6.32% to chloramphenicol (C) and cefotaxime (CTX), 2.53% to cefepime (FEP) and 1.26% to imipenem (IPM) and gentamicin (CN). Thirteen (16.5%) isolates were found with a high multiple antibiotic resistance (MAR) index. The observed MAR index could be due to the contamination of water sources with antibiotics used in the surrounding areas. It throws a potential risk to the local population from antimicrobial-resistant infections that can lead to serious side effects such as organ failures. [ABSTRACT FROM AUTHOR]

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Abstract 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.

Detecting pathogenic bacteria and their phenotypes including microbial resistance is crucial for preventing infection, ensuring food safety, and promoting environmental protection. Raman spectroscopy offers rapid, seamless, and label-free identification, rendering it superior to gold-standard detection techniques such as culture-based assays and polymerase chain reactions. However, its practical adoption is hindered by issues related to weak signals, complex spectra, limited datasets, and a lack of adaptability for detection and characterization of bacterial pathogens. This review focuses on addressing these issues with recent Raman spectroscopy breakthroughs enabled by machine learning (ML), particularly deep learning methods. Given the regulatory requirements, consumer demand for safe food products, and growing awareness of risks with environmental pathogens, this study emphasizes addressing pathogen detection in clinical, food safety, and environmental settings. Here, we highlight the use of convolutional neural networks for analyzing complex clinical data and surface enhanced Raman spectroscopy for sensitizing early and rapid detection of pathogens and analyzing food safety and potential environmental risks. Deep learning methods can tackle issues with the lack of adequate Raman datasets and adaptability across diverse bacterial samples. We highlight pending issues and future research directions needed for accelerating real-world impacts of ML-enabled Raman diagnostics for rapid and accurate diagnosis and surveillance of pathogens across critical fields.

Xiaosu Wang,&ast; Dan Zhang,&ast; Zheng Dai, Jingdan Yu, Qichao Li, Litao Zhang Department of Clinical Laboratory, Wuhan Asia General Hospital, Wuhan Asia General Hospital Affiliated to Wuhan University of Science and Technology, Wuhan, Hubei Province, 430056, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Litao Zhang, Email dandanzhangnihao@126.comIntroduction: W. confusa has been known to cause various human infections. It is naturally resistant to vancomycin and is difficult to identify using traditional methods, which may lead to misidentification and delay treatment.Case Presentation: We present a case of a 42-year-old male patient with gastrointestinal bleeding and coronary heart disease who developed sepsis caused by Weissella confusa. The patient’s blood cultures showed the presence of gram-positive coccobacilli, later identified as W. confusa through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing revealed that W. confusa had low minimum inhibitory concentrations (MICs) for some drugs (eg, ampicillin) and higher MICs for others (eg, cefotaxime). Empirical treatment with vancomycin was initially started, but after obtaining the identification and susceptibility results, the treatment was switched to meropenem combined with daptomycin, resulting in a successful outcome.Conclusion: Weissella confusa bacteremia is relatively rare, and accurate pathogenic diagnosis is essential for effective clinical treatment.Keywords: Weissella confusa, bacterial identification, 16S rRNA, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.

Introduction: In this work, we target the analysis and the characterization of bioaerosols species present in medical emergency of north Algerian hospital, were we consider in four operating rooms, two preoperative and resuscitation rooms. Materials and methods: Passive technique was applied for the collection of bacterial and fungal samples in indoor air, for three days from 16 to 18 January 2018. Two techniques were then chosen for the bacterial identification, the Analytical Profile Index (API) system and Matrix Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Results: It has been found that fungal contamination was highest in neurosurgery block, 103 CFU/m3 , whereas the highest bacterial contamination, 2645 CFU/m3 , was noted in postoperative room. The most predominant identified bacteria were Gram-positive cocci. Conclusion: The high contamination in bioaerosols and the types of bacteria identified in the premises studied increase the risk of contracting a nosocomial infection, hence the importance of daily disinfection and sterilization of hospitals area in order to reduce bioaerosols dangerous effects on human health

The recent advent of third-generation sequencing technologies brings promise for better bacterial identification. However, the commercial applications of the current sequencing technologies are still constrained since their high cost and error rate of this sequencing technology. Thus, strategies for time-saving bacterial identification with low cost and high accuracy are urgently needed. In this study, we evaluated the performance of the Nanopore long-read sequencer and the Illumina platform alone or in combination with different data volume (0.5–80×) after assembly, to ascertain whether the incomplete genomes enable accurate bacterial identification. The results showed that the average nucleotide identity value of hybrid assembly and Illumina assembly does not change much as the amount of data increases. In addition, the ANI value reached a plateau at 5×, while the ANI values of Nanopore-only assemblies increased in the range of 5–30×. Since the assembly process was time-consuming, this study compared the accuracy of the Nanopore long-read sequencer in species identification with various sets of the reads (0.5–80×), to further confirm the minimum amount of data required for accurate bacteria identification. Without assembly, it enabled rapid and accurate bacterial identification with low coverage of Nanopore raw reads (0.5×). The results of study suggested the potential of Nanopore sequencing in rapid bacteria identification using low-depth raw reads (0.5×) without assembly, and provided data basis in developing new strategy for bacterial identification. [ABSTRACT FROM AUTHOR]

Lobsters and crayfishes have social, economic and ecological significance in several regions around the world. These crustaceans have complex digestive systems and open circulatory systems colonized by symbiotic microorganisms that play essential roles in the health, nutrition and disease of crustaceans, attracting considerable interest. The importance of the microbiota for the successful cultivation of animals in captivity is known, however, there is a lack of information about the symbiotic microbiota in different species of lobsters. The aim of this study was to summarize global information on microbiota studies of anatomical sites of lobsters and crayfishes looking for patterns of bacteria that colonize their intestine, hepatopancreas and haemolymph. A systematic review was conducted using "Methodi Ordinatio". Twenty-five significant articles, from 12 countries, were identified for analysis from the Scopus database. The crustacean genera found were Cherax , Pacifastacus and Procambarus (crayfish); Homarus , Nephrops and Panulirus (lobsters). The intestinal, hepatopancreas and haemolymph microbiota show a wide variety of bacteria influenced by infections affecting the individual, stress caused by changes in the environment, availability and quality of food, period of the year, stage of development and the animal's sex, among other aspects. The common core bacterial microbiota among crustaceans consist of the genera Acinetobacter , Aeromonas , Candidatus Bacilloplasma, Candidatus Hepatoplasma, Citrobacter , Hafnia , Photobacterium , Pseudomonas , Shewanella and Vibrio. The results broadened our understanding of the microbiota in the intestinal and circulatory systems of crustaceans, the flaws for and needs for standardization of analysis techniques, and the use of a multi-specialized approach for the protection and management of lobsters as a fisheries resource. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Probiotics are microorganisms that exert strain-specific health-promoting effects on the host. Τhey are employed in the production of functional dairy or non-dairy food products; still, their detection in these complex matrices is a challenging task. Several culture-dependent and culture-independent methods have been developed in this direction; however, they present low discrimination at the strain level. Here, we developed a multiplex PCR assay for the detection of two potential probiotic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum L125 and Lp. pentosus L33, in monocultures and yogurt samples. Unique genomic regions were identified via comparative genomic analysis and were used to produce strain-specific primers. Then, primer sets were selected that produced distinct electrophoretic DNA banding patterns in multiplex PCR for each target strain. This method was further implemented for the detection of the two strains in yogurt samples, highlighting its biotechnological applicability. Moreover, it can be applied with appropriate modifications to detect any bacterial strain with available WGS. [ABSTRACT FROM AUTHOR]

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA’s Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 103 and 1 × 105 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus-related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

Using the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method for bacterial diagnosis, rapid urine sample preparation can reduce time relapsing of diagnosis and improve discriminatory power in coinfection cases. We aimed to evaluate rapid urine preparation procedures before MALDI-TOF MS application using dog clinical urine samples in comparison with standard microbiological diagnostic methods by agreement analysis. We determined the frequency and distribution of bacteria and bacterial resistance and their correlations to clinical history. Three experimental procedures comprising direct centrifugation, 10% sodium dodecyl sulfate digestion, and ultrasonic preparation were performed for method validation and sensitivity. Sterile urine containing Escherichia coli and/or Staphylococcus aureus were used as simulated samples. By ultrasonic preparation, the microorganisms could be detected 1.46–1.51 × 105 CFU, which was considered the most suitable technique. This preparation was significantly consistent with the routine method based on data from Hospital Information Systems for 50 urine samples from canine cystitis. By standard protocol, Enterobacteriaceae and Staphylococcus pseudintermedius were found in most of the 155 urine samples with cystitis. Extended-spectrum beta-lactamase-producing Enterobacteriaceae was found in 25–30% of the samples. Imipenem resistance was found in 70% of Acinetobacter baumannii cases; almost all were resistant to second-generation fluoroquinolones and tetracyclines. The most efficient antibiotic for treating bacterial urinary tract infection was amoxicillin plus clavulanic acid. A. baumannii and Pseudomonas aeruginosa were susceptible to pradofloxacin. Prolonged urine catheterization was linked to lower urinary tract infections by Enterobacter spp., which also correlated with chronic kidney disease. [ABSTRACT FROM AUTHOR]

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Introduction: Microbial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRITOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria. Methods: We constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures. Results: For validation, we used 58 sequence-confirmed strains and the CLOSTRITOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates. Discussion: We describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS. [ABSTRACT FROM AUTHOR]

The provision of potable drinking water is of public health concern, especially in developing countries. This study aimed to test the bacteriological quality of well water in El-Qalubia governorate, Egypt. Untreated well water samples were collected from thirteen sites all over the governorate. The most probable number of tests (presumptive and confirmed tests) revealed that water is unpotable for drinking. Five pathogenic microorganisms were isolated from samples. These isolates were identified by biochemical and phytic tests as Klebsiella Pneumonia, Enterobacter sp., Pseudomonas aeruginosa, Citrobacter braaki, and E. coli. Antibiotic susceptibility test for these isolates confirmed their multidrug resistance. Thus, this study has shown a high level of bacterial contamination in all the samples. Hence, the need for good maintenance and hygienic practices by households to reduce the risk of disease outbreaks from the organisms encountered in this study. [ABSTRACT FROM AUTHOR]

The present study was aimed to isolate and identify the different bacterial population from diabetic wound and to investigate the antibacterial efficacy of green synthesized silver nanoparticles used as topical application. Silver nanoparticle was synthesized by Saraca asoca bark extract. In this investigation, different bacterial species were identified by light microscopic and scanning electron microscopic methods, biochemical method, and using molecular marker like 16SrRNA. The bactericidal activity of synthesized AgNPs was analyzed by measuring zone of inhibition on agar, MIC, and MBC. The most prevalent bacterial species were Pantoea agglomerans; Staphylococcus gallinarum; Staphylococcus saprophyticus; Bacillus firmus; Bacillus cereus; Bacillus paramycoides; and Staphylococcus sciuri. A differential bactericidal activity of nanoparticles was observed on agar plate zone of inhibition and it was 65–80% more effective on Staphylococcus gallinarum (28 ± 2 mm), Bacillus firmus (26 ± 2 mm), and Pantoea agglomerans (12 ± 1.5 mm) than the nomal sulphadiazine drug and common antibiotics. So it is concluded that green synthesized silver nanoparticles can be used as topical formulation of nontoxic bactericidal agent on diabetic wound against multidrug resistant bacterial population. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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%. [ABSTRACT FROM AUTHOR]

[Objectives] The paper was to establish a molecular biological method for identification of bacterial strains. [Methods] The thalli of standard bacterial strains existing in the laboratory were collected and genomic DNA was extracted for amplification of 16S rDNA and gyrB gene. The 16S rDNA and gyrB gene sequences were obtained after sequencing. Sequences were aligned and analyzed via EzBioCloud and NCBI database, and phylogenetic trees were constructed to determine the species relationship of strains. Meantime, they were compared with known strains. [Results] This method could identify 5 standard strains accurately to the species level. The 16S rDNA and gyrB gene sequences were aligned and analyzed in EzBioCloud database and NCBI database. The strain with the max score was consistent with the known strain. And the query cover and ident were both above 99%. [Conclusions] The established molecular biological method for identification of bacterial strains by 16S rDNA and gyrB gene has good accuracy, which effectively solves the problem that the laboratory identification of bacteria relies on traditional methods and the accuracy can not be guaranteed, and further improves the identification ability of laborator)' bacterial strains. [ABSTRACT FROM AUTHOR]

Abstract 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.

ABSTRACT The detection and accurate identification of bacterial species in clinical samples are crucial for diagnosis and appropriate antibiotic treatment. To date, sequencing of the 16S rRNA gene has been widely used as a complementary molecular approach when identification by culture fails. The accuracy and sensitivity of this method are highly affected by the selection of the 16S rRNA gene region targeted. In this study, we assessed the clinical utility of 16S rRNA reverse complement PCR (16S RC-PCR), a novel method based on next-generation sequencing (NGS), for the identification of bacterial species. We investigated the performance of 16S RC-PCR on 11 bacterial isolates, 2 polymicrobial community samples, and 59 clinical samples from patients suspected of having a bacterial infection. The results were compared to culture results, if available, and to the results of Sanger sequencing of the 16S rRNA gene (16S Sanger sequencing). By 16S RC-PCR, all bacterial isolates were accurately identified to the species level. Furthermore, in culture-negative clinical samples, the rate of identification increased from 17.1% (7/41) to 46.3% (19/41) when comparing 16S Sanger sequencing to 16S RC-PCR. We conclude that the use of 16S RC-PCR in the clinical setting leads to an increased sensitivity of detection of bacterial pathogens, resulting in a higher number of diagnosed bacterial infections, and thereby can improve patient care. IMPORTANCE The identification of the causative infectious pathogen in patients suspected of having a bacterial infection is essential for diagnosis and the start of appropriate treatment. Over the past 2 decades, molecular diagnostics have improved the ability to detect and identify bacteria. However, novel techniques that can accurately detect and identify bacteria in clinical samples and that can be implemented in clinical diagnostics are needed. Here, we demonstrate the clinical utility of bacterial identification in clinical samples by a novel method called 16S RC-PCR. Using 16S RC-PCR, we reveal a significant increase in the number of clinical samples in which a potentially clinically relevant pathogen is identified compared to the commonly used 16S Sanger method. Moreover, RC-PCR allows automation and is well suited for implementation in a diagnostic laboratory. In conclusion, the implementation of this method as a diagnostic tool is expected to result in an increased number of diagnosed bacterial infections, and in combination with adequate treatment, this could improve clinical outcomes for patients.

Microbial induced carbonate precipitation (MICP) through the ureolysis metabolic pathway is one of the most studied topics in biocementation due to its high efficiency. Although excellent outcomes have proved the potential of this technique, microorganisms face some obstacles when considering complicated situations in the real field, such as bacterial adaptability and survivability issues. This study made the first attempt to seek solutions to this issue from the air, exploring ureolytic airborne bacteria with resilient features to find a solution to survivability issues. Samples were collected using an air sampler in Sapporo, Hokkaido, a cold region where sampling sites were mostly covered with dense vegetation. After two rounds of screening, 12 out of 57 urease-positive isolates were identified through 16S rRNA gene analysis. Four potentially selected strains were then evaluated in terms of growth pattern and activity changes within a range of temperatures (15°C–35°C). The results from sand solidification tests using two Lederbergia strains with the best performance among the isolates showed an improvement in unconfined compressive strength up to 4–8 MPa after treatment, indicating a high MICP efficiency. Overall, this baseline study demonstrated that the air could be an ideal isolation source for ureolytic bacteria and laid a new pathway for MICP applications. More investigations on the performance of airborne bacteria under changeable environments may be required to further examine their survivability and adaptability.

Introduction: We compared the performance of real-time PCR with culture-based methods for identifying bacteria in sputum samples from patients with chronic obstructive pulmonary disease (COPD) in three studies. Methods: This was an exploratory analysis of sputum samples collected during an observational study of 127 patients (AERIS; NCT01360398), phase 2 study of 145 patients (NTHI-004; NCT02075541), and phase 2b study of 606 patients (NTHI-MCAT-002; NCT03281876). Bacteria were identified by culture-based microbiological methods in local laboratories using fresh samples or by real-time PCR in a central laboratory using frozen samples. Haemophilus influenzae positivity with culture was differentiated from H. haemolyticus positivity by microarray analysis or PCR. The feasibility of bacterial detection by culture-based methods on previously frozen samples was also examined in the NTHI-004 study. Results: Bacterial detection results from both culture-based and PCR assays were available from 2,293 samples from AERIS, 974 from the NTHI-004 study, and 1736 from the NTHI-MCAT-002 study. Quantitative real-time PCR (qPCR) showed higher positivity rates than culture for H. influenzae (percentages for each study: 43.4% versus 26.2%, 47.1% versus 23.6%, 32.7% versus 10.4%) and Moraxella catarrhalis (12.9% versus 6.3%, 19.0% versus 6.0%, 15.5% versus 4.1%). In the NTHI-004 and NTHI-MCAT-002 studies, positivity rates were higher with qPCR for Streptococcus pneumoniae (15.6% versus 6.1%, 15.5% versus 3.8%); in AERIS, a lower rate with qPCR than with culture (11.0% versus 17.4%) was explained by misidentification of S. pseudopneumoniae/mitis isolates via conventional microbiological methods. Concordance analysis showed lowest overall agreement for H. influenzae (82.0%, 75.6%, 77.6%), due mainly to culture-negative/qPCR-positive samples, indicating lower sensitivity of the culture-based methods. The lowest positive agreement (culture-positive/ qPCR-positive samples) was observed for S. pneumoniae (35.1%, 71.2%, 71.2%). Bacterial load values for each species showed a proportion of culture negative samples with a load detected by qPCR; for some samples, the loads were in line with those observed in culture-positive samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples. Discussion: Real-time PCR on frozen sputum samples has enhanced sensitivity and specificity over culture-based methods, supporting its use for the identification of common respiratory bacterial species in patients with COPD. [ABSTRACT FROM AUTHOR]

16S rRNA gene sequence is the most common housekeeping genetic marker to study bacterial phylogeny and taxonomy. Therefore, 16S rRNA gene sequencing has the potential to identify novel bacteria and diagnose bacteria. This study compared 16S rRNA gene sequencing with conventional PCR for bacterial identification and disease diagnosis. The bacterial community in healthy and diseased hosts was analyzed by 16S rRNA gene sequencing. 16S rRNA gene sequencing is more sensitive than conventional PCR in detecting bacteria. Moreover, 16S rRNA gene sequencing is adequate to identify novel bacteria. 16S rRNA gene sequencing demonstrated that most pathogenic bacteria persist in diseased or healthy hosts in different abundance. Pathogenic bacteria, such as well-known chicken pathogen Avibacterium paragallinarum , Ornithobacterium rhinotracheale , and Gallibacterium anatis , were identified as indicator species of diseased samples. Alpha diversity analysis showed that the healthy group species is significantly higher than in the diseased groups. Beta diversity analysis also demonstrated differences between healthy and infected groups. The study concluded that 16S rRNA gene sequencing is a more sensitive method for detecting pathogens, and microbiota analysis can distinguish between healthy and diseased samples. Eventually, 16S rRNA gene sequencing has represented the potential in human and animal clinical diagnosis and novel bacterial identification. • 16S rRNA gene sequencing efficiently identifies novel bacteria and detects bacteria in clinical samples. • As a diagnosis method, 16S rRNA gene sequencing is quantifiable and more effective than conventional PCR. • 16S rRNA gene sequencing efficiently identified indicator species in diseased individuals. [ABSTRACT FROM AUTHOR]

Probiotics are microorganisms that exert strain-specific health-promoting effects on the host. Τhey are employed in the production of functional dairy or non-dairy food products; still, their detection in these complex matrices is a challenging task. Several culture-dependent and culture-independent methods have been developed in this direction; however, they present low discrimination at the strain level. Here, we developed a multiplex PCR assay for the detection of two potential probiotic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum L125 and Lp. pentosus L33, in monocultures and yogurt samples. Unique genomic regions were identified via comparative genomic analysis and were used to produce strain-specific primers. Then, primer sets were selected that produced distinct electrophoretic DNA banding patterns in multiplex PCR for each target strain. This method was further implemented for the detection of the two strains in yogurt samples, highlighting its biotechnological applicability. Moreover, it can be applied with appropriate modifications to detect any bacterial strain with available WGS.

IntroductionWe compared the performance of real-time PCR with culture-based methods for identifying bacteria in sputum samples from patients with chronic obstructive pulmonary disease (COPD) in three studies.MethodsThis was an exploratory analysis of sputum samples collected during an observational study of 127 patients (AERIS; NCT01360398), phase 2 study of 145 patients (NTHI-004; NCT02075541), and phase 2b study of 606 patients (NTHI-MCAT-002; NCT03281876). Bacteria were identified by culture-based microbiological methods in local laboratories using fresh samples or by real-time PCR in a central laboratory using frozen samples. Haemophilus influenzae positivity with culture was differentiated from H. haemolyticus positivity by microarray analysis or PCR. The feasibility of bacterial detection by culture-based methods on previously frozen samples was also examined in the NTHI-004 study.ResultsBacterial detection results from both culture-based and PCR assays were available from 2,293 samples from AERIS, 974 from the NTHI-004 study, and 1736 from the NTHI-MCAT-002 study. Quantitative real-time PCR (qPCR) showed higher positivity rates than culture for H. influenzae (percentages for each study: 43.4% versus 26.2%, 47.1% versus 23.6%, 32.7% versus 10.4%) and Moraxella catarrhalis (12.9% versus 6.3%, 19.0% versus 6.0%, 15.5% versus 4.1%). In the NTHI-004 and NTHI-MCAT-002 studies, positivity rates were higher with qPCR for Streptococcus pneumoniae (15.6% versus 6.1%, 15.5% versus 3.8%); in AERIS, a lower rate with qPCR than with culture (11.0% versus 17.4%) was explained by misidentification of S. pseudopneumoniae/mitis isolates via conventional microbiological methods. Concordance analysis showed lowest overall agreement for H. influenzae (82.0%, 75.6%, 77.6%), due mainly to culture-negative/qPCR-positive samples, indicating lower sensitivity of the culture-based methods. The lowest positive agreement (culture-positive/qPCR-positive samples) was observed for S. pneumoniae (35.1%, 71.2%, 71.2%). Bacterial load values for each species showed a proportion of culture-negative samples with a load detected by qPCR; for some samples, the loads were in line with those observed in culture-positive samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples. In the NTHI-004 study, of fresh samples that tested culture-positive, less than 50% remained culture-positive when tested from freeze/thawed samples.DiscussionReal-time PCR on frozen sputum samples has enhanced sensitivity and specificity over culture-based methods, supporting its use for the identification of common respiratory bacterial species in patients with COPD.

IntroductionMicrobial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization–time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRI-TOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria.MethodsWe constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures.ResultsFor validation, we used 58 sequence-confirmed strains and the CLOSTRI-TOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates.DiscussionWe describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS.

Shrimp activity is associated with the impact of bacterial communities. Therefore, this research aimed to evaluate the influence of the bacterial microbiota on the organic matter content of the soil of the shrimp lagoon in La Segua-Ecuador. Starting from a descriptive approach, the field research method and documentary review were used. In total, 25 soil samples were collected in 5 quadrants of 100 m2. The bacterial DNA was extracted by using the Powersoil® kit and the identification of the strains was carried out with the 16SrDNA gene. The organic matter content was determined by Walkley-Black titration. The genus Bacillus was predominant in the bacterial strains; moreover, individuals of the genera Exiguobacterium, Acinetobacter, Prolinoborus, Arthrobacter Planococcus were identified with more than 99% homology for all cases. It was concluded that the organic matter content is suitable for shrimp farming. [ABSTRACT FROM AUTHOR]