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Mycoplasma hyopneumoniae's slow growth makes enumeration challenging using the traditional colony-forming unit (CFU) method. We introduced an innovative CFU-slide culture (CFU-SC) approach to address this issue. CFU-SC allows accurate counting of M. hyopneumoniae microcolonies, ranging from 7 to 50 µm, using high magnification. While comparing the accuracy of enumeration among CFU, CFU-SC, quantitative polymerase chain reaction (qPCR), and colour change unit (CCU), we found a robust positive correlation between qPCR and CFU-SC with colourCCU method for quantifying M. hyopneumoniae. Newly isolated strains exhibited the lowest Pearson correlation coefficient (r = 0.52) when comparing CFU and real-time PCR, while the highest coefficient (r = 0.86) was observed for the type strain J and new isolates using real-time PCR and CFU-SC. A consensus growth pattern was observed among all quantification methods, except CFU, between newly isolated strains and the type strain J. Notably, new isolates of M. hyopneumoniae showed no senescence phase after 228 h of cultivation, differing from the type strain J. The disparity in growth rate and pattern between new isolates and the type strain J is evident in the smaller agar microcolonies (7-10 µm) of the isolates, contrasting with the larger colonies (100-200 µm) of type strain J., (© The Author(s) 2025. Published by Oxford University Press on behalf of Applied Microbiology International.)

Zebrafish larvae are used to model the pathogenesis of multiple bacteria. This transparent model offers the unique advantage of allowing quantification of fluorescent bacterial burdens (fluorescent pixel counts [FPC]) in vivo by facile microscopical methods, replacing enumeration of bacteria using time-intensive plating of lysates on bacteriological media. Accurate FPC measurements require laborious manual image processing to mark the outside borders of the animals so as to delineate the bacteria inside the animals from those in the culture medium that they are in. Here, we have developed an automated ImageJ/Fiji-based macro that accurately detects the outside borders of Mycobacterium marinum -infected larvae.

Objectives: Early detection of treatment failure is essential to improve the management of drug-resistant tuberculosis (DR-TB). We evaluated the molecular bacterial load assay (MBLA) in comparison to standard diagnostic tests for monitoring therapy of patients affected by drug-resistant TB., Methods: The performance of MBLA in tracking treatment response in a prospective cohort of patients with pulmonary MDR/RR- and pre-XDR/XDR-TB was compared with mycobacterial culture, mycobacterial DNA detection using GeneXpert (Xpert) and microscopy detection of sputum acid-fast-bacilli., Results: Mycobacterium tuberculosis culture conversion was used as the read-out for treatment responses. The MBLA was most concordant during the early phase of treatment, detecting changes in bacterial load with similar accuracy to microscopy and outperforming Xpert. When considering all timepoints, concordance with MGIT results was 72.1% for MBLA, 57.4% for Xpert and 76.7% for microscopy. The AUC for culture conversion was higher for MBLA (0.88, CI 0.84-0.95) than for Xpert (0.78, CI 0.72-0.85) and microscopy (0.77, CI 0.71-0.83)., Conclusions: MBLA was superior in the early identification of successful culture conversion compared to microscopy and Xpert and could be a useful biomarker to evaluate novel entities in Phase IIA early-bactericidal-activity drug trials regardless of the degree of M. tuberculosis drug resistance., Competing Interests: Declaration of Competing Interest J.H. received a honorarium for lectures from Chiesi, Böhringer Ingelheim, and Sanofi, which have nothing to do with the content of this manuscript. C.L. has received an honorarium for consultation service to INSMED, a company that produced liposomal amikacin as an inhalation suspension for the treatment of NTM-PD outside of the scope of this work. He received speakers’ honoraria from INSMED, GSK, Gilead, Astra Zeneca, MedUpdate and MedUpdateEurope outside of the scope of this study. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Tuberculosis (TB) remains a major cause of infectious disease mortality globally, with significant underdiagnosis perpetuating transmission. Tongue swab analysis has emerged as a promising non-invasive method for pulmonary TB diagnosis. This study evaluates the diagnostic accuracy of the TB-EASY quantitative PCR (qPCR) assay using tongue swab specimens., Methods: In this prospective multicentre study, conducted across seven designated TB hospitals in China, 729 participants were included in the analysis. Tongue swabs were tested using the new TB-EASY assay from Hugobiotech, while sputum samples were analysed by Xpert MTB/RIF (Xpert), smear and culture tests. Diagnostic performance was compared to a composite microbiological reference standard (MRS)., Results: The TB-EASY assay demonstrated high diagnostic accuracy, with sensitivity and specificity of 89.6% and 96.2% compared to sputum Xpert, and 87.4% and 98.0% compared to the MRS. Sensitivity varied by bacterial load, ranging from 100% in high-load cases to 70.4% in very-low-load cases. The assay demonstrated robust performance in diverse epidemiological settings., Conclusions: The TB-EASY qPCR assay using tongue swabs offers a reliable, non-invasive diagnostic alternative for pulmonary TB, especially where sputum collection is challenging. Its potential for wider use in high TB burden settings warrants further validation in community-based studies. Limitations include potential overestimation of sensitivity due to the selection of symptomatic patients and the use of sputum Xpert rather than Xpert Ultra. Additionally, the performance in non-sputum-producing patients remains untested, and the cost-effectiveness should be further evaluated to assess the feasibility of its implementation., Competing Interests: Conflict of interest: Swabs, DNA extraction kits and TB-EASY assay materials were donated by Hugobiotech Co., Ltd, Beijing, China. The authors have no financial involvement with any organisation or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright ©The authors 2025.)

Introduction. Bloodstream infections (BSIs) are one of the most serious infections investigated by microbiologists. However, the time to detect a BSI fails to meet the rapidity required to inform clinical decisions in real time. Gap Statement. Blood culture (BC) is considered the gold standard for diagnosing bloodstream infections. However, the time to blood culture positivity can be lengthy. Underpinning this is the reliance on bacteria replicating to a high concentration, which is necessary for the detection using routine blood culture systems. To improve the diagnosis and management of patients with BSIs, more sensitive detection methods are required. Aim. The study aimed to answer key questions addressing the delay in BSI detection and whether the time to BSI detection could be expedited using a Scattered Light Integrated Collection (SLIC) device. Methodology. A proof-of-concept study was conducted to compare the time to positivity (TTP) of Gram-negative BCs flagging positive on BacT/ALERT with an SLIC device. An SLIC device was utilized to compare the TTP of the most prevalent BSI pathogens derived from nutrient broth and BC, the influence of bacterial load on TTP and the TTP directly from whole blood. Additionally, the overall turnaround time (TAT) of SLIC was compared with that of a standard hospital workflow. Results. Most pathogens tested took significantly longer to replicate when derived from BC than from nutrient medium. The median TTP of Gram-negative BC on BacT/ALERT was 13.56 h with a median bacterial load of 6.4×10 9  c.f.u. ml -1 . All pathogens (7/7) derived from BC at a concentration of 10 5  c.f.u. ml -1 were detectable in under 70 min on SLIC. Decreasing Escherichia coli BC concentration from 10 5 to 10 2  c.f.u. ml -1 increased the TTP of SLIC from 15 to 85 min. Direct BSI detection from whole blood on SLIC demonstrated a 76% reduction in TAT when compared with the standard hospital workflow. Conclusion. An SLIC device significantly reduced the TTP of common BSI pathogens. The application of this technology could have a major impact on the detection and management of BSI.

The bacterial load (BL), or total viable count, of aerobes can be measured using micro-respirometry, %O 2 -μR, in which the consumption of dissolved O 2 is monitored with respect to incubation time, t . In %O 2 -μR the 'bioreactor' often comprises a canonical plastic tube with a small %O 2 sensor; it is simple, fast and accurate and used in automated, commercial instruments for measuring BL. Here we show that it is also possible to measure BL using a new form of micro-respirometry, %CO 2 -μR, in which the production of CO 2 in the growth medium is monitored. In %CO 2 -μR, the 'bioreactor' is the same as that used in %O 2 -μR, but with a small 3D printed, colour-based %CO 2 indicator set in its base and its apparent absorbance, A ', is measured at any t , as it is related to the %CO 2 dissolved in the inoculated growth medium. Under aerobic conditions, different inoculations of the facultative anaerobe, E. coli , of different concentrations (10 1 -10 8 colony forming units (CFU) per mL) are used to generate a series of A ' vs . t profiles, and a straight-line calibration curve. Statistical comparative analysis of the results generated in the above %CO 2 -μR study, to those generated for the same system but using a commercial %O 2 -μR system, is used to demonstrate method equivalence. A study of the same system, under anaerobic conditions, using %CO 2 -μR, shows that %CO 2 -μR is suitable for measuring the BL of anaerobes. The potential of %CO 2 -μR for measuring the bacterial load of CO 2 -generating aerobes and anaerobes is discussed briefly.

Background and Objectives: Management of skin cancer comprises a substantial proportion of general practitioner (GP) workload in Australia. Flap and graft procedures below the knee have an increased risk of infection. Antibiotic resistance is a threat to global health, and any decision about antibiotic prophylaxis must balance adverse outcomes of antibiotic use with patient morbidity. This study will investigate the effectiveness of two interventions to prevent surgical site infection (SSI) after below-knee surgery: (1) 450 mg of clindamycin preoperatively and postoperatively; and (2) preoperative chlorhexidine wash and nasal mupirocin., Method: This prospective randomised controlled trial will be conducted across three skin cancer clinics over nine months, with 155 participants. Consecutive patients presenting for below-knee flap and graft procedures will be eligible to participate. The primary outcome is superficial SSI in the first 30 days following excision. Secondary outcomes include adverse effects (anaphylaxis, skin irritation and foreign body reaction) and patterns of antibiotic resistance., Results: As this is a study protocol paper, there are no results available to present., Discussion: As this is a study protocol paper, there are no results to be discussed.

Background: We examined whether the time to positivity (TTP) and growth and detection plot graph (GDPG) created by the automated blood culture system can be used to determine the bacterial load in bacteremic patients and its potential association correlation with disease severity., Methods: Known bacterial inocula were injected into the blood culture bottles. The GDPGs for the specific inocula were downloaded and plotted. A cohort of 30 consecutive clinical cultures positive for S. aureus and E. coli was identified. Bacterial load was determined by comparing the GDPG with the "standard" curves. Variables associated with disease severity were compared across 3 bacterial load categories (< 100, 100-1000, > 1000 CFU/mL)., Results: S. aureus growth was sensitive to the blood volume obtained whereas E. coli growth was less so. A 12-hour delay in sample transfer to the microbiology laboratory resulted in a decrease in TTP by 2-3 h. Mean TTP was 15 and 10 h for S. aureus and E. coli, respectively, which correlates with > 1000 CFU/mL and 500-1000 CFU/ml. For S. aureus, patients with a bacterial load > 100 CFU/mL had a higher mortality rate, (OR for death = 9.7, 95% CI 1.6-59, p = 0.01). Bacterial load > 1000 CFU/mL had an odds ratio of 6.4 (95% CI1.2-35, p = 0.03) to predict an endovascular source. For E. coli bacteremia, we did not find any correlations with disease severity., Conclusion: GDPG retrieved from the automated blood culture system can be used to estimate bacterial load. S.aureus bacterial load, but not E.coli, was associated with clinical outcome., (© 2024. The Author(s).)

Background: Time-dependent changes in cell populations during acute bacterial infections remain unclear. We assessed time-dependent changes in fluorescent light intensity of the neutrophil area (NE-SFL) and fluorescent light distribution width index of the neutrophil area (NE-WY) and their association with sepsis and bacteremia., Methods: Patients with acute bacterial infections were enrolled in this prospective, observational cohort study. Blood samples were collected from all patients at the onset of bacterial infections (day 0) and on days 1 and 3. Microbiological evaluation included the examination of blood bacterial load using PCR. Cell population data were assessed using an automated hematology analyzer (Sysmex series XN-2000)., Results: Forty-three participants with acute bacterial infections were enrolled in the study. Twenty-five participants developed definite sepsis. All the participants improved after the onset of infection. NE-WY levels showed significant time-dependent changes in participants with sepsis, peaking on day 0 and significantly decreasing until day 3, whereas these changes were not statistically significant for NE-SFL. A significant correlation with the Sequential Organ Failure Assessment score was observed with NE-WY and NE-SFL in the entire cohort on days 0 and 1. However, only NE-WY showed a significant correlation with blood bacterial load on days 0 and 1., Conclusion: This study demonstrated that NE-WY elevation in sepsis peaked earlier than NE-SFL, which may partly reflect the early bacterial invasion into circulation. These findings advocate caution in interpreting cell population data values as sepsis biomarkers and propose the potential of NE-WY as a therapeutic indicator., Competing Interests: Declaration of competing interest Hideki Niimi received research funding on a contract basis and joint re-search funding from Sysmex Corporation. Rinako Yoshida and Yosuke Iwasaki were employees of the Sysmex Corporation. The authors declare that this study received funding from Sysmex Corporation. The funder had the following involvement in the study: study design, analysis, interpretation of data. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Background: High-frequency jet ventilation (HFJV) is used in pneumological endoscopy for rigid, diagnostic, and therapeutic bronchoscopies. It is unclear to what extent the unobstructed flow of respiratory gas from the patient's lungs causes microbial contamination of the surrounding air., Material and Methods: After the start of the HFJV (15 min) in 16 rigid bronchoscopies, airborne pathogen measurements were taken directly at the distal endoscope outlet, at examiner height (40 cm above the endoscope outlet), at a 2 m distance from the endoscope in the room and at the supply air outlet of the examination room using an RCS air sampler. The number and type of pathogens isolated in the air samples were then determined, as well as germs in the bronchoalveolar lavage fluid (BALF) from the patient's lungs., Results: An increased bacterial density (136 and 114 CFU/m 3 ) was detected directly at the distal end of the endoscope and at examiner height at a distance of 40 cm, which decreased significantly with increasing distance from the bronchoscope (98 CFU/m 3 at a distance of 2 m and 82 CFU/m 3 at the supply air outlet). The most frequently detected bacteria were Staphylococcus spp., Micrococcus spp. and Bacillus spp. In the BALF, pathogens could only be cultivated in four of 16 samples, but the same pathogens were detected in the BALF and the ambient air., Conclusion: When performing a rigid bronchoscopy, in which patients are mechanically ventilated in a controlled manner using an open HFJV system, there is an increased pathogen load in the ambient air and therefore a potential risk for the examiner., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Background: Split-thickness skin graft (STSG) 1 integration rates are susceptible to improvement. Infection and/or biofilm should be appropriately addressed prior to grafting to improve the likelihood of graft-take. Incorporating technological aids such as fluorescence (FL) 2 imaging (MolecuLight®), which accurately locates areas of bacterial loads above 10 4 CFU/gr, for graft site assessment and preparation could yield better outcomes., Methods: This single-center, prospective observational study included adult burn patients with previously infected wounds that had been deemed clinically and microbiologically clean and were therefore candidates for grafting. Prior to grafting, a FL imaging assessment (blinded to the surgical team) localized areas positive for moderate-high bacterial loads (>10 4 CFU/gr). Intra-operatively, a standard swab sample from the recipient site was collected by the surgical team. Postoperatively, areas positive/negative for FL and areas of graft take and failure were overlapped and measured (cm 2 ) over a 2D schematic. The performance and accuracy of FL imaging and swab sampling in relation to graft outcomes were assessed., Results: 38 patients were enrolled in the study. The mean total body surface area (TBSA) 3 involvement was 14.5 ± 12.4 % [range 0.8 - 40.2 %]. 25/38 of the subjects enrolled had complete graft take while 13 had partial graft losses. There were no total losses. FL-imaging was positive in 100 % of losses versus 31 % (4/13) of the swab microbiology. FL-imaging was found to have a sensitivity of 86 %, specificity of 98 %, PPV of 72 %, NPV of 99 %, and an accuracy of 94 % for predicting any type or range of graft loss in the entire cohort. Meanwhile, the sensitivity of microbiology from swab samples was 30 %, with a specificity of 76 %., Conclusions: FL imaging is an accurate method for assessing recipient sites and predicting the outcome of a skin graft among burn patients. These findings suggest that FL imaging can inform better decision-making surrounding grafts that may lead to better outcomes., Level of Evidence: Level IIA, Therapeutic study., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: High bacterial burden stalls wound healing and can quickly progress to infection and sepsis in complex, older-adult patients in long-term care (LTC) or skilled nursing facilities (SNFs)., Objective: To investigate the outcomes of point-of-care fluorescence (FL) imaging (MolecuLight i:X) of bacterial loads, which are frequently asymptomatic, to inform customized wound treatment plans for patients in LTC/SNFs., Methods: In this retrospective pre/postinterventional cohort study, the authors compared the healing and infection-associated outcomes of 167 pressure injuries from 100 Medicare beneficiaries before and after implementation of FL imaging., Results: Most patient demographics and wound characteristics did not differ significantly between the standard-of-care (SOC; n = 71 wounds) and FL (n = 96 wounds) cohorts. Significantly more wounds (+71.0%) healed by 12 weeks in the FL cohort (38.5%) versus the SoC cohort (22.5%). Wounds in the FL cohort also healed 27.7% faster (-4.8 weeks), on average, and were 1.4 times more likely to heal per Kaplan-Meier survival analysis (hazard ratio = 1.40; 95% CI, 0.90-2.12). Infection-related complications decreased by 75.3% in the FL cohort, and a significant shift from largely systemic to topical antibiotic prescribing was evidenced., Conclusions: Fluorescence-imaging-guided management of wounds significantly improved healing and infection outcomes in highly complex and multimorbid patients in LTC/SNFs. Proactive bacterial infection management via local treatments was enabled by earlier, objective detection. These reported outcome improvements are comparable to randomized controlled trials and cohort studies from less compromised, selectively controlled outpatient populations. Fluorescence imaging supports proactive monitoring and management of planktonic and biofilm-encased bacteria, improving patient care in a complex, real-world setting., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

A new innovative method, MICA Legionella, allows for the automatic enumeration of Legionella pneumophila in domestic water samples in 2 days, with a detection limit of 2 CFU per test portion. Here we show that it gives equivalent results to those obtained by the French standard method NF T90-431 in 7 to 15 days., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Legionella pneumophila is a water-borne bacterium that can cause Legionnaires' disease. Legiolert® (IDEXX, USA) is a low-labour liquid culture assay for the detection and enumeration of L. pneumophila (SG1-15) from water., Aim: To analyse concordance between Legiolert and ISO 11731:2017 plate culture method (membrane filtration and culture on selective agars) using hospital water samples (N = 100)., Methods: Incubation was at 39 °C and 36 °C, respectively, for seven days, followed by most-probable enumeration for Legiolert and subculturing and serogrouping of suspected Legionella colonies, with plate culture., Findings: L. pneumophila (SG1-15) was isolated from 25 out of 100 samples when using Legiolert or plate culture. Fourteen additional Legiolert samples tested positive for L. pneumophila; analysis of the same samples by plate culture was negative (12 out of 14) or yielded only Legionella rubrilucens (two out of 14; confirmed via matrix-assisted ionization/desorption time-of-flight mass spectrometry). L. pneumophila was not captured from Quanti-Tray/Legiolert pouch wells of these positive samples after subculture of puncture aliquots on buffered charcoal yeast-extract agar. Both methods in concordance did not detect L. pneumophila in 61 out of 100 samples., Conclusion: Legiolert and plate culture are both satisfactory methods to detect L. pneumophila from water samples, and both to detect isolated L. pneumophila in 25% of the sample population. Legiolert provides a faster time to result, and is less resource-demanding and labour-intensive; however, there may be a low risk of cross-reactivity with other organisms. Both methods are suitable for the analysis of water in healthcare settings, where the monitoring of L. pneumophila is imperative in preventing cases of Legionnaires' disease., (Copyright © 2023 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC 2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC 2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC 2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10 -1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC 2 dye, to determine the viable number of cells, it was found that the DiOC 2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC 2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We examined the correlation between three different methods of Mycobacterium tuberculosis quantification: time to positivity (TTP), log 10 CFU, and an assay to detect differentially detectable M. tuberculosis (DD Mtb) from three different prospective studies. Participants with DD Mtb have significantly more variation in the CFU/TTP correlation than participants with no DD Mtb ( P < 0.001). This may impact the design of early bactericidal activity studies that use TTP as the primary outcome., Competing Interests: The authors declare no conflict of interest.

Background: Many Gram-negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks, but standardized laboratory detection methods for these organisms have not been established., Aim: This study aimed to establish laboratory testing methodologies for six waterborne pathogens: Acinetobacter spp., Burkholderia spp., Cupriavidus spp., Delftia acidovorans, Elizabethkingia spp. and Stenotrophomonas maltophilia., Methods: Water samples were spiked by UK Health Security Agency laboratories and sent to the Glasgow Royal Infirmary laboratory for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure that the methodology could identify organisms from a mixed culture. Volumes of 100 mL were filtered under negative pressure on to culture media and incubated at 30 °C and 37 °C. The incubation time was 7 days, with plates read on days 2, 5 and 7. Further identification of colonies was undertaken using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)., Findings: Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar, chocolate bacitracin agar and pseudomonas selective agar. The optimal temperature for isolation was 30 °C. The optimal incubation time was 5 days, and MALDI-TOF MS identified all test species reliably., Conclusion: The methodology described was able to detect the six tested waterborne pathogens reliably, and can be utilized by laboratories involved in testing water samples during outbreak investigations., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

This study aimed to investigate the prognostic value of a novel droplet digital polymerase chain reaction (DDPCR) assay in sepsis patients. In this prospective cohort study, univariable and multivariable Cox regressions were used to assess risk factors for 28-day mortality. We also monitored pathogen load together with clinical indicators in a subgroup of the cohort. A total of 107 sepsis patients with positive baseline DDPCR results were included. Detection of poly-microorganisms [adjusted hazard ratio (HR) = 3.19; 95% confidence interval (CI) = 1.34-7.62; P = 0.009], high Charlson Comorbidity Index (CCI) score (adjusted HR = 1.14; 95% CI = 1.01-1.29; P = 0.041), and Sequential Organ Failure Assessment (SOFA) score (adjusted HR = 1.18; 95% CI = 1.05-1.32; P = 0.005) at baseline were independent risk factors for 28-day mortality while initial pathogen load was not associated (adjusted HR = 1.17; 95% CI = 0.82-1.66; P = 0.385). Among 63 patients with serial DDPCR results, an increase in pathogen load at days 6-8 compared to baseline was a risk factor for 28-day mortality ( P = 0.008). Also, pathogen load kinetics were significantly different between day-28 survivors and nonsurvivors ( P = 0.022), with a decline overtime only in survivors and an increase from days 3 and 4 to days 6-8 in nonsurvivors. Using DDPCR technique, we found that poly-microorganisms detected and increased pathogen load a week after sepsis diagnosis were associated with poor prognosis.IMPORTANCEThis prospective study was initiated to explore the prognostic implications of a novel multiplex PCR assay in sepsis. Notably, our study was the largest cohort of sepsis with droplet digital polymerase chain reaction pathogen monitoring to date, allowing for a comprehensive evaluation of the prognostic significance of both pathogen species and load. We found that detection of poly-microorganisms was an independent risk factors for 28-day mortality. Also, pathogen load increase 1 week after sepsis diagnosis was a risk factor for 28-day mortality, and differential pathogen load kinetics were identified between day-28 survivors and nonsurvivors. Overall, this study demonstrated that pathogen species and load were highly correlated with sepsis prognosis. Patients exhibiting conditions mentioned above face a more adverse prognosis, suggesting the potential need for an escalation of antimicrobial therapy.Registered at ClinicalTrials.gov (NCT05190861)., Competing Interests: The authors declare no conflict of interest.

Heterotrophic bacteria are commonly found in water samples. While these Heterotrophic Bacterial/Plate Counts (HPC) do not necessarily indicate a health hazard, high counts provide a good indication of the efficiency of water disinfection and integrity of distribution systems. The aim of this study was to compare the PetrifimTM AC method to the pour plate technique for the testing of HPC in water samples. Artificially contaminated (192 samples) and natural water samples (25) were processed using two methods. Both methods accurately detected high, medium and low counts of HPC, producing average Z scores between -2 and +2. Paired-wise student t-test and correlation coefficient showed nonsignificant differences between the results of two methods. Acceptable repeatability and reproducibility was obtained using both the methods. Uncertainty of measurement for PetrifilmTM AC and pour plate method was found to be 2.9% and 5.4%, respectively. PetrifilmTM AC proved to be robust at 33°C and 37°C. In conclusion, PetrifimTM AC, which is easy to process, read, and less time consuming, proved to be comparable to the conventional pour plate method in establishing HPC in water. In addition, PetrifimTM AC requires less space for the processing and incubation, generate small volume of waste for disposal, and requires no equipment, except for the incubator., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages., One-Sentence Summary: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Smear microscopy for acid-fast bacilli visualization is important to assess the infectivity rate in patients with pulmonary tuberculosis (PTB), but it has limited sensitivity; hence, it is important to find an alternative strategy. The aim of our study was to compare the fluorescence microscopy grading by Auramine O phenol staining technique of respiratory samples with the cyclic threshold (Ct) values of GeneXpert Ultra (Mycobacterium tuberculosis/rifampicin [MTB/RIF]) and assess the diagnostic efficacy of GeneXpert Ultra (MTB/RIF) compared to microscopy in suspected cases of PTB., Methods: The study was conducted in the Mycobacteriology Laboratory, Department of Microbiology, in Kasturba Hospital, Manipal. The study was a prospective, single-centered, cross-sectional study. Four hundred and fifty-two respiratory samples were included in the study. An optimal Ct cutoff value for ruling smear-positivity and smear-negativity and the mean Ct cutoff value were calculated. Clinical and radiological data from the requisition forms were assessed. IBM SPSS statistics software version 22 was used. The correlation between GeneXpert Ultra (MTB/RIF) Ct values and smear status was calculated by polychoric correlation. The extended McNemar's test was used to find the association between the variables., Results: GeneXpert Ultra (MTB/RIF) yielded a higher positivity rate of 22.2% compared to smear microscopy 17.2%. Ct value and smear grading yielded a positive correlation (P = 0.8681; P < 0.05). GeneXpert Ultra (MTB/RIF) yielded nontuberculous mycobacteria in five undetected cases and speciated as Mycobacterium abscessus complex., Conclusions: Our study confirms the GeneXpert Ultra (MTB/RIF) Ct value levels as a predictor of smear positivity., (Copyright © 2024 Copyright: © 2024 International Journal of Mycobacteriology.)

Aspiration pneumonia can occur in perioperative and older patients, and various oral care methods have been used to prevent it. To validate the effective oral care methods, measuring bacterial counts before and after oral care is necessary. However, isolating and quantifying viable bacteria from those that are inactivated by agents used in oral care is not possible. In this study, we developed a novel method, Delayed real-time PCR (DR-PCR), that can quantify only viable bacteria from mixed samples of viable and dead bacteria. This method takes advantage of the fact that dead bacteria do not grow but viable bacteria do. When the samples were incubated in a liquid medium for 4 hours, the higher the percentage of viable bacteria, the higher the rate of increase in the number of bacteria. This method showed that povidone‑iodine mouthwashing reduced the number of viable bacteria to approximately 1/4 of that before mouthwashing. Although DR-PCR is slightly more time consuming than real-time PCR, it is effective for studying changes in bacterial counts before and after oral care., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: A pilot clinical study to evaluate the use of propidium monoazide PCR (PMA-PCR) in quantifying a reduction of bacterial load after antiseptic use on the canine oral mucosa and skin, comparison of quantitative PCR (qPCR) to PMA-PCR, and comparison of patterns seen between PCR methods and bacterial culture., Animals: Client-owned dogs (n = 10) undergoing general anesthesia and intravenous catheter placement., Procedures: The oral mucosa and antebrachial skin of each dog underwent swabs for culture, qPCR, and PMA-PCR before and after antiseptic preparation of each site. Reduction in bacterial load between sampling times was evaluated for each quantification method., Results: All testing methods found a significant decrease in bacterial load from oral mucosa after antiseptic preparation (culture P = .0020, qPCR P = .0039, PMA-PCR P = .0039). PMA-PCR had a significantly greater reduction of bacterial load after preparation than qPCR (P = .0494). Only culture detected a significant reduction after preparation of the skin (culture P = .0039, qPCR P = .3125, PMA-PCR P = .0703)., Clinical Relevance: PMA-PCR was able to quantify a reduction of bacterial load after antiseptic preparation of the high-bacterial load environment, with a pattern similar to that of culture, and was more specific than qPCR for detecting viable bacterial load. The results of this study support the use of PMA-PCR for antiseptic effectiveness studies performed on a high-bacterial load environment, such as canine oral mucosa.

Background: Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms., Objectives: We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT., Methods: We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log 10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses., Results: In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log 10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms-especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log 10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log 10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load., Conclusion: Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT., Trial Registration Number: ClinicalTrials.gov NCT02694497., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

To predict bacterial population behavior in food, statistical models with specific function form have been applied in the field of predictive microbiology. Modelers need to consider the linear or non-linear relationship between the response and explanatory variables in the statistical modeling approach. In the present study, we focused on machine learning methods to skip definition of primary and secondary structure model. Support vector regression, extremely randomized trees regression, and Gaussian process regression were used to predict population growth of Escherichia coli O157 at 15 and 25 °C without defining the primary and secondary models. Furthermore, the support vector regression model was applied to predict small population of bacteria cells with probability theory. The model performance of the machine learning models were nearly equal to that of the current statistical models. Machine learning models have a potential for predicting bacterial population behavior., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The improvement of cell enumeration methods for the counting of Escherichia coli (E. coli) is important as E. coli gains in popularity as a basis for biopharmaceutical applications. In the biopharmaceutical industry, enumerating, characterizing, and dosing the accurate number of cells is imperative. In this work, we demonstrate the utilization of a chip-based image cytometer using a thin-gap, low volume counting chamber consumable to directly enumerate E. coli in bright field and fluorescence, and measure their viability using SYTOX™ Green. The total E. coli particles can be counted accurately label-free by adjusting the focus and targeting the linear range of the instrument. The E. coli are stained with SYTOX™ Green to count the membrane-compromised dead bacterial cells in the green fluorescence channel, while the total cells are counted using the bright field channel. Optimization of the system settings, image focus, cell counting range, and staining conditions have yielded a precise, rapid, and accurate E. coli cell enumeration and viability measurement., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To assess the predictive value of swab cultures of cryopreserved skull flaps during cranioplasties for surgical site infections (SSIs)., Methods: A retrospective review was conducted of consecutive patients who underwent delayed cranioplasties with cryopreserved autografts between 2009 and 2017. The results of cultures obtained from swabs and infected surgical sites were assessed. The accuracy, sensitivity, and specificity of swab cultures for SSIs were evaluated., Results: The study included 422 patients categorized into two groups, swab and nonswab, depending on whether swab cultures were implemented during cranioplasties. The overall infection rate was 7.58%. No difference was seen in infection rates between groups. There were 18 false-positive and no true-positive swab culture results. All bacteria between swab cultures and SSI cultures were discordant. Meanwhile, there were 19 false-negative swab cultures. The results showed high specificity but low sensitivity for swab cultures to predict SSI occurrence and the pathogens., Conclusions: Owing to low accuracy and sensitivity, swab cultures of cryopreserved autografts should not be routinely performed during delayed cranioplasties., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study, we report that the addition of a growth enhancer mix consisting of trehalose, sodium pyruvate, magnesium chloride, and 1× trace mineral supplement improved growth of microorganisms from chlorinated secondary effluent in the base medium with Colilert-18. Improving growth of chlorine stressed microorganisms from secondary effluent is crucial to decreased detection time from 18 to 8 h., (Copyright © 2021. Published by Elsevier B.V.)

A host-guest colorimetric strategy is described for the detection of Listeria monocytogenes (L. monocytogenes). The optical probes were self-assembled based on the supramolecular interactions between the carbonyl groups of cucurbit[7]uril portals and gold nanoparticles (CB[7]-AuNPs). Aptamer and urease modified magnetic nanoparticles were used to specifically recognize and binding to L. monocytogenes, simultaneously hydrolyzing urea to produce ammonium ion (NH 4 + ) that can reverse CB[7] induced AuNPs aggregation. In the presence of L. monocytogenes, the above-mentioned magnetic conjugates preferentially bind to the bacterial surface, which results in blocking the catalytic active sites, thus inhibiting the production of ammonium ions. The normalized absorbance ratio of A 700 nm /A 525 nm was proportional to the L. monocytogenes concentration ranging from 10 to 10 6  cfu·mL -1 , and the visual determination can be done down to 10 cfu·mL -1 . For spiked food samples analyzed without pre-enrichment, recoveries of 98.4% to 99.3% were achieved could be verified and RSD were less than 10%. This work may offer a broad prospect for sensitive and specific determination  of pathogens., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

An ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of Lactobacillus rhamnosus GG (LGG). The N/O co-doped three-dimensional hierarchical porous graphitic (THPG) carbon was synthesized by a one-step synthesis of polyaniline hydrogel, and followed by simple carbonization and chemical activation procedures. Because of the unique structure design, the obtained THPG carbon networks possess an ultra-large specific surface area of 4859 m 2 g -1 along with a class of highly graphitic carbons. The results offer an enormous surface area and excellent electrical conductivity for label-free electrochemical immunosensing of probiotic L. rhamnosus strain. Under optimal conditions, the immunosensor showed a good linear relationship between peak current and concentration of LGG (R 2  = 0.9976), with a detection limit of 2 CFU mL -1 . Furthermore, this label-free immunosensor also shows good specificity, long-term stability, and reliability, and could be applied to detect probiotic LGG in dairy products and drinks with satisfactory results. The present protocol was shown to be quite promising for practical screening and functional evaluation of probiotic products containing LGG. A ultrasensitive label-free electrochemical immunosensor based on THPG carbon was fabricated for detection of Lactobacillus rhamnosus GG., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation., Competing Interests: The authors have declared that no competing interests exist.

This study developed a novel method for the rapid detection of Escherichia coli ( E. coli ) O157:H7 on a microfluidic platform. First, the concentration of bacteria in a sample was determined with the adenosine triphosphate (ATP) method. Then, the specific detection of E. coli was achieved in a microfluidic chip by the immune-microsphere technique. The influences of the culture time, flow rate and capture time on the detection of the target bacteria were investigated systematically. Generally, with increasing capture time, more bacteria could be captured by the microspheres, which had a positive effect on bacterial detection. Furthermore, the sensitivity and specificity of the method were also tested. The results showed that this method could specifically detect E. coli with a sensitivity as high as 49.1 cfu/μL; the consumption of bacteria was 1 μL, and the reagent was at the microliter level. The testing time can be controlled within one and a half hours, and the cost of testing was approximately RMB 10. The method described in this article is simple and accurate and has great application value in bacterial detection for medical diagnostics.

Use of flow cytometry (FCM) for bacteria quantification is growing in the food industry. We report here a FCM method using a double-staining LDS751/SYTO24 for the quantification of probiotic Bacillus viable cells and its spores, with potential application for the control of commercial product specifications., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Neonatal sepsis accounts for a large proportion of neonatal deaths in sub-Saharan Africa. The lack of access to diagnostic testing and excessively long turnaround times to result contributes to delays in sepsis identification and initiation of appropriate treatment. This study aims to evaluate the novel InTrays COLOREX Screen and extended-spectrum beta-lactamase for rapid identification of bacterial pathogens causing sepsis and detection of resistance., Methods: Neonates with suspected sepsis admitted to the Harare Central Hospital were prospectively enrolled. One blood culture was collected and incubated using the BacT/ALERT automated system. Positive blood cultures with potential pathogens identified by Gram stain were inoculated on the InTray COLOREX Screen and extended-spectrum beta-lactamase culture plates., Results: A total of 216 neonates with suspected sepsis were recruited. Pathogens were isolated from blood cultures in 56 (25.9%) neonates of which 54 were Klebsiella pneumoniae. All K. pneumoniae were resistant to ceftriaxone and 53 (98%) were resistant to gentamicin. Sensitivity and specificity for ceftriaxone-resistant K. pneumoniae detection using InTrays were 100%. InTrays results were interpretable as early as 5-10 hours (median 7 hours, interquartile range 7-7) post blood culture positivity enabling rapid identification and notification of result and leading to a 60% reduction in time to result from blood culture collection., Conclusions: This study shows that the implementation of a novel culture method was feasible and reduced turnaround times for results by 60% compared with standard microbiologic techniques. An impact on patient outcomes and cost-effectiveness of this method needs to be demonstrated., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The detection and identification of bacteria currently rely on enrichment steps such as bacterial culture and nucleic acid amplification to increase the concentration of target analytes. These steps increase assay time, cost and complexity, making it difficult to realize a truly rapid point-of-care test. Here we report the development of an electrical assay that uses electroactive RNA-cleaving DNAzymes (e-RCDs) to identify specific bacterial targets and subsequently release a DNA barcode for transducing a signal onto an electrical chip. Integrating e-RCDs into a two-channel electrical chip with nanostructured electrodes provides the analytical sensitivity and specificity needed for clinical analysis. The e-RCD assay is capable of detecting 10 CFU (equivalent to 1,000 CFU ml -1 ) of Escherichia coli selectively from a panel containing multiple non-specific bacterial species. Clinical evaluation of this assay using 41 patient urine samples demonstrated a diagnostic sensitivity of 100% and specificity of 78% at an analysis time of less than one hour compared with the several hours needed for currently used culture-based methods., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Systemic diseases have been associated with oral health and gut microbiota. We examined the association between oral health and the community composition and structure of the adherent colonic gut microbiota., Methods: We obtained 197 snap-frozen colonic biopsies from 62 colonoscopy-confirmed polyp-free individuals. Microbial DNA was sequenced for the 16S rRNA V4 region using the Illumina MiSeq, and the sequences were assigned to the operational taxonomic unit based on SILVA. We used a questionnaire to ascertain tooth loss, gum disease, and lifestyle factors. We compared biodiversity and relative abundance of bacterial taxa based on the amount of tooth loss and the presence of gum disease. The multivariable negative binomial regression model for panel data was used to estimate the association between the bacterial count and oral health. False discovery rate-adjusted P value (q value) < .05 indicated statistical significance., Results: More tooth loss and gum disease were associated with lower bacterial alpha diversity. The relative abundance of Faecalibacterium was lower (q values < .05) with more tooth loss. The association was significant after adjusting for age, ethnicity, obesity, smoking, alcohol use, hypertension, diabetes, and the colon segment. The relative abundance of Bacteroides was higher in those with gum disease., Conclusions: Oral health was associated with alteration in the community composition and structure of the adherent gut bacteria in the colon. The reduced anti-inflammatory Faecalibacterium in participants with more tooth loss may indicate systemic inflammation. Future studies are warranted to confirm our findings and investigate the systemic role of Faecalibacterium., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated <60-min generation times. The relatively short generation times observed in the spleen were in contrast to an overall decrease in bacterial burden for some species, suggesting that the rate of immune-mediated clearance exceeded replication. Furthermore, bacterial generation times measured in the murine spleen approximated those measured during growth in human serum cultures. Together, these findings provide insight into the infection kinetics of six medically important species during bacteremia. IMPORTANCE Bloodstream infections are a global public health problem. The goal of this work was to determine the replication characteristics of Gram-negative bacterial species in the host following bloodstream infection. The number of bacteria in major organs is likely determined by a balance between replication rates and the ability of the host to clear bacteria. We selected a cohort of six species from three families that represent common causative agents of bloodstream infections in humans and determined their replication rates in a murine bacteremia model. We found that the bacteria grow rapidly in the spleen, demonstrating that they can obtain the necessary nutrients for growth in this environment. However, the overall number of bacteria decreased in most cases, suggesting that killing of bacteria outpaces their growth. Through a better understanding of how bacteria replicate during bloodstream infections, we aim to gain insight into future means of combating these infections.

Background: The ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15)., Results: The QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets., Conclusions: No false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation., (© 2021. The Author(s).)

High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

For the detection of Vibrio bacteria, a kit involving two-step method was developed. In the in first step, a specific media was added in the water sample which selectively promote the growth of vibrios and inhibit the growth of other bacteria. The second step involved addition of dye-based sensor (already developed in our previous work) in the sample which detect the active Vibrio and changed the colour of the sample to red/pink. The vibrio detection kit was optimised on five different species of Vibrio (V. cholerae, V. parahaemolyticus, V. campbellii, V. harveyi and V. proteolyticus) and two negative control bacteria (Escherichia coli and Bacillus subtilis). The kit was further evaluated on aquaculture pond water and probiotics used in aquaculture farms. It successfully estimated Vibrio concentration of all the five strains in aquaculture ponds. The negative control bacteria and probiotics were not sensed by the kit. Hence, the kit developed here is perfect for the detection of Vibrio, especially in aquaculture farms., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination. IMPORTANCE There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples. Reporting a contaminant species as a relevant pathogen may cause unnecessary antibiotic treatment or even falsely classify a noninfectious condition as a bacterial infection. Yet, there are few studies on how to filter contamination in clinical microbiology. Here, we demonstrate that sequencing of extraction controls will not reveal the full spectrum of contaminants that could occur in the associated clinical samples. Only the most abundant contaminant species were consistently detected, and we present how this can be used to set sample specific thresholds for reliable identifications. We believe this work can facilitate the implementation of 16S deep sequencing in diagnostic laboratories. The new data we provide on the patterns of microbial DNA contamination is also important for microbiome research.

The U.S. FDA Food Safety Modernization Act Preventive Controls for Human Food Rule underlines the importance of an effective environmental monitoring (EM) program. EM is used to determine harborage sites of microorganisms on processing equipment, assess effectiveness of sanitation programs, and prevent transmission of foodborne pathogens. This study characterizes commercially-available polyurethane foam (PUF) and cellulose (CELL) EM tools for their efficacy in the release of foodborne pathogens from their sponge matrices. Specifically, the objectives of this study were to 1) compare the ability of EM tools to release microorganisms into a recovery eluent, 2) characterize EM tool performance at decreasing inoculum concentrations, and 3) assess the impact of various operators during the processing of EM samples. Two bacteria (Listeria monocytogenes, Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus [TV]) were compared at decreasing inoculum levels utilizing two elution techniques (mechanical stomacher, manually by operator), and across six operators. Data indicated that EM tool material composition impacted the release of microorganisms (p = 0.0001), where the PUF EM tool released TV more readily than the CELL EM tool. Conversely, the decreasing inoculum levels did not statistically differ in the release of microorganisms from the EM tool matrices. In addition, no significant difference was found between the machine stomacher and manual elution by human operator or between operators. Overall, the study provides a detailed characterization of two commercially-available EM tools, and the differences identified in this study can be used to improve the effectiveness of EM programs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: In resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB., Methods: A subset of 138 PLHIV from the 'SOUTH' study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity., Results: A total of 81 patients (58.7%) were males, median age 34 (IQR 29  ̶ 40) years, median CD4 cell count of 180 (IQR 68  ̶ 345) cells/μL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0  ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04  ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03  ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient's colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74  ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79  ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8., Conclusion: Baseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R 2  = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R 2  = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.

Bacterial colonization has been already demonstrated in heart valve tissues of patients without cardiovascular infections. However, the evidence of a valvular microbiome is still scarce. The next-generation sequencing method was carried out on 34 specimens of aortic (n = 20) and mitral valves (n = 14) explanted from 34 patients having neither evidence nor history of infectious diseases, particularly infective endocarditis. While no bacteria were demonstrated using standard culture methods, bacterial deoxyribonucleic acid (DNA) sequences were found using next-generation sequencing in 15/34 (44%) cases. Escherichia coli was present in 6 specimens and was the most frequently identified bacterium. There was a trend towards a higher rate of bacterial DNA positivity in specimens of calcific valves than in those of non-calcific valves (10/17 vs 5/17, P = 0.17). Based on a quantitative test, E. coli accounted for 0.7% ± 1% in calcific valvular tissue and 0.3% ± 0.3% in non-calcific valvular tissue (P = 0.2), and for 11% ± 27% in the valvular tissue of diabetic patients and 0.3% ± 1% in the valvular tissue of non-diabetic patients (P = 0.08). Detection of bacterial DNA in non-endocarditis valvular tissues could be a relatively common finding. There could be an association between the valvular microbiome and certain models of valve degeneration and common metabolic disorders., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

The bacterium Escherichia coli is commonly associated with the presence of faecal contamination in environmental samples, and is therefore subject to statutory surveillance. This is normally done using a culture-based methodology, which can be slow and laborious. Nucleic acid amplification for the detection of E. coli DNA sequences is a significantly more rapid approach, suited for applications in the field such as a point of sample analysis, and to provide an early warning of contamination. An existing, high integrity qPCR method to detect the E. coli ybbW gene, which requires almost an hour to detect low quantities of the target, was compared with a novel, isothermal RPA method, targeting the same sequence but achieving the result within a few minutes. The RPA technique demonstrated equivalent inclusivity and selectivity, and was able to detect DNA extracted from 100% of 99 E. coli strains, and exclude 100% of 30 non-target bacterial species. The limit of detection of the RPA assay was at least 100 target sequence copies. The high speed and simple, isothermal amplification chemistry may indicate that RPA is a more suitable methodology for on-site E. coli monitoring than an existing qPCR technique., (© 2020 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB&#95;YepM&#95;ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH 2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 10 2 to 1.05 × 10 7  CFU mL -1 , detection limit of 3 CFU mL -1 , and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.