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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 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.

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.

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.

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.)

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.)

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.

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.

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.)

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.

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.

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.)

Although a number of different methods have been used to quantify soil bacteria, identifying the optimal method(s) for soil bacterial abundance is still in question. No single method exists for undertaking an absolute microbial count using culture-dependent methods (CDMs) or even culture-independent methods (CIMs). This study investigated soil storage and pretreatment methods for optimal bacterial counts. Appropriate storage temperature (4°C) and optimal pretreatment methods (sonication time for 3 min and centrifugation at 1400 g) were necessary to preserve bacterial cell viability and eliminate interference from soil particles. To better estimate soil bacterial numbers under various cellular state and respiration, this study also evaluated three CDMs (i.e., colony forming unit, spotting, and most probable number (MPN) and three CIMs (i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA quantitation). Each counting method was tested using 72 soil samples collected from a local arable farm site at three different depths (i.e., 10-20, 90-100, and 180-190 cm). Among all CDMs, MPN was found to be rapid, simple, and reliable. However, the number of bacteria quantified by MPN was 1-2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA quantitation method appeared to overestimate soil bacterial numbers, which may be attributed to DNA from dead bacteria and free DNA in the soil matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead)., Competing Interests: The authors have declared that no competing interests exist.

Pork is one of the most globally eaten meats and the pig production chain contributes significantly to the water footprint of livestock production. However, very little knowledge is available about the on-farm factors that influence freshwater use in the pig production chain. An experiment was conducted to quantify the effect of three different washing treatments on freshwater use, bacterial levels [(total bacterial counts; TBC), Enterobacteriaceae and Staphylococcus] and cleaning time in washing of pens for weaning pigs. Three weaner rooms were selected with each room having 10 pens and a capacity to hold up to 14 pigs each. Pigs were weaned and kept in the pens for 7 weeks. Finally, the pens were cleaned before the next batch of pigs moved in. The washing treatments used were power washing and disinfection (WASH); presoaking followed by power washing and disinfection (SOAK), and presoaking followed by detergent, power washing and disinfection (SOAK + DETER). A water meter was used to collect water use data and swab samples were taken to determine the bacterial levels. The results showed that there was no overall effect of washing treatments on water use. However, there was an effect of treatment on the washing time (p<0.01) with SOAK and SOAK+DETER reducing the washing time per pen by 2.3 minutes (14%) and 4.2 minutes (27%) compared to WASH. Nonetheless, there was an effect of sampling time (before or after washing) (p<0.001) on the levels of TBC and Staphylococcus, but no effect was seen on Enterobacteriaceae levels. Thus, the washing treatments used in this study had no effect on the water use of the pork production chain. Although there was no difference in both water use and bacterial load, from a producer perspective, presoaking and detergent use can save time and labour costs, so this would be the preferred option., Competing Interests: The authors have declared that no competing interests exist.

Background: There is evidence that bacterial colonisation in chronic obstructive pulmonary disease (COPD) is associated with increased neutrophilic airway inflammation. This study tested the hypothesis that different bacterial phyla and species cause different inflammatory profiles in COPD patients., Methods: Sputum was analysed by quantitative polymerase chain reaction (qPCR) to quantify bacterial load and 16S rRNA gene sequencing to identify taxonomic composition. Sputum differential cell counts (DCC) and blood DCC were obtained at baseline and 6 months. Patients were categorised into five groups based on bacterial load defined by genome copies/ml of ≥ 1 × 10 4 , no colonisation and colonisation by Haemophilus influenzae (H. influenzae), Moraxella catarrhalis (M. catarrhalis), Streptococcus pneumoniae (S. pneumoniae), or > 1 potentially pathogenic microorganism (PPM)., Results: We observed an increase in sputum neutrophil (%), blood neutrophil (%) and neutrophil-lymphocyte ratio (NLR) in patients colonised with H. influenzae (82.6, 67.1, and 3.29 respectively) compared to those without PPM colonisation at baseline (69.5, 63.51 and 2.56 respectively) (p < 0.05 for all analyses), with similar findings at 6 months. The bacterial load of H. influenzae and Haemophilus determined by qPCR and 16s rRNA gene sequencing respectively, and sputum neutrophil % were positively correlated between baseline and 6 months visits (p < 0.0001, 0.0150 and 0.0002 with r = 0.53, 0.33 and 0.44 respectively)., Conclusions: These results demonstrate a subgroup of COPD patients with persistent H. influenzae colonisation that is associated with increased airway and systemic neutrophilic airway inflammation, and less eosinophilic airway inflammation.

Quantification of phenotypic heterogeneity present amongst bacterial cells can be a challenging task. Conventionally, classification and counting of bacteria sub-populations is achieved with manual microscopy, due to the lack of alternative, high-throughput, autonomous approaches. In this work, we apply classification-type convolutional neural networks (cCNN) to classify and enumerate bacterial cell sub-populations (B. subtilis clusters). Here, we demonstrate that the accuracy of the cCNN developed in this study can be as high as 86% when trained on a relatively small dataset (81 images). We also developed a new image preprocessing algorithm, specific to fluorescent microscope images, which increases the amount of training data available for the neural network by 72 times. By summing the classified cells together, the algorithm provides a total cell count which is on parity with manual counting, but is 10.2 times more consistent and 3.8 times faster. Finally, this work presents a complete solution framework for those wishing to learn and implement cCNN in their synthetic biology work., Competing Interests: The authors have declared that no competing interests exist.

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC-MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models.

Populations of genetically identical bacteria are phenotypically heterogeneous, giving rise to population functionalities that would not be possible in homogeneous populations. For instance, a proportion of non-dividing bacteria could persist through antibiotic challenges and secure population survival. This heterogeneity can be studied in complex environmental or clinical samples by spreading the bacteria on agar plates and monitoring time to growth resumption in order to infer their metabolic state distribution. We present ColTapp, the Colony Time-lapse application for bacterial colony growth quantification. Its intuitive graphical user interface allows users to analyze time-lapse images of agar plates to monitor size, color and morphology of colonies. Additionally, images at isolated timepoints can be used to estimate lag time. Using ColTapp, we analyze a dataset of Staphylococcus aureus time-lapse images including populations with heterogeneous lag time. Colonies on dense plates reach saturation early, leading to overestimation of lag time from isolated images. We show that this bias can be corrected by taking into account the area available to each colony on the plate. We envision that in clinical settings, improved analysis of colony growth dynamics may help treatment decisions oriented towards personalized antibiotic therapies.

Background: Urinary tract infections (UTI) account for millions of office visits and approximately 400 000 hospital admissions every year in the United States; as a result, the cost burden of UTI in the USA is estimated at approximately $2.8 billion. There is a great deal of interest in finding newer, faster, and more reliable methods for diagnosing UTI as compared to the standard urine culture., Methods: An automated fluorescent microbial cell counter was used to compare urine samples found to be positive for Escherichia coli UTI via cell culturing (n = 11) with UTI-negative samples (n = 10)., Results: Patients with a positive urine culture had significantly higher cell count results using the microbial cell counter (1.01 × 10 8 cells/mL) as compared to the negative samples (2.35 × 10 6 cells/mL; P = .0022)., Conclusions: These observations suggest that automated microbial cell counters may serve as a rapid, objective method for the detection of bacteriuria in urine samples submitted for evaluation of suspected UTI., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals Inc.)

We counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS in the low optical density (OD) range (OD = 0.05-1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration c b of the E. coli cells in the range of c b = 0.06 to 1.26 × 10 9 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of c b = 0.06 to 1.26 × 10 9 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of c b = 0.06 to 0.50 × 10 9 cells/mL.

Liposomes have been widely applied in bioanalytical assays. Most liposomes used bare negative charges to prevent non-specific binding and increase colloidal stability. Here, in contrast, highly stable, positively charged liposomes entrapping the fluorescent dye sulforhodamine B (SRB) were developed to serve as a secondary, non-specific label' and signal amplification tool in bioanalytical systems by exploiting their electrostatic interaction with negatively charged vesicles, surfaces, and microorganisms. The cationic liposomes were optimized for long-term stability (> 5 months) and high dye entrapment yield. Their capability as secondary, non-specific labels was first successfully proven through electrostatic interactions of cationic and anionic liposomes using dynamic light scattering, and then in a bioassay with fluorescence detection leading to an enhancement factor of 8.5 without any additional surface blocking steps. Moreover, the cationic liposomes bound efficiently to anionic magnetic beads were stable throughout magnetic separation procedures and could hence serve directly as labels in magnetic separation and purification strategies. Finally, the electrostatic interaction was exploited for the direct, simple, non-specific labeling of gram-negative bacteria. Isolated Escherichia coli cells were chosen as models and direct detection was demonstrated via fluorescent and chemiluminescent liposomes. Thus, these cationic liposomes can be used as generic labels for the development of ultrasensitive bioassays based on electrostatic interaction without the need for additional expensive recognition units like antibodies, where desired specificity is already afforded through other strategies. Graphical abstract.

The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication., Competing Interests: The authors have declared that no competing interests exist.

Background: Airways of Cystic Fibrosis (CF) patients are Nitric Oxide (NO) deficient which may contribute to impaired lung function and infection clearance. Mycobacterium abscessus (M. abscessus) infection prevalence is increasing in CF patients and is associated with increased morbidity and mortality. Here, we assess the safety and efficacy of intermittent inhaled NO (iNO) as adjuvant therapy in CF patients with refractory M. abscessus lung infection., Methods: A prospective, open-label pilot study of iNO (160 ppm) administered five times/day during hospitalization (14 days), and three times/day during ambulatory treatment (7 days) was conducted. The primary outcome was safety measured by NO-related adverse events (AEs). Secondary outcomes were six-minute walk distance (6MWD), forced expiratory volume in 1 s (FEV 1 ), and M. abscessus burden in airways., Results: Nine subjects were recruited. INO at 160 ppm was well-tolerated and no iNO-related SAEs were observed during the study. Mean FEV1 and 6WMD were increased relative to baseline during NO treatment. M. abscessus culture conversion was not achieved, but 3/9 patients experienced at least one negative culture during the study. Mean time to positivity in M. abscessus culture, and qPCR analysis showed reductions in sputum bacterial load. The study was not powered to achieve statistical significance in FEV1, 6WMD, and bacterial load., Conclusions: Intermittent iNO at 160 ppm is well tolerated and safe and led to increases in mean 6MWD and FEV1. INO exhibited potential antibacterial activity against M. abscessus. Further evaluation of secondary endpoints in a larger cohort of CF patients is warranted to demonstrate statistical significance., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Bloodstream infections (BSIs) are a major cause of morbidity and mortality in paediatric patients. For fast and accurate diagnosis, blood culture (BC) is the reference standard. However, the procedure for blood sampling in paediatric patients, particularly the optimal blood volume, is the subject of controversy stemming from a lack of knowledge of the bacterial load and because of several obstacles such as low intravascular volume and the risk of causing anaemia., Aims: The aim of this narrative review is to summarize current knowledge on blood sampling in paediatric patients for BC purposes, in particular blood volume and number and type of BC bottles needed for reasonable future guidelines/recommendations., Sources: A comprehensive literature search of PubMed, including all publications in English, was performed in June 2019 using the search terms 'blood culture', 'blood volume', 'bloodstream infection', 'diagnostic', 'paediatric' and/or 'sepsis'., Content: The amount of inoculated blood determines the sensitivity, specificity and time to positivity of a BC, and low-level bacteraemia (≤10 cfu/mL) in paediatric patients is presumed to be more common than reported. Current approaches for 'adequate' blood volume for paediatric BC are mainly weight- or age-dependent. Of these recommendations, the scheme devised by Gaur and colleagues seems most appropriate and calls for a sample of 1-1.5 mL for children weighing <11 kg and 7.5 mL for a patient weight of 11-17 kg to be drawn into one BC bottle. Inclusion of a more detailed grading in the weight range 4-14 kg, as published by Gonsalves and colleagues, might be useful., Implications: This review could be important for future guidelines on paediatric BC collection and thus could contribute to improving patient management and lowering the economic and global health burden associated with BSI. Furthermore, upcoming molecular-based approaches with low sample volumes might be an interesting alternative., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectroscopy (MS) has revolutionized microbiology by allowing clinicians and scientists to rapidly identify microbes at genus and species levels. The present study extensively assesses the suitability and reliability of MALDI-ToF biotyping of 14 different aerobic and anaerobic bacterial species as pure and mixed cultures. Reliable identification at species level was possible from biomaterial of older colonies and even frozen biomaterial, although this was species dependent. Using standard instrument settings and direct application of biomaterial onto the MALDI-ToF target plates, it was determined that the cell densities necessary for completely reliable identification of pure cultures varied between 2.40 × 10 8 and 1.10 × 10 10 viable cell counts (VCCs) per mL, depending on the species. Evaluation of the mixed culture algorithm of the Bruker Biotyper ® software showed that the performance of the algorithm depends greatly on the targeted species, on their phylogenetic distance, and on their ratio of VCC per mL in the mixed culture. Hence, the use of MALDI-ToF-MS with incorporation of the mixed culture algorithm of the software is a useful pre-screening tool for early identification of contaminants, but due to the great variability in performance between different species and the usually unknown percentage of the possible contaminant in the mixture, it is advisable to combine this method with other microbiology methods.

Lactobacillus rhamnosus is a gram-positive, rod-shaped bacterium and is commonly used as a probiotic to maintain intestinal health. Recently, surveillance of Lactobacillus bacteremia was conducted using a biochemical test and conventional PCR assay; however, these assays are unable to quantify the target and might yield a false positive result. In this study, we developed an L. rhamnosus-specific quantitative PCR assay, which yields accurate and reproducible results on the basis of the specificity of a TaqMan probe targeting the unique 16S rDNA sequence of L. rhamnosus. The assay specifically detected the target bacterium, L. rhamnosus, and no nonspecific signals were generated under the assay conditions. With genomic DNA from the cells of L. rhamnosus (10 1 to 10 6 cells), the threshold cycle values showed a linear dependence (R 2 = 0.9993). This L. rhamnosus-specific quantitative PCR assay can advance the research into the effects of this microorganism on microflora, microbial infections, and on the host.

Mycoplasma genitalium is a common sexually transmitted infection with a propensity to acquire resistance to commonly used antimicrobial therapies. Bacterial load has been linked to patient symptoms and the success of treatment. In this study, we demonstrate methodology to estimate load from routine diagnostic assays using the ResistancePlus MG test (SpeeDx Pty Ltd., Australia). The method gave comparable quantitation to an M. genitalium -specific 16S rRNA quantitative PCR (qPCR; Spearman r  = 0.94) for the samples analyzed ( n  = 499, including urine and swab types as detailed below) and was, therefore, employed to analyze typical load levels for samples in a diagnostic laboratory (total of 1,012 tests). When stratified by sample type, female urine (median, 826 genomes/ml) had the lowest load. This was significantly lower than median loads for all other sample types (male urine [6.91 × 10 3 genomes/ml], anal swabs [5.50 × 10 3 ], cervical swabs [8.15 × 10 3 ], endocervical swabs [3.97 × 10 3 ], and vaginal swabs [6.95 × 10 3 ]) ( P  < 0.0001). There were no significant differences in load estimates between the other sample types. Reproducibility of load estimates conducted on the same samples was high (r > 0.85). In conclusion, this methodology to provide load estimates for M. genitalium can be easily integrated into routine diagnostic laboratory workflow. Given the association between organism load, symptoms, and treatment success, load assessment has future diagnostic potential., (Copyright © 2019 American Society for Microbiology.)

Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-short TUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads., (Copyright © 2019 American Society for Microbiology.)

Background: The aim of this study was to establish a set of assessment methods suitable for evaluating the complex indoor environment of hospital wards and to ascertain the composition of bacteria and microbial ecology of hospital wards., Methods: Colony-forming units (CFUs), PM2.5 detection, real-time PCR, and adenosine triphosphate (ATP) bioluminescence assay were employed to evaluate the complexity of indoor air in 18 wards of nine departments in a hospital and two student dormitories in a university. Subsequently, the microbial samples were quantified and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)., Results: Although the studied indices were relatively independent, the PM2.5 content was correlated with bacterial CFUs determined by passive sedimentation method, bacterial and fungal counts measured by real-time PCR, and ATP bioluminescence assay. The composition of microorganisms in the air of hospital wards differed from that in the air of student dormitories. The dominant genera in hospital wards were Staphylococcus (39.4%), Micrococcus (21.9%), Corynebacterium (11.7%), Kocuria (4.4%), Bacillus (2.9%), Streptococcus (1.6%), Moraxella (1.6%), and Enterococcus (1.3%), and the microbial ecology differed between Respiration Dept. III and other hospital departments. Additionally, 11.1 and 27.3% of bacteria in hospital wards and student dormitories were not identified, respectively., Conclusions: Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators. There may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections.

Effective methods to detect viable Mycobacterium tuberculosis , the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of M. tuberculosis is the gold standard, which depends on initial sample processing with N -acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), chemicals that compromise M. tuberculosis viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of M. tuberculosis viability following NALC-NaOH treatment of M. tuberculosis H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of M. tuberculosis reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log 10 at 23°C ( P  = 0.018) and 0.72 ± 0.08 log 10 at 30°C ( P  = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log 10 CFU/ml at 23°C ( P  < 0.001) and 0.85 ± 0.01 log 10 CFU/ml at 30°C ( P  < 0.001), respectively. The reduction in the viable count was reflected by a corresponding increase in the time to positivity of the mycobacterial growth indicator tube (MGIT) liquid culture: 1.2 days at 23°C ( P  < 0.001) and 1.1 days at 30°C ( P  < 0.001). This NaOH-induced M. tuberculosis viability loss was replicated in clinical sputum samples, with the bacterial load dropping by 0.65 ± 0.17 log 10 from 5.36 ± 0.24 log 10 eCFU/ml to 4.71 ± 0.16 log 10 eCFU/ml for untreated and treated sputa, respectively. Applying the model of Bowness et al. (R. Bowness, M. J. Boeree, R. Aarnoutse, R. Dawson, et al., J Antimicrob Chemother 70:448-455, 2015, https://doi.org/10.1093/jac/dku415) revealed that the treated MGIT time to culture positivity of 142 ± 7.02 h was equivalent to 4.86 ± 0.28 log 10 CFU, consistent with the MBLA-measured bacterial load. Our study confirms the contribution of NALC-NaOH treatment to the loss of viable bacterial counts. Tests that obviate the need for decontamination may offer an alternative option for the accurate detection of viable M. tuberculosis and treatment response monitoring., (Copyright © 2019 American Society for Microbiology.)

Aerobic composting is a bacteria-driven process to degrade and recycle wastes. This study quantified the kinetics of bacterial growth and decay during pig manure-wheat straw composting, which may provide insights into microbial reaction mechanisms and composting operations. First, a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) method was developed to quantify the viable bacteria concentration of composting samples. The optimal PMA concentration and light exposure time were 100 μM and 8 min respectively. Subsequently, the concentrations of total and decayed bacteria were quantified. Viable and decayed bacteria coexisted during the entire composting period (experiments A and B), and the proportion of viable bacteria finally fell to only 35.1%. At the beginning, bacteria grew logarithmically and decayed rapidly. Later, the bacterial growth in experiment A remained stable, while that of experiment B was stable at first and then decomposed. The duration of the stable stage was positively related to the soluble sugar content of composting materials. The logarithmic growth and rapid decay of bacteria followed Monod equations with a specific growth (0.0317 ± 0.0033 h -1 ) and decay rate (0.0019 ± 0.0000 h -1 ). The findings better identified the bacterial growth stages and might enable better prediction of composting temperatures and the degree of maturation., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: Lipoarabinomannan (LAM) is a major antigen of Mycobacterium tuberculosis (MTB). In this report, we evaluated the ability of a novel immunoassay to measure concentrations of LAM in sputum as a biomarker of bacterial load prior to and during treatment in pulmonary tuberculosis (TB) patients., Methods and Findings: Phage display technology was used to isolate monoclonal antibodies binding to epitopes unique in LAM from MTB and slow-growing nontuberculous mycobacteria (NTM). Using these antibodies, a sandwich enzyme-linked immunosorbent assay (LAM-ELISA) was developed to quantitate LAM concentration. The LAM-ELISA had a lower limit of quantification of 15 pg/mL LAM, corresponding to 121 colony-forming units (CFUs)/mL of MTB strain H37Rv. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. Two clinical studies were performed between the years 2013 and 2016 in Manila, Philippines, in patients without known human immunodeficiency virus (HIV) coinfection. In a case-control cohort diagnostic study, sputum specimens were collected from 308 patients (aged 17-69 years; 62% male) diagnosed as having pulmonary TB diseases or non-TB diseases, but who could expectorate sputum, and were then evaluated by smear microscopy, BACTEC MGIT 960 Mycobacterial Detection System (MGIT) and Lowenstein-Jensen (LJ) culture, and LAM-ELISA. Some sputum specimens were also examined by Xpert MTB/RIF. The LAM-ELISA detected all smear- and MTB-culture-positive samples (n = 70) and 50% (n = 29) of smear-negative but culture-positive samples (n = 58) (versus 79.3%; 46 positive cases by the Xpert MTB/RIF), but none from non-TB patients (n = 56). Among both LAM and MGIT MTB-culture-positive samples, log10-transformed LAM concentration and MGIT time to detection (TTD) showed a good inverse relationship (r = -0.803, p < 0.0001). In a prospective longitudinal cohort study, 40 drug-susceptible pulmonary TB patients (aged 18-69 years; 60% male) were enrolled during the first 56 days of the standard 4-drug therapy. Declines in sputum LAM concentrations correlated with increases of MGIT TTD in individual patients. There was a 1.29 log10 decrease of sputum LAM concentration, corresponding to an increase of 221 hours for MGIT TTD during the first 14 days of treatment, a treatment duration often used in early bactericidal activity (EBA) trials. Major limitations of this study include a relatively small number of patients, treatment duration up to only 56 days, lack of quantitative sputum culture CFU count data, and no examination of the correlation of sputum LAM to clinical cure., Conclusions: These results indicate that the LAM-ELISA can determine LAM concentration in sputum, and sputum LAM measured by the assay may be used as a biomarker of bacterial load prior to and during TB treatment. Additional studies are needed to examine the predictive value of this novel biomarker on treatment outcomes., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MK, TO, RI, RH and KK are employees of Otsuka Pharmaceutical Co., Ltd. YL is an employee of Otsuka Pharmaceutical Development & Commercialization, Inc., which is a subsidiary of Otsuka Pharmaceutical Co., Ltd. During the conduct of the clinical studies, MTG was an employee of Otsuka (Philippines) Pharmaceutical Inc., which is a subsidiary of Otsuka Pharmaceutical Co., Ltd. The rest of the authors declare no conflicts of interest associated with this manuscript. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Background: Neutrophil extracellular traps (NETs) are innate defense mechanisms that are also implicated in the pathogenesis of organ dysfunction. However, the role of NETs in pediatric sepsis is unknown., Methods: Infant (2 weeks old) and adult (6 weeks old) mice were submitted to sepsis by intraperitoneal (i.p.) injection of bacteria suspension or lipopolysaccharide (LPS). Neutrophil infiltration, bacteremia, organ injury, and concentrations of cytokine, NETs, and DNase in the plasma were measured. Production of reactive oxygen and nitrogen species and release of NETs by neutrophils were also evaluated. To investigate the functional role of NETs, mice undergoing sepsis were treated with antibiotic plus rhDNase and the survival, organ injury, and levels of inflammatory markers and NETs were determined. Blood samples from pediatric and adult sepsis patients were collected and the concentrations of NETs measured., Results: Infant C57BL/6 mice subjected to sepsis or LPS-induced endotoxemia produced significantly higher levels of NETs than the adult mice. Moreover, compared to that of the adult mice, this outcome was accompanied by increased organ injury and production of inflammatory cytokines. The increased NETs were associated with elevated expression of Padi4 and histone H3 citrullination in the neutrophils. Furthermore, treatment of infant septic mice with rhDNase or a PAD-4 inhibitor markedly attenuated sepsis. Importantly, pediatric septic patients had high levels of NETs, and the severity of pediatric sepsis was positively correlated with the level of NETs., Conclusion: This study reveals a hitherto unrecognized mechanism of pediatric sepsis susceptibility and suggests that NETs represents a potential target to improve clinical outcomes of sepsis.

Here we report a bacteria detection method based on a flow cytometric bead system and 16S rRNA-targeted oligonucleotide probes. Polymerase chain reaction (PCR) was first used to acquire bacterial DNA including bacteria-specific sequences. Half of the resulting target DNA was then captured by a capture probe immobilized on a magnetic microbead (MB) surface. The other half of the target DNA was hybridized with a fluorescence-labeled signal probe. In this manner, a sandwich DNA hybridization involving a MB-based capture probe, the target DNA, and a signal probe was realized. The MB carriers modified with reporter dye were analyzed one by one by flow cytometry through a capillary. Using PCR amplicons and this flow cytometric bead system, a detection limit of 180 cfu mL -1 was achieved, along with high selectivity that permitted the discrimination of different targets when challenged with control bacteria targets and multiplexing capabilities that enabled the simultaneous detection of two kinds of bacteria. Given these advantages, the developed method can be used for the highly sensitive and specific PCR amplicon analysis of DNA extracted from a fresh bacterial culture, as well as multiplex target analysis. Graphical abstract The flow cytometric bead system with 16S rRNA-targeted oligonucleotide probes for bacteria detection developed in this work. This system is highly specific and sensitive, with a detection limit of 180 cfu mL -1 bacteria.

Background: Invasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR)., Aims: Our aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity., Methods: The National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples., Results: Three-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p < .10-4; Cohen's κ 0.08 for blood and OR 49.0; 95% CI 19.1-133.4; p<10-4; Cohen's κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14-23 and mean 29.6, range 16-39; p<10-17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025-7.215), however 60% of deaths would have remained undiagnosed with the use of culture only., Conclusions: In conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD., Competing Interests: SG received a scholarship from GSK for meningococcal epidemiology; GSK did not participate in study design, data analysis, manuscript writing and in any other aspects related to the present work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors have no other conflict of interest.

In this study, the ability to discriminate viable from dead cells of Mycobacterium tuberculosis complex (MTC), using an ethidium monoazide (EMA) treatment-dependent viable bacteria selection PCR kit was examined. Detection of dead bacteria was possible for bacterial concentrations in the range 1.5 × 10 7 -3.0 × 10 7 CFU/mL, which was equivalent to McFarland No. 0.05-0.10. There was a significant difference between the results for viable and dead bacteria, and the sensitivity and specificity of this method for culture-negative samples from patients were 83% and 100%, respectively. To the best of our knowledge, this is the first successful selective detection of DNA from viable cells of MTC by EMA-PCR, using the viable bacteria selection kit for PCR (gram-positive), an EMA treatment kit. We believe that application of this method could promote earlier discharge of patients undergoing tuberculosis treatment by discriminating dead from viable cells.

The aim of this study was to evaluate the abatement power of oral microbial loading of a new gel formulation based on the complex silver-2-mercaptobenzoate, chlorhexidine digluconate and didecyldimethylammonium chloride (named ADC) through polymerase chain reaction (PCR). The study sample consists of a group of 20 patients with chronic periodontal disease. Patients were over 25 years of age and did not undergo surgical or non-surgical periodontal treatment in the previous 6 months. The study sample was allotted into two groups of 10 patients each, homogeneous by age and sex. The test group received a bottle containing ADC gel, while the control group received an identical one containing placebo, similar to ADC in consistence, colour, taste and odour. Sub-gingival samples of four sites, one in each quadrant, of greatest probing depth in each patient were used. Microbiological analyses were performed at baseline and at day 15. Paired t test was performed to detect statistical significant reduction in total bacterial loading and oral pathogens in the study groups. The analysis showed a statistically significant reduction in the total bacterial loading evaluated pre- and post-treatment ( P = 0.029) in the study groups. In the control group, the decrease in total bacterial loading was not significant ( P = 0.279). Clinically, ADC gel does not have any side effects and discomfort such as pain, burning, tingling sensation or numbness and produces no adverse reactions in time. Our study aimed to evaluate the efficacy of a new chemical formulation with antibacterial properties to use for daily oral hygiene with a preliminary study. Our results showed a statistically significant reduction in total bacterial loading after treatment, but the limitations of our study do not allow us to demonstrate the clinical efficacy of the ADC gel.

Objective: To assess the impact of chlorhexidine gluconate (CHG) bathing on skin bacterial burden in neonates., Study Design: In this prospective observational study, arm and groin skin bacterial growth was measured in 40 CHG-exposed and nonexposed neonates admitted to the NICU. Exposed neonates received 2% CHG baths per protocol for central line-associated bloodstream infection (CLABSI) prevention or Staphylococcus aureus decolonization., Results: Forty neonates were enrolled, 18 of whom were CHG-exposed. Mean baseline Gram-positive (GP) bacterial burden was 2.19 log CFU/ml on the arm and 1.81 log CFU/ml on the groin. Bacterial burden decreased after the first bath, but returned to baseline by 72 h. Residual skin CHG concentration declined over time, with a corresponding increase in GP bacterial burden., Conclusions: CHG bathing reduces skin bacterial burden, but burden returns to baseline after 72 h. Twice weekly CHG bathing may be inadequate to suppress skin bacterial growth in hospitalized neonates.

Background: The aim was to evaluate the value of organ-specific weighted incidence antibiogram (OSWIA) percentages for bacterial susceptibilities of Gram-negative bacteria (GNB) collected from intra-abdominal infections (IAIs) during SMART 2010-2014., Methods: We retrospectively calculated the OSWIA percentages that would have been adequately covered by 12 common antimicrobials based on the bacterial compositions found in the appendix, peritoneum, colon, liver, gall bladder and pancreas., Results: The ESBL positive rates were 65.7% for Escherichia coli, 36.2% for Klebsiella pneumoniae, 42.9% for Proteus mirabilis and 33.1% for Klebsiella oxytoca. Escherichia coli were mainly found in the appendix (76.8%), but less so in the liver (32.4%). Klebsiella pneumoniae constituted 45.2% of the total liver pathogenic bacteria and 15.2-20.8% were found in 4 other organs, except the colon and appendix (< 10%). The percentages of Pseudomonas aeruginosa infections were higher in the gall bladder, intra-abdominal abscesses, pancreas and colon (10.2-13.2%) and least (5.4%) in the appendix. The susceptibilities of hospital acquired (HA) and community acquired (CA) IAI isolates from appendix, gall bladder and liver showed ≥80% susceptibilities to amikacin (AMK), imipenem (IPM), piperacillin-tazobactam (TZP) and ertapenem (ETP), while the susceptibility of isolates in abscesses and peritoneal fluid showed ≥80% susceptibility only to amikacin (AMK) and imipenem (IPM). In colon CA IAI isolates susceptibilities did not reach 80% for AMK and ETP, and in pancreatic IAIs susceptibilities of HA GNBs did not reach 80% to AMK, TZP and ETP, and CA GNBs to IMP and ETP. In addition, besides circa 80% susceptibility of HA and CA IAI isolates from appendix to cefoxitin (FOX), IAI isolates from all other organs had susceptibilities between 7.6 and 67.9% to all cephalosporins tested, 28.3-75.2% to fluoroquinolones and 7.6-51.0% to ampicillin-sulbactam (SAM), whether they were obtained from CA or HA infections., Conclusion: The calculated OSWIA susceptibilities were specific for different organs in abdominal infections.

Platelet transfusions carry greater risks of infection, sepsis, and death than any other blood product, owing primarily to bacterial contamination. Many patients may be at particular risk, including critically ill patients in the intensive care unit. This narrative review provides an overview of the problem and an update on strategies for the prevention, detection, and reduction/inactivation of bacterial contaminants in platelets. Bacterial contamination and septic transfusion reactions are major sources of morbidity and mortality. Between 1:1000 and 1:2500 platelet units are bacterially contaminated. The skin bacterial microflora is a primary source of contamination, and enteric contaminants are rare but may be clinically devastating, while platelet storage conditions can support bacterial growth. Donor selection, blood diversion, and hemovigilance are effective but have limitations. Biofilm-producing species can adhere to biological and non-biological surfaces and evade detection. Primary bacterial culture testing of apheresis platelets is in routine use in the US. Pathogen reduction/inactivation technologies compatible with platelets use ultraviolet light-based mechanisms to target nucleic acids of contaminating bacteria and other pathogens. These methods have demonstrated safety and efficacy and represent a proactive approach for inactivating contaminants before transfusion to prevent transfusion-transmitted infections. One system, which combines ultraviolet A and amotosalen for broad-spectrum pathogen inactivation, is approved in both the US and Europe. Current US Food and Drug Administration recommendations advocate enhanced bacterial testing or pathogen reduction/inactivation strategies (or both) to further improve platelet safety. Risks of bacterial contamination of platelets and transfusion-transmitted infections have been significantly mitigated, but not eliminated, by improvements in prevention and detection strategies. Regulatory-approved technologies for pathogen reduction/inactivation have further enhanced the safety of platelet transfusions. Ongoing development of these technologies holds great promise.

Background: Chlorhexidine and parachlorometaxylenol (PCMX) are antiseptics recommended for surgical hand antisepsis. To our knowledge, PCMX has not been evaluated for bactericidal efficacy "in vivo., Methods: We conducted a randomized, double-blind, controlled crossover trial to compare the bacterial loads on fingertips and fingernails under laboratory conditions after use of antiseptic test products, including chlorhexidine digluconate 4%, PCMX 3%, and a reference solution of propan-1-ol 60% (P-1). We assessed bacterial load after a prewash with soft soap, immediately after application of an antiseptic, and 3 hours after application and wearing of sterile, powder-free gloves. Our procedures followed those specified by European Norm (EN) 12791 for evaluating surgical hand antiseptics and using cotton swab for fingertips and fingernails., Results: Chlorhexidine digluconate 4% and PCMX 3% did not decrease bacterial load on the hands. The bactericidal performances of chlorhexidine digluconate 4% and PCMX 3% did not differ significantly. Chlorhexidine digluconate 4% and PCMX 3% increased bacterial load on the fingertips after participants had worn gloves for 3 hours. Fingernails had greater bacterial loads than skin on the fingertips., Conclusions: Chlorhexidine digluconate 4% and PCMX 3% had similar bactericidal efficacy, but they failed to meet the EN 12791 efficacy standard. Fingernails should be a particular focus of antisepsis in preparation for surgery.The trial was registered at ClinicalTrials.gov (ID: NCT02500758).

Background: Escherichia coli (E. coli) is one of the best-known zoonotic bacterial species, which pathogenic strain can cause infections in humans and animals. However, existing technologies or methods are deficient for quickly on-site identifying infection of E. coli before they breakout. Herein, we present an ultrasensitive and on-site method for counting E. coli using magnetic nanoparticle (MNP) probe under a dark-field in 30 min., Results: The antibodies functionalized MNP, binding to E. coli to form a golden ring-like structure under a dark-field microscope, allowing for counting E. coli. This method via counting MNP-conjugated E. coli under dark-field microscope demonstrated the sensitivity of 6 CFU/μL for E. coli detection. Importantly, due to the advantages such as time-saving (only 30 min) and almost free of instrument (only require a portable microscope), our MNP-labeled dark-field counting strategy has the potential of being a universal tool for on-site quantifying a variety of pathogens with size ranges from a few hundreds of nanometers to a few micrometers., Conclusion: In summary, the MNP-labeled dark-field counting strategy is a rapid, simple, sensitive as well as low-cost assay strategy, which has the potential of being a universal tool for on-site quantification of micrometer-size pathogens like E. coli.

Contaminated surfaces serve as an important reservoir for Clostridium difficile transmission. Current strategies to detect environmental contamination of C. difficile rely heavily on culture, and often only indicate presence versus absence of spores. The goal of this study was to compare quantitative PCR (qPCR) to culture for the detection and quantification of C. difficile from inert surfaces. First, we compared the limit of detection (LOD) of a 16S rRNA gene and toxin B gene qPCR assay for detection of C. difficile in solution. Second, we compared the LODs of 16S rRNA gene qPCR versus culture for detection of C. difficile from surfaces. Solution experiments were performed by direct seeding of spores into neutralizing broth, whereas surface experiments involved seeding of spores onto plastic test surfaces, and recovery using sponge swabs. Both experiments were conducted using spores expressing short (NAP1) and long (NAP4) hair lengths. Combining data from both strains, the overall LOD for C. difficile cells in solution was 1.4 cells for 16S rRNA gene and 23.6 cells for toxin B gene qPCR (p<0.001). The overall LOD for C. difficile cells from surfaces was 17.1 cells for 16S rRNA gene qPCR and 54.5 cells for culture (p = 0.05), and was not statistically different between strains for each method (p = 0.52). Overall, the proportion of C. difficile cells recovered from surfaces was good when detected by 16S rRNA gene qPCR and culture (qPCR: 76%, culture: 67%, p = 0.36), but, 16S rRNA gene qPCR was capable of detecting lower levels of surface contamination. Future work attempting to measure the presence of C. difficile on environmental surfaces should consider using qPCR., Competing Interests: The authors have declared that no competing interests exist.

Background/purposes: Routine cell number determination for specific Lactobacillus strain by cultivation requires at least 4-7 days. Thus rapid and specific cell number determine methods such as strain-specific quantitative PCR (qPCR) are valuable. However, qPCR method is vulnerable to difficult PCR target such as dimer/secondary structure forming sequence., Methods: In this study, a two-component, "Ct contrast" approach was applied to strain-specific qPCR system following the development of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) strain-specific PCR with random amplification of polymorphic DNA (RAPD)-derived strain-specific sequences., Results: The quantitative range of the NTU 101 strain-specific qPCR system was 3.0 × 10 1 to 3.0 × 10 5 copies for pure cultures, and 3.0 × 10 2 to 3.0 × 10 5 copies for multi-strain or unknown food samples. The results of spike in test and real sample testing suggested that non-specific weak background signals did not compromise test specificity, and demonstrated the potential of the NTU 101 strain-specific qPCR system in food samples., Conclusion: The two-component, "Ct contrast" approach is useful for qPCR discrimination when no ideal PCR target is available or the variance of the target site is unpredictable. The Ct contrast approach might provide a simple and robust solution for other challenging qPCR targets., (Copyright © 2017. Published by Elsevier B.V.)

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.

Background: Brucellosis is one of the neglected zoonotic diseases in humans. The serological methods based on antibody detections are unable to detect the effectiveness of treatment in humans as antibodies persist for long time in humans even after therapy. Therefore, we developed qPCR technique to overcome such discrepancy and device a rapid and efficient test for both diagnosis and follow up of the brucellosis affected individuals., Methods: High risk suspected individuals with positive serology (RBPT, STAT and iELISA) and PCR were mainly analyzed for DNA quantification by qPCR assay. The bcsp-31 gene, a shared gene of Brucella species was amplified by genus specific primers and cloned to pGEMT™ easy vector and the cloned plasmid were used to construct a standard curve (R 2 =0.99, efficiency=1.98) over 7 orders of magnitude with sensitivity of ≈10 copy number. The assay was found 100% specific., Results: Overall 85 individuals were found positive out of 188. Out of them, 23 serological, PCR and qPCR positive individuals were recommended for 45days therapy according to WHO regimen (Doxycycline and Rifampin) and each case was further followed by qPCR. The mean threshold cycle (C q ) before treatment was 26.05±0.347 (3940.5copies/μl), which increased significantly to 32.7±0.66 (259.13copies/μl) on 4th week during treatment, 35.12±3.12 (38.52copies/μl) at 6th week on day of treatment completion, 35.6±0.66 (34.21copies/μl) on 21st day after treatment depicting a significant reduction in DNA load over the course of treatment. Serological follow up showed that only 3 individuals had decreased STAT titre but no change in RBPT results. Out of 17 symptomatic individuals under therapy, 10 improved clinically, 5 improved clinically with persistent weakness and 2 had no effect of therapy., Conclusion: The study suggests that qPCR is more useful and rapid test to follow treated individuals than serology., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Growth rates, interactions between community members, stochasticity, and immigration are important drivers of microbial community dynamics. In sequencing data analysis, such as network construction and community model parameterization, we make implicit assumptions about the nature of these drivers and thereby restrict model outcome. Despite apparent risk of methodological bias, the validity of the assumptions is rarely tested, as comprehensive procedures are lacking. Here, we propose a classification scheme to determine the processes that gave rise to the observed time series and to enable better model selection., Results: We implemented a three-step classification scheme in R that first determines whether dependence between successive time steps (temporal structure) is present in the time series and then assesses with a recently developed neutrality test whether interactions between species are required for the dynamics. If the first and second tests confirm the presence of temporal structure and interactions, then parameters for interaction models are estimated. To quantify the importance of temporal structure, we compute the noise-type profile of the community, which ranges from black in case of strong dependency to white in the absence of any dependency. We applied this scheme to simulated time series generated with the Dirichlet-multinomial (DM) distribution, Hubbell's neutral model, the generalized Lotka-Volterra model and its discrete variant (the Ricker model), and a self-organized instability model, as well as to human stool microbiota time series. The noise-type profiles for all but DM data clearly indicated distinctive structures. The neutrality test correctly classified all but DM and neutral time series as non-neutral. The procedure reliably identified time series for which interaction inference was suitable. Both tests were required, as we demonstrated that all structured time series, including those generated with the neutral model, achieved a moderate to high goodness of fit to the Ricker model., Conclusions: We present a fast and robust scheme to classify community structure and to assess the prevalence of interactions directly from microbial time series data. The procedure not only serves to determine ecological drivers of microbial dynamics, but also to guide selection of appropriate community models for prediction and follow-up analysis.