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Background: Studying long-term treatment outcomes of TB is time-consuming and impractical. Early and reliable biomarkers reflecting treatment response and capable of predicting long-term outcomes are urgently needed., Objectives: To develop a pharmacometric multistate model to evaluate the link between potential predictors and long-term outcomes., Methods: Data were obtained from two Phase II clinical trials (TMC207-C208 and TMC207-C209) with bedaquiline on top of a multidrug background regimen. Patients were typically followed throughout a 24 week investigational treatment period plus a 96 week follow-up period. A five-state multistate model (active TB, converted, recurrent TB, dropout, and death) was developed to describe observed transitions. Evaluated predictors included patient characteristics, baseline TB disease severity and on-treatment biomarkers., Results: A fast bacterial clearance in the first 2 weeks and low TB bacterial burden at baseline increased probability to achieve conversion, whereas patients with XDR-TB were less likely to reach conversion. Higher estimated mycobacterial load at the end of 24 week treatment increased the probability of recurrence. At 120 weeks, the model predicted 55% (95% prediction interval, 50%-60%), 6.5% (4.2%-9.0%) and 7.5% (5.2%-10%) of patients in converted, recurrent TB and death states, respectively. Simulations predicted a substantial increase of recurrence after 24 weeks in patients with slow bacterial clearance regardless of baseline bacterial burden., Conclusions: The developed multistate model successfully described TB treatment outcomes. The multistate modelling framework enables prediction of several outcomes simultaneously, and allows mechanistically sound investigation of novel promising predictors. This may help support future biomarker evaluation, clinical trial design and analysis., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: Pharmacodynamic parameters evaluated under conditions that simulate an infection site volume and microbial load might reveal hidden risks of resistance selection and subsequent treatment failure. The study aimed to investigate the predictive potential of MICs determined at various conditions on the antimicrobial effect and emergence of resistance., Methods: We assessed meropenem MICs (microdilution: 0.2 mL, 5 × 105 cfu/mL; macrodilution: 2 mL, 5 × 105 cfu/mL), MICHVs (220 mL, 5 × 105 cfu/mL), MICHIs (0.2 mL, 5 × 107 cfu/mL) and MICHVIs (220 mL, 5 × 107 cfu/mL) for five Klebsiella pneumoniae strains and analysed these values alongside the results of experiments in a dynamic in vitro model. A clinically relevant meropenem dosing regimen was simulated and the starting bacterial inocula were 106 and 108 cfu/mL., Results: The effectiveness of meropenem agreed with MICHVs for the 106 cfu/mL inoculum and with MICHIs or MICHVIs for the 108 cfu/mL inoculum. Strains characterized as resistant according to these values grew during meropenem exposure, and resistant mutants were selected., Conclusions: Our results suggest that MICHV-based parameters may be suitable for predicting antibacterial effects and the risk of resistance development when the inoculum is 106 cfu/mL, while MICHI- or MICHVI-based parameters are suitable for these purposes when the inoculum is 108 cfu/mL. Also, the correlation between resistance selection and the MICHI-based parameter was as high as one that corresponds with a mutant prevention concentration (MPC)-based parameter; this suggests that the MPC can be replaced by the more easily determined alternative parameter MICHI., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Adaptive platform trials can be more efficient than classic trials for developing new treatments. Moving from culture-based to simpler- or faster-to-measure biomarkers as efficacy surrogates may enhance this advantage. We performed a systematic review of treatment efficacy biomarkers in adults with tuberculosis. Platform trials can span different development phases. We grouped biomarkers as: α, bacterial load estimates used in phase 2a trials; β, early and end-of treatment end points, phase 2b-c trials; γ, posttreatment or trial-level estimates, phase 2c-3 trials. We considered as analysis unit (biomarker entry) each combination of biomarker, predicted outcome, and their respective measurement times or intervals. Performance metrics included: sensitivity, specificity, area under the receiver-operator curve (AUC), and correlation measures, and classified as poor, promising, or good. Eighty-six studies included 22 864 participants. From 1356 biomarker entries, 318 were reported with the performance metrics of interest, with 103 promising and 41 good predictors. Group results were: α, mycobacterial RNA and lipoarabinomannan (LAM) in sputum, and host metabolites in urine; β, mycobacterial RNA and host transcriptomic or cytokine signatures for early treatment response; and γ, host transcriptomics for recurrence. A combination of biomarkers from different categories could help in designing more efficient platform trials. Efforts to develop efficacy surrogates should be better coordinated., Competing Interests: Potential conflicts of interest. R. A. is employed by Quiagen. J. G. M. is employed by Janssen Pharmaceuticals. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bacterial killing in patients with tuberculosis (TB) relapse was compared to that in patients achieving cure, measured by TB molecular bacterial load assay (TB-MBLA) or mycobacteria growth indicator tube (MGIT) time to positivity (TTP). TB-MBLA in 4 relapsed patients was significantly different compared to 132 cured patients after 2 weeks of treatment; MGIT TTP showed a significant difference from week 8., Competing Interests: Potential conflicts of interest. N. E. N., D. M., W. S., G. K., L. T. M., D. K., K. R., P. P. J. P., M. J. B., S. H. G., M. H., and N. H. report an EDCTP grant through the PanACEA Consortium. Additionally, S. H. G. received a UK Medical Research Council grant through his institution, and N. H. has received a research grant from Beckman Coulter and equipment from Cepheid, as research support to his institution. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: Bone marrow culture (BMC) is the reference standard for typhoid fever diagnosis. We studied the additional yield of BMC over blood culture (BC) and the relationship between quantitative BMC counts and severe disease., Methods: Hospitalised Vietnamese patients with suspected typhoid fever were prospectively investigated with a BC, BMC, faecal culture and quantitative BMC counts., Results: Salmonella typhi was isolated in 195 of 231 patients: from BC and BMC in 144 (73.8%), from BMC alone in 33 (16.9%), from BC alone in 12 (6.2%) and from faeces alone in 6 (3.1%). In 167 patients the median extracellular count of S. typhi was 2.5 cfu/mL (interquartile range [IQR] 0-10) and the intracellular count was 10.5 cfu/mL (IQR 2-42) with a ratio of 1.3 bacteria/cell (IQR 0.6-2.5). The median count of intracellular bacteria in 24 patients with severe disease was 46 bacteria/cell (IQR 9-105) compared with 6.5 bacteria/cell (IQR 2-34) in 143 with non-severe disease (p=0.005). The intracellular BMC count was negatively correlated with the peripheral white cell count and positively correlated with hepatomegaly, splenomegaly, aspartate transaminase, a positive BC and the fever clearance time following treatment with azithromycin, ofloxacin or a combination of the two., Conclusions: BMC gave a moderate additional yield over BC. Intracellular BMC counts may reflect the bacterial load in typhoid fever., (© The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Vaccines against Staphylococcus aureus have eluded researchers for >3 decades while the burden of staphylococcal diseases has increased. Early vaccine attempts mainly used rodents to characterize preclinical efficacy, and all subsequently failed in human clinical efficacy trials. More recently, leukocidin AB (LukAB) has gained interest as a vaccine antigen. We developed a minipig deep surgical wound infection model offering 3 independent efficacy readouts: bacterial load at the superficial and at the deep-seated surgical site, and dissemination of bacteria. Due to similarities with humans, minipigs are an attractive option to study novel vaccine candidates. With this model, we characterized the efficacy of a LukAB toxoid as vaccine candidate. Compared to control animals, a 3-log reduction of bacteria at the deep-seated surgical site was observed in LukAB-treated minipigs and dissemination of bacteria was dramatically reduced. Therefore, LukAB toxoids may be a useful addition to S. aureus vaccines and warrant further study., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Host-symbiont interactions may form obligatory or facultative associations that are context dependent. Long-term studies on microbiome composition from wild populations should assess the temporal and spatial dynamics of host-microbe associations. We characterized the temporal and spatial variation in the bacterial microbiome composition in six populations of the social spider Stegodyphus dumicola for 2.5 years, using 16S rRNA gene amplicon sequencing of whole spiders. Individuals within a nest exhibit highly similar microbiomes, which remain stable over several generations and are not predictably affected by seasonal variation in temperature or humidity. This stability in nest microbiome is likely due to social transmission, whereas drift-like processes during new nest foundations explain variation in host microbiomes between nests. This is supported by the lack of obligate symbionts (i.e. no symbionts are present in all spider individuals). Quantitative PCR analyses showed that the bacterial load of individual spiders is stable in healthy nests but can increase dramatically in perishing nests. These increases are not driven by specific bacterial taxa but likely caused by loss of host immune control under deteriorating conditions. Spider nests show an annual survival rate of approximately 45%, but nest death is not correlated to microbiome composition, and the bacteria found in S. dumicola are not considered to be high virulence pathogens., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Aim: The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow's milk., Methods and Results: A copper ions treatment was evaluated in naturally contaminated cow's milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical-chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively)., Conclusion: With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow's milk., Significance and Impact of the Study: This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow's milk, information that could be useful as a tool for MAP control., (© 2020 The Society for Applied Microbiology.)

Aims: This study evaluated the microbiological quality and safety of minimally processed parsley sold in southeastern Brazilian food markets., Methods and Results: One hundred samples were submitted to the enumeration of Enterobacteriaceae by plating on MacConkey agar. Colonies of Enterobacteriaceae were randomly selected and identified by MALDI-TOF MS. Samples were also tested for Listeria monocytogenes and Salmonella sp. The mean count of Enterobacteriaceae was 6·0 ± 1·0 log CFU per gram, while 18 genera (including 30 species) of bacteria belonging to this family were identified. Salmonella and L. monocytogenes were not detected, while L. innocua was found in two samples and L. fleischmannii was found in one sample. Moreover generic Escherichia coli was found in three samples, all from different brands of minimally processed parsley., Conclusions: Even though microbial pathogens were not isolated, a variety of indicator micro-organisms were identified, including vegetable spoilers and species capable of causing human opportunistic infections. These results suggest hygienic failures and/or lack of temperature control during processing and storage of these ready-to-eat products., Significance and Impact of Study: This study highlights the need for control measures during the production chain of minimally processed parsley in order to reduce microbial contamination and the risks of foodborne diseases., (© 2020 The Society for Applied Microbiology.)

Myeloid-derived suppressor cells (MDSCs) increase in tuberculosis (TB) and may be targets for host-directed therapy (HDT). In this study, we use flow cytometry to analyze the effects of cyclooxygenase-2 inhibitors (COX-2i) on monocytic (M)-MDSCs in blood from TB patients attending a clinical trial of COX-2i. The effects of COX-2i on M-MDSCs and mycobacterial uptake were also studied by an in vitro mycobacterial infection model. We found that M-MDSC frequencies correlated with TB disease severity. Reduced M-MDSC (P = 0.05) and IDO (P = 0.03) expression was observed in the COX-2i group. We show that peripheral blood-derived M-MDSCs successfully internalized Mycobacterium bovis and that in vitro mycobacterial infection increased COX-2 (P = 0.002), PD-L1 (P = 0.01), and Arginase-1 (P = 0.002) expression in M-MDSCs. Soluble IL-1β, IL-10, and S100A9 were reduced in COX-2i-treated M-MDSCs cultures (P < 0.05). We show novel data that COX-2i had limited effect in vivo but reduced M-MDSC cytokine production in vitro. The relevance of COX-2i in a HDT strategy needs to be further explored., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Background: The Chlamydia trachomatis bacterial load could have impact on transmission and sequelae. This is the first study providing comparison of C. trachomatis load at 3 anatomic sites estimated by cycle quantification (Cq) values., Methods: Data from 7900 C. trachomatis-positive samples were included (2012-2018). Cq value was used as an inversely proportional measure for C. trachomatis load. Multivariable linear regression analyses assessed differences in mean Cq values., Results: Vaginal swabs had the lowest Cq values (31.0) followed by urine (32.5), anorectal swabs (34.0), and oropharyngeal swabs (36.8) (P < .001). Men and women had similar oropharyngeal (36.4 vs 37.3; P = .13) and anorectal (34.2 vs 33.9; P = .19) Cq values. Men (32.2) and women (30.7) aged <25 years had lower urogenital Cq values than men (32.8) and women (31.9) aged ≥25 years (P < .001). HIV-positive patients had higher urogenital Cq values than HIV-negative patients (33.8 vs 32.6; P < .03)., Conclusions: Men and women have a similar C. trachomatis load at extragenital locations arguing for similar transmission potential and clinical relevance. Older patients and HIV-coinfected patients had lower C. trachomatis load, suggesting exposure to previous C. trachomatis infections potentially leading to partial immunity reducing load., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Chlamydiae are pathogenic intracellular bacteria that cause a wide variety of diseases throughout the globe, affecting the eye, lung, coronary arteries and female genital tract. Rather than by direct cellular toxicity, Chlamydia infection generally causes pathology by inducing fibrosis and scarring that is largely mediated by host inflammation. While a robust immune response is required for clearance of the infection, certain elements of that immune response may also damage infected tissue, leading to, in the case of female genital infection, disease sequelae such as pelvic inflammatory disease, infertility and ectopic pregnancy. It has become increasingly clear that the components of the immune system that destroy bacteria and those that cause pathology only partially overlap. In the ongoing quest for a vaccine that prevents Chlamydia-induced disease, it is important to target mechanisms that can achieve protective immunity while preventing mechanisms that damage tissue. This review focuses on mouse models of genital Chlamydia infection and synthesizes recent studies to generate a comprehensive model for immunity in the murine female genital tract, clarifying the respective contributions of various branches of innate and adaptive immunity to both host protection and pathogenic genital scarring., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

This study evaluated the effect of T regulatory cells (Treg cells) and the impact of BCG vaccination history of donors using an in vitro model of Mycobacterium tuberculosis H37Ra infection of peripheral blood mononuclear cells (PBMCs). PBMCs from donors with or without prior BCG vaccination were depleted of Treg cells (PBMCs-Tregs) or not depleted with Treg cells (PBMCs + Tregs) were infected up to 8 days with Mtb H37Ra. Cell aggregates were smaller in PBMCs-Tregs compared to PBMCs + Tregs at day 8 post-infection. Mtb CFUs were higher in the PBMCs-Tregs compared to PBMCs + Tregs at days 3, 5 and 8. The levels of IL-17, IFN-γ (at days 3 and 5), and TNF-α and IL-6 (at day 3) were lower in PBMCs-Tregs compared to PBMCs + Tregs. In contrast, the levels of IL-10 and IL-4 cytokines were higher at day 3 in PBMCs-Tregs compared to PBMCs + Tregs. BCG vaccination status of donors had no impact on the mycobacterial culture, level of cytokines and immune cell populations. This study shows that depletion of Tregs in human PBMCs infected with Mtb H37Ra in vitro leads to a shift from a Th1 to a Th2 cytokine rich environment that supports the survival of Mtb in this model., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS..)

Background: PixeeMo™ is a compact instrument that enables bacterial cell counting using microfluidic chips instead of counting of colonies on culture media. Chips containing electrodes, based on fluid, electric filtering and sorting technology (FES), allow the selection of bacterial cells from other components in the sample. In the United States (US), surface water or ground water affected by surface water must be treated to reduce the total microbial load to less than 500 CFU/mL. In Japan, drinking water regulations limit the total bacterial load to 100 CFU/mL., Objective: To validate the PixeeMoTM aerobic bacteria method based on the Japanese regulation in the range of 30-300 CFU/mL in drinking water., Method: PixeeMoTM aerobic bacteria method was compared to the Standard Method for the Examination of Water and Wastewater (SMEWW) 9215B (2017) using naturally contaminated drinking water., Results: The maximum repeatability standard deviation of the PixeeMoTM method was 14.8%. The difference of mean log10 values between the PixeeMoTM and SMEWW 9215B methods ranged from -0.015 to 0.258. Similar results were obtained in the independent laboratory study., Conclusions: The PixeeMoTM method is equivalent to that of the SMEWW 9215B methods. The product consistency and stability study demonstrated no significant difference within the expiration date. The robustness study confirmed that there was no effect within the expected range. The instrument variation study also demonstrated no significant difference among the data of three PixeeMoTM instruments., Highlights: Total counts of bacteria in drinking water can be determined accurately within 1 h with PixeeMoTM., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aim: Public health promotion of fruit and vegetables as healthier sources of nutrition in Nigeria has led to an increase in consumption of fruit and vegetables, particularly in the form of ready-to-eat (RTE) salads. However, the continual association of drug-resistant bacteria with fruit and vegetables creates an additional challenge for consumer safety. Hence this study was carried out to determine the prevalence of multidrug-resistant bacteria (MRB) in, and characterization of MRB isolated from, RTE salads., Methods and Results: RTE vegetable salad samples were randomly selected for bacterial analysis with Wei's Urn iterative randomization technique and a One Way Analysis of Variance performed with Kruskal-Wallis test. Total viable bacteria count (TVC) was performed with pour-plate technique. Bacterial colonies from Petri plates were tested for multidrug resistance with Kirby Bauer disc diffusion test and prevalence/counts of MRB, as well as multiple antibiotic resistance indices (MAR), was/were subsequently deduced. Mean prevalence of MRB in all RTE salad samples was estimated at 54·38% while mean counts of MRB were estimated at 6·83 ± 6·42 log 10 CFU per g. Prevalence of bacterial resistance to all antibiotics tested ranged from 5·92 to 100·00%. Mean MAR obtained for all RTE salad samples was estimated at 0·51 (recommended limit = 0·20) indicating that RTE salads were a potential source of MRB, with significant health risk. MRB isolated from RTE salads included Proteus vulgaris strain ATU 243, Bacillus thuringiensis strain AND 236, Citrobacter freundii strain ABC 2 and Serratia marcescens strain ADJ 212., Conclusion: This study showed that MRB constitute a significant proportion of the bacterial community present in RTE salads served in Nigerian restaurants., Significance and Impact of the Study: Our study provided empirical evidence which showed that RTE salads served in Nigerian restaurants were a potential source of MRB, with significant health risk., (© 2020 The Society for Applied Microbiology.)

Background: Laparoscopic sleeve gastrectomy (LSG) is a well established treatment for severe obesity and type 2 diabetes. Although the gut microbiota is linked to the efficacy of LSG, the underlying mechanisms remain elusive. The effect of LSG for morbid obesity on the gut microbiota and bile acids was assessed here., Methods: Severely obese subjects who were candidates for LSG were included and followed until 6 months after surgery. The composition and abundance of the microbiota and bile acids in faeces were assessed by 16S ribosomal RNA sequencing, quantitative PCR and liquid chromatography-mass spectrometry., Results: In total, 28 patients with a mean(s.d.) BMI of 44·2(6·6) kg/m 2 were enrolled. These patients had achieved excess weight loss of 53·2(19·0) per cent and showed improvement in metabolic diseases by 6 months after LSG, accompanied by an alteration in the faecal microbial community. The increase in α-diversity and abundance of specific taxa, such as Rikenellaceae and Christensenellaceae, was strongly associated with reduced faecal bile acid levels. These changes had a significant positive association with excess weight loss and metabolic alterations. However, the total number of faecal bacteria was lower in patients before (mean(s.d.) 10·26(0·36) log 10 cells per g faeces) and after (10·39(0·29) log 10 cells per g faeces) operation than in healthy subjects (10·83(0·27) log 10 cells per g faeces)., Conclusion: LSG is associated with a reduction in faecal bile acids and greater abundance of specific bacterial taxa and α-diversity that may contribute to the metabolic changes., (© 2020 BJS Society Ltd Published by John Wiley & Sons Ltd.)

Neutrophils can shape adaptive immunity; however, their role in vaccine-induced protection against infections in vivo remains unclear. Here, we tested their role in the clinically relevant polysaccharide conjugate vaccine against Streptococcus pneumoniae (pneumococcus). We antibody depleted neutrophils during vaccination, allowed them to recover, and 4 weeks later challenged mice with pneumococci. We found that while isotype-treated vaccinated controls were protected against an otherwise lethal infection in naive mice, full protection was lost upon neutrophil depletion. Compared to vaccinated controls, neutrophil-depleted mice had higher lung bacterial burdens, increased incidence of bacteremia, and lower survival rates. Sera from neutrophil-depleted mice had less antipneumococcal IgG2c and IgG3, were less efficient at inducing opsonophagocytic killing of bacteria by neutrophils in vitro, and were worse at protecting naive mice against pneumococcal pneumonia. In summary, neutrophils are required during vaccination for optimal host protection, which has important implications for future vaccine design against pneumococci and other pathogens., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Lymphocyte activation gene 3 (LAG-3) is one of the immune checkpoint molecules, negatively regulating the T-cell reactions. The present study investigated the role of LAG-3 in sepsis-induced T-lymphocyte disability., Methods: Mice sepsis was induced by cecal ligation and puncture (CLP). LAG-3 expression on some immune cells were detected 24 hours after CLP. LAG-3 knockout and anti-LAG-3 antibody were applied to investigate the effects on the survival, bacterial clearance. Cytokine levels, T-cell counts, and the presence of apoptosis (in blood, spleen, and thymus) were also determined. In vitro T-cell apoptosis, interferon γ secretion, and proliferation were measured. The expression of interleukin 2 receptor on T cells was also determined after CLP., Results: LAG-3 was up-regulated on CD4+/CD8+ T, CD19+ B, natural killer, CD4+CD25+ regulatory T cells and dendritic cells. Both LAG-3 knockout and anti-LAG-3 antibody had a positive effect on survival and on blood or peritoneal bacterial clearance in mice undergoing CLP. Cytokine levels and T-cell apoptosis decreased in anti-LAG-3 antibody-treated mice. Induced T-cell apoptosis decreased, whereas interferon γ secretion and proliferation were improved by anti-LAG-3 antibody in vitro. Interleukin 2 receptor was up-regulated on T cells in both wild-type and LAG-3-knockout mice undergoing CLP., Conclusions: LAG-3 knockout or anti-LAG-3 antibody blockade protected mice undergoing CLP from sepsis-associated immunodysfunction and may be a new target for the treatment., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood., Methods: Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters., Results: Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 μmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters., Conclusions: Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Aims: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms., Methods and Results: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12&#95;Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma&#95;BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma&#95;BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type., Conclusion: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection., Significance and Impact of the Study: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus., (© 2020 The Society for Applied Microbiology.)

Cellular immunity is evolutionarily conserved in invertebrates and vertebrates. In insects, cellular immune response is provided by the hemocytes, and its molecular mechanisms are currently not fully understood. Here, we identified a CD109 antigen-like gene ( HaCD109 ) from Helicoverpa armigera which is highly expressed in the hemocytes of larvae. Stimulation by Escherichia coli and chromatography beads significantly upregulated HaCD109 expression. In vivo HaCD109 silencing significantly increased bacterial load in larval hemolymphs and reduced the hemocyte spread. 20-Hydroxyecdysone (20E) can induce HaCD109 expression through its receptors, EcR and USP. In vivo HaCD109 silencing nearly abolished 20E-induced bacterial clearance and hemocyte spread. These results suggested that HaCD109 plays an important role in cellular immunity, and the 20E-induced cellular immune response in H. armigera requires HaCD109 involvement. Our study contributes to the understanding of regulatory mechanisms for innate immune response and provides new insights into the interaction between innate immunity and steroid hormone signaling.

The ready-to-eat products can be contaminated during processing by pathogen or spoilage bacteria, which persist in the industrial environment. Some bacterial species are able to form biofilms which protect them from environmental conditions. To check the bacterial contamination of the surfaces in the food industries, the professionals must regularly use surface sampling methods to detect the pathogen such as Listeria monocytogenes or the spoilage such as Pseudomonas fluorescens. In 2010, we designed and carried out a European survey to collect surface sampling information to detect or enumerate L. monocytogenes in food processing plants. A total of 137 questionnaires from 14 European Union Member States were returned. The outcome of this survey showed that the professionals preferred friction sampling methods with gauze pad, swab and sponges versus contact sampling methods. After this survey, we compared the effectiveness of these three friction sampling methods and the contact plates, as recommended in the standard EN ISO 18593 that was revised in 2018, on the recovery of L. monocytogenes and of P. fluorescens in mono-specie biofilms. This study showed no significant difference between the effectiveness of the four sampling methods to detach the viable and culturable bacterial population of theses mono-specie biofilms., (© FEMS 2020.)

Aims: Faecal microbiota transplantation (FMT) consists of the infusion of faeces from a healthy donor to the gastrointestinal tract of a recipient patient to treat disease associated with alterations in gut microbiota. The objective of this article was to describe laboratory workflow of an FMT laboratory to provide tips for preparing the faecal suspensions to be infused., Methods and Results: Twenty-stool solutions obtained from ten donors were prepared using two different protocols: magnet plate emulsion (MPE) and Seward Stomacher TM Emulsion (SSE). We evaluated parameters such as preparation time, handiness, and aerobic and anaerobic microbial count. For three donors, we monitored bacterial counts after defrosting at different time-points. MPE requires more time than SSE. In terms of microbial load, both methods showed similar values, with small and statistically differences (P ≤ 0·05) regarding anaerobes in favour of SSE. Frozen aliquots showed the same bacterial load values after defrosting., Conclusion: Although both methods allow an easy and available preparation of a stool suspension, SSE seems more suitable, particularly for stool banking. Aerobic and anaerobic species are preserved with both protocols; and safety for laboratory operators is guaranteed., Significance and Impact of the Study: In recent years, FMT has become a fascinating and interesting subject. Nevertheless, there are no real guidelines describing laboratory facilities and procedures. This paper aims to be a useful and simple guide to increase the number FMT centres as much possible., (© 2019 The Society for Applied Microbiology.)

Pediatric burn injuries are vulnerable to severe complications, most often infection, making prompt and precise diagnosis of bacterial bioburden vital to preventing detrimental consequences and optimizing patients' outcomes. Currently, burn wounds are assessed for infection via examining the clinical signs and symptoms of infection, which can be confirmed by swab culture analysis. While the former approach is subjective and experience-dependant, the latter technique is susceptible to missing subsurface, biofilm-associated colonization, and any peripheral bacterial burden, and also delays confirmation by up to 5 days. The MolecuLight i:X is a handheld, noncontact fluorescence imaging device, which can reveal real-time information about clinically significant levels of bacteria and their biodistribution in surface and subsurface burn wound tissues. We conducted a single-center observational study to assess the device efficacy in identifying critical bacterial levels in pediatric burn wounds and to test the children's compliance and the overall feasibility of the device integration into the current diagnostic practice. Ten patients with 16 wounds were recruited and assessed for the presence or absence of clinical signs and symptoms of infection and the presence or absence of bacterial fluorescence on images, with swabs taken to confirm findings. Results demonstrate the device's ability to visualize clinically significant bacterial burden and to localize distribution of pathogens. All clinicians agreed on the high compliance with the device and high feasibility of incorporating the device into routine wound assessments. The results of this study may pave the way toward including bacterial fluorescence imaging into the standard diagnostic algorithm for pediatric burn population., (© American Burn Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bacteria coordinate cellular behaviors using a cell-cell communication system termed quorum sensing. In Vibrio harveyi, the master quorum sensing transcription factor LuxR directly regulates >100 genes in response to changes in population density. Here, we show that LuxR derepresses quorum sensing loci by competing with H-NS, a global transcriptional repressor that oligomerizes on DNA to form filaments and bridges. We first identified H-NS as a repressor of bioluminescence gene expression, for which LuxR is a required activator. In an hns deletion strain, LuxR is no longer necessary for transcription activation of the bioluminescence genes, suggesting that the primary role of LuxR is to displace H-NS to derepress gene expression. Using RNA-seq and ChIP-seq, we determined that H-NS and LuxR co-regulate and co-occupy 28 promoters driving expression of 63 genes across the genome. ChIP-PCR assays show that as autoinducer concentration increases, LuxR protein accumulates at co-occupied promoters while H-NS protein disperses. LuxR is sufficient to evict H-NS from promoter DNA in vitro, which is dependent on LuxR DNA binding activity. From these findings, we propose a model in which LuxR serves as a counter-silencer at H-NS-repressed quorum sensing loci by disrupting H-NS nucleoprotein complexes that block transcription., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Symbiosis between intracellular bacteria (endosymbionts) and animals are widespread. The alphaproteobacterium Wolbachia pipientis is known to maintain a variety of symbiotic associations, ranging from mutualism to parasitism, with a wide range of invertebrates. Wolbachia infection might deeply affect host fitness (e.g. reproductive manipulation and antiviral protection), which is thought to explain its high prevalence in nature. Bacterial loads significantly influence both the infection dynamics and the extent of bacteria-induced host phenotypes. Hence, fine regulation of bacterial titers is considered as a milestone in host-endosymbiont interplay. Here, we review both environmental and biological factors modulating Wolbachia titers in arthropods., (© FEMS 2019.)

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading global cause of death from an infectious agent. Mycobacteria thrive within their host Mϕs and presently, there is no animal model that permits combined in vitro and in vivo study of mycobacteria-host Mϕ interactions. Mycobacterium marinum (Mm), which causes TB in aquatic vertebrates, has become a promising model for TB research, owing to its close genetic relatedness to Mtb and the availability of alternative, natural host aquatic animal models. Here, we adopted the Xenopus laevis frog-Mm surrogate infection model to study host Mϕ susceptibility and resistance to mycobacteria. Mϕ differentiation is regulated though the CSF-1 receptor (CSF-1R), which is activated by CSF-1 and the unrelated IL-34 cytokines. Using combined in vitro and in vivo approaches, we demonstrated that CSF-1-Mϕs exacerbate Mm infections, are more susceptible to mycobacterial entry and are less effective at killing this pathogen. By contrast, IL-34-Mϕs confer anti-Mm resistance in vivo, are less susceptible to Mm entry and more effectively eliminate internalized mycobacteria. Moreover, we showed that the human CSF-1- and IL-34-Mϕs are likewise, respectively, susceptible and resistant to mycobacteria, and that both frog and human CSF-1-Mϕs are more prone to the spread of mycobacteria and to being infected by Mm-laden Mϕs than the respective IL-34-Mϕ subsets. This work marks the first report describing the roles of these Mϕ subsets in mycobacterial disease and may well lead to the development of more targeted anti-Mtb approaches., (©2019 Society for Leukocyte Biology.)

Background: ZTI-01 (fosfomycin for injection) is an epoxide antibiotic with a differentiated mechanism of action (MOA) inhibiting an early step in bacterial cell wall synthesis. ZTI-01 has broad in vitro spectrum of activity, including multidrug-resistant Gram-negative pathogens, and is being developed for treatment of complicated urinary tract infection (cUTI) and acute pyelonephritis (AP) in the United States., Methods: Hospitalized adults with suspected or microbiologically confirmed cUTI/AP were randomized 1:1 to 6 g ZTI-01 q8h or 4.5 g intravenous (IV) piperacillin-tazobactam (PIP-TAZ) q8h for a fixed 7-day course (no oral switch); patients with concomitant bacteremia could receive up to 14 days., Results: Of 465 randomized patients, 233 and 231 were treated with ZTI-01 and PIP-TAZ, respectively. In the microbiologic modified intent-to-treat (m-MITT) population, ZTI-01 met the primary objective of noninferiority compared with PIP-TAZ with overall success rates of 64.7% (119/184 patients) vs 54.5% (97/178 patients), respectively; treatment difference was 10.2% (95% confidence interval [CI]: -0.4, 20.8). Clinical cure rates at test of cure (TOC, day 19-21) were high and similar between treatments (90.8% [167/184] vs 91.6% [163/178], respectively). In post hoc analysis using unique pathogens typed by pulsed-field gel electrophoresis, overall success rates at TOC in m-MITT were 69.0% (127/184) for ZTI-01 versus 57.3% (102/178) for PIP-TAZ (difference 11.7% 95% CI: 1.3, 22.1). ZTI-01 was well tolerated. Most treatment-emergent adverse events, including hypokalemia and elevated serum aminotransferases, were mild and transient., Conclusions: ZTI-01 was effective for treatment of cUTI including AP and offers a new IV therapeutic option with a differentiated MOA for patients with serious Gram-negative infections., Clinical Trial Registration: NCT02753946., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

The current vaccines against brucellosis, namely Brucella abortus strains 19 and RB51, prevent infection in animals but pose potential risks like virulence and attenuation reversal. In this milieu, although subunit vaccination using a single potent immunogen of B. abortus, e.g. BP26 or Omp25 or L7/L12 etc., appears as a safer alternative, nonetheless it confers inadequate protection against the zoonosis compared to attenuated vaccines. Hence, we have investigated the prophylactic potential of a combined subunit vaccine (CSV) comprising the BP26, Omp25 and L7/L12 antigens of B. abortus, in mice model. Sera obtained from CSV immunized mice groups showed heightened IgG titers against all the three components and exhibited specificity upon immunoblotting, reiterating their authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen revealed a predominant Th2 immune response in CSV immunized mice group. However, on assessing the levels of Th1-dependent (IFN-γ and TNF-α) and Th2-dependent (IL-4 and IL-10) cytokines in different formulations, prominent IFN-γ levels were elicited in CSV immunized mice. Further, upon infection with virulent B. abortus 544, the combined subunit vaccinated mice displayed superior degree of protection (Log10 reduction) than the individual vaccines; however, B. abortus S19 showed the highest protection. Altogether, this study suggests that co-immunization of three B. abortus immunogens as a CSV complements and triggers a mixed Th1/Th2 immune response leading to superior degree of protection against pathogenic B. abortus 544 infection., (© FEMS 2020.)

Background: Searching for new strategies to defeat Pseudomonas aeruginosa is of paramount importance. Previous works in vitro showed that peptidoglycan recycling blockade disables AmpC-dependent resistance and enhances susceptibility against cell-wall-targeting immunity. Our objective was to validate these findings in murine models.This study shows for the first time in different murine models of infection that blocking the peptidoglycan recycling in Pseudomonas aeruginosa causes an important virulence impairment and disables AmpC-mediated resistance, being hence validated as a promising therapeutic target., Methods: Wildtype PAO1, recycling-defective AmpG and NagZ mutants, an AmpC hyperproducer dacB mutant, and their combinations were used to cause systemic/respiratory infections in mice. Their survival, bacterial burden, inflammation level, and effectiveness of ceftazidime or subtherapeutic colistin to treat the infections were assessed., Results: Inactivation of AmpG or NagZ significantly attenuated the virulence in terms of mice mortality, bacterial load, and inflammation. When inactivating these genes in the dacB-defective background, the β-lactam resistance phenotype was abolished, disabling the emergence of ceftazidime-resistant mutants, and restoring ceftazidime for treatment. Subtherapeutic colistin was shown to efficiently clear the infection caused by the recycling-defective strains, likely due to the combined effect with the mice cell-wall- targeting immunity., Conclusions: This study brings us one step closer to new therapies intended to disable P. aeruginosa AmpC-mediated resistance and dampen its virulence, and strongly support the interest in developing efficient AmpG and/or NagZ inhibitors., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Wound infections cause inflammation, tissue damage, and delayed healing that can lead to invasive infection and even death. The efficacy of systemic antibiotics is limited due to poor tissue penetration that is especially a problem in burn and blast wounds where the microcirculation is disrupted. Topical administration of antimicrobials is an attractive approach because it prevents infection and avoids systemic toxicity, while hydrogels are an appealing vehicle for topical drug delivery. They are easy to apply to the wound site by being injectable, the drug release properties can be controlled, and their many characteristics, such as biodegradation, mechanical strength, and chemical and biological response to stimuli can be tailored. Hydrogels also create a moist wound environment that is beneficial for healing. The purpose of this study was to formulate an agarose hydrogel that contains high concentrations of minocycline or gentamicin and study its characteristics. Subsequently, the minocycline agarose hydrogel was tested in a porcine burn model and its effect as a prophylactic treatment was studied. The results demonstrated that 0.5% agarose in water was the optimal concentration in terms of viscosity and pH. Bench testing at room temperature demonstrated that both antibiotics remained stable in the hydrogel for at least 7 days and both antibiotics demonstrated sustained release over the time of the experiment. The porcine burn experiment showed that prophylactic treatment with the agarose minocycline hydrogel decreased the burn depth and reduced the number of bacteria as efficiently as the commonly used silver sulfadiazine cream., (© American Burn Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The phenomenon of attenuated antibacterial activity at inocula above those utilized for susceptibility testing is referred to as the inoculum effect. Although the inoculum effect has been reported for several decades, it is currently debatable whether the inoculum effect is clinically significant. The aim of the present review was to consolidate currently available evidence to summarize which β-lactam drug classes demonstrate an inoculum effect against specific bacterial pathogens. Review of the literature showed that the majority of studies that evaluated the inoculum effect of β-lactams were in vitro investigations of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae and Staphylococcus aureus. Across all five pathogens, cephalosporins consistently displayed observable inoculum effects in vitro, whereas carbapenems were less susceptible to an inoculum effect. A handful of animal studies were available that validated that the in vitro inoculum effect translates into attenuated pharmacodynamics of β-lactams in vivo. Only a few clinical investigations were available and suggested that an in vitro inoculum effect of cefazolin against MSSA may correspond to an increased likeliness of adverse clinical outcomes in patients receiving cefazolin for bacteraemia. The presence of β-lactamase enzymes was the primary mechanism responsible for an inoculum effect, but the observation of an inoculum effect in multiple pathogens lacking β-lactamase enzymes indicates that there are likely multiple mechanisms that may result in an inoculum effect. Further clinical studies are needed to better define whether interventions made in the clinic in response to organisms displaying an in vitro inoculum effect will optimize clinical outcomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)