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

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

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

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.)

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10 -13  M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 10 4  CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 10 5  CFU/mL without enrichment and 1.0 × 10 0  CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 10 5 to 1.0 × 10 8  CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bacillus cereus, a common soil bacterium, has been shown to act as a biogeochemical indicator for concealed mineralisations, e.g., vein-type Au deposits. Field and cultivation-free detection of Bacillus cereus in the presence of Au 3+ and other metal ions is significantly important but still almost blank in current biogeochemical prospecting of gold mine system. Herein, a self-established simple approach was slightly improved to make silver nanoparticles (Ag NPs) rapidly concentrated on every bacterial cell, and highly strong and distinct surface-enhanced Raman scattering (SERS) signals of Bacillus cereus free from any native fluorescence have been obtained in a so called 'mixing-and-measuring' manner. Furthermore, SERS was used for the first time to our knowledge to investigate the impacts of different concentrations of metal ions on Bacillus cereus, and successfully utilized for distinguishing Au 3+ ions from other species. A more convincing multi-Raman criterion based on Raman bands, and further the entire Raman spectrum in combination with statistical analysis (e.g., principal component analysis (PCA)) were found capable of detecting spectral differences of Bacillus cereus in the presence of metal ions (Au 3+ , Ag + , Cu 2+ and Zn 2+ ) with different concentrations. An interesting phenomenon has been found that except for Au 3+ ions, the highest permissive concentration of other metal ions for the detected Bacillus cereus is up to 10 μg/mL possibly due to their resistance to Au. The results also indicate that an effective biogeochemical exploration technique of SERS spectral response may be developed, where Bacillus cereus spore counts are measured in the field and used as a pre-screening method to target areas useful for further sampling and complete geochemical analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hunting is currently a very popular activity, and interest in game meat is increasing. However, only limited research is available on the bacterial quality and safety of moose (Alces alces) and white-tailed deer (Odocoileus virginianus) harvested by hunters. Poor hunting hygiene can spread bacteria onto the carcasses, and inadequate chilling of the carcasses may increase the bacterial load on the carcass surface. We studied the bacterial contamination level on carcasses of 100 moose and 100 white-tailed deer shot in southern Finland. Hunters eviscerated carcasses in the field and skinned them in small slaughter facilities. During the sampling, same person visited 25 facilities located in 12 municipalities of four provinces. Moose carcasses had mean mesophilic aerobic bacteria (MAB), Enterobacteriaceae (EB) and Escherichia coli (EC) values of 4.2, 2.6 and 1.2 log 10  cfu/cm 2 , respectively, while deer carcass values were 4.5, 1.5 and 0.7 log 10  cfu/cm 2 , respectively. Moose carcasses were significantly more contaminated with EB and EC than deer carcasses. High bacterial counts (MAB>5.0 log 10  cfu/cm 2 and EB > 2.5 log 10  cfu/cm 2 ) on the carcasses were associated with the smallest facilities having only one room. The outdoor temperature and days between hunting and sampling affected the bacterial counts. High EB counts on the carcasses indicated a gut hit. Male gender was significantly more contaminated by EC and meat-borne pathogenic bacteria: Campylobacter spp., Salmonella spp., enteropathogenic Yersinia spp., stx-harbouring EC (STEC) and Listeria monocytogenes. STEC (28/200) and L. monocytogenes (20/200) were the most commonly detected bacteria by PCR. L. monocytogenes isolates of different sequence types (ST7, 18, 29, 37, 249, 412, 451 and 611) belonged to serotypes 1/2a (seven isolates) and 4b (three isolates). The virulence gene ail was detected in four Yersinia enterocolitica biotype 1A isolates and one Yersinia kristensenii isolate. The bacterial counts on the moose and deer carcasses varied highly, and more attention should be paid to hunting hygiene and training of hunters. Game meat may be a source of meat-borne pathogens, and close attention should therefore be paid when handling and preparing game., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Enzymatically-switchable fluorescent substrates, such as the commercially available 4-methyl umbelliferones (4-MU) are used as standard indicators of enzymatic activity for the detection of various microorganisms and pathogens. However, a major disadvantage of 4-MU is its relatively high pKa leading to only partial dissociation of the fluorescent anion under the conditions where the enzymes are most effective (pH 6-6.5). Here we present a method for new, enzymatically-switchable, fluorescent substrates with improved photo-physico/chemical properties. The lead derivative, 4-AAU, shows excellent solubility in aqueous media (0.81 mg/mL) when compared to 4-MU (0.16 mg/mL), significantly improved quantum yield and wider dynamic range of its fluorescence properties. The corresponding bacterial substrate β-4-AAUG showed superior selectivity in the detection of clinically relevant amounts of E. coli, Enterococcus and K. pneumonia (1 CFU). The fluorescence intensity of β-4-AAUG was almost 5 times higher than that of the standard, the detection was possible in reasonably short time (∼ 2.5 h) and with excellent sensitivity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This article reports the detection of Salmonella spp. based on M13 bacteriophage in a capacitive flow injection system. Salmonella-specific M13 bacteriophage was immobilized on a polytyramine/gold surface using glutaraldehyde as a crosslinker. The M13 bacteriophage modified electrode can specifically bind to Salmonella spp. via the amino acid groups on the filamentous phage. An alkaline solution was used to break the binding between the sensing surface and the analyte to allow renewable use up to 40 times. This capacitive system provided good reproducibility with a relative standard deviation (RSD) of 1.1%. A 75 µL min -1 flow rate and a 300 µL sample volume provided a wide linear range, from 2.0 × 10 2 to 1.0 × 10 7 cfu mL -1 , with a detection limit of 200 cfu mL -1 . Bacteria concentration can be analyzed within 40 min after the sample injection. When applied to test real samples (raw chicken meat) it provided good recoveries (100-111%). An enrichment process was also explored to increase the bacteria concentration, enabling a quantitative detection of Salmonella spp. This biosensor opens a new opportunity for the detection of pathogenic bacteria using bacteriophage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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: The early diagnosis of infection or sepsis in burns are important for patient care. Globally, a large number of burn centres advocate quantitative cultures of wound biopsies for patient management, since there is assumed to be a direct link between the bioburden of a burn wound and the risk of microbial invasion. Given the conflicting study findings in this area, a systematic review was warranted., Methods: Bibliographic databases were searched with no language restrictions to August 2015. Study selection, data extraction and risk of bias assessment were performed in duplicate using pre-defined criteria. Substantial heterogeneity precluded quantitative synthesis, and findings were described narratively, sub-grouped by clinical question., Results: Twenty six laboratory and/or clinical studies were included. Substantial heterogeneity hampered comparisons across studies and interpretation of findings. Limited evidence suggests that (i) more than one quantitative microbiology sample is required to obtain reliable estimates of bacterial load; (ii) biopsies are more sensitive than swabs in diagnosing or predicting sepsis; (iii) high bacterial loads may predict worse clinical outcomes, and (iv) both quantitative and semi-quantitative culture reports need to be interpreted with caution and in the context of other clinical risk factors., Conclusion: The evidence base for the utility and reliability of quantitative microbiology for diagnosing or predicting clinical outcomes in burns patients is limited and often poorly reported. Consequently future research is warranted., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Background: Prevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque., Methods: Thirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person's correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated., Results: When relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia., Conclusion: To check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study we report a method for the rapid and sensitive estimation of bacterial cell concentration in solution based on a colorimetric enzyme/gold nanoshells conjugate system. The CTAB capped gold nanoshells are electrostatically attracted by both the bacterial surface and the enzyme β-galactosidase. The preferential binding of cationic (CTAB)-functionalized gold nanoshells to the more negative bacterial surfaces leaves active β-galactosidase in solution, providing an enzyme-amplified colorimetric response of the binding event. A progressive increase in the enzyme activity is evidenced by the conversion of the yellow-orange CPRG substrate into the red chromophore chlorophenol red, which can be correlated with increasing bacterial cell numbers. Using this strategy, the quantification of bacteria at concentrations as low as 10 bacteria/mL of solution has been achieved. The present method of bacterial cell load assessment offers a distinct potential advantage over other conventional methods such as plate counting in terms of ease of operation, rapidity, high sensitivity and quantitative detection of bacterial cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Verocytotoxigenic Escherichia coli (VTEC) is associated with a wide spectrum of disease from mild self-limiting diarrhoea to haemolytic uremic syndrome. Contaminated drinking water is accepted as an important route of transmission in Ireland as elsewhere however established methods for detection of VTEC in drinking water have limitations. We describe a sensitive and rapid method for detection of VTEC from large volumes (20 to 30L) of drinking water based on filtration, enrichment culture of filters and real-time PCR detection of VTEC virulence and O antigen determinants from enrichments. The method has potential applications for other waterborne pathogens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A next-generation diagnostic tool for bacterial vaginosis, consisting of quantitative and/or qualitative molecular criteria, has not yet been identified. The optimal diagnostic tool should not only diagnose bacterial vaginosis in diverse populations, but should also detect early signs of transition to dysbiosis. We evaluated a tool based on log10-transformed qPCR data for Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus vaginalis, Lactobacillus genus, Atopobium vaginae and Gardnerella vaginalis in vaginal specimens of 426 African women to detect dysbiosis and predict transition to dysbiosis. G. vaginalis (p = 0.204) and A. vaginae (p = 0.001) were more commonly present in women who evolved to an intermediate (Nugent 4-6) or bacterial vaginosis score (Nugent 7-10) compared to women who continued to have a normal Nugent score. The combination of G. vaginalis, A. vaginae and Lactobacillus genus counts performed best for diagnostic accuracy for bacterial vaginosis--sensitivity 93.4% and specificity 83.6%; and for predictive accuracy for bacterial vaginosis--sensitivity 79% and specificity 52%. L. crispatus combinations did not perform well. We conclude that a triple-G. vaginalis-A. vaginae-Lactobacillus genus-qPCR tool holds promise for research in sub-Saharan Africa or when developed as a next-generation clinical diagnostic modality for bacterial vaginosis, ideally engineered as a rapid assay., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Yersinia pestis 201 contains 4 plasmids pPCP1, pMT1, pCD1 and pCRY, but little is known about the effects of these plasmids on the dissemination of Y. pestis. We developed a plasmid-based luxCDABE bioreporter in Y. pestis 201, Y. pestis 201-pCD1(+), Y. pestis 201-pMT1(+), Y. pestis 201-pPCP1(+), Y. pestis 201-pCRY(+), Y. pestis 201-p(-) and Yersinia pseudotuberculosis Pa36060 strains, and investigated their dissemination by bioluminescence imaging during primary septicemic plague in a mouse model. These strains mainly colonized the livers and spleens shortly after intravenous inoculation. Y. pestis 201-pMT1(+) appeared to have a stronger ability to survive in the livers, spleens and blood, and to be more virulent than other plasmid-deficient strains. Y. pestis 201-pPCP1(+) appeared to have a stronger ability to colonize lungs than other plasmid-deficient strains. Pa36060 has the strongest ability to colonize intestines and lungs. Y. pestis 201 has the strongest ability to survive in blood, and the strongest virulence. These results indicated that the plasmid pMT1 was an important determinant in the colonization of livers, spleens and blood, whereas the plasmid pPCP1 appeared to correlate with the colonization in lungs. The resistance to killing in mouse blood seemed to be the critical factor causing animal death., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™ (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 10(4) GE/μL (1.2 × 10(5) ; 1 to 6.1 × 10(6) DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies., (© 2013 The Authors. Clinical Microbiology and Infection published by John Wiley & Sons Ltd on behalf of the European Society of Clinical Microbiology and Infectious Disease.)

The rise in antimicrobial resistance has become a serious global health problem. Restrictive use of antibiotics seems the only option to temper this accession since research in new antibiotics has halted. Antimicrobial stewardship programmes rely on quick access to susceptibility data. This study evaluated the concept of bacterial cell count monitoring as a fast method to determine susceptibility. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains were tested for amoxicillin/piperacillin and gentamicin by three conventional methods (VITEK2(®) , Etest(®) and broth-macrodilution). Bacterial cell count monitoring reliably predicted susceptibility after 90 min for Escherichia coli and after 120 min for Pseudomonas aeruginosa and Staphylococcus aureus without any minor, major or very major discrepancies. Time-to-result was reduced by 74%, 83% and 76%, respectively. Bacterial cell count monitoring shows great potential for rapid susceptibility testing., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

Diarrheal diseases represent a tremendous health burden in low-resource countries affecting child mortality. The main sources of diarrheal diseases are water source contamination and fecal-oral transmission. A major obstacle in disease control is the ability to identify and monitor water source quality for potential infectious contamination. We explore a technique for real-time surveillance of coliform bacteria contamination in water sources which is of modest cost and does not require electricity. Specifically, we used body heat as a source for thermal regulation in contrast to traditional incubation for the enumeration of coliforms on 3M petrifilm™ Escheriachia coli/coliform count plates. Our data support that the body heat incubation technique is a promising strategy for water source surveillance in low resource settings., (Published by Elsevier B.V.)

Campylobacter jejuni is worldwide recognized as a human foodborne pathogen. It is widely present in poultry meat and slaughterhouses, but little is known about its fate during the processing of poultry meat preparations. In stress conditions, this pathogen can enter into a viable but non-culturable state, where quantitative PCR (qPCR) becomes more convenient for its detection. In this study, two different pairs of primers, targeting the rpoB and the hipO genes, were compared for its detection and quantification by PCR. Two calibration curves were prepared: one for the meat samples and the other for the environmental samples. rpoB primers showed higher sensitivity with a quantification limit of 1 log cfu/g or ml. Microbial Assessment Scheme (MAS) was used to select the Critical Sampling Locations (CSLs) along the poultry processing line. Forty-six out of 48 samples were positive by qPCR after enrichment (t = 48 ) while only 6 samples were positive by ISO 10272-1:2006. Forty-three samples showed positive signal without enrichment (t = 0 h), however only 16 samples could be quantified. These results showed the high prevalence of C. jejuni in the poultry industry and the need for new, rapid and sensitive techniques, such as qPCR, for the detection and quantification of C. jejuni in meat and environmental samples., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 10(6) CFU/ml of background microflora were 93-104 and 92-99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi-mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi-mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4°C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Jeotgal or jeot, a traditional Korean salted and fermented food, is made by adding 20-30% (w/w) salt to various types of seafood. To develop a more complete overview of the bacterial community present in jeotgal, 610 pure colonies were isolated from Myeolchi-jeot and Saeu-jeot, the most commonly consumed varieties of jeotgal, which are made with anchovy (Engraulis japonicas) and tiny shrimp (Acetes japonicas), respectively. The bacterial isolates were identified by 16S rDNA sequence analysis. A total of 104 species comprising 47 genera and 31 previously unknown species were identified. Eleven genera were isolated from both jeotgal samples, including species in the genera Staphylococcus, Bacillus, Halomonas, and Kocuria, with Staphylococcus spp. constituting the highest number. The most populous genus detected in Myeolchi-jeot was Bacillus and its relatives, while the most populous in Saeu-jeot was Staphylococcus. These were isolated from both jeotgal samples, but their proportion in the bacterial community may be influenced by matrix composition and fermentation parameters. Among the proteolytic isolates, although Virgibacillus halodenitrificans KM2100 and Staphylococcus spp. maintained their growth in 20% NaCl, protease activities were not detected in these conditions. This suggests that bacteria are not the major source of the proteolytic enzyme involved in protein hydrolysis in high-salt-containing jeotgal. However, the Staphylococcus spp. isolated from Saeu-jeot were too numerous for us to ignore their possible role in jeotgal fermentation. Staphylococcus spp. may not be hugely involved in proteolysis, but they may play a significant role in the ripening of jeotgal. Bacteria of the genus Bacillus and its relatives and of the genus Staphylococcus may be the major organisms involved in jeotgal fermentation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)