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For separation of deoxyribonucleic acid (DNA), positively charged amino-modified magnetic nanoparticles (MN) can effectively adsorb negatively charged DNA through electrostatic interaction. However, the reported preparation of amino-modified MN is usually tedious and time-consuming. Therefore, a simple synthesis method of amino-modified MN is necessary for DNA extraction. Herein, a novel polyethyleneimine-coated MN (PMN) was fabricated by one-pot hydrothermal synthesis for high-efficient DNA extraction. The fabricated PMN showed numerous exposed amino groups, which not only could effectively capture DNA through electrostatic interaction, but also limited the aggregation of PMN during application. Under optimized adsorption conditions, the maximum adsorption capacity of PMN for DNA could reach 192.4 μg m g -1 . Shigella flexneri (S. flexneri) has the highest mortality rate among Shigella species and has been selected as target model pathogenic bacteria. Based on the optimized extraction conditions, PMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated for detection of S. flexneri. The limit of detection of the proposed strategy was 2.4 × 10 2 CFU mL -1 and obviously lower than the commercial kit. To prove the practicability, the PMN-based MSPE combined qPCR strategy was successfully used in the determination of S. flexneri in spiked real sample, and the recovery values were in the range of 95.1 % to 102.1 % for apple juice, 60.4 % to 85.7 % for pickled vegetable, 97.8 % to 99.5 % for pig liver and 95.1 % to 102.1 % for pig colon, respectively. Therefore, we believe that the resultant PMN have great potential to become a universal magnetic adsorbent for high-efficient DNA extraction from complex biological samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The gastrointestinal diseases caused by Helicobacter pylori (H. pylori) infection made the accurate detection of H. pylori infection more important. Non-invasive methods, such as molecular diagnostic methods, had become a promising method for detection of H. pylori. Stool samples combined with loop-mediated isothermal amplification (LAMP), showed potential practicability for real-time detection. However, complex nucleic acid extraction steps were required to remove the large numbers of amplification inhibitors in stool samples before LAMP reaction. And the limited number of H. pylori made the detection with long reaction time and low sensitivity. The problems mentioned above were urgently to be solved., Results: In this study, we proposed a strategy for ultra-rapid sensitive detection of H. pylori in stool samples by hydrogel LAMP (hLAMP) without extraction. The hydrogel was combined with stool samples after simple thermal cracking, and amplification spaces were formed in its nanopore structures by nano-localization. The LAMP reaction was accelerated by nano space-localization. Besides, this method based on hLAMP could specifically and sensitively detect as low as 100 CFU/mL H. pylori within 40 min from sampling to result due to good anti-inhibition effect on complex samples of hydrogel. The whole process involved sample simple disposal for 10 min and LAMP reaction for 30 min. Furthermore, the excellent anti-inhibition mechanism of hydrogel was discussed, and the mechanism of hydrogel accelerating LAMP was explored., Significance: This is the first application of that hydrogel and LAMP systematically combined to detect H. pylori in stool samples. The developed method had been verified in actual clinical applications that the accuracy rate reached 88.9 % compared with routine histopathology. And it also provided a potential idea for the diagnosis and prevention of H. pylori., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An electrochemical sensor has received much attention due to its importance for early infection identification, hinting at its critical relevance in diagnostic applications. For the detection of field-isolated strains of Pasteurella multocida, this paper reports the development and fabrication of a DNA-based electrochemical biosensor by integrating zinc oxide (ZnO) nanorods (NRs) into an electrochemical paper-based analytical device (ePAD). One significant improvement over the state-of-the-art features of the sensor is the using paper, an economically viable substrate that can be manufactured in large numbers. These sensors, being categorized under precision instruments with an ecologically friendly design, are ideal for mass production of eco-sensitive substrates. The biosensor is more sensitive and specific as compared to the conventional PCR-based sensing techniques. Moreover, the biosensor exploits the inherent properties of ZnO-NRs to amplify the signal output, whereas ePAD limits detection cost. In addition, a probe targeting the KMT gene of P. multocida was first characterized using conventional PCR and then immobilized onto the ZnO nanorods-modified ePAD surface, which improved the biosensor's ability for the rapid and selective genetic material of the pathogen detection. In addition, the sensor was validated using the methods of Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LSV). The reported biosensor it provides Linear Response of 0.001-10 μg/ml with LOD 0.001 μg/ml within a response time of 30 s. Subsequently, the biosensor reveals fast kinetics of the reaction as a powerful tool for timely and accurate diagnostics, pointing to its contribution to further development in biosensing technologies in the diagnostic area of infectious diseases at hospitals and clinics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe 3 O 4 ). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH 2 layer could help Arg-CSP graft onto Fe 3 O 4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe 3 O 4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH 2 could disintegrate in buffer with high concentration of PO 4 3- for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe 3 O 4 -LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Developing an alternative and benign method for DNA extraction is imperative due to the high cost and potential harms associated with conventional techniques. Investigation of Ionic liquid (IL) as a solvent for DNA storage and stability revealed the ability of IL to assist DNA processes. IL-based aqueous biphasic system emerges as a comprehensive extraction platform capitalizing on the task-specificity of ILs and the wide applicability of ABS for biomolecule extractions. Therefore, it is beneficial to optimize an IL-based ABS specifically for DNA extraction, taking into account the fundamental interactions between the IL and DNA., Results: The primary objective was to design ABS consisting of Ammonium based ILs, and Potassium phosphate buffer as the salting-out agent for the partitioning of salmon sperm DNA. The analysis focused on optimizing biocompatible anions for the extraction. Moreover, the stability of the DNA in the IL rich phases was analysed to validate the method. The proposed process was then employed for extracting plasmid DNA from bacteria, demonstrating results comparable to those obtained with a commercially available kit. Further validation using agarose gel electrophoresis and transformation of the extracted DNA into E.coli were conducted, producing promising outcomes. Although there is room for improvement in terms of recovery of DNA and reusability of ABS, the described approach is comparable with the conventional one while being cost-effective, and showcases a noticeable and convincing link to eco-friendly processes., Significance: There is limited literature on IL-based ABS for DNA extraction, and the existing studies predominantly concentrate on systems derived from Cholinium ILs. However, their high hydrophilicity limits the choice of the second-phase forming component to polymers for the formation of ABS. Ammonium ILs efficiently form biphasic systems with various available salting-out agents, and biocompatible anions are introduced to mitigate the toxicity of the ILs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The success of polymerase chain reaction (PCR) depends on the quality of deoxyribonucleic acid (DNA) templates. This study developed a cost-effective and eco-friendly DNA extraction system utilizing poly(3,4-dihydroxyphenylalanine)-modified cellulose paper (polyDOPA@paper). PolyDOPA@paper was prepared by oxidatively self-polymerizing DOPA under weak alkaline conditions and utilizing the adhesive property of polyDOPA on different materials. Compared to the uncoated cellulose paper, polyDOPA coating significantly enhances DNA adsorption owing to its abundant amino, carboxyl, and hydroxyl moieties. The DNA extraction mechanism using polyDOPA@paper was discussed. The maximum adsorption capacity of polyDOPA@paper for DNA was 20.7 μg cm -2 . Moreover, an automated extraction system was designed and fabricated using 3D printing technology. The device simplifies the operation and ensures the reproducibility and consistency of the results. More importantly, it eliminates the need for specialized training of operators. The feasibility of the polyDOPA@paper-based automated extraction system was evaluated by quantitatively detecting Escherichia coli in spiked milk samples via a real-time PCR. The detection limit was 10 2 cfu mL -1 . The results suggest that the system would have significant potential in detecting pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Efficient separation of deoxyribonucleic acid (DNA) through magnetic nanoparticles (MN) is a widely used biotechnology. Hedgehog-inspired MNs (HMN) possess a high-surface-area due to the distinct burr-like structure of hedgehog, but there is no report about the usage of HMN for DNA extraction. Herein, to improve the selection of MN and illustrate the performance of HMN for DNA separation, HMN and silica-coated Fe 3 O 4 nanoparticles (Fe 3 O 4 @SiO 2 ) were fabricated and compared for the high-efficient separation of pathogenic bacteria of DNA. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are typical Gram-negative and Gram-positive bacteria and are selected as model pathogenic bacteria. To enhance the extraction efficiency of two kinds of MNs, various parameters, including pretreatment, lysis, binding and elution conditions, have been optimized in detail. In most separation experiments, the DNA yield of HMN was higher than that of Fe 3 O 4 @SiO 2 . Therefore, a HMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated and used to detect pathogenic bacteria in real samples. Interestingly, the HMN-based MSPE combined qPCR strategy exhibited high sensitivity with a limit of detection of 2.0 × 10 1 CFU mL -1 for E. coli and 4.0 × 10 1 CFU mL -1 for S. aureus in orange juice, and 2.8 × 10 2 CFU mL -1 for E. coli and 1.1 × 10 2 CFU mL -1 for S. aureus in milk, respectively. The performance of the proposed strategy was significantly better than that of commercial kit. This work could prove that the novel HMN could be applicable for the efficient separation of DNA from complex biological samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The advent of 16S RNA profiling and shotgun metagenomics has enabled a holistic approach to the study of the skin microbiome composition. Despite the interesting findings in this rapidly developing scientific area, the big question remains: What role does the microbiome play in skin physiology? To begin answering this question, we employed an integrative methodology for microbiome and metabolome analysis of skin surface samples collected from the volar forearm of healthy infants aged 3-6-months. Whereas the infant skin metabolome was dominated by amino acids, lipids, and xenobiotics, the primary phyla of the microbiome were Firmicutes, Actinobacteria, and Proteobacteria. Zooming in on the species level revealed a large contribution of commensals belonging to the Cutibacterium and Staphylococcus genera, including Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus. This heterogeneity was further highlighted when combining the microbiome with metabolome data. Integrative analyses delineated the coexistence of three distinct metabolite‒microbe clusters: one dominated by Cutibacterium linked to hydrophobic elements of the skin barrier, one associating Staphylococcus genus with amino acids relevant to the water holding capacity and pH regulation of the skin surface, and one characterized by Streptococcus and independent of any particular metabolomic profile., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

From 2015 to 2017, 3197 interpretable Bordetella polymerase chain reaction (PCR) tests were performed for 2760 children presenting to our tertiary university hospital. Requests mainly came from the emergency department (62%) and for children older than 1 year (68%). Only 32 PCR (1%) results were positive, mainly in children younger than 1 year (n = 29/32, 90.6%; p<0.001). When focusing on the PCR indications in 2017, we found the requests were mainly based on nonspecific respiratory symptoms and were clinically unjustified in 383 cases (39%). Pediatricians overused Bordetella PCR in clinical practice. They should reserve their requests for cases of young children with symptoms suggestive of respiratory illness and/or incomplete pertussis immunization., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest with the subject of this study., (Copyright © 2021 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.)

The robust and precise nucleic acid detection platform enormously influences the clinical diagnosis of human and plant pathogens, drastically affecting disease pandemic control. CRISPR-based nucleic acid detection tools have been successfully applied for rapid and sensitive nucleic acid detection. However, the T-rich protospacer adjacent motif (PAM), specificity, and sensitivity of the CRISPR-based nucleic acid detection tools limited its wide application. We first developed a new Cas12c-based nucleic acid detection platform (Cas12c-DETECTOR), recognizing a 5'-TG PAM and showing high sensitivity and specificity on examined targets. Our results indicate that Cas12c-DETECTOR coupling with the optimized single-guide RNA (sgRNA) can be applied to specifically identity single nucleotide polymorphism (SNP). Moreover, combined with pre-amplification and lateral flow strips or the visual fluorescence detection method, Cas12c-DETECTOR can be used to diagnose human and plant pathogens in practice. Therefore, our findings illustrated that Cas12c-DETECTOR is a robust, sensitive, precise, and practiced nucleic acid detection platform., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: To evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods., Methods: Sputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies., Results: FluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance., Conclusions: The FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Reliable, fast, and affordable diagnosis for tuberculosis (TB) remains a challenge to reduce disease incidence in resource-poor countries. Tests based on nucleotide sequences that are signature to Mycobacterium tuberculosis have the potential to make a positive impact on case detection rates, which can eventually help control TB. Using extensive comparative bioinformatics approach, we mined the genome for M. tuberculosis-specific genes and identified four genes so-called signature sequence (SS). With <25% homology with other known genes/proteins of mycobacterial/nonmycobacterial origin in various databases, these SS genes are ideal targets for species-specific identification. Sputum from suspected patients was liquefied using novel complete liquefying reagent, and DNA was isolated. Samples from patients (n = 417), reporting to TB clinics at two different hospitals, which met our inclusion criteria, were collected for this study. A small number (n = 143) was used for initial standardization, and the remaining patient samples (n = 274) were evaluated by SS and compared with smear microscopy, GeneXpert, culture, and clinical outcome. An overwhelming sensitivity of 97.0%, significantly higher than GeneXpert (95.0%), was seen. SS could pick all smear-negative, but culture-positive samples, along with other culture-negative samples; some of the latter were declared clinically positive. Our results yielded superior sensitivity and specificity through conventional PCR., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: The gut microbiota is known to play a key role in autoimmune diseases., Objectives: To identify and compare the characteristics in the gut microbial composition of patients with alopecia areata (AA) and healthy controls (HCs)., Methods: In a cross-sectional discovery cohort, we enrolled 33 patients with AA and 35 HCs from the same geographic location in Shanghai, China. The 16S rRNA gene sequencing and bioinformatic analyses were conducted to analyze DNA extracted from the subjects., Results: The α-diversity of the AA group demonstrated no statistically significant differences compared with the HC group (P > 0.05). However, the overall gut microbial communities in the AA group were distinct from the HCs (P = 0.0096). We also adopted a random forest model to select three AA-associated OTU biomarkers: OTU1237(Achromobacter), OTU257(Megasphaera), and OTU1784(Lachnospiraceae Incertae Sedis)., Conclusion: The overall gut microbial composition for AA was distinct from that of HCs. The gut microbial markers we identified may potentially be used for earlier diagnosis and as therapeutic targets., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Amplicon sequencing approaches have been widely used in food bacterial ecology. However, choices regarding the methodology can bias results. In this study, bacterial communities associated with cold-smoked salmon products and their processing plant surfaces were monitored via sequencing of the V3-V4 region of the 16S rRNA gene. The impact of DNA extraction protocols, sampling methods (swabbing or sponging) and surface materials on bacterial communities were investigated. α and β diversity analyses revealed that DNA extraction methods mainly influence the observed cold-smoked salmon microbiota composition. Moreover, different DNA extraction methods revealed significant differences in observed community richness and evenness. β-Proteobacteria: Photobacterium, Serratia and Firmicutes: Brochothrix, Carnobacterium and Staphylococcus were identified as the dominant genera. Surface microbiota richness, diversity and composition were mainly affected by cleaning and disinfection procedures but not by DNA extraction methods. Surface community richness and evenness appeared higher when sampled by sponging compared to swabbing. β-diversity analyses highlighted that surface topology, cleaning and disinfection and sampling devices seemed to affect the bacterial community composition. The dominant surface bacteria identified were mainly Flavobacteriaceae, β-Proteobacteria and γ-Proteobacteria described as fish spoilers such as Acinetobacter, Pseudomonas and Shewanella. DNA extraction and sampling methods can have an impact on sequencing results and the ecological analysis of bacterial community structures. This study confirmed the importance of methodology standardization and the need for analytical validation before 16S rDNA metabarcoding surveys., (Copyright © 2020. Published by Elsevier Ltd.)

Antimicrobial resistance in Neisseria gonorrhoeae persists as a major public health concern globally. We developed and evaluated a multiplex assay that relied on high-resolution melting (HRM) technology as a rapid, simple, and cost-effective method for simultaneously detecting and identifying different molecular markers associated with extended-spectrum cephalosporins (ESCs) and azithromycin (AZM) resistance in N. gonorrhoeae. Forty-eight well-characterized N. gonorrhoeae clinical isolates were selected for initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the antimicrobial resistance determinants related to ESCs and AZM resistance. Specificity and cross-reactivity were assessed by testing 15 nongonococcal strains. Then, the assay was validated on 218 archived DNA specimens that had been sequenced using whole-genome sequencing technology. Compared with whole-genome sequencing, these assays had a sensitivity of 98.6%, with a specificity of 99.2%. For further validation of the assay's performance, a total of 338 samples (156 clinical isolates and 182 clinical specimens) were screened using the multiplex HRM assay. The results showed good concordance with the results of PCR sequencing. Given its rapidity (within 90 minutes), ease of performing, and low cost (<$1.00 per sample), this method may be applied to large-scale epidemiologic programs for increasing surveillance of ESCs and AZM resistance in N. gonorrhoeae., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: The yield of mycobacteria is shown to be very low in pleural effusions as it is a pauci-bacillary disease. The present study looked at the yield of mycobacterium tuberculosis (MTB) in terms of GeneXpert for acid fast bacilli (AFB) and culture using a medical thoracoscopy guided biopsy and analysed whether the yield increases in more complicated effusions., Materials and Methods: This is a retrospective analysis of patients who underwent medical thoracoscopy for tubercular pleural effusions at our institute over the last 5-years. Patients who had no or minimal thin septations were considered as simple effusions and were subjected to semi-rigid thoracoscopy (n = 61). While patients who had multiple loculations and thick septations were considered as complicated effusions and were subjected to rigid thoracoscopy (n = 64). We considered granuloma on a biopsy as the standard for diagnosis of Tuberculosis (TB). Xpert MTB/RIF and The BACTEC MGIT was used for culture., Results: Out 125 patients with granulomatous inflammation on biopsy, 56 (44.8%) were positive for either GeneXpert or culture for MTB. Only GeneXpert was positive in 43 and only culture was positive in 13. Amongst 61 patients with simple effusion, 14 had either GeneXpert for AFB or AFB culture being positive and 9 out of these patients had GeneXpert for MTB detected on biopsy sample. Only culture was positive in 5 patients. In complicated pleural effusion group either GeneXpert or culture for mycobacterium was positive in 42 (65.6%) out of 64 patients. Only GeneXpert was positive in 34 and culture alone was positive in 8 patients., Conclusion: The yield of MTB increases as the pleural effusion becomes more complicated. GeneXpert in a biopsy sample is a useful marker for MTB yield especially in a complicated effusion., Competing Interests: Declaration of competing interest All authors have none to declare., (Copyright © 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Hidradenitis suppurativa (HS) is a chronic inflammatory disease of the skin associated with specific lesional dysbiotic features. We studied the microbiome of clinically unaffected typical HS sites (armpits, inguinal folds, and gluteal clefts) in 60 patients with HS and 17 healthy controls. A total of 192 samples obtained by swabbing were analyzed by bacterial cultures. Of these, 116 randomly selected samples were studied by 16S rRNA gene amplicon sequencing. Patients and controls showed similar characteristics, except for smoking (87% vs. 6%, respectively). HS skinfolds were characterized by an increased abundance of anaerobes, predominantly Prevotella, but also Actinomyces, Campylobacter ureolyticus, and Mobiluncus, contrasting with a lower abundance of skin commensals such as Staphylococcus epidermidis, a major component of the skin microbiome; Kocuria; and Micrococcus luteus. The following three independent factors were associated with an abundance of high anaerobes by multivariate analysis: samples originating from patients with HS patients (P = 2.1 × 10 -4 ); body mass index (P = 5 × 10 -5 ); and the sampling site, the gluteal cleft being the most anaerobic area, followed by inguinal folds and axilla (P = 3 × 10 -6 ). The microbiome of clinically unaffected HS skinfolds is reminiscent, albeit to a minor extent, of the microbiome of chronic suppurative HS lesions and may fuel inflammation at a preclinical stage of the disease., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Fusobacterium nucleatum. nucleatum (Fn.n) is associated with colorectal carcinoma. A highly sensitive fluorescence quenching-recovery detection platform based on rolling circle amplification and hairpin molecular beacon technology for the specific analysis of Fn.n, hairpin MB being used to achieve double quenching. First, a specific recognition sequence target in the padlock probe was designed based on the nusG specific gene of Fn.n. The padlock probe then formed circular DNA using the ligase. After digestion, linear amplification was achieved by the phi29 DNA polymerase and the RCA primer, and a large amount of amplified products was obtained. Subsequently, the amplification products hybridized with the signal probe, leading to the opening of the hairpin structure in MBs. As a result, FAM at 5' end and BHQ-1 at 3' end became separated, which allowed for the recovery of the fluorescence signal. The proposed method showed high sensitivity and specificity for the detection of Fn.n genomic DNA (LOD as low as 0.7 ng L -1 ) and performed well in the identification of this bacteria in real samples., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although rapid, highly sensitive molecular diagnostics tests are useful for diagnosing fluoroquinolone-resistant tuberculosis (TB), results of molecular testing versus conventional sequential phenotypic drug susceptibility testing (pDST) are frequently discordant. This article determined the discordance rate of levofloxacin (LFX) resistance results, obtained via MeltPro TB molecular testing, versus pDST of clinical TB isolates collected in Beijing, China, between January and December 2018. Isolates with discordant results were further subjected to LFX minimal inhibitory concentration (MIC) determinations and DNA sequence analysis to explore causes of discordance. Of 571 total TB cases, 126 (22.1%) were identified as LFX resistant using the MeltPro TB assay. However, 34 of these 126 LFX-resistant isolates yielded LFX-susceptible test results using pDST, for an overall discordance rate of 27.0%. LFX MICs mainly clustered around the critical LFX concentration, with 7 (21.2%) and 13 (39.4%) of isolates exhibiting MICs of 2.0 and 4.0 mg/L, respectively. The most prevalent LFX resistance mutations associated with discordant results were involved in DNA gyrase subunit A amino acid substitutions Ala90Val (13, 39.4%) and Asp94Ala (11, 33.3%). Notably, more than one-quarter of isolates deemed LFX resistant via the MeltPro assay were scored as LFX susceptible on the basis of pDST results. Ultimately, highly discordant LFX-resistance test results were associated with specific gyrA mutations in isolates with MICs approaching the critical LFX concentration., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: Legionella is a bacterial species able to cause influenza-like illness (Pontiac fever) or severe pneumonia (Legionnaires disease, LD). We assessed Legionella presence and concentration in composting facilities in The Netherlands., Methods: A total of 142 samples from 23 green waste composting facilities were screened for Legionella DNA using qPCR., Results: Of 142 samples, Legionella spp. DNA was detected in 97 (68%), and the subspecies L. pneumophila and L. longbeachae in 33 (23%) and one (0.7%) samples, respectively. Legionella was observed in samples from all composting facilities. The concentration of Legionella spp. DNA ranged from 10 3 to 10 5 genomic units (GU)/gram. Compost temperature was negatively correlated with the presence (odds ratio 0.67, 95% CI 0.50-0.92 per 10 degrees increase) and concentration (geometric mean ratio 0.90, 95% CI 0.83-0.97 per 10 degrees) of Legionella spp. Average humidity in the week prior to sampling was negatively correlated with the L. pneumophila concentration (geometric mean ratio 0.73, 95% CI 0.56-0.96 per increase in 10% of humidity)., Discussion: This study suggests that composting facilities can be regarded as reservoirs of Legionella in The Netherlands, but additional studies should target if such facilities represent a human health risk., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The escalating problem of the dissemination of carbapenemase-producing bacteria (CPB) has gained worldwide attention. The prompt diagnosis of CPB and precise identification of carbapenemases are imperative to enable specific antibiotic therapy and control the spread of these bacteria. The present study was designed to assess the performance of five important assays for the detection of carbapenemases. The modified carbapenem inactivation method (mCIM), CARBA-5, GeneXpert Carba-R, BD MAX Check-Points CPO, and GeneFields CPE assays were evaluated with an international collection of 159 bacterial isolates, including 93 CPB and 66 non-CPB isolates. The overall accuracy/sensitivity/specificity for carbapenemase detection were 100% (95% CI, 97.7%-100%)/100% (95% CI, 96.1%-100%)/100% (95% CI, 94.6%-100%) for mCIM, 98.7% (95% CI, 95.5%-99.9%)/97.9% (95% CI, 92.5%-99.7%)/100% (95% CI, 94.6%-100%) for CARBA-5, 96.9% (95% CI, 92.8%-99%)/95.7% (95% CI, 89.4%-98.8%)/98.5% (95% CI, 91.8%-99.9%) for GeneXpert Carba-R, 94.3% (95% CI, 89.5%-97.4%)/90.3% (95% CI, 82.4%-95.5%)/100% (95% CI, 94.6%-100%) for BD MAX Check-Points CPO, and 86.2% (95% CI, 79.8%-91.1%)/77.4% (95% CI, 67.6%-85.5%)/98.5% (95% CI, 91.8%-100%) for GeneFields CPE. Interestingly, mCIM and CARBA-5 assays showed 100% accuracy/sensitivity/specificity for detection of the target genes. Furthermore, all the other assays showed comparable high accuracy (96.9% to 100%), sensitivity (100%), and specificity (96.4% to 100%) for the detection of the target genes. On the basis of these results, a new scheme was proposed for their efficient application. These results confirmed the high sensitivity of the evaluated assays, and the proposed scheme is reliable and improves the overall sensitivity and specificity of the assays., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection and may influence pregnancy outcome., Aims and Objectives: This study was conducted to assess Chlamydial infection during pregnancy by PCR., Materials and Methods: Study group consists of patients who are attending the antenatal clinics. Endocervical swabs were collected from 300 patients., Results: Off the 300 samples tested, 29 were positive as per PCR which used CT F : 5' CGT GTC GGC AAT CCT GCT GAT 3' and CT R : 5' GTC GAT AAC ATA GTC ACG ATA GTC 3'as the primers., Conclusion: This suggests there is a prevalence of Chlamydia trachomatis in our population which is 10%. Hence, it should be noted as a significant public health problem especially among sexually active young women of child bearing age. Timely detection and prompt treatment of Chlamydial infection during pregnancy can eliminate its adverse outcomes., Competing Interests: None

Campylobacter upsaliensis is an enteropathogenic bacterium in animals, and is also rarely isolated from humans, where it can cause enteritis and bacteremia. This report describes the first case of isolation of C. upsaliensis from an infected giant hepatic cyst. This bacterium could not be cultured from abscess punctuate in a usual Campylobacter-selection medium (charcoal cefoperazone deoxycholate agar medium), because of high concentration of cefoperazone as a selection agent. It could not identified by matrix-assisted laser desorption ionization-time of flight mass spectrum. Rather, it was identified as C. upsaliensis by whole genome sequencing, including by multilocus sequence typing., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Acinetobacter baumannii isolates belonging to international clonal lineage (IC) II are often multidrug-resistant and are the predominant cause of nosocomial outbreaks. While many studies have investigated the genetic and functional basis of antimicrobial resistance of these strains, few have examined specific virulence characteristics such as biofilm formation or overall pathogenic potential. Here, we analyzed biofilm formation and the associated mechanisms in A. baumannii clinical isolates from Japan belonging to the IC II lineage. Draft whole-genome sequence data for each of the isolates was analyzed to detect biofilm-associated genes, including csu (pili) and bfmS/R (two-component regulatory system), and transcription of these genes was evaluated using reverse transcription quantitative PCR. Biofilm formation was measured by crystal violet staining assay. csu operon genes showed some variation in prevalence among the isolates, with an overall prevalence of 73.7% (14/19). The biofilms formed by csu operon-positive isolates were significantly more mature than those of csu operon-negative isolates, supporting the importance of the csu operon in biofilm formation by A. baumannii. However, there was substantial variation among the csu operon-positive isolates, indicating the influence of other factors in biofilm formation. Furthermore, transcriptional levels of csu operon genes were highly divergent, with comprehensive analysis indicating that regulatory factors other than bfmS/R were involved. Our findings are a first step towards understanding the mechanisms of biofilm formation by A. baumannii IC II strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype., Methods: CPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006-2015. We confirmed the presence of the carbapenemase genes (bla IMP , bla VIM , bla KPC , bla NDM , and bla OXA-48 ) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment., Results: The CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 bla IMP-1 or 1 bla NDM-5 . The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight bla IMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid., Conclusion: The CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Bordetella pertussis (B. pertussis) infection occasionally causes severe respiratory infections in children. Potential association between virulence-associated gene alleles and severe clinical outcomes has been suggested; however, frequencies of these alleles in pediatric patients with severe pertussis have not been clarified. We retrospectively tested stored respiratory samples collected from B. pertussis-positive patients by polymerase chain reaction targeting for major virulence-associated genes; fimbrae (fim) serotype 3 (fim3), pertactin (prn), pertussis toxin A (ptxA), and pertussis toxin promotor (ptxP). Based on the identified sequences, multilocus sequence typing (MLST) was conducted. Association of gene allele frequency and clinical outcomes such as management in pediatric intensive care unit, intubation, and mortality was analyzed. Out of 25 patients with available samples, the most prevalent allele for each virulence-associated gene was fim3A (17/21, 83%), ptxA1 (20/23, 87%), prn2 (13/16, 81%), and ptxP3 (14/17, 82%). In the study, total of 3 MLST types were identified; MLST-1 from 3 patients (19%), MLST-2 from 10 patients (63%), and MLST-4 from 3 patients (19%). Detection rate of the major MLST type; MLST-2 was significantly higher for patients who required intubation compared to those who did not (6/6, 100% vs 4/10, 40%; p = 0.034). In conclusion, MLST-2 was the most common MLST type in B. pertussis isolated from Japanese children with severe pertussis. Further studies investigating its causal association with disease severity is warranted., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Escherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance., Methods: Phylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates., Results: The most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains., Conclusion: Commensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cases of tick-borne diseases are increasing in the United States, and new tick-borne pathogen species causing human illness are being discovered. The specific etiology is generally difficult to diagnose based on clinical signs and symptoms alone, because of their generalized nature and often lack of a known tick bite. For some infections, such as Lyme disease and spotted fever group rickettsioses, serology remains the most appropriate laboratory diagnostic tool, but for others such as anaplasmosis, ehrlichiosis, and babesiosis, direct detection in the blood is preferred for rapid diagnosis. In Kentucky, USA, the area served by our laboratory, the most commonly reported tick-borne illnesses include spotted fever group rickettsiosis, ehrlichiosis and Lyme disease, but of these three diseases, only ehrlichiosis is well-suited for direct detection using PCR methods during the acute stage of illness. We report here the validation of a duplex real-time PCR assay using whole blood specimens on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. This method allows for rapid and sensitive detection of acute infections with Ehrlichia spp. and Anaplasma phagocytophilum using whole blood specimens. We included A. phagocytophilum to monitor emergence of this pathogen in Kentucky, since surrounding states have reported many more cases than Kentucky., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Q fever prosthetic valve endocarditis in association with antiphospholipid antibody syndrome (APS) in systemic lupus erythematosus (SLE) has not been previously reported. Here, we report a 22-year-old Saudi female diagnosed with SLE and APS. She had mitral valve replacement with bio-prosthesis five years earlier for Libman-Sack endocarditis. She presented with two months' history of fever, cough, palpitations, and progressive shortness of breath. A transthoracic echocardiogram showed a degenerative mitral valve prosthesis with a large mass causing severe obstruction. Open heart surgery revealed multiple masses on the mitral valve. PCR from the resected tissues was positive for Coxiella burnetii DNA. Q fever serology showed phase two IgG 1:2048, phase one IgG 1:512, and IgM 1:1024. The valve was replaced with a bio-prosthesis. She was well at 12 months of follow-up., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Vitiligo is impacted by environmental triggers. We studied the contribution of the microbiome in FH mice, in which depigmentation is mediated by tyrosinase-reactive T cells. The mice received oral antibiotics and were monitored for depigmentation. The microbiome was studied in fecal and skin samples using 16S rRNA analysis. The resulting T-cell distributions were evaluated. In untreated mice, pigment loss did not expand to the pelage, whereas mice in the ampicillin group were approximately 1/3 depigmented at 30 weeks. In contrast to models of autoimmunity that are less dependent on IFN-γ, ampicillin but not neomycin treatment correlated with accelerated disease and reduced bacteria in the fecal pellets. Modified cytokine patterns in the tissue and serum suggest a response that transcends the gut. Ampicillin-induced depigmentation was accompanied by gut but not skin dysbiosis, and reduced T cell numbers in both sites. Neomycin induced a redistribution of gut T cells and an accumulation of skin regulatory T cells. This treatment spurred a Bacteroides-dominated population of fecal bacteria. Reduced diversity is prominent particularly after ampicillin treatment, when the gut is dominated by Pseudomonas species. In line with current concepts relating the microbiome and the immune system, we predict that dietary measures might promote skin health and delay vitiligo onset., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Tuberculous meningitis (TBM) is difficult to diagnose. Digital PCR (dPCR) is a novel method which can quantify trace nucleic acids. This study sought to evaluate the diagnostic accuracy of dPCR analysis of cerebrospinal fluid (CSF) for TBM., Methods: We collected CSF specimens from hospitalized TBM and non-TBM patients. Total CSF DNA was purified and the concentrations of Mycobacterium tuberculosis insert sequence 6110 (IS6110) and gyrase subunit B (gyrB) were quantified using droplet dPCR. The receiver operating characteristic curves of dPCR were established and the diagnostic performances were obtained. We also compared the sensitivity of dPCR with routine diagnostic tests., Results: A total of 101 patients were recruited, 68 of whom suffered from TBM (26 definite, 34 probable and eight possible TBM) and 33 from non-TBM. The sensitivity of IS6110-dPCR assay for total TBM was higher than that of gyrB-dPCR assay (57.4% (44.8-69.3%) vs. 22.1% (12.9-33.8%)), and there was no significant difference for specificity between them (97.0% (84.2-99.9%) vs. 100% (89.4-100.0%)). The sensitivity of IS6110-dPCR in definite TBM was higher than that in probable and possible TBM (73.1% vs. 52.9% and 25.0%, respectively). IS6110-dPCR assay showed a higher sensitivity than smear microscopy (53.3% vs. 6.7%), mycobacterial culture (50.0% vs. 12.5%), IS6110-quantitative PCR (53.1% vs. 21.9%) and Xpert MTB/RIF (70.4% vs. 29.6%). Long anti-tuberculosis treatment time was found to be significantly associated with negative dPCR results., Conclusion: CSF IS6110-dPCR assay is a rapid and sensitive molecular test, which has the potential to be used to enhance the diagnosis of TBM., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: Preterm birth (PTB) is a major cause of neonatal mortality. The vaginal microbiome is associated with PTB, but results vary across racial/ethnic populations. Some evidence suggests gestational age affects this association. We investigated these associations in a novel population, conducting a post hoc analysis assessing if associations differed between women swabbed at different gestational ages., Methods: We compared vaginal microbiomes from women with PTB (n = 25) to a random sample of women with term births (n = 100) among participants in the Pregnancy Outcomes, Maternal and Infant Study, conducted in Lima, Peru. Using DADA2, we identified taxa from 16S DNA sequencing and used Dirichlet multinomial mixture models to group into community state types (CSTs)., Results: If gestational age at sampling was not considered, no CST (diverse, Lactobacillus-dominated or Lactobacillus iners-dominated), was associated with PTB. Among women sampled before 12 weeks' gestation, women with Lactobacillus-dominated CSTs were less likely to have a PTB than those with a diverse CST. Among those swabbed between 12 and 16 weeks' gestation, the reverse was true., Conclusions: Our study supports previous literature suggesting that what constitutes a healthy vaginal microbiome varies by race/ethnicity. Longitudinal studies are necessary to disentangle effects of vaginal microbiome differences over gestation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A-26-year-old man was admitted to our hospital with diffuse abdominal pain, nausea, and vomiting. He had a history of malignant nephrosclerosis, for which he had been receiving peritoneal dialysis (PD) for the past 14 months. His PD effluent was cloudy and turbid (white blood cell count, 10,528/μL; neutrophils 95.2%). A Gram-negative coccobacillus was isolated from peritoneal fluid culture. However, the organism could not be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS, bioMérieux), but was identified as Moraxella osloensis by the 16S rRNA gene sequencing. He was successfully treated with intraperitoneal cefazolin therapy for 3 weeks without removing the intra-abdominal catheter. A literature review revealed three previous case reports all of which were diagnosed by MALDI Biotyper (Bruker Daltonics), suggesting that the identification of M. osloensis may vary depending on the type of MALDI-TOF MS system. In conclusion, we experienced a case of M. osloensis infection in a PD patient, which was successfully treated by antibiotic treatment, without removing the PD catheter., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We previously reported the distribution of Achromobacter spp. (species and Sequence Types (ST)) in our French Cystic Fibrosis (CF) centre. In the present study we collected 109 Achromobacter isolates (1/patient) from 9 other French CF Centres for species identification, antimicrobial susceptibility testings and Multilocus-Sequence-Typing (MLST) analysis. Ten species were detected, A. xylosoxidans being the most predominant one (73.4% of the isolates). Piperacillin-tazobactam, ceftazidime, imipenem, meropenem and ciprofloxacin were respectively active against 88, 70, 79, 72 and 23% of the isolates. Among the 79 A. xylosoxidans isolates, 46 STs were detected. Interestingly, ST 137, recovered in 4 centres (5 patients), was previously detected in our centre (2 patients). The strains from the 7 patients belonged to the same pulsotype (pulsed-field-gel-electrophoresis analysis) and harboured acquired resistance to meropenem, ceftazidime, ciprofloxacin, and except for 2 isolates, to imipenem and piperacillin-tazobactam. This is the first description in France of a circulating multiresistant A. xylosoxidans strain., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Salmonella is a widely distributed, extremely harmful bacteria, the presence of which requires confirmation via an on-site visual biosensor. In this study, we constructed a label-free, cascade amplification visualization biosensor for the sensitive and rapid detection of Salmonella enterica subsp. enterica serovar typhimurium based on the RDTG principle (recombinase polymerase amplification (RPA), duplex-specific enzyme (DSN) cleavage, terminal deoxynucleotidyl transferase (TdT) extension and G-quadruplexes output). Following DNA extraction of Salmonella spp., the first step in the construction involved the recognition and amplification of nucleic acids, carried out by RPA, to achieve the first signal amplification within 10 min. This RPA product was then specifically cleaved by DSN to produce a large number of small double-stranded DNA (dsDNA) products with 3'-OH within 15 min to achieve the second signal amplification. Thereafter, TdT was employed to empower these small 3'-OH dsDNA products to extend and produce a large number of long G-rich single-stranded DNAs (ssDNAs) within 20 min, thus realizing the third signal increase. These long G-rich ssDNA products displayed a color change that could be directly observed through the naked eye by adding H 2 O 2 /3,3',5,5'-tetramethylbenzidine (TMB). The RDTG biosensor for the detection of Salmonella spp. has several advantages, including a low limit of 6 cfu/mL. It is an isothermal-free instrument, simple to operate, with a rapid detection time of less than 1.5 h. Furthermore, it can be visually characterized and quantified by a microplate reader to detect Salmonella spp., in food and environmental samples, and it has broad application prospects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microbacterium species are coryneform gram-positive rods that are widely distributed in the environment and have been recently recognized as rare pathogens in humans. However, information about the epidemiologic and clinical characteristics of Microbacterium species is scarce. We herein reported an 11-year-old girl with acute leukemia who was found to have catheter-related bloodstream infection in her neutropenic phase. Gram-positive bacilli repeatedly grew on the blood cultures and were later confirmed by 16S rRNA analysis as Microbacterium paraoxydans. A literature review found available clinical courses in 21 cases (7 pediatric cases) of Microbacterium spp. bacteremia. Our case and those in literature suggested that Microbacterium spp. bacteremia often occurs in patients with indwelling central venous catheters; the literature review further suggested that removal of central venous catheters is required in most cases and that 16S rRNA sequence was useful in identifying in detail the species of Microbacterium., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen., Methods: Here we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling. This assay was performed in a single closed tube at 39°C within 30min. A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling. For comparison, the real-time PCR (RT-PCR) was also performed with DNA extraction in parallel., Results: The sensitivity of the internally controlled RAA assay was 10 1 copies or 10CFU/ml per reaction in detecting plasmid DNA or B. pertussis strain. The optimum concentration of the IAC plasmid was determined to be 100 copies, and the introduction of IAC effectively reduced the occurrence of false negatives. Compared to the RT-PCR, RAA results with DNA extraction obtained 100% sensitivity and specificity, and the RAA results with heat-treated DNA showed 85.96% sensitivity and 100% specificity., Conclusion: With the advantages of 45min turn-around time and simple steps of DNA purification, this assay could become a useful diagnostic tool for Bordetella pertussis detection and is potentially suitable for point-of-care identification to guide prompt clinical treatment., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is considered to be a potential zoonotic pathogen and meat is one of the sources of MAP exposure for humans. MAP has been shown to be relatively resistant to different food processing methods, but there is a lack of information about the effects of ripening and fermentation processes on MAP survival in meat. Our results demonstrate that a short ripening process during teewurst production did not reduce MAP counts and viable mycobacteria were detected even during 4 weeks of storage. Although no viable MAP was recovered during the dry fermented sausage production process, there was no reduction in MAP count detected by real time PCR during production and storage of both sausages. Although the impact of foodborne exposure to viable MAP and/or mycobacterial components has not yet been clearly determined, the consumption of raw fermented meat products may be considered as a possible route of MAP transmission to humans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: Hardware-related infection remains a major problem in patients with neurostimulation systems. The role of bacterial colonization and the formation of biofilm on the surface of implanted devices remain unclear. Here, we analysed the incidence of bacterial DNA on the surface of implantable pulse generators (IPGs) using 16S rRNA gene sequencing in a consecutive series of patients who underwent routine IPG replacement without clinical signs of infection., Patients and Methods: We included 36 patients who underwent scheduled replacement surgery of 44 IPGs. The removed IPGs were processed and whole genomic DNA was extracted. The detection of bacterial DNA was carried out by Polymerase Chain Reaction (PCR) using universal bacterial primers targeting the 16S rRNA gene. The DNA strands were analysed by single-strand conformation polymorphism (SSCP) analysis., Results: Indications for chronic neurostimulation were Parkinson disease, tremor, dystonia, neuropathic pain and peripheral artery occlusion disease. Mean age of patients at the time of implantation was 48 ± 17.6 years. The mean interval between implantation and replacement of the IPG was 24.8 months. PCR/SSCP detected bacterial DNA of various species in 5/36 patients (13.9%) and in 5/44 pacemakers (11.4%), respectively. There was no evidence of clinical infection or wound healing impairment during follow-up time of 45.6 ± 19.6 months., Conclusion: Bacterial DNA can be detected on the surface of IPGs of neurostimulation systems in patients without clinical signs of infection by using PCR techniques. It remains unclear, similar to other permanently implanted devices, which mechanisms and processes promote progression to the point of overt infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microbial food spoilage is responsible for significant economic losses. Brochothrix thermosphacta is one of the major bacteria involved in the spoilage of meat and seafood. Its growth and metabolic activities during food storage result in the production of metabolites associated with off-odors. In this study, we evaluated the genotypic and phenotypic diversity of this species. A collection of 161 B. thermosphacta strains isolated from different foods, spoiled or not, and from a slaughterhouse environment was constituted from various laboratory collections and completed with new isolates. A PCR test based on the rpoB gene was developed for a fast screening of B. thermosphacta isolates. Strains were typed by MALDI-TOF MS, rep-PCR, and PFGE. Each typing method separated strains into distinct groups, revealing significant intra-species diversity. These classifications did not correlate with the ecological origin of strains. The ability to produce acetoin and diacetyl, two molecules associated with B. thermosphacta spoilage, was evaluated in meat and shrimp juices. The production level was variable between strains and the spoilage ability on meat or shrimp juice did not correlate with the substrate origin of strains. Although the B. thermosphacta species encompasses ubiquitous strains, spoiling ability is both strain- and environment-dependent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The presence of Borrelia theileri in Argentina is confirmed after recording the spirochete from a bovine in northern Argentina. The analysis of sequences of the flagellin gene (fla) and length of Borrelia spp. specimens on thick blood films shows that the local isolate clusters within a well-supported clade with B. theileri isolates from different geographical origins, confirming the presence of B. theileri in Argentina. The mean length of 30 specimens of B. theileri was 12.89 μm (standard deviation 2.88 μm, range 9.35-20.16 μm). The only known vector of Borrelia theileri in northern Argentina is the cattle tick Rhipicephalus microplus, therefore Borrelia infection should be regarded as a potential complication of other cattle tick-borne diseases such as babesiosis, especially on cattle introduced from areas free of R. microplus. The possibility of serologic cross-reaction with B. theileri must not be minimized in studies of other spirochaetes in the R. microplus infested region of Argentina., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

DNA purification is a critical step in the processing of samples for molecular diagnosis and/ or identification of pathogens via polymerase chain reaction (PCR). Especially when handling vectors like ticks, purifying the DNA always poses a challenge. In this study, we compared factors that may have an influence on DNA extraction namely commercially available DNA extraction kits vs alkaline hydrolysis for DNA extraction. The methods were applied to questing Ixodes (I.) ricinus ticks and Borrelia cultures of defined cell concentrations. A total of 69 questing I. ricinus ticks were collected. From 34 ticks, total DNA was extracted using a commercial DNA extraction kit. Thirty-five ticks were treated with 1.25% ammonium hydroxide (NH 4 OH). Six ticks from each batch were placed in 70% ethanol (EtOH) for one week prior to DNA extraction to see the effect of EtOH preservation on total DNA yield. DNA yield was estimated in field-collected ticks using conventional PCR targeting the Ixodes Cytochrome C oxidase (coi) gene and in cultured Borrelia isolates using quantitative real-time PCR (qPCR) targeting the FlaB encoding gene of Borrelia. Column DNA extraction yielded slightly better results than NH 4 OH treatment when tested in a PCR targeting a tick-specific coi gene (96% PCR-positive vs 86% PCR-positive results, respectively). EtOH preservation had a slightly negative effect on DNA yield and - again - slightly stronger PCR products were observed by commercial kit extraction. A Shapiro-Wilk test conducted revealed a significance-level of 90% for both the methods, indicating a normal distribution of the values generated by BioNumerics quantification. A two-sided t-test conducted revealed a significant (p < 0.01) mean difference between the methods. Similarly, qPCR on cultured specimen DNA of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s.) (B31) with different concentrations revealed a better yield for kit extraction in comparison to NH 4 OH treatment; a difference of approximately 3 Ct-values was ascertained between extraction methods. A one-sided t-test showed a significant difference between the methods at lower concentration of Borrelia i.e. better extraction with a commercial kit at lower borrelial DNA concentration, while at higher concentration (10 6 cells per ml) the difference was not significant., (Copyright © 2019. Published by Elsevier GmbH.)

Previous studies have shown independently that the skin and gut microbiota are closely associated with atopic dermatitis (AD); however, the microbiota across different habitats of AD patients as an integrated community has not been characterized. In the present study, we comparatively analyzed the structure and function of the microbial communities in the skin, oral cavity, and gut of 172 AD patients and 120 healthy controls through 16S ribosomal RNA gene amplicon sequencing. The skin and oral cavity, but not the gut, of AD patients demonstrated differential reduction in the microbial diversity, and these were distinctly correlated with disease severity. Different degrees of shifts in the community structure were found among different habitats, and the lineage distance between the skin and oral microbiota of AD patients was closer than that observed in the controls. The different habitats of AD patients exhibited site-specific alterations at the genus level, and many oral-specific microbes of AD showed opposing directions of enrichment in the skin and oral cavity. Most interestingly, an inverse association in the functional pathways was found between the skin and oral microbiota of AD patients. Additionally, the alterations of the microbiota in different body sites of AD patients were differentially affected by age., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)