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bioRxiv2018

Water quality and microbial load: a double-threshold identification procedure intended for space applications

During longer-lasting future space missions, water renewal by ground-loaded supplies will become increasingly expensive and unmanageable for months. Space exploration by self-sufficient space-crafts is thus demanding the development of culture-independent microbiological methods for in-flight water monitoring to counteract possible contamination risks. In this study, we aimed at evaluating microbial load data assessed by selected techniques with current or promising perspectives in space applications (i.e., HPC, ATP-metry, qPCR, flow cytometry), through the analysis of water sources with constitutively different contamination levels (i.e., chlorinated and unchlorinated tap waters, groundwaters, river waters, wastewaters). Using a data-driven double-threshold identification procedure, we identified and presented new alternative standards of water quality based on the assessment of the total microbial load. Our approach is suitable to provide an immediate alert of microbial load peaks, thus enhancing the crew responsiveness in case of unexpected events due to water contamination and treatment failure. Finally, the backbone dataset could help in managing water quality and monitoring issues for both space and Earth-based applications.

microbiology↗

bioRxiv2018

High-dose rifamycins enable shorter oral treatment in a murine model of Mycobacterium ulcerans disease

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical skin and soft tissue infection that is associated with disability and social stigma. The mainstay of BU treatment is an eight-week course of 10 mg/kg rifampin (RIF) and 150 mg/kg streptomycin (STR). Recently, the injectable STR has been shown to be replaceable with oral clarithromycin (CLR) for smaller lesions for the last four weeks of treatment. A shorter, all-oral, highly efficient regimen for BU is needed, as the long treatment duration and indirect costs currently burden patients and health systems. Increasing the dose of RIF or replacing it with the more potent rifamycin drug rifapentine (RPT) could provide such a regimen. Here, we performed a dose-ranging experiment of RIF and RPT in combination with CLR over four weeks of treatment in a mouse model of M. ulcerans disease. A clear dose-dependent effect of RIF on both clinical and microbiological outcomes was found, with no ceiling effect observed with tested doses up to 40 mg/kg. RPT-containing regimens were more effective on M. ulcerans. All RPT-containing regimens achieved culture negativity after only four weeks while only the regimen with the highest RIF dose (40 mg/kg) did so. We conclude that there is dose-dependent efficacy of both RIF and RPT and that a ceiling effect is not reached with the current standard regimen used in the clinic. A regimen based on higher rifamycin doses that are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

microbiology↗

bioRxiv2018

Finally, Bulk Typing of Bacterial Species down to Strain Level using ON-rep-seq

Despite the massive developments within culture-independent methods for detection and quantification of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (< 24h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation takes less than 5h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting the random error of reads within peaks of LCp generates a set ([~]10 contigs per sample; 300bp - 3Kb) of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation. With benchmarked 288 isolates identified on a single flow cell and a theoretical throughput to evaluate over 1000 isolates, our method allows for detailed bacterial identification for less than 2$ per sample at very high speed.

microbiology↗

bioRxiv2018

Chromatic Bacteria - A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria

Differential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The here constructed plasmids are available to the community and provide a valuable tool to investigate bacteria-bacteria, bacteria-host, and bacteria-environmental interactions.

microbiology↗

bioRxiv2018

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.\n\nResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~108, which is ~2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.\n\nConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

microbiology↗

bioRxiv2018

Quantitative proteomics of the 2016 WHO Neisseria gonorrhoeae reference strains surveys vaccine candidates and antimicrobial resistance determinants

The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally due to the repercussions on reproductive health, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n=15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles, previously described in regard to phenotypic and reference genome characteristics, and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 901 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635, WHO_F_00745, WHO_F_01139, WHO_F_01144, and WHO_F_01226, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics databank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.

microbiology↗

bioRxiv2018

Hydrogenotrophic methanogens of the mammalian gut: functionally similar, thermodynamically different. A modelling approach

Methanogenic archaea occupy a functionally important niche in the gut microbial ecosystem of mammals. Our purpose was to quantitatively characterize the dynamics of methanogenesis by integrating microbiology, thermodynamics and mathematical modelling. For that, in vitro growth experiments were performed with pure cultures of key methanogens from the human and ruminant gut, namely Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanobacterium formicium. Microcalorimetric experiments were performed to quantify the methanogenesis heat flux. We constructed an energetic-based mathematical model of methanogenesis. Our model captured efficiently the dynamics of methanogenesis with concordance correlation coefficients of 0.94 for CO2, 0.99 for H2 and 0.97 for CH4. Together, experimental data and model enabled us to quantify metabolism kinetics and energetic patterns that were specific and distinct for each species despite their use of analogous methane-producing pathways. Then, we tested in silico the interactions between these methanogens under an in vivo simulation scenario using a theoretical modelling exercise. In silico simulations suggest that the classical competitive exclusion principle is inapplicable to gut ecosystems and that kinetic information alone cannot explain gut ecological aspects such as microbial coexistence. We suggest that ecological models of gut ecosystems require the integration of microbial kinetics with nonlinear behaviours related to spatial and temporal variations taking place in mammalian guts. Our work provides novel information on the thermodynamics and dynamics of methanogens. This understanding will be useful to construct new gut models with enhanced prediction capabilities and could have practical applications for promoting gut health in mammals and mitigating ruminant methane emissions.

microbiology↗

bioRxiv2018

Virulence gene profiles and phylogeny of Shiga toxin-positive Escherichia coli strains isolated from FDA regulated foods during 2010-2017

Illnesses caused by Shiga toxin-producing Escherichia coli (STECs) can be life threatening, such as hemolytic uremic syndrome (HUS). The STECs most frequently identified by USDAs Microbiological Data Program (MDP) carried toxin gene subtypes stx1a and/or stx2a. Here we describe the genome sequences of 331 STECs isolated from foods regulated by the FDA 2010-2017, determining their genomic identity, serotype, sequence type, virulence potential, and prevalence of antimicrobial resistance. Isolates were selected from the MDP archive, routine food testing by field labs (ORA), food testing by a contract company, and our laboratory (ORS). Only 276 (83%) were confirmed as STECs by in silico analysis. Foods from which STECs were recovered included cilantro (6%), spinach (25%), lettuce (11%), and flour (9%). Phylogenetic analysis using core genome MLST revealed these STEC genomes were highly variable, with some clustering associated with ST types and serotypes. We detected 95 different sequence types (ST); several ST were previously associated with HUS: ST21 and ST29 (O26:H11), ST11 (O157:H7), ST33 (O91:H14), ST17 (O103:H2), and ST16 (O111:H-). in silico virulome analyses showed ~ 51% of these strains were potentially pathogenic [besides stx gene they also carried eae (25%) or 26% subA (26%)]. Virulence gene prevalence was also determined: stx1 only (19%) -variants a and c; stx2 only (66%) - variants a, b, c, d, e, and g; and stx1/sxt2 (15%). Our data form a new WGS database that can be used to support food safety investigations and monitor the recurrence/emergence of E. coli in foods.\n\nImportanceShiga toxin-producing Escherichia coli (STECs) are associated with foodborne outbreaks worldwide; however, surveillance has not previously included genomic analyses for phylogenetics, prevalence, or potential virulence. We constructed the first genomic database of isolates from FDA-regulated foods to help monitor the emergence of new pathogenic STECs. Although only ~30 STECs were isolated per year, 50% of these carried markers associated with pathogenesis either a combination of eae plus stx, or subA plus stx. Moreover, those strains also carried virulence genes associated with severe illnesses. Here we showed that WGS enabled comparisons across isolates to establish phylogeny, help in identification of antibiotic resistance by monitoring the presence of antimicrobial resistance genes, and determined the presence of known virulence genes that have been linked with illnesses. Future food safety investigations will benefit from improved source tracking and risk assessments made possible by these analyses and new WGS database.

microbiology↗

bioRxiv2018

PUMA: A tool for processing 16S rRNA taxonomy data for analysis and visualization

Microbial community profiling and functional inference via 16S rRNA analysis is quickly expanding across various areas of microbiology due to improvements to technology. There are numerous platforms for producing 16S rRNA taxonomic data which often vary in file and sequence formatting, creating a common barrier in microbiome studies. Additionally, many of the methods for analyzing and visualizing this sequencing data each require their own specific formatting. As a result, efficient and reproducible comparative analysis of taxonomic data and corresponding metadata in multiple programs remains a challenge in the investigation of microbial communities. PUMA, the Program for Unifying Microbiome Analysis, alleviates this problem in microbiome studies by allowing users to take advantage of numerous 16S rRNA taxonomic identification platforms and analysis tools in an efficient manner. PUMA accepts sequencing results from several taxonomic identification platforms and then automates configuration of data and file types for analysis and visualization via many popular tools. The protocol accomplishes this by producing a variety of properly configured, annotated, and altered files for both analysis and visualization of taxonomic community profiles and inferred functional profiles. PUMA provides an easy and flexible interface to accommodate for a variety of users to produce all files needed for all-inclusive analysis of targeted amplicon sequencing studies. PUMA is an unprecedented open-source solution for unifying multiple microbiome analysis softwares and uses an adaptable implementation with the potential to improve and consolidate the state of microbiome research.\n\nBody/Findings

microbiology↗

bioRxiv2018

Antimicrobial Resistance Surveillance Among Gram Negative Bacterial Isolates from Patients in Khartoum State Hospitals

BackgroundAntimicrobial resistance (AMR) among Gram-negative bacilli is a global health problem. Surveillance of AMR is required to advise on empirical antimicrobial therapy. This study aimed at evaluating the frequency and the AMR patterns of Gram-negative isolates from patients treated in eight hospitals in Khartoum State, Sudan.\n\nMethodsA cross-sectional laboratory based study was conducted over six months period at the microbiology department, Soba University Hospital, Khartoum State, Sudan. All Gram-negative isolates from blood, urine, wound, and sputum during the period of study were included.\n\nResultsA total of 734 Gram-negative bacilli were isolated. Klebsiella spp. 249 (34%) was the most frequently encountered one, followed by Pseudomonas spp. 153(21%), E.coli 123(17%), Acinetobacter spp. 75 (10%), Burkholderia cepacia 42(6%), Proteus spp. 28(4%) Enterobacter spp. 28(4%), Stenotrophomonas maltophilia 21(2.8%), and others gram-negative bacilli 15(2.2%) The analysis of the antimicrobial susceptibility patterns showed that 134 (22.3%) isolates were multidrug resistant to three or more classes of antibiotics including cephalosporins, {beta}-lactam-{beta}-lactamase inhibitor group, quinolones, aminoglycosides and carbapenems.\n\nConclusionThis high level of resistance among Gram-negative bacilli in Khartoum state hospitals is alarming. The local health authorities are prompted to step up infection control program and introduce the concept of antimicrobial stewardship in Khartoum State hospitals.

microbiology↗

bioRxiv2018

Bacterial community composition of recycled irrigation water of a NFT-type experimental system, with and without a slow sand filter

The bacterial community composition (BCC) of recycled irrigation freshwater was monitored on a Nutrient Film Technique (NFT)-type experimental hydroponic system. Two identical NFT systems in a greenhouse were used to grow tomato plants. One was connected to a slow sand filter (SSF), and one was used as control. DNA was isolated from irrigation freshwater, and molecular methods were carried out to characterise the BCC. These included terminal-restriction fragment length polymorphism (T-RFLP), cloning and 454 pyrosequencing. A total number of 291 442 trimmed sequences, of 211 bp average length. A strong differentiation of the bacterial community composition in recycled irrigation water was triggered by the activity of the SSF. Phylogenetic affiliation revealed that Bacilli, Alpha- and Gammaproteobacteria, and Nitrospira were differentially abundant during filtration with the SSF. This study showed that SSF modified the relative amount of a set of bacterial genera. These included Pseudomonas, Bacillus, Flavobacterium, Burkholderia and Azospirillum. These bacteria have been previously described as plant growth promoting rhizobacteria (PGPR). The presence, increased or exclusive, in Water ssf of bacteria such as Bacteroidetes, Gemmatimonadetes, Nitrospira, Firmicutes, Alpha-, Beta-, Gamma- and Deltaproteobacteria, was associated with an increased biomass in plants. Such findings are promising for future applications of a combined system NFT-SSF: NFT guarantees a controlled closed environment for the growth of plants, while the SSF secures the microbiological balance of recycled irrigation water.

microbiology↗

bioRxiv2018

Klebsiella quasipneumonaiae provides a window into carbapenemase gene transfer, plasmid rearrangements and nosocomial acquisition from the hospital environment

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

microbiology↗

bioRxiv2018

Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings.\n\nImportanceNanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

microbiology↗

bioRxiv2018

A counterselectable sucrose sensitivity marker permits efficient and flexible mutagenesis in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus; GBS) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations such as single base pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrate lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.\n\nImportanceThe ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single base pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

microbiology↗

bioRxiv2019

Automation for the identification of Pseudomonas aeruginosa: Comparison of TDR-300B, VITEK(R)2, and VITEK(R)-MS

Quick and precise methods have always been needed in the medical field to correctly identify the agent of infection. Automated systems for diagnosis of infectious pathogen such Pseudomonas aeruginosa from critical patients with infections in the Intensive Care Unit is essential. This study aimed to compare the capability of automated biochemistry-based identification system, TDR-300B and VITEK(R)2, to the one of Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-TOF), VITEK(R)-MS, in the identification of P. aeruginosa.\n\nSamples were P. aeruginosa isolates collection from Laboratory of Microbiology, Faculty of Medicine and Health Science UNIKA Atma Jaya. These isolates were refreshed; one single colony of oxidase-positive Gram-negative rods was further inoculated in TDR-300B NF-64 cards and VITEK(R)2 GN cassettes. The bacterial identification was also carried out using VITEK(R)-MS as gold-standard. Positivity of TDR-300B and VITEK(R)2 in the identification of P. aeruginosa was 87.09% (27/31) and 90.32% (28/31) in the species level, and 87.09%, 96.77% in the genus level respectively. The congruity of TDR-300B/ VITEK(R)2 in the species and genus level was 83.87% and 87.09%. When compared to VITEK(R)-MS, congruence of VITEK(R)2 was 93.30% (24/26) and TDR-300B was 80.76% (21/26). Sensitivity value for TDR-300B and VITEK(R)2 was high, 95.45% and 100%, positive predictive value and accuracy were lower in TDR-300B than VITEK(R)2; Fisher exact value was >0.05, thus there were no significant differences in the capability of TDR-300B and VITEK(R)2 in the identification of P. aeruginosa.

microbiology↗

bioRxiv2019

Carriage of a single strain of non-toxigenic Corynebacterium diphtheriae biovar Belfanti (Corynebacterium belfantii) in four patients with cystic fibrosis

Cystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission by Corynebacterium diphtheriae has not been reported so far. The aim of this article was to investigate the possibility of a cross transmission of C. diphtheriae biovar Belfanti between four patients of a CF center. C. diphtheriae biovar belfanti (now formally called C. belfantii) isolates were collected from four patients in a single CF care center over a 6 years period and analyzed by microbiological methods and whole genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient had the same strain on two consecutive samplings nine months apart. The strain was non-toxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. Transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrates colonization of CF patients by C. diphtheriae biovar Belfanti (C. belfantii) and shows persistence and transmission of a unique strain during at least six years in a single CF patient care center.\n\nPrevious meeting presentationsThe information in this work was not previously presented in any meeting.\n\nAccession numbersThe genomic sequence data generated in this work were submitted to the European Nucleotide Archive and are available from the International Nucleotide Sequence Database Collaboration (NCBI/ENA/DDBJ) databases under project accession number PRJEB28372 and run data accession numbers ERR2757916 to ERR2757921.

microbiology↗

bioRxiv2019

Prediction of pyrazinamide resistance in Mycobacterium tuberculosis using structure-based machine learning approaches.

SynopsisO_ST_ABSBackgroundC_ST_ABSPyrazinamide is one of four first-line antibiotics used to treat tuberculosis, however antibiotic susceptibility testing for pyrazinamide is challenging. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, an enzyme that converts pyrazinamide into its active form. MethodsWe curated a dataset of 664 non-redundant, missense amino acid mutations in pncA with associated high-confidence phenotypes from published studies and then trained three different machine learning models to predict pyrazinamide resistance. All models had access to a range of protein structural-, chemical- and sequence-based features. ResultsThe best model, a gradient-boosted decision tree, achieved a sensitivity of 80.2% and a specificity of 76.9% on the hold-out Test dataset. The clinical performance of the models was then estimated by predicting the binary pyrazinamide resistance phenotype of 4,027 samples harboring 367 unique missense mutations in pncA derived from 24,231 clinical isolates. ConclusionsThis work demonstrates how machine learning can enhance the sensitivity/specificity of pyrazinamide resistance prediction in genetics-based clinical microbiology workflows, highlights novel mutations for future biochemical investigation, and is a proof of concept for using this approach in other drugs.

microbiology↗

bioRxiv2019

Bacteriophage to Combat Biofilms in Hospital Drains

BackgroundAccording to the World Health Organization, nearly 15% of all hospitalized patients worldwide acquire nosocomial infections. A particular area of concern for bacterial build up in hospitals is sink drains. The moist, microbiologically active environment of drains promotes the formation of biofilms that are difficult to target with standard chemical disinfectants. Bacteriophages, however, show potential to be used as disinfecting agents in hospital drains. Not only do bacteriophages increase in titer as they infect, spreading to hard-to-reach surfaces, phages have been shown to degrade the extracellular matrix of biofilms and gain access to underlying bacteria. This research explores the potential of bacteriophages to eradicate biofilms in an environment modeling a sink drain by comparing the efficacy, range, and durability of bacteriophage to a chemical disinfectant.\n\nMethodsE. coli biofilms were grown in M9 minimal media placed in sink P-traps assigned one of three treatments: chemical disinfectant, bacteriophage, or deionized water (control). Biofilms were quantified at five time points -- 1, 12, 24, 36, and 48 hours -- using the crystal violet assay.\n\nResultsBoth chemical disinfectant and bacteriophage significantly decreased the optical densities of biofilms (p < 0.001***). P-traps treated with bacteriophages showed more uniform destruction of biofilm across P-trap compared to chemical disinfectant (p < 0.01**). A trend may suggest that over time bacteriophage became more effective at reducing biofilm than chemical disinfectant.\n\nConclusionThis work highlights the potential of bacteriophage as an alternative to conventional chemical disinfectants for biofilm control in settings such as hospital drains.\n\nImportanceNosocomial infections prolong hospital stay, costing the U.S. healthcare system $5-10 billion annually. An increasing number of reports demonstrate that sink drains -- reservoirs for multidrug resistant bacteria -- may be a source of hospital-related outbreaks. Recent studies have elucidated the mechanism of dispersal of bacteria from contaminated sinks to patients, but limited data are available identifying disinfecting methods for hospital drains. Not only did this study demonstrate that bacteriophages could reduce biofilms on sink drains just as effectively as a commercial disinfectant, it showed that phages tended to spread more thoroughly across P-traps and may work for longer. With hand-washing an imperative activity for disease prevention, hospital sinks should remain clean. This work explores an alternative disinfecting method for hospital sink drains.

microbiology↗