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bioRxiv2018

A decay effect of bacterial growth rate associated with genome reduction

Bacterial growth is an important topic in microbiology and of crucial importance to better understand living cells. Bacterial growth dynamics are quantitatively examined using various methods to determine the physical, chemical or biological features of growing populations. Due to methodological differences, the exponential growth rate, which is a parameter that is representative of growth dynamics, should be differentiated. This study experimentally verified the differentiation in growth rates attributed to different methodologies, and demonstrated that the most popular method, optical turbidity, led to the determination of a lower growth rate in comparison to the methods based on colony formation and ATP abundance, due to a decay effect of reading OD600 during a population increase. Accordingly, the logistic model, which is often applied to growth data reading the OD600, was revised by introducing a new parameter: the decay rate, to compensate for the lowered estimation in growth rates. The modified logistic model not only presented an improved goodness of fit in comparison to the original model but also led to an intriguing finding of a correlation between genome reduction and the decay rate. The decay effect seemed to be partially attributed to the decrease in cell size accompanied by a population increase and was medium dependent. In summary, the present study provides not only a better theoretical tool for the high-throughput studies on bacterial growth dynamics linking with experimental data using optical turbidity to the theoretical analysis with biological importance, but also a valuable insight for understanding the genome evolution and fitness increase in microbial life.

microbiology↗

bioRxiv2018

Reducing blood culture contamination using an initial specimen diversion device

False positive blood cultures result from contamination, consuming microbiological laboratory resources and causing unnecessary antibiotic treatment and lengthened hospitalizations. Skin sterilization has been shown to reduce contamination; however, bacteria do not only colonize the surface of human skin, but are also found in deeper tissues, requiring additional techniques to reduce contamination. An initial specimen diversion device diverts the initial 1-2 ml of blood so as to remove any potentially contaminated skin plug, thus potentially further reducing culture contamination. The device has been associated with a reduction in culture contamination over short study periods in certain populations. However, more study is needed to understand whether the effect continues over longer periods of time and in hospitalized patients. Thus, in this prospective, controlled pragmatic study, cultures were obtained from hospitalized patients using the initial specimen diversion device, with cultures taken using standard methods serving as control. In total, 671 blood cultures were obtained: 207 cultures were taken using an initial specimen diversion device, with 2 (1.0%) contaminated cultures and 464 cultures were taken without the device, with 24 (5.2%) contaminated cultures (p < 0.008). No significant difference was shown in the rate of true positive cultures. Thus, use of a diversion device was associated with reduced culture contamination in hospitalized patients over a six month period without concomitant reduction in true positive cultures. This intervention may result in a reduction in costs, antibiotic use and duration of hospitalization.

microbiology↗

bioRxiv2018

Evaluation of Two Sigma Transwab(R) Systems for Maintenance of Viability of Pathogenic Candida spp. Using the Clinical and Laboratory Standards Institute M40-2 A2 Standard

A key aspect to routine microbiology processes include the retrieval, transport and maintenance of specimens. Swab transport systems (STS) can be utilised for their low cost, ease of use and their ability to recover and maintain specimens over long durations. An increase in healthcare complications due to fungal infections raises the requirement for STS to efficiently recover and preserve pathogens of yeast origin. The Clinical and Laboratory Standards Institute (CLSI) M40-A2 protocol is used to assess the compliance of STS to a quality control standard but at present does not include the recovery of yeast. The aim of this study was to compare the results of two commercial STS and their ability to recover and maintain viability of five clinical and reference strains of Candida spp., including C. auris, when stored at room temperature and 4{degrees}C, over 48 h, using the qualitative roll plate method. Findings from this study indicate that the STS used in this study are suitable for the collection and maintenance of the Candida spp. tested, and is very suitable for the recovery of clinical C. auris.

microbiology↗

bioRxiv2018

Evaluation of whole genome sequencing for the identification and typing of Vibrio cholerae

Epidemiological and microbiological data on Vibrio cholerae isolated between 2004 and 2017 (n=836) and held in the Public Health England culture archive were reviewed. The traditional biochemical species identification and serological typing results were compared with the genome derived species identification and serotype for a sub-set of isolates (n=152). Of the 836 isolates, 750 (89.7%) were from faecal specimens, 206 (24.6%) belonged to serogroup O1 and seven (0.8%) were serogroup O139, and 792 (94.7%) isolates from patients reporting recent travel abroad, most commonly to India (n=209) and Pakistan (n=104). Of the 152 isolates of V. cholerae speciated by kmer identification, 149 (98.1%) were concordant with the traditional biochemical approach. Traditional serotyping results were 100% concordant with the whole genome sequencing (WGS) analysis for identification of serogroups O1 and O139 and Classical and El Tor biotypes. ctxA was detected in all isolates of V. cholerae O1 El Tor and O139 belonging to sequence type (ST) 69, and in V. cholerae O1 Classical variants belonging to ST73. A phylogeny of isolates belonging to ST69 from UK travellers clustered geographically, with isolates from India and Pakistan located on separate branches. Moving forward, WGS data from UK travellers will contribute to global surveillance programs, and the monitoring of emerging threats to public health and the global dissemination of pathogenic lineages. At the national level, these WGS data will inform the timely reinforcement of direct public health messaging to travellers and mitigate the impact of imported infections and the associated risks to public health.

microbiology↗

bioRxiv2018

The Metabolic Capability and Phylogenetic Diversity of Mono Lake During a Bloom of the Eukaryotic Phototroph Picocystis strain ML

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur naturally. In Spring of 2016 Mono Lake, a hyperalkaline lake in California, was near the height of a rare bloom of the algae Picocystis strain ML and at the apex of a multi-year long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics during an intense natural bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from surface to 25 m depth during June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis associated chloroplast were found at 40-50 % relative abundance, greater than values recorded previously. Despite the presence of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectible limits below 15 m depth, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, metagenomic and metatranscriptomic data suggested a loss of sulfate reducing microorganisms throughout the lakes water column. Gene transcripts associated with Photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production may occur at extremely low light levels at depth within the lake. Oxygenic photosynthesis under low light conditions, in the absence of potential grazing by the brine shrimp Artemia, may allow for a cryptic redox cycle to occur in an otherwise anoxic setting at depth in the lake with the following effects: enhanced productivity, reduced grazing pressure on Picocystis, and an exacerbation of bloom.\n\nIMPORTANCEMono Lake, California provides habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists about Mono Lake to understand how the microbial community responds to drought, bloom, and what genetic functions are lost in the water column. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest these microorganisms seek refuge at lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank which could restore the microbial community as a bloom subsides. Our work also sheds light on the activity of the halotolerant algae Picocystis strain ML during a bloom at Mono Lake, its ability to potentially produce oxygen via photosynthesis even under extreme low-light conditions, and how the remainder of the microbial community responds.

microbiology↗

bioRxiv2018

Next-Generation Sequencing Could be a Promising Diagnostic Approach for Pathogen Detection: Pathogenic Analysis of Pediatric Bacterial Meningitis by Next-Generation Sequencing Technology Directly from Cerebrospinal Fluid Specimens

BackgroundBacterial meningitis remains one of the major challenges in infectious diseases, leading to sequel in many cases. A prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bacterial meningitis. Although various targeted tests for cerebrospinal fluid (CSF) samples are available, it is a big problem for the identification of etiology of bacterial meningitis.\n\nMethodsHere we describe the use of unbiased sequence analyses by next-generation sequencing (NGS) technology for the identification of infectious microorganisms from CSF samples of pediatric bacterial meningitis patients in the Department of Infectious Diseases from Beijing Childrens Hospital.\n\nResultsIn total, we had 99 bacterial meningitis patients in our study, 55 (55.6%) of these were etiologically confirmed by clinical microbiology methods. Combined with NGS, 68 cases (68.7%) were etiologically confirmed. The main pathogens identified in this study were Streptococcus pneumoniae (n=29), group B streptococcus (n=15), Staphylococcus aureus (n=7), Escherichia coli (n=7). In addition, two cases with cytomegalovirus infection and one with Taenia saginata asiatica were confirmed by NGS.\n\nConclusionsNGS could be a promising alternative diagnostic approach for critically ill patients suffering from bacterial meningitis in pediatric population.\n\nSummaryWe conducted the study for the identification of microorganisms by next-generation sequencing directly from CSF samples of pediatric bacterial meningitis patients. And the study showed that NGS could be a promising alternative diagnostic approach for bacterial meningitis in pediatric population.

microbiology↗

bioRxiv2018

Understanding trivial challenges of microbial genomics: An assembly example

The perceived \"simplicity\" of bacterial genomics (these genomes are small and easy to assemble) feeds the decentralized state of the field where computational analysis standards have been slow to evolve. This situation has a historical explanation. In cases of human, mouse, fly, worm and other model organisms there have been large sustained multinational genome sequencing efforts and analysis consortia such as the 1,000 genomes, ENCODE, modENCODE, GTEx and others. These resulted in development and proliferation of common tools, workflows, and data standards. This is not the case in microbiology. After the development of highly parallel sequencing methodologies in mid-2000s bacterial genomes no longer required initiatives of such scale. The flipside of this is the extreme heterogeneity of approaches to many well established microbial genomic analysis problems such as genome assembly. While competition amongst different methods is good, we argue that the quality of data analyses will improve if cutting edge tools are more accessible and microbiologists become more computationally savvy. Here we use genome assembly as an example to highlight current challenges and to provide a possible solution.

microbiology↗

bioRxiv2018

Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease

Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The non-invasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.

microbiology↗

bioRxiv2018

The washing machine as a reservoir for transmission of ESBL-producing Klebsiella oxytoca in newborns

During the period from April 2012 to May 2013, 13 newborns and one child in a paediatric hospital ward in Germany were found to be colonised with a distinct clinical clone of an (extended spectrum {beta}-lactamase) (ESBL)-producing Klebsiella oxytoca. This clone was specific to this hospital and had not been previously isolated in Germany.\n\nA source-tracking analysis was carried out to identify the source and transmission pathways of the ESBL-producing K. oxytoca clone. A systematic environmental survey of the ward and an audit of the procedures for cleaning and disinfecting surfaces, instruments, incubators, and washing machines was performed. Microbiological samples were obtained from environmental surfaces. Risk factors were analysed for epidemiological linkage.\n\nIsolates of an ESBL-producing K. oxytoca were found in the detergent drawer and on the rubber door seal of a washing machine and in two sinks. These strains were typed by pulsed-field-gel-electrophoresis (PFGE) and compared with the isolates from the newborns and their clothing and found to be identical. The retrospective analysis demonstrated that only newborns who had worn clothes that had been washed in the washing machine were colonised with the identical clone. After the washing machine was taken out of use, no further cases were detected over the following 4-year period.\n\nWe conclude that washing machines are potential reservoirs and vectors for transmission of Enterobacteriaceae, and likely other bacteria.\n\nImportanceWashing machines should be further investigated as possible sites for horizontal gene transfer (ESBL-/carbapenemase-genes) and cross-contamination of clinically important Gram-negative strains. Particularly in the healthcare sector, the knowledge of possible (re-) contamination of laundry (patients clothes, staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and control nosocomial infections. To date, the potential of the washing machine as a source and vector of antibiotic-resistant gram-negative bacteria causing an outbreak in a clinical setting has not been investigated. This report shows that sampling of washing machines should be included in environmental audits associated with outbreak control management, and conditions for the laundering of baby 64 clothing should be reviewed.

microbiology↗

bioRxiv2018

The unsolved problem of otitis media in indigenous populations: A systematic review of upper respiratory and middle ear microbiota in indigenous children

BackgroundOtitis media (OM) imposes a great burden of disease in indigenous populations around the world, despite a variety of treatment and prevention programs. Improved understanding of the pathogenesis of OM in indigenous populations is required to advance treatment and reduce prevalence. We conducted a systematic review of the literature exploring upper airway and middle ear microbiota in relation to OM in indigenous children.\n\nMethodsPapers targeting microbiota in relation to OM in children <18 years indigenous to Australia, New Zealand, North America, and Greenland were sought. MEDLINE, CINAHL, EMBASE, Cochrane Library, and Informit databases were searched using key words. Two independent reviewers screened titles, abstracts, and then full-text papers against inclusion criteria according to PRISMA guidelines.\n\nResultsTwenty-five papers considering indigenous Australian, Alaskan and Greenlandic children were included. There were high rates of nasopharyngeal colonization with the three main otopathogens (Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis) in indigenous children with OM. Middle ear samples had lower rates of otopathogen detection, although detection rates increased when molecular methods were used. Pseudomonas aeruginosa and Staphylococcus aureus were commonly detected in middle ear discharge of children with chronic suppurative OM. There was significant heterogeneity between studies, particularly in microbiological methods, which were largely limited to culture-based detection of the main otopathogens.\n\nConclusionsThere are high rates of otopathogen colonization in indigenous children with OM. Chronic suppurative OM appears to be associated with a different microbial profile. Beyond the main otopathogens, the data are limited. Further research is required to explore the entire upper respiratory tract/ middle ear microbiota in relation to OM, with the inclusion of healthy indigenous peers as controls.

microbiology↗

bioRxiv2018

Infection of highly insecticide-resistant malaria vector Anopheles coluzzii with an environmentally friendly entomopathogenic bacteria Chromobacterium violaceum reduces its survival, blood feeding propensity and fecundity

This is now a concern that malaria eradication will not be achieved without the introduction of novel control tools. Microbiological control might be able to make a greater contribution to vector control in the future. Here, we studied the impact of Chromobacterium violaceum infections isolated from wild caught Anopheles gambiae s.l. mosquitoes in Burkina Faso on mosquito survival, blood feeding and fecundity propensy. C. violaceum kills pyrethroid resistant mosquitoes An. coluzzii (LT80 ~ at 108 bacteria cell/ml of sugar meal). Interestingly, this bacterium had other negative effects on mosquito lifespan by significantly reducing (~59%, P<0.001) the mosquito feeding willingness from day 4-post infection to 9-day post infection. Moreover, C.violaceum considerably jeopardized the mosquito egg laying and hatching of mosquitoes by ~77.93% and ~22 % respectively. Mosquitoes infected with C. violaceum also showed significantly higher retention rates of immature eggs and follicles. These data showed important entomopathogenic properties of Burkina Faso C. violaceum strains. However, additional studies as the sequencing of C. violaceum genome and the potential toxins secreted will certainly provide useful information render it a potential candidate for the biological control strategies of malaria.

microbiology↗

bioRxiv2018

Effects of endophyte infected tall fescue seed and red clover isoflavones on rumen microbial populations, fiber fermentation, and volatile fatty acids in vitro

Negative impacts of endophyte-infected Lolium arundinaceum (Darbyshire) (tall fescue) are responsible for over $2 billion in losses to livestock producers annually. While the influence of endophyte-infected tall fescue has been studied for decades, mitigation methods have not been clearly elucidated. Isoflavones found in Trifolium pretense (red clover) have been the subject of recent research regarding tall fescue toxicosis mitigation. Therefore, the aim of this study was to determine the effect of ergovaline and red clover isoflavones on rumen microbial populations, fiber degradation, and volatile fatty acids (VFA) in an in vitro system. Using a dose of 1.10 mg x L-1, endophyte-infected or endophyte-free tall fescue seed was added to ANKOM fiber bags with or without 2.19 mg of isoflavones in the form of a control, powder, or pulverized tablet, resulting in a 2 x 3 factorial arrangements of treatments. Measurements of pH, VFA, bacterial taxa, as well as the disappearance of neutral detergent fiber (aNDF), acid detergent fiber (ADF), and crude protein (CP) were taken after 48 h of incubation. aNDF disappearance values were significantly altered by seed type (P = 0.003) and isoflavone treatment (P = 0.005), and ADF disappearance values were significantly different in a seed x isoflavone treatment interaction (P [≤] 0.05). A seed x isoflavone treatment interaction was also observed with respect to CP disappearance (P [≤] 0.05). Seventeen bacterial taxa were significantly altered by seed x isoflavone treatment interaction groups (P [≤] 0.05), six bacterial taxa were increased by isoflavones (P [≤] 0.05), and eleven bacterial taxa were altered by seed type (P [≤] 0.05). Due to the beneficial effect of isoflavones on tall fescue seed fiber degradation, these compounds may be viable options for mitigating fescue toxicosis. Further research should be conducted to determine physiological implications as well as microbiological changes in vivo.

microbiology↗

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↗