Effect of Incubation Temperature on Antibiotic Resistance and Virulence Factors of Acinetobacter baumannii ATCC 17978.

Acinetobacter baumannii is a notorious opportunistic pathogen that is prevalent mainly in hospital settings. The ability of A. baumannii to adapt and to survive in a range of environments has been a key factor for its persistence and success as an opportunistic pathogen. In this study, we investigated the effect of temperature on the clinically relevant phenotypes displayed by A. baumannii at 37°C and 28°C. Surface-associated motility was significantly reduced at 28°C, while biofilm formation on plastic surfaces was increased at 28°C. Decreased susceptibility to aztreonam and increased susceptibility to trimethoprim-sulfamethoxazole were observed at 28°C. No differences in virulence, as assayed in a Galleria mellonella model, were observed. Proteomic analysis showed differential expression of 629 proteins, of which 366 were upregulated and 263 were downregulated at 28°C. Upregulation of the Csu and iron uptake proteins at 28°C was a key finding for understanding some of the phenotypes displayed by A. baumannii at 28°C.

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract.

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.

MicroRNA abundance is altered in synaptoneurosomes during prion disease.

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

Molecular Signatures of Immune Activation and Epithelial Barrier Remodeling Are Enhanced during the Luteal Phase of the Menstrual Cycle: Implications for HIV Susceptibility.

UNLABELLED: The variable infectivity and transmissibility of HIV/SHIV has been recently associated with the menstrual cycle, with particular susceptibility observed during the luteal phase in nonhuman primate models and ex vivo human explant cultures, but the mechanism is poorly understood. Here, we performed an unbiased, mass spectrometry-based proteomic analysis to better understand the mucosal immunological processes underpinning this observed susceptibility to HIV infection. Cervicovaginal lavage samples (n = 19) were collected, characterized as follicular or luteal phase using days since last menstrual period, and analyzed by tandem mass spectrometry. Biological insights from these data were gained using a spectrum of computational methods, including hierarchical clustering, pathway analysis, gene set enrichment analysis, and partial least-squares discriminant analysis with LASSO feature selection. Of the 384 proteins identified, 43 were differentially abundant between phases (P < 0.05, ≥2-fold change). Cell-cell adhesion proteins and antiproteases were reduced, and leukocyte recruitment (interleukin-8 pathway, P = 1.41E-5) and extravasation proteins (P = 5.62E-4) were elevated during the luteal phase. LASSO/PLSDA identified a minimal profile of 18 proteins that best distinguished the luteal phase. This profile included cytoskeletal elements and proteases known to be involved in cellular movement. Gene set enrichment analysis associated CD4(+) T cell and neutrophil gene set signatures with the luteal phase (P < 0.05). Taken together, our findings indicate a strong association between proteins involved in tissue remodeling and leukocyte infiltration with the luteal phase, which may represent potential hormone-associated mechanisms of increased susceptibility to HIV. IMPORTANCE: Recent studies have discovered an enhanced susceptibility to HIV infection during the progesterone-dominant luteal phase of the menstrual cycle. However, the mechanism responsible for this enhanced susceptibility has not yet been determined. Understanding the source of this vulnerability will be important for designing efficacious HIV prevention technologies for women. Furthermore, these findings may also be extrapolated to better understand the impact of exogenous hormone application, such as the use of hormonal contraceptives, on HIV acquisition risk. Hormonal contraceptives are the most widely used contraceptive method in sub-Saharan Africa, the most HIV-burdened area of the world. For this reason, research conducted to better understand how hormones impact host immunity and susceptibility factors important for HIV infection is a global health priority.

Complement opsonization of HIV-1 results in decreased antiviral and inflammatory responses in immature dendritic cells via CR3.

Immature dendritic cells (iDCs) in genital and rectal mucosa may be one of the first cells to come into contact with HIV-1 during sexual transmission of virus. HIV-1 activates the host complement system, which results in opsonization of virus by inactivated complement fragments, for example, iC3b. We investigated antiviral and inflammatory responses induced in human iDCs after exposure to free HIV-1 (F-HIV), complement-opsonized HIV-1 (C-HIV), and complement and Ab-opsonized HIV-1 (CI-HIV). F-HIV gave rise to a significantly higher expression of antiviral factors such as IFN-ß, myxovirus resistance protein A, and IFN-stimulated genes, compared with C-HIV and CI-HIV. Additionally, F-HIV induced inflammatory factors such as IL-1ß, IL-6, and TNF-α, whereas these responses were weakened or absent after C-HIV or CI-HIV exposure. The responses induced by F-HIV were TLR8-dependent with subsequent activation of IFN regulatory factor 1, p38, ERK, PI3K, and NF-κB pathways, whereas these responses were not induced by C-HIV, which instead induced activation of IFN regulatory factor 3 and Lyn. This modulation of TLR8 signaling was mediated by complement receptor 3 and led to enhanced infection. The impact that viral hijacking of the complement system has on iDC function could be an important immune evasion mechanism used by HIV-1 to establish infection in the host.

Highly Pathogenic H5N1 and Novel H7N9 Influenza A Viruses Induce More Profound Proteomic Host Responses than Seasonal and Pandemic H1N1 Strains.

Influenza A viruses (IAV) are important human and animal pathogens with potential for causing pandemics. IAVs exhibit a wide spectrum of clinical illness in humans, from relatively mild infections by seasonal strains to acute respiratory distress syndrome during infections with some highly pathogenic avian influenza (HPAI) viruses. In the present study, we infected A549 human cells with seasonal H1N1 (sH1N1), 2009 pandemic H1N1 (pdmH1N1), or novel H7N9 and HPAI H5N1 strains. We used multiplexed isobaric tags for relative and absolute quantification to measure proteomic host responses to these different strains at 1, 3, and 6 h post-infection. Our analyses revealed that both H7N9 and H5N1 strains induced more profound changes to the A549 global proteome compared to those with low-pathogenicity H1N1 virus infection, which correlates with the higher pathogenicity these strains exhibit at the organismal level. Bioinformatics analysis revealed important modulation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response in infection. Cellular fractionation and Western blotting suggested that the phosphorylated form of NRF2 is not imported to the nucleus in H5N1 and H7N9 virus infections. Fibronectin was also strongly inhibited in infection with H5N1 and H7N9 strains. This is the first known comparative proteomic study of the host response to H7N9, H5N1, and H1N1 viruses and the first time NRF2 is shown to be implicated in infection with highly pathogenic strains of influenza.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Based Peptide Mass Fingerprinting.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

Tatumella saanichensis sp. nov., isolated from a cystic fibrosis patient.

Polyphasic taxonomic analysis was performed on a clinical isolate (NML 06-3099T) from a cystic fibrosis patient, including whole-genome sequencing, proteomics, phenotypic testing, electron microscopy, chemotaxonomy and a clinical investigation. Comparative whole-genome sequence analysis and multilocus sequence analysis (MLSA) between Tatumella ptyseos ATCC 33301T and clinical isolate NML 06-3099T suggested that the clinical isolate was closely related to, but distinct from, the species T. ptyseos. By 16S rRNA gene sequencing, the clinical isolate shared 98.7 % sequence identity with T. ptyseos ATCC 33301T. A concatenate of six MLSA loci (totalling 4500 bp) revealed < 93.9 % identity between T. ptyseos ATCC 33301T, other members of the genus and the clinical isolate. A whole-genome sequence comparison between NML 06-3099T and ATCC 33301T determined that the average nucleotide identity was 76.24 %. The overall DNA G+C content of NML 06-3099T was 51.27 %, consistent with members of the genus Tatumella. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis, NML 06-3099T had a genus-level match, but not a species-level match, to T. ptyseos. By shotgun proteomics, T. ptyseos ATCC 33301T and NML 06-3099T were found to have unique proteomes. The two strains had similar morphologies and multiple fimbriae, as observed by transmission electron microscopy, but were distinguishable by phenotypic testing. Cellular fatty acids found were typical for members of the Enterobacteriaceae. NML 06-3099T was susceptible to commonly used antibiotics. Based on these data, NML 06-3099T represents a novel species in the genus Tatumella, for which the name Tatumella saanichensis sp. nov. is proposed (type strain NML 06-3099T = CCUG 55408T = DSM 19846T).

Complement opsonization of HIV-1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells.

Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and ß7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and ß7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery.

Sequence-level and dual-phase identification of Salmonella flagellum antigens by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Forty-three reference strains involving the 24 most common serovars of Salmonella enterica were examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.

A systems biology examination of the human female genital tract shows compartmentalization of immune factor expression.

Many mucosal factors in the female genital tract (FGT) have been associated with HIV susceptibility, but little is known about their anatomical distribution in the FGT compartments. This study comprehensively characterized global immune factor expression in different tissue sites of the lower and upper FGT by using a systems biology approach. Tissue sections from the ectocervix, endocervix, and endometrium from seven women who underwent hysterectomy were analyzed by a combination of quantitative mass spectrometry and immunohistochemical staining. Of the >1,000 proteins identified, 281 were found to be differentially abundant in different tissue sites. Hierarchical clustering identified four major functional pathways distinguishing compartments, including innate immune pathways (acute-phase response, LXR/RXR) and development (RhoA signaling, gluconeogenesis), which were enriched in the ectocervix/endocervix and endometrium, respectively. Immune factors important for HIV susceptibility, including antiproteases, immunoglobulins, complement components, and antimicrobial factors, were most abundant in the ectocervix/endocervix, while the endometrium had a greater abundance of certain factors that promote HIV replication. Immune factor abundance is heterogeneous throughout the FGT and shows unique immune microenvironments for HIV based on the exposure site. This may have important implications for early events in HIV transmission and site-specific susceptibility to HIV in the FGT.

The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts.

BACKGROUND: The presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays. RESULTS: Quantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether there was a corresponding difference in protein expression levels. Initial experiments indicated that about 2% of the total proteins characterized were expressed at different levels in isolates with or without the prophage. Some of these proteins regulated by the presence of CJIE1 were associated with virulence or regulatory functions. Additional experiments were conducted using C. jejuni isolates with and without CJIE1 grown on four different media: Mueller Hinton (MH) media containing blood; MH media containing 0.1% sodium deoxycholate, which is thought to result in increased expression of virulence proteins; MH media containing 2.5% Oxgall; and MHwithout additives. These experiments provided further evidence that CJIE1 affected protein expression, including virulence-associated proteins. They also demonstrated a general bile response involving a majority of the proteome and clearly showed the induction of almost all proteins known to be involved with iron acquisition. The data have been deposited to the ProteomeXchange with identifiers PXD000798, PXD000799, PXD000800, and PXD000801. CONCLUSION: The presence of the CJIE1 prophage was associated with differences in protein expression levels under different conditions. Further work is required to determine what genes are involved in causing this phenomenon.

High pH reversed-phase chromatography as a superior fractionation scheme compared to off-gel isoelectric focusing for complex proteome analysis.

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off-gel IEF (OG-IEF) and high pH RP (Hp-RP) column chromatography have both been successfully utilized as a first-dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12-fraction replicate analysis, Hp-RP resulted in more peptides and proteins identified than OG-IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp-RP. This leads to a more uniform distribution of total and unique peptides for Hp-RP across all fractions collected. These results suggest that fractionation by Hp-RP over OG-IEF is the better choice for typical complex proteome analysis.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 ΔtolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and ΔtolA OMVs, where ΔtolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and ΔtolA OMVs confirmed that ΔtolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies.

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

Influence of dapivirine vaginal ring use on cervicovaginal immunity and functional microbiome in adolescent girls.

OBJECTIVE: The antiretroviral-based dapivirine vaginal ring reduced HIV risk among women in phase III clinical trials. However, limited data exists on the impact of dapivirine on the vaginal microenvironment in adolescents. DESIGN: A comprehensive metaproteomics approach was used to assess host proteome and microbiome changes in cervicovaginal mucus with dapivirine ring use in adolescents enrolled in the MTN-023/IPM 030 (MTN-023) trial. METHODS: Participants were randomized 3 : 1 to use dapivirine or placebo vaginal rings monthly for 6 months. Cervicovaginal samples from a subset of 35 participants (8 placebo, 27 dapivirine) were analyzed. RESULTS: Mass spectrometry analysis identified 405 human and 2467 bacterial proteins belonging to 15 unique genera. The host proteome belonged to many functional pathways primarily related to inflammation. When stratified by study treatment arm, 18 (4.4%) and 28 (6.9%) human proteins were differentially abundant (adjusted P < 0.05) between baseline and follow-up in the placebo and dapivirine arms, respectively. The vaginal microbiome was predominantly composed of Lactobacillus, Gardnerella, and Prevotella. Although bacterial taxa did not differ by arm or change significantly, Lactobacillus crispatus increased (P < 0.001) and Lactobacillus iners decreased (P < 0.001) during the 6-month follow-up. There were no significant differences in bacterial functions by arm or time in the trial. Protected vaginal sex significantly associated with decreased neutrophil inflammatory biomarkers and may be associated with changes in bacterial taxa and metabolism. CONCLUSION: Condom use may associate with differences to inflammation and bacterial function but dapivirine ring use does not, thereby supporting the mucosal safety profile of this vaginal ring for adolescents.

Phenotypic and Multi-Omics Characterization of Escherichia coli K-12 Adapted to Chlorhexidine Identifies the Role of MlaA and Other Cell Envelope Alterations Regulated by Stress Inducible Pathways in CHX Resistance.

Chlorhexidine (CHX) is an essential medicine used as a topical antiseptic in skin and oral healthcare treatments. The widespread use of CHX has increased concerns regarding the development of antiseptic resistance in Enterobacteria and its potential impact on cross-resistance to other antimicrobials. Similar to other cationic antiseptics, resistance to CHX is believed to be driven by three membrane-based mechanisms: lipid synthesis/transport, altered porin expression, and increased efflux pump activity; however, specific gene and protein alterations associated with CHX resistance remain unclear. Here, we adapted Escherichia coli K-12 BW25113 to increasing concentrations of CHX to determine what phenotypic, morphological, genomic, transcriptomic, and proteomic changes occurred. We found that CHX-adapted E. coli isolates possessed no cross-resistance to any other antimicrobials we tested. Scanning electron microscopy imaging revealed that CHX adaptation significantly altered mean cell widths and lengths. Proteomic analyses identified changes in the abundance of porin OmpF, lipid synthesis/transporter MlaA, and efflux pump MdfA. Proteomic and transcriptomic analyses identified that CHX adaptation altered E. coli transcripts and proteins controlling acid resistance (gadE, cdaR) and antimicrobial stress-inducible pathways Mar-Sox-Rob, stringent response systems. Whole genome sequencing analyses revealed that all CHX-resistant isolates had single nucleotide variants in the retrograde lipid transporter gene mlaA as well as the yghQ gene associated with lipid A transport and synthesis. CHX resistant phenotypes were reversible only when complemented with a functional copy of the mlaA gene. Our results highlight the importance of retrograde phospholipid transport and stress response systems in CHX resistance and the consequences of prolonged CHX exposure.

Pregnancy associates with alterations to the host and microbial proteome in vaginal mucosa.

PROBLEM: Pregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood. METHOD OF STUDY: Here, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women. RESULTS: Mass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P < .05) between pregnant and non-pregnant women, including proteins involved in angiogenesis (P = 3.36E-3), cell movement of phagocytes (P = 1.34E-6), and permeability of blood vessels (P = 1.27E-4). The major bacterial genera identified were Lactobacillus, Gardnerella, Prevotella, Megasphaera, and Atopobium. Pregnant women had higher levels of Lactobacillus species (P = .017) compared with non-pregnant women. Functional pathway analysis indicated that pregnancy associated with changes to bacterial metabolic pathway involved in energy metabolism, which were increased in pregnant women (P = .035). CONCLUSION: Overall, pregnant women showed differences in the cervicovaginal proteome and microbiome that may be important for HIV infection risk.

Detection of Antimicrobial Resistance Using Proteomics and the Comprehensive Antibiotic Resistance Database: A Case Study.

PURPOSE: Antimicrobial resistance (AMR), especially multidrug resistance, is one of the most serious global threats facing public health. The authors proof-of-concept study assessing the suitability of shotgun proteomics as an additional approach to whole-genome sequencing (WGS) for detecting AMR determinants. EXPERIMENTAL DESIGN: Previously published shotgun proteomics and WGS data on four isolates of Campylobacter jejuni are used to perform AMR detection by searching the Comprehensive Antibiotic Resistance Database, and their detection ability relative to genomics screening and traditional phenotypic testing measured by minimum inhibitory concentration is assessed. RESULTS: Both genomic and proteomic approaches identify the wild-type and variant molecular determinants responsible for resistance to tetracycline and ciprofloxacin, in agreement with phenotypic testing. In contrast, the genomic method identifies the presence of the ß-lactamase gene, blaOXA-61 , in three isolates. However, its corresponding protein product is detected in only a single isolate, consistent with results obtained from phenotypic testing.