Functional OmpA of Salmonella Typhimurium provides protection from lysosomal degradation and inhibits autophagic processes in macrophages.

Our previous study showed that OmpA-deficient Salmonella Typhimurium (STM) failed to retain LAMP-1, quit Salmonella-containing vacuole (SCV) and escaped to the host cytosol. Here we show that the cytosolic population of STM ΔompA sequestered autophagic markers, syntaxin17 and LC3B in a sseL-dependent manner and initiated lysosomal fusion. Moreover, inhibition of autophagy using bafilomycinA1 restored its intracellular proliferation. Ectopic overexpression of OmpA in STM ΔsifA restored its vacuolar niche and increased interaction of LAMP-1, suggesting a sifA-independent role of OmpA in maintaining an intact SCV. The OmpA extracellular loops impaired the LAMP-1 recruitment to SCV and caused bacterial release into the cytosol of macrophages, but unlike STM ΔompA, they retained their outer membrane stability and didn't activate the lysosomal degradation pathway aiding in their intra-macrophage survival. Finally, OmpA extracellular loop mutations protected the cytosolic STM ΔsifA from the lysosomal surveillance, revealing a unique OmpA-dependent strategy of STM for its intracellular survival.

Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda.

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Anti-OmpA antibodies as potential inhibitors of Acinetobacter baumannii biofilm formation, adherence to, and proliferation in A549 human alveolar epithelial cells.

Outer membrane protein A (OmpA) is a critical virulence factor in Acinetobacter baumannii, influencing adhesion, biofilm formation, host immune response, and host cell apoptosis. We investigated the invasion of A549 alveolar epithelial cells by A. baumannii and examined how anti-OmpA antibodies impact these interactions. OmpA was expressed and purified, inducing anti-OmpA antibodies in BALB/c mice. The potential toxicity of OmpA was evaluated in mice by analyzing histology from six organs. A549 cells were exposed to A. baumannii strains 19606 and a clinical isolate. Using cell culture and light microscopy, we scrutinized the effects of anti-OmpA sera on serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells. The viability of A549 cells was assessed upon exposure to live A. baumannii and anti-OmpA sera. OmpA-induced antibody demonstrated potent bactericidal effects on both strains of A. baumannii. Both strains formed biofilms, which were reduced by anti-OmpA serum, along with decreased bacterial adherence, internalization, and proliferation in A549 cells. Anti-OmpA serum improved the survival of A549 cells post-infection. Pre-treatment with cytochalasin D hindered bacterial internalization, highlighting the role of actin polymerization in invasion. Microscopic examination revealed varied interactions encompassing adherence, apoptosis, membrane alterations, vacuolization, and damage. A549 cells treated with anti-OmpA serum exhibited improved structures and reduced damage. The findings indicate that A. baumannii can adhere to and proliferate within epithelial cells with OmpA playing a pivotal role in these interactions, and the complex nature of these interactions shapes the intricate course of A. baumannii infection in host cells.

Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles.

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Anorectal lymphogranuloma venereum among men who have sex with men: a 3-year nationwide survey, France, 2020 to 2022.

BackgroundIn France, lymphogranuloma venereum (LGV) testing switched from universal to selective testing in 2016.AimTo investigate changes in LGV-affected populations, we performed a nationwide survey based on temporarily reinstated universal LGV testing from 2020 to 2022.MethodsEach year, during three consecutive months, laboratories voluntarily sent anorectal Chlamydia trachomatis-positive samples from men and women to the National Reference Centre for bacterial sexually transmitted infections. We collected patients' demographic, clinical and biological data. Genovars L of C. trachomatis were detected using real-time PCR. In LGV-positive samples, the ompA gene was sequenced.ResultsIn 2020, LGV positivity was 12.7% (146/1,147), 15.2% (138/907) in 2021 and 13.3% (151/1,137) in 2022 (p > 0.05). It occurred predominantly in men who have sex with men (MSM), with rare cases among transgender women. The proportion of HIV-negative individuals was higher than that of those living with HIV. Asymptomatic rectal LGV increased from 36.1% (44/122) in 2020 to 52.4% (66/126) in 2022 (p = 0.03). Among users of pre-exposure prophylaxis (PrEP), LGV positivity was 13.8% (49/354) in 2020, 15.6% (38/244) in 2021 and 10.9% (36/331) in 2022, and up to 50% reported no anorectal symptoms. Diversity of the LGV ompA genotypes in the Paris region increased during the survey period. An unexpectedly high number of ompA genotype L1 variant was reported in 2022.ConclusionIn rectal samples from MSM in France, LGV positivity was stable, but the proportion of asymptomatic cases increased in 2022. This underscores the need of universal LGV testing and the importance of continuous surveillance.

Immunoinformatics design of multi-epitope vaccine using OmpA, OmpD and enterotoxin against non-typhoidal salmonellosis.

BACKGROUND: Non-typhoidal Salmonella (NTS) is one of the important bacteria that cause foodborne diseases and invasive infections in children and elderly people. Since NTS infection is difficult to control due to the emergence of antibiotic-resistant species and its adverse effect on immune response, the development of a vaccine against NTS would be necessary. This study aimed to develop a multi-epitope vaccine against the most prevalent serovars of NTS (Salmonella Typhimurium, Salmonella Enteritidis) using an immunoinformatics approach and targeting OmpA, OmpD, and enterotoxin (Stn). RESULTS: Initially, the B cell and T cell epitopes were predicted. Then, epitopes and suitable adjuvant were assembled by molecular linkers to construct a multi-epitope vaccine. The computational tools predicted the tertiary structure, refined the tertiary structure and validated the final vaccine construct. The effectiveness of the vaccine was evaluated via molecular docking, molecular dynamics simulation, and in silico immune simulation. The vaccine model had good binding affinity and stability with MHC-I, MHC-II, and toll-like receptors (TLR-1, 2, 4) as well as activation of T cells, IgM, IgG, IFN-γ and IL-2 responses. Furthermore, after codon optimization of the vaccine sequence, this sequence was cloned in E. coli plasmid vector pET-30a (+) within restriction sites of HindIII and BamHI. CONCLUSIONS: This study, for the first time, introduced a multi-epitope vaccine based on OmpA, OmpD and enterotoxin (Stn) of NTS that could stimulate T and B cell immune responses and produced in the prokaryotic system. This vaccine was validated in-silico phase which is an essential study to reduce challenges before in vitro and in vivo studies.

Overexpression of Pasteurella multocida OmpA induces transcriptional changes and its possible implications for the macrophage polarization.

Pasteurella multocida (P. multocida) is a highly infectious, zoonotic pathogen. Outer membrane protein A (OmpA) is an important virulence component of the outer membrane of P. multocida. OmpA mediates bacterial biofilm formation, eukaryotic cell infection, and immunomodulation. It is unclear how OmpA affects the host immune response. We estimated the role of OmpA in the pathogenesis of P. multocida by investigating the effect of OmpA on the immune cell transcriptome. Changes in the transcriptome of rat alveolar macrophages (NR8383) upon overexpression of P. multocida OmpA were demonstrated. A model cell line for stable transcription of OmpA was constructed by infecting NR8383 cells with OmpA-expressing lentivirus. RNA was extracted from cells and sequenced on an Illumina HiSeq platform. Key gene analysis of genes in the RNA-seq dataset were performed using various bioinformatics methods, such as gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, Gene Set Enrichment Analysis, and Protein-Protein Interaction Analysis. Our findings revealed 1340 differentially expressed genes. Immune-related pathways that were significantly altered in rat alveolar macrophages under the effect of OmpA included focal adhesion, extracellular matrix and vascular endothelial growth factor signaling pathways, antigen processing and presentation, nucleotide oligomerization domain-like receptor and Toll-like receptor signaling pathways, and cytokine-cytokine receptor interaction. The key genes screened were Vegfa, Igf2r, Fabp5, P2rx1, C5ar1, Nedd4l, Gas6, Cxcl1, Pf4, Pdgfb, Thbs1, Col7a1, Vwf, Ccl9, and Arg1. Data of associated pathways and altered gene expression indicated that OmpA might cause the conversion of rat alveolar macrophages to M2-like. The related pathways and key genes can serve as a reference for OmpA of P. multitocida and host interaction mechanism studies.

Multilocus VNTR analysis-ompA typing of Chlamydia trachomatis isolates in Tehran, Iran.

INTRODUCTION: This study is the first to describe the genetic diversity of C. trachomatis strains derived from patients with signs and symptoms of genitourinary infections admitted to Tehran health centers and hospitals using the high-resolution genotyping method, multilocus variable-number tandem-repeat analysis with ompA sequencing (MLVA)-ompA. METHODS: One hundred and sixty-seven urogenital specimens were collected from October 2019 to July 2020. Specimens were inoculated to cell culture and examined for the presence of C. trachomatis isolates by microscopic valuation. Out of 167 samples, 19 (11.3%) viable C. trachomatis organisms were isolated in cell culture. Eighteen isolates were successfully genotyped by MLVA-ompA analysis. RESULTS: The most prevalent ompA genotypes were E, D, F and G, comprising 42%, 26.3% and 21% and 10.5% of isolates, respectively. Other genotypes were not detected from any of the samples. Out of the 18 fully genotyped isolates, 10 different MLVA-ompA genotypes were obtained. The most prevalent MLVA-ompA genotypes were 8.6.1-E (33.3%) and 8.5.2-D (16.6%). Genotype 8.6.1-E was common in both females and males. CONCLUSIONS: Our results showed that MLVA-ompA analysis was more discriminatory than ompA typing alone and, therefore, a suitable complement to ompA. Using this method, dominant genotypes in the community and transmission patterns in sexual networks could be identified. The high diversity of C. trachomatis strains in Tehran may be due to the low level of public health and awareness, and future studies are needed.

Targeting outer membrane protein A (OmpA) - inhibitory effect of 2'-hydroxychalcone derivatives on Acinetobacter baumannii and Candida albicans dual-species biofilm formation.

Biofilm production facilitates microbial colonization of wounds and catheters. Acinetobacter baumannii produces high levels of biofilm and causes difficult-to-treat nosocomial infections. Candida albicans is another strong biofilm producer which may facilitate A. baumannii adhesion by providing hyphae-mediated OmpA-binding sites. Here we tested the potential of 2'-hydroxychalcones to inhibit dual-species biofilm production of A. baumannii and Candida spp., and further predicted the mechanism of structure-related difference in activity. The results suggest that 2'-hydroxychalcones exhibit potent activity against Candida spp./A. baumannii dual-species biofilm production. Particularly active was trifluoromethyl-substituted derivative (p-CF3), which decreased C. albicans/A. baumannii biomass produced on vein-indwelling parts of the central venous catheterization set by up to 99%. Further, higher OmpA-binding affinity was also calculated for p-CF3, which together with demonstrated significant ompA-downregulating activity, suggests that superior antibiofilm activity of this chalcone against the tested dual-species community of A. baumannii is mediated through the OmpA.

Electrochemical Detection of ompA Gene of C. sakazakii Based on Glucose-Oxidase-Mimicking Nanotags of Gold-Nanoparticles-Doped Copper Metal-organic Frameworks.

The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA, tDNA) gene of Cronobacter sakazakii (C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10-15 to 1.0 × 10-9 mol L-1 to tDNA with a detection limit (LOD) of 0.42 fmol L-1 under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection of ompA gene segments in total DNA extracts from C. sakazakii with a broad linear range of 5.4-5.4 × 105 CFU mL-1 and a LOD of 0.35 CFU mL-1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection of ompA gene segments in real samples with recovery between 87.5% and 107.3%.

Genetic Characterization of Non-Lymphogranuloma venereum Chlamydia trachomatis Indicates Distinct Infection Transmission Networks in Spain.

Chlamydia trachomatis infection is an important public health problem. Our objective was to assess the dynamics of the transmission of this infection, analysing the distribution of circulating ompA genotypes and multilocus sequence types of C. trachomatis in Spain as a function of clinical and epidemiological variables. During 2018 and 2019, we genetically characterized C. trachomatis in tertiary hospitals in six areas in Spain (Asturias, Barcelona, Gipuzkoa, Mallorca, Seville and Zaragoza), with a catchment population of 3.050 million people. Genotypes and sequence types were obtained using polymerase chain reaction techniques that amplify a fragment of the ompA gene, and five highly variable genes (hctB, CT058, CT144, CT172 and pbpB), respectively. Amplicons were sequenced and phylogenetic analysis was conducted. We obtained genotypes in 636/698 cases (91.1%). Overall and by area, genotype E was the most common (35%). Stratifying by sex, genotypes D and G were more common among men, and genotypes F and I among women (p < 0.05). Genotypes D, G and J were more common in men who have sex with men (MSM) than in men who have sex with women (MSW), in whom the most common genotypes were E and F. The diversity index was higher in sequence typing (0.981) than in genotyping (0.791), and the most common sequence types were ST52 and ST108 in MSM, and ST30, ST148, ST276 and ST327 in MSW. Differences in genotype distribution between geographical areas were attributable to differences in population characteristics. The transmission dynamics varied with sexual behaviour: the predominant genotypes and most frequent sequence types found in MSM were different to those detected in MSW and women.

Therapeutic strategies against potential antibiofilm targets of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is the causative agent of various hospital-acquired infections. Biofilm formation is one of the various antimicrobial resistance (AMR) strategies and is associated with high mortality and morbidity. Hence, it is essential to review the potential antibiofilm targets in A. baumannii and come up with different strategies to combat these potential targets. This review covers different pathways involved in the regulation of biofilm formation in A. baumannii like quorum sensing (QS), cyclic-di-GMP signaling, two-component system (TCS), outer-membrane protein (ompA), and biofilm-associated protein (BAP). A newly discovered mechanism of electrical signaling-mediated biofilm formation and contact-dependent biofilm modulation has also been discussed. As biofilm formation and its maintenance in A. baumannii is facilitated by these potential targets, the detailed study of these targets and pathways can bring light to different therapeutic strategies such as anti-biofilm peptides, natural and synthetic molecule inhibitors, QS molecule degrading enzymes, and other strategies. These strategies may help in suppressing the lethality of biofilm-mediated infections. Targeting essential proteins/targets which are crucial for biofilm formation and regulation may render new therapeutic strategies that can aid in combating biofilm, thus reducing the recalcitrant infections and morbidity associated with the biofilm of A. baumannii.

Mosaic Evolution of Beta-Barrel-Porin-Encoding Genes in Escherichia coli.

Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid adaptation, although it remains unknown whether it is a common evolutionary mechanism for the porins or outer membrane proteins in Gram-negative bacteria. In this study, we investigated the beta-barrel (ß-barrel) porin-encoding genes in Escherichia coli that were reported under positive Darwinian selection. Besides fhuA that was found with ingenic local recombination previously, we identified four other genes, i.e., lamB, ompA, ompC, and ompF, all showing the similar mosaic evolution patterns. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of bacteriophages. For each of the porin families, mosaic recombination leads to unique combinations of the variable regions with different sequence patterns, generating diverse protein groups. Structural modeling indicated a conserved global topology among the different porins, with the extracellular surface varying a lot due to individual or combinatorial variable regions. The conservation of global tertiary structure would ensure the channel activity, while the wide diversity of variable regions may represent selection to avoid the invasion of phages, antibiotics or immune surveillance factors. Our study identified multiple bacterial porin genes with mosaic evolution. We hypothesize that this could be generalized strategy for outer membrane proteins to both maintain normal life processes and evade the attack of unfavored factors rapidly. IMPORTANCE Microevolution studies can disclose more elaborate evolutionary mechanisms of genes, appearing especially important for genes with multifaceted function such as those encoding outer membrane proteins. However, in most cases, the gene is considered as a whole unit, and the evolutionary patterns are disclosed. Here, we report that multiple bacterial porin proteins follow mosaic evolution, with local ingenic recombination combined with spontaneous mutations based on positive Darwinian selection, and conservation for most structural regions. This could represent a common mechanism for bacterial outer membrane proteins. The variable regions within each porin family showed large coincidence with the binding sites of bacteriophages, antibiotics, and immune factors and therefore would represent effective targets for the development of new antibacterial agents or vaccines.

Combination of BauA and OmpA elicit immunoprotection against Acinetobacter baumannii in a murine sepsis model.

AIMS: Acinetobacter baumannii causes severe nosocomial infections and is a difficult-to-treat pathogen due to the development of multidrug-resistant (MDR) strains. Vaccines and antibody therapy represent alternative promising strategies for the control of infections caused by A. baumannii or its MDR strains. OmpA and BauA have been assigned as protective antigens. However, the efficacy of the combination of these antigens is yet to be investigated. In this study, we targeted two critical antigens of A. baumannii (BauA and OmpA) to enhance immunoprotecting against A. baumannii. METHODS AND RESULTS: The recombinant BauA and OmpA were expressed and purified. The purified proteins were administered to BALB/c mice alone and in combination. Immune sera were assessed against BauA, OmpA and two constructs harboring immunogenic loops of these antigen. The mice were then challenged with a clinical isolate of A. baumannii. Indirect ELISA confirmed significant antibody rise to the antigens. The immunogenic loops were detected in the hybrid construct. The specific sera detected OmpA, BauA and constructs harboring immunogenic loops of these antigen with different affinities. A significant decrease in the bacterial loads was noted in the spleen, liver, and lungs of the immunized mice groups. However, the group received combination of BauA and OmpA showed lower bacterial burden in the spleen and liver. CONCLUSIONS: Combination of BauA and OmpA enhances immunoprotection against A. baumannii infections.

Mannheimia haemolytica OmpH binds fibrinogen and fibronectin and participates in biofilm formation.

Mannheimia haemolytica is the causal agent of the shipping fever in bovines and produces high economic losses worldwide. This bacterium possesses different virulence attributes to achieve a successful infection. One of the main virulence factors expressed by a pathogen is through adhesion molecules; however, the components participating in this process are not totally known. The present work identified a M. haemolytica 41 kDa outer membrane protein (Omp) that participates in bacterial adhesion. This protein showed 100% identity with the OmpH from M. haemolytica as determined by mass spectrometry and it interacts with sheep fibrinogen. The 41 kDa M. haemolytica OmpH interacts with bovine monocytes; a previous incubation of M. haemolytica with a rabbit hyperimmune serum against this Omp diminished 45% cell adhesion. The OmpH was recognized by serum from bovines affected by acute or chronic pneumonia, indicating its in vivo expression; moreover, it showed immune cross-reaction with the serum of rabbit infected with Pasteurella multocida. The OmpH is present in biofilms and previous incubation of M. haemolytca with rabbit serum against this protein diminished biofilm, indicating this protein's participation in biofilm formation. M. haemolytica OmpH is proposed as a relevant immunogen in bovine pneumonia protection.

Evaluation the reactivity of a peptide-based monoclonal antibody derived from OmpA with drug resistant pulsotypes of Acinetobacter baumannii as a potential therapeutic approach.

BACKGROUND: Acinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed a novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A. baumannii. METHODS: Peptide derived from A. baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into BALB/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, western blotting, indirect immunofluorescence (IFA), and in vitro opsonophagocytosis assays were used to evaluate the reactivity of generated mAb. RESULTS: The anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (1.94 × 10-9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased the bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement. CONCLUSIONS: The peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as a novel therapeutic approach.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples.

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.

Extracellular Vesicles from 50,000 Generation Clones of the Escherichia coli Long-Term Evolution Experiment.

Extracellular vesicles (EVs) are critical elements of cell-cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation.

Microbiota-Derived Extracellular Vesicles Detected in Human Blood from Healthy Donors.

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host-microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.