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Carbapenem resistant Acinetobacter baumannii has evolved as the most troublesome microorganism with multiple virulence factors. Biofilm formation, porins, micronutrient capturing mechanism and quorum sensing, provide protection against desiccation, host-pathogen killing and enhance its persistence. The conservation of these factors between colonizing and pathogenic carbapenem resistant A. baumannii has been barely investigated. We studied biofilm formation, desiccation survival, motility and hemolysis in pathogenic carbapenem resistant A. baumannii and colonizer carbapenem resistant A. baumannii from the hospital environment. The virulent genes pgaA, csuE, bap, ompA, abaI, pilA and bauA were detected by simplex-PCR and Quantitative Real-Time PCR was done for expressional studies. In-vivo survival percentage was studied by Galleria mellonella (wax moth) killing assay. Phenotypic characterization revealed that the biofilm formation and desiccation survival proportion was significantly higher in colonizer carbapenem resistant A. baumannii (p < 0.05). Twitching motility was found comparable (mean 0.5 to 1.5 cm). Surface associated motility varied widely. None showed hemolysis. The csuE, bap, ompA, abaI, pilA and bauA genes were detected in almost all the pathogenic and colonizer carbapenem resistant A. baumannii isolates while none harboured pgaA gene. The expression of bap, ompA and bauA gene was found significantly higher in pathogenic carbapenem resistant A. baumannii while expression of csuE and abaI gene was comparable in both. Overexpression of pilA gene was seen in those with higher surface associated motility. Pathogenic carbapenem resistant A. baumannii showed significantly higher pathogenicity in-vivo, as 100% of larvae died on 4th day post-infection. In conclusion high level expression of outer membrane proteins (ompA) and siderophores is significantly associated with the pathogenicity in carbapenem resistant A. baumannii isolated from infections, which can be a differentiating point from the colonizers. Clinical Trial: Not Applicable [ABSTRACT FROM AUTHOR]

Abstract Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results: The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions: In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment. [ABSTRACT FROM AUTHOR]

Background: Rickettsial infections are often neglected and poorly recognized by physicians in many tropical and subtropical regions. Despite a number of recent reports describing rickettsial diseases in new locations and the discovery of new rickettsiae, medical science and research have largely neglected the diagnosis and antimicrobial treatment of rickettsial infections in subtropical and tropical areas; thus, much remains to be discovered. This study aimed to detect and characterize spotted fever group (SFG) rickettsiae in ixodid ticks infesting domestic ruminants in Khartoum State. Methods: Polymerase chain reaction targeting both genes that encode for citrate synthase (gltA) and outer membrane protein (ompA) was performed for the presence of SFG rickettsia followed by sequence and phylogenetic analysis. Results: Of the 202 ticks examined for the presence of SFG rickettsia, gltA gene was detected in 4 samples (2%). Furthermore, gltA‐positive samples were used to amplify the ompA gene, in which only two samples yielded positive results. Sequence and phylogenetic analysis of the positive samples revealed four different species of SFG rickettsiae: Rickettsia aeschlimannii, Rickettsia rhipicephali, Rickettsia massiliae and Rickettsia raoultii. Conclusions: These results indicated the presence of SFG rickettsia in Sudanese ticks. This also indicates that humans have an opportunity to acquire these infections. It is important to keep in mind the need for careful consideration of rickettsial infections in individuals with a fever of unknown origin. [ABSTRACT FROM AUTHOR]

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Background: The objective of this study lies in identifying the dominant genotype of C. trachomatis isolated in Republic of Korea (ROK) between 2017 and 2018. Methods: A total of 504 clinical cervicovaginal swabs from patients were collected and inoculated on McCoy cell monolayers to isolate Chlamydial agents. C. trachomatis isolates were analyzed by sequencing of its ompA gene. Results: A total of 54 C. trachomatis isolates were obtained. The constructed phylogenetic tree revealed the genotypes of isolates are D/Ep6 (48, 88.89%), D/Ep6-like (5, 9.26%), and Ja (1, 1.85%). The C. trachomatis D/Ep6-like have one single nucleotide polymorphism (SNP) compared to C. trachomatis D/Ep6, leading to E870K amino acid change. Conclusions: Phylogenetic analysis demonstrated globally rare C. trachomatis D/Ep6 was dominant in the ROK from 2017 to 2018. Findings in this study act as a keystone, bridging past and present molecular epidemiology of C. trachomatis in context of ROK. [ABSTRACT FROM AUTHOR]

Surfactants are molecules derived primarily from petroleum that can reduce the surface tension at interfaces. Their slow degradation is a characteristic that could cause environmental issues. This and other factors contribute to the allure of biosurfactants today. Progress has been made in this area of research, which aims to satisfy the need for effective surfactants that are not harmful to the environment. In previous studies, we demonstrated the surface tension activity of the Escherichia coli transmembrane protein OmpA. Here, we carried out DNA shuffling on ompA to improve its interfacial activity. We evaluated changes in interfacial tension when exposing mutants to a water-oil interface to identify the most promising candidates. Two mutants reached an interfacial tension value lower (9.10 mN/m and 4.24 mN/m) than the original protein OmpA (14.98 mN/m). Since predicted isoelectric point values are far from neutral pH, the charge of the protein was a crucial factor in explaining the migration of proteins towards the interface. Low molecular weight mutants did not exhibit a significant difference in their migration to the interface.

Enterobacteriaceae pathogens such as Escherichia coli, Salmonella sp., Shigella sp., Proteus sp., and Klebsiella pneumoniae cause a wide range of gastrointestinal and other mucosal infections. These bacteria acquire antibiotic resistance very quickly and evolve into multi-drug resistant strains thereby making the treatment very difficult. The outer membrane proteins (OMPs) in Enterobacteriaceae are potential vaccine candidates owing for their high immunogenicity and amino acid conservation. The OmpA is one such protein which need to be investigated for the development of a potential subunit vaccine against multiple infections casued by the pathogens of Enterobacteriaceae. To investigate this, we expressed and purified the highly conserved OmpA of S. typhimurium and studied the antibody mediated cross reactivity with the other Enterobacteriaceae pathogens. This was validated through dot ELISA performed with the hyperimmune sera raised against rOmpA of S. typhimurium. We further analyzed the sequence of OmpA protein and clearly understood that the B-cell epitopes in the protein are highly conserved are responsible for cross reactivity among the Enterobacteriaceae pathogens. This work led to findings that provide strong evidence for the application of OmpA in broad-spectrum subunit vaccine against enteric infections.

Enterobacteriaceae pathogens such as Escherichia coli, Salmonella sp., Shigella sp., Proteus sp., and Klebsiella pneumoniae cause a wide range of gastrointestinal and other mucosal infections. These bacteria acquire antibiotic resistance very quickly and evolve into multi-drug resistant strains thereby making the treatment very difficult. The outer membrane proteins (OMPs) in Enterobacteriaceae are potential vaccine candidates owing for their high immunogenicity and amino acid conservation. The OmpA is one such protein which need to be investigated for the development of a potential subunit vaccine against multiple infections casued by the pathogens of Enterobacteriaceae. To investigate this, we expressed and purified the highly conserved OmpA of S. typhimurium and studied the antibody mediated cross reactivity with the other Enterobacteriaceae pathogens. This was validated through dot ELISA performed with the hyperimmune sera raised against rOmpA of S. typhimurium. We further analyzed the sequence of OmpA protein and clearly understood that the B-cell epitopes in the protein are highly conserved are responsible for cross reactivity among the Enterobacteriaceae pathogens. This work led to findings that provide strong evidence for the application of OmpA in broad-spectrum subunit vaccine against enteric infections. [ABSTRACT FROM AUTHOR]

Background: The first pathogen to be designated a “red-alert” human pathogen is Acinetobacter baumannii, which is on the list of infections that must be treated urgently with new antibiotics. Infections due to this bacterium are on the rise, especially in patients admitted to hospital intensive care units. It can create biofilms on both biotic and abiotic surfaces. Objectives: This study aimed to detect biofilm formation by A. baumannii phenotypically and genotypically. Materials and Methods: A total of 250 samples were subjected to bacterial identification using the VITEK-2 compact system, which showed 42 A. baumannii isolates. Biofilm formation was phenotypically investigated using the microtiter plate method. Results: The results revealed three stages of biofilm formation: 5 (11.6%) nonbiofilm, 13 (30.2%) weak biofilm, 15 (34.9%) moderate, and 10 (23.3%) strong biofilm formation. The isolates from intensive care unit (ICU) patients had strong, moderate, weak, and nonforming biofilm ability in higher rates of biofilm producers compared with the isolates from samples of hospital wards. The polymerase chain reaction (PCR) products showed genotypically positive results as follows: PapII 12 (31.5%), OmpA 11 (28.9%), and LuxR 8 (21%) out of 38 positive samples of A. baumannii for all genes. Conclusion: Isolates of A. baumannii appeared in different stages of biofilm formation with a higher percentage rate in the ICU compared with hospitalized patients. The PCR products for isolates of A. baumannii showed that PapII, OmpA, and LuxR showed positive results.

ABSTRACT The discovery of Replication Competent Circular DNA molecules in mammalian cells and tissues is being linked to debilitating diseases, such as multiple sclerosis (MS), bovine spongiform encephalopathy (BSE), and colorectal cancer (CRC). These circular DNA molecules, otherwise known as bovine meat and milk factors (BMMFs) and Slow Progressive Hidden INfections of variable (X) latency (SPHINX), bear significant (80%) sequence similarity with the plasmids of Acinetobacter baumannii strains. Nanostructures, such as bacterial outer membrane vesicles (OMVs) serve as vehicles for transporting biomolecular cargo and have the potential to facilitate interkingdom lateral mobility of DNA. Strengthening the proposed hypothesis, this study demonstrates that OMVs derived from A. baumannii DS002 carrying four plasmids and genome (pTS236) of phage, AbDs1, successfully reached different parts of the body, including the central nervous system, following the injection of fluorescein isothiocyanate (FITC)-labeled OMVs into experimental mice. Out of the four OMV-associated plasmids, three (pTS4586, pTS9900, and pTS134338) were identified within the lumen, and the fourth one (pTS11291) was found on the surface of OMVs. In addition to the indigenous plasmids, the phage-encoded protein, Orf96, anchored on the surface of the OMVs by establishing a strong interaction with the OMV-associated porin, OmpA. Intriguingly, a subset of labeled OMVs, when incubated with Neuro2A cells, translocated across the membrane and reached to the cytoplasmic space of the cells. Collectively, the experimental evidence presented herein underscores the promising potential of OMVs as vehicles for delivering molecular cargo containing plasmids and phage genomes to diverse mammalian tissues and cells.IMPORTANCESeveral independent studies have demonstrated the existence of replication competent circular DNA molecules of bacterial and viral origin in mammalian cells and tissues. However, studies about their origin and lateral mobility to mammalian cells are scarce. Our work describes the existence of circular DNA, similar to that of DNA molecules identified in mammalian cells, OMVs derived from soil isolate of A. baumannii DS002. Furthermore, the work also provides visual evidence that demonstrates the passage of labeled OMVs to different organs of experimental mice within hours after intravenously administering OMVs into experimental mice. Some of the labeled OMVs have even crossed the membrane of Neuro2A, suggesting the existence of interkingdom horizontal mobility between bacteria and mammals.

Abstract All living organisms produce only one enantiomer, so we found that all natural compounds are presented in enantiomerically pure form. Asymmetric synthesis is highly spread in medicinal chemistry because enantiomerically pure drugs are highly applicable. This study initially demonstrated the feasibility of a good idea for the asymmetric synthesis of α-alkylated carbonyl compounds with high enantiomeric purity ranging from 91 to 94% using different quinazolinone derivatives. The structure of all compounds was confirmed via elemental analysis and different spectroscopic data and the enantioselectivity was determined via HPLC using silica gel column. The synthesized compounds’ mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-beta-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed that compound 6a has a broad biocidal activity and this in-vitro study was in line with the in-silico results. Overall, the formulated compound 6a can be employed as antimicrobial agent without any toxicity with high bioavailability in medical applications.

All living organisms produce only one enantiomer, so we found that all natural compounds are presented in enantiomerically pure form. Asymmetric synthesis is highly spread in medicinal chemistry because enantiomerically pure drugs are highly applicable. This study initially demonstrated the feasibility of a good idea for the asymmetric synthesis of α-alkylated carbonyl compounds with high enantiomeric purity ranging from 91 to 94% using different quinazolinone derivatives. The structure of all compounds was confirmed via elemental analysis and different spectroscopic data and the enantioselectivity was determined via HPLC using silica gel column. The synthesized compounds' mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-beta-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed that compound 6a has a broad biocidal activity and this in-vitro study was in line with the in-silico results. Overall, the formulated compound 6a can be employed as antimicrobial agent without any toxicity with high bioavailability in medical applications. [ABSTRACT FROM AUTHOR]

The plague, caused by Yersinia pestis, is a natural focal disease and the presence of Y. pestis in the environment is a critical ecological concern worldwide. The role of Y. pestis phages in the ecological life cycle of the plague is crucial. Previously, a temperature-sensitive phage named vB_YpM_HQ103 was isolated from plague foci in Yunnan province, China. Upon infecting the EV76 strain of Y. pestis, vB_YpM_HQ103 exhibits lysogenic behavior at 21 °C and lytic behavior at 37 °C. Various methods including continuous passage lysogenic tests, in vitro lysis tests, comparative genomic assays, fluorescence quantitative PCR and receptor identification tests were employed to demonstrate that the lysogenic life cycle of this phage is applicable to wild Y. pestis strains; its lysogeny is pseudolysogenic (carrying but not integrating), allowing it to replicate and proliferate within Y. pestis. Furthermore, we have identified the outer membrane protein OmpA of Y. pestis as the receptor for phage infection. In conclusion, our research provides insight into the characteristics and receptors of a novel Y. pestis phage infection with a pseudolysogenic cycle. The findings of this study enhance our understanding of Y. pestis phages and plague microecology, offering valuable insights for future studies on the conservation and genetic evolution of Y. pestis in nature.

Chlamydia psittaci is a bacterium that infects several species of birds and mammals. It is the causal agent of avian chlamydiosis and psittacosis in humans and it is globally distributed. Chlamydia psittaci is one of the main zoonotic pathogens transmitted by birds. In Argentina, there has been limited research on the prevalence and genetic variability of C. psittaci. The aim of this study was to detect and genotype C. psittaci using molecular techniques in birds living in Buenos Aires City, Argentina, during the period 2012–2015. A descriptive, observational, retrospective and cross-sectional study was carried out. A total of 983 bird samples submitted for diagnosis of avian chlamydiosis were analyzed. The frequency of C. psittaci was 12.54% and 7.89% in Psittaciformes and Columbiformes, respectively. A 348 bp region of the ompA gene was sequenced in positive samples. Molecular genotyping was performed through a Bayesian phylogenetic analysis. Of the 983 bird samples, 83 were positive for C. psittaci and 44 could be sequenced. The genotypes found were A, B, and E. Despite the high levels of host specificity, we found six psittacids with genotype B and one pigeon with genotype A, reflecting the affiliative interaction between Psittaciformes and Columbiformes. This study represents the first survey reporting the presence of C. psittaci in birds within the largest and most populous city in Argentina.

Abstract Background Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. Results The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. Conclusion The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp. infection.

Typhoid fever is a serious concern precisely in developing nations. Still investigators are exploring a better conjugate partner for Vi-polysaccharide to develop a more effective vaccine for typhoid fever. Here, we cloned and expressed S. Typhi outer membrane protein A (OmpA). The conjugation of Vi-polysaccharide with OmpA was carried out by the carbodiimide (EDAC) method employing ADH as a linker. Total Ig and IgG generated against OmpA, and Vi polysaccharide was quantified by ELISA. Vi polysaccharide alone induced very low levels of Vi polysaccharide antibody. Vi-OmpA conjugate (Vi-conjugate) elicited a robust immune response compared to Vi polysaccharide alone and showed booster response. Further, IgG was only evoked by Vi-OmpA conjugate, not with Vi polysaccharide alone. OmpA antibody induction in both the Vi-OmpA conjugate and OmpA were similar level. Taken together, we show that OmpA as a carrier protein conjugated to Vi polysaccharide is immunogenic. We predict OmpA antibodies will contribute protection along with antibodies generated by Vi-polysaccharide. Past and current literature supports that OmpA is highly conserved protein not only among Salmonellae but entire Enterobacteriacea family with 96–100% identity.

ABSTRACT: Understanding the characteristics of bacteriophages is crucial for the optimization of phage therapy. In this study, the biological and genomic characteristics of coliphage LHE83 were determined and its synergistic effects with different types of antibiotics against E. coli E82 were investigated. Phage LHE83 displayed a contractile tail morphology and had a titer of 3.02 × 109 pfu/mL at an optimal MOI of 0.01. Meanwhile, phage LHE83 exhibited good physical and chemical factors tolerance. The 1-step growth analysis revealed a latent period of approx. 10 min with a burst size of 87 pfu/infected cell. Phage LHE83 belongs to the genus Dhakavirus. Its genome consists of 170,464 bp with a 40% GC content, and a total of 268 Open Reading Frames (ORF) were predicted with no detected virulent or resistant genes. ORF 213 was predicted to encode the receptor binding protein (RBP) and confirmed by the antibody-blocking assay. Furthermore, a phage-resistant strain E. coli E82R was generated by co-culturing phage LHE83 with E. coli E82. Genomic analysis revealed that OmpA served as the receptor for phage LHE83, which was further confirmed by phage adsorption assay using E. coli BL21ΔOmpA, E. coli BL21ΔOmpA: OmpA and E. coli BL21:OmpA strains. Additionally, a synergistic effect was observed between phage LHE83 and spectinomycin against the drug-resistant strain E. coli E82. These results provide a theoretical basis for understanding the interactions between phages, antibiotics, and host bacteria, which can assist in the clinical application of phages and antibiotics against drug-resistant bacteria.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections. [ABSTRACT FROM AUTHOR]

Background: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. Results: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. Conclusion: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp. infection. [ABSTRACT FROM AUTHOR]

Typhoid fever is a serious concern precisely in developing nations. Still investigators are exploring a better conjugate partner for Vi-polysaccharide to develop a more effective vaccine for typhoid fever. Here, we cloned and expressed S. Typhi outer membrane protein A (OmpA). The conjugation of Vi-polysaccharide with OmpA was carried out by the carbodiimide (EDAC) method employing ADH as a linker. Total Ig and IgG generated against OmpA, and Vi polysaccharide was quantified by ELISA. Vi polysaccharide alone induced very low levels of Vi polysaccharide antibody. Vi-OmpA conjugate (Vi-conjugate) elicited a robust immune response compared to Vi polysaccharide alone and showed booster response. Further, IgG was only evoked by Vi-OmpA conjugate, not with Vi polysaccharide alone. OmpA antibody induction in both the Vi-OmpA conjugate and OmpA were similar level. Taken together, we show that OmpA as a carrier protein conjugated to Vi polysaccharide is immunogenic. We predict OmpA antibodies will contribute protection along with antibodies generated by Vi-polysaccharide. Past and current literature supports that OmpA is highly conserved protein not only among Salmonellae but entire Enterobacteriacea family with 96–100% identity. [ABSTRACT FROM AUTHOR]

The aim of this research is to study the distribution, species diversity, and genetic variability of rickettsiae in Dermacentor spp. ticks from Western Siberia and Northern Kazakhstan. Thus, samples from 571 Dermacentor ticks (406 individuals of Dermacentor reticulatus, 136 Dermacentor nuttalli, 21 Dermacentor marginatus, and 8 Dermacentor silvarum) have been examined for the presence of Rickettsia spp. DNA using nested PCR. The rickettsial species have been determined by species-specific PCR and/or sequencing of gltA gene fragments. For a number of R. raoultii samples, the sequences of the ompA (3266 bp) and ompB (4852 bp) gene fragments have been additionally determined. The examined ticks carry DNA of four Rickettsia species and a new genotype Rickettsia spp. Kos-97-Dr, which cannot be assigned to any known species. All tick species are most commonly infected with R. raoultii; the infection rate varies from 47.0 to 86.8%. Rickettsia sibirica DNA has been found in 16.1–45.7% of D. nuttalli from three sites in the Republic of Altai. DNAs of Rickettsia aeschlimannii, Rickettsia aeschlimannii-like, "Candidatus Rickettsia tarasevichiae," and a new genotype Rickettsia sp. Kos-97-Dr have been found sporadically in D. reticulatus and D. marginatus. Based on analysis of the gltA gene fragment, seven haplotypes of R. raoultii have been identified; four of them correspond to previously described genotypes. The analysis of long fragments of the ompA and ompB genes of R. raoultii samples revealed the presence of three genetic groups corresponding to different genotypes for the gltA gene. Thus, Dermacentor spp. carry both typical and atypical species of Rickettsia spp. The existence of natural foci of Siberian tick typhus in the Republic of Altai has been demonstrated. Based on the analysis of the variable surface protein genes and conserved gltA gene, the existence of three genetic groups of R. raoultii has been shown. [ABSTRACT FROM AUTHOR]

Ling Tang,&ast; Hui Wang,&ast; Kangli Cao,&ast; Yajuan Li, Tingting Li, Ying Huang, Yuanhong Xu Department of Clinical Laboratory, First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yuanhong Xu, Email xyhong1964@163.comPurpose: Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium that is predominantly associated with liver abscesses in global diabetic patients. High levels of glucose in the surrounding of K. pneumonia increase its pathogenicity including capsular polysaccharide (CPS) and fimbriae. Other important virulent factors include outer membrane protein A (ompA) and regulator mucoid phenotype A (rmpA). The objective of this investigation was to elucidate the effects of high glucose on rmpA and ompA gene expression and serum resistance of K. pneumoniae causing liver abscess.Patients and Methods: The clinical history of 57 patients suffering from K. pneumoniae-caused liver abscesses (KLA) was acquired and their clinical and laboratory manifestations in the presence or absence of diabetes were analyzed. The antimicrobial susceptibility, serotypes, and virulence genes were tested. Clinical isolates of 3 serotype-K1 hypervirulent K. pneumoniae (hvKP) were used to detect the effect of exogenous high glucose on rmpA, ompA, and clbB genes expression, and bacterial serum resistance.Results: KLA patients with diabetes showed higher C-reactive protein (CRP) compared to non-diabetic KLA patients. Furthermore, the diabetic group showed increased incidences of sepsis and invasive infections, and their length of hospital stay was also prolonged. Pre-incubation of K. pneumoniae in high glucose (0.5%) concentration up-regulated rmpA, ompA, and clbB genes expression. However, cAMP supplementation, which was inhibited by environmental glucose, reversed the increase of rmpA and ompA in a cAMP-dependent manner. Moreover, hvKP strains incubated in high glucose also exhibited enhanced protection from serum killing.Conclusion: High glucose levels reflected by poor glycemic control has increased gene expression of rmpA and ompA in hvKP by the cAMP signaling pathway and enhanced its resistance to serum killing, thus providing a new and reasonable explanation for the high incidences of sepsis and invasive infections in KLA patients with diabetes.Keywords: Klebsiella pneumoniae, liver abscess, diabetes, rmpA, ompA, serum resistance

Abstract 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- $$\gamma$$ 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. Graphical abstract

Avian chlamydiosis is an acute or chronic bacterial disease of birds. Chlamydia psittaci is the primary agent of the disease. It is also an important zoonotic pathogen. Chlamydia avium and Chlamydia gallinacea have also been recognized as potential causative agents of the disease. Clinical signs of this disease can vary in severity. Asymptomatic infections of Chlamydia have commonly been reported in various birds worldwide. In this study, we investigated the distribution of Chlamydia species in healthy psittacine birds in Korea. A total of 263 samples (pharyngeal/cloacal swabs and faeces) were collected from psittacine birds of 26 species in five zoos, five parrot farms and seven parrot cafes between 2020 and 2021. Ages of these birds had a wide range (1 month to 30 years). During sample collection, no bird showed any clinical signs indicating diseases such as chlamydiosis. Samples were tested for the presence of Chlamydia spp. using real‐time PCR assays. Chlamydia spp. were detected in 168 (63.9%) samples and C. psittaci was detected in 96 (36.5%) samples. However, C. avium and C. gallinacea were not detected. There were no significant differences in the prevalence of asymptomatic infections in birds among three types of housing environments. Regarding ompA genotypes, 87 C. psittaci‐positive samples had genotype A based on sequence analysis (n = 28) and genotype‐specific real‐time PCR (n = 59). Other positive samples were untyped (n = 9). Overall findings showed high prevalence of asymptomatic infections of C. psittaci in psittacine birds in Korea, posing a significant hazard to public health. [ABSTRACT FROM AUTHOR]

Edwardsiella anguillarum is a bacterium that commonly infects cultivated eels. Outer membrane protein A (OmpA) emulsified with Freund's adjuvant has been shown to be an effective fishery vaccine against this pathogen. However, the specific roles of OmpA in the vaccine have not been fully explored. In this study, we performed RNA-seq in the liver of a European eel (Anguilla anguilla) after challenge with E. anguillarum in eels previously immunized with an OmpA subunit vaccine. Our aim was to elucidate the differentially alternative splicing (DAS) and differentially expressed long noncoding RNAs (DE-lncRNAs) using a genome-wide transcriptome. The results showed after that at 28 days post-immunization, eels challenged with E. anguillarum (Con_inf) exhibited severe pathological changes in the liver. In contrast, the OmpA infused eels (OmpA_inf group) showed infiltrated lymphocytes, while Freund's adjuvant-inoculated eels (FCIA_inf group) showed edema of hepatocytes and blood coagulation. The relative percent survival (RPS) was 77.7% and 44.4% for OmpA_inf and FCIA_inf compared to the Con_inf group. We identified 37 DE-lncRNAs and 293 DAS genes between OmpA_inf and FCIA_inf. Interactions between DAS gene-expressed proteins indicated that 66 expressed proteins formed 20 networks. Additionally, 33 DE-lncRNAs interacted with 194 target genes formed 246 and 41 networks in co-expression and co-location. Taken together, our findings demonstrate that the OmpA subunit vaccine elicits a higher RPS and provides novel insights into the role of OmpA through DAS genes and DE-lncRNAs perspective. These results are significant for the development of fishery subunit vaccines. [ABSTRACT FROM AUTHOR]

ABSTRACT Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299–356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299–356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299–356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299–356. KJΔOmpA299–356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299–356. Next, the involvement of the three σ factors in the ΔompA299–356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299–356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299–356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal β-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to β-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.

ABSTRACT Increasing antibiotic resistance of Neisseria gonorrhoeae, the causative agent of gonorrhea, is a growing global concern that has renewed vaccine development efforts. The gonococcal OmpA protein was previously identified as a vaccine candidate due to its surface exposure, conservation, stable expression, and involvement in host–cell interactions. We previously demonstrated that the transcription of ompA can be activated by the MisR/MisS two-component system. Interestingly, earlier work suggested that the availability of free iron also influences ompA expression, which we confirmed in this study. In the present study, we found that iron regulation of ompA was independent of MisR and searched for additional regulators. A DNA pull-down assay with the ompA promoter from gonococcal lysates obtained from bacteria grown in the presence or absence of iron identified an XRE (Xenobiotic Response Element) family member protein encoded by NGO1982. We found that an NGO1982 null mutant of N. gonorrhoeae strain FA19 displayed a reduced level of ompA expression compared to the wild-type (WT) parent strain. Given this regulation, and the capacity of this XRE-like protein to regulate a gene involved in peptidoglycan biosynthesis (ltgA), along with its presence in other Neisseria sp., we termed the NGO1982-encoded protein as NceR (Neisseria cell envelope regulator). Critically, results from DNA-binding studies indicated that NceR regulates ompA through a direct mechanism. Thus, ompA expression is subject to both iron-dependent (NceR) and -independent (MisR/MisS) pathways. Hence, levels of the vaccine antigen candidate OmpA in circulating gonococcal strains could be influenced by transcriptional regulatory systems and the availability of iron. IMPORTANCE Herein, we report that the gene encoding a conserved gonococcal surface-exposed vaccine candidate (OmpA) is activated by a heretofore undescribed XRE family transcription factor, which we term NceR. We report that NceR regulation of ompA expression in N. gonorrhoeae is mediated by an iron-dependent mechanism, while the previously described MisR regulatory system is iron-independent. Our study highlights the importance of defining the complexity of coordinated genetic and physiologic systems that regulate genes encoding vaccine candidates to better understand their availability during infection.

Background: Outer membrane protein A (OmpA) is the major virulence factor of Acinetobacter baumannii and plays a wide role in the pathogenesis and antimicrobial resistance of A. baumannii. Dendritic cells (DCs) are the most effective antigen‐presenting cells and play a crucial role in regulating the immune response to multiple antigens and immune sentries. We aimed to study the role and molecular mechanisms of OmpA‐induced mouse bone marrow‐derived dendritic cells (BMDCs) autophagy in the immune response of A. baumannii. Methods: First, purified A. baumannii OmpA was assessed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot. OmpA effect on BMDCs viability was evaluated by MTT assay. BMDCs were pretreated with autophagy inhibitor chloroquine or transfected with overexpression plasmids (oe‐NC or oe‐PI3K). Then BMDCs apoptosis, inflammatory cytokines, protein kinase B (PI3K)/mammalian target of rapamycin (mTOR) pathway, and autophagy‐related factors levels were evaluated. Results: SDS‐PAGE and western blot verified the successful purification of OmpA. BMDCs viability repressed gradually with the increase of OmpA concentration. OmpA treatment of BMDCs led to apoptosis and inflammation in BMDCs. OmpA caused incomplete autophagy in BMDCs, and light chain 3 (LC3), Beclin1, P62, and LC3II/I levels were significantly elevated with the increase of the time and concentration of OmpA treatment. Chloroquine reversed OmpA effects on autophagy in BMDCs, that was, LC3, Beclin1, and LC3II/I levels were reduced, while P62 level was elevated. Furthermore, chloroquine reversed OmpA effects on apoptosis and inflammation in BMDCs. PI3K/mTOR pathway‐related factor expression was affected by OmpA treatment of BMDCs. After overexpression of PI3K, these effects were reversed. Conclusions: A. baumannii OmpA induced autophagy in BMDCs involving the PI3K/mTOR pathway. Our study may provide a novel therapeutic target and theoretical basis for treating infections caused by A. baumannii. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background The genus Rickettsia contains the lineages spotted fever group (SFG), typhus group (TG), and transitional group (TRG). The spotted fever group Rickettsia (SFGR) is transmitted by ticks. The tick species Dermacentor nuttalli is considered the main vector carrying SFGR in Inner Mongolia. Studying the genetic diversity and population structure of Rickettsia is essential for developing effective control strategies and predicting evolutionary trends of Rickettsia. Methods In 2019 we collected 408 D. nuttalli in the Inner Mongolia Autonomous Region, detected the percentage of Rickettsia-positive specimens, and characterized the haplotypes. From the Rickettsia-positive ticks, the gltA and ompA genes were extracted, amplified, and sequenced. Results Ten haplotypes of the gltA gene and 22 haplotypes of the ompA gene were obtained. The phylogenetic analysis showed that the haplotypes G1–G7 and G9 of the gltA gene cluster with Rickettsia raoultii, while G8 and G10 cluster with Rickettsia sibirica. Haplotypes O1–O15, O18 and O20–O22 of the ompA gene cluster with R. raoultii, while O16 and O19 cluster with R. sibirica. The average haplotype diversity was 0.3 for gltA and 0.7 for ompA. The average nucleotide diversity was greater than 0.05. Neutrality tests were nonsignificant for Tajima’s D results and Fu’s Fs results. The fixation index values (F ST) showed that the degree of genetic differentiation between most sampled populations was small (F ST 0.05) or large (F ST > 0.15) degree of differentiation. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that between populations. The mismatch analysis of Rickettsia showed double peaks. Conclusions We found two Rickettsia spp. (R. raoultii and R. sibirica). The high genetic disparity of Rickettsia allows for easy adaption to different environments. Genetic differentiation between populations is small, and Rickettsia populations do not show a geographically differentiated structure. The high rates of retention and infection of Rickettsia in D. nuttalli together with the animal husbandry exchange in Inner Mongolia gradually led to the harmonization of genetic characteristics of Rickettsia across various regions. Overall, the significant genetic diversity and geographical structure of Rickettsia in D. nuttalli are critical for SFGR control. Graphical Abstract

• BamA, a two-domain OMP with a 16-stranded barrel, harbor L4 as the longest loop. • The designed antigen consisted of 478 aa showed antigen probability of 0.7793. • The novel antigen could elicit protective antibodies against BamA, OmpA and Omp34. • No identical peptides were found in the human proteome for the novel antigen. • The designed construct was safe in regard to allergenicity and toxicity. Acinetobacter baumannii , is among the highest priority bacteria according to the WHO categorization which necessitate the exploration of alternative strategies such as vaccination. OmpA, BamA, and Omp34 are assigned as appropriate antigens to serve in vaccine development against this pathogen. Experimentally validated exposed epitopes of OmpA and Omp34 along with selected exposed epitopes predicted by an integrative in silico approach were represented by the barrel domain of BamA as a scaffold. Among the 8 external loops of BamA, 5 loops were replaced with selected loops of OmpA and Omp34. The designed antigen was analyzed regarding the physicochemical properties, antigenicity, epitope retrieval, topology, structure, and safety. BamA is a two-domain OMP with a 16-stranded barrel in which L4, L6, and L7 were the longest loops of BamA in order. The designed antigen consisted of 478 amino acids with antigen probability of 0.7793. The novel antigen was a 16-stranded barrel. No identical 8-meric peptides were found in the human proteome against the designed antigen sequence. The designed construct was safe regarding the allergenicity, toxicity, and human proteome reactivity. The designed antigen could develop higher protection against A. baumannii in comparison to either OmpA, BamA, or Omp34 alone. [ABSTRACT FROM AUTHOR]

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

ABSTRACT: 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.

The limited delivery of cargoes at the cellular level is a significant challenge for therapeutic strategies due to the presence of numerous biological barriers. By immobilizing the Buforin II (BUF-II) peptide and the OmpA protein on magnetite nanoparticles, a new family of cell-penetrating nanobioconjugates was developed in a previous study. We propose in this study to extend this strategy to silica nanoparticles (SNPs) and silanized fullerenol (F) as nanostructured supports for conjugating these potent cell-penetrating agents. The same molecule conjugated to distinct nanomaterials may interact with subcellular compartments differently. On the obtained nanobioconjugates (OmpA-SNPs, BUF-II-PEG12-SNPs, OmpA-F, and BUF-II-PEG12-F), physicochemical characterization was performed to evaluate their properties and confirm the conjugation of these translocating agents on the nanomaterials. The biocompatibility, toxicity, and internalization capacity of nanobioconjugates in Vero cells and THP-1 cells were evaluated in vitro. Nanobioconjugates had a high internalization capacity in these cells without affecting their viability, according to the findings. In addition, the nanobioconjugates exhibited negligible hemolytic activity and a low tendency to induce platelet aggregation. In addition, the nanobioconjugates exhibited distinct intracellular trafficking and endosomal escape behavior in these cell lines, indicating their potential for addressing the challenges of cytoplasmic drug delivery and the development of therapeutics for the treatment of lysosomal storage diseases. This study presents an innovative strategy for conjugating cell-penetrating agents using silica nanoparticles and silanized fullerenol as nanostructured supports, which has the potential to enhance the efficacy of cellular drug delivery.

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. [ABSTRACT FROM AUTHOR]

The World Health Organization has classified multidrug-resistant (MDR) Acinetobacter baumannii as a significant threat to human health, necessitating the urgent discovery of new antibacterial drugs to combat bacterial resistance. Outer membrane protein A of A. baumannii (AbOmpA) is an outer membrane-anchored β-barrel-shaped pore protein that plays a critical role in bacterial adhesion, invasion, and biofilm formation. Therefore, AbOmpA is considered a key virulence factor of A. baumannii . Herein, we screened three phage display peptide libraries targeting AbOmpA and identified several peptides. Among them, P92 (amino acid sequence: QMGFMTSPKHSV) exhibited the highest binding affinity with AbOmpA, with a KD value of 7.84 nM. In vitro studies demonstrated that although P92 did not directly inhibit bacterial growth, it significantly reduced the invasion and adhesion capabilities of multiple clinical isolates of MDR A. baumannii and concentration-dependently inhibited biofilm formation by acting on OmpA. Furthermore, the polymerase chain reaction results confirmed a significant positive correlation between the antibacterial effect of P92 and OmpA expression levels. Encouragingly, P92 also displayed remarkable therapeutic efficacy against A. baumannii infection in various models, including an in vitro cell infection model, a mouse skin infection model, and a mouse sepsis model. These results highlight P92 as a novel and highly effective antimicrobial molecule specifically targeting the virulence factor AbOmpA., Competing Interests: The authors declare no conflict of interest.

ABSTRACT The presence or absence of BlsA, a protein with a blue light-sensing flavin domain in the genomes of Acinetobacter species has aroused curiosity about its roles in the regulation of bacterial lifestyle under light. Genomic and transcriptomic analyses revealed the loss of BlsA in several multidrug-resistant (MDR) A. baumannii strains as well as the light-mediated induction of blsA, along with a possible BlsA-interacting partner BipA. Their direct in vivo interactions were verified using a bacterial two-hybrid system. The results demonstrated that the C-terminal region of BipA could bind to the C-terminal residues of BlsA under blue light at 23°C but not at 37°C. Genetic manipulations of blsA and bipA revealed that the coexistence of BlsA and BipA was required to induce the light-dependent expression of ompA in A. baumannii ATCC 17978 at 23°C. The same phenomenon occurred in the BlsA-deficient MDR strain in our functional complementation assay; however, the underlying molecular mechanism remains poorly understood. BlsA-modulated amounts of OmpA, the most abundant porin, in the outer membrane affected the membrane integrity and permeability of small molecules. Dark conditions or the deletion of ompA made the membrane more permeable to lipophilic ethidium bromide (EtBr) but not to meropenem. Interestingly, light illumination and low temperature conditions made the cells more sensitive to meropenem; however, this bactericidal effect was not noted in the blsA mutant or in the BlsA-deficient MDR strains. Light-mediated cell death and the reduction of biofilm formation at 23°C were abolished in the blsA mutant strain, suggesting multifaceted roles of BlsA in A. baumannii strains. IMPORTANCE Little is known about the functional roles of BlsA and its interacting partners in Acinetobacter species. Intriguingly, no BlsA homolog was found in several clinical isolates, suggesting that BlsA was not required inside the host because of the lack of blue light and the warm temperature conditions. As many chromophore-harboring proteins interact with various partners to control light-dependent cellular behaviors, the maintenance of blsA in the genomes of many Acinetobacter species during their evolution may be beneficial when fluctuations occur in two important environmental factors: light and temperature. Our study is the first to report the novel protein partner of BlsA, namely, BipA, and its contribution to multiple phenotypic changes, including meropenem resistance and biofilm formation. Rapid physiological acclimation to changing light or temperature conditions may be possible in the presence of the light-sensing BlsA protein, which may have more interacting partners than expected.

ABSTRACT OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of β-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA299-356, an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJΔOmpA299-356 was more susceptible to β-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA299-356-mediated increase in β-lactam susceptibility (abbreviated as “ΔOmpA299-356 phenotype”). KJΔOmpA299-356 displayed a lower ceftazidime (CAZ)-induced β-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 β-lactamases-null mutant, and KJ2ΔOmpA299-356, a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable β-lactam susceptibility. Both lines of evidence indicate that decreased β-lactamase activity contributes to the ΔOmpA299-356 phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA299-356, focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA299-356. Deletion of the nagA gene from the chromosome of KJΔOmpA299-356 restored β-lactam susceptibility and CAZ-induced β-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA299-356 contributes to the ΔOmpA299-356 phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA299-356. The deletion mutant construction, β-lactam susceptibility, and β-lactamase activity analysis demonstrated that σP, but not σE, was involved in the ΔOmpA299-356 phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σP regulon. The involvement of the σP-NagA-L1/L2 regulatory circuit in the ΔOmpA299-356 phenotype was manifested. IMPORTANCE Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed. The linkage between porin deficiency and antibiotic resistance increase has been reported widely, with a rationale for blocking antibiotic influx. In this study, a link between porin defects and β-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel σP-NagA-L1/L2 regulatory circuit is triggered due to the loss of the OmpA-PG interaction. This study extends the understanding on the porin defect and antibiotic susceptibility. Porin defects may cause opposite impacts on antibiotic susceptibility, which is dependent on the involvement of the defect. Blocking the porin channel role can increase antibiotic resistance; in contrast, the loss of porin structure role may increase antibiotic susceptibility.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We report molecular detection of Rickettsia africae in Amblyomma ovale ticks from Nicaragua and a novel rickettsial strain in an A. triste tick. Of 146 ticks from dogs, 16.4% were Rickettsia PCR positive. The presence of Rickettsia spp. in human-biting ticks in Nicaragua may pose a public health concern.

As one of the most common bacterial infections globally, urinary tract infections (UTI)s affect the bladder and kidneys of many the bladders and kidneys of many. Along with gram-negative bacteria, Escherichia coli (E. coli) causes nearly 40% of nosocomial UTIs, 25% of recurrent infections, and between 80 to 90% of community-acquired infections. Proteomics, commonly used to study changes in protein expression of organisms, can be used to explore candidate biomarkers useful for the diagnosis of pathological conditions. Here, protein profiles of samples from patients diagnosed with E. coli-induced UTI were compared to identify distinctive proteins. Extracted proteins from bacteria from patients’ urine samples were separated into excisable spots using 2D-gel electrophoresis. The gels were then analyzed using Progenesis SameSpot software to select uniquely expressed protein spots, excised, and analyzed by LC/MS. The results were then compared against a database of known proteins. We identified two proteins, outer membrane protein A (OmpA) and RNA polymerase-binding transcription factor (DksA), involved in the survival of E. coli in the harsh environment of the host. We suggest their use as a part of a battery of possible biomarkers proteins for E. coli-induced UTI, and suggest that their overexpression is possibly associated with the stage of infection, early or late.

Background: The genus Rickettsia contains the lineages spotted fever group (SFG), typhus group (TG), and transitional group (TRG). The spotted fever group Rickettsia (SFGR) is transmitted by ticks. The tick species Dermacentor nuttalli is considered the main vector carrying SFGR in Inner Mongolia. Studying the genetic diversity and population structure of Rickettsia is essential for developing effective control strategies and predicting evolutionary trends of Rickettsia. Methods: In 2019 we collected 408 D. nuttalli in the Inner Mongolia Autonomous Region, detected the percentage of Rickettsia-positive specimens, and characterized the haplotypes. From the Rickettsia-positive ticks, the gltA and ompA genes were extracted, amplified, and sequenced. Results: Ten haplotypes of the gltA gene and 22 haplotypes of the ompA gene were obtained. The phylogenetic analysis showed that the haplotypes G1–G7 and G9 of the gltA gene cluster with Rickettsia raoultii, while G8 and G10 cluster with Rickettsia sibirica. Haplotypes O1–O15, O18 and O20–O22 of the ompA gene cluster with R. raoultii, while O16 and O19 cluster with R. sibirica. The average haplotype diversity was 0.3 for gltA and 0.7 for ompA. The average nucleotide diversity was greater than 0.05. Neutrality tests were nonsignificant for Tajima's D results and Fu's Fs results. The fixation index values (FST) showed that the degree of genetic differentiation between most sampled populations was small (FST < 0.05), whereas some populations showed a medium (FST > 0.05) or large (FST > 0.15) degree of differentiation. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that between populations. The mismatch analysis of Rickettsia showed double peaks. Conclusions: We found two Rickettsia spp. (R. raoultii and R. sibirica). The high genetic disparity of Rickettsia allows for easy adaption to different environments. Genetic differentiation between populations is small, and Rickettsia populations do not show a geographically differentiated structure. The high rates of retention and infection of Rickettsia in D. nuttalli together with the animal husbandry exchange in Inner Mongolia gradually led to the harmonization of genetic characteristics of Rickettsia across various regions. Overall, the significant genetic diversity and geographical structure of Rickettsia in D. nuttalli are critical for SFGR control. [ABSTRACT FROM AUTHOR]