Role Of Outer Membrane Proteins Research Articles

Bacterial efflux pump OMPs as vaccine candidates against Multidrug-Resistant Gram-negative bacteria.

The emergence and propagation of bacteria resistant to antimicrobial drugs is a serious public health threat worldwide. The current antibacterial arsenal is becoming obsolete and the pace of drug development is decreasing, highlighting the importance of investment in alternative approaches to treat or prevent infections caused by antimicrobial-resistant bacteria. A significant mechanism of antimicrobial resistance employed by Gram-negative bacteria is the overexpression of efflux pumps that can extrude several compounds from the bacteria, including antimicrobials. The overexpression of efflux pump proteins has been detected in several multidrug resistant (MDR) Gram-negative bacteria, drawing attention to these proteins as potential targets against these pathogens. This review will focus on the role of outer membrane proteins (OMPs) from efflux pumps as potential vaccine candidates against clinically relevant MDR Gram-negative bacteria, discussing advantages and pitfalls. Additionally, we will explore the relevance of efflux pump OMP diversity and the possible impact of vaccination on microbiota.

Analysis of Alternative Splicing and Long Noncoding RNAs After the Edwardsiella anguillarum Infected the Immunized European Eels (Anguilla anguilla) Revealed the Role of Outer Membrane Protein A in OmpA Subunit Vaccine.

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 28days 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.

The Role of Outer Membrane Proteins in UPEC Antimicrobial Resistance: A Systematic Review.

Uropathogenic Escherichia coli (UPEC) are one of the most common agents of urinary tract infection. In the last decade, several UPEC strains have acquired antibiotic resistance mechanisms and some have become resistant to all classes of antibiotics. UPEC outer membrane proteins (OMPs) seem to have a decisive role not only in the processes of invasion and colonization of the bladder mucosa, but also in mechanisms of drug resistance, by which bacteria avoid killing by antimicrobial molecules. This systematic review was performed according to the PRISMA guidelines, aiming to characterize UPEC OMPs and identify their potential role in antimicrobial resistance. The search was limited to studies in English published during the last decade. Twenty-nine studies were included for revision and, among the 76 proteins identified, seven were associated with antibiotic resistance. Indeed, OmpC was associated with β-lactams resistance and OmpF with β-lactams and fluoroquinolone resistance. In turn, TolC, OmpX, YddB, TosA and murein lipoprotein (Lpp) were associated with fluoroquinolones, enrofloxacin, novobiocin, β-lactams and globomycin resistances, respectively. The clinical implications of UPEC resistance to antimicrobial agents in both veterinary and human medicine must propel the implementation of new strategies of administration of antimicrobial agents, while also promoting the development of improved antimicrobials, protective vaccines and specific inhibitors of virulence and resistance factors.

Salmonella Typhimurium outer membrane protein A (OmpA) renders protection from nitrosative stress of macrophages by maintaining the stability of bacterial outer membrane.

Bacterial porins are highly conserved outer membrane proteins used in the selective transport of charged molecules across the membrane. In addition to their significant contributions to the pathogenesis of Gram-negative bacteria, their role(s) in salmonellosis remains elusive. In this study, we investigated the role of outer membrane protein A (OmpA), one of the major outer membrane porins of Salmonella, in the pathogenesis of Salmonella Typhimurium (STM). Our study revealed that OmpA plays an important role in the intracellular virulence of Salmonella. An ompA deficient strain of Salmonella (STM ΔompA) showed compromised proliferation in macrophages. We found that the SPI-2 encoded virulence factors such as sifA and ssaV are downregulated in STM ΔompA. The poor colocalization of STM ΔompA with LAMP-1 showed that disruption of SCV facilitated its release into the cytosol of macrophages, where it was assaulted by reactive nitrogen intermediates (RNI). The enhanced recruitment of nitrotyrosine on the cytosolic population of STM ΔompAΔsifA and ΔompAΔssaV compared to STM ΔsifA and ΔssaV showed an additional role of OmpA in protecting the bacteria from host nitrosative stress. Further, we showed that the generation of greater redox burst could be responsible for enhanced sensitivity of STM ΔompA to the nitrosative stress. The expression of several other outer membrane porins such as ompC, ompD, and ompF was upregulated in STM ΔompA. We found that in the absence of ompA, the enhanced expression of ompF increased the outer membrane porosity of Salmonella and made it susceptible to in vitro and in vivo nitrosative stress. Our study illustrates a novel mechanism for the strategic utilization of OmpA by Salmonella to protect itself from the nitrosative stress of macrophages.

Treponema pallidum outer membrane proteins: current status and prospects.

The outer membrane proteins (OMPs) of Treponema pallidum subsp. pallidum (T. pallidum), the etiological agent of the sexually transmitted disease syphilis, have long been a hot research topic. Despite many hurdles to studying the pathogen, especially the inability to manipulate T. pallidum in vitro genetically, considerable progress has been made in elucidating the structure, pathogenesis and functions of T. pallidum OMPs. In this review, we integrate this information to garner fresh insights into the role of OMPs in the diagnosis, pathogenicity and vaccine development of T. pallidum. Collectively, the essential scientific discussions herein should provide a framework for understanding the current status and prospects of T. pallidum OMPs.

Roles of OmpA in Type III Secretion System-Mediated Virulence of Enterohemorrhagic Escherichia coli.

Outer membrane proteins are commonly produced by gram-negative bacteria, and they have diverse functions. A subgroup of proteins, which includes OmpA, OmpW and OmpX, is often involved in bacterial pathogenesis. Here we show that OmpA, rather than OmpW or OmpX, contributes to the virulence of enterohemorrhagic Escherichia coli (EHEC) through its type III secretion system (T3SS). Deletion of ompA decreased secretion of the T3SS proteins EspA and EspB; however, the expression level of the LEE genes that encode a set of T3SS proteins did not decrease. The ompA mutant had less abilities to form A/E lesions in host epithelial cells and lyse human red blood cells than the parent strain. Moreover, the virulence of an ompA mutant of Citrobacter rodentium (traditionally used to estimate T3SS-associated virulence in mice) was attenuated. Mice infected with the ompA mutant survived longer than those infected with the parent strain. Furthermore, mice infected with ompA developed symptoms of diarrhea more slowly than mice infected with the parent strain. Altogether, these results suggest that OmpA sustains the activity of the T3SS and is required for optimal virulence in EHEC. This work expands the roles of outer membrane proteins in bacterial pathogenesis.

Functional Characterization of Outer Membrane Proteins (OMPs) in Xenorhabdus nematophila and Photorhabdus luminescens through Insect Immune Defense Reactions.

Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic bacterial symbionts that produce toxic proteins that can interfere with the immune system of insects. Herein, we show that outer membrane proteins (OMPs) could be involved as bacterial virulence factors. Purified totals OMPs of both bacterial species were injected into fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for cellular defenses fluctuations in total haemocyte counts (THC) and granulocyte percentage and for the humoral defenses protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities at specific time intervals. Changes in the expression of the three inducible antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with OMPs of both bacterial species had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially activated insect defensive enzymes post-injection, the degree of activation varying with enzyme type. The AMPs, attacin, cecropin, and spodoptericin were up-regulated by OMP injections compared with the normal larvae. The expression of these three AMPs was maximal at four hours post injection (hpi) with P. luminescens OMPs treatment. Expression of the three AMPs in X. nematophila treated insects was irregular and lower than in the P. luminescens OMPs treatment. These findings provide insights into the role of OMPs of entomopathogenic nematode bacterial symbionts in countering the physiological defenses of insects.

Role of outer membrane proteins in virulence of Klebsiella ozaenae and antibiotic sensitivity

In the present study, the association between outer membrane proteins loss, virulence and antibiotic sensitivity was investigated in K. ozaenae. An outer membrane proteins deficient K. ozaenae was derived from local clinical K. ozaenae isolate. Virulence was assessed by comparing the clearance of viable complete K. ozaenae with that outer membrane proteins deficient K. ozaenae in the spleen, liver, appendix, and blood of injected laboratory mice. Antibiotic susceptibility was tested by disc diffusion method. It was recorded significant differences at a P value of ⩽0.05 between the logarithm of the viable number of the complete K. ozaenae and logarithm of the viable number of outer membrane proteins deficient K. ozaenae that retrieved from the spleen, liver, appendix, and blood. It was observed that the logarithm of the viable number of complete K. ozaenae increased gradually during 18, 24, 48 and 72 hours compared with the logarithm of the viable number of outer membrane proteins deficient K. ozaenae which increases during 18-24 hours, and then decreased in all organs. Susceptibility tests showed that complete K. ozaenae isolate was resistant to most used antibiotics including amoxicillin, cephalothin, ceftazidime, cefotoxime, amikacin, tetracycline, naldixic acid, erythromycin, trimethoprim and sensitive to only imipenem and gentamicin. Whereas outer membrane deficient K. ozaenae isolate was resistant to all antibiotics used in our study.

Distinct role of outer membrane protein A in the intrinsic resistance of Acinetobacter baumannii and Acinetobacter nosocomialis

Acinetobacter baumannii outer membrane protein A (AbOmpA) contributes to the intrinsic resistance of A. baumannii through the OmpA-like domain. The present study investigated the role of Acinetobacter nosocomialis OmpA (AnOmpA) in the intrinsic resistance of A. nosocomialis and compared it with the role of AbOmpA. The minimal inhibitory concentrations (MICs) of antimicrobial agents against wild-type A. nosocomialis ATCC 17903, ∆AnompA mutant, and single-copy AnompA-complemented strains were determined by performing E-test or agar dilution. Single-copy ompA cross-complemented strains were constructed by cross-inserting AnompA and AbompA open reading frames (ORFs) along with their native promoters into ∆AbompA and ∆AnompA mutant strains, respectively, and the MICs of antimicrobial agents against these strains were determined. The ∆AnompA mutant of A. nosocomialis was more susceptible to colistin (20.0-fold) and gentamicin (4.8-fold) than the wild-type strain. The MICs of gentamicin and tetracycline against the ∆AnompA mutant did not decrease in the presence of an efflux pump inhibitor. The MIC of trimethoprim against the ∆AnompA mutant harbouring PAbompA and AbompA ORF increased by >4.0-fold compared with that against the wild-type strain. However, the MICs of all the tested antimicrobial agents were similar against the wild-type A. baumannii ATCC 17978 and ∆AbompA mutant harbouring PAnompA and AnompA ORF. These results indicate that AnOmpA contributes to the intrinsic resistance of A. nosocomialis similar to AbOmpA. However, AbOmpA and AnOmpA perform different roles in the intrinsic resistance of trimethoprim.

The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides

Bacterial resistance to classical antibiotics is emerging worldwide. The number of infections caused by multidrug resistant bacteria is increasing and becoming a serious threat for human health globally. In particular, Gram-negative pathogens including multidrug resistant Escherichia coli are of serious concern being resistant to the currently available antibiotics. All Gram-negative bacteria are enclosed by an outer membrane which acts as an additional protection barrier preventing the entry of toxic compounds including antibiotics and antimicrobial peptides (AMPs). In this study we report that the outer membrane component lipopolysaccharide (LPS) plays a crucial role for the antimicrobial susceptibility of E. coli BW25113 against the cationic AMPs Cap18, Cap11, Cap11-1-18m2, melittin, indolicidin, cecropin P1, cecropin B, and the polypeptide antibiotic colistin, whereas the outer membrane protease OmpT and the lipoprotein Lpp only play a minor role for the susceptibility against cationic AMPs. Increased susceptibility toward cationic AMPs was found for LPS deficient mutants of E. coli BW25113 harboring deletions in any of the genes required for the inner part of core-oligosaccharide of the LPS, waaC, waaE, waaF, waaG, and gmhA. In addition, our study demonstrates that the antimicrobial activity of Cap18, Cap11, Cap11-1-18m2, cecropin B, and cecropin P1 is not only dependent on the inner part of the core oligosaccharide, but also on the outer part and its sugar composition. Finally, we demonstrated that the antimicrobial activity of selected Cap18 derivatives harboring amino acid substitutions in the hydrophobic interface, are non-active against wild-type E. coli ATCC29522. By deleting waaC, waaE, waaF, or waaG the antimicrobial activity of the non-active derivatives can be partially or fully restored, suggesting a very close interplay between the LPS core oligosaccharide and the specific Cap18 derivative. Summarizing, this study implicates that the nature of the outer membrane component LPS has a big impact on the antimicrobial activity of cationic AMPs against E. coli. In particular, the inner as well as the outer part of the core oligosaccharide are important elements determining the antimicrobial susceptibility of E. coli against cationic AMPs.

Molecular epidemiology of carbapenem-resistant Pseudomonas aeruginosa in an endemic area: comparison with global data.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is an endemic problem in certain countries including Greece. CRPA and multidrug-resistant P. aeruginosa (MDRPA) firstly emerged in our region during the 80s, right after the launch of imipenem and meropenem as therapeutic agents against P. aeruginosa infections. The role of outer membrane protein (Opr) inactivation has been known to contribute to imipenem resistance since many years, while efflux overexpression systems have been mainly associated with meropenem resistance. Among carbapenemases, metallo-β-lactamases (MBL) and mostly Verona integron-mediated (VIM) MBL's have played the most crucial role in CRPA emergence. VIM-2 and VIM-4 producing CRPA, usually belonging to clonal complexes (CC) 111 and 235 respectively, have most frequently been isolated. BlaVIM-2 and blaVIM-4 are usually associated with a class 1 integron. VIM-17 also has appeared in Greece. On the other hand, other VIM subtypes detected in a global level, such as VIM-3, VIM-5, VIM-6, VIM-7, VIM-11, VIM-14, VIM-15, VIM-16 and VIM-18 have not yet emerged in Greece. However, new VIM subtypes will probably emerge in the future. In addition, MBL carbapenemases other than VIM, detected worldwide have not yet appeared. A single CRPA isolate producing KPC has emerged in our region several years ago. The study of the molecular basis of Opr deficiency and efflux overexpression remains a challenge for the future. In this article, we review the molecular epidemiology of CRPA in an endemic area, compared to global data.

Short communication: Roles of outer membrane protein W (OmpW) on survival and biofilm formation of Cronobacter sakazakii under neomycin sulfate stress

Cronobacter sakazakii is associated with severe infections including sepsis, neonatal meningitis, and necrotizing enterocolitis. Antibiotic resistance in Cronobacter species has been documented in recent years, but the genes involved in resistance in Cronobacter strains are poorly understood. In this study, we determined the role of outer membrane protein W (OmpW) on survival rates, morphologic changes, and biofilm formation between wild type (WT) and an OmpW mutant strain (ΔOmpW) under neomycin sulfate stress. Results indicated that the survival rates of ΔOmpW were significantly reduced after half minimum inhibitory concentration (½ MIC) treatment compared with the WT strain. Filamentation of C. sakazakii cells was observed after ½ MIC treatment in WT and ΔOmpW, and morphologic injury, including cell disruption and leakage of cells, was more predominant in ΔOmpW. Under ½ MIC stress, the biofilms of WT and ΔOmpW were significantly decreased, but decreasing rates of biofilm formation in mutant strain were more predominant compared with WT strain. This is the first report to determine the role of OmpW on survival, morphological changes, and biofilm formation in C. sakazakii under neomycin sulfate stress. The findings indicated that OmpW contributed to survival and reduction of morphological injury under neomycin sulfate stress. In addition, enhancing biofilm formation in ΔOmpW may be an alternative advantage for adaptation to neomycin sulfate stress.

Role of outer membrane proteins in serum resistance of A. actinomycetemcomitans and A. aphrophilus

ABSTRACTThe aim of this project was to study how outer membrane proteins contribute to serum resistance in A. actinomycetemcomitans and A. aphrophilus.Genes encoding Omp100 (A. actinomycetemcomitans) and OmpA (A. actinomycetemcomitans and A. aphrophilus) were knocked out through homologous recombination.Serum resistance was tested by incubating the bacterial strains in normal human serum (NHS; 50%) for 2h at 37°C. As control heat-inactivated (56°C) NHS was used. LC-MS/MS analysis was used to identify proteins in selected bands from SDS-PAGE.Whereas Omp100 appeared not to contribute significantly to serum resistance, we observed that the ompA mutants of both species had a markedly reduced capability to survive in 50% NHS. In presence of Mg-EGTA the ompA mutants survived clearly better in NHS suggesting that OmpA may act through the classical pathway of the complement system. Interestingly, when incubating A. actinomycetemcomitans ompA mutants in NHS, derivatives were obtained that had regained serum resistance. LC-MS/MS analysis revealed that these strains produced high levels of an OmpA-like protein (76% amino acid identity).Further experiments to investigate how OmpA and the OmpA-like protein may interact with complement system are being conducted.

Role of outer membrane protein T in pathogenicity of avian pathogenic Escherichia coli

An outer membrane protein T (OmpT) could play a vital role in the pathogenesis of the neonatal meningitis Escherichia coli (NMEC) in human and animals. However, whether ompT plays a role in avian pathogenic E. coli (APEC) infection remains unclear. In this study we evaluated the potential of ompT in APEC pathogenesis. An ompT gene was deleted from APEC mutant strain (TW-XM) was constructed and characterized. The inactivation of ompT reduced significantly the adherence and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells at the rates of 43.8% and 28.8% respectively, compared with the wild strain TW-XM. Further studies showed that deletion of ompT gene reduced the bacterial virulence with 15.2-fold in ducklings and 9.7-fold in mouse models based on the measurement of the LD50. Furthermore, experimental infection of animals revealed that, loss of ompT showed reduced APEC colonization and invasion capacity in brains, lungs and blood by 2-fold, 1.96-fold, and 1.7-fold, respectively, compared with the wild-type strain TW-XM. These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that the loss of ompT significantly decreased the expression levels of ompA, fimC and tsh in the mutant strain ΔOmpT, when compared with TW-XM (p<0.01). Collectively, our data showed that inactivation of ompT decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and tsh, which may justify that, ompT is implicated in APEC pathogenicity.

Interaction of Complement System Components with Bacterial Outer Membrane Proteins and Presentation of Techniques Useful in Studying these Interactions

The complement (C) system participates in the immune response against pathogenic microorganisms. C3 protein plays a crucial role because its activation supports the course of the three pathways of the C cascade: the alternative pathway (AP), the classical pathway (CP), and the lectin pathway (LP) leading to the destruction of microorganisms and limitation of the infection. This paper attempts to highlight the role of outer membrane proteins (OMPs) in C components binding, as there are porins able to alter the course of C activation. Additionally, we present molecular methods adapted to C3 activation in human serum upon a contact with Salmonella cells and their OMPs. We demonstrate our achievements by using Salmonella strains as an exemplary microorganism of the public importance. The most severe outcomes of the presence of vital Salmonella rods in human body is sepsis. Therefore, it is desirable to do research on the C evasion mechanisms developed by pathogens, to understand the principles of bacteremia caused by other pathogens. We propose four techniques that may be useful in studying interactions between bacterial surfaces and C components: electrophoretic resolution of OMPs under denaturing and non-denaturing conditions, western blotting, and sandwich ELISA. Keywords: BN-PAGE, C3, outer membrane protein, Salmonella, sandwich ELISA, SDS-PAGE.

The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

Outer membrane Protein A plays a role in pathogenesis of Acinetobacter nosocomialis

ABSTRACTAcinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of human infections. However, the specific virulence factors of this microorganism have not yet been determined. We investigated the role of outer membrane protein A (OmpA) in the pathogenesis of A. nosocomialis. A ΔompA mutant of the A. nosocomialis ATCC 17903T strain was constructed using markerless gene deletion. The ΔompA mutant displayed reduced biofilm formation in polystyrene tubes and reduced adherence to A549 cells in comparison to the wild-type strain. These virulence traits of the ΔompA mutant strain were restored when the ompA gene was complemented. Cytotoxicity was not significantly different between the wild-type strain and the ΔompA mutant when A549 cells were infected with bacteria or treated with outer membrane vesicles (OMVs). However, OMVs from the wild-type strain induced cytotoxicity in HEp-2 cells, whereas OMVs from the ΔompA mutant did not induce cytotoxicity. Proteomic analysis of OMVs revealed that OmpA influenced the distribution of envelope and periplasmic proteins. Overall, this study is the first report that links OmpA to A. nosocomialis pathogenesis, and highlights OmpA as a putative target to develop anti-virulence agents or vaccines against A. nosocomialis infection.

A study of outer membrane protein and molecular epidemiology of carbapenem-resistant Acinetobacter baumannii

To study the characteristics of the distribution and drug resistance of Acinetobacter baumannii, and the epidemiology of the main strains among wards and hospitals, and to investigate the role of outer membrane protein in producing resistance against carbapenems. 145 Acinetobacter baumannii strains were collected from July 2013 to July 2014 from Huangdao Hospital Affiliated to Qingdao University and 401st Army Hospital. Antimicrobial susceptibility test was carried out with K-B disk diffusion method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used for DNA typing and test of homology. The carO gene of outer membrane protein was amplified by PCR, and the outer membrane proteins were extracted by ultrasonication and ultracentrifuge method from 30 randomly selected carbapenem-resistant Acinetobacter baumannii and 17 carbapenem-sensitive strains. Sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) was used to analyze the expressions of outer membrane proteins. 145 Acinetobacter baumannii strains were generally resistant to 16 common antimicrobial agents, with the highest susceptibility rate of 79.3% for minocycline, followed by susceptibility rate of 40.7% for amikacin. There were 128 carbapenem-resistant strains (resistance rate of 88.3%), 137 multidrug-resistant strains and 126 extensively drug-resistant strains. The detection rates of carO gene were 97.7% (125/128) and 17.6% (3/17) in carbapenem-resistant and sensitive strains respectively. Around position of relative molecular mass 47,000, 16 of 17 sensitive isolates were expressed this protein, while 20 of 30 resistant ones were detected nothing or fade; 13 of 17 sensitive isolates were expressed around position of relative molecular mass 37,000 and 29,000 while 25 were detected nothing or fade around position of relative molecular mass 37,000 and 23 were detected nothing or fade around position of relative molecular mass 29,000 in 30 resistant ones. 145 Acinetobacter baumannii were classified into 8 types based on ERIC-PCR electrophoresis patterns, and the major prevalence types were type A (71 strains) and type E (37 strains). Drug resistance of clinically isolated Acinetobacter baumannii is a serious problem in two hospitals; drug-resistant strains are spread and epidemic among wards and hospitals. The carO gene of outer membrane protein is widespread in carbapenem-resistant Acinetobacter baumannii. The loss or fading of outer membrane protein may play an important role in Acinetobacter baumannii resistance to carbapenems drugs.

Role of Outer Membrane Protein H in the Pathogenesis of Avian Pasteurella multicida Strain C48-3

The previous study has indicated that the targeted inactivation of ompH gene decreased the lethality of serotype A Pasteurella multocida C48-3 in mice after intraperitoneal injection. The ompH gene was cloned from P. multocida C48-3 and expressed in Escherichia coli M15,and immune serum was generated by immunizing rabbit with the purified recombinant protein OmpH. Differences of adhesion ability,serum resistance and antiphagocytic activity between the wild-type strain C48-3,mutant strain ΔompH and its complemented strain C48-3C were compared by biological experiments. The adhesion of wild-type strain and complemented strain to CEF cells was inhibited significantly by treatment with immune sera as compared to the treatment with normal serum. The mutant strain was sensitive to the bactericidal action of chicken serum,whereas the wild-type strain and complemented strain were both resistant. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum,whereas the wild-type strain and complemented strain were both resistant. In addition,the rabbit antiserum against OmpH promoted the phagocytosis of C48-3 and C48-3C by mouse peritoneal macrophages,but mutant strain was not affected. These results demonstrate that the OmpH is a major virulence factor of P. multocida.

Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii.

Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA) and outer membrane vesicles (OMVs) in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.