Outer Membrane Protein Profiles Research Articles

Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, β-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

Proteomic profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures

Leptospirosis is a global zoonotic disease affecting humans, domestic, and wild animals. Leptospira are typically shed in the urine of reservoir hosts which persist in suitable environments where incidental host transmission occurs after direct contact with infected urine or contaminated environments. Interestingly, serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 show divergent disease severity in the hamster model; JB197 causes severe acute infection while HB203 causes persistent chronic infection. Historically, serovar Hardjo was limited to culture at 29 °C, but utilization of HAN media allows propagation from host tissues at 37 °C. Here, the proteome of strains JB197 and HB203 were characterized after culture from experimentally challenged hamsters at 29 °C and 37 °C. Comparative analyses of JB197 and HB203 samples cultured at 29 °C yielded 425 significantly differentially expressed (DE) proteins, while strains at 37 °C yielded 613 DE proteins including prominent outer membrane proteins and known virulence factors. In agreement, membrane protein GO terms were identified by STRING network analyses along with numerous metabolic KEGG pathways consistent with condition differences. Within strain, JB197 cultured at 29 °C vs 37 °C identified 529 DE proteins, while HB203 identified 524 DE proteins. Investigating differential protein profiles provide insights into strain specific behaviors with implications for better understanding host-pathogen interactions, disease transmission, and response to environmental conditions which can contribute to vaccine development, diagnostic improvement, and ultimately leptospirosis control. SignificanceLeptospirosis is a devastating zoonotic disease affecting humans, wild and domestic animals around the globe. Different species and serovars of Leptospira can affect various animal host species differently; for instance, a serovar that is asymptomatic in the rat may cause severe disease in a dog or human. These differences in host response are not only found at the species and serovar level for Leptospira, but also at the strain level. A prime example comes from strains JB197 and HB203, both species L. borgpetersenii, both serovar Hardjo. Interestingly, JB197 causes a severe acute infection in the hamster while HB203 causes an asymptomatic chronic infection. Understanding these unique relationships between pathogen and host species is important, especially in the context of prevention technologies such as vaccine design, where the strain of Leptospira used as a bacterin might have different efficiencies in different hosts. In this study, proteomic profiles of strains JB197 and HB203 were analyzed, and results revealed diverse protein expression profiles of outer membrane proteins, as well as proteins functioning in motility and growth.

Intraspecifična karakterizacija izolata Vibrio alginolyticus prikupljenih iz sustava akvakulture i morskih biotopa u Tunisu korištenjem PCR-RFLP, ECP i OMPS profiliranja

A total of 54 presumptive Vibrio alginolyticus strains isolated from a variety of Tunisian marine biotopes, such as seawater, sediment samples from some bathing areas, and aquaculture farms, were characterized and identified by several biochemical tests. DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, extracellular products (ECP) and outer membrane protein (OMP) profiling were used to evaluate their usefulness as a tool to investigate the Vibrio alginolyticus diversity within this complex group. Results showed that there is great heterogeneity in the diversity observed via the PCR-RFLP method related to the number of genotypes generated by the two enzymes SduI and FaqI tested. This heterogeneity was observed not only according to the origin (seawater, sediment, fish and bivalve aquaculture farms) but also within the same type of sample. The two other methods, ECP and OMP resulted in 32 and 26 profiles, respectively. The discriminatory index determined in this study highlighted the good ability of the PCR-RFLP method to discriminate similar V. alginolyticus strains.

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4g/0.5g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.

Polymerase chain reaction-based typing methods and protein profiling analysis of Acinetobacter baumannii isolated from environmental and clinical sources from South India.

Acinetobacter baumannii is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental A. baumannii isolates.

P-Aminobenzoic acid inhibits the growth of soybean pathogen Xanthomonas axonopodis pv. glycines by altering outer membrane integrity.

p-Aminobenzoic acid (pABA) is an environmentally friendly bioactive metabolite synthesized by Lysobacter antibioticus. This compound showed an unusual antifungal mode of action based on cytokinesis inhibition. However, the potential antibacterial properties of pABA remain unexplored. In this study, pABA showed antibacterial activity against Gram-negative bacteria. This metabolite inhibited growth (EC50 = 4.02 mM), and reduced swimming motility, extracellular protease activity, and biofilm formation in the soybean pathogen Xanthomonas axonopodis pv. glycines (Xag). Although pABA was previously reported to inhibit fungal cell division, no apparent effect was observed on Xag cell division genes. Instead, pABA reduced the expression of various membrane integrity-related genes, such as cirA, czcA, czcB, emrE, and tolC. Consistently, scanning electron microscopy observations revealed that pABA caused major alternations in Xag morphology and blocked the formation of bacterial consortiums. In addition, pABA reduced the content and profile of outer membrane proteins and lipopolysaccharides in Xag, which may explain the observed effects. Preventive and curative applications of 10 mM pABA reduced Xag symptoms in soybean plants by 52.1% and 75.2%, respectively. The antibacterial properties of pABA were studied for the first time, revealing new insights into its potential application for the management of bacterial pathogens. Although pABA was previously reported to show an antifungal mode of action based on cytokinesis inhibition, this compound inhibited Xag growth by altering the outer membrane's integrity. © 2023 Society of Chemical Industry.

Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants

BackgroundMoraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi.ResultsPhylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins.ConclusionsThere are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains.

Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn’s disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

The role of uspE in virulence and biofilm formation by Histophilus somni

UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™<KAN-2> Tnp Transposome™ that were biofilm deficient. The 2336::TnuspE mutant was highly attenuated in mice, the electrophoretic profile of its lipooligosaccharide (LOS) indicated the LOS was truncated, and the mutant was significantly more serum-sensitive compared to the wildtype strain. In addition to forming a deficient biofilm, exopolysaccharide (EPS) production was also compromised, but the electrophoretic profile of outer membrane proteins was not altered. RNA sequence analysis revealed that the transcription levels of some stress response chaperones, transport proteins, and a large number of ribosomal protein genes in 2336::TnuspE were significantly differentially regulated compared to strain 2336. Therefore, uspE may differentially function in direct and indirect expression of H. somni genes, but its attenuation may be linked to poor biofilm formation and rapid clearance of the bacteria resulting from a compromised LOS structure. Our results support that uspE is a global stress regulatory gene in H. somni.

The CRISPR-Cas System Is Involved in OmpR Genetic Regulation for Outer Membrane Protein Synthesis in Salmonella Typhi.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.

Repurposing of the Tamoxifen Metabolites to Combat Infections by Multidrug-Resistant Gram-Negative Bacilli.

The development of new strategic antimicrobial therapeutic approaches, such as drug repurposing, has become an urgent need. Previously, we reported that tamoxifen presents therapeutic efficacy against multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli in experimental infection models by modulating innate immune system cell traffic. The main objective of this study was to analyze the activity of N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen, three major metabolites of tamoxifen, against these pathogens. We showed that immunosuppressed mice infected with A. baumannii, P. aeruginosa, or E. coli in peritoneal sepsis models and treated with tamoxifen at 80 mg/kg/d for three days still reduced the bacterial load in tissues and blood. Moreover, it increased mice survival to 66.7% (for A. baumannii and E. coli) and 16.7% (for P. aeruginosa) when compared with immunocompetent mice. Further, susceptibility and time-kill assays showed that N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen exhibited minimum inhibitory concentration of the 90% of the isolates (MIC90) values of 16 mg/L, and were bactericidal against clinical isolates of A. baumannii and E. coli. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of A. baumannii and E. coli without affecting their outer membrane proteins profiles. Together, these data showed that tamoxifen metabolites presented antibacterial activity against MDR A. baumannii and E. coli, and may be a potential alternative for the treatment of infections caused by these two pathogens.

Identification of Immunodominant Outer Membrane Proteins of Fusobacterium necrophorum from Severe Ovine Footrot By MALDI-TOF Mass Spectrometry

The aim of this study was to identify the immunodominant outer membrane proteins (OMPs) of Fusobacterium necrophorum from sheep affected with severe foot-rot. The OMP profile of ovine strains of F. necrophorum has not been well studied. We analyzed the OMP profile of the most frequent lktA variant JKS-F3 of F. necrophorum associated with severe ovine foot-rot with lesion score 4 in order to identify its major immunodominant OMPs. Electrophoretic separations of extracted OMPs showed a number of spots in two-dimensional electrophoretic gels. Two immunoreactive proteins of size around 43 kDa were identified through western blotting using hyperimmune sera raised in rabbits. These two immunogenic OMPs were analyzed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) which revealed that these two OMPs of lktA variant JKS-F3 of F. necrophorum showed 46 and 42 percent protein sequence coverage and scores of 125 and 114, respectively, with the reported 43 kDa outer membrane protein of F. necrophorum strain H05, a putative porin having properties similar to pore-forming proteins of anaerobic Gram-negative bacteria. These identified immunogenic OMPs will contribute to our understanding of the pathogenic role played by this organism in ovine foot-rot and could be exploited to devise an effective control strategy through development of an OMP-based recombinant vaccine to mitigate foot-rot in sheep and goats.

Development of novel iron-regulated Pasteurella multocida B: 2 bacterin and refinement of vaccine quality in terms of minimum variation in particle size and distribution vis-a-vis critical level of iron in media

To enhance the qualitative bacterial biomass per unit of media and to overcome the limitations of the existing haemorrhagic septicaemia (HS) vaccines, a comprehensive study was undertaken encompassing the role of iron on the bacterial biomass of Pasteurella multocida B: 2 to vaccine development. Trypsin digested hydrochloric acid-treated sheep blood (THSB) as a novel iron rich supplement had been devised for the first time for augmenting the qualitative bacterial biomass per unit of media which was evident with growth kinetic study. The higher recovery of iron from THSB became evident via atomic absorbance spectrophotometry. The critical level of iron in the media as well as mode of iron supplementation showed a major impact on the outer membrane protein profile of P. multocida B:2 and variation in droplet size and particle-size distribution of formulated vaccine. Immune response study against iron-regulated bacterin adjuvanted with aluminum hydroxide gel in mouse model showed that 3% THSB supplementation of casein sucrose yeast (CSY) not only augmented the growth of P. multocida B:2 significantly but conferred highest pre-challenged ELISA IgG titer and protection against pasteurellosis. Thus, THSB supplementation of CSY can resolve existing up-scaling and immunogenic potential problems of HS vaccine production.

The Antimicrobial Peptide Human Beta-Defensin 2 Inhibits Biofilm Production of Pseudomonas aeruginosa Without Compromising Metabolic Activity.

Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 – independent of its chiral state – significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.

Transport of bacterial cell (E. coli) from different recharge water resources in porous media during simulated artificial groundwater recharge

Commonly used recharge water resources for artificial groundwater recharge (AGR) such as secondary effluent (SE), river water and rainfall, are all oligotrophic, with low ionic strengths and different cationic compositions. The dwelling process in recharge pond imposed physiologic stress on Escherichia coli (E. coli) cells, in all three types of investigated recharge water resources and the cultivation of E. coli under varying recharge water conditions, induced changes in cell properties. During adaptation to the recharge water environment, the zeta potential of cells became more negative, the hydrodynamic diameters, extracellular polymeric substances content and surface hydrophobicity decreased, while the cellular outer membrane protein profiles became more diverse. The mobility of cells altered in accordance with changes in these cell properties. The E. coli cells in rainfall recharge water displayed the highest mobility (least retention), followed by cells in river water and finally SE cells, which had the lowest mobility. Simulated column experiments and quantitative modeling confirmed that the cellular properties, driven by the physiologic state of cells in different recharge water matrices and the solution chemistry, exerted synergistic effects on cell transport behavior. The findings of this study contribute to an improved understanding of E. coli transport in actual AGR scenarios and prediction of spreading risk in different recharge water sources.

Prevalence and Some Possible Mechanisms of Colistin Resistance Among Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa.

Background and AimThe emergence of colistin-resistant strains is considered a great threat for patients with severe infections. Here, we investigate the prevalence and some possible mechanisms of colistin resistance among multidrug-resistant (MDR) and extensively drug-resistant (XDR) Pseudomonas aeruginosa (P. aeruginosa).MethodsAntimicrobial susceptibility was performed using disc diffusion methods while colistin resistance was detected by agar dilution method. Possible mechanisms for colistin resistance were studied by detection of mcr-1 and mcr-2 genes by conventional PCR, detection of efflux mechanisms using Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP), studying outer membrane protein profile and Lipopolysaccharide (LPS) profile of resistant isolates.ResultsIt was found that MDR and XDR represented 96% and 87% of the isolated P. aeruginosa, respectively, and colistin resistance represented 21.3%. No isolates were positive for mcr-2 gene while 50% of colistin-resistant isolates were positive for mcr-1. Efflux mechanisms were detected in 3 isolates. Protein profile showed the presence of a band of 21.4 KDa in the resistant strains which may represent OprH while LPS profile showed differences among colistin-resistant mcr-1 negative strains, colistin-resistant mcr-1 positive strains and susceptible strains.ConclusionThe current study reports a high prevalence of colistin resistance and mcr-1 gene in P. aeruginosa strains isolated from Egypt that may result in untreatable infections. Our finding makes it urgent to avoid unnecessary clinical use of colistin.

Comparative Study on A Novel Pathogen of European Seabass. Diversity of Aeromonas veronii in the Aegean Sea.

Aeromonas veronii is an emerging pathogen causing severe pathology and mortalities in European seabass aquaculture in the Aegean Sea, Mediterranean. More than 50 strains of the pathogen were characterized biochemically and genetically in order to study the epidemiology of the disease, as well as the phylogeny and virulence of the bacterium. Based on the phenotypic characteristics, the isolates form three groups consisting of: (a) the West Aegean Sea, non-motile, non-pigment-producing strains, (b) the West Aegean Sea, motile, and pigment-producing strains and (c) the East Aegean Sea motile strains that produce minute amounts of pigment. All strains were highly similar at the genomic level; however, the pattern of West/East geographic origin was reflected in biochemical properties, in general genomic level comparison and in the putative virulent factors studied. Type VI secretion system was not detected in the western strains. The outer membrane protein (OMP) profile which contains proteins that are putative antigenic factors, was very similar between strains from the different areas. Although most of the OMPs were detected in all strains with great sequence similarity, diversification according to geographic origin was evident in known antigenic factors such as the maltoporin LamB. A systematic comparative analysis of the strains is presented and discussed in view of the emergence of A. veronii as a significant pathogen for the Mediterranean aquaculture.

Natural outer membrane permeabilizers boost antibiotic action against irradiated resistant bacteria

BackgroundThis study sought to develop new strategies for reverting the resistance of pathogenic Gram-negative bacilli by a combination of conventional antibiotics, potent permeabilizers and natural beta lactamase inhibitors enhancing the activity of various antibiotics.MethodsThe antibiotic susceptibility in the presence of natural non-antibacterial tested concentrations of phytochemicals (permeabilizers and natural beta lactamase inhibitors) was performed by disk diffusion and susceptibility assays. Thymol and gallic acid were the most potent permeabilizers and facilitated the passage of the antibiotics through the outer membrane, as evidenced by their ability to cause LPS release, sensitize bacteria to SDS and Triton X-100.ResultsThe combination of permeabilizers and natural beta lactamase inhibitors (quercetin and epigallocatechin gallate) with antibiotics induced greater susceptibility of resistant isolates compared to antibiotic treatment with beta lactamase inhibitors alone. Pronounced effects were detected with 24.4 Gy in vitro gamma irradiation on permeability barrier, beta lactamase activity, and outer membrane protein profiles of the tested isolates.ConclusionsThe synergistic effects of the studied natural phytochemicals and antibiotics leads to new clinical choices via outer membrane destabilization (permeabilizers) and/or inactivation of the beta lactamase enzyme, which enables the use of older, more cost-effective antibiotics against resistant strains.

The anthelmintic oxyclozanide restores the activity of colistin against colistin-resistant Gram-negative bacilli

Due to the significant increase in antimicrobial resistance in Gram-negative bacilli (GNB), development of non-antimicrobial therapeutic alternatives, which can be used together with the few and non-optimal available antimicrobial agents such as colistin, has become an urgent need. In this context, dysregulation of the bacterial cell wall could be a therapeutic adjuvant to the activity of colistin. The aim of this study was to analyse the activity of oxyclozanide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) GNB. Three Col-S reference strains and 13 clinical isolates (1 Col-S, 12 Col-R) of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were studied. Microdilution assays and time-kill curves were performed to examine the activity of oxyclozanide in combination with colistin. The outer membrane protein (OMP) profile, membrane permeability and cell wall structure of Col-S and Col-R A. baumannii, P. aeruginosa and K.pneumoniae in the presence of oxyclozanide were assessed by SDS-PAGE, fluorescence microscopy and transmission electron microscopy, respectively. Oxyclozanide in combination with colistin increased the activity of colistin against Col-S and Col-R A. baumannii, P. aeruginosa and K. pneumoniae. Time-kill curves showed synergistic activity between oxyclozanide and colistin against these bacterial isolates. Moreover, Col-R A. baumannii, P. aeruginosa and K. pneumoniae in the presence of oxyclozanide presented greater permeability and disruption of their cell wall than Col-S strains, without modification of their OMP profile. These data suggest that combination of oxyclozanide and colistin may be a new alternative for the treatment of Col-R GNB infections.