Porin Expression Research Articles

Genetic Characterization of Multidrug-Resistant Acinetobacter baumannii and Synergy Assessment of Antimicrobial Combinations.

Background/Objectives: A. baumannii is a prominent nosocomial pathogen due to its drug-resistant phenotype, representing a public health problem. In this study, the aim was to determine the effect of different antimicrobial combinations against selected multidrug-resistant (MDR) or extensive drug-resistant (XDR) isolates of A. baumannii. Methods: MDR or XDR A. baumannii isolates were characterized by assessing genes associated with drug resistance, efflux pumps, porin expression, and biofilm formation. The activities of antimicrobial combinations including tigecycline, ampicillin/sulbactam, meropenem, levofloxacin, and colistin were evaluated using checkerboard and time-to-kill assays on isolates with different susceptibility profiles and genetic characteristics. Results: Genetic characterization of MDR/XDR strains (n = 100) included analysis of OXA-24/40 gene carbapenemase (98%), genes encoding aminoglycoside-modifying enzymes (44%), and parC gene mutations (10%). AdeIJK, AdeABC, and AdeFGH efflux pumps were overexpressed in 17-34% of isolates. Omp33-36, OmpA, and CarO membrane porins were under-expressed in 50-76% of isolates; CarO was overexpressed in 22% of isolates. Isolates showed low biofilm production (11%). Synergistic activity was observed with levofloxacin-ampicillin/sulbactam and meropenem-colistin, which were able to inhibit bacterial growth. Conclusions: Genetic characteristics of A. baumannii were highly variable among the strains. Synergistic activity was observed with meropenem-colistin and levofloxacin-ampicillin/sulbactam combinations in the checkerboard method, but not in the time-to-kill assays. These discrepancies among both methods indicate that further studies are needed to determine the best therapeutic combination for treating infections by A. baumannii.

TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection.

Exploration of Antimicrobial Peptides in the Treatment of Gentamicin-Resistant Klebsiella pneumoniae Infection.

The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) and the decline of effective antibiotics lead to the urgent need for new antibacterial agents. The aim of this study is to investigate the therapeutic effect of antimicrobial peptides against gentamicin-resistant (RT) K. pneumoniae and to screen effective antimicrobial peptides. In this study, the RT strains were induced by gradient gentamicin, and the RT strains were selected by detecting the expression levels of efflux pump genes, porin genes, and biofilm formation genes of the strains combined with their effects on the cells. Then the effects of four antimicrobial peptides on the efflux pump activity, biofilm formation level and cell condition after infection were detected to explore the effects of antimicrobial peptides on RT strains. Finally, the RT strain was used to induce a mouse model of pneumonia, and the four antimicrobial peptides were used to treat pneumonia mice for in vivo experiments. The pathological changes in lung tissues in each group were detected to explore the antimicrobial peptide with the most significant effect on the RT strain in vivo. The results showed that the minimal inhibitory concentrations of the RT strains (strain C and strain I) were significantly higher than those of the wild-type strain, and the expression of efflux pump, porin and biofilm formation genes was significantly increased. The antimicrobial peptides could effectively inhibit the biofilm formation and efflux pump protein function of the RT strains. In addition, the antimicrobial peptides showed promising antibacterial effects both in vitro and in vivo. Our study provided a theoretical basis for the treatment of gentamicin resistant K. pneumoniae infection with antimicrobial peptides, and found that KLA was significantly superior to LL37, Magainin I, KLA and Dermaseptin (10 μg/mL in cells, 50 μg in mice).

In Silico Molecular Analysis of Carbapenemase-Negative Carbapenem-Resistant Pseudomonas aeruginosa Strains in Greece.

To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD porin, and overexpression of the efflux pump MexAB-OprM. Twelve carbapenemase-negative carbapenem-resistant Pseudomonas aeruginosa strains, isolated from patients hospitalized at the University Hospital of Larissa, Central Greece, during 2023, which belonged to various sequence types (STs), were selected and were studied focusing on the characterization of their β-lactamases, on changes to OprD and its regulator MexT proteins, and on alterations to the MexAB-OprM regulator proteins encoded by the mexR, nalC, and nalD genes. Whole genome sequencing analysis revealed the presence of β-lactamase encoding genes, with blaPAO present in all isolates. Additionally, seven different genes of the oxacillinase family (blaOXA-35, blaOXA-50, blaOXA-395, blaOXA-396, blaOXA-486, blaOXA-488, blaOXA-494) were identified, with each strain harboring one to three of these. Regarding the OprD, five strains had truncated structures, at Loop 2, Loop 3, Loop 4, and Loop 9, while the remaining strains carried previously reported amino acid changes. Further, an additional strain had a truncated MexR; whereas, two other strains had totally modified NalC sequences. The active form of MexT, responsible for the downregulation of OprD production, as the intact sequence of the NalD protein, was found in all the strains studied. It is concluded that the truncated OprD, MexR, and NalC proteins, detected in eight strains, probably led to inactive proteins, contributing to carbapenem resistance. However, four strains carried known modifications in OprD, MexR, and NalC, as previously reported in both susceptible and resistant strains, a finding that indicates the complexity of carbapenem resistance in P. aeruginosa.

469 Diverse Role of blaCTX-M and Porins in Mediating Ertapenem Resistance Among Carbapenem Resistant Enterobacterales

OBJECTIVES/GOALS: In this study, we aim to report the role of porins and blaCTX-M β-lactamases among Escherichia coli and Klebsiella pneumoniae, focusing on emerging carbapenem resistant Enterobacterales (CRE) subtypes, including non-carbapenemase producing Enterobacterales (NCPE) and ertapenem-resistant but meropenem-susceptible (ErMs) strains. METHODS/STUDY POPULATION: Whole genome sequencing was conducted on 76 carbapenem-resistant isolates across 5 hospitals in San Antonio, U.S. Among these, NCP isolates accounted for the majority of CRE (41/76). Identification and antimicrobial susceptibility testing (AST) results were collected from the clinical charts. Repeat speciation was determined through whole genome sequencing (WGS) analysis and repeat AST, performed with microdilution or ETEST®. Minimum inhibitory concentrations (MIC) were consistent with Clinical and Laboratory Standards Institute (CLSI M100, ED33). WGS and qPCR were used to characterize the resistome of all clinical CRE subtypes, while western blotting and liquid chromatography with tandem mass spectrometry (LC-MS-MS) were used to determine porin expression and carbapenem hydrolysis, respectively. RESULTS/ANTICIPATED RESULTS: blaCTX-Mwas found to be most prevalent among NCP isolates (p = 0.02). LC-MS/MS analysis of carbapenem hydrolysis revealed that blaCTX-M-mediated carbapenem hydrolysis, indicating the need to reappraise the term, “non-carbapenemase (NCP)®” for quantitatively uncharacterized CRE strains harboring blaCTX-M. Susceptibility results showed that 56% of all NCPE isolates had an ErMs phenotype (NCPE vs. CPE, p < 0.001), with E. coli driving the phenotype (E. coli vs. K. pneumoniae, p < 0.001). ErMs strains carrying blaCTX-M, had 4-fold more copies of blaCTX-M than ceftriaxone-resistant but ertapenem-susceptible isolates (3.7 v. 0.9, p < 0.001). Immunoblot analysis demonstrated the absence of OmpC expression in NCP-ErMs E. coli, with 92% of strains lacking full contig coverage ofompC. DISCUSSION/SIGNIFICANCE: Overall, this work provides evidence of a collaborative effort between blaCTX-M and OmpC in NCP strains that confer resistance to ertapenem but not meropenem. Clinically, CRE subtypes are not readily appreciated, potentially leading to mismanagement of CRE infected patients. A greater focus on optimal treatments for CRE subtypes is needed.

Evaluating Porins OmpK35 and OmpK36 mRNA Expression in Ciprofloxacin-Resistant Klebsiella pneumoniae

Evaluating Porins OmpK35 and OmpK36 mRNA Expression in Ciprofloxacin-Resistant Klebsiella pneumoniae

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Impact of high-intensity exercise training (HIIT) versus moderate continuous training (MCT) on skeletal muscle alterations in HFrEF: a substudy of the SMART-EX trial

Abstract Background Skeletal muscle dysfunction contributes to exercise intolerance in chronic heart failure. The randomized multicenter Study of Myocardial Recovery After Exercise Training in Heart Failure (SMART-EX) has shown that high-intensity exercise training (HIIT) but not moderate continuous training (MCT) reduced the left ventricular end diastolic diameter. Both training modalities significantly increased exercise tolerance as determined by cardiopulmonary exercise testing (CPET). However, the underlying molecular alterations within the skeletal muscle with respect to different exercise training modalities are largely unknown. Methods In patients with chronic heart failure with reduced ejection fraction (HFrEF) who participated in the SMART-EX skeletal muscle substudy needle biopsies from the vastus lateralis muscle were performed before and after 12 weeks of supervised HIIT, MCT, or recommendation of regular exercise (RRE) to allow molecular assessment of markers for muscle catabolism, inflammation and energy supply by RT-PCR and western blotting. Echocardiography and CPET were performed in all patients. Changes over time were tested within each group. Results Skeletal muscle biopsies at baseline and follow-up were available in 31 patients (HIIT n=9; MCT n=9; RRE n=13). The majority of patients were male (87%). Median left ventricular ejection fraction was 29%. Median improvement in peak VO2 was 2.0 (IQR -0.4-2.8) in HIIT vs. 1.4 (IQR -0.9-3.4) in MCT vs. 0.0 (IQR -2.9-2.1) ml/min/kg in RRE. This was statistically not significant. HIIT reduced mRNA levels of the E3 ligases of the ubiquitine proteasome system MuRF1 by -72% (p<0.01) and MafBx -45% (p=0.09) with no change in MCT (MuRF1 -31%, p=0.23; MafBx -47%, p=0.21) and RRE (MuRF1 -8%, p=0.20; MafBx -6%, p=0.46). There was no change in mRNA expression of the inflammatory markers interleukin 6 (HIIT 12%; MCT 29%; RRE -35%; n.s.) and TNFα (HIIT -9%; MCT 20%; RRE 27%; n.s.) in either group. The mRNA expression of the mitochondrial F1 subunit of complex V (ATP5F1) significantly increased in HIIT by 14% (p<0.05) with no change in MCT (-3%, p=0.75) and RRE (2%, p=0.76). The mRNA expression of mitochondrial complex I subunits NDUFB8 and NDUFB6 remained unchanged in all groups. Western blot analysis of porin expression, which is a marker of total mitochondrial content, revealed a significant increase in HIIT by 69% (p<0.05) whereas MCT (21%, p=0.18) and RRE (4%, p=0.79) remained unchanged. Conclusion High-intensity interval training in HFrEF for 12 weeks resulted in reduced markers of protein degradation via the ubiquitine proteasome system and increased markers of mitochondrial content and energy supply via mitochondrial complex V whereas moderate continuous exercise training had only minor (non-significant) effects. These molecular changes are probably one reason why exercise capacity improved in patients of the SMART-EX trail.

Recent findings in molecular reactions of E. coli as exposed to alkylated, nano- and ordinary chitosans

The antibacterial effects of chitosan have been widely studied, but the underlying molecular mechanisms are not fully understood. We investigated the molecular responses of Escherichia coli MG1655 cell, a model gram-negative bacterium, upon exposure to chitosan (Cs), alkylated Cs (AlkCs), and chitosan nanoparticles (CsNPs). Nine target genes involved in relevant signaling pathways (ompF, ompC, ompA, mrcA, mrcB, mgtA, glnA, kdpA, lptA) were selected for analysis. A significant reduction in the expression of mrcA, mgtA, glnA, and lptA genes was observed in the cells treated with Cs. Those treated with Cs, AlkCs, and CsNPs revealed an increase in ompF gene expression, but the expression level was lower in the cells treated with AlkCs and CsNPs compared to Cs. This increase in porin expression suggests compromised membrane integrity and disrupted nutrient transport. In addition, the changes in the expression of mgtA, kdpA, and glnA are related to different effects on membrane permeability. The higher expression in the genes mrcA and mrcB is associated with morphological changes of cells treated with AlkCs and CsNPs. These findings contribute to our understanding of the molecular mechanisms underlying chitosan-induced stress responses and provide insights for the development of safer antimicrobial compounds in the future.

Molecular Characterization of Class A-ESBLs, Class B-MBLs, Class C-AmpC, and Class D-OXA Carbapenemases in MDR Acinetobacter baumannii Clinical Isolates in a Tertiary Care Hospital, West Bengal, India.

Background Acinetobacter calcoaceticus baumannii (ACB) complex has become a major concern nowadays because of its increasing involvement in several severe infections associated with catheter-related bloodstream and urinary tract infections, ventilator-associated pneumonia, cerebrospinal shunt-related meningitis, and wound infections. Multiple drug-resistant (MDR) ACB cases have been described with an increasing trend where at least it is resistant to a minimum of three antimicrobial groups. The mortality rate associated with A. baumannii is significantly higher than all Acinetobacterspp. isolates with the most prevalence seen in India and Thailand. The rapid spread of high resistance to most potent antimicrobial drugs is due to its ability to incorporate resistance determinants despite multifactorial reasons such as alteration in permeability of cell membrane by either losing expression of outer membrane porins or excess production of efflux pumps. This study aims to characterize resistance determinants responsible for MDR at the genetic level and emphasizes the use of genotyping in routine diagnosis as genotype analysis is reliable and valid. Methodology A total of 289 ACB complex clinical isolates were included in this study. The study for species-level identification of A. baumannii was conducted at the Department of Microbiology, IQ City Medical College Hospital, Durgapur, West Bengal. In addition, the detection of encoded genes associated with class A-extended spectrum beta-lactamases (i.e., CTX-M, KPC, SHV, and TEM genes), class B-metallo-β-lactamases (i.e., IMP, NDM, and VIM genes), Class C-AmpC cephalosporinase, and classD-OXA carbapenemases (i.e., blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235was done using real-time polymerase chain reaction. Results All 289 non-repetitive ACB complex clinical isolates were confirmed as A. baumannii, of which 277 (96%) isolates were MDR. There were no findings of blaCTX-M, blaKPC, blaSHV, blaTEM, blaIMP, blaVIM, blaAmpC, blaOXA-10/11, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-143, and blaOXA-235 genes in our study. However, there were four (1.44%) positive findings of the blaNDM gene. All MDR isolates (n = 277) were positive for the blaOXA-51 gene. In addition, blaOXA-23 was positive in 269 (97.12%) isolates. Conclusions The oxacillinase production corresponding to blaOXA-23 and blaOXA-51 were the most prevalent antibiotic resistance determinants among MDR A. baumanniiin our study. Four (1.44%) isolates had the multiple genes blaOXA-51, blaOXA-23, and blaNDM that shows the coexistence of diverse genetic elements involved in MDR A. baumannii, resulting in total resistance except for a few potent drugs such as colistin and tigecycline. Genotyping is helpful in determining the contribution of the isolates in understanding their association with encoded genes, which, in turn, helps in designing effective surveillance and control strategies in the management of such MDR isolates.

Nontypeable Haemophilus influenzae released from biofilm residence by monoclonal antibody directed against a biofilm matrix component display a vulnerable phenotype

Bacterial biofilms contribute significantly to pathogenesis, recurrence and/or chronicity of the majority of bacterial diseases due to their notable recalcitrance to clearance. Herein, we examined kinetics of the enhanced sensitivity of nontypeable Haemophilus influenzae (NTHI) newly released (NRel) from biofilm residence by a monoclonal antibody against a bacterial DNABII protein (α-DNABII) to preferential killing by a β-lactam antibiotic. This phenotype was detected within 5 min and lasted for ~ 6 h. Relative expression of genes selected due to their known involvement in sensitivity to a β-lactam showed transient up-regulated expression of penicillin binding proteins by α-DNABII NTHI NRel, whereas there was limited expression of the β-lactamase precursor. Transient down-regulated expression of mediators of oxidative stress supported similarly timed vulnerability to NADPH-oxidase sensitive intracellular killing by activated human PMNs. Further, transient up-regulated expression of the major NTHI porin aligned well with observed increased membrane permeability of α-DNABII NTHI NRel, a characteristic also shown by NRel of three additional pathogens. These data provide mechanistic insights as to the transient, yet highly vulnerable, α-DNABII NRel phenotype. This heightened understanding supports continued validation of this novel therapeutic approach designed to leverage knowledge of the α-DNABII NRel phenotype for more effective eradication of recalcitrant biofilm-related diseases.

Combined metabolomics and transcriptomics analysis reveals the mechanism of antibiotic resistance of Salmonella enterica serovar Typhimurium after acidic stress

Drug-resistant Salmonella is widely distributed in the meat production chain, endangering food safety and public health. Acidification of meat products during processing can induce acid stress, which may alter antibiotic resistance. Our study investigated the effects of acid stress on the antibiotic resistance and metabolic profile of Salmonella Typhimurium, and explored the underlying mechanisms using metabolomic and transcriptomic analysis. We found that acid-stressed 14028s was more sensitive to small molecule hydrophobic antibiotics (SMHA) while more resistant to meropenem (MERO). Metabolomic analysis revealed that enhanced sensitivity to SMHA was correlated with increased purine metabolism and tricarboxylic acid cycle. Transcriptomic analysis revealed the downregulation of chemotaxis-related genes, which are also associated with SMHA sensitivity. We also found a significant downregulation of the ompF gene, which encodes a major outer membrane protein OmpF of Salmonella. The decreased expression of OmpF porin hindered the influx of MERO, leading to enhanced resistance of the bacteria to the drug. Our findings contribute to greatly improve the understanding of the relationship between Salmonella metabolism, gene expression, and changes in drug resistance after acid stress, while providing a structural framework for exploring the relationship between bacterial stress responses and antibiotic resistance.

Reduced porin expression with EnvZ-OmpR, PhoPQ, BaeSR two-component system down-regulation in carbapenem resistance of Klebsiella Pneumoniae based on proteomic analysis.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has proven to be an urgent threat to human health. Proteomics (TMT/LC-MS/MS) and bioinformatics approaches were employed to explore the potential mechanisms underlying carbapenem resistance. Proteomic profiling of CRKP and susceptible KP (sKP) isolates revealed the involvement of outer membrane, beta-lactam resistance pathway, and two-component systems (TCSs) in carbapenem resistance. 27 CRKP strains and 27 susceptible Klebsiella pneumoniae strains were isolated from inpatients at the Second Xiangya Hospital, China to verify the mechanisms. Modified carbapenem inactivation method (mCIM) and PCR of common carbapenem resistance genes confirmed that 77.8% (21/27) of CRKP isolates were carbapenemase-producing. Porin decrease in CRKP isolates was found by SDS-PAGE and mRNA levels of major porins (OmpK35 and OmpK36). RT-qPCR detection of two-component systems (envZ, ompR, phoP, phoQ, baeS and baeR) revealed down-regulation of EnvZ-OmpR, PhoPQ, BaeSR TCSs. Expression of the TCSs, except ompR, were closely correlated with OMPs with the R-value >0.7. Together, this study reaffirmed the significance of the β-lactam resistance pathway in CRKP based on proteomic analysis. OmpK35/36 porin reduction and the controversial downregulation of EnvZ-OmpR, PhoPQ, and BaeSR TCSs were confirmed in carbapenem resistance of Klebsiella pneumoniae.

Resistance mechanisms in Gram-negative bacteria

Enterobacterales resistant to carbapenems or producing extended-spectrum β-lactamases (ESBL) and non-fermenters resistant to carbapenems present resistance to many of the antimicrobials commonly used in clinical practice, and have been recognized by the World Health Organization as a critical priority for the development of new antimicrobials. In this review, the main mechanisms of resistance of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia to β-lactams, quinolones, aminoglycosides and polymyxins will be addressed. Updated information will be presented on the importance in resistance of antimicrobial modification mechanisms (including class C or extended-spectrum β-lactamases, carbapenemases and aminoglycoside-modifying enzymes), permeability alterations due to porin or lipopolysaccharide expression disorders, production of active efflux pumps, target alterations or protection, and expression of two-component systems.

​Cloning and Expression Studies of the Major Outer Membrane Protein (OmpH) Gene of Pasteurella multocida P52 in Prokaryotic Vector

Background: The present study was aimed at cloning and expression of the major outer membrane porin (ompH) gene of P. multocida serotype B:2 P52, the strain used in vaccines against HS in India. The Porin H (OmpH) is major immunodominant outer membrane protein in the envelope of Pasteurella multocida. Methods: The gene encoding OmpH was amplified by PCR, cloned and expressed in prokaryotic expression vector. The recombinant OmpH protein was expressed as a protein with E. coli BL21 (DE3) cells transformed with recombinant plasmid pQE30-Xa-ompH. Result: The expressed protein was purified from E. coli and characterized by SDS-PAGE and western blot analysis. The fusion recombinant protein eluted had a molecular mass of about 34 kDa. The expressed recombinant protein was confirmed with western blot analysis using RGS-His antibody and anti-P. multocida serum raised against whole cell lysate.

Cefotaxime Exposure Selects Mutations within the CA-Domain of envZ Which Promote Antibiotic Resistance but Repress Biofilm Formation in Salmonella.

ABSTRACTCephalosporins are important beta lactam antibiotics, but resistance can be mediated by various mechanisms including production of beta lactamase enzymes, changes in membrane permeability or active efflux. We used an evolution model to study how Salmonella adapts to subinhibitory concentrations of cefotaxime in planktonic and biofilm conditions and characterized the mechanisms underpinning this adaptation. We found that Salmonella rapidly adapts to subinhibitory concentrations of cefotaxime via selection of multiple mutations within the CA-domain region of EnvZ. We showed that changes in this domain affect the ATPase activity of the enzyme and in turn impact OmpC, OmpF porin expression and hence membrane permeability leading to increased tolerance to cefotaxime and low-level resistance to different classes of antibiotics. Adaptation to cefotaxime through EnvZ also resulted in a significant cost to biofilm formation due to downregulation of curli. We assessed the role of the mutations identified on the activity of EnvZ by genetic characterization, biochemistry and in silico analysis and confirmed that they are responsible for the observed phenotypes. We observed that sublethal cefotaxime exposure selected for heterogeneity in populations with only a subpopulation carrying mutations within EnvZ and being resistant to cefotaxime. Population structure and composition dynamically changed depending on the presence of the selection pressure, once selected, resistant subpopulations were maintained even in extended passage without drug.IMPORTANCE Understanding mechanisms of antibiotic resistance is crucial to guide how best to use antibiotics to minimize emergence of resistance. We used a laboratory evolution system to study how Salmonella responds to cefotaxime in both planktonic and biofilm conditions. In both contexts, we observed rapid selection of mutants within a single hot spot within envZ. The mutations selected altered EnvZ which in turn triggers changes in porin production at the outer membrane. Emergence of mutations within this region was repeatedly observed in parallel lineages in different conditions. We used a combination of genetics, biochemistry, phenotyping and structural analysis to understand the mechanisms. This data show that the changes we observe provide resistance to cefotaxime but come at a cost to biofilm formation and the fitness of mutants changes greatly depending on the presence or absence of a selective drug. Studying how resistance emerges can inform selective outcomes in the real world.

Differential Gene Expression of Efflux Pumps and Porins in Clinical Isolates of MDR Acinetobacter baumannii.

Background: Acinetobacter baumannii is an opportunistic pathogen associated with healthcare infections and high mortality rates in intensive care units all over the globe. Porins and efflux pumps over-expression have been reported as contributing factors in escalating drug resistance and rendering treatment ineffective. In this study, we investigated the mechanisms of multidrug resistance (MDR) in A. baumannii clinical isolates. Methods: A total of 30 A. baumannii isolates were included in the present study from Nehru Hospital (PGIMER-Chandigarh) located in North India. Kirby Bauer disk diffusion assay and MIC were performed to determine the antimicrobial susceptibility pattern. Screening of beta-lactamases was performed using PCR. Relative gene expression of four RND, one MATE efflux pump, and two outer membrane proteins were determined using RT-PCR. Molecular typing of 22 isolates was carried out using MLST Oxford scheme. Results: CarO porin genes showed over-expression in 63% isolates followed by adeGandabeM efflux pump downregulation/underexpression (<0.5 fold), suggesting the carbapenem-susceptible phenotypic nature of the isolates. High prevalence of VIM-2, NDM-1, and OXA-23 genes was observed in A. baumannii isolates. Interestingly, NDM-1 and OXA-58 were traced in 10 and3 A. baumannii isolates respectively; 13 of 22 (59%) isolates showed novel Sequence Types (STs) in the Multi-Locus Sequence Typing (MLST) analysis. ST 1087 was most commonly found ST among all others (16 STs). Conclusions: This study indicated a possible role of carO porin genes and adeG (RND) andabeM (MATE) efflux pumps in carbapenem susceptibility of A. baumannii. New STs were also reported in the majority of the isolates.

Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.

Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria. Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels. Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE. Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates.