Alterations In Outer Membrane Proteins Research Articles

Activity of meropenem/vaborbactam and comparators against non-carbapenemase-producing carbapenem-resistant Enterobacterales isolates from Europe.

BackgroundCarbapenem-resistant Enterobacterales (CRE) isolates have disseminated worldwide. CREs usually produce a carbapenemase; however, some isolates are negative for known carbapenemases. In this study, we evaluated the activity of meropenem/vaborbactam and comparators against CREs without a carbapenemase (nonCP CREs) collected from European hospitals from 2016 to 2019.Materials and methods23 043 Enterobacterales clinical isolates were collected in 41 hospitals located in 20 countries. Susceptibility (S) testing was performed using the broth microdilution method. CLSI/EUCAST (2021) interpretive criteria were used. 978 CREs were identified with MICs >2 mg/L to meropenem or imipenem. Whole-genome sequencing was performed on each CRE isolate. 125 isolates were negative for carbapenemase genes, including blaKPC, blaNDM, blaIMP, blaVIM and blaOXA-48-like. NonCP CRE isolates were analysed for the presence of other β-lactamases, multilocus sequence types (ST) and mutations in outer membrane protein (OMP) sequences.ResultsMost nonCP CRE were Klebsiella pneumoniae (KPN; n = 97/125). 84.0% of nonCP CRE (n = 105) were from Poland, including 88 KPN. The most common β-lactamase was blaCTX-M-15 in 92/125 isolates. OMP disruptions or alterations were noted among 76 KPN. Among KPN isolates that had MLST typing, 30 belonged to ST11, 18 to ST152 and 17 to ST147, while 13 other STs were observed. Susceptibility to meropenem/vaborbactam was 96.0/97.6% (CLSI/EUCAST) while meropenem was 2.4/8.0%S.ConclusionsMeropenem/vaborbactam had potent in vitro activity against CRE isolates that lacked known carbapenemases. Resistance mechanisms observed among nonCP CREs included acquired β-lactamases and OMP alterations. These results indicate that meropenem/vaborbactam may be a useful treatment for infections caused by nonCP CREs.

Changes in Outer Membrane Proteins of Benzalkonium Chloride Adapted Pseudomonas Aeruginosa and Mutations in GyrA and ParC Genes

AbstractBackground: Benzalkonium Chloride (BC) is widely used in hospitals, industry and cosmetics. Adaptation of Pseu-domonas aeruginosa to BC was increased. This adaptation may lead to the emergence of cross-resistance to other disin-fectants and antibiotics. Little attention has been focused on the resistant mechanisms.Aim of Study: Examination of mutations in the Quinolone-resistance-Determining Region (QRDR) of GyrA and ParC genes and alterations of Outer-Membrane Proteins (OMPs) in Benzalkonium Chloride (BC)-adapted Pseudomonas aeru-ginosa isolates.Material and Methods: GyrA and ParC genes of the BC-adapted P. aeruginosa isolate and the wild-type strain ATCC 15442 were amplified by Polymerase-Chain-Reaction (PCR) and sequenced. OMPs of these isolates were also analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore-sis (SDS-PAGE) in the presence and absence of permeabilizer disodium Ethylenediamine-Tetraacetate (EDTA) to investigate the variations.Results: The results manifested a single mutation in both GyrA (Thr-83-Ile) and ParC (Ser-87-Leu). In SDS-PAGE, one band with molecular weight of 52.23kDa was detected in all samples. Seven bands with molecular weights of 71.44, 38.48, 36.01, 33.03, 26.17, 21.14 and 6.35kDa were stable in both ATCC 15442 and treated isolate. Also, seven bands with molecular weights of 120.83, 90.89, 79.72, 44.58, 28.91, 16.12 and 12.49kDa were newly induced in the treated isolate.Conclusions: These results revealed that mutations in GyrA and ParC genes, and alteration of OMPs contributed to the adaptation of P. aeruginosa to BC, co-operating with efflux pump over-expression.

Development and analysis of furazolidone-resistant Escherichia coli mutants.

Furazolidone-resistant mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe-Arg-β-Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS-PAGE. The frequency of mutation ranged from <9.6×10(-10) to 9.59×10(-7) . Neither an effect on efflux pumps inhibited by Phe-Arg-β-Naphtylamyde nor cross-resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase-encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone-resistant mutants, together with the lack of cross-resistance with unrelated antimicrobial agents.

An outbreak of infections caused by extensively drug-resistant Klebsiella pneumoniae strains during a short period of time in a Chinese teaching hospital: epidemiology study and molecular characteristics

In this study, we comprehensively described the clinical risk factors, outcome, epidemiology, and molecular basis associated with an outbreak of extensively drug-resistant KPC-2–producing Klebsiella pneumoniae involving 15 patients in a teaching hospital from May 1 to June 27, 2013. Most of the patients were elderly and received long-term hospital treatment, and 40.0% (6/15) of them were dead. All strains carried blaKPC-2, rmtB, blaCTX-M-65, blaSHV-11, oqxA, oqxB, and aac(6′)-Ib-cr and even harbored additional other resistance genes, such as armA, blaCTX-M-1, blaTEM-1. blaKPC-2, rmtB, and blaCTX-M-65 were located on the same ~54.2-kb plasmid, and conjugation experiments further proved the cotransferable characteristic. Alterations of outer membrane proteins were confirmed by sodium dodecyl sulfate - polyacrylamide gelelectrophoresis and sequencing, which can lead to a drastic change in the permeability of cells. All isolates belonged to the clone complex 258, spreading rapidly across the world. Our study demonstrated that a high degree of awareness and surveillance of those drug resistance determinants is urgently needed.

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica

ObjectivesTo study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. MethodsAntimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. ResultsFive Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2–4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. ConclusionsEfflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gram-negative clinical isolates.

Acinetobacter baumannii: a universal threat to public health?

Acinetobacter spp. are non-fermentative, strictly aerobic, Gram-negative microorganisms with a confusing taxonomic history. The Acinetobacter baumannii–Acinetobacter calcoaceticus complex is the species most commonly isolated from clinical specimens. It is ubiquitous in nature and has been found as part of the normal skin, throat and rectal flora as well as in food and body lice. It colonises patients in Intensive Care Units and contaminates inanimate hospital surfaces and devices as well as wounds, including war injuries. Although a frequent coloniser, Acinetobacter can be the cause of severe and sometimes lethal infections, mostly of nosocomial origin, predominantly ventilator-associated pneumonia. Bacteraemic infections are rare but may evolve to septic shock. Acinetobacter also emerges as a cause of nosocomial outbreaks and is characterised by increasing antimicrobial multiresistance. Antibiotic use, especially carbapenems and third-generation cephalosporins, is recognised as the most important risk factor for multiresistance. Described resistance mechanisms include hydrolysis by β-lactamases, alterations in outer membrane proteins and penicillin-binding proteins, and increased activity of efflux pumps. Today, Acinetobacter resistant to carbapenems, aminoglycosides and fluoroquinolones presents a challenge to the clinician. However, sulbactam, tigecycline and colistin represent the current therapeutic approaches, which are associated with satisfactory efficacy.

Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens

To characterize the mechanisms of fluoroquinolone resistance in urinary tract pathogens exhibiting a multiple antibiotic resistance phenotype as well as a high-level resistance to fluoroquinolones. Nineteen Escherichia coli urinary tract infection pathogens exhibiting high-level resistance to fluoroquinolones were characterized in this study. Alterations in outer membrane proteins (OMPs) and lipopolysaccharide (LPS) were analysed by PAGE. Changes to the quinolone resistance-determining regions (QRDRs) of GyrA and ParC were determined by PCR and DNA sequencing. The presence of the qnrA gene was determined by PCR amplification. Ciprofloxacin uptake was determined spectrophotometrically using the quinolone accumulation assay. OMP analysis showed decreased expression, the absence of certain proteins or the presence of proteins with altered molecular weights when compared with wild-type strains. Most isolates possessed a smooth LPS phenotype. Isolates had double mutations in GyrA codons 83 and 87, in addition to a ParC alteration at Ser-80/Glu-84. Isolates accumulated varying levels of ciprofloxacin, and upon the addition of carbonyl cyanide m-chlorophenylhydrazone, increased accumulation was observed in all instances. E. coli isolates with a rough LPS phenotype appeared to accumulate higher levels of ciprofloxacin compared with those with a smooth LPS phenotype expressing OmpF normally, or even those not possessing OmpF. All E. coli isolates tested demonstrated active efflux of ciprofloxacin. Plasmid-mediated quinolone resistance (presence of the qnrA gene) was observed in 36.8% of isolates. A combination of target gene alterations, altered OM permeability, presence of the qnrA gene and active efflux appear to act together to produce a high-level, multiresistance phenotype.

Outer membrane protein alterations and blaTEM-1 variants: their role in beta-lactam resistance in Klebsiella pneumoniae.

The aim of the study was to characterize the genetic basis of resistance to selected beta-lactam antibiotics in two clinical isolates of Klebsiella pneumoniae. K. pneumoniae strains were isolated from two hospitalized patients. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin but susceptible to all the other cephalosporins tested. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. PCR assays and DNA sequencing showed that the cefoxitin-susceptible strain contained a novel blaTEM-1 variant downstream of the strong Pa/Pb promoter. SDS-PAGE analysis of the outer membrane proteins (OMPs) did not identify OmpK35 and suggested reduced expression of OmpK36 in this strain. Following passage in non-selective media, expression of OmpK36 was restored with a concomitant increase in cefuroxime susceptibility. A similar experimental approach identified blaTEM-1C in the cefoxitin-resistant K. pneumoniae strain. This strain was deficient in OmpK35 and OmpK36; absence of the latter protein was due to the presence of IS1 in the ompK36 regulatory region. Resistance to selected beta-lactams in two clinical isolates of K. pneumoniae was due to interplay between the expression of OMPs and TEM-1.

Mechanism of quinolone resistance in anaerobic bacteria

Several recently developed quinolones have excellent activity against a broad range of aerobic and anaerobic bacteria and are thus potential drugs for the treatment of serious anaerobic and mixed infections. Resistance to quinolones is increasing worldwide, but is still relatively infrequent among anaerobes. Two main mechanisms, alteration of target enzymes (gyrase and topoisomerase IV) caused by chromosomal mutations in encoding genes, or reduced intracellular accumulation due to increased efflux of the drug, are associated with quinolone resistance. These mechanisms have also been found in anaerobic species. High-level resistance to the newer broad-spectrum quinolones often requires stepwise mutations in target genes. The increasing emergence of resistance among anaerobes may be a consequence of previous widespread use of quinolones, which may have enriched first-step mutants in the intestinal tract. Quinolone resistance in the Bacteroides fragilis group strains is strongly correlated with amino acid substitutions at positions 82 and 86 in GyrA (equivalent to positions 83 and 87 of Escherichia coli). Several studies have indicated that B. fragilis group strains possess efflux pump systems that actively expel quinolones, leading to resistance. DNA gyrase seems also to be the primary target for quinolones in Clostridium difficile, since amino acid substitutions in GyrA and GyrB have been detected in resistant strains. To what extent other mechanisms, such as mutational events in other target genes or alterations in outer-membrane proteins, contribute to resistance among anaerobes needs to be further investigated.

Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics.

Our objective was to determine whether strains of Pseudomonas aeruginosa can adapt to growth in increasing concentrations of the disinfectant benzalkonium chloride (BKC), and whether co-resistance to clinically relevant antimicrobial agents occurs. Attempts were made to determine what phenotypic alterations accompanied resistance and whether these explained the mechanism of resistance. Strains were serially passaged in increasing concentrations of BKC in static nutrient broth cultures. Serotyping and genotyping were used to determine purity of the cultures. Two strains were examined for cross-resistance to other disinfectants and antibiotics by broth dilution MIC determination. Alterations in outer membrane proteins and lipopolysaccharide (LPS) expressed were examined by SDS-PAGE. Cell surface hydrophobicity and charge, uptake of disinfectant and proportion of specific fatty acid content of outer and cytoplasmic membranes were determined. Two P. aeruginosa strains showed a stable increase in resistance to BKC. Co-resistance to other quaternary ammonium compounds was observed in both strains; chloramphenicol and polymyxin B resistance were observed in one and a reduction in resistance to tobramycin observed in the other. However, no increased resistance to other biocides (chlorhexidine, triclosan, thymol) or antibiotics (ceftazidime, imipenem, ciprofloxacin, tobramycin) was detected. Characteristics accompanying resistance included alterations in outer membrane proteins, uptake of BKC, cell surface charge and hydrophobicity, and fatty acid content of the cytoplasmic membrane, although no evidence was found for alterations in LPS. Each of the two strains had different alterations in phenotype, indicating that such adaptation is unique to each strain of P. aeruginosa and does not result from a single mechanism shared by the whole species.

Acid stress upregulated outer membrane proteins in clinical isolates ofNeisseria gonorrhoeae, but not most commensalNeisseria

Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.Key words: Neisseria gonorrhoeae, commensal Neisseria, acid stress, outer membrane proteins.

Acid stress upregulated outer membrane proteins in clinical isolates of &lt;i&gt;Neisseria gonorrhoeae&lt;/i&gt;, but not most commensal &lt;i&gt;Neisseria&lt;/i&gt;

Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.

Impaired imipenem uptake associated with alterations in outer membrane proteins and lipopolysaccharides in imipenem-resistant Shigella dysenteriae.

Three imipenem-resistant mutants were obtained from a clinical isolate (C152) of Shigella dysenteriae by selection with increasing concentrations of imipenem. Resistance to imipenem was associated with resistance to several other beta-lactam antibiotics. The penicillin-binding protein (PBP) patterns of the resistant and the wild-type strains were comparable. The permeability of the outer membrane proteins (OMPs) of the most resistant mutant, IM16, was lower than that of the parent strain C152 when imipenem and arabinose were used as test solutes. This mutant had lower levels of both the major OMPs of M(r) 43,000 and 38,000. There were also differences in the patterns of lipopolysaccharide (LPS) of the mutants and the wild-type strain. The mutant IM16 had less short-chain LPS than the parent C152. Increasing imipenem resistance was also associated with a concomitant decrease in the level of 2-keto-3-deoxyoctonate, a component of the core region of LPS.

Resistance of Pseudomonas aeruginosa to imipenem induced by eluates from siliconized latex urinary catheters is related to outer membrane protein alterations.

The activity of imipenem against Pseudomonas aeruginosa HUS-3 decreased by 16 times in the presence of substances eluted from siliconized latex urinary catheters (SLUCs). SLUCs did not inactivate imipenem or increase beta-lactamase activity. The outer membrane of P. aeruginosa HUS-3 grown in the presence of eluate lacked an OprD-like protein and expressed a new 50-kDa protein. The decreased activity of imipenem against P. aeruginosa in the presence of SLUCs is related to the loss of an OprD-like protein and the expression of a new outer membrane protein.

Alterations in outer membrane proteins identified in a clinical isolate of Aeromonas salmonicida subsp. salmonicida

Alterations in outer membrane proteins identified in a clinical isolate of <i>Aeromonas salmonicida</i> subsp. <i>salmonicida</i>

Impermeability to quinolones in gram-positive and gram-negative bacteria

The initial step in the accumulation of fluoroquinolone antimicrobial agents is binding to cell surface components reduced by lowered pH and divalent cations. Uptake into gram-negative and gram-positive bacteria is by simple diffusion. Entry through the outer membrane occurs preferentially for most agents by the porin route but a second process using the self-promoted uptake pathway is active especially for more hydrophobic agents. Fluoroquinolones bind to vesicles of phospholipid which may be the initiating step in cross-cytoplasmic membrane diffusion. An active efflux system has been described in Escherichia coli with evidence supporting its presence in several other bacteria. Total upset is not altered by a resistant gyrase. Resistant isolates associated with reduced total quinolone accumulation due to lowered uptake have been described for laboratory mutants and clinical isolates. Most but not all of these have had alterations in outer membrane proteins. A functionally dominant resistance gene has been cloned from resistant Staphylococcus aureus and codes for a highly hydrophobic protein most likely membrane associated. This gene is expressed in Escherichia coli and specifies resistance especially to hydrophilic quinolones, possibly by altered accumulation.

Cross resistance between oxytetracycline and oxolinic acid in Aeromonas salmonicida associated with alterations in outer membrane proteins

Oxytetracycline resistant mutants of Aeromonas salmonicida isolated from mutation frequency experiments showed decreased susceptibility to oxolinic acid. Outer membrane preparations of these resistant mutant strains revealed a major protein, with a molecular mass of approximately 37 kDa, which was not present in significant quantities in the parent strain.

Cross resistance between oxytetracycline and oxolinic acid inAeromonas salmonicidaassociated with alterations in outer membrane proteins

Oxytetracycline resistant mutants of Aeromonas salmonicida isolated from mutation frequency experiments showed decreased susceptibility to oxolinic acid. Outer membrane preparations of these resistant mutant strains revealed a major protein, with a molecular mass of approximately 37 kDa, which was not present in significant quantities in the parent strain.

A Novel Chromosomal TEM Derivative and Alterations in Outer Membrane Proteins Together Mediate Selective Ceftazidime Resistance in Escherichia coli

A clinical Escherichia coli isolate (MG32) resistant to ceftazidime but susceptible to other third-generation cephalosporins was previously examined and found to harbor TEM-1 and exhibit alterations in outer membrane proteins. Reexamination of this isolate revealed the additional presence of a novel TEM-1 derivative, now designated TEM-12. Evaluation of ceftazidime and cefotaxime minimum inhibitory concentrations for isogenic derivatives demonstrated a major role for TEM-12 in the decreased susceptibility observed. This was selectively enhanced for ceftazidime resistance by the altered porins of E. coli MG32. TEM-12 appeared identical to TEM-101, an in vitro TEM derivative, in both isoelectric point (pI 5.25) and substrate profile. Hybridization and cloning of the TEM-12 determinant revealed that, unlike other TEM derivatives, TEM-12 is chromosomally encoded, not plasmid-encoded.

Role of the Outer Membrane for Quinolone Resistance in Enterobacteria

Quinolone-resistant clones were selected from clinical Escherichia coli, Citrobacter freundii and Serratia marcescens isolates in a frequency ranging from 10(-8) to 10(-6). The outer membrane proteins of quinolone-resistant E. coli clones remained unaltered, as was the case for 10 of 11 C. freundii and 4 of 11 S. marcescens clones ('nal B' type). There was no strong relation between alterations of outer membrane proteins and cross-resistance with chemically unrelated compounds such as tetracycline or chloramphenicol; however, tetracycline resistance was observed in some C. freundii clones with unaltered outer membrane proteins ('mar A'). Most of the quinolone-resistant S. marcescens clones can be considered 'nor B' or 'nor C' mutants due to their cross-resistance with other compounds, their altered outer membrane proteins and changes of lipopolysaccharide. In a few cases, subinhibitory quinolone concentrations caused alterations of outer membrane proteins in S. marcescens during mid log phase without development of resistance.