Changes In Outer Membrane Proteins Research Articles

Chlorhexidine leads to the evolution of antibiotic-resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a major public-health concern. We evaluate chlorhexidine role in selection of resistant Pseudomonas aeruginosa mutants and their antibiotic cross-resistance. Mutation frequency and mutation rate after short-term exposure to sub-inhibitory concentrations of chlorhexidine were compared to those after spontaneous chlorhexidine-exposure, in P. aeruginosa PAO1 strain. Chlorhexidine-resistant mutants were generated, either by serial passage in increasing chlorhexidine concentrations or by single exposure to lethal chlorhexidine concentration. The generated mutants were tested for cross-resistance to different antibiotics, by determination of minimum inhibitory concentrations (MIC). The accompanied phenotypic changes in membrane permeability, outer membrane proteins (OMP), and efflux function were evaluated. The effect of exposure to chlorhexidine on MexAB-OprM, MexEF-oprN, and MexXY efflux pumps expression was investigated. No significant change was recorded between the mutation frequencies and mutation rates after short-term exposure to sub-inhibitory concentrations of chlorhexidine and after spontaneous chlorhexidine-exposure, in P. aeruginosa PAO1 strain. Twelve stable mutants, with ≥ eight-fold increase in chlorhexidine MIC, were generated. Several mutants showed increase in the MIC of colistin, cefepime, ceftazidime, meropenem, ciprofloxacin, and amikacin; seven mutants expressed meropenem cross-resistance. This was accompanied by decreased outer membrane permeability and changes in OMP. Using efflux pump inhibitor, chlorhexidine resistance was reverted in most isolates. Exposure to sub-inhibitory concentration of chlorhexidine induced the expression of MexXY efflux pump. Some resistant mutants had overexpressed MexXY efflux pump. Chlorhexidine can select P. aeruginosa strains with antibiotic cross-resistance. This necessitates implementing special protocols for chlorhexidine use and re-evaluation of its benefit versus risk in personal-care products.

First genome sequence of Chilean Brucella canis SCL strain provides insights on the epidemiology and virulence factors, explaining differences between geographical origins

Brucella canis is the etiological agent of canine brucellosis, a worldwide neglected zoonosis that constitutes one of the major infectious causes of infertility and reproductive failure in dogs. Although genomic information available for this pathogen has increased in recent years, here we report the first genome sequencing of a B. canis strain in Chile, and the differences in virulence genes with other B. canis strains. Genome assembly produced a total length of 3,289,216 bp, N50 of 95,163 and GC% of 57.27, organized in 54 contigs in chromosome I, and 21 contigs in chromosome II. The genome annotation identified a total of 1981 CDS, 3 rRNA and 36 tRNA in chromosome I, and 1113 CDS and 10 tRNA in chromosome II. There is little variation between the different strains and the SCL isolate. Phylogenetic analysis showed that the Chilean SCL strain is closely related to B. canis and B. suis strains. Small differences were found when compared to the Serbian isolate, but all strains shared the same recent common ancestor. Finally, changes in the sequence of some virulence factors showed that the SCL strain is similar to other South American B. canis strains. This work sequenced and characterized the complete genome of B. canis strain SCL, evidencing the complete presence of all the genes of the vir B operon, and minor changes in outer membrane proteins and in the urease operon. Our data suggest that B. canis was introduced from North America and then spread throughout the South American continent. How to cite: Borie C, Bravo C, Dettleff P, et al. First genome sequence of Chilean Brucella canis SCL strain provides insights on the epidemiology and virulence factors, explaining differences between geographical origins Electron J Biotechnol 2021;49. https://dx.doi.org/10.1016/j.ejbt.2020.10.002 .

The synergy of resveratrol and alcohol against Helicobacter pylori and underlying anti-Helicobacter pylori mechanism of resveratrol.

To determine individual antibacterial and synergistic antibacterial effects of resveratrol and alcohol against Helicobacter pylori 26695 in vitro, and to elucidate the underlying mechanism of action of resveratrol against H. pylori. The minimum inhibitory concentrations (MICs) and time-killing curve of resveratrol and alcohol were determined. Transcriptome analysis by RNA sequencing was used to elucidate the underlying mechanism of action of resveratrol against H. pylori. Our results showed that the MICs of resveratrol and alcohol against H. pylori 26695 are about 64μgml-1 and 4% (v/v) respectively. The synergy was found: resveratrol at concentration of 64μgml-1 in combination with alcohol at concentration of 4% (v/v) showed >10000-fold decrease in the mount of viable bacteria compared with resveratrol and alcohol used alone. Transcriptome analysis showed 152 genes were downregulated and 111 genes were upregulated in the presence of resveratrol. Genes involved in protein translation (17·1%), outer membrane proteins (OMPs) (9·9%) and transports (11·2%) comprise 38·2% of the downregulated genes. In comparison, genes involved in redox (13·5%), pathogenesis and motility (9·9%) and iron homeostasis (4·5%) comprise 27·9% of the upregulated genes. The synergy of resveratrol and alcohol against H. pylori was found in this study. The underlying mechanism of action of resveratrol against H. pylori may be mainly attributed to its inhibitory effect on translation, OMPs, transports, ATP synthase and possible oxidative damage. Our study provides a global insight into the anti-H. pylori mechanism of resveratrol. Both resveratrol and alcohol can contribute to inhibition of ribosomes, changes in OMPs and oxidative damage, which may be the explanations of synergistic effect against H. pylori elicited by resveratrol and alcohol.

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.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Synergistic anti-bacterial and proteomic effects of epigallocatechin gallate on clinical isolates of imipenem-resistant Klebsiella pneumoniae

Imipenem-resistant Klebsiella pneumoniae (IRKP) were used to explore the synergistic anti-bacterial and proteomic effects of imipenem alone or in combination with epigallocatechin gallate (EGCg). The minimal inhibitory concentrations (MICs) of EGCG for 12 clinically isolated IRKP strains ranged from 300 to 650 μg/ml. Each of the 12 IRKP strains experienced a 4- to 64-fold reduction in the MIC of imipenem upon co-incubation with 0.25 × MIC level of EGCg. The time-kill method was used on the 12 IRKP clinical isolates to evaluate the bactericidal activities of imipenem alone or with EGCg. Compared to imipenem alone, EGCg with imipenem demonstrated enhanced bactericidal activity. Two-dimensional polyacrylamide gel electrophoresis identified eight down-regulated and four up-regulated proteins in the IRKP strain upon exposure to 1 × MIC of EGCg. Analysis of the outer membrane protein profiles of IRKP cultures treated with EGCg revealed unique changes in outer membrane proteins. In addition, scanning electron microscopic analysis demonstrated the presence of cells with wrinkled surfaces containing perforations and irregular rod-shaped forms after treatment with EGCg or imipenem. These studies demonstrate that EGCg can synergize the bacterial activity of imipenem and differentially stimulate the expression of various proteins in IRKP.

Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa

The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results demonstrated that the rhamnolipid at concentrations below and above CMC provoked a multi-component response of the bacterial cells without affecting their growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F, Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa. In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine.

Evaluation of the contemporary occurrence rates of metallo-β-lactamases in multidrug-resistant Gram-negative bacilli in Japan: Report from the SENTRY Antimicrobial Surveillance Program (1998–2002)

Metallo-β-lactamases (MβL) were initially characterized in Japan, usually of the IMP-type, and found in Pseudomonas aeruginosa (PSA), Acinetobacter spp. (ACB), or Serratia marcescens (SM). The number of MβL types has increased worldwide, but geographic dissemination within Japan has appeared limited. This study compares baseline levels of MβL resistance from two 22-center studies (1996–1997) to the longitudinal sample (3 sites) of Japanese isolates from the SENTRY Antimicrobial Surveillance Program (1998–2002). All minimal inhibitory concentration results were determined by reference methods. A total of 26.8% PSA, 3.4% ACB, and 3.1% Enterobacteriaceae (enterobacters and SM) with resistance to monitored carbapenems (CARB) (minimal inhibitory concentration, ≥8 μg/mL) were screened for MβL production by disk approximation tests (EDTA and 2-MPA inhibitors), CARB hydrolysis by enzyme extracts, and selected PCR primers for known MβL types. All MβL-positive strains (10) were sequenced to determine enzyme identification. Clonality in each center was determined by automated ribotyping and PFGE. The CARB susceptibility rates in PSA decreased (80.7% to 62.0%) over the monitored interval (1998–2002), but varied by medical center location. Among CARB-resistant isolates, 10.8% were attributed to MβL strains (1.1% of all PSA tested). MβL identification showed the following: five PSA (three IMP-1, two IMP-2), four SM (one IMP-1, two IMP-1 + OXA-1, and one IMP-11). Also a single ACB had an IMP-1. Eight of 10 MβL isolations occurred between 2000 and 2002; four occurred in 2002. BRL42715, an AMP-C inhibitor, confirmed AMP-C–mediated resistance in 87.3% of PSA, and outer membrane protein changes were also discovered by membrane studies. Prior results (22 sites, 1997–1998) showed CARB resistance at 22.4–25.6% and 0.5–0.9% MβLs (IMP-1) overall; it was slightly elevated in this SENTRY Program sample. In conclusion, MβL-producing strains from several species persist in Japan, but represent a distinct minority of all CARB-resistant strains (1998–2002). Although MβL rates appear generally stable in Japan, continued surveillance for these mechanisms seems to be a prudent practice, because of the mobility of the genetic determinants and the emergence of novel enzyme types, especially among the Enterobacteriaceae.

The effect of starvation stress on the porin protein expression of Escherichia coli in lake water.

The aim was to identify changes in outer membrane proteins (omps), OmpA, OmpC and OmpF, in Escherichia coli under starvation conditions in lake water microcosms studied at different temperatures. Escherichia coli was incubated in lake water microcosms at a variety of temperatures and the omps studied using quantitative densitometric analysis of protein bands of sodium dodecyl sulphate gels of omp preparations. The amount of OmpF increased over the incubation period relative to that of OmpC whereas the relative abundance of OmpA declined, most notably at 25 and 37 degrees C. This change was linked to changes in peak cell volume as determined by cell measurements. Major changes to the omps of E. coli accompany the adaptation of the organism to starvation conditions in lake water microcosms. Prolonged starvation affects the relative amounts of outer membrane porins. This study furthers the understanding of the role played by changes in the omp composition in E. coli during survival in lake water environments.

Selection of Klebsiella pneumoniae Mutants with High-Level Cefotaxime Resistance during Growth in Serum Containing Therapeutic Concentrations of Cefotaxime

Background: In a previous investigation of the genetic characterization of extended-spectrum β-lactamases (ESBLs) in Klebsiella pneumoniae from Zagreb, Croatia, 20 strains were found to produce SHV-2 β-lactamase. Those strains displayed varying degrees of β-lactam resistance and a wide range of β-lactamase activity. We concluded that more resistant isolates were hyperproducers of SHV-2 β-lactamase. Methods: In this investigation, we tried to develop hyperproducing variants from 8 low-level SHV-2 β-lactamase-producing Klebsiella strains by subculturing them in serum containing therapeutic concentrations of cefotaxime (CTX). Results: In most cases, there was a moderate increase in CTX resistance (twofold to threefold), except in one strain which displayed a 16-fold increase in the minimum inhibitory concentration (MIC) of CTX after incubation in the serum. That strain showed a marked increase in enzyme activity as well. The strains with a moderate increase in CTX MIC did not produce more enzyme after exposure to the serum, except for one strain which had a threefold rise in β-lactamase activity after exposure to serum. Conclusions: In this investigation, it was established that the mutants with high-level CTX resistance developed very quickly in the biological fluids containing therapeutic concentrations of CTX. It is reasonable to expect that a similar process occurs in patients infected with an ESBL-producing K. pneumoniae strain during antibiotic treatment. Since most of the high-level CTX-resistant mutants did not have a marked rise in β-lactamase activity after exposure to serum, it is possible that the elevated resistance was due to some other mechanism, such as reduced penicillin-binding protein affinity, changes in outer membrane proteins or efflux by multidrug efflux pumps.

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg+) Bordetella bronchiseptica cultures. Congo red binding and urease activity of Bvg+ B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as filamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (> or = 26 nmol ml-1 of CR and < or = 2.6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO4 20 mmol l-1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO4 5 mmol l-1 cultures) could be distinguished. CR binding assay and urease activity are specific and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP profiles. The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production.

Outer membrane changes in Pseudomonas stutzeri resistant to chlorhexidine diacetate and cetylpyridinium chloride

Changes in outer membrane proteins (OMP) and lipopolysaccharide (LPS) in cells of strains of Pseudomonas stutzeri sensitive and resistant to chlorhexidine diacetate (CHA) or cetylpyridinium chloride (CPC) have been examined. Four of five CHA-resistant strains had alterations in OMP profiles, including the expression of two additional protein bands. All the CPC-resistant strains had altered OMP profiles but the changes varied from strain to strain. Loss of the fastest-migrating bands was observed in the LPS from CHX-resistant strains. In strain JM 302R with high-level CHX resistance (minimal inhibitory concentration 100 mg/l as opposed to 2.5 mg/l in the parent-strain, JM 302), all the fast-migrating bands were lost and the strain showed ‘cross-resistance’ to polymyxin, gentamicin and ethylenediamine tetraacetic acid. It is however, possible that the altered LPS patterns reflect a response to alterations in other components rather than being directly associated per se with the enhanced resistance.

High-level expression of chromosomally encoded SHV-1 beta-lactamase and an outer membrane protein change confer resistance to ceftazidime and piperacillin-tazobactam in a clinical isolate of Klebsiella pneumoniae.

We describe Klebsiella pneumoniae 15571, a clinical isolate resistant to ceftazidime MIC = 32 microg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 microg/ml). K. pneumoniae 15571 expresses a single beta-lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector in Escherichia coli, this bla(SHV-1) gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described bla(SHV-1) genes. Outer membrane protein (OMP) analysis of K. pneumoniae 15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in K. pneumoniae 44NR, a low-level ampicillin-resistant strain that also expresses a chromosomally determined bla(SHV-1). Crude beta-lactamase enzyme extracts from K. pneumoniae 15571 produced roughly 200-fold more beta-lactamase activity than K. pneumoniae 44NR. Northern hybridization analysis revealed that this difference was explainable by quantifiable differences in transcription of the bla(SHV-1) gene in the two strains. Primer extension analysis of bla(SHV-1) mRNA from K. pneumoniae 15571 and 44NR indicated that the transcriptional start sites were identical in the two strains. DNA sequencing of the promoter regions upstream of the of bla(SHV-1) open reading frames in the two K. pneumoniae strains revealed an A-->C change in the second position of the -10 region in K. pneumoniae 44NR compared to that in 15571. Site-directed mutagenesis of the cloned K. pneumoniae 15571 bla(SHV-1), in which the A in the second position of the 15571 -10 region was changed to a C, resulted in a substantial lowering of the MIC of ampicillin. When the levels of beta-lactamase enzyme expression in E. coli were compared, the bla(SHV-1) downstream of the altered -10 region produced 17-fold less beta-lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a combination of increased enzyme production and minor OMP changes and that levels of chromosomally encoded SHV-1 beta-lactamase production can vary substantially with a single-base-pair change in promoter sequence.

Colonial Opacity Variations among the Choleragenic Vibrios

Cultures of Vibrio cholerae 01, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the new V. cholerae serogroup O139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors. By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished. In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique. These (and older) observations prompted the questions: (1) why is a V. cholerae colony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-beta-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in 0139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions. Colonial variations in choleragenic vibrios are phenotypically multifactorial. The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.

Serum survival and plasmid possession by strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow, carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.

Outer Membrane Protein Changes During Bacteroid Development Are Independent of Nitrogen Fixation and Differ Between Indeterminate and Determinate Nodulating Host Plants ofRhizobium leguminosarum

The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular basis of the development of bacterium to bacteroid, we wanted to know whether these outer membrane protein differences are conserved in various plant-Rhizobium combinations, For this purpose we developed a faster assay in which cell lysates instead of isolated cell envelopes were used to analyze these outer membrane changes, With this method the previously described low levels of antigen groups II and III in isolated bacteroid cell envelopes were confirmed, Moreover the described decrease in antigen groups II and III was also found in bacteroids of Rhizobium leguminosarum by. viciae with a mutated nifA or nifK gene as well as in the non-fixing pea mutant FN1 inoculated with the wild-type strain 248, This indicates that the decrease in the antigen levels is not restricted to effective nodules, The results also showed that the decrease in antigen group II not only occurs in bacteroids from pea, but also in bacteroids from vetch, broadbean, white clover, and common bean, Antigen group III, however, remained present in bacteroids from common bean, It is concluded that the changes in antigen group II are not restricted to a specific cross-inoculation group but represent a general phenomenon in the rhizobial bacteroid differentiation process, Of the tested plants, the decrease in antigen group III was not found in bacteroids from common bean and appeared to be restricted to bacteroids from indeterminate nodules. Therefore one should expect that at least two molecular mechanisms are responsible for these outer membrane protein changes and that elucidation of these mechanisms will contribute to our understanding of bacteroid development.

Persistence mechanisms in Pseudomonas aeruginosa from cystic fibrosis patients undergoing ciprofloxacin therapy.

The mechanisms of persistence to ciprofloxacin in nine sets of Pseudomonas aeruginosa strains isolated during ciprofloxacin therapy of chronic lung infections in cystic fibrosis patients were studied. Low to moderate levels of ciprofloxacin resistance developed in each case. Each set of pretherapy ciprofloxacin-susceptible, during-therapy ciprofloxacin-resistant, and posttherapy ciprofloxacin-susceptible isolates were shown to be genotypically related by using a radiolabeled epidemiological gene probe. All ciprofloxacin-resistant isolates were found to have altered susceptibilities to both nalidixic acid and various chemically unrelated antibiotics. Analysis of possible resistance mechanisms showed that the strains had altered outer membrane protein or lipopolysaccharide profiles. Complementation of possible DNA gyrase mutations with a plasmid-borne, wild-type Escherichia coli gyrA gene indicated that altered DNA gyrase was at least partly responsible for ciprofloxacin resistance in all strains tested. Attempts to generate ciprofloxacin-susceptible revertants in vitro showed that in some strains reversion was rapid in the absence of ciprofloxacin, while in other strains it was not possible to generate revertants. These data indicate that persistence of Pseudomonas aeruginosa to ciprofloxacin involves changes in DNA gyrase and is associated with pleiotropic changes in outer membrane proteins and lipopolysaccharide.

Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid

Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 micrograms/ml) but fluoroquinolone-resistant (8 to 16 micrograms/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy. As compared with the susceptible E. coli Q1 isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5- to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid. A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E. coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E. coli Q1 and Q2 with similar amounts of nalidixic acid. Selection from E. coli Q1 on norfloxacin of one-step resistant mutants resembling E. coli Q2 was unsuccessful. From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E. coli Q2.

β-Lactamase expression and outer membrane protein changes in cefpirome-resistant and ceftazidime-resistant Gram-negative bacteria

Twenty-five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) and five strains of Pseudomonas aeruginosa were exposed to various concentrations of cefpirome or ceftazidime in agar. Mutants with a greater than four-fold increase in the MIC were examined for changes in beta-lactamase expression and outer membrane protein (OMP) profile. Both agents selected mutants with decreased susceptibility to the selecting antibiotic and other beta-lactams at a frequency of 10(-7)-10(-8). The MICs of all beta-lactams were higher for the resistant mutants of E. cloacae and P. aeruginosa than for the other species. Both agents selected mutants expressing derepressed class I beta-lactamase, but this was more common with ceftazidime. Only a few mutants of P. aeruginosa and E. cloacae had an MIC of cefpirome that was above the recommended breakpoint concentration. Some mutant strains of Enterobacteriaceae lacked an OMP of molecular size similar to OmpF, but the MIC of cefpirome was below the breakpoint concentration for all these strains.

Analysis of acquired ciprofloxacin resistance in a clinical strain of Pseudomonas aeruginosa

Decreasing susceptibility to ciprofloxacin was investigated in sequential clinical isolates of Pseudomonas aeruginosa from a patient on ciprofloxacin therapy. All isolates were verified as the same strain by DNA probe. MICs of all quinolones tested were 16- to 32-fold higher for the posttherapy isolates; nonquinolone MICs were unchanged. The isolates were compared by analyses of outer membrane proteins and lipopolysaccharide composition, antimicrobial susceptibilities, measurement of accumulation of ciprofloxacin, and inhibition of DNA gyrase activity by ciprofloxacin and nalidixic acid. No significant changes in outer membrane proteins or ciprofloxacin accumulation were observed; however, both posttherapy isolates lost the long chain O-polysaccharide component of lipopolysaccharide. Preparations of DNA gyrase from the quinolone-resistant posttherapy isolates were 16- to 32-fold less sensitive to inhibition of supercoiling by ciprofloxacin and nalidixic acid than was gyrase from the pretherapy isolate. Inhibition studies on combinations of heterologous gyrase subunits showed that decreased inhibition was conferred by the resistant gyrase A subunits. Thus, acquired resistance to ciprofloxacin in this strain involved an alteration in the A subunit of DNA gyrase and was associated with changes in lipopolysaccharide.