Ciprofloxacin-resistant Mutants Research Articles

DinB (DNA polymerase IV), ImuBC and RpoS contribute to the generation of ciprofloxacin-resistance mutations in Pseudomonas aeruginosa

We investigated the role(s) of the damage-inducible SOS response dinB and imuBC gene products in the generation of ciprofloxacin-resistance mutations in the important human opportunistic bacterial pathogen, Pseudomonas aeruginosa. We found that the overall numbers of ciprofloxacin resistant (CipR) mutants able to be recovered under conditions of selection were significantly reduced when the bacterial cells concerned carried a defective dinB gene, but could be elevated to levels approaching wild-type when these cells were supplied with the dinB gene on a plasmid vector; in turn, firmly establishing a role for the dinB gene product, error-prone DNA polymerase IV, in the generation of CipR mutations in P. aeruginosa. Further, we report that products of the SOS-regulated imuABC gene cassette of this organism, ImuB and the error-prone ImuC DNA polymerase, are also involved in generating CipR mutations in this organism, since the yields of CipR mutations were substantially decreased in imuB- or imuC-defective cells compared to wild-type. Intriguingly, we found that the mutability of a dinB-defective strain could not be rescued by overexpression of the imuBC genes. And similarly, overexpression of the dinB gene either only modestly or else failed to restore CipR mutations in imuB- or imuC-defective cells, respectively. Combined, these results indicated that the products of the dinB and imuBC genes were acting in the same pathway leading to the generation of CipR mutations in P. aeruginosa. In addition, we provide evidence indicating that the general stress response sigma factor σs, RpoS, is required for mutagenesis in this organism and is in part at least modulating the dinB (DNA polymerase IV)-dependent mutational process. Altogether, these data provide further insight into the complexity and multifaceted control of the mutational mechanism(s) contributing to the generation of ciprofloxacin-resistance mutations in P. aeruginosa.

Genomic Characterization of Ciprofloxacin Resistance in Laboratory-Derived Mutants of Vibrio parahaemolyticus

The quinolone ciprofloxacin is a broad-spectrum bactericidal antibiotic used for human medicine as well as the aquaculture industry. The emergence of ciprofloxacin-resistant Vibrio parahaemolyticus strains is currently a global public health concern. However, the mechanism of ciprofloxacin resistance in V. parahaemolyticus is not yet fully clarified. We generated mutants with decreased ciprofloxacin susceptibility using in vitro selection and investigated genes associated with ciprofloxacin resistance on a genetic level. Our selection process yielded mutants that possessed altered minimal inhibitory concentrations (MICs) for ciprofloxacin and other unrelated antibiotics. These included Ser83Ile mutations in GyrA and Val461Glu in ParE as well as mutations in the resistance nodulation cell division (RND) family transporter gene vmeD and the putative TetR family regulator gene vp0040 upstream of the vmeCD operon. Measurements of steady-state mRNA levels revealed that the ciprofloxacin-resistant mutants overexpressed vmeCD. Further, the introduction of the vp0040 mutated allele from H512 into the sensitive parental strain increased the MIC for ciprofloxacin 31.25-fold. Taken together, these results indicated that ciprofloxacin resistance in these mutants was due to the quinolone resistance determining region mutation as well as overexpression of vmeCD caused by a loss of vp0040 gene repression. This also accounted for the presence of the multidrug resistance phenotype for these mutant strains since RND efflux system can export structurally unrelated antibiotics.

Inactivation of UmuC Protein Significantly Reduces Resistance to Ciprofloxacin and SOS Mutagenesis in Escherichia coli Mutants Harboring Intact umuD Gene

Background: Ciprofloxacin induces SOS response and mutagenesis by activation of UmuD’2C (DNA polymerase V) and DinB (DNA polymerase IV) in Escherichia coli, leading to antibiotic resistance during therapy. Inactivation of DNA polymerase V can result in the inhibition of mutagenesis in E. coli. Objectives: The aim of this research was to investigate the effect of UmuC inactivation on resistance to ciprofloxacin and SOS mutagenesis in E. coli mutants. Methods: Ciprofloxacin-resistant mutants were produced in a umuC- genetic background in the presence of increasing concentrations of ciprofloxacin. The minimum inhibitory concentration of umuC-mutants was measured by broth dilution method. Alterations in the rifampin resistance-determing region of rpoB gene were assessed by PCR amplification and DNA sequencing. The expression of SOS genes was measured by quantitative real-time PCR assay. Results: Results showed that despite the induction of SOS response (overexpression of recA, dinB, and umuD genes) following exposure to ciprofloxacin in E. coli umuC mutants, resistance to ciprofloxacin and SOS mutagenesis significantly decreased. However, rifampicin-resistant clones emerged in this genetic background. One of these clones showed mutations in the rifampicin resistance-determining region of rpoB (cluster II). The low mutation frequency of E. coli might be associated with the presence and overexpression of umuD gene, which could somehow limit the activity of DinB, the location and type of mutations in the β subunit of RNA polymerase. Conclusions: In conclusion, for increasing the efficiency of ciprofloxacin against Gram-negative bacteria, use of an inhibitor of umuC, along with ciprofloxacin, would be helpful.

Exposure to Sublethal Ciprofloxacin Induces Resistance to Ciprofloxacin and Cross-Antibiotics, and Reduction of Fitness, Biofilm Formation, and Apx Toxin Secretion in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 μg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

Resistance to widely-used disinfectants and heavy metals and cross resistance to antibiotics in Escherichia coli isolated from pigs, pork and pig carcass

A total of Escherichia coli isolated from pigs (n = 643), pork (n = 111) and pig carcasses (n = 110) in Thailand during 2008–2018 were included. Susceptibilities to antibiotics, disinfectants (i.e. triclosan (TCS), chlorhexidine digluconate (CHX), benzalkonium chloride (BKC), glutaraldehyde (GLU), formaldehyde (FOR)) and heavy metals (i.e. zinc chloride and copper sulfate) were examined. The E. coli isolates susceptible to all antibiotics (n = 24) were included for in vitro exposure experiment. The effect of phenylalanine arginine β-naphthylamide (PAβN) on the susceptibilities was determined. The results showed that the majority of E. coli (89%) from pigs, pig carcass and pork were multidrug resistant strains. The MICs of all disinfectants and heavy metals, except triclosan were clustered under 1 or 2 concentrations, indicating that there was no development or development at limited degree of resistance to these substances. TCS MICs for E. coli isolated in 2011–2014 were significantly higher than that of the strains in 2008–2010. The weak correlation between biocide MICs (BKC, GLU, TCS, CHX and CuSO4) and antibiotic resistance was observed (p < 0.05). Exposure to triclosan, benzalkonium chloride and chlorhexidine digluconate selected for spontaneous-resistant mutants exhibited cross resistance to at least one antibiotic. The cross resistance was observed between TCS and all eight antimicrobial drugs; BKC and chloramphenicol/ciprofloxacin/sulfamethoxazole/tetracycline; and CHX and ciprofloxacin/gentamicin/streptomycin. The presence of PAβN restored MICs of chloramphenicol and trimethoprim in some BKC- and TCS-spontaneous resistant mutants, highlighting the important role of multidrug efflux system as cross-resistance mediated mechanism. The presence of PAβN, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine could not restore ciprofloxacin MIC in some ciprofloxacin-resistant mutant derivatives with no mutation in neither gyrA nor parC, suggesting the existence of proton motive force-independent mechanisms. In conclusions, the widely-used disinfectants and heavy metals play an important role as non-antibiotic selective pressure for emergence and spread of AMR. Therefore, susceptibilities to disinfectants/heavy metals should be routinely monitored.

Stationary phase selects predominantly ciprofloxacin resistant strains of Salmonella Typhimurium with mutation in gyrA

This study was focused on determination the mutation frequency leading to ciprofloxacin resistance as well as mechanism of resistance to ciprofloxacin and changes in the ability to form a biofilm at various growth stages of S. Typhimurium. The mutation frequency leading to resistance (resistance index RI) was expressed as the mean number of resistant colonies divided by the mean of the total number of viable cells per culture. The resistance index (RI) was increased linearly over time and it had the highest value in the death phase. The mutations in the Quinolone Resistance Determining sources Regions (QRDR) of gyrA in S. Typhimurium strain were identified. All mutations identified in this study were substitutions in the QRDR of the gyrA gene, Serine 83 (Ser 83) and Aspartate 87 (Asp 87). The Ser 83 mutations were found already in exponential phase and their occurrence increased at other growth stages. Point mutations in Asp-87 were found only in later growth stages. The Ser 83 mutations occurred in all resistant strain obtained from stationary phase, with substitutions by Phenylalanine (Phe). Detection of gyrA mutations was carried out by AS-PCR-RFLP assay. This occurrence correlated with the increased expression of efflux pumps in mutant strains. The highest over-expression of efflux pump was found in resistant strains obtained from 96 h of cultivation (death phase). Ciprofloxacin resistant mutants formed a biofilm to a greater extent compared to the parental strain. Our data strengthen opinion that stressful environment conditions selects for antibiotic resistant mutants and in case of Salmonella spp. could contribute to improved ability to form biofilm.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ± 0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ± 4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ± 0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ± 0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

Expression of TolC and Organic Solvent Tolerance of Escherichia Coli Ciprofloxacin Resistant Mutants.

AcrAB-TolC is a major efflux pump in Escherichia coli. It was reported that tolC is overexpressed and involves in improving the organic solvent tolerance level in Escherichia colimarR mutants that are resistant to several antibiotics, such as ciprofloxacin. Low and intermediate levels resistance did not improve organic solvent tolerance. Thus, it was decided to measure tolC expression and organic solvent tolerance in high level ciprofloxacin resistant mutants. tolC expression was measured by real time PCR and organic solvent tolerance assay was conducted by counting bacterial colonies on LBGMg agar. Results showed that tolC expression was increased significantly (P<0.05) and organic solvent tolerance was slightly improved in high resistant mutants. It was concluded that high organic solvent tolerance may need higher expression of tolC.

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

Pseudomonas aeruginosa develops Ciprofloxacin resistance from low to high level with distinctive proteome changes

Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. Different Ciprofloxacin concentrations selected different low level resistant mutants, and high level resistant mutants emerged from low level resistant mutants if stressed further by Ciprofloxacin. A deep quantitative proteomic study of the Ciprofloxacin resistant mutants uncovered the cellular pathways that supported such resistances. The two low level resistant mutants had different molecular mechanisms. One was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, and the other was probably due to iron and polyamine uptake and DNA repair. High level of resistance involved the mexCD-oprJ efflux pump and the downregulation of PQS quorum sensing. Other pathways might also have contributed to high level resistance, like the arginine deiminase pathway, catalase, peroxidase, protein degradation and DNA repair. The intracellular Ciprofloxacin concentration assay indicated that only the mexCD-oprJ overexpressed mutants had low drug accumulation. This study provided a comprehensive overview of the proteomic landscape in the evolution of Ciprofloxacin resistance in P. aeruginosa, and might have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa. Data are available via ProteomeXchange with identifier PXD004560. Biological significancePseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. We found that Ciprofloxacin resistance developed from low to high level. Two different low levels resistant molecular mechanisms were discovered from different mutants selected by different Ciprofloxacin concentrations, one was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, the other was probably due to iron, polyamine, and DNA repair. High level of Ciprofloxacin resistance all involved the efflux pump, mexCD-oprJ, and the downregulation of quorum sensing. The findings of this study provided insights into the evolution of Ciprofloxacin resistance in P. aeruginosa and should have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa.

Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants.

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.

Study the Expression of ompf Gene in Esherichia coli Mutants.

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5' end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription.

Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni.

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright © 2016 John Wiley & Sons, Ltd.

Searching for the Optimal Predictor of Ciprofloxacin Resistance in Klebsiella pneumoniae by Using In Vitro Dynamic Models.

There is growing evidence of applicability of the hypothesis of the mutant selection window (MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-based pharmacokinetic variables are preferable to the respective MIC-based variables as interstrain predictors of resistance. To examine the predictive power of the ratios of the area under the curve (AUC24) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of three Klebsiella pneumoniae strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant K. pneumoniae mutants were intensively enriched at an AUC24/MIC ratio of 60 to 360 h (AUC24/MPC ratio from 2.5 to 15 h) but not at the lower or higher AUC24/MIC and AUC24/MPC ratios, in accordance with the MSW hypothesis. AUC24/MPC and AUC24/MIC relationships with areas under the time courses of ciprofloxacin-resistant K. pneumoniae (AUBCM) were bell shaped. These relationships predict highly variable "antimutant" AUC24/MPC ratios (20 to 290 h) compared to AUC24/MIC ratios (1,310 to 2,610 h). These findings suggest that the potential of the AUC24/MPC ratio as an interstrain predictor of K. pneumoniae resistance is lower than that of the AUC24/MIC ratio.

Predictors of bacterial resistance using in vitro dynamic models: area under the concentration-time curve related to either the minimum inhibitory or mutant prevention antibiotic concentration.

To examine the predictive power of the ratios of the 24 h AUC (AUC24) to the mutant prevention concentration (MPC) and the MIC, the selection of ciprofloxacin-resistant mutants of Pseudomonas aeruginosa was studied in an in vitro dynamic model. Four clinical isolates of P. aeruginosa with MPC/MIC ratios from 5.6 to 32 were exposed to twice-daily ciprofloxacin for 3 days over a 100- to 200-fold range of the AUC24/MIC ratio. The emergence of P. aeruginosa resistance to ciprofloxacin was concentration dependent: mutants resistant to 2-16 × MIC were enriched at antibiotic concentrations between the MIC and MPC, but not at concentrations below the MIC or above the MPC. Both AUC24/MIC and AUC24/MPC relationships with the area under the bacterial mutant concentration-time curve (AUBCM) were bell-shaped. These relationships predict highly variable 'anti-mutant' AUC24/MIC and AUC24/MPC ratios: e.g. with mutants resistant to 2 × MIC the ratios ranged from 220 to 1100 and from 7 to 180 h, respectively. Using combined data for the four studied organisms, correlations between AUBCM and AUC24/MIC or AUC24/MPC were established (r(2) = 0.75 and 0.65, respectively). Much stronger correlation was observed between AUC24/MIC and the area between the cut-off level at 10(8) cfu/mL and the time-kill curve (ABBC) as an integral index of the antimicrobial effect of ciprofloxacin on the parental strains (r(2) = 0.93). Findings obtained with ciprofloxacin-exposed P. aeruginosa are consistent with the mutant selection window hypothesis. AUC24/MIC and AUC24/MPC relationships with resistance were more bacterial strain specific than AUC24/MIC relationships with fluoroquinolone-induced killing of susceptible cells.

Bacterial antibiotic resistance studies using in vitro dynamic models: Population analysis vs. susceptibility testing as endpoints of mutant enrichment

Emergence of bacterial antibiotic resistance is usually characterised either by population analysis or susceptibility testing. To compare these endpoints in their ability to demonstrate clear relationships with the ratio of 24-h area under the concentration–time curve (AUC24) to the minimum inhibitory concentration (MIC), enrichment of ciprofloxacin-resistant mutants of four clinical isolates of Pseudomonas aeruginosa was studied in an in vitro dynamic model that simulates mono-exponential pharmacokinetics of ciprofloxacin over a wide range of the AUC24/MIC ratios. Each organism was exposed to twice-daily ciprofloxacin for 3 days. Amplification of resistant mutants was monitored by plating on media with 2×, 4×, 8× and 16× MIC of ciprofloxacin. Population analysis data were expressed by the area under the bacterial mutant concentration–time curve (AUBCM). Changes in P. aeruginosa susceptibility were examined by daily MIC determinations. To account for the different susceptibilities of P. aeruginosa strains, post-exposure MICs (MICfinal) were related to the MICs determined with the starting inoculum (MICinitial). For each organism, AUC24/MIC relationships both with AUBCM and MICfinal/MICinitial were bell-shaped, but the latter were more strain-specific than the former. Using combined data on all four isolates, AUBCM showed a better correlation than MICfinal/MICinitial (r2=0.75 vs. r2=0.53). The shift of MICfinal/MICinitial relative to AUBCM vs. AUC24/MIC curves resulted in a weak correlation between AUBCM and MICfinal/MICinitial (r2=0.41). These data suggest that population analysis is preferable to susceptibility testing in bacterial resistance studies and that these endpoints should not be considered interchangeable.

Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates.

The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates

This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.

Increase of multidrug efflux pump expression in fluoroquinolone-resistant Salmonella mutants induced by ciprofloxacin selective pressure

Multidrug-resistant foodborne pathogens are a leading public health concern, as antimicrobial resistance can lead to therapeutic failure. In this study, a ciprofloxacin-susceptible Salmonella Istanbul (Sal10-FC-KU12) was isolated from chicken meat obtained from a market in Korea to induce ciprofloxacin-resistant mutants (SalML, SalMM, and SalMH). Minimum inhibitory concentrations (MICs) of 12 antibiotics were measured in the presence or absence of an efflux pump inhibitor. Expression levels of efflux pump-related genes (acrB, acrF, marA, ramA, rob, and soxS) were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Elevated MICs for the derived mutants were shown to result from the action of the efflux pump, with increased expression of marA, ramA, and acrB compared with the wild-type strain. The results of this study suggest that continued use of ciprofloxacin might induce the emergence of Salmonella mutants resistant not only to fluoroquinolones, but also to several other classes of antimicrobials.