Ciprofloxacin-susceptible Strains Research Articles

Relative quantification of the recA gene for antimicrobial susceptibility testing in response to ciprofloxacin for pathogens of concern

Antimicrobial resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antimicrobial susceptibility tests (AST) rely on organism growth rates that result in prolonged time-to-answer for slow growing organisms. Changes in the cellular transcriptome can be rapid in the presence of stressors such as antibiotic pressure, providing the opportunity to develop AST towards transcriptomic signatures. Here, we show that relative quantification of the recA gene is an indicator of pathogen susceptibly when select species are challenged with relevant concentrations of ciprofloxacin. We demonstrate that ciprofloxacin susceptible strains of Y. pestis and B. anthracis have significant increases in relative recA gene expression after 15 min of exposure while resistant strains show no significant differences. Building upon this data, we designed and optimized seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, response and housekeeping genes, respectively, for multiple biothreat and ESKAPE pathogens. Final evaluation of all seven duplex assays tested against 124 ciprofloxacin susceptible and resistant strains, including Tier 1 pathogens, demonstrated an overall categorical agreement compared to microbroth dilution of 97% using a defined cutoff. Testing pathogen strains commonly associated with urinary tract infections in contrived mock sample sets demonstrated an overall categorical agreement of 96%. These data indicate relative quantification of a single highly conserved gene accurately determines susceptibility for multiple bacterial species in response to ciprofloxacin.

Quinolone Resistance in Biofilm-Forming Klebsiella pneumoniae-Related Catheter-Associated Urinary Tract Infections: A Neglected Problem

Background: Klebsiella pneumoniae is a bacterium that commonly causes urinary tract infections (UTIs) in hospital settings. The widespread and improper usage of quinolones has increased the resistance rates against these broad-spectrum antibiotics. Objectives: This study aimed to examine the connection between the ability to form biofilms and fluoroquinolone resistance in K. pneumoniae isolated from catheter-associated UTIs. Methods: A total of 110 nonduplicative K. pneumoniae-related catheter-associated UTIs were isolated from three large educational hospitals in Babol, north of Iran. The minimal inhibitory concentration (MIC) of ciprofloxacin was calculated for each detected isolate using the agar dilution procedure. Biofilm production was investigated by a 96-well flat-bottom microtiter plate. The prevalence of gyrA, parC, qnrA, qnrS, acc (6’)-Ib-cr, qepA, qnrB, oqxA, and oqxB genes was evaluated using polymerase chain reaction (PCR). Results: Overall, 28.2% of the strains were resistant to imipenem and considered carbapenem-resistant K. pneumoniae (CRKp). Ciprofloxacin resistance was observed in 66.4%. Moreover, 70% of the isolates produced biofilm. Biofilm production was significantly higher in ciprofloxacin-resistant compared to ciprofloxacin-susceptible strains (P-value < 0.05). Molecular distribution of resistance genes in the 68-fluoroquinolone resistance-Kp strains showed that the prevalence of gyrA, parC, qnrA, qnrS, acc (6’)-Ib-cr, qepA,, qnrB, oqxA, and oqxB genes was 39.7%, 42.6%, 5.9%, 54.4%, 69.1%, 94.1%, 41.2%, 69.1%, and 83.8%, respectively. Conclusions: Our study highlights the high prevalence of plasmid-mediated quinolone resistance genes in clinical samples of K. pneumoniae in the studied region, which is alarming given the possibility of the spread of these pathogens and the few treatments available for infections brought on by multidrug-resistant strains. Moreover, the study characterizes particular mutations in the parC and gyrA genes that cause quinolone resistance.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

BackgroundCampylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates.MethodsA total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping.ResultsOut of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates.ConclusionSignificant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

The mRNA expression of ompF, invA and invE was associated with the ciprofloxacin-resistance in Salmonella.

Salmonella developed drug-resistance under durative antibiotic pressures pressure. The widespread prevalence of Salmonella has been associated with not only drug-resistance but also pathogenicity. Outer membrane porin proteins (OMPs) are critical for the drug resistance of bacteria. Virulence genes in Salmonella pathogenicity islands (SPIs) play key roles in the virulence of bacteria. In this study, we analyzed the expression levels of three critical genes in ciprofloxacin-resistant strains and ciprofloxacin-susceptible strains of Salmonella, including outer membrane porin protein F (ompF), virulence genes invA and invE. In the clinical ciprofloxacin-resistant strains of Salmonella, the expression level of ompF was decreased. Meanwhile, the expression levels of invA and invE were decreased except for only one strain, indicating generally decreased virulence. These results were also verified with ciprofloxacin-induced resistant strains. Thus, it was informative for understanding the drug-resistance in Salmonella. Monitoring drug-resistance and virulence relevant genes would be significant in the prevention and control of salmonellosis.

The Role of Ciprofloxacin Resistance and Extended-spectrum beta-lactamase (ESBL) Positivity in Infective Complications Following Prostate Biopsy.

To evaluate ciprofloxacin resistance (CR) and extended-spectrum beta-lactamase (ESBL) positivity in the rectal flora, antibiotic prophylaxis received, and post-biopsy infectious complications in patients undergoing prostate biopsy. Rectal swab samples collected from 99 patients suspected of prostate cancer two days before prostate biopsy were tested for microbial susceptibility and ESBL production. All patients were given standard ciprofloxacin and ornidazole prophylaxis. Ten days post-biopsy, the patients were contacted by phone and asked about the presence of fever and/or symptoms of urinary tract infection. Escherichia coli (E.coli) was the most common isolate detected in 82 (75%) of the rectal swab samples. Ciprofloxacin resistance was detected in 33% and ESBL positivity in 22% of the isolated E.coli strains. No microorganisms other than E.coli were detected in blood, urine, and rectal swab cultures of patients who developed post-biopsy complications. CR E.coli strains also showed resistance to other antimicrobial agents. The lowest resistance rates were to amikacin (n = 2, 7.4%) and nitrofurantoin (n = 1, 3.7%). Seven patients (7.6%) developed infectious complications. There was no significant difference in probability of hospitalization between patients with CR strains (14.3%) and those with ciprofloxacin-susceptible strains (14.3% vs. 4.7%; p = 0.194). However, strains that were both CR and ESBL-positive were associated with significantly higher probability of hospitalization compared to ciprofloxacin-susceptible strains (28.6% vs. 3.8%; p = 0.009). The higher rate of infectious complications with CR and ESBL-positive strains suggests that the agents used for antibiotic prophylaxis should be reevaluated. It is important to consider local resistance data when using extended-spectrum agents to treat patients presenting with post-biopsy infectious complications.

Proteomic comparison of three clinical diarrhoeagenic drug-resistant Escherichia coli isolates grown on CHROMagar™STEC media

Shiga-toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterize the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one resistant to ciprofloxacin] and one STEC) cultured on CHROMagar™STEC solid media after minimal laboratory passage. We identified 4767 unique peptides from 1630 protein group across all three clinical E. coli strains. Label-free proteomic analysis allowed the identification of virulence and drug resistance proteins that were unique to each of the clinical isolates compared in this study. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain while several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. Parallel reaction monitoring assays validated the presence of biologically relevant proteins across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convenient means to quantify virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria. SignificanceThrough the use of quantitative proteomics, we have characterized the virulence and antimicrobial resistance attributes of three clinically isolated, pathogenic E. coli strains cultured on solid media. Our results provide new, quantitative data on the expressed proteomes of these tellurite-resistant, diarrhoeagenic E. coli strains and reveal a subset of antimicrobial resistance and virulence proteins that are differentially abundant between these clinical strains. Our quantitative proteomics-based approach should thus have applicability in microbiological diagnostic labs for the identification of pathogenic/drug resistant E. coli in the future.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

BackgroundEnterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh.MethodsA total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE).ResultsAmong 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 μg/ml and 32μg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains.ConclusionEmergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Case Series of Imported Enteric Fever at a Referral Center in Tokyo, Japan: Antibiotic Susceptibility and Risk Factors for Relapse.

Owing to the increase in Salmonella strains with decreased fluoroquinolone susceptibility in the endemic areas, we have been treating enteric fever with intravenous ceftriaxone empirically since 2007. In this study, we reevaluated our treatment protocol. This retrospective cohort study was conducted at a single institute in Tokyo, Japan, between January 2006 and December 2013. Enteric fever was defined as isolation of Salmonella Typhi or Salmonella Paratyphi A, B, and C from the blood and/or stool of patients with fever. Of the 35 patients with imported enteric fever, 28 (80%) had returned from south Asia. Ciprofloxacin-susceptible strains were detected in only 12% of the cases. The isolates showed excellent susceptibility to ampicillin (91%), chloramphenicol (94%), ceftriaxone (97%), and azithromycin (97%). One case of Salmonella Paratyphi B was excluded, and of the remaining 34 patients, 56% were treated with ceftriaxone alone, 26% with ceftriaxone then fluoroquinolone, and 9% with levofloxacin alone. The overall relapse rate was 6.1%; however, among those receiving ceftriaxone monotherapy, the relapse rate was 11% (N = 2). The relapse group was characterized by longer times to treatment initiation (P = 0.035) and defervescence (> 7 days) after treatment initiation (P = 0.022). In such cases, we recommend that ceftriaxone treatment be continued for > 4 days after defervescence or be changed to fluoroquinolone if the strains are found to be susceptible to prevent relapse. Furthermore, ampicillin and chloramphenicol, which are no longer prescribed, may be reconsidered as treatment options in Asia.

Genetic diversity and clonal characteristics of ciprofloxacin-resistant meningococcal strains in China.

The purpose of the present study was to identify the clonal characteristics and gyrA gene diversity of ciprofloxacin-resistant meningococcal strains in China. One hundred and forty-one ciprofloxacin-resistant and 103 ciprofloxacin-susceptible meningococcal strains were selected for multilocus sequence typing. Of these, 54 ciprofloxacin-resistant and 42 ciprofloxacin-susceptible strains were selected for gyrA gene sequencing. Of the three clonal complexes prevalent in China, serogroup A of ST-5 complex (CC5) and serogroup C/B strains of CC4821 had a high proportion of ciprofloxacin resistance, whereas CC11 serogroup W strains were all susceptible. Nucleotide and amino acid sequences of the gyrA gene among ciprofloxacin-resistant strains showed more diversity than those among ciprofloxacin-susceptible strains. All ciprofloxacin-resistant strains had a T91I mutation and the ciprofloxacin-susceptible strains had no T91I mutation. Phylogenetic analysis showed that the gyrA gene sequences of CC4821 serogroup B/C strains, CC11 serogroup W, CC1 serogroup A, ciprofloxacin-susceptible CC5 serogroup A and reference strains had high similarity. By contrast, the ciprofloxacin-resistant CC5 serogroup A strains had a highly conserved gyrA gene sequence which was different (94.8% similarity) from that in the above strains. The results of our investigation showed that the high proportion of ciprofloxacin resistance in Neisseria meningitidis is associated with certain sequence types (STs) or clonal complexes (CCs). The prevalence of certain CCs with a high proportion of ciprofloxacin resistance can facilitate the spread of ciprofloxacin resistance.

Mutant prevention concentrations of ciprofloxacin against urinary isolates of Escherichia coli and Klebsiella pneumoniae

The aim of this work was to study the ability of ciprofloxacin to restrict the development of resistant mutants of Escherichia coli and Klebsiella pneumoniae through determination of the Mutant Prevention Concentration (MPC). We studied 140 strains of E. coli and 86 strains of K. pneumoniae with different profiles of sensitivity to fluoroquinolones and extended spectrum beta lactamase (ESBL) production. The MPCs were determined using an inoculum of 1010 CFU/ml in Mueller-Hinton agar plates with serial concentrations of ciprofloxacin. Ciprofloxacin-susceptible ESBL-producing strains showed a higher MPC for ciprofloxacin (P <0.001) than ciprofloxacin-susceptible non ESBL-producing strains, while ciprofloxacin-resistant ESBL-producing and non ESBL-producing strains did not significantly differ. The presence of qnr variants was associated with elevated MPCs. This was observed for both tested organisms. Our study helps to explain the frequent finding of resistance to fluoroquinolones in ESBL-producing strains. Consequently, the use of concentrations of ciprofloxacin higher than the MIC in order to prevent the recovery and growth of resistant mutants is recommended.

Antibiotic Resistance in Human Chronic Periodontitis Microbiota

Patients with chronic periodontitis (CP) may yield multiple species of putative periodontal bacterial pathogens that vary in their antibiotic drug susceptibility. This study determines the occurrence of in vitro antibiotic resistance among selected subgingival periodontal pathogens in patients with CP. Subgingival biofilm specimens from inflamed deep periodontal pockets were removed before treatment from 400 adults with CP in the United States. The samples were cultured, and selected periodontal pathogens were tested in vitro for susceptibility to amoxicillin at 8 mg/L, clindamycin at 4 mg/L, doxycycline at 4 mg/L, and metronidazole at 16 mg/L, with a post hoc combination of data for amoxicillin and metronidazole. Gram-negative enteric rods/pseudomonads were subjected to ciprofloxacin disk-diffusion testing. Overall, 74.2% of the patients with CP revealed subgingival periodontal pathogens resistant to at least one of the test antibiotics. One or more test species, most often Prevotella intermedia/nigrescens, Streptococcus constellatus, or Aggregatibacter actinomycetemcomitans, were resistant in vitro to doxycycline, amoxicillin, metronidazole, or clindamycin, in 55%, 43.3%, 30.3%, and 26.5% of the patients with CP, respectively. Fifteen percent of patients harbored subgingival periodontal pathogens resistant to both amoxicillin and metronidazole, which were mostly either S. constellatus (45 individuals) or ciprofloxacin-susceptible strains of Gram-negative enteric rods/pseudomonads (nine individuals). Patients with CP in the United States frequently yielded subgingival periodontal pathogens resistant in vitro to therapeutic concentrations of antibiotics commonly used in clinical periodontal practice. The wide variability found in periodontal pathogen antibiotic-resistance patterns should concern clinicians empirically selecting antibiotic treatment regimens for patients with CP.

Antibiotic resistance in human peri‐implantitis microbiota

Because antimicrobial therapy is often employed in the treatment of infectious dental implant complications, this study determined the occurrence of in vitro antibiotic resistance among putative peri-implantitis bacterial pathogens. Submucosal biofilm specimens were cultured from 160 dental implants with peri-implantitis in 120 adults, with isolated putative pathogens identified to species level, and tested in vitro for susceptibility to 4mg/l of doxycycline, 8mg/l of amoxicillin, 16mg/l of metronidazole, and 4mg/l of clindamycin. Findings for amoxicillin and metronidazole were combined post-hoc to identify peri-implantitis species resistant to both antibiotics. Gram-negative enteric rods/pseudomonads were subjected to ciprofloxacin disk diffusion testing. One or more cultivable submucosal bacterial pathogens, most often Prevotella intermedia/nigrescens or Streptococcus constellatus, were resistant in vitro to clindamycin, amoxicillin, doxycycline, or metronidazole in 46.7%, 39.2%, 25%, and 21.7% of the peri-implantitis subjects, respectively. Only 6.7% subjects revealed submucosal test species resistant in vitro to both amoxicillin and metronidazole, which were either S.constellatus (one subject) or ciprofloxacin-susceptible strains of gram-negative enteric rods/pseudomonads (seven subjects). Overall, 71.7% of the 120 peri-implantitis subjects exhibited submucosal bacterial pathogens resistant in vitro to one or more of the tested antibiotics. Peri-implantitis patients frequently yielded submucosal bacterial pathogens resistant in vitro to individual therapeutic concentrations of clindamycin, amoxicillin, doxycycline, or metronidazole, but only rarely to both amoxicillin and metronidazole. Due to the wide variation in observed drug resistance patterns, antibiotic susceptibility testing of cultivable submucosal bacterial pathogens may aid in the selection of antimicrobial therapy for peri-implantitis patients.

Molecular analysis of ciprofloxacin resistance and clonal relatedness of clinical Escherichia coli isolates from haematology patients receiving ciprofloxacin prophylaxis

Widespread use of fluoroquinolones has led to increased levels of resistance in clinical isolates of Escherichia coli. We investigated the evolution of ciprofloxacin susceptibility and molecular epidemiology of clinical E. coli isolates in haematology patients receiving ciprofloxacin prophylaxis on the population and individual patient level. From August 2006 through December 2007 we collected all E. coli isolates (n = 404) from surveillance and infection-site cultures from 169 haematology patients receiving ciprofloxacin prophylaxis. Analysis of the gyrase A (gyrA) gene was performed by denaturing gradient gel electrophoresis (DGGE) in 364 isolates and clonal relatedness was determined by the single-enzyme amplified fragment length polymorphism (seAFLP) technique in 162 isolates. One hundred of these isolates were also subjected to qnrA analysis. The average number of samples per patient was 2.4 (maximum 20) and 122 (30%) of 404 E. coli isolates were resistant to ciprofloxacin. In 124 patients only ciprofloxacin-susceptible strains were detected. DGGE revealed 11 different gyrA sequence patterns and, based on AFLP analysis, there was evidence of selection of ciprofloxacin-resistant strains under antibiotic pressure, as well as the occurrence of genetically indistinguishable ciprofloxacin-resistant and -susceptible E. coli isolates within one patient. Clonal dissemination of ciprofloxacin-resistant E. coli was observed, but did not predominate. The genetic evolution of clinical E. coli isolates in haematology patients receiving ciprofloxacin prophylaxis is characterized by selection of ciprofloxacin-resistant strains. However, we did find evidence for de novo resistance mutation in ciprofloxacin-susceptible E. coli in individual patients under selective pressure.

Decreased quinolone susceptibility in high percentage of Enterobacter cloacae clinical isolates caused only by Qnr determinants

The prevalence of qnr genes was similar in the ciprofloxacin-resistant and ciprofloxacin-susceptible Enterobacter cloacae isolates, and no amino acid change in quinolone resistance-determining regions was found in any of the 21 qnr-positive but ciprofloxacin-susceptible strains. Qnr may thus be present before the development of higher-level resistance from target mutations.

Phylogenetic Background and Virulence Genotype of Ciprofloxacin‐Susceptible and Ciprofloxacin‐ResistantEscherichia coliStrains of Human and Avian Origin

Previous studies have suggested that fluoroquinolone-resistant strains of Escherichia coli that infect humans probably emerged as a consequence of using fluoroquinolones in poultry. This study aims to provide further insight into the possible avian origin of fluoroquinolone-resistant extraintestinal pathogenic E. coli (ExPEC) strains that infect humans. We compared the phylogenetic backgrounds, virulence gene profiles, and genetic relatedness of 125 ExPEC strains recovered from humans (61 were ciprofloxacin susceptible and 64 were ciprofloxacin resistant) and 113 E. coli strains recovered from poultry (47 were ciprofloxacin susceptible and 66 were ciprofloxacin resistant). Ciprofloxacin-resistant strains of both human and avian origin harbored fewer virulence genes than did ciprofloxacin-susceptible strains, but ciprofloxacin-resistant strains from humans were found to be clearly distinct from ciprofloxacin-resistant avian strains, based on their phylogenetic backgrounds and virulence gene profiles. The phylogenetic background of ciprofloxacin-susceptible and ciprofloxacin-resistant strains of human origin was not different, and no shift from the phylogenetic group B2 toward other groups was detected in association with ciprofloxacin resistance. No genetic relatedness was observed among human and avian strains that belonged to the major virulence profile (traT-iucD-iutA). Our results did not support the hypothesis of an avian origin for the ciprofloxacin-resistant human ExPEC strains analyzed. Nevertheless, prudent use of fluoroquinolones in both human and veterinary medicine is recommended.

Molecular characterizations of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida

To analyze the genetic mechanisms of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida, 27 clinical isolates (comprising 11 carbapenem- and ciprofloxacin-resistant strains, 13 carbapenem-resistant and ciprofloxacin-susceptible strains, and 3 carbapenem- and ciprofloxacin-susceptible strains) were collected from different patients. Carbapenem resistance was examined by polymerase chain reaction (PCR) and DNA sequencing for metallo-beta-lactamase (MBL) and integrase genes (IntI-1 and IntI-3), and by reverse transcriptase-PCR (RT-PCR) for expression of the porin gene (oprD). Ciprofloxacin resistance was characterized by PCR and DNA sequencing for mutations in the quinoloneresistance determining regions of the gyrA and parC genes. The blaIMP-1 MBL and intI-1 and/or intI-3 genes were detected in all carbapenem-resistant strains, and decreased expression of the oprD gene as compared to carbapenemsusceptible strains was observed in several strains. All the 11 strains with ciprofloxacin minimal inhibitory concentrations (MICs) of > or =64 mg/l had substitution in GyrA (Thr83Ile), and one (ciprofloxacin MIC of 512 mg/l) of these strains also had substitution in ParC (Ser87Leu). Overproduction of the efflux pump was observed in 10 of the 11 ciprofloxacin-resistant strains. We concluded that the production of IMP-1 type MBL was the most critical factor in developing high-level resistance to carbapenems, and mutations in the target proteins and overproduction of the efflux pump synergistically contribute to the acquisition of high-level resistance to ciprofloxacin in clinical isolates of P. putida.

Effects of Low-Level Ciprofloxacin Challenge in theIn VitroDevelopment of Ciprofloxacin Resistance inCampylobacter jejuni

The effects on MIC values and the selection of different base substitutions in the quinolone resistance determining region (QRDR) of gyrA were studied on initially ciprofloxacin-susceptible Campylobacter jejuni strains by challenging them to 0.125 mg/L of ciprofloxacin. This ciprofloxacin challenge selected variants with ciprofloxacin MIC levels up to 32 mg/L. Repeated experiments under identical conditions resulted in different responses in MIC levels and alterations in the QRDR of gyrA. A characteristic outcome to ciprofloxacin challenges was the appearance of double peaks in the sequencing chromatograms of QRDR. This finding suggested the coexistence of subpopulations possessing Thr86 --> Ile and/or Asp90 --> Asn mutations alongside the unmutated parent population. In some cases, bacterial variants expressing ciprofloxacin-resistant phenotypes possessed no mutations in their QRDR. These variants were prone to regain susceptibility to ciprofloxacin rapidly after the removal of the selection pressure, whereas the QRDR-mutated variants persisted over several subcultivations in a medium without ciprofloxacin. In conclusion, a low ciprofloxacin concentration of 0.125 mg/L selects a variety of QRDR mutations and also a QRDR-independent resistance mechanism, which may coexist with each other in a C. jejuni population. Persistent ciprofloxacin challenge selects Thr86 --> Ile and/or Asp90 --> Asn mutants.

Identification and control of an outbreak of ciprofloxacin-susceptible EMRSA-15 on a neonatal unit

We report the identification and control of an outbreak of a ciprofloxacin-susceptible strain of UK epidemic meticillin-resistant Staphylococcus aureus (EMRSA)-15 on a neonatal unit (NNU). All babies were screened for MRSA on admission using ciprofloxacin-containing media which did not detect the outbreak strain. The first identified case was a premature baby who developed MRSA bacteraemia with associated tibial osteomyelitis and multiple subcutaneous abscesses. The outbreak strain was subsequently identified in the nasopharyngeal secretions of a second child who was not clinically infected. Screening of all patients on the NNU using non-ciprofloxacin-media identified two other colonised babies. All four patient isolates were EMRSA-15, spa type t022, SCCmec IV, Panton-Valentine leucocidin (PVL) negative, indistinguishable by pulsed-field gel electrophoresis and susceptible to all non-beta-lactam antimicrobials tested. The outbreak strain was cultured from four of 48 environmental sites in a communal milk-expressing room. Unsupervised movement of mothers to and from the milk-expressing room may have contributed to the outbreak. Control measures included cohort isolation of affected babies, improved environmental cleaning, increased emphasis on hand hygiene and education of mothers. Ciprofloxacin-containing media should be used with caution for MRSA screening in settings where ciprofloxacin-susceptible strains (including community-associated MRSA) are increasing in prevalence.

In vitro activity of garenoxacin tested against a worldwide collection of ciprofloxacin-susceptible and ciprofloxacin-resistant Enterobacteriaceae strains (1999–2004)

Garenoxacin is a novel des-F(6)-quinolone with a chemical structure that lacks the C6 position fluorine and has a unique difluoromethoxy substitution at position C8. This study evaluated the in vitro activity of garenoxacin tested against a large collection of Enterobacteriaceae collected worldwide. The bacterial isolates were consecutively collected from more than 70 medical centers from bloodstream, respiratory, urinary tract, and skin and soft tissue infections. The isolates were tested against garenoxacin, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, and many other oral and parenteral antimicrobial agents. All isolates were susceptibility tested by broth microdilution methods according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and interpretative criteria. A garenoxacin-susceptible breakpoint of ≤2 μg/mL was applied for comparison purposes only. The fluoroquinolones were the most active oral compounds with overall susceptibility rates of 87.5% to 90.0%. Garenoxacin showed in vitro activity comparable with that of ciprofloxacin and the other fluoroquinolones against Escherichia coli (MIC 50, ≤0.03 μg/mL; 87.3% susceptible), Klebsiella spp. (MIC 50, 0.12 μg/mL; 90.4–94.5% susceptible), Enterobacter spp. (MIC 50, 0.12 μg/mL; 85.6–86.5% susceptible), Salmonella spp. (MIC 50, 0.06 μg/mL; 99.7% susceptible), and Shigella spp. (MIC 50, ≤0.03 μg/mL; 99.9% susceptible), which accounted for 87.0% of the Enterobacteriaceae strains tested. Garenoxacin was highly active against ciprofloxacin-susceptible strains (98.1% susceptibility), whereas ciprofloxacin-resistant strains were generally resistant to all other fluoroquinolones and also showed high rates of resistance to other orally administered antimicrobials. In conclusion, garenoxacin in vitro activity was similar to that of currently marketed fluoroquinolones and superior to other orally administered antimicrobial agents (cephalosporins, amoxicillin/clavulanate, and trimethoprim/sulfamethoxazole) when tested against more than 45 000 globally collected Enterobacteriaceae.

Update on fluoroquinolone resistance in Helicobacter pylori: new mutations leading to resistance and first description of a gyrA polymorphism associated with hypersusceptibility

Helicobacter pylori eradication by standard therapy is decreasing due to clarithromycin and metronidazole resistance. Fluoroquinolones are valuable drugs for alternative therapy, but their activity needs to be updated. We determined minimum inhibitory concentrations (MICs) of the newly marketed fluoroquinolones (levofloxacin, moxifloxacin and gatifloxacin) and assessed the prevalence of resistance in 128 H. pylori strains isolated in 2004–2005. The quinolone resistance-determining region (QRDR) of gyrA was sequenced for all strains. Gatifloxacin MICs (MIC 50 = 0.25 mg/L) were two- to four-fold lower than those of the other fluoroquinolones. The prevalence of resistance (ciprofloxacin MIC > 1 mg/L) was 17.2% (22 strains). All resistant strains harboured one gyrA mutation at codons 86, 87 or 91, including three new mutations (Asp86Asn, Thr87Ile and Asn87Tyr). Ciprofloxacin-susceptible strains were devoid of such gyrA mutations, but harboured a polymorphism at codon 87 that distinguished 18 isolates (17%) with a Thr87 like the reference strain J99 from 88 strains with Asn87 like the reference strain 26695. Strains with Thr87 were four-fold more susceptible to nalidixic acid, pefloxacin, ciprofloxacin and levofloxacin and were equally susceptible to moxifloxacin and gatifloxacin. The high rate of quinolone resistance in H. pylori requires the use/implication of a ‘test and treat’ strategy that can confidently rely on QRDR gyrA sequencing.