AdeR-AdeS mutations & overexpression of the AdeABC efflux system in ciprofloxacin-resistant Acinetobacter baumannii clinical isolates

Background & objectives: Overexpression of efflux pumps is a cause of acquired resistance to fluoroquinolones in Acinetobacter baumannii. The present study was done to investigate the presence and overexpression of AdeABC efflux system and to analyze the sequences of AdeR-AdeS regulatory system in ciprofloxacin-resistant A. baumannii isolates. Methods: Susceptibility of 50 clinical A. baumannii isolates to ciprofloxacin, imipenem, ceftazidime, cefepime and gentamicin antimicrobials was evaluated by agar dilution method. Isolates were screened for the evidence of active efflux pump. Isolates were also examined for adeR-adeS and adeB efflux genes by polymerase chain reaction (PCR). The adeR and adeS regulatory genes were sequenced to detect amino acid substitutions. Expression of adeB was evaluated by quantitative reverse-transcriptase PCR. Results: There were high rates of resistance to ciprofloxacin (88%), ceftazidime (88%), cefepime (74%) and imipenem (72%) and less resistance rate to gentamicin (64%). Phenotypic assay showed involvement of active efflux in decreased susceptibility to ciprofloxacin among 16 isolates. The 12.27-fold increase and 4.25-fold increase were found in adeB expression in ciprofloxacin-full-resistant and ciprofloxacin-intermediate-resistant isolates, respectively. Several effective mutations, including A91V, A136V, L192R, A94V, G103D and G186V, were detected in some domains of AdeR-AdeS regulators in the overexpressed ciprofloxacin-resistant isolates. Interpretation & conclusions: The results of this study indicated that overexpression of the AdeABC efflux pump was important to reduce susceptibility to ciprofloxacin and cefepime in A. baumannii that, in turn, could be triggered by alterations in the AdeR-AdeS two-component system. However, gene expression alone does not seem adequate to explain multidrug resistance phenomenon. These results could help plan improved active efflux pump inhibitors.

Molecular basis of high-level ciprofloxacin resistance in Neisseria gonorrhoeae strains from Shandong Province, China

In the study, the ciprofloxacin resistance rate was 100%. High-level ciprofloxacin resistance rate was 63.55%. Sixteen different mutation patterns involved in the formation of ciprofloxacin resistance were identified. The most prevalent were patterns P7 (25.2%), P8 (15.0%), P9 (11.2%), P1 (10.3%), and P5 (10.3%). All of the 107 NG isolates analyzed for mutations in the study have demonstrated a change of Ser-91 → Phe in the gyrA gene, and all except one have demonstrated a change in position 95 of the amino acid sequence. All of the 68 high-level QRNG isolates had double mutations in gyrA gene combined with a single or two mutations in parC gene. It is most important that a new mutation site of Ile-97 → Met in gyrA and a new mutation of Leu-106 → Ile in parC were found in the study, both leading to high-level ciprofloxacin resistance (MIC values, 8 µg/mL, 32 µg/mL, respectively). Therefore, we confim that gyrA mutations are necessary for the fluoroquinolone resistance phenotype and parC mutations are correlated intimately with high-level fluoroquinolone resistance. In China fluoroquinolone resistance in Neisseria gonorrhoeae strains is very serious and the new mutation sites in the fluoroquinolone resistance-determining regions emerge more and more quickly. Hence, in China fluoroquinolones, which are used to treat gonorrhoea presently, should be substituted by a new antibiotics.

Change of Antimicrobial Susceptibility among Escherichia coli Strains Isolated from Female Patients with Community-Onset Acute Pyelonephritis

PURPOSE: There is a concern on which antimicrobials are appropriate as empirical agents for community-onset acute pyelonephritis (APN) in regions where the fluoroquinolone resistance rate is high, such as in Korea. MATERIALS AND METHODS: Three hundred and two strains of E. coli in 2001-2002 and 349 strains in 2008-2009 were isolated from the urine cultures of female adult APN patients, and the antimicrobial susceptibility was compared according to each study period. All the patients were classified as uncomplicated or complicated APN, and a subgroup analysis was done thereafter. RESULTS: The E. coli strains isolated in 2008-2009 showed improved susceptibility to trimethoprim-sulfamethoxazole compared to those isolated in 2001-2002. However, the third generation cephalosporin and gentamicin susceptibility was worsened. Of the 232 isolates from the uncomplicated APN patients, there was no difference between the two different time periods. On the other hand, of the 419 isolates from the complicated APN patients, the susceptibility to third generation cephalosporin, gentamicin and ciprofloxacin was significantly worsened. CONCLUSION: The antimicrobial susceptibility of E. coli changed over the study period, however, this change occurred mainly in the complicated APN patients. In Korea, ciprofloxacin is still useful as an empirical agent for uncomplicated APN patients, but this is not the case for patients with complicated APN because of high resistance rate to ciprofloxacin in these patients. For the complicated APN patients, the rate of resistance to ciprofloxacin is already more than 30%.

Quinolone-resistant Shigella flexneri Isolated in a Patient Who Travelled to India

We report a recent case in which ciprofloxacin-resistant Shigella flexneri was isolated from a 23-yr-old female patient with a history of travel to India. Prior to her admission to our internal medicine department, she experienced symptoms of high fever and generalized weakness from continuous watery diarrhea that developed midway during the trip. S. flexneri was isolated from the stool culture. Despite initial treatment with ciprofloxacin, the stool cultures continued to show S. flexneri growth. In the susceptibility test for antibiotics of the quinolone family, the isolate showed resistance to ciprofloxacin (minimum inhibitory concentration [MIC], 8 microg/mL), norfloxacin (MIC, 32 microg/mL), ofloxacin (MIC, 8 microg/mL), nalidixic acid (MIC, 256 microg/mL), and intermediate resistance to levofloxacin (MIC, 4 microg/mL). In molecular studies for quinolone resistance related genes, plasmid borne-quinolone resistance genes such as qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, and oqxAB were not detected. Two mutations were observed in gyrA (248C-->T, 259G-->A) and 1 mutation in parC (239G-->T). The molecular characteristics of the isolated S. flexneri showed that the isolate was more similar to the strains isolated from the dysentery outbreak in India than those isolated from Korea.

Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi.

Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 μg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe), whereas double mutations (Ser 83→Phe and Asp 87→Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.

Causative Organisms and Antibiotics Sensitivity in Community Acquired Acute Pyelonephritis / 대한신장학회지

PURPOSE: Ciprofloxacin has been used as one of choice antibiotics for the empirical treatment of community-acquired acute pyelonephritis (APN), of which E-coli is the main causative bacterium. Recently, however, some studies show that ciprofloxacin-resistant E.coli is increasing in APN. In this study, authors aimed to re-evaluate ciprofloxacin as one of initial antibiotics for APN according to the survey of the patients' clinical and microbiologic characteristics. METHOD: In this single center, the records of patients diagnosed as APN were reviewed retrospectively who visited emergency room or out-patient clinic from Aug. 01, 2005 to Jul. 31, 2007. RESULT: A total of 303 patients were enrolled. The proportion of males to females was 1:9.4, the average age was 49.3+/-19 years old. Complicated APNs were 121 out of 303 patients, diabetes ranked highest, 52.5% (64/121). According to the urine culture analysis, the patients with isolated microorganism reached up to 45.2% (137/303) and E.coli occupied 94.2% out of total. As the initial antibiotics, ciprofloxacin was most commonly used (64.4%, 195/303), next was the 3rd generation cephalosporin (23.8%, 72/303). The analysis of antibiotics sensitivity demonstrated ciprofloxacin susceptibility was 81.4%, whereas high over 90% in the 3rd generation cephalosporin. When the ciprofloxacin-resistant E.coli was isolated, the patients tended to have prolonged duration of antibiotics treatment, a higher proportion of complicated APNs and of treatment non-responders. CONCLUSION: For the APNs, ciprofloxacin is clinically effective initial antibiotics; however, it should be carefully used especially in case of the patients having combined underlying disease or the aged.

Emerging antimicrobial resistance, plasmid profile and pulsed-field gel electrophoresis pattern of the endonuclease-digested genomic DNA of Neisseria gonorrhoeae

Resistant gonococci are very prevalent in many countries, particularly in Asia. This study was conducted to determine the trend of resistance, the effect of decreasing the ciprofloxacin susceptibilities of gonococci on the prevalence of penicillinase-producing N. gonorrhoeae (PPNG), and to compare the epidemiology of strains with the previous studies. A total of 602 strains of gonococci were isolated from prostitutes in 1997-1999. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. For epidemiologic analysis, vplasmid analysis and pulsed-field gel electrophoresis (PFGE) were performed. The proportion of PPNG remained high (79%), and the strains with decreased susceptibility to ciprofloxacin increased significantly from 67% in 1997 to 84% in 1999. Compared to our previous study, the PFGE patterns were similar, while the proportion of strain with the 3.2-MDa plasmid markedly decreased. In conclusion, a rapid increase in ciprofloxacin-nonsusceptible strains may suggest difficulties in the treatment of gonococcal infections in the near future with the drug. The recent decrease of PPNG with the 3.2-MDa plasmid may suggest that there is an epidemiological change in gonococcal infections, and the prevalence of related PFGE patterns suggests the dissemination of a few clones among the high risk populations.