Minimum Inhibitory Concentrations Of Ciprofloxacin Research Articles

Microbial virulence, molecular epidemiology and pathogenic factors of fluoroquinolone-resistant Haemophilus influenzae infections in Guangzhou, China

BackgroundFluoroquinolone-resistant Haemophilus influenzae (FRHI) has been reported worldwide but remain unclear in China.MethodsA total of 402 H. influenzae isolates collected from 2016 to 2017 were included. Antimicrobial susceptibility on 10 antibiotics was performed, and minimum inhibitory concentration of ciprofloxacin- and nalidixic acid-resistant strains were further determined by E-test strips, with risk factors also evaluated. Strains with resistance or reduced susceptibility to ciprofloxacin were subjected to sequencing of the quinolone resistance-determining regions (QRDR) and plasmid-mediated quinolone resistance genes by sequencing, with multi-locus sequence typing.Results2.2% of H. influenzae strains were non-susceptible (7/402, 1.7%) or susceptible (2/402, 0.5%) to ciprofloxacin but NAL-resistant by E-test, and multidrug resistance was more common in fluoroquinolones non-susceptible H. influenzae group (p = 0.000). Infection risk factors included invasive procedure (p = 0.011), catching cold/previous contact with someone who had a cold (p = 0.019), fluoroquinolones use during previous 3 months (p = 0.003). With none of mutations obtained in gyrB, parE and other plasmid-mediated quinolone resistance genes, 7 and 4 strains were found for Ser-84-Leu substitutions in gyrA and one amino acid substitution in the QRDR of gyrA linked with one amino acid substitution in the QRDR of parC, respectively. In addition, five sequence types (ST) were identified, with ST1719 firstly found.ConclusionsFor the first time, this study has reported the incidence, risk factors, molecular determinants on fluoroquinolones resistance and ST of FRHI strains in mainland China, representing the first evidence of mutation of gyrA and parC in China and the new ST1719 worldwide.

Minimum Inhibitory Concentration of Ciprofloxacin against Pseudomonas Aeruginosa in the Presence of the Efflux Inhibitor Phenylalanine-arginine Beta-naphthylamide

Introduction: Pseudomonas aeruginosa is a gram-negative, opportunistic pathogen causing infections in patients staying in the hospital and is resistant to multiple drugs. This study investigated the resistance to ciprofloxacin by the efflux system of Pseudomonas aeruginosa.Materials and Methods: For this purpose, the inhibitor of the efflux system phenylalanine-arginine beta-naphthylamide was used. In this study, 135 isolates of Pseudomonas aeruginosa were collected from the hospitalized patients of Imam Khomeini Hospital and outpatient clinics in Urmia during a ten-month period from June 2015 to March 2016. These isolates were re-identified by standard microbiological and biochemical methods. Finally, 51 isolates were selected for antibiotic susceptibility testing.Results: According to the antibiogram test, the Pseudomonas aeruginosa isolates exhibited highest resistance against ciprofloxacin (90.2%), tobramycin (88.2%), and gentamycin (86.3%) and the highest sensitivity towards colistin (76.4%), and imipenem (72.5%). The 51 isolates, which were selected for the minimum inhibitory concentration test, had multi-drug resistance regulators.Conclusion: The discovery and development of the efflux system inhibitors is an important strategy to deal with bacterial infections.

Trends in antibiotic susceptibility of enteric fever isolates from South India, 2002–2013

BackgroundEnteric fever is endemic in India. Trends in antibiotic resistance in Salmonella enterica subspecies enterica serovars Typhi and Paratyphi A isolates over the past 12 years were studied. MethodsA retrospective analysis of consecutive blood culture isolates of Salmonella Typhi and Salmonella Paratyphi A was performed from 2002 to 2013. Antibiotic susceptibility testing was carried out for ampicillin, chloramphenicol, cotrimoxazole, nalidixic acid (NA), ciprofloxacin and ceftriaxone by disc diffusion. The minimum inhibitory concentration of ciprofloxacin and azithromycin was determined using E-test strips. Mantel-Haenszel extended chi-square test was used for analysis of trends across years. ResultsThree thousand two hundred ninety-six Salmonella spp. were isolated; of which, 1905 were identified as Salmonella Typhi (58%) and 1393 as Salmonella Paratyphi A (42%). Multidrug resistance (chloramphenicol, ampicillin and cotrimoxazole) was relatively stable throughout the study period. NA resistance increased from 18% in 2007 to 100% in 2013 among Salmonella Paratyphi A isolates and from 67% to 82% among Salmonella Typhi isolates. Complete susceptibility to ceftriaxone and azithromycin was observed in this study. ConclusionsKnowledge of the local patterns of resistance would help in appropriate therapy for enteric fever. With increasing rates of fluoroquinolone resistance in our hospital setting, it is probably prudent to revert back to the first-line agents for treatment and save azithromycin and third-generation cephalosporins for difficult and non-responsive cases.

Fluoroquinolone Resistance Mechanisms in Shigella Isolates in Shanghai, China, Between 2010 and 2015.

This study aimed to investigate the antimicrobial susceptibility of Shigella isolated in Shanghai, China and to determine the genetic basis of its resistance to fluoroquinolones. A total of 402 strains of Shigella, including 145 Shigella flexneri and 257 Shigella sonnei isolates, were analyzed. The Kirby-Bauer disk diffusion method was used to determine the susceptibility of the strains to 13 antimicrobials. Minimum inhibitory concentration of ciprofloxacin was determined by E-test. Mutations within the quinolone resistance-determining regions (QRDRs) of gyrA and parC and in the plasmid-mediated quinolone resistance (PMQR) genes, including qnrA, qnrB, qnrS, and aac (6')-Ib-cr, were detected by polymerase chain reaction. All the products were then sequenced. Most of the Shigella isolates were found to be resistant to nalidixic acid (96.4%), streptomycin (96.4%), ampicillin (86.2%), tetracycline (79.8%), and sulfamethoxazole/trimethoprim (80.6%). S. flexneri isolates showed a significantly higher resistance to cefepime (33.6%), ciprofloxacin (54.2%), norfloxacin (34.1%), and levofloxacin (12.1%) compared with that observed for the S. sonnei strains (χ2 analysis, p < 0.05). Three mutations (Ser83, Asp87, and His211) in gyrA and one mutation (Ser80) in parC were detected. Of 257 S. sonnei isolates, 11.7% possessed gyrA mutations and 2% had parC mutations. Of 145 S. flexneri isolates, 98.6% possessed gyrA mutations and 97.9% had parC mutations. The plasmid-mediated resistance genes of qnrS and aac (6')-Ib-cr were detected among 17 strains (4.2%). The mutation percentage within the QRDR of S. flexneri was as high as 98.6 in gyrA and 97.9 in parC. The significant abundance of mutations within QRDRs conferred high levels of fluoroquinolone resistance. Moreover, the PMQR genes, particularly qnrS, played an important role in the decreased susceptibility of Shigella to fluoroquinolones.

Distribution and Expression of Efflux Pump Gene and Antibiotic Resistance in Acinetobacter baumannii

Background: Multidrug efflux pump is a ubiquitous mechanism of drug resistance in bacterial pathogens, such as Acinetobacter baumannii, which is mediated by integral membrane transport proteins. The purpose of the current study was to analyse the distribution of adeG and its role in resistance to ciprofloxacin in A. baumannii isolates from two hospitals of Tehran. Methods: Fifty-one isolates of A. baumannii were recovered from Shahid Motahari and Milad hospitals of Tehran, from July 2016 to March 2017. Identification of A. baumannii was confirmed by phenotypically and molecular methods. Antibiotic susceptibility testing was prepared by the Kirby-Bauer method. Resistance to ciprofloxacin was confirmed by determination of minimum inhibitory concentration (MIC). Efflux pumps inhibitor was used for phenotypic detection of active efflux pumps. Real-time polymerase chain reaction (PCR) was used to analyze adeG gene over expression. Results: Fifty-one isolates of A. baumannii were confirmed by phonotypical and molecular methods. Results of antibiotic susceptibility testing confirmed resistance to ciprofloxacin in all isolates. Results of conventional PCR showed that all of the isolates had the adeG gene. The MIC of ciprofloxacin was decreased by 4 to 32 folds or more in 88% of resistant isolates after adding the efflux pump inhibitor. Over expression of AdeG efflux pump gene was confirmed by real-time PCR in 34 strains. Conclusions: In conclusion, multidrug-resistant A. baumannii were prevalent in the studied isolates. Carbonyl cyanide3-chlorophenylhydrazone could reverse the susceptibility to ciprofloxacin in A. baumannii and was associated with overexpression of the adeG gene.

A study on the relationship between changes in serum hs-CRP levels and Chinese ischemic stroke subclassification

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissues of full-term healthy fetuses after cesarean section and then cultured. Cells in the third passage were chosen for the use of experiment after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the following experiment. Cells were divided into 0, 10, 100 and 1,000 ng/ml lipopolysaccharide (LPS) groups by use of the random number table (similarly hereinafter). Cells were cultured with culture medium containing mesenchymal stem cells (MSCs) after being treated with medium containing corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12 and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay (ELISA). Ninety blood agar culture plates were divided into ciprofloxacin control group (CC), ciprofloxacin + supernatant group (CS), and ciprofloxacin + supernatant + LL-37 antibody group (CSL), with 30 blood agar culture plates in each group. Blood agar culture plates in group CC were coated with 1.5 × 10 8 colony forming unit (CFU)/ml bacteria solution prepared with normal saline. Blood agar culture plates in group CS were coated with 1.5 × 10 8 CFU/ml bacteria solution prepared with normal saline and hUCMSC culture supernatant (cultured by MSC culture medium, the same below) in double volume of normal saline. Blood agar culture plates in group CSL were coated with 1.5 × 10 8 CFU/ml bacteria solution prepared with normal saline, hUCMSC culture supernatant in double volume of normal saline, and 2.6 μL of LL-37 antibodies at the concentration of 2 μg/ml. At PCH 12, 24 and 48, 10 blood agar culture plates were taken out from each group to observe the distribution of SA colonies on blood agar culture plates and to measure diameters of zones of inhibition of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA in each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in group CS and group CSL at PCH 12, 24 and 48 were calculated, with the synergistic effect evaluated. Data were processed with factorial design ANOVA, one way ANOVA, LSD- t test, Kruskal-Wallis test and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in cell culture supernatant in 10 ng/ml LPS group, 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 0 ng/ml LPS group (with t values ranging from 11.22 to 33.36, p values all below .01); the content of LL-37 in cell culture supernatant in either 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 10 ng/ml LPS group (with t values ranging from 2.24 to 18.73, p &lt; .05 or p &lt; .01); the content of LL-37 in cell culture supernatant in 1,000 ng/ml LPS group was higher than that in 100 ng/ml LPS group (with t values ranging from 12.46 to 14.70, p values all below .01). (2) At PCH 12, 24 and 48, the bacterial colonies in groups CC, CS and CSL became integrated over time. In CC group, diameters of zones of inhibition of ciprofloxacin at PCH 12, 24 and 48 were 26 mm, 24 mm and 23 mm respectively, with no obvious changes. At PCH 12, 24 and 48, diameters of zones of inhibition of ciprofloxacin in groups CS and CSL were 82 mm, 71 mm, 68 mm and 74 mm, 59 mm, 56 mm respectively, which were significantly larger than those in group CC. (3) At each PCH, MIC of ciprofloxacin against SA in group CC was significantly higher than that in groups CS and CSL respectively (with Z values ranging from 6.22 to 6.71, p values all below .01); MIC of ciprofloxacin against SA in group CSL was significantly higher than that in group CS (with Z values all equal to 6.72, p values all below .01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24 and 48 were 0.011, 0.032, 0.032 and 0.122, 0.350, 0.350, respectively. The results indicated that hUCMSC culture supernatant had a synergistically antibacterial effect when combined with ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is improved with increase of LPS concentration. The combination of hUCMSC culture supernatant with ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA effectively. Once LL-37 is neutralized, the synergistically antibacterial effect of hUCMSC culture supernatant is decreased thereby.

The effect of exposure to sub-inhibitory concentrations of hypochlorite and quaternary ammonium compounds on antimicrobial susceptibility of Pseudomonas aeruginosa

Pseudomonas is a group of medically important species that inhabit a wide range of niches, including hospital environments. Controversies have emerged about the possible link between improper use of disinfectants and the emergence of antibiotic resistance in bacteria. The aim of this study was to assess the effect of exposure of antibiotic-susceptible Pseudomonas isolates to sub-inhibitory concentrations of 2 disinfectants-didecyldimonium chloride and sodium hypochlorite-on their antibiotic susceptibility patterns. This study involved 50 Pseudomonas isolates. The antibiotic susceptibility patterns of the isolates were assessed using broth microdilution method. The minimal inhibitory concentrations (MICs) of each antibiotic were compared before and after exposure to sub-inhibitory concentrations of didecyldimonium chloride and sodium hypochlorite. After overnight incubation with sub-inhibitory concentrations of sodium hypochlorite, a statistically significant increase was observed in the MICs of colistin (P = .012), ceftazidime (P < .001), amikacin (P < .001), meropenem (P < .001), gentamicin (P < .001), piperacillin-tazobactam (P = .003), and ciprofloxacin (P = .004). In contrast, exposure to sub-inhibitory concentrations of didecyldimonium chloride showed a statistically significant increase in the MICs of amikacin (P < .001), gentamicin (P < .001), meropenem (P = .041), and ciprofloxacin (P = .008). The use of suboptimal concentrations of sodium hypochlorite and didecyldimonium chloride can lead to the evolution of antibiotic-resistant Pseudomonas strains.

Susceptibility of ruminal bacteria isolated from large and small ruminant to multiple conventional antibiotics

The pivotal aim of the present context was to isolate diversified group of bacteria from the ruminants and to evaluate their antibiogram pattern against 22 antibiotics of 14 different classes. The bacterial isolates from small and large ruminant (sheep, cattle and calves) were isolated from the rumen based on various colonies morphology, and subjected for preliminary antibiotics susceptibility assay using disc diffusion method. The most sensitive isolates (based on zone of inhibition) were selected for determining the minimum inhibitory concentration (MIC) of each antibiotic ranging from 1 to 256 μg/mL. Results revealed the concentration dependent growth inhibitory property of antibiotics a species-specific process. The maximum tolerable concentration (MTC) of each antibiotic was further determined using disc diffusion method, and results exhibited that the tolerance nature of ruminal isolates to antibiotics is a species-specific mechanism. Based on the MIC and MTC values of antibiotics, amikacin, ciprofloxacin, and amoxicilline were observed to be the most potent antibiotics in terms of inhibiting the growth of ruminal isolates. In brief, the findings of the current study showed that despite the overexploitation of antibiotics as additives in the animal's feed, most of the ruminal isolates are sensitive to multiple conventional antibiotics tested. The growth inhibitory trait of antibiotics proves these antimicrobials a propitious agent against the pathogenesis of ruminal isolates in livestock.

The clinical significance of silent mutations with respect to ciprofloxacin resistance in MRSA.

BackgroundThe aim of this study was to investigate the genotypic differences between different sequence type MRSA isolates, especially focusing on silent rpoB474 mutations and the relationship between such mutations and ciprofloxacin resistance.MethodsSeventy-nine MRSA isolates were obtained for antibiotic susceptibility tests and molecular study.ResultsAmong these isolates, we found that the MIC50, MIC90, and minimum inhibitory concentration (MIC) range of ciprofloxacin were much higher for the isolates without the rpoB474 mutation than for isolates with the rpoB474 mutation. A total of 87.5% of the isolates with the rpoB474 mutation were susceptible to ciprofloxacin, but none of the isolates without the rpoB474 mutation were susceptible to ciprofloxacin. For 27 MRSA isolates without rpo474 silent mutation but with gyrA86/126 silent mutation, all of them belonged to SCCmec III, and had high ciprofloxacin MIC levels. For another 44 MRSA isolates with rpo474 silent mutation but without gyrA86/126 silent mutation, all of them showed low ciprofloxacin MIC levels, all of them belonged to either SCCmec IV or V. Furthermore, MRSA ciprofloxacin resistance was found to be associated with the mutations gyrA S84L/parC S80F or gyrA S84L, and S85P/parC S80Y.ConclusionMost occurrences of this rpoB474 silent mutation were found in community acquired-MRSA (CA-MRSA) isolates with susceptibility to most antibiotics, especially for ciprofloxacin and vice versa. Thus, this mutation may help to differentiate the different microbiologic characteristics of MRSA clinical isolates.

Campylobacter Prevalence and Quinolone Susceptibility in Feces of Preharvest Feedlot Cattle Exposed to Enrofloxacin for the Treatment of Bovine Respiratory Disease.

Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. Data on resistance in relation to fluoroquinolone use in beef cattle are scarce. This cross-sectional study of preharvest cattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Twenty fresh fecal samples were collected from each of 10 pens, in each of five feedlots, 1-2 weeks before harvest. Feces were cultured for Campylobacter using selective enrichment and isolation methods. Genus and species were confirmed via PCR. Minimum inhibitory concentrations (MICs) of ciprofloxacin and nalidixic acid were determined using a micro-broth dilution method and human breakpoints. Antimicrobial use within each pen was recorded. Data were analyzed using generalized linear mixed-models (prevalence) and survival analysis (MICs). Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots (p < 0.01). Campylobacter coli was the most common species (55.1%; 150/272), followed by Campylobacter hyointestinalis (42.6%; 116/272). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%; 175/256) and for ciprofloxacin (65.6%; 168/256). Distributions of MICs for nalidixic acid (p ≤ 0.01) and ciprofloxacin (p ≤ 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease.

In Vitro Susceptibility of Ciprofloxacin-Resistant Methicillin-Resistant Staphylococcus aureus to Ototopical Therapy.

Objective Despite the rising incidence of methicillin-resistant Staphylococcus aureus (MRSA) otologic infections, choice of treatment remains controversial. Only fluoroquinolone-containing ototopical preparations are approved by the US Food and Drug Administration for middle ear application. Furthermore, American Academy of Otolaryngology-Head and Neck Surgery Foundation guidelines advocate ototopical monotherapy for both tympanostomy tube otorrhea and acute otitis externa. Unfortunately, MRSA may be ciprofloxacin resistant. This causes confusion regarding antibiotic selection, because susceptibility profiles reflect a minimum inhibitory concentration (MIC), referenced against systemic, not ototopical, drug delivery dosing. The goal of this study is to determine the ciprofloxacin MIC for ciprofloxacin-resistant MRSA isolates from otologic infections and compare that value to the expected drug concentration achieved by fluoroquinolone ear drops and determine MRSA genotype for each isolate. Study Design In vitro assay with retrospective medical record review. Setting Tertiary care university hospital. Subjects and Methods Thirty otologically sourced ciprofloxacin-resistant MRSA isolates collected from adult and pediatric patients. MICs were calculated by broth dilution method. Isolates underwent multilocus sequence typing and polymerase chain reaction for arcA and Panton-Valentine leukocidin to establish the genotype. Results MICs ranged from 16 to 1025 µg/mL. There was a relationship between MIC and genotype; of the 7 isolates with an MIC value greater than 512 µg/mL, 6 were sequence type (ST)8. Conclusion These findings support the practice of ototopical monotherapy for patients with uncomplicated ciprofloxacin-resistant MRSA otitis externa. However, they raise concern that ototopical therapy may not be adequate to treat highly resistant strains of MRSA infecting the middle ear space.

Preparation of an optimized ciprofloxacin-loaded chitosan nanomicelle with enhanced antibacterial activity

Objective: The objective of this study was to evaluate the effect of lipid structure on physicochemical properties of chitosan-fatty acid nanomicelles and prepare an optimum ciprofloxacin-loaded formulation from these conjugates which could enhance the antibacterial effects of drug against some important pathogens like P. aeruginosa.Significance: Nowadays, resistance in infectious diseases is a growing worldwide concern. Nanocarriers can increase the therapeutic index and consequently reduce the antibiotic resistance. By site-specific delivery of drug, the adverse effects of broad-spectrum antibiotics such as ciprofloxacin would be reduced.Methods: Fatty acid grafted chitosan conjugates were synthetized in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The effects of fatty acid type (stearic acid, palmitic acid, and linoleic acid) on physicochemical properties of conjugates were investigated. Ciprofloxacin was encapsulated in nanomicelles by thin film hydration method. Also, the preparation process was optimized with a central composite design. The antibacterial effect of optimum formulation against P. aeruginosa, K. pneumoniae, and S. pneumoniae species was determined.Results: All conjugates were synthetized with high yield values and the substitution degrees ranged between 2.13 and 35.46%. Ciprofloxacin was successfully encapsulated in nanomicelles. The optimum formulation showed high drug loading (≈ 19%), with particle size of about 260 nm and a sustained release profile of ciprofloxacin. The minimum inhibitory concentrations of ciprofloxacin in optimum formulation against P. aeruginosa and K. pneumoniae species were 4 and 2 times lower in comparison with the free drug, respectively.Conclusions: The antibacterial effect of ciprofloxacin was improved by encapsulation of drug in chitosan nanomicelles.

Does ciprofloxacin prophylaxis during chemotherapy induce intestinal microflora resistance to ceftazidime in children with cancer?

To determine the susceptibility and minimal inhibitory concentrations (MIC) of ceftazidime, the commonly used empirical antibiotic in patients with febrile neutropenia, in Escherichia coli and Klebsiella pneumoniae isolated from the intestinal microflora of pediatric patients with cancer, who received ciprofloxacin prophylaxis during chemotherapy, children younger than 18 years with acute lymphoblastic leukemia or lymphoma scheduled to undergo chemotherapy were randomized to receive oral ciprofloxacin 20mg/kg/day or placebo from the beginning of their chemotherapy. Rectal swab cultures were taken before (R0) and at 1 (R1), 2 (R2), and 3 (R3) weeks during the intervention. The antimicrobial susceptibilities and MICs of ceftazidime and ciprofloxacin were determined via the E test. Of the total 87 patients enrolled, 44 received ciprofloxacin and 43 placebo. A total of 350 isolates were obtained, 62, 49, 46 and 22 from the ciprofloxacin group and 68, 54, 38 and 11 from the placebo group at R0, R1, R2 and R3, respectively. The percentages of ceftazidime susceptibility did not show significantly greater decreases from R0 to R1-R3 in the ciprofloxacin group compared to the placebo group. The MIC50s of ceftazidime showed significantly greater increases after ciprofloxacin prophylaxis during R1-R3 compared to R0 in the intervention group compared to the placebo group (R0, 0.12 vs. 0.12; R1, 0.19 vs. 0.12; R2, 0.19 vs. 0.12 and R3, 0.38 vs. 0.09μg/mL, respectively). Due to the increasing MIC50 of ceftazidime over time after ciprofloxacin prophylaxis, the use of ceftazidime in patients who have previously had ciprofloxacin prophylaxis needs to be closely monitored.

Mutations in the gyrA, parC, and mexR genes provide functional insights into the fluoroquinolone-resistant Pseudomonas aeruginosa isolated in Vietnam.

IntroductionPseudomonas aeruginosa has many mechanisms of resistance to fluoroquinolones. The main mechanism is to change the effect of two enzymes that open the DNA helix – the enzyme DNA gyrase (gyrA) and the topoisomerase IV (parC). In addition, mutations that render the MexAB-oprM pump (mexR) dysfunctional, leading to its overexpression, also enhance resistance to fluoroquinolones. In this study, we aim to detect point mutations of gyrA, parC, and mexR genes that are predicted to be associated with fluoroquinolone resistance in 141 fluoroquinolone-resistant clinical isolates of P. aeruginosa isolated in Vietnam during 2013–2016.MethodsWe tested minimum inhibitory concentrations (MICs) of fluoroquinolone antibiotics in 141 clinical isolates of P. aeruginosa using the VITEK 2 Compact System, followed by PCR assay, to detect and clone the fluoroquinolone resistance-determining region (FRDR) of gyrA, parC, and mexR. Point mutations were analyzed through Sanger sequencing, and the correlation between genetic mutations and phenotypic resistance of 141 clinical isolates was undertaken.ResultsFluoroquinolone-resistant substitution mutations such as Ile for Thr83 and Met for Thr133 in gyrA, Leu for Ser87 in parC, and Val for Glu126 in the repressor of mexR were mainly detected. Comparative analytical data indicated that amino acid alterations within the gyrA and parC genes are highly associated with resistance to ciprofloxacin (CIP) and levofloxacin (LEV) in the isolates, whereas alterations in the efflux regulatory mexR gene are not highly consistent with resistance in these isolates. Moreover, fluoroquinolone-resistant clinical isolates of P. aeruginosa were mainly isolated from pus and sputum specimens.ConclusionIn clinical isolates of P. aeruginosa, a high correlation was observed between MICs of CIP and LEV and alterations in gyrA and parC genes. However, mutations occurring in mexR did not highly correlate with the antibiotic resistance of the bacterium.

Chromosomal mutations that accompany qnr in clinical isolates of Escherichia coli

We examined 13 qnr-positive and 14 qnr-negative clinical isolates of Escherichia coli for mutations previously seen in a qnr-containing laboratory strain exposed to supra minimum inhibitory concentrations (MICs) of ciprofloxacin. Among the qnr-positive strains, those with ciprofloxacin MICs of ≥ 2 µg/mL had at least one mutation in gyrA. Mutations in parC were present in strains with a ciprofloxacin MIC of ≥ 128 µg/mL. The 6 most ciprofloxacin-resistant strains contained additional plasmid-mediated quinolone resistance determinants. aac(6')-Ib-cr was found in 5 of the 6 strains. Eleven of the 13 strains had alterations in MarR, 9 in SoxR, and 5 had mutations in AcrR. All had elevated expression of at least one efflux pump gene, predominantly acrA (92% of the strains), followed by mdtE (54%) and ydhE (46%). Nine had functionally silent alterations in rfa, two had mutations in gmhB, and one of these also had a mutation in surA. An E. coli with ciprofloxacin MIC of 1024 µg/mL contained 4 different plasmid-mediated quinolone resistance determinants as well as gyrA, parC, parE and pump overexpression mutations. Nine of the 14 qnr-negative strains had mutations in topoisomerase genes with a ciprofloxacin MIC of 0.25 to 256 µg/mL. The three most resistant strains also had mutations in parE. Twelve had alterations in MarR, 10 in SoxR and 5 in AcrR. Ten of the 14 strains had elevated expression of efflux pumps with acrA (71.4%), followed by ydhE (50%) and mdtE (14.3%). A diversity of resistance mechanisms occurs in clinical isolates with and without qnr genes.

Real-Time PCR Targeting Mosaic penA XXXIV for Prediction of Extended-Spectrum Cephalosporins Susceptibility in Clinical Neisseria Gonorrheae Isolates

BackgroundAntimicrobial-resistant Neisseria gonorrheae (NG) is a global public health problem, resulting in limited empirical treatment options. Due to increasing minimum inhibitory concentrations (MICs) of ESCs against NG in the US, it is critical that susceptibility to ESCs be monitored. Since few laboratories routinely perform culture and susceptibility testing for NG, there is a need for a rapid test to predict susceptibility to ESCs. More than 98% of isolates with decreased susceptibility to cefixime (CFM) in the US carry mosaic penA XXXIV. In this study, we developed a multiplex real-time PCR for mosaic penA XXXIV and previously validated gyrA to predict ESCs MICs and ciprofloxacin (CIP) susceptibility.Methods150 NG isolates with known cefpodoxime (CPD), CFM, ceftriaxone (CRO) and CIP MICs were obtained from Neisseria Reference Laboratory at University of Washington and CDC Antimicrobial Resistance Bank. DNA extracted from culture was used in multiplex HybProbe real-time PCR on Lightcycler 480. gyrA was genotyped by melt curve and served as internal control, while presence of mosaic penA XXXIV was detected by selective amplification.ResultsAll 32 (100%) CIP-susceptible and 118 (100%) CIP-resistant isolates, as determined by Clinical and Laboratory Standards Institute breakpoints, demonstrated wild-type and Ser91 mutant gyrA genotype, respectively. Melt curve genotyping demonstrated mosaic penA XXXIV melt patterns in 66/68 (97%) isolates with at least one ESC MIC above alert value set forth by the CDC (CPD and CFM MICs ≥0.25 µg/ml; CRO MIC ≥0.125), while all 82 (100%) isolates with ESC MICs under alert values did not amplify. The first of the 2 false-negative isolates had MICs above alert values for all ESCs tested and harbored IX mosaic type, while the second one had CRO MIC above alert value and harbored XII mosaic type. Both of these mosaic types did not share homology with mosaic penA XXXIV in the region targeted by the assay.ConclusionThe mosaic penA XXXIV assay demonstrated 97% sensitivity and 100% specificity in predicting alert ESCs MIC values among clinical isolates tested, and was successfully multiplexed with gyrA assay. Clinical utility of this assay may be limited due to false negativity in isolates with non-XXXIV mosaic types, but it could serve as a useful surveillance tool for XXXIV mosaic.Disclosure R. Humphries, Roche: Consultant, Consulting fee

INFLUENCE OF ANTIBIOTICS ON BIOFILMS OF PSEUDOMONAS AERUGINOSA STRAINS WITH DIFFERENT LEVEL OF CYCLIC DIGUNOSINMONOPHOSPHATE

Aim of the study was to determine the effect of antibiotics on the formation process and mature biofilm of strains of P. aeruginosa with different levels of cyclic diguanosine monophosphate (c-di-GMP). Methods. As test microorganisms, strains P. aeruginosa PA01, P. aeruginosa PA01 Δ wspF (elevated level of c-diGMP) and P. aeruginosa PA01 pJN2133 (reduced level of c-di-GMP) were used. Cultivation was performed in test tubes to determine minimum inhibitory concentrations of antibiotics (MICs) or 96-well flat-bottomed Nuclon plates in LB medium at 37 °C for 24 hours when studying biofilm formation. To assess the effect of the antibiotics on mature biofilm, they were added to the wells one day after the start of the incubation, removing previously plankton cells. The amount of plankton cells was evaluated spectrophotometrically, biofilm mass was determined by the crystalline violet staining method. Results. After preliminary assessment of susceptibility of the strains to a broad range of antibiotics by the disco-diffuse Kirby-Bauer method, streptomycin, ciprofloxacin and cefepime were selected for study, all of the strains tested were susceptible. Minimum inhibitory concentrations of ciprofloxacin and streptomycin were similar for all the strains of P. aeruginosa and were 0.15 and 10 μg/ml, respectively. P. aeruginosa PA01 pJN2133 (MIC 12.5 μg/ml) was more sensitive to cefepime. MIC of this antibiotic for the other two strains was 20 μg/ml. With the addition of antibiotics at the beginning of the cultivation, they effectively reduced number of plankton cells (by 70–90%) and mass of biofilms (by 40–70%). In this case, there was no difference between strains with different content of c-di-GMP. When exposed to mature biofilms, ciprofloxacin suppressed the formation of persistent cells, but reduced the mass of biofilm only in P. aeruginosa PA01 pJN2133. Cefepime and streptomycin reduced the content of persisters only in concentrations of 5 and 10 μg/ml and had no effect on biofilms. Conclusions. The decreased content of c-di-GMP in P. aeruginosa cells increases the sensitivity of biofilms to antimicrobial drugs. Ciprofloxacin is the most promising antibiotic for the development of combined agents that can prevent the formation of biofilms and destroy them.

Nanoparticles of Chitosan Loaded Ciprofloxacin: Fabrication and Antimicrobial Activity.

Purpose: Chitosan is a natural mucoadhesive polymer with antibacterial activity. In the present study, chitosan (CS) nanoparticles were investigated as a vehicle for delivery of antibiotic, ciprofloxacin hydrochloride.Methods: Ionotropic gelation method was used for preparation chitosan nanoparticles. The effects of various factors including concentration of CS, concentration of tripolyphosphate (TPP), and homogenization rate on the size of nanoparticles were studied. The effects of various mass ratios of CS to ciprofloxacin hydrochloride on the encapsulation efficiency of nanoparticles were assessed.Results: The particles prepared under optimal condition of 0.45% CS concentration, 0.45% TPP concentration and homogenizer rate at 6000 rpm, had 72 nm diameter. In these particles with 1:0.5 mass ratio of CS to ciprofloxacin hydrochloride, the encapsulation efficiency was 23%. The antibacterial activity of chitosan nanoparticles and ciprofloxacin-loaded nanoparticles against E.coli and S.aureus was evaluated by calculation of minimum inhibitory concentration (MIC). Results showed that MIC of ciprofloxacin loaded chitosan nanoparticles was 50% lower than that of ciprofloxacin hydrochloride alone in both of microorganism species. Nanoparticles without drug exhibited antibacterial activity at higher concentrations and MIC of them against E.coli and S.aureus was 177 and 277 µg/ml, respectively.Conclusion: Therefore chitosan nanoparticles could be applied as carrier for decreasing the dose of antibacterial agents in the infections.

Frequency of efflux pump genes mediating ciprofloxacin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates

Efflux pumps are well known as a key role to fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus (MRSA). In this study, among 60 clinical MRSA isolates, 42 isolates (70%) were resistant to ciprofloxacin. MRSA were isolated to detect efflux genes including norA, norB, norC, mepA, sepA, mdeA, qacA/B and smr. Isolates subjected to PCR detection and DNA sequence analysis for these genes. PCR detection showed that 42 isolates (70%) contained at least one efflux pump gene. Among ciprofloxacin-resistant isolates, mdeA and qacA/B genes were found with the highest (61.7%) and lowest (3.3%) frequency, respectively. We also observed that the highest minimum inhibitory concentrations of ciprofloxacin in the presence of mdeA+mepA+norA-C+sepA+smr combination. This type of combination may have the greatest impact on resistance to ciprofloxacin. Finally, compared to previous studies, our study demonstrates that prevalence of ciprofloxacin resistance has been increasing among MRSA clinical isolates.

Effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells and ciprofloxacin on Staphylococcus aureus in vitro

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. Cells in the third passage were used in the experiments after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the experiments. Cells were divided into 0, 10, 100, and 1 000 ng/mL lipopolysaccharide (LPS) groups according to the random number table (the same dividing method below). Cells were cultured with culture medium of mesenchymal stem cells (MSCs) after being treated with medium containing the corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12, and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay. Ninety blood agar plates were divided into ciprofloxacin control group (CC), ciprofloxacin+ supernatant group (CS), and ciprofloxacin+ supernatant+ LL-37 antibody group (CSL), with 30 blood agar plates in each group. Blood agar plates in group CC were coated with 1.5×10(8) colony forming unit (CFU)/mL bacteria solution prepared with normal saline. Blood agar plates in group CS were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline and culture supernatant of hUCMSCs (cultured by culture medium of MSCs, the same below) in double volume of normal saline. Blood agar plates in group CSL were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline, culture supernatant of hUCMSCs in double volume of normal saline, and 2.6 μL LL-37 antibody in the concentration of 2 μg/mL. At PCH 12, 24, and 48, 10 blood agar plates of each group were harvested to observe the distribution of SA colony on blood agar plate and to measure the diameter of bacterial inhibition ring of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA of each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were calculated, and the effect of synergy was evaluated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD-t test, Kruskal-Wallis test, and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in culture supernatant of cells in 10, 100, and 1 000 ng/mL LPS groups was higher than that in 0 ng/mL LPS group (with t values from 11.22 to 33.36, P values below 0.01); the content of LL-37 in culture supernatant of cells in 100 and 1 000 ng/mL LPS groups was higher than that in 10 ng/mL LPS group (with t values from 2.24 to 18.73, P<0.05 or P<0.01); the content of LL-37 in culture supernatant of cells in 1 000 ng/mL LPS group was higher than that in 100 ng/mL LPS group (with t values from 12.46 to 14.70, P values below 0.01). (2) At PCH 12, 24, and 48, the bacterial colonies in groups CC, CS, and CSL began to integrate over time. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in group CC were 26, 24, and 23 mm, respectively, with no obvious change. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in groups CS and CSL were 82, 71, 68 mm, and 74, 59, 56 mm, respectively, significantly longer than those of group CC. (3) At each PCH, the MIC of ciprofloxacin against SA was significantly higher in group CC than in groups CS and CSL (with Z values from 6.22 to 6.71, P values below 0.01); the MIC of ciprofloxacin against SA was significantly higher in group CSL than in group CS (with Z values all equal to 6.72, P values below 0.01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were 0.011, 0.032, 0.032, and 0.122, 0.350, 0.350, respectively. The results indicated that culture supernatant of hUCMSCs had synergistically antibacterial effect on ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is increased with increase of LPS concentration. Combination of culture supernatant of hUCMSCs and ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA. Once LL-37 is neutralized, the synergistically antibacterial effect of culture supernatant of hUCMSCs is decreased.