Minimum Inhibitory Concentrations Of Ciprofloxacin Research Articles

Surveillance of Fluoroquinolone Resistance in Wisconsin: Geographic Variation and Impact of Revised CLSI Breakpoints

Objective: Many clinical microbiology laboratories procure antimicrobial susceptibility testing data using guidelines established by Clinical and Laboratory Standards Institute (CLSI). When necessary, CLSI revises interpretive breakpoints in efforts to improve clinical correlation, with two revisions relative to fluoroquinolone agents occurring in 2019. The purpose of this investigation was to determine the impact of fluoroquinolone breakpoint revisions on Wisconsin clinical isolates of Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. Design: Multi-center laboratory surveillance, with testing at a single location utilizing standardized media and susceptibility testing protocols.Methods: From the Surveillance of Wisconsin Organisms for Trends in Antimicrobial Resistance and Epidemiology (SWOTARE) program, levofloxacin and ciprofloxacin minimum inhibitory concentration (MIC) values for 1911, 1521, and 1463 Wisconsin isolates of E. coli, P. mirabilis, and P. aeruginosa, respectively, were determined by broth microdilution testing. In separate data analyses, all MIC data were interpreted using CLSI breakpoints published prior to 2019, then secondarily by using CLSI breakpoints published since 2019 (which reflect lower breakpoints for both levofloxacin and ciprofloxacin resistance). Findings were further stratified by Wisconsin Department of Health Services region.Results: Up to 3.2% decreased statewide fluoroquinolone susceptibility was observed for E. coli isolates, while 5.1% and 6.3% decreases in levofloxacin susceptibility were noted for P. aeruginosa and P. mirabilis isolates, respectively, when revised breakpoints were applied. E. coli isolates from the Western region and P. mirabilis isolates from the Southeastern region demonstrated significant shifts toward decreased fluoroquinolone susceptibility upon application of revised breakpoints. Northern region P. mirabilis isolates exhibited consistently decreased fluoroquinolone susceptibility.Conclusions: Fluoroquinolone resistance has been underreported in Wisconsin as a whole, yet geographic variability continues to exist. Targeted annual surveillance is important to identify and monitor resistance trending. Compilations of SWOTARE surveillance data can be utilized to predict the impact of future CLSI interpretive breakpoint revisions in Wisconsin.

Isolation of persister cells within the biofilm and relative gene expression analysis of type II toxin/antitoxin system in Pseudomonas aeruginosa isolates in exponential and stationary phases

Chronic infections and treatment failure are concerning issues in patients with Pseudomonas aeruginosa infections. Persister cell formation in biofilm is considered a key reason for antibiotic resistance and treatment failure. In this study, expression of type II toxin/antitoxin (TA) system genes (relBE, Xre-COG5654, vapBC and Xre-GNAT) in persister cells of biofilm was evaluated in the presence of the antibiotics ciprofloxacin and colistin during exponential and stationary phases. The impact of antibiotics on biofilm persister cell formation during exponential and stationary phases of P. aeruginosa strains was examined by colony count at different time intervals in the presence of 5-fold minimum inhibitory concentration (MIC) of ciprofloxacin and colistin. Furthermore, expression of relBE, Xre-COG5654, vapBC and Xre-GNAT genes in P. aeruginosa strains undergoing antibiotic treatment for 3.5 h during exponential and stationary phases was examined by RT-qPCR. Formation of persister cells was observed in biofilms formed by P. aeruginosa strains in the presence of 5×MIC of ciprofloxacin and colistin compared with the control group after 3.5 h of incubation both during exponential and stationary phases. The number of biofilm persister cells was higher in stationary phase than in exponential phase. According to the findings of RT-qPCR, ciprofloxacin and colistin may induce persister cell formation by enhancing the expression of type II TA systems during stationary and exponential phases. The result of this study indicate that ciprofloxacin and colistin have the potential to increase persister cells formation in biofilms by influencing the expression of type II TA systems.

1066. In Vitro and in Vivo Antimicrobial Activity of Cefiderocol and Comparators against Achromobacter spp

Background Achromobacter spp. is intrinsically resistant to multiple antibiotics, and the treatment options are limited. Cefiderocol (CFDC), a siderophore cephalosporin approved in US and EU, is active against a wide variety of aerobic Gram-negative bacteria, including carbapenem-resistant strains. In this study, in vitro and in vivo antibacterial activity of CFDC against Achromobacter spp. was evaluated.MethodsA total of 334 global isolates collected by IHMA from 39 countries in 2015-2019 were used. Minimum inhibitory concentrations (MICs) of CFDC and comparators were determined by broth microdilution method using iron-depleted CAMHB or CAMHB, respectively, as recommended by CLSI guidelines. In vivo efficacy of CFDC was compared with meropenem (MEM), piperacillin-tazobactam (PIP/TAZ), ceftazidime (CAZ), and ciprofloxacin (CIP) in a neutropenic murine lung infection model (n=5), and compared with MEM in a immunocompetent rat lung infection model (n=3-7) caused by 2 A. xylosoxydans. In the murine model, treatment was given 2, 5, and 8 hours post-infection, and the numbers of viable cfu in lungs were determined 24 hours post-infection. In the rat model, the humanized PK in plasma resulting from CFDC 2 g every 8 h (3-h infusion) or meropenem 1 g every 8 h (0.5-h infusion) were recreated via continuous intravenous infusion for 4 days, following which cfu in lungs were determined.ResultsCFDC showed in vitro activity with MIC50/90 of 0.06/0.5 µg/mL against 334 Achromobacter spp. Only 7 isolates (2.1%) had MICs > 4 µg/mL. These were the lowest values among all compound tested (Table). In the murine model, CFDC caused > 1.5 log10 decrease of viable cfu in lungs at 100 mg/kg dose (%fT >MIC: < 50%) from baseline control against both of strains (CFDC MIC: 0.5 and 2 µg/mL) (P< 0.05). No decrease of cfu in lungs was observed for the comparators at 100 mg/kg (MEM, PIP/TAZ, CAZ, and CIP MICs were >16, >64, >32, and >8 µg/mL, respectively). In the rat model, humanized CFDC dosing reduced the viable cfu by >1 log10 CFU/lung compared with baseline controls (P< 0.05). MEM showed no significant activity.In vitro activity of CFDC and comparator agents against Achromobacter spp. 334 Achromobacter spp. isolates collected from 2015 and 2019. The majority of isolates tested were A. xylosoxidans (312/334; 93.4%), followed by A. insolitus (11/334; 3.3%), Achromobacter sp. (8/334; 2.4%), A. denitrificans (2/334; 0.6%), and A. piechaudii (1/334; 0.3%).ConclusionCFDC showed potent in vivo efficacy reflecting in vitro activity against A. xylosoxidans. The results suggested that CFDC has the potential to be an effective therapeutic option for Achromobacter spp. infections.Disclosures Ryuichiro Nakai, MSc, Shionogi TechnoAdvance Research & Co., Ltd. (Employee) Ayaka makino, BSc, Shionogi TechnoAdvance Research & Co., Ltd. (Employee) Toriko Yoshitomi, -, Shionogi TechnoAdvance Research & Co., Ltd. (Employee) Rio Nakamura, BSc, Shionogi TechnoAdvance Research & Co., Ltd. (Employee) Meredith Hackel, PhD MPH, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Daniel F. Sahm, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Yoshinori Yamano, PhD, Shionogi (Employee)

Sub-Inhibitory Concentrations of Chlorhexidine Induce Resistance to Chlorhexidine and Decrease Antibiotic Susceptibility in Neisseria gonorrhoeae.

Objectives: Chlorhexidine digluconate (chlorhexidine) and Listerine® mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in Neisseria gonorrhoeae. We performed in vitro challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials.Methods: A customized morbidostat was used to subject N. gonorrhoeae reference strain WHO-F to dynamically sustained Listerine® or chlorhexidine pressure for 18 days and 40 days, respectively. Cultures were sampled twice a week and minimal inhibitory concentrations (MICs) of Listerine®, chlorhexidine, ceftriaxone, ciprofloxacin, cefixime and azithromycin were determined using the agar dilution method. Isolates with an increased MIC for Listerine® or chlorhexidine were subjected to whole genome sequencing to track the evolution of resistance.Results: We were unable to increase MICs for Listerine®. Three out of five cultures developed a 10-fold increase in chlorhexidine MIC within 40 days compared to baseline (from 2 to 20 mg/L). Increases in chlorhexidine MIC were positively associated with increases in the MICs of azithromycin and ciprofloxacin. Low-to-higher-level chlorhexidine resistance (2–20 mg/L) was associated with mutations in NorM. Higher-level resistance (20 mg/L) was temporally associated with mutations upstream of the MtrCDE efflux pump repressor (mtrR) and the mlaA gene, part of the maintenance of lipid asymmetry (Mla) system.Conclusion: Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in N. gonorrhoeae. This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.

Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt.

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.

Occurrence of Point Mutations in gyrA and parC Genes of Ciprofloxacin-Resistant Pseudomonas aeruginosa Isolated from Burn Infections

The spread of antibiotic resistant bacteria is a worldwide problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed, through molecular techniques, to detect point mutations in chromosomal genes responsible for the quinolones class of antibiotics resistance. A total of 52 isolates from burn infections were identified using specific primers for P. aeruginosa 16S rDNA. Ciprofloxacin minimum inhibitory concentrations (MIC) were estimated using the agar dilution assay. DNA sequences of the quinolone resistance-determining regions of gyrA and parC were determined for detecting the mutations found in these genes and the relations among the isolates by constructing phylogenetic trees. The results revealed that only 43 (82.7%) of isolates were P. aeruginosa, of which 31 (72.06%) were resistant to different concentrations of ciprofloxacin, ranging between 4 and &gt;32 µg/ml. Twenty six isolates were selected for sequencing, including sensitive, intermediately resistant, and highly resistant to ciprofloxacin. The ciprofloxacin sensitive isolates did not exert any amino acid alterations in gyrA or parC genes; however, a single intermediately resistant isolate had a single mutation at each gene. Of the total resistant isolates (20), 6 isolates had no mutations at different MIC levels, While 14 isolates had Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC, only five isolates had a second mutation, namely Asp-87-Asn, in gyrA. The phylogenetic analysis of the studied groups showed divergence from the P. aeruginosa PAO1 and PAO1OR reference strains due to increased mutations and polymorphisms in studied isolates. In conclusion, P. aeruginosa occurrence was increased in burn infections and the fluoroquinolones in current use are not as effective as before; the main resistance mechanism in local clinical isolates of P. aeruginosa is mutations, where the main target of fluoroquinolones is gyrA gene.

Wide spread and diversity of mutation in the gyrA gene of quinolone-resistant Corynebacterium striatum strains isolated from three tertiary hospitals in China

BackgroundCorynebacterium striatum was confirmed to be an important opportunistic pathogen, which could lead to multiple-site infections and presented high prevalence of multidrug resistance, particularly to quinolone antibiotics. This study aimed to investigate the mechanism underlying resistance to quinolones and the epidemiological features of 410 quinolone-resistant C. striatum clinical strains isolated from three tertiary hospitals in China.MethodsA total of 410 C. striatum clinical strains were isolated from different clinical samples of patients admitted to three tertiary teaching hospitals in China. Antibiotic susceptibility testing was performed using the microdilution broth method and pulsed-field gel electrophoresis (PFGE) was used for genotyping. Gene sequencing was used to identify possible mutations in the quinolone resistance-determining regions (QRDRs) of gyrA.ResultsIn total, 410 C. striatum isolates were sensitive to vancomycin, linezolid, and daptomycin but resistant to ciprofloxacin. Depending on the antibiotic susceptibility testing results of 12 antimicrobial agents, the 410 C. striatum strains were classified into 12 resistant biotypes; of these, the three biotypes R1, R2, and R3 were dominant and accounted for 47.3% (194/410), 21.0% (86/410), and 23.2% (95/410) of the resistant biotypes, respectively. Mutations in the QRDRs ofgyrA were detected in all quinolone-resistant C. striatum isolates, and 97.3% of the isolates (399/410) showed double mutations in codons 87 and 91 of the QRDRs of gyrA. Ser-87 to Phe-87 and Asp-91 to Ala-91 double mutation in C. striatum was the most prevalent and accounted for 72.2% (296/410) of all mutations. Four new mutations in gyrA were identified in this study; these included Ser-87 to Tyr-87 and Asp-91 to Ala-91 (double mutation, 101 isolates); Ser-87 to Val-87 and Asp-91 toGly-91 (double mutation, one isolate); Ser-87 to Val-87 and Asp-91 to Ala-91 (double mutation, one isolate); and Ser-87 to Ile-87 (single mutation, one isolate). The minimum inhibitory concentration of ciprofloxacin for isolates with double (96.5%; 385/399) and single (72.7%; 8/11) mutations was high (≥ 32 µg/mL). Based on the PFGE typing results, 101 randomly selected C. striatum strains were classified into 50 genotypes (T01-T50), including the three multidrug-resistant epidemic clones T02, T06, and T28; these accounted for 14.9% (15/101), 5.9% (6/101), and 11.9% (12/101) of all genotypes, respectively. The multidrug-resistant T02 clone was identified in hospitals A and C and persisted from 2016 to 2018. Three outbreaks resulting from the T02, T06, and T28 clones were observed among intensive care unit (ICU) patients in hospital C between April and May 2019.ConclusionsQuinolone-resistant C. striatum isolates showed a high prevalence of multidrug resistance. Point mutations in the QRDRs of gyrA conferred quinolone resistance to C. striatum, and several mutations in gyrA were newly found in this study. The great clonal diversity, high-level quinolone resistance and increased prevalence among patients susceptible to C. striatum isolates deserve more attention in the future. Moreover, more thorough investigation of the relationship between quinolone exposure and resistance evolution in C. striatum is necessary.

Nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis from male urethritis in Japan: Comparison with the first surveillance report

The Urogenital Sub-committee and the Surveillance Committee of the Japanese Society of Chemotherapy, The Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology conducted the second nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis. In this second surveillance study, clinical urethral discharge specimens were collected from patients with urethritis in 26 hospitals and clinics from May 2016 to July 2017. Based on serial cultures, the minimum inhibitory concentration (MIC) could be determined for 41 isolates; the MICs (MIC90) of ciprofloxacin, levofloxacin, tosufloxacin, sitafloxacin, doxycycline, minocycline, erythromycin, clarithromycin, azithromycin and solithromycin were 2 μg/ml (2 μg/ml), 1 μg/ml (0.5 μg/ml), 0.25 μg/ml (0.25 μg/ml), 0.125 μg/ml (0.063 μg/ml), 0.125 μg/ml (0.125 μg/ml), 0.25 μg/ml (0.25 μg/ml), 0.031 μg/ml (0.031 μg/ml), 0.25 μg/ml (0.125 μg/ml), and 0.016 μg/ml (0.008 μg/ml), respectively. In summary, this surveillance project did not identify any strains resistant to fluoroquinolone, tetracycline, or macrolide agents in Japan. In addition, the MIC of solithromycin was favorable and lower than that of other antimicrobial agents. However, the MIC of azithromycin had a slightly higher value than that reported in the first surveillance report, though this might be within the acceptable margin of error. Therefore, the susceptibility of azithromycin, especially, should be monitored henceforth.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

Validation of Pefloxacin for detection of fluoroquinolone (FQ) resistance among Salmonella Typhi with special reference to GyrB mutations.

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml-1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella.Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species.Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations.Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005-2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30μg), ciprofloxacin (5μg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi.Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml-1) and non-susceptible (≥0.125 µg ml-1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type.Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

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

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

Characteristics of Quinolone Resistance in Multidrug-Resistant Acinetobacter baumannii Strains Isolated from General Hospitals

Background: Acinetobacter baumannii is the causative agent in various types of hospital-acquired infections, including respiratory, urinary tract, and wound infections. Objectives: This study investigated the primary mechanisms underlying quinolone resistance in A. baumannii strains, isolated from samples collected from general hospitals. Methods: Ninety-eight strains of A. baumannii were isolated from clinical specimens from general hospitals from 2017 – 2019. Antimicrobial susceptibility, efflux pump inhibition tests, multilocus sequence typing (MLST), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses were conducted on 64 strains, and the blaoxa-51-like gene sequence was detected. Results: In the antimicrobial susceptibility test, 78.1% (n = 50) of the strains exhibited resistance to ciprofloxacin, a quinolone antibiotic, and 57.8% (n = 37) strains were multidrug resistant (MDR). For 18 strains, the minimum inhibitory concentration of ciprofloxacin reduced in presence of an efflux pump inhibitor. Sequence analysis revealed that in 50 strains of A. baumannii, the codon for serine (TCA) in gyrA was replaced by that for leucine (TTA), whereas in 43 strains, the codon for serine (TCG) in parC was replaced by that for leucine (TTG). Multilocus sequence typing analysis confirmed 18 sequence types, and allelic number analysis showed the presence of nine gyrB alleles, with gyrB3 showing the highest frequency (62.5%). Conclusions: The findings of this study will be useful in improving treatment efficiency and preventing the spread of A. baumannii (both MDR and non-MDR strains).

Antimicrobial Susceptibility Pattern of Salmonella spp. Isolated from Enteric Fever Patients in Nepal.

Introduction: Enteric fever, a systemic infection caused by Salmonella enterica Typhi and S. enterica Paratyphi is one of the most common infections in developing countries such as Nepal. Aside from irrational practices of antibiotic use, mutations in chromosomal genes encoding DNA gyrase and Topoisomerase IV and by plasmid mediated quinolone resistant (PMQR) genes are suggested mechanisms for the development of resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. Regardless of high endemicity of enteric fever in Nepal, there is paucity of studies on prevalence and drug-resistance of the pathogen. Therefore, this study aimed to assess the antibiotic susceptibility pattern of Salmonella isolates and determine the minimum inhibitory concentration of ciprofloxacin. Methods: A total of 1298 blood samples were obtained from patients with suspected enteric fever, attending Sukraraj Tropical and Infectious Disease Hospital (STIDH) during March–August, 2019. Blood samples were inoculated immediately into BACTEC culture bottles and further processed for isolation and identification of Salmonella Typhi and S. Paratyphi. Axenic cultures of the isolates were further subjected to antimicrobial susceptibility testing (AST) by using the modified Kirby–Bauer disc diffusion method based on the guidelines by CLSI. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by agar-dilution method. Results: Out of 1298 blood cultures, 40 (3.1%) were positive for Salmonella spp. among which 29 (72.5%) isolates were S. Typhi and 11 (27.5%) isolates were S. Paratyphi A. In AST, 12.5% (5/40), 15% (6/40) and 20% (8/40) of the Salmonella isolates were susceptible to nalidixic acid, ofloxacin and levofloxacin, respectively, whereas none of the isolates were susceptible to ciprofloxacin. The MIC value for ciprofloxacin ranged from 0.06-16 µg/mL in which, respectively, 5% (2/40) and 52.5% (21/40) of the isolates were susceptible and resistant to ciprofloxacin. None of the isolates showed multidrug-resistance (MDR) in this study. Conclusion: This study showed high prevalence of quinolone-resistant Salmonella spp., while there was marked re-emergence of susceptibilities to traditional first option drugs. Hence, conventional first-line-drugs and third-generation cephalosporins may find potential usage as the empirical drugs for enteric fever. Although our reporting was free of MDR strains, extensive surveillance, augmentation of diagnostic facilities and treatment protocol aided by AST report are recommended for addressing the escalating drug-resistance in the country.

Clinical Impact of Revised Ciprofloxacin Breakpoint in Patients with Urinary Tract Infections by Enterobacteriaceae.

In 2018, the Clinical and Laboratory Standards Institute (CLSI) revised ciprofloxacin (CIP)-susceptible breakpoint for Enterobacteriaceae from ≤1 μg/mL to ≤0.25 μg/mL, based on pharmacokinetic-pharmacodynamic (PK-PD) analysis. However, clinical data supporting the lowered CIP breakpoint are insufficient. This retrospective cohort study evaluated the clinical outcomes of patients with bacteremic urinary tract infections (UTIs) caused by Enterobacteriaceae, which were previously CIP-susceptible and changed to non-susceptible. Bacteremic UTIs caused by Enterobacteriaceae with CIP minimal inhibitory concentration (MIC) ≤ 1 μg/mL were screened, and then patients treated with CIP as a definitive treatment were finally included. Patients in CIP-non-susceptible group (MIC = 0.5 or 1 μg/mL) were compared with patients in CIP-susceptible group (MIC ≤ 0.25 μg/mL). Primary endpoints were recurrence of UTIs within 4 weeks and 90 days. A total of 334 patients were evaluated, including 282 of CIP-susceptible and 52 of CIP-non-susceptible. There were no significant differences in clinical outcomes between two groups. In multivariate analysis, CIP non-susceptibility was not associated with recurrence of UTIs. CIP non-susceptibility based on a revised CIP breakpoint, which was formerly susceptible, was not associated with poor clinical outcomes in bacteremic UTI patients were treated with CIP, similar to those of the susceptible group. Further evaluation is needed to guide the selection of definitive antibiotics for UTIs.

Overexpression of mfpA Gene Increases Ciprofloxacin Resistance in Mycobacterium smegmatis.

Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, but FQ-resistant mutants can be selected rapidly. Although mutations in the DNA gyrase are the principal cause of this resistance, pentapeptide proteins have been found to confer low-level FQ resistance in Gram-negative bacteria. MfpA is a pentapeptide repeat protein conserved in mycobacterial chromosomes, where it is adjacent to a group of four highly conserved genes termed a conservon. We wished to characterize the transcriptional regulation of the mfpA gene and relate its expression to ciprofloxacin resistance in M. smegmatis. Reverse transcription PCR showed that mfpA gene is part of an operon containing the conservon genes. Using a transcriptional fusion, we showed that a promoter was located 5′ to the mfpEA operon. We determined the promoter activity under different growth conditions and found that the expression of the operon increases slightly in late growth phases in basic pH and in subinhibitory concentrations of ciprofloxacin. Finally, by cloning the mfpA gene in an inducible vector, we showed that induced expression of mfpA increases the ciprofloxacin Minimal Inhibitory Concentration. These results confirm that increased expression of the mfpA gene, which is part of the mfpEA operon, increases ciprofloxacin resistance in M. smegmatis.

Characterization of Ciprofloxacin Resistance Levels: Implications for Ototopical Therapy.

Ciprofloxacin-resistant pathogens are inhibited by high concentrations of ciprofloxacin found in commercially-available ototopical solutions. Ciprofloxacin-resistant pathogens in otitis media are currently treated with ototopical ciprofloxacin suspensions. This is done irrespective of laboratory-reported ciprofloxacin susceptibility, under the assumption that the high concentration of ciprofloxacin applied topically is sufficient to overcome antimicrobial resistance. We evaluated 34 ciprofloxacin-resistant isolates consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium spp., and Turicella otitidis. Ciprofloxacin minimum inhibitory concentration (MIC) assays and clinical ototopical solution minimum bactericidal concentration (CMBC) assays were performed. Amongst the ciprofloxacin-resistant isolates, ciprofloxacin MICs ranged from 8 to 256 mcg/ml (mean: 87.1 mcg/ml) and CMBCs ranged from 23.4 to 1500 mcg/ml (mean: 237.0 mcg/ml). There were no significant differences with respect to MIC in comparing P. aeruginosa versus Corynebacterium spp. (mean: 53.3 versus 55.2, p = 0.86), S. aureus versus P. aeruginosa (mean: 128.0 versus 53.3, p = 0.34), and S. aureus versus Corynebacterium spp. (mean: 128.0 versus 55.2, p = 0.09). The correlation between ciprofloxacin MIC and CMBC was poor (Pearson's r = -0.08, p = 0.75). Ciprofloxacin-resistant pathogens commonly recovered from otitis media exhibit highly variable ciprofloxacin MIC and CMBC levels. Ciprofloxacin was able to inhibit growth in all isolates tested at MIC levels less than or equal to 256 mcg/ml; however, CMBC's up to 1500 mcg/ml were observed within that same group. The clinical relevance of these in vitro MICs is unclear due in part to higher bactericidal concentrations (CMBC) in several strains. Our results suggest that treatment failures may be due to a combination of factors rather than high-level resistance alone.

Evaluation and validation of Biolog OmniLog® system for antibacterial activity assays.

Minimal inhibitory concentration of antimicrobials, determined by the broth microdilution method, requires visual assessment or absorbance measurement using a spectrophotometer. Both procedures are usually performed manually, requiring the presence of an operator to assess the plates at specific time point. To increase the throughput of antimicrobial susceptibility testing, and concurrently convert into an automatic assay, the Biolog OmniLog® system was validated for a new, label-free application using standard 96-well microplates. OmniLog was evaluated for its signal strength to ensure that the signal intensity, detected and measured by the system's camera, was satisfactory. Variability due to the plate location inside the OmniLog incubator, as well as variation between wells, was investigated. Then the system was validated by determining the minimal inhibitory concentration of ciprofloxacin, piperacillin and linezolid against a selected Gram-negative and Gram-positive strains. No significant difference was observed in relation to position of the plates within the system. Plate edge effects were noticeable, thus the edge wells were not included in further experiments. Minimal inhibitory concentration results were comparable to those obtained by conventional protocol as well as to values defined by the Clinical Laboratory Standards Institute or published in the literature.

Complete genome sequence of a ciprofloxacin-resistant Salmonella Kentucky ST198 strain from a chicken carcass in South Korea

ObjectivesHuman infection by ciprofloxacin-resistant Salmonella enterica serovar Kentucky sequence type 198 (ST198) has been reported in the USA, Europe and Korea. In this study, we report the complete genome sequence of the first Salmonella Kentucky ST198 strain isolated from a chicken carcass in South Korea. MethodsThe recovered Salmonella Kentucky, designated as K13SK002, was isolated from a chicken carcass in 2013. Genomic DNA was sequenced using a combination of 20-kb PacBio SMRTbell library and PacBio RS II. Antimicrobial susceptibility testing was performed and minimum inhibitory concentration (MIC) values were determined. Antimicrobial resistance and virulence genes were investigated in silico using ResFinder and VirulenceFinder tools, respectively, available at the Center for Genomic Epidemiology server. ResultsThe genome of K13SK002 consists of contiguous sequences (contigs) with a total length of 4 847 849 bp and a GC content of 52.20%. We detected a total of 4352 protein-coding sequences, 85 tRNA genes and 22 rRNA genes. MICs for ampicillin, ciprofloxacin, gentamicin, streptomycin, sulfisoxazole and tetracycline were 64, 16, 16, 64, >256 and 128 μg/mL, respectively. We found six antimicrobial resistance genes, however no plasmids and genes associated with adherence, toxins and exoenzyme were found. Ciprofloxacin-resistant Salmonella Kentucky K13SK002 was found to have mutations in DNA gyrase A (S83F and D87Y). ConclusionThis is the first report of the complete genome sequence of a Salmonella Kentucky ST198 strain isolated from a chicken carcass in South Korea. This genome sequence provides useful information on the genomic features associated with virulence and antimicrobial resistance in Salmonella Kentucky ST198.

Fluoroquinolone Can Be an Effective Treatment Option for Acute Pyelonephritis When the Minimum Inhibitory Concentration of Levofloxacin for the Causative Escherichia coli Is ≤16 mg/L.

The purpose of this study was to determine whether the fluoroquinolone (FQ) minimum inhibitory concentration (MIC) for the causative agent Escherichia coli influences the clinical response of FQ treatment at 72 h in patients with community-acquired acute pyelonephritis (CA-APN). We prospectively collected the clinical data of women with CA-APN from 11 university hospitals from March 2010 to February 2012 as well as E. coli isolates from the urine or blood. In total, 78 patients included in this study received FQ during the initial 72 h, and the causative E. coli was detected. The clinical response at 72 h was significantly higher in patients with a levofloxacin MIC ≤ 16 mg/L than in those with an MIC > 16 mg/L (70.4% vs. 28.6%, p = 0.038). No difference was observed in clinical response at 72 h based on ciprofloxacin MIC. To summarize, FQ can be an effective treatment option for CA-APN when levofloxacin MIC against E. coli is ≤16 mg/L.