MICs Of Ciprofloxacin Research Articles

A novel alanine to serine substitution mutation in SoxS induces overexpression of efflux pumps and contributes to multidrug resistance in clinical Escherichia coli isolates.

The purpose of this study was to describe a putative role for a novel soxS mutation in contributing to multiple-antibiotic resistance in canine fluoroquinolone-associated MDR (FQ-MDR) Escherichia coli. This soxS mutation was discovered in canine faecal E. coli isolates during a study investigating the effect of oral fluoroquinolone administration on faecal E. coli in healthy dogs. We determined via quantitative real-time RT-PCR that both soxS and acrB were overexpressed in the clinical soxS Ala-12→Ser (soxS(A12S)) mutants and this may account for their FQ-MDR phenotype. We validated the FQ-MDR phenotype of the clinical isolates by reconstructing the WT and the soxS(A12S) mutation in the E. coli soxS null mutant JW4023 (soxS::kn) via allelic exchange. The JW4023 soxS(A12S) derivative showed an increase in MICs of ciprofloxacin, enrofloxacin and chloramphenicol compared with the JW4023 derivative in which the WT soxS had been restored. The soxS and acrB genes were overexpressed in the JW4023 soxS(A12S) mutant compared with JW4023 with WT soxS. A similar overexpression of efflux pump genes and an increase in antibiotic resistance were observed upon stimulation with paraquat to resemble the phenotype of the clinical soxS(A12S) isolates. Our data suggest that the soxS(A12S) substitution mutation is selected in clinical isolates when dogs are exposed to a fluoroquinolone and that this mutation contributes to the FQ-MDR phenotype of E. coli isolates.

Nalidixic Acid Susceptibility Test for Screening Salmonella Isolates of Reduced Susceptibility/Higher Minimum Inhibitory Concentration to Ciprofloxacin

Enteric fever is the major diagnosis among febrile patients in Nepal with yearly increase in nalidixic acid resistance and reduced ciprofloxacin susceptibility among Salmonella isolates. This study was carried out to evaluate the validity of nalidixic acid resistance as an indicator of reduced susceptibility of Salmonella isolates to ciprofloxacin. In this study, 999 blood specimens collected from suspected enteric fever patients visiting B&B Hospital were processed by standard microbiological techniques. Isolates were identified by biochemical tests and serotyping. Antibiotic susceptibility test was performed by Kirby Bauer disc diffusion method and CLSI recommended interpretive criteria. MIC of ciprofloxacin was determined by agar dilution method. Isolation rate of Salmonella species was 6.21%. Among 62 Salmonella isolates, 51 were S. typhi, 10 were S. paratyphi A and one isolate was S. paratyphi B. Only one isolate of S. typhi was multi-drug resistant. Resistance to ceftriaxone, cefixime and azithromycin was nil. On disc diffusion test, 55 isolates were resistant to nalidixic acid. Fifty-seven isolates were found to have increased (>0.125mg/ml) MIC of ciprofloxacin with the clinical and laboratory standards institute breakpoints. Nalidixic acid resistance showed a predictive value of 100% for ciprofloxacin resistance. Screening with nalidixic acid disc had a sensitivity of 100% and a specificity of 71.43% for the determination of decreased ciprofloxacin susceptibility. Before using ciprofloxacin for the treatment of enteric fever, appropriate identification of Salmonella isolates with reduced ciprofloxacin susceptibility is essential to limit the possible treatment failure and further development of highly resistant strains.DOI: http://dx.doi.org/njst.v15i2.12122Nepal Journal of Science and Technology Vol. 15, No.2 (2014) 97-104

Novel synthetic bis-indolic derivatives with antistaphylococcal activity, including against MRSA and VISA strains.

We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3'-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time-kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.

Resistance Phenotypes and Genotypes of Salmonella enterica subsp. enterica Isolates from Feed, Pigs, and Carcasses in Brazil

Salmonella enterica subsp. enterica plays a role as a foodborne pathogen worldwide. The consumption of contaminated pork has been associated with human salmonellosis and the increase in antimicrobial resistance among Salmonella from pigs and pork products is a concern. A total of 225 Salmonella isolates from feed mills, the lairage environment, and the intestinal contents of pigs and carcasses were investigated for their antimicrobial susceptibility. A MIC for ciprofloxacin was screened by agar dilution, and antimicrobial resistance genes were investigated by PCR assays. Among the tested isolates, 171 (76%) showed resistance to at least one antimicrobial agent, and 91 (40.4%) were multiresistant. Resistance occurred most frequently to tetracycline (54.5%), sulfonamides (39.6%), and streptomycin (33.7%). Thirty-two (94.1%) nalidixic acid-resistant isolates exhibited decreased susceptibility to ciprofloxacin. The resistance genes found were blaTEM (ampicillin), tet(A) (tetracycline), tet(B) (tetracycline/minocycline), sul1, sul2, and sul3 (sulfonamides), catA1 (chloramphenicol), floR (florfenicol/chloramphen- icol), strA and strB (streptomycin), aph(3')-Ia (kanamycin), aac (3)-IIa and aac (3)-IVa (apramycin/gentamicin), aadA variant (streptomycin/spectinomycin), and dfrA1 (trimethoprim). Salmonella isolates from pig feces and carcasses displayed a higher frequency of resistance to most antimicrobials tested than isolates from feed mills. Common resistance gene profiles were found in isolates from the lairage and the intestinal content of pigs and carcasses, demonstrating that resistance genes selected on farms may be found in pork.

Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

ObjectiveTo establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. MethodsEight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. ResultsEighty isolates with a nalidixic acid MIC of <32mg/L (NALS) and a ciprofloxacin MIC of >0.064mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n=34), mutation in gyrB at codon 464, NAL MIC of 3–12mg/L and CIP MIC of 0.064–0.5mg/L.; and Group C, mutation in gyrA at codon 83 (n=2) NAL MIC of 16mg/L and CIP MIC of 0.25–0.38mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. ConclusionThe use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

Co-existence of plasmid-mediated quinolone resistance determinants and mutations in gyrA and parC among fluoroquinolone-resistant clinical Enterobacteriaceae isolated in a tertiary hospital in Warsaw, Poland

Plasmid-mediated quinolone resistance (PMQR) determinants and the distribution of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC were investigated in 215 ciprofloxacin-resistant (MIC>1mg/L) clinical Enterobacteriaceae collected during a 6-month prospective study in a tertiary hospital in Warsaw, Poland. PMQR determinants were present in 49 isolates (22.8%), among which aac(6′)-Ib-cr and qnrB1 predominated (85.7% and 26.5%, respectively). Mutations in gyrA and parC QRDRs were detected among 89.8% of isolates (MIC≥4mg/L). Changes in Ser-83, Ala-84 and Asp-87 in GyrA and Ser-80 and Glu-84 in ParC were detected. Five isolates with ciprofloxacin MICs in the range 1.5–16mg/L were found to have unaltered QRDRs, with PMQR as the only fluoroquinolone (FQ) resistance trait detected. The remaining 44 PMQR-positive isolates were found to carry altered QRDRs. Three substitutions (two in GyrA and one in ParC) were detected in 23 isolates, whilst 8 isolates carried four mutations (two in GyrA and two in ParC). One isolate of Klebsiella pneumoniae with two amino acid substitutions in the ParC QRDR in the presence of aac(6′)-Ib-cr and qnrB1 had a ciprofloxacin MIC of 16mg/L. The results presented here show that FQ resistance in these clinical Enterobacteriaceae is a complex interplay between PMQR determinants and mutations in gyrA and parC rather than a single stepwise accumulation of mutations in the gyrase and topoisomerase subunits. In addition, these results show the role of PMQR determinants in promoting QRDR mutations and the acquisition of high-level FQ resistance in clinical settings.

Molecular characterization of fluoroquinolone resistance in nontypeable Haemophilus influenzae clinical isolates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7,267 H. influenzae isolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates studied, two mutations producing changes in two GyrA residues (Ser84, Asp88) and/or two ParC residues (Ser84, Glu88) were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n = 15) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 μg/ml, while those with three or more mutations (n = 15) had MICs of 4 to 16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment.

MLST typing of antimicrobial-resistant Propionibacterium acnes isolates from patients with moderate to severe acne vulgaris

Molecular typing data on antimicrobial-resistant Propionibacterium strains are limited in the literature. We examined antimicrobial resistance profiles and the underlying resistance mechanisms in Propionibacterium spp. isolates recovered from patients with moderate to severe acne vulgaris in Greece. The clonallity of the resistant Propionibacterium acnes isolates was also investigated.Propionibacterium spp. isolates were detected using Tryptone-Yeast Extract-Glucose (TYG) agar plates supplemented with 4% furazolidone. Erythromycin, clindamycin, vancomycin, penicillin, co-trimoxazole, doxycycline, minocycline and ciprofloxacin MICs were determined using the gradient strip method. Erythromycin, clindamycin and tetracycline mechanisms of resistance were determined using PCR and sequencing of the domain V of 23S rRNA and 16S rRNA, as well as the presence of the ermX gene. Typing was performed using the multi locus sequence typing (MLST) methodology.Seventy nine isolates from 76 patients were collected. Twenty-three isolates (29.1%) exhibited resistance to erythromycin and clindamycin, while two additional isolates (2.5%) were resistant only to erythromycin. Resistance to tetracycline was not detected. The underlying molecular mechanisms were point mutations A2059G and A2058G. MLST typing of the P. acnes resistant isolates revealed that lineage type IA1 (ST-1, 3 and 52) prevailed (12/18; 66.7%), whilst lineage type IA2 (ST-2 and 22) accounted for five more isolates (27.8%). Susceptible isolates were more evenly distributed between ST types.Propionibacterium spp. from moderate to severe acne vulgaris in Greece are frequently resistant to erythromycin/clindamycin but not to tetracyclines, mainly due to the point mutations A2059G and A2058G. P. acnes resistant isolates were more clonally related than susceptible ones and belonged to a limited number of MLST types.

Mutant prevention concentrations of levofloxacin, pazufloxacin and ciprofloxacin for A. baumannii and mutations in gyrA and parC genes.

Fluoroquinolones are antimicrobial agents that are widely used clinically, but the increasing resistance of Acinetobacter baumannii (A. baumannii) to these agents is a matter of concern. We investigated mutant prevention concentrations (MPCs) of three fluoroquinolones, levofloxacin (LVX), pazufloxacin (PAZ) and ciprofloxacin (CIP). We analyzed an A. baumannii standard strain (ATCC19606) for mutation prevention indices (MPIs), MPCs and mutant selection windows as well as MICs of CIP, PAZ and LVX and compared the derived values with 34 A. baumannii strains collected in hospitals. In addition, A. baumannii standard strain (ATCC19606) fluoroquinolone-resistant mutants were investigated for gyrA and parC gene mutations. MPCs of CIP, prevention antibiotics concentration and LVX for A. baumannii ATCC19606 were 12.8, 5.6 and 2.8 μg ml(-1) and their MPIs were 16, 8 and 4, respectively. Clinically isolated A. baumannii strains had CIP, PAZ and LVX MPC value ranges of 1-8, 1-16 and 0.5-2 μg ml(-1) and their MPIs were 8, 8 and 4 μg ml(-1). Single gyrA mutations (Ser(83)-Leu(83)) occurred in 18 resistant strains (48.7%) and single parC mutations (Ala(79)-Asp(79) or (Ser(80)-Leu(80)) occurred in 8 resistant strains (21.6%), whereas gyrA and parC double mutations occurred in 2 (5.4%) of the resistant strains. MPC and MPI values of LVX were lower than that of CIP and PAZ. Single gyrA and parC mutations accounted for the majority of mutations (n=24), whereas double mutations occurred only in two strains.

Spread of Streptococcus pneumoniae serotype 8-ST63 multidrug-resistant recombinant Clone, Spain.

Since 2004, a total of 131 isolates of Streptococcus pneumoniae multidrug-resistant invasive serotype 8 have been detected in Spain. These isolates showed resistance to erythromycin, clindamycin, tetracycline, and ciprofloxacin. All isolates were obtained from adult patients and shared a common genotype (sequence type [ST]63; penicillin-binding protein 1a [pbp1a], pbp2b, and pbp2x gene profiles; ermB and tetM genes; and a ParC-S79F change). Sixty-eight isolates that required a ciprofloxacin MIC ≥16 μg/mL had additional gyrA gene changes. Serotype 8-ST63 pbp2x sequences were identical with those of antimicrobial drug-susceptible serotype 8-ST53 isolates. Serotype 8-ST63 pbp2b sequences were identical with those of the multidrug-resistant Sweden 15A-ST63 clone. Recombination between the capsular locus and flanking regions of an ST53 isolate (donor) and an ST63 pneumococcus (recipient) generated the novel 15A-ST63 clone. One recombination point was upstream of pbp2x and another was within pbp1a. A serotype 8-ST63 clone was identified as a cause of invasive disease in Spain.

The effects of the natural enzyme, Pectinex Ultra SP-L, on human cell cultures and bacterial biofilms in vitro.

BackgroundPectinex Ultra SP-L (Pectinex) is a microbial-derived enzyme that is used in the food industry and that has been shown to inhibit bacterial biofilms. It has been suggested that Pectinex may be useful in the management of biofilm-related bacterial infections and therefore warrants further investigation in this regard. The aim of this study was to investigate the cytotoxicity of Pectinex on cervical adenocarcinoma cells (HeLa), lymphocytes and neutrophils. Cell viability and morphology were assessed using an in vitro spectrophotometric MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay and polarization-optical transmitted light differential interference contrast microscopy. This study also investigated the antibacterial and antibiofilm actions of Pectinex, alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined using p-iodo-nitrotetrazolium violet staining of bacterial cultures and regrowth of subcultures. Biofilm biomass and cell viability were quantified spectrophotometrically after staining with crystal violet and MTT.ResultsThe IC50 (±SEM) of Pectinex was 193.9 (±22.2) PGU/ml for HeLa cells, 383.4 (±81.5) and 629.6 (±62.8) PGU/ml for fMLP-stimulated and non-stimulated lymphocytes respectively, and 245.9 (±9.4) and 529.7 (±40.7) PGU/ml for fMLP-stimulated and non-stimulated neutrophils, respectively. Induced morphological features characteristic of apoptosis and necrosis included cell membrane blebs and vacuolization in HeLa cells, clumping in lymphocytes, as well as shrunken rounded cells, apoptotic bodies and debris in all cultures. Pectinex (7.42 – 950 PGU/ml−1) was not bactericidal. In clinical cultures of Staphylococcus aureus, co-administration of Pectinex was associated with a 28.0% increase in both the MIC and MBC of amoxicillin-clavulanate. In clinical cultures of P. aeruginosa, there was an 89.0% and 92.8% increase in the MIC and MBC of ciprofloxacin, respectively. Pectinex ≤ 118.75 PGU/ml−1 and incubation periods ≥ 6 h were associated with increased biomass and cell viability in S. aureus or P. aeruginosa biofilms.ConclusionsPectinex appeared to antagonize the antibacterial effects of amoxicillin-clavulanate and ciprofloxacin and furthermore demonstrated significant cytotoxicity. It was therefore deemed unsuitable for the management of either planktonic or biofilm phenotypes of S. aureus or P. aeruginosa.

Sesquiterpene Derivatives and Steroids from the Sponge Dactylospongia elegans Collected from the South China Sea

Sponges of the genus Dactylospongia have been widely investigated for their biologically active compounds. The majority of the reported metabolites are sesquiterpene quinones [1, 2], as well as a few macrolides [3]. For example, smenospongine, a sesquiterpene aminoquinone isolated from the Indonesian sponge D. elegans, showed multifaceted antitumor activity on leukemia cells [4]. Sponge Material. The sponge of Dactylospongia elegans was collected in April 2006 from Meishan coral reef, Sanya, China, and was authenticated (RMNH POR. 5235) by Dr. Nicole J. de Voogd, Netherlands Centre for Biodiversity Naturalis, The Netherlands. A voucher specimen (HN-SY-2006WMS-18) was deposited in the Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of China, Qingdao, China. Extraction and Isolation. The sponge Dactylospongia elegans (0.7 kg, dry weight) was extracted exhaustively with 95% ethanol (2000 mL 5) at room temperature. The solvent-free EtOAc extract (1.0 g) was subjected to column chromatography on silica gel and eluted with EtOAc in petroleum (0–100%, gradient) to yield six fractions. The subfractions were then isolated by successive column chromatography on silica gel and Sephadex LH-20 and purified by semi-preparative HPLC, leading to the isolation of compounds (1/2) (50.6 mg), (3) (2.4 mg), (4) (4.2 mg), (5) (1.8 mg), and (6) (8.5 mg). On the basis of extensive spectroscopic analyses and by comparison with those reported in the literature, the structures of compounds 1–6 were established as dactyltronic acids (1/2) [5, 6], dictyoceratin C (3) [7], polyfibrospongol A (4) [8], 3 -hydroxycholesta-5,8-dien-7-one (5) [9], and pregna-1,20-dien-3-one (6) [10]. Compounds 3, 5, and 6 were obtained from the genus Dactylospongia for the first time. The antibacterial activity in vitro of the isolated compounds was evaluated against a panel of pathogenic bacterial strains, Staphylococcus aureus, S. albus, Bacillus subtilis, B. cereus, Micrococcus tetragenus, Kocuria rhizophila, Vibrio parahemolyticus, V. anguillarum, and Pseudomonas putida, by using a standard screening protocol [11]. The results indicated that dactyltronic acids 1/2 showed pronounced antibacterial activity against V. parahemolyticus with a MIC value of 3.45 M (the positive control ciprofloxacin MIC = 1.25 M). Pregna-1,20-dien-3-one (6) also exhibited activity toward B. cereus with a MIC value of 4.19 M (ciprofloxacin MIC = 1.25 M). However, compounds 3, 4, and 5 only showed weak antibacterial activity.

Overexpression of the novel MATE fluoroquinolone efflux pump FepA in Listeria monocytogenes is driven by inactivation of its local repressor FepR.

Whereas fluoroquinolone resistance mainly results from target modifications in gram-positive bacteria, it is primarily due to active efflux in Listeria monocytogenes. The aim of this study was to dissect a novel molecular mechanism of fluoroquinolone resistance in this important human pathogen. Isogenic L. monocytogenes clinical isolates BM4715 and BM4716, respectively susceptible and resistant to fluoroquinolones, were studied. MICs of norfloxacin and ciprofloxacin were determined in the presence or in the absence of reserpine (10 mg/L). Strain BM4715 was susceptible to norfloxacin (MIC, 4 mg/L) and ciprofloxacin (MIC, 0.5 mg/L) whereas BM4716 was highly resistant to both drugs (MICs 128 and 32 mg/L, respectively). Reserpine was responsible for a 16-fold decrease in both norfloxacin and ciprofloxacin MICs against BM4716 suggesting efflux associated resistance. Whole-genome sequencing of the strains followed by comparative genomic analysis revealed a single point mutation in the gene for a transcriptional regulator, designated fepR (for fluoroquinolone efflux protein regulator) belonging to the TetR family. The frame-shift mutation was responsible for the introduction of a premature stop codon resulting in an inactive truncated protein. Just downstream from fepR, the structural gene for an efflux pump of the MATE family (named FepA) was identified. Gene expression was quantified by qRT-PCR and demonstrated that fepA expression was more than 64-fold higher in BM4716 than in BM4715. The clean deletion of the fepR gene from BM4715 was responsible for an overexpression of fepA with resistance to norfloxacin and ciprofloxacin, confirming the role of FepR as a local repressor of fepA. In conclusion, we demonstrated that overexpression of the new MATE efflux pump FepA is responsible for fluoroquinolone resistance in L. monocytogenes and secondary to inactivation of the FepR repressor.

Multi-drug resistance and reduced susceptibility to ciprofloxacin among Salmonella enterica serovar Typhi isolates from the Middle East and Central Asia.

Typhoid fever is common in developing countries, with an estimated 120 million infections and 700 000 annual deaths, worldwide. Fluoroquinolones have been the treatment of choice for infection with multidrug-resistant (MDR) Salmonella enterica serovar Typhi (S. Typhi). However, alarming reports of fluoroquinolone-resistance and failure of typhoid fever treatment have recently been published. To determine the proportion of S. Typhi isolates with reduced susceptibility to ciprofloxacin (RSC) from six countries in the Middle East and Central Asia, 968 S. Typhi isolates collected between 2002 and 2007 from Egypt, Uzbekistan, Pakistan, Qatar, Jordan and Iraq were tested for antibiotic susceptibility to five antibiotics using the disc-diffusion method. MDR was defined as resistance to amicillin, chloramphenicol and trimethoprim-sulfamethoxazole. The E-test was employed to determine the MIC of ciprofloxacin only. Nalidixic acid resistance was evaluated as a marker for RSC. Interpretations were made according to CLSI guidelines. MDR strains were considerably more prevalent in Iraq (83%) and Pakistan (52%) compared with the other countries studied (13–52%). Nearly all isolates were susceptible (99.7%) to ceftriaxone. RSC was detected in a total of 218 isolates (22%), mostly from Iraq (54/59, 92%), Uzbekistan (98/123, 80%), Qatar (23/43, 54%) and Pakistan (31/65, 47%). Many of these (21%) were also MDR. Use of nalidixic acid resistance as an indicator for RSC was 99% sensitive and 98% specific. This study reinforces the need for routine antimicrobial susceptibility surveillance of enteric fever isolates and close review of current therapeutic policies in the region.

QnrS1 structure-activity relationships.

Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and gyrase protection was assessed. qnrS1 and 11 qnrS1 alleles with site-directed Ala mutations in loop B were expressed in Escherichia coli BL21(DE3) and proteins were purified by affinity chromatography. Ciprofloxacin MICs were determined with and without IPTG. Gyrase DNA supercoiling was measured with and without ciprofloxacin IC50 and with various concentrations of QnrS1 proteins. Wild-type QnrS1 and QnrS1 with Asn-110→Ala and Arg-111→Ala substitutions increased the ciprofloxacin MIC 12-fold in BL21(DE3), although QnrS1 with Gln-107→Ala replacement increased it 2-fold more than wild-type did. However, QnrS1 with Ala substitutions at His-106, Val-108, Ser-109, Met-112, Tyr-113, Phe-114, Cys-115 and Ser-116 increased ciprofloxacin MIC 1.4- to 8-fold less than wild-type QnrS1. Induction by 10-1000 μM IPTG increased ciprofloxacin MICs for all mutants, reaching values similar to those for wild-type. Purified wild-type and mutated proteins differed in protection of gyrase from ciprofloxacin action. Wild-type QnrS1 produced complete protection of gyrase supercoiling from ciprofloxacin (1.8 μM) action at 0.05 nM and half protection at 0.5 pM, whereas QnrS1 with Ala replacements that conferred the least increase in ciprofloxacin MICs also required the highest QnrS1 concentrations for protection. Key individual residues in QnrS1 loop B affect ciprofloxacin resistance and gyrase protection from ciprofloxacin action, supporting the concept that loop B is key for interaction with gyrase necessary for quinolone resistance.

SalmonellaSubtypes with Increased MICs for Azithromycin in Travelers Returned to the Netherlands

Antimicrobial susceptibility was analyzed for 354 typhoidal Salmonella isolates collected during 1999–2012 in the Netherlands. In 16.1% of all isolates and in 23.8% of all isolates that showed increased MICs for ciprofloxacin, the MIC for azithromycin was increased. This resistance may complicate empirical treatment of enteric fever.

Prevalence and characterisation of quinolone resistance mechanisms in Salmonella spp.

The study was focused on characterisation of quinolone resistance mechanisms in Salmonella isolated from animals, food, and feed between 2008 and 2011. Testing of Minimal Inhibitory Concentrations revealed 6.4% of 2680 isolates conferring ciprofloxacin resistance. Simultaneously 37.7% and 40.8% were accounted for, respectively, nalidixic acid and ciprofloxacin Non Wild-Type populations. Amplification and sequencing of quinolone resistance determining region of topoisomerases genes in 44 isolates identified multiple amino-acid substitutions in gyrA at positions Ser83 (N=22; →Leu, →Phe, →Tyr), Asp87 (N=22; →Asn, →Gly, →Tyr) and parC (Thr57Ser, N=23; Ala141Ser, N=1). No relevant mutations were identified in gyrB and parE. Twelve patterns combining one or two substitutions were related to neither serovar nor ciprofloxacin MIC. In 92 isolates suspected for plasmid mediated quinolone resistance two qnr alleles were found: qnrS1 (or qnrS3; N=50) and qnrB19 (or qnrB10; N=24). Additionally, two isolates with chromosomally encoded mechanisms carried qnrS1 and qnrS2. All tested isolates were negative for qnrA, qnrC, qnrD, qepA, aac(6′)-Ib-cr. Both chromosomal and plasmid mediated quinolone resistance determinants were found in several Salmonella serovars and Pulsed Field Gel Electrophoresis was used to assess phylogenetic similarity of selected isolates (N=82). Salmonella Newport was found to accumulate quinolone resistance determinants and the serovar was spreading clonally with either variable gyrA mutations, qnrS1/S3, or qnrB10/B19. Alternatively, various determinants are dispersed among related S. Enteritidis isolates. Antimicrobial selection pressure, multiple resistance determinants and scenarios for their acquisition and spread make extremely difficult to combat quinolone resistance.

The association between occurrence of plasmid-mediated quinolone resistance and ciprofloxacin resistance in Escherichia coli isolates of different origins

This study was performed to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and characterize the ciprofloxacin resistance in Escherichia coli isolated from different sources in China. PMQR determinants were detected by PCR amplification and sequencing in 2297 E. coli isolates randomly collected from animals, food and humans during 2004 to 2011. MICs of ciprofloxacin were determined by agar dilution method. Of the 2297 E. coli isolates, 43.6% harbored at least one PMQR gene. The most common PMQR gene was oqxAB (29.3%), followed by qnr (13.6%), aac(6′)-Ib-cr (11.6%), and qepA (3.3%). 12.0% isolates carried two or more PMQR genes. The prevalence of PMQR genes in food animal isolates increased over time, from 38.7% in 2004 to 69.8% in 2011. The prevalence of PMQR/ciprofloxacin resistance among isolates from pig, chicken, duck, companion animals, animal food and human volunteers were 65.2%/69.6%, 42.4%/60.0%, 59.4%/65.0%, 28.6%/57.5%, 29.3%/25.6%, and 14.0/8.7%, respectively. Most isolates carrying qnr along showed susceptible to ciprofloxacin, and only 21.6% the isolates exhibited resistance to ciprofloxacin, which was significantly lower than those carrying other PMQR genes (65.2–89.9%) and those that do not (43.1%) (p<0.01). In conclusion, high frequency of ciprofloxacin resistance and PMQR genes was observed among E. coli isolates of different origins in China, with oqxAB being the most frequent. qnr-positive E. coli isolates have relatively low ciprofloxacin resistance rate compared with other PMQR determinants-carrying isolates and PMQR-negative isolates.

Fluoroquinolone Susceptibility Testing of Salmonella enterica: Detection of Acquired Resistance and Selection of Zone Diameter Breakpoints for Levofloxacin and Ofloxacin

Fluoroquinolones (e.g., ciprofloxacin) have become a mainstay for treating severe Salmonella infections in adults. Fluoroquinolone resistance in Salmonella is mostly due to mutations in the topoisomerase genes, but plasmid-mediated quinolone resistance (PMQR) mechanisms have also been described. In 2012, the Clinical and Laboratory Standards Institute (CLSI) revised the ciprofloxacin interpretive criteria (breakpoints) for disk diffusion and MIC test methods for Salmonella. In 2013, the CLSI published MIC breakpoints for Salmonella to levofloxacin and ofloxacin, but breakpoints for assigning disk diffusion results to susceptible (S), intermediate (I), and resistant (R) categories are still needed. In this study, the MICs and inhibition zone diameters for nalidixic acid, ciprofloxacin, levofloxacin, and ofloxacin were determined for 100 clinical isolates of nontyphi Salmonella with or without resistance mechanisms. We confirmed that the new levofloxacin MIC breakpoints resulted in the highest category agreement (94%) when plotted against the ciprofloxacin MICs and that the new ofloxacin MIC breakpoints resulted in 92% category agreement between ofloxacin and ciprofloxacin. By applying the new MIC breakpoints in the MIC zone scattergrams for levofloxacin and ofloxacin, the following disk diffusion breakpoints generated the least number of errors: ≥28 mm (S), 19 to 27 mm (I), and ≤18 mm (R) for levofloxacin and ≥25 mm (S), 16 to 24 mm (I), and ≤15 mm (R) for ofloxacin. Neither the levofloxacin nor the ofloxacin disk yielded good separation of isolates with and without resistance mechanisms. Further studies will be needed to develop a disk diffusion assay that efficiently detects all isolates with acquired resistance to fluoroquinolones.