MICs Of Ciprofloxacin Research Articles

Synthesis and biologic activities of some novel heterocyclic chalcone derivatives

We synthesized 36 chalcone-like (E)-3-(substitutedphenyl)-1-hetrylprop-2-en-1-ones by condensing 2-acetylfuran/2-acetylpyrrole with substituted benzaldehydes under basic conditions. Of the 36 molecules synthesized, 10 are new to the literature. Bio-evaluation studies of these molecules revealed that compounds 5, 9, 15, 25, and 29 were potent NorA efflux pump inhibitors against Staphylococcus aureus by reducing MIC of ciprofloxacin fourfold, while compounds 11, 21, 25, and 26 showed promising anticancer activity in all four tested cancer cell lines (HL-60, MOLT-4, PC-3, and HeLa). Compound 25 emerged as a very good potentiator of ciprofloxacin against multidrug resistant S. aureus and also showed promising anticancer activity. The present communication describes syntheses, bio-evaluation, and structure-related activity of the (E)-3-(substitutedphenyl)-1-hetrylprop-2-en-1-ones.

Expression of efflux pump gene lde in ciprofloxacin‐resistant foodborne isolates of Listeria monocytogenes

The expression of efflux pump gene lde in ciprofloxacin resistant (Cip(R) ) and susceptible strains of Listeria monocytogenes collected from retail food samples was investigated. For two Cip(R) strains, the MICs of ciprofloxacin decreased four- to eightfold in the presence of reserpine; however, no significant alterations were observed with naturally sensitive isolates. Overexpression of the lde gene induced by ciprofloxacin was observed in two resistant isolates. The present findings indicate that expression of lde and the MICs of ciprofloxacin are well correlated with the presence and absence of reserpine, suggesting that Lde might be involved in ciprofloxacin resistance of L. monocytogenes.

Concentration-resistance relationships with Pseudomonas aeruginosa exposed to doripenem and ciprofloxacin in an in vitro model

To compare the abilities of doripenem and ciprofloxacin to restrict the enrichment of resistant Pseudomonas aeruginosa, multiple antibiotic dosing regimens were simulated in an in vitro model at comparable ratios of the 24 h AUC (AUC24) to the MIC. Three clinical isolates of ciprofloxacin-resistant P. aeruginosa (MIC of doripenem 1 mg/L, MIC of ciprofloxacin 4 mg/L) were exposed to thrice-daily doripenem or twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios from 50 to 170 h (doripenem) and from 55 to 180 h (ciprofloxacin). Doripenem- and ciprofloxacin-resistant mutants were enriched at antibiotic concentrations that fell into the mutant selection window for ≥ 45% and ≥ 60% of the dosing interval, respectively. The anti-mutant effects of doripenem and ciprofloxacin expressed by the area under the bacterial mutant concentration-time curve (AUBCM) depended on the AUC24/MIC ratio, and the AUBCM-log AUC24/MIC relationships were antibiotic specific. Based on AUC24/MIC relationships with AUBCM, a greater anti-mutant potential was predicted for doripenem compared with ciprofloxacin.

Coordinate Hyperproduction of SmeZ and SmeJK Efflux Pumps Extends Drug Resistance in Stenotrophomonas maltophilia

A Stenotrophomonas maltophilia mutant that coordinately hyper-expresses three resistance nodulation division-type efflux pump genes, smeZ, smeJ, and smeK, has been identified. SmeZ is responsible for elevating aminoglycoside MICs; SmeJ and SmeK are jointly responsible for elevating tetracycline, minocycline, and ciprofloxacin MICs and conferring levofloxacin resistance. One clinical isolate with this same phenotype was identified from a sample of six, and the isolate also coordinately hyper-expresses smeZ and smeJK, confirming the clinical relevance of our findings.

Identification of a novel insertion sequence element associated with carbapenem resistance and the development of fluoroquinolone resistance in Acinetobacter radioresistens

Sir, Acinetobacter radioresistens, although sometimes isolated on the skin of healthy humans, rarely causes serious illness. In a recent study, A. radioresistens was isolated from blood and urine cultures, but made up ,1% of the total Acinetobacter spp. isolated, while other studies found a higher incidence of A. radioresistens when only looking at Acinetobacter bloodstream isolates, where bacteraemia is most often associated with indwelling devices. Compelling evidence suggests that the origin of OXA-23, the most commonly acquired oxacillinase in Acinetobacter baumannii, is A. radioresistens, to which this enzyme is intrinsic. In A. baumannii, blaOXA-23 mediates carbapenem resistance when overexpressed and this is caused by the insertion element ISAba1, which is located upstream where it provides a strong promoter; however, this has not been found in A. radioresistens, so the mechanism of blaOXA-23 mobilization is still not fully understood. Despite possessing OXA-23, carbapenem resistance is rarely described in A. radioresistens and has only been reported when the additional acquired carbapenemases IMP-1 and OXA-58 were present. In this study, we investigated carbapenem resistance and the development of fluoroquinolone resistance in two A. radioresistens isolates that were recovered from a patient 17 days apart. The primer sequences used in this investigation are shown in Table S1 (available as Supplementary data at JAC Online). The ISAcra1-blaOXA-23 nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank nucleotide sequence database under accession no. JQ326202. Isolate F1244 was isolated from a blood culture of a patient with a catheter-related bloodstream infection. Ciprofloxacin was used for treatment. Isolate A1474 was recovered 17 days later from a sputum sample. Species identification was confirmed by rpoB sequencing and the isolates were typed by rep-PCR (DiversiLab) as previously described. The isolates showed 98% similarity in their rep-PCR patterns and confirms their clonality. The only difference we found between the isolates was in their fluoroquinolone susceptibility: isolate F1244 was susceptible to ciprofloxacin, levofloxacin and moxifloxacin, whereas isolate A1474 was resistant (Table 1). Carbapenem MICs were investigated by Etest and showed that both isolates were carbapenem resistant (Table 1). The isolates were negative for other blaOXA genes often associated with Acinetobacter and we investigated the expression of the intrinsic blaOXA-23 by qRT–PCR. qRT–PCR was performed three times in triplicate using freshly prepared RNA and cDNA as previously described, with rpoB as a reference gene. This revealed that F1244 and A1474 expressed similar levels of blaOXA-23 and that it was overexpressed .100-fold when compared with the carbapenem-susceptible control strain A. radioresistens SH164 (data not shown). PCR to detect ISAba1 adjacent to blaOXA-23 proved negative. Analysis of the A. radioresistens genome sequence revealed putative O-sialoglycoproteinand ATPase-encoding genes flanking blaOXA-23. The primer pair OXA-23-up/OXA-23-down was designed to amplify and sequence the region between these flanking genes and revealed an 700 bp insertion in the 5′ region of blaOXA-23. Sequencing revealed a novel insertion element in a noncoding region 62 bp upstream of the blaOXA-23 start codon. This insertion element was submitted to the IS Database (http://www-is. biotoul.fr/) and termed ISAcra1. ISAcra1 is a 732 bp element that encodes a predicted 220 amino acid transposase that is flanked by 15 bp inverted repeats and a 7 bp target site duplication. The element belongs to the IS1595 family and the transposase showed 47% amino acid identity to ISFtu3 from Francisella tularensis. The blaOXA-23 gene and putative promoter region was amplified and cloned into shuttle plasmid pWH1266 and transformed into carbapenem-susceptible A. baumannii ATCC 17978 and A. radioresistens SH164, leading to carbapenem resistance (Table 1). We found a single nucleotide difference between the isolates in gyrA, leading to a Ser-83 Phe amino acid substitution in isolate A1474. In A. baumannii, Ser-83 in GyrA is the most commonly altered amino acid associated with low-level fluoroquinolone resistance (typically ciprofloxacin MICs of 4–32 mg/L). We did not detect any difference in parC between the two isolates. We have previously found efflux to be associated with the development of fluoroquinolone resistance. To test for an efflux phenotype, agar dilution MICs were performed with the following commonly effluxed substrates: tetracycline, chloramphenicol, rifampicin, gentamicin, erythromycin and the dye rhodamine 6-G. Agar dilution MICs did not show any difference in MICs between isolates F1244 and A1474 against these substrates, suggesting efflux was not selected (see Table S2 available as Supplementary data at JAC Online). In conclusion, this study demonstrates that carbapenem resistance was mediated through overexpression of the intrinsic blaOXA-23 and was associated with the novel ISAcra1. Fluoroquinolone resistance developed during ciprofloxacin therapy and was associated with a gyrA mutation. These data show for the first time the ability of A. radioresistens to develop fluoroquinolone resistance during therapy. Additionally, ISAcra1

Multidrug-ResistantSalmonella enterica, Democratic Republic of the Congo

Multidrug-Resistant<i>Salmonella enterica</i>, Democratic Republic of the Congo

Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia

A previous study identified an association between high MICs of quaternary ammonium compounds (QACs) and antibiotic resistance. The current aim was to investigate the genetic background of this association. Of 153 Escherichia coli clinical strains, seven were selected for their low or high MICs of antibiotics and/or QACs. Integron resistance gene contents were identified by sequencing after PCR amplification. The genes encoding the efflux pump AcrA/TolC and its regulatory regions marA, marO, marR, soxS and rob were sequenced. The gene expression of acrA, tolC, marA, marOR, soxS and rob was assessed by quantitative real-time PCR. MICs in the presence and absence of the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PAβN) were compared. Of the seven strains, five were resistant to amoxicillin, amoxicillin/clavulanic acid and/or co-trimoxazole (trimethoprim/sulfamethoxazole) and/or had high MICs of ciprofloxacin and QACs. Four of the five harboured a class 1 integron (intI1). In three of these four strains, the presence of dfrA/sul1 and qacEΔ1 gene cassettes correlated with resistance to co-trimoxazole and high MICs of QACs. In all of the five strains, overexpression of tolC, marOR and soxS was always associated with higher MICs of antibiotics and/or QACs. PAβN reduced the MICs of ciprofloxacin and QACs, suggesting that extrusion of ciprofloxacin and QACs from bacteria depends on the AcrAB-TolC system. To our knowledge, this report is the first to describe dual involvement of the AcrAB-TolC system and class 1 integrons in clinical E. coli strains.

Increase in fluoroquinolone non-susceptibility among clinical Streptococcus pyogenes in Belgium during 2007-10

To study the temporal evolution of fluoroquinolone non-susceptibility among Streptococcus pyogenes during 2007-10 in Belgium. S. pyogenes (n = 4690) recovered from patients with tonsillopharyngitis or skin, wound or invasive infections were screened for fluoroquinolone non-susceptibility. A selection of fluoroquinolone-non-susceptible strains was investigated for resistance mechanisms: reserpine-sensitive efflux and mutations in topoisomerase genes parC and gyrA. Clonality was determined by emm typing. Fluoroquinolone non-susceptibility (ciprofloxacin MIC ≥2 mg/L) was identified in 535 (11.4%) of 4690 S. pyogenes recovered during 2007-10 in Belgium. The proportion of fluoroquinolone-non-susceptible S. pyogenes increased significantly from 4.3% (2008) to 10.9% (2009) to 21.6% (2010) and coincided with a significant increase in emm6 strains among fluoroquinolone-non-susceptible S. pyogenes. Ciprofloxacin MICs of 2-8 mg/L correlated with first-step ParC substitutions. Two high-level fluoroquinolone-resistant S. pyogenes strains (ciprofloxacin MICs 32 mg/L) showed second-step substitutions in GyrA (Ser-81→Phe or Tyr) in addition to first-step mutations in parC. Reserpine-sensitive efflux was not observed. We report an unprecedented increase in fluoroquinolone-non-susceptible S. pyogenes in Belgium, a country with high quinolone use, as well as emergence of two high-level fluoroquinolone-resistant S. pyogenes strains with second-step mutations in gyrA, warning us of the need for more prudent use of fluoroquinolones and for continued resistance surveillance.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus

To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

Impact of low-level fluoroquinolone resistance genes qnrA1, qnrB19 and qnrS1 on ciprofloxacin treatment of isogenic Escherichia coli strains in a murine urinary tract infection model

To study the impact of qnrA1, qnrB19 and qnrS1 on the ciprofloxacin treatment of urinary tract infection (UTI). From a wild-type (wt) Escherichia coli UTI isolate, three isogenic strains were constructed carrying low-level ciprofloxacin resistance genes qnrA1, qnrB19 or qnrS1 (ciprofloxacin MIC range: 0.19-0.38 mg/L). Time-kill studies were performed for all four isogenic strains at the following concentrations: 1×, 2×, 4×, 8× and 16× MIC. Ciprofloxacin serum and urine pharmacokinetics was determined to calculate a murine dose equivalent (AUC(24)) to the standard human dose of 500 mg twice daily, which corresponded to 0.2 mg/mouse four times daily. In the murine UTI model, mice infected with each of the isogenic qnr strains or the wt strain were treated with ciprofloxacin (0.2 mg/mouse) or saline (only the E. coli wt) subcutaneously four times daily for 3 days starting 24 h after bacterial inoculation. In vitro, the strains responded to ciprofloxacin concentrations of 4-16× MIC by several log(10) reductions. In vivo, despite ciprofloxacin reaching urine concentrations far exceeding the MICs for the strains (500 mg/L), ciprofloxacin was significantly less efficient at reducing the urine and bladder bacterial counts of qnrA1-, qnrB19- and qnrS1-positive strains compared with the ciprofloxacin-treated wt strain (P < 0.05). None of the four strains infected the kidneys well, with median cfu counts of <1 log(10). Although qnr genes only confer low levels of resistance to ciprofloxacin, a reduced bactericidal activity of ciprofloxacin was observed in both urine and bladder in the murine model of UTI.

Characterization of CTX-M-14- and CTX-M-15-producing Escherichia coli of porcine origin

Sir, The emergence of extended-spectrum b-lactamase (ESBL)producing Escherichia coli among human patients is largely due to the spread of CTX-M, especially the clonal spread of sequence type (ST) 131 E. coli producing CTX-M-15. In recent years ESBLproducing E. coli have also been detected in food and animals and ST131 has been detected in E. coli of animal origin. To our knowledge, E. coli ST131 producing CTX-M-15 has been detected in companion animals, but not in other animals or in meat. Major questions include whether animals can be a reservoir of the STs that have been associated with ESBL-producing E. coli causing infections in humans, and whether animals and meat can be a reservoir of ESBL genes that can be transferred to some of the pathogenic E. coli STs (e.g. ST131) in the human intestine. Recently Agerso et al. reported on the prevalence of cephalosporinase-producing E. coli from Danish pig (n1⁄4786), Danish pork (n1⁄4153) and imported pork (n1⁄4173) samples taken in 2009. CTX-M-1-producing E. coli were most common among the ESBL-producing E. coli from pigs and pork, whereas a few isolates were producers of CTX-M-14 and CTX-M-15; whether these types are established in pigs is unknown. In the present study, one CTX-M-14-producing E. coli isolate from imported pork, six CTX-M-14-producing E. coli from Danish pigs and two CTX-M-15-producing E. coli from Danish pigs originating from the cephalosporinase prevalence study mentioned above were characterized for PFGE profile, multilocus sequence typing (MLST) ST, resistance profile, phylotype, clonal group A status, virulence genes and transfer of blaCTX-M genes (Table 1). PFGE with XbaI was performed on the nine E. coli isolates. MLST was performed using seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) (http://mlst. ucc.ie/mlst/dbs/Ecoli). The MICs of chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline and trimethoprim for the isolates were determined (Trek Diagnostic, East Grinstead, UK). Results were interpreted according to the EUCAST (www.EUCAST.org) clinical breakpoint except for results for nalidixic acid, tetracycline, streptomycin and sulfamethoxazole, which were interpreted according to CLSI. E. coli ATCC 25922 was used for quality control. The phylogenetic background (A, B1, B2 or D) of the isolates was determined by PCR. Results obtained allowed classification of the isolates into the four major E. coli phylogenetic lineages or non-typeable isolates. Isolates classified as phylogroup D were tested for the presence of single-nucleotide polymorphisms in fumB and gyrB, which has been associated with a clonal group of extraintestinal pathogenic E. coli (ExPEC), designated clonal group A. Furthermore, the isolates were investigated for the ExPEC-related virulence genes kpsM II, papA, papC, iutA, sfaS, focG, afa and dra. In vitro matings were performed with a rifampicin-resistant mutant of an ST131 B2 E. coli of Danish pork origin as recipient. Three selected transconjugants from each mating were examined for the presence of the transferred blaCTX-M gene by PCR. Furthermore, PFGE was performed on the three selected transconjugants, the donor and the recipient. The isolates had different PFGE types (≤80% identical) and were not clonally related to any of the 80 ESBL-producing E. coli blood isolates of human origin from 2009 obtained in our BioNumerics database (data not shown). The nine CTX-M-producing E. coli isolates in the present study had nine different STs, of which ST48, ST101, ST117 and ST167 had previously been detected in human patients (Table 1). – 8 Six of the nine isolates were resistant to other classes of antimicrobial agents beside the b-lactams (Table 1). Urinary tract infections are most often associated with phylogroup B2 and to some extent with phylogroup D. None of the nine isolates belonged to phylogroup B2 (Table 1). However, blaCTX-M-14and blaCTX-M-15-producing E. coli isolates from patients with infections belonging to phylogroup A, B1 and D have been detected. – 5 Two of the tested E. coli belonged to phylogroup D, but not to the clonal group A. A previous Danish study of phylogroup D E. coli of porcine origin likewise failed to identify any clonal group A isolates. Isolate 2700-1 (CTX-M-14) was positive for focG (F1C fimbriae) and iutA (aerobactin siderophore) and isolate 2469-1 (CTX-M-14) was positive for papA/papC (P fimbriae) and iutA, which defined both isolates as ExPEC isolates according to the molecular definition.

Prevalence of the oqxAB gene complex in Klebsiella pneumoniae and Escherichia coli clinical isolates

To investigate the epidemiology and genetic elements of the efflux pump gene, oqxAB, in clinical isolates of Escherichia coli and Klebsiella pneumoniae. The prevalence of the oqxAB gene in a total of 308 consecutive clinical isolates of E. coli, K. pneumoniae and other Klebsiella spp. was investigated by PCR amplification. The full-length oqxAB gene was sequenced. Conjugation experiments were carried out in oqxAB-positive E. coli strains. Of 136 E. coli strains, 71.3% and 70.6% were non-susceptible to ciprofloxacin and levofloxacin, respectively, while 66.9% and 50.7% of the 154 K. pneumoniae were non-susceptible to these two quinolones. oqxA and oqxB were present in 6.6% of E. coli and in all K. pneumoniae strains. The oqxA and oqxB sequences of E. coli and randomly selected K. pneumoniae strains showed high similarity to the original oqxAB sequence of pOLA52. One or two amino acid substitutions were detected in the OqxA or OqxB sequences from two E. coli strains and were designated oqxA2, oqxB2 and oqxB3. oqxAB was transferred in seven of nine oqxAB-positive E. coli strains, and transconjugants showed 2- to 256-fold increases in ciprofloxacin and levofloxacin MICs relative to that for the recipient. Variants of the oqxAB gene, oqxA2, oqxB2 and oqxB3, were identified in two E. coli strains. The oqxAB gene was present in all K. pneumoniae strains studied and is likely located on the chromosome, thus identifying the genome of K. pneumoniae as a possible reservoir of oqxAB.

Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks

The antimicrobial susceptibility of 103 Riemerella anatipestifer isolates obtained from ducks during 2008 and 2010 in Southern China, to 23 antimicrobial agents was investigated using the agar dilution method. The MIC50 and MIC90 values of streptomycin, kanamycin, gentamicin, apramycin, amikacin, neomycin, nalidixic acid and sulfadimidine were high (32–≥128μg/ml) among the 103 R. anatipestifer isolates. However, relatively low MIC90 values (8μg/ml) of ampicillin and florfenicol were observed among these isolates. The presence of resistance genes and integrons was determined using PCR. The genes blaTEM-1, aph(3′)-VII, aadA1, aadA2, aac(3′)-IV, aac(3′)-IIc, aac(6′)-Ib, cat2, cmlA, floR, tet(A), tet(B), tet(C), sul1, and sul2 were detected in 1, 2, 24, 35, 11, 4, 67, 16, 26, 10, 6, 1, 9, 36 and 2 isolates, respectively. Twenty isolates contained one or two class 1 integrons carrying aadA2 or aac(6′)-II-catB3-aadA1 gene cassette(s). Mutation analysis of the quinolone resistance-determining regions (QRDRs) of 43 R. anatipestifer isolates with nalidixic acid MICs≥32μg/ml, showed that the most prevalent mutations in gyrA were those resulting in the amino acid exchanges Ser83-Ile (n=37), followed by Asp87-His (n=7) and Ser83-Arg (n=5). Point mutations in parC (Arg120-Glu) were observed in 5 isolates with a ciprofloxacin MIC of >16μg/ml. No plasmid-mediated quinolone resistance gene was detected. PFGE analysis showed that the clonal spread of multi-drug resistant R. anatipestifer isolates occurred in the same farm or between different farms. Our results reported, for the first time, the mechanism of quinolone resistance in R. anatipestifer.

Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009

Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum β-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ≥ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.

English

The quinolone-resistance determining-region of&nbsp;gyrA&nbsp;was PCR-amplified and sequenced in seven fluoroquinolone-resistant&nbsp;Pseudomonas aeruginosa&nbsp;(Nalidixic acid MICs ranging 8 to128 &micro;g/ml, ciprofloxacin MICs ranging 8 to 32 &micro;g/ml and&nbsp; levofloxacin MICs ranged from 32 to 64 &micro;g/ml) isolated from poultry that died of septicaemic clinical diseases in Ibadan, Oyo State, South Western Nigeria. The seven isolates were also multidrug resistant to various combinations of the commonly used antibiotics like streptomycin, ampicillin, tetracycline, kanamycin, neomycin and chloramphenicol. The entire seven isolates possessed the&nbsp;gyrA&nbsp;mutation encoding the histidine to tyrosine conversion at amino acid 150 (H150Y). Additional substitutions observed included: aspartic acid to tyrosine substitution at amino acid 87 (D87Y) in two isolates, aspartic acid to glycine D87G substitution in three isolates, another simultaneous seven substitutions: aspartic acid to alanine, D87A; alanine to proline, A62P; tyrosine to isoleucine, Y83I; methionine to leucine, M92L; leucine to methionine, L98M; L128M; and aspartic acid to proline D148P&nbsp; in one of the isolate, whereas one of the seven isolates possessed only the H150Y substitution. The H150Y is typical of the quinolone resistant&nbsp;P. aeruginosa&nbsp;isolated from septic poultry in Nigeria, regardless of other resistance- patterns exhibited to other commonly used antibiotics.&nbsp; This study associate&nbsp;gyrA&nbsp;mutations with fluoroquinolone resistance in&nbsp;P. aeruginosa&nbsp;isolated from septicaemic poultry in Nigeria, where fluoroquinolone use in livestock is not strictly regulated and misuse/abuse of most antibiotics is rampart. &nbsp; Key words:&nbsp;Pseudomonas aeruginosa,&nbsp;resistance, fluoroquinolone, poultry, Oyo state, Nigeria.

No Decrease in Susceptibility to NVC-422 in Multiple-Passage Studies with Methicillin-Resistant Staphylococcus aureus, S. aureus, Pseudomonas aeruginosa, and Escherichia coli

Twenty-five serial passages of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus and 50 passages of methicillin-resistant Staphylococcus aureus resulted in no significant increase in NVC-422 MICs, while ciprofloxacin MICs increased 256-fold for E. coli and 32-fold for P. aeruginosa and S. aureus. Mupirocin, fusidic acid, and retapamulin MICs for MRSA increased 64-, 256-, and 16-fold, respectively. No cross-resistance to NVC-422 was observed with mupirocin-, fusidic acid-, and retapamulin-resistant strains.

Effect of 1-(1-naphthylmethyl)-piperazine on antimicrobial agent susceptibility in multidrug-resistant isogenic and veterinary Escherichia coli field strains

The objective of this study was to evaluate the interaction of the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP) when combined with different families of antimicrobial agents against isogenic strains and multidrug-resistant (MDR) Escherichia coli field strains isolated from animals. Laboratory isogenic strains of E. coli with different levels of expression of efflux pumps were used as quality controls. Ten MDR E. coli strains were collected from healthy animals in a cross-sectional study in four commercial dairy farms. The MICs of florfenicol, ciprofloxacin, tetracycline and ampicillin were determined by a serial microdilution method in Luria-Bertani broth in the presence or absence of NMP. NMP used with ampicillin exerted no effect on the isogenic or field strains. In most of the field MDRE. coli strains and in an acrAB-overexpressing (AG112) isogenic strain, the MICs of florfenicol, ciprofloxacin and tetracycline decreased at least fourfold when the antimicrobial was combined with the highest NMP concentrations. In the wild-type strain (AG100), there were no decreases of more than twice the MIC, whilst in strain AG100A, an efflux pump-deficient strain, the MIC did not change, regardless of the concentration of NMP used with these three antimicrobials. Thus, ampicillin was not affected by the efflux pump mechanism, whereas ciprofloxacin, tetracycline and florfenicol were shown to be substrates of efflux pumps, with a consequent significant reduction in MICs. Resistance could not be completely reversed in the E. coli field strains by NMP, probably because other resistance mechanisms were also present. However, in strain AG112, the MIC results demonstrated that NMP expressed an important synergistic activity with florfenicol. The reduction in florfenicol MIC value was sufficient to reverse antimicrobial resistance completely for AG112.

Evaluation of quinolone resistance–determining region mutations and efflux pump expression in Neisseria meningitidis resistant to fluoroquinolones

Three Neisseria meningitidis serotype B strains displaying elevated ciprofloxacin MIC values (0.06 and 0.25 μg/mL) and a GyrA alteration at T91I have been described in the United States by Wu et al. [Wu et al., (2009) Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America. N. Engl. J. Med. 360:886–892] and were further evaluated here by reference broth microdilution against fluoroquinolones (FQ) and other antimicrobial agents. Additional quinolone resistance–determining region (QRDR) mutations and alterations in structure and expression of the mtrCDE efflux system, including its intergenic regions, were also analyzed. Two strains showed ciprofloxacin and levofloxacin MIC values at 0.25 μg/mL, and 1 strain had a ciprofloxacin MIC at 0.06 μg/mL. In addition to T91I, the 2 strains displaying higher FQ MIC values also possessed a T173A alteration on GyrA. All components of the efflux pump mtrCDE (also associated with rifampin resistance) were intact, excluding the presence of insertions/deletions within the pump operon. The promoter region (mtrR) was fully sequenced and was distinct from FQ-susceptible controls. Isolates with higher ciprofloxacin MIC values had identical promoter region, whereas the isolate displaying a lower ciprofloxacin MIC value possessed a different sequence. Expression experiments showed discrepancies of the mRNA in pump components. The most remarkable difference was for the outer membrane protein encoded by mtrE that was hyperexpressed (>3600× elevated compared to control) in the strain displaying a ciprofloxacin MIC value of 0.06 μg/mL. These results confirm that T91I alteration on GyrA had an important role in elevating ciprofloxacin MIC values; however, additional resistance determinants, including other gyrA mutations, also contribute to higher FQ MIC levels. Alterations in expression of mtrCED pump appear to have minimal influence on ciprofloxacin resistance, and this finding was supported by low rifampin susceptible-level results (MIC, ≤0.008 to 0.03 μg/mL).

Deletion of Tn AbaR23 Results in both Expected and Unexpected Antibiogram Changes in a Multidrug-Resistant Acinetobacter baumannii Strain

Since the 2006 discovery of the Acinetobacter baumannii strain AYE AbaR1 resistance island, similar elements have been reported in numerous members of this species. As AbaR1 is distantly related to Tn7, we have renamed it TnAbaR1. TnAbaR transposons are known to carry multiple antibiotic resistance- and efflux-associated genes, although none have been experimentally studied en bloc. We deleted the TnAbaR transposon in A. baumannii A424, which we have designated TnAbaR23, and characterized independent deletion mutants DCO163 and DCO174. The NotI pulsed-field gel electrophoresis (PFGE) profile of strain DCO174 was consistent with targeted deletion of TnAbaR23 alone, but strain DCO163 apparently harbored a second large genomic deletion. Nevertheless, "subtractive amplification" targeting 52 TnAbaR and/or resistance-associated loci yielded identical results for both mutants and highlighted genes lost relative to strain A424. PCR mapping and genome sequencing revealed the entire 48.3-kb sequence of TnAbaR23. Consistent with TnAbaR23 carrying two copies of sul1, both mutants exhibited markedly increased susceptibility to sulfamethoxazole. In contrast, loss of tetAR(A) resulted in only a minor and variable increase in tetracycline susceptibility. Despite not exhibiting a growth handicap, strain DCO163 was more susceptible than strain DCO174 to 9 of 10 antibiotics associated with mutant-to-mutant variation in susceptibility, suggesting impairment of an undefined resistance-associated function. Remarkably, despite all three strains sharing identical gyrA and parC sequences, the ciprofloxacin MIC of DCO174 was >8-fold that of DCO163 and A424, suggesting a possible paradoxical role for TnAbaR23 in promoting sensitivity to ciprofloxacin. This study highlights the importance of experimental scrutiny and challenges the assumption that resistance phenotypes can reliably be predicted from genotypes alone.

Detection of mutations in quinolone-resistant determining regions in clinical isolates of Escherichia coli from Saudi Arabia

Since the use of fluoroquinolone antibiotic in clinical practice was introduced about two decades ago, quinolone-resistant E. coli strains are being isolated with increasing frequency. This study devotes to determine the resistance rate of fluoroquinolones for 112 Escherichia coli isolates from Prince Salman Hospital, Riyadh and to detect the mutations in the quinolone resistance-determining region (QRDR) of gyrA and parC in the fluoroquinolones resistant isolates. The resistance rate of ciprofloxacin for E. coli isolates was 37.5%. We have determined QRDR of the gyrA and parC genes of ciprofloxacin resistant E. coli (MIC 2-64 mg/L). A single mutation in gyrA (Ser TCG-83 → → → →Leu TTG) was detected in the isolate with a reduced susceptibility to ciprofloxacin (MIC 2 mg/L). Three mutations were detected in E. coli isolates with ciprofloxacin MIC ≥16 mg/L. Double mutations in gyrA (Ser-83 → → → →Leu and Asp-87 →Asn) and a single mutation in parC (Ser-80 →Ile). This study suggests, that in clinical isolates of E. coli , DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.