Coli Clinical Isolates Research Articles

Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae

Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.

Susceptibility to Alternative Oral Antimicrobial Agents in Relation to Sequence Type ST131 Status and Coresistance Phenotype among Recent Escherichia coli Isolates from U.S. Veterans

The rising prevalence of resistance to first-line antimicrobial agents in Escherichia coli, which has paralleled the emergence of E. coli sequence type ST131, has created a need for alternative oral options for use in treating outpatients with infections such as cystitis and chronic prostatitis. Accordingly, we determined susceptibility to six alternative oral agents (azithromycin, chloramphenicol, doxycycline, fosfomycin, minocycline, and rifampin) by Etest or disk diffusion for 120 recently obtained E. coli clinical isolates from Veterans Affairs Medical Centers across the United States. Isolates were randomly selected in three subgroups of 40 isolates each based on coresistance to fluoroquinolones with and without extended-spectrum cephalosporins (ESCs). Results were stratified according to trimethoprim-sulfamethoxazole (TMP-SMZ) phenotype. Overall, the prevalence of susceptible (or susceptible plus intermediate) isolates varied by agent, with rifampin being lowest (0%), fosfomycin highest (98 to 99%), and others in the mid-range (37 to 88%). Substantial proportions of isolates (15 to 27%) yielded intermediate results for azithromycin, chloramphenicol, doxycycline, and minocycline. Among isolates resistant (versus susceptible) to fluoroquinolones with or without ESCs, susceptibility to the above four agents declined significantly among non-ST131 isolates but not ST131 isolates. In contrast, in the presence of resistance to TMP-SMZ, susceptibility to azithromycin, doxycycline, and minocycline was significantly reduced among both ST131 and non-ST131 isolates. These findings identify potential alternative oral agents for use with E. coli isolates resistant to fluoroquinolones, ESCs, and/or TMP-SMZ and suggest that determination of ST131 status could help guide initial antimicrobial selection, pending susceptibility results.

Role of CSE1034 in Escherichia coli Biofilm Destruction

Present study was conducted to assess the effect of ethylenediamine tetraacetic acid (EDTA); a non antibiotic adjuvant, and CSE1034, a novel antibiotic adjuvant entity in biofilm destruction of Escherichia coli, and comparing the efficacy with other drugs. We first determined the susceptibility of six antimicrobial agents against planktonic cultures, as well as sessile cells of E. coli clinical isolates, using the Clinical and Laboratory Standards Institute (CLSI) method. Subsequently, effects of EDTA alone, and drugs on bacterial curli production, adhesion, and in-vitro biofilm destruction were studied. The percentage of biofilm persistence was determined with spectrophotometry. The structural damage of biofilms was studied by scanning electron microscope. It was found that, among the drugs used, CSE1034 was the most effective against all of the E. coli clinical isolates, with MIC and MBEC values ranging from 32-64 μg/ml and 256-512 μg/ml, respectively. Exposure of clinical isolates with EDTA alone caused inhibitions of curli formation and bacterial adhesion at 4 to 5 mM. Further, EDTA treatment of preformed biofilm caused complete biofilm destruction at 8-10 mM. Interestingly, CSE1034 due to presence of 10 mM EDTA led to enhanced antibacterial, as well as biofilm destruction activities. Results of spectrophotometric analysis and scanning electron microscopy revealed that approximately 92% biofilms were eradicated by the CSE1034, and not by comparator drugs. The results indicate that the CSE1034 appears to be most effective drug of choice for the treatment of infections caused by E. coli.

New Delhi Metallo-β-Lactamase 4–producingEscherichia coliin Cameroon

New Delhi Metallo-β-Lactamase 4–producing<i>Escherichia coli</i>in Cameroon

Fluoroquinolone-resistant extraintestinal Escherichia coli clinical isolates representing the O15:K52:H1 clonal group from humans and dogs in Australia

Antimicrobial-resistant extraintestinal pathogenic Escherichia coli (ExPEC) impact both human and veterinary medicine. One ExPEC clonal group that has become increasingly multidrug-resistant is serotype O15:K52:H1. Accordingly, we sought O15:K52:H1 strains among fluoroquinolone-resistant (FQ(r)) E. coli clinical isolates from humans (n=582) and dogs (n=120) in Australia. The phylogenetic group D isolates (267/702; 38%) were screened for O15:K52:H1-specific single-nucleotide polymorphisms (SNPs) in fumC and the O15 rfb variant. The 34 so-identified O15:K52:H1 isolates (33 human, 1 canine) underwent antimicrobial susceptibility profiling, virulence genotyping, and macrorestriction profiling. Although susceptibility profiles varied, the 34 isolates were closely related by pulsed-field gel electrophoresis and exhibited typical O15:K52:H1-associated virulence profiles (complete pap operon, F16 papA allele, papG allele II, iha, fimH, sat, fyuA, iutA, kpsMII, ompT). The canine isolate closely resembled human isolates. Thus, O15:K52:H1 strains contribute to the FQ(r) ExPEC population in Australia and may potentially be transferred between humans and dogs.

Virulence of Escherichia coli Clinical Isolates in a Murine Sepsis Model in Relation to Sequence Type ST131 Status, Fluoroquinolone Resistance, and Virulence Genotype

Escherichia coli sequence type ST131 (O25b:H4) has emerged over the past decade as a globally disseminated, multidrug-resistant pathogen. Unlike traditional antimicrobial-resistant E. coli, ST131 derives from virulence-associated phylogenetic group B2 and exhibits extraintestinal virulence factors. This, plus preliminary evidence of virulence in experimental animals, has suggested that ST131's epidemic emergence may be due to high virulence potential, compared with other E. coli types. To test this hypothesis, we compared a large number of matched ST131 and non-ST131 E. coli clinical isolates, both fluoroquinolone resistant and susceptible, plus isolates from classic extraintestinal pathogenic E. coli (ExPEC) sequence types (STs) and case report ST131 household transmission isolates, for virulence in a mouse subcutaneous sepsis model. Overall, in mice, the study isolates produced a wide range of lethality and clinical illness. However, neither ST131 status nor fluoroquinolone phenotype correlated with this diversity of illness severity, which occurred within each of the 6 study groups. In contrast, multiple known or suspected ExPEC virulence genes, including pap (P fimbriae), vat (vacuolating toxin), kpsM II (group 2 capsule), ibeA (invasion of brain endothelium), and clbB/N (colibactin synthesis), plus molecularly defined ExPEC status, were significantly associated with virulence. These findings point away from ST131 isolates as having higher virulence potential compared with other E. coli types in causing invasive extraintestinal infections and suggest instead that ST131's epidemiological success may reflect enhanced fitness for upstream steps in pathogenesis or in colonization and transmission. Additionally, the extensive within-ST virulence diversity suggests an opportunity to compare closely related strains to identify the responsible genetic determinants.

SoxS Increases the Expression of the Zinc Uptake System ZnuACB in an Escherichia coli Murine Pyelonephritis Model

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.

Emergence of Ertapenem Resistance in an Escherichia coli Clinical Isolate Producing Extended-Spectrum β-Lactamase AmpC

Escherichia coli isolate MEV, responsible for a bloodstream infection, was resistant to penicillins, cephalosporins, and ertapenem. Molecular and biochemical characterization revealed the production of a novel, chromosome-borne, extended-spectrum AmpC (ESAC) β-lactamase with a Ser-282 duplication and increased carbapenemase activity. This study demonstrates for the first time that chromosome-borne ESAC β-lactamases can contribute to the emergence of ertapenem resistance in E. coli clinical isolates.

Evaluation of antimicrobial susceptibility for β-lactams against clinical isolates from 51 medical centers in Japan (2008)

This antimicrobial resistance surveillance study was performed in 51 medical centers in Japan over an 11-year period. The susceptibilities of 4228 strains including Escherichia coli (491 strains), Klebsiella spp. (462 strains), Enterobacter spp. (459 strains), Citrobacter freundii (358 strains), indole-positive Proteus spp. (386 strains), Serratia spp. (443 strains), Acinetobacter spp. (327 strains), Pseudomonas aeruginosa (473 strains), oxacillin-susceptible Staphylococcus aureus (481 strains), and coagulase-negative staphylococci (CoNS; 348 strains) were tested with 7 β-lactams (cefepime, cefpirome, ceftazidime, cefoperazone/sulbactam, imipenem, and piperacillin for Gram-negative bacteria, or oxacillin for Gram-positive bacteria). No resistance to these β-lactams (with the exception of ceftazidime) was found in oxacillin-susceptible S. aureus and CoNS. Of the E. coli clinical isolates, 24.6% were resistant to piperacillin, whereas 3.5% or less (cefpirome = 4.5%) were resistant to other β-lactam agents. Klebsiella spp. isolates were more susceptible to imipenem (99.6%), cefepime (98.7%), ceftazidime (98.5%), cefpirome (97.6%), and cefoperazone/sulbactam (97.6%). Isolates of Enterobacter spp., C. freundii, and Serratia spp. were susceptible to imipenem, cefepime, and cefpirome as well. The sensitivities of these organisms against cefepime and cefoperazone/sulbactam were 100%. Acinetobacter spp. isolates were less resistant to cefoperazone/sulbactam (0.6% resistance), imipenem (0.9%), and ceftazidime (2.8%) compared with other β-lactam antibiotics tested. Isolates of P. aeruginosa were more susceptible to piperacillin (9.1% resistance), cefoperazone/sulbactam (9.5%), and cefepime (6.6%) compared with ceftazidime (10.8%), cefpirome (16.3%), and imipenem (23.5%). The proportion of strains resistant to β-lactam antimicrobials has decreased compared with data from 2006 ( Diagn. Microbiol. Infect. Dis. 60:177–183), reflecting the reduced consumption of β-lactams in Japan.

Diagnostic Accuracy Assessment of Sensititre and Agar Disk Diffusion for Determining Antimicrobial Resistance Profiles of Bovine Clinical Mastitis Pathogens

Determining the accuracy and precision of a measuring instrument is pertinent in antimicrobial susceptibility testing. This study was conducted to predict the diagnostic accuracy of the Sensititre MIC mastitis panel (Sensititre) and agar disk diffusion (ADD) method with reference to the manual broth microdilution test method for antimicrobial resistance profiling of Escherichia coli (n = 156), Staphylococcus aureus (n = 154), streptococcal (n = 116), and enterococcal (n = 31) bovine clinical mastitis isolates. The activities of ampicillin, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin, the penicillin-novobiocin combination, pirlimycin, and tetracycline were tested against the isolates. Diagnostic accuracy was determined by estimating the area under the receiver operating characteristic curve; intertest essential and categorical agreements were determined as well. Sensititre and the ADD method demonstrated moderate to highly accurate (71 to 99%) and moderate to perfect (71 to 100%) predictive accuracies for 74 and 76% of the isolate-antimicrobial MIC combinations, respectively. However, the diagnostic accuracy was low for S. aureus-ceftiofur/oxacillin combinations and other streptococcus-ampicillin combinations by either testing method. Essential agreement between Sensititre automatic MIC readings and MIC readings obtained by the broth microdilution test method was 87%. Essential agreement between Sensititre automatic and manual MIC reading methods was 97%. Furthermore, the ADD test method and Sensititre MIC method exhibited 92 and 91% categorical agreement (sensitive, intermediate, resistant) of results, respectively, compared with the reference method. However, both methods demonstrated lower agreement for E. coli-ampicillin/cephalothin combinations than for Gram-positive isolates. In conclusion, the Sensititre and ADD methods had moderate to high diagnostic accuracy and very good essential and categorical agreement for most udder pathogen-antimicrobial combinations and can be readily employed in veterinary diagnostic laboratories.

Composición química del aceite esencial de Tagetes elliptica Smith "chincho" y actividades antioxidante, antibacteriana y antifúngica

El presente trabajo de investigación tuvo por finalidad determinar cualitativamente los componentes químicos, así como las actividades antibacteriana, antifúngica y antioxidante del aceite esencial de las hojas de Tagetes elliptica Smith “chincho”, oriundo de la provincia de Huaraz, región Ancash; el cual se obtuvo por el método de destilación por arrastre con vapor de agua. Posteriormente se determinaron las constantes fisicoquímicas, y luego se hizo la determinación de su composición química por cromatografía de gases / espectometría de masas (CG/EM). Por los métodos de difusión en agar y dilución en agar se determinaron las actividades antimicrobiana y concentración mínima inhibitoria (CMI), respectivamente, frente a los siguientes microorganismos: S. aureus ATCC 25933, S. epidermidis (cepa clínica), B. subtilis (cepa ambiental), E. coli (cepa clínica), P. aeruginosa ATCC 27853, Klebsiella (cepa clínica) y C. albicans ATCC 10231. En las pruebas realizadas, el aceite esencial evidenció actividad antibacteriana frente a cinco cepas, mostrándose Klebsiella resistente debido a que presentó un halo de inhibición menor de 18 mm; frente a Candida albicans ATCC 10231, demostró actividad antifúngica significativa. La CMI frente a S. aureus, E. coli y P. aeruginosa fue de 7,62 x 10-1 μg/mL; para S. epidermidis 97,5 x 10-1 μg/mL; para B. subtilis 390 μg/mL x 10-1 y para C. albicans 780 μg/mL x 10-1. Respecto a la actividad antioxidante, se realizaron dos pruebas de captación de radicales libres denominadas: captación del radical difenilpicrilhidrazilo (DPPH) y captación del radical superóxido producido por pirogalol; obteniéndose los siguientes resultados: IC50=97,5 μg/mL e IC50= 47,5 μg/mL; respectivamente, los cuales no fueron significativos, teniendo en cuenta los patrones de Trolox y Vitamina C, respectivamente.

Use of fimH Single-Nucleotide Polymorphisms for Strain Typing of Clinical Isolates of Escherichia coli for Epidemiologic Investigation

Strain typing methods that compare electrophoresis banding patterns are commonly used but are difficult to standardize and poorly portable. Multilocus sequence typing (MLST) is a sequence-based alternative, but it is not practical for large-scale epidemiological studies. In the present study, the usefulness of fimH single-nucleotide polymorphisms (SNPs) for Escherichia coli typing was explored. fimH SNPs were determined for 345 E. coli clinical isolates (including 3 reference strains) and compared to PCR-based ECOR (E. coli reference collection) phylogrouping. The fimH gene could be amplified for 316 (92%) of the 345 isolates. fimH SNP analysis found 46 distinct terminal groups in the nucleotide sequence-based phylogenetic tree (fimH types). A subset of the E. coli isolates (162 clinical isolates and the 3 reference strains) were compared by fimH type, PCR phylogroup, and MLST. These isolates fell into 27 fimH types and 18 MLST clonal complexes (CCs) that contained 2 to 28 isolates per complex. The combination of PCR phylogroup and fimH type corresponded to a single CC for 113 (68%) isolates and 2 or 3 CCs for the other 52 (32%) isolates. We propose that the combination of PCR phylogrouping and fimH SNP analysis may be a useful method to type a large collection of clinical E. coli isolates for epidemiologic studies.

Constitutive SoxS Expression in a Fluoroquinolone-Resistant Strain with a Truncated SoxR Protein and Identification of a New Member of themarA-soxS-robRegulon,mdtG

Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.

Activity of Temocillin against KPC-Producing Klebsiella pneumoniae and Escherichia coli

Temocillin, a 6-α-methoxy derivative of ticarcillin, is currently approved for treatment of infections due to members of the Enterobacteriaceae in Belgium and the United Kingdom. It is stable against hydrolysis by most β-lactamases, including extended-spectrum β-lactamases (ESBLs) and AmpC-type β-lactamases, with studies reporting MICs at which 90% of bacteria are inhibited (MIC90s) between 16 and 32 μg/ml (3, 4, 8). Temocillin is thus drawing attention as a potential alternative to carbapenems in treatment of infections caused by the Enterobactericeae producing these broad-spectrum β-lactamases. Carbapenem-resistant Klebsiella pneumoniae producing KPC-type β-lactamase has emerged in recent years and caused hospital outbreaks of serious infections in the United States and other parts of the world (7). Furthermore, KPC-type β-lactamase is increasingly identified in other species of the Enterobacteriaceae as well, including Escherichia coli. One concerning recent phenomenon is the occurrence of urinary tract infections due to KPC-producing organisms at nursing homes (10). Currently, the limited treatment options for infections due to KPC-producing organisms include colistin and tigecycline. Concern over nephrotoxicity due to colistin limits its use outside closely monitored settings, whereas tigecycline does not achieve a therapeutic concentration in urine (2). Furthermore, emergence of resistance to these agents has recently been recorded for the Enterobacteriaceae (5, 6). The present study was conducted to evaluate the in vitro activities of temocillin against clinical isolates of K. pneumoniae and E. coli producing KPC-type β-lactamase. A total of 33 KPC-producing clinical isolates (30 K. pneumoniae isolates and 3 E. coli isolates) were used. KPC production was confirmed by an ertapenem resistance phenotype, a positive modified Hodge test, and positive PCR for the KPC structural gene. The isolates were collected from hospitals in three states in the United States. The MIC of temocillin was determined by the standard agar dilution method (1). Temocillin was provided by Eumedica (Brussels, Belgium). In addition, MICs of temocillin against an E. coli isogenic clone producing KPC-3 were tested to determine the direct effect of KPC production on the temocillin MIC. E. coli ATCC 25922 was used as the control strain. Table ​Table11 summarizes the results. For K. pneumoniae, the MICs ranged between 16 μg/ml and 64 μg/ml (MIC at which 50% of bacteria were inhibited = 32 μg/ml; MIC90 = 32 μg/ml). The E. coli clinical isolates had MICs between 8 and 16 μg/ml. E. coli DH10B both with and without the cloning vector pBCSK− (Stratagene, La Jolla, CA) encoding blaKPC-3 had an MIC of 8 μg/ml. An inoculum effect was not observed at 105 CFU, whereas a mild inoculum effect averaging within a twofold MIC difference was seen with K. pneumoniae when 106 CFU was inoculated (Table ​(Table1).1). This result was in line with those of a previous study documenting a modest inoculum effect of temocillin for non-KPC-producing isolates (9). The frequencies of mutants of representative clinical isolates that grew at their MICs and at 2× MICs were calculated to be approximately 1 × 10−10 and 0 for K. pneumoniae and 3 × 10−10 and 1 × 10−10 for E. coli, respectively. TABLE 1. Susceptibilities of KPC-producing K. pneumoniae and E. coli isolates to temocillin Currently, the British Society for Antimicrobial Chemotherapy (BSAC) is the only organization that defines temocillin MIC breakpoints for the Enterobacteriaceae. The BSAC defines temocillin susceptibilities at ≤8 and ≤32 μg/ml in systemic and urinary tract infections, respectively (http://www.bsac.org.uk/). One gram of temocillin is known to achieve a peak serum concentration of approximately 160 μg/ml, with serum binding of 85% and a half-life of 4 to 5 h (9). The urinary concentration after a 500-mg dose is approximately 500 μg/ml (9). These pharmacokinetic properties of temocillin make it a potential alternative treatment option for mild to moderate urinary tract infections caused by KPC-producing members of the Enterobacteriaceae.

Unreliable Extended-Spectrum β-Lactamase Detection in the Presence of Plasmid-Mediated AmpC in Escherichia coli Clinical Isolates

The emergence of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated AmpC (pAmpC) enzymes in Escherichia coli raises concern regarding accurate laboratory detection and interpretation of susceptibility testing results. Twenty-six cefpodoxime ESBL screen-positive, cefoxitin-resistant E. coli clinical isolates were subjected to clavulanate ESBL confirmatory testing employing disk augmentation, Etest, and the BD Phoenix NMC/ID-132 panel. Phenotypic pAmpC production was assessed by boronic acid disk augmentation. ESBL and pAmpC genes were detected by gene amplification and sequencing. ESBL genes (SHV and/or CTX-M-type genes) were detected in only 7/26 ESBL screen-positive isolates. Of 23 aminophenylboronic acid screen-positive isolates, pAmpC genes were detected in 20 (CMY-2 or FOX-5 genes). High incidences of false-positive ESBL confirmatory results were observed for both clavulanate disk augmentation (9/19) and BD Phoenix (5/19). All were associated with the presence of pAmpC genes with or without TEM-1. Etest performed poorly, as the majority of interpretations were nondeterminable. In addition, false-negative ESBL confirmatory results were observed in isolates possessing concomitant ESBL and pAmpC genes for Etest (four of five), BD Phoenix (three of five), and disk augmentation (one of five). The results indicate poor performance of currently employed ESBL confirmatory methods in the setting of concomitant pAmpC. Some isolates with pAmpC and ESBL genes fell within the susceptible category to extended-spectrum cephalosporins, raising concern over currently employed breakpoints.

Mechanisms Accounting for Fluoroquinolone Resistance in Escherichia coli Clinical Isolates

Fluoroquinolone MICs are increased through the acquisition of chromosomal mutations in the genes encoding gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), increased levels of the multidrug efflux pump AcrAB, and the plasmid-borne genes aac(6')-Ib-cr and the qnr variants in Escherichia coli. In the accompanying report, we found that ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs for fluoroquinolone-resistant E. coli clinical isolates were very high and widely varied (L. Becnel Boyd, M. J. Maynard, S. K. Morgan-Linnell, L. B. Horton, R. Sucgang, R. J. Hamill, J. Rojo Jimenez, J. Versalovic, D. Steffen, and L. Zechiedrich, Antimicrob. Agents Chemother. 53:229-234, 2009). Here, we sequenced gyrA, gyrB, parC, and parE; screened for aac(6')-Ib-cr and qnrA; and quantified AcrA levels in E. coli isolates for which patient sex, age, location, and site of infection were known. We found that (i) all fluoroquinolone-resistant isolates had gyrA mutations; (ii) approximately 85% of gyrA mutants also had parC mutations; (iii) the ciprofloxacin and norfloxacin MICs for isolates harboring aac(6')-Ib-cr ( approximately 23%) were significantly higher, but the gatifloxacin and levofloxacin MICs were not; (iv) no isolate had qnrA; and (v) approximately 33% of the fluoroquinolone-resistant isolates had increased AcrA levels. Increased AcrA correlated with nonsusceptibility to the fluoroquinolones but did not correlate with nonsusceptibility to any other antimicrobial agents reported from hospital antibiograms. Known mechanisms accounted for the fluoroquinolone MICs of 50 to 70% of the isolates; the remaining included isolates for which the MICs were up to 1,500-fold higher than expected. Thus, additional, unknown fluoroquinolone resistance mechanisms must be present in some clinical isolates.

Evaluation of antimicrobial susceptibility for β-lactams using the Etest method against clinical isolates from 100 medical centers in Japan (2006)

This antimicrobial resistance surveillance study was performed in 100 medical centers. Susceptibility testing (Etest; AB BIODISK, Solna, Sweden) of 9152 strains including Escherichia coli (991 strains), Klebsiella spp. (1000 strains), Enterobacter spp. (971 strains), Citrobacter spp. (803 strains), indole-positive Proteae spp. (834 strains), Serratia spp. (902 strains), Acinetobacter spp. (874 strains), Pseudomonas aeruginosa (992 strains), oxacillin-susceptible Staphylococcus aureus (984 strains), and coagulase-negative staphylococci (CoNS; 801 strains) was performed with 7 β-lactams (cefepime, cefpirome, ceftazidime, cefoperazone/sulbactam, imipenem and piperacillin for Gram-negative bacteria, or oxacillin for Gram-positive bacteria). No strain resistance to these β-lactams (except for ceftazidime) was found in oxacillin-susceptible S. aureus and CoNS. Of the E. coli clinical isolates, 17.1% were resistant to piperacillin, whereas 2.9% or less (cefpirome = 2.9%) were resistant to other β-lactam agents. Klebsiella spp. strains were more susceptible to imipenem (99.9%), cefepime (99.2%), ceftazidime (98.6%), and cefpirome (98.3%). Isolates of Enterobacter spp., Citrobacter spp., indole-positive Proteae, and Serratia spp. were susceptible to imipenem, cefepime, and cefpirome as well. Acinetobacter spp. strains were least resistant to cefoperazone/sulbactam (0.7% resistance), imipenem (2.6%), cefepime (6.6%), and ceftazidime (7.7%) compared with other β-lactam antibiotics tested. Isolates of P. aeruginosa were more susceptible to ceftazidime (8.7% resistance), cefoperazone/sulbactam (9.8%), and cefepime (8.9%) than piperacillin (11.9%), cefpirome (16.2%), and imipenem (12.4%). The percentage of imipenem-resistant P. aeruginosa was approximately 13% in clinical isolates in Japan. The proportion of strains resistant to β-lactam antimicrobials has been decreasing compared with data from 2004, suggesting that reduced consumption of β-lactams has reflected the decreased rates of resistant bacterial isolates in Japan.

Regulation of P-Fimbrial Phase Variation Frequencies in Escherichia coli CFT073

Adherence of uropathogenic Escherichia coli to host tissue is required for infection and is mediated by fimbriae, such as pyelonephritis-associated pili (Pap). Expression of P fimbriae is regulated by phase variation, and to date, phase transition frequencies have been measured only for pap regulatory region constructs integrated into the E. coli K-12 chromosome. The aim of this work was to measure P phase transition frequencies in clinical isolates for the first time, including frequencies for the sequenced strain E. coli CFT073. P fimbriation and associated phase transition frequencies were measured for two E. coli clinical isolates and compared with levels for homologous pap constructs in E. coli K-12. Fimbriation and off-to-on transition frequencies were always higher in the clinical isolate. It was concluded that the regulatory inputs controlling papI expression are likely to be different in E. coli CFT073 and E. coli K-12 as (i) phase variation could be stimulated in E. coli K-12 by induction of papI and (ii) the level of expression of a papI::gfp(+) fusion was higher in E. coli CFT073 than in E. coli K-12. Furthermore, phase transition frequencies for the two E. coli CFT073 pap clusters were shown to be different depending on the culture conditions, indicating that there is a hierarchy of expression depending on signal inputs.

DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.

Molecular characterization of an Escherichia coli clinical isolate that produces both metallo- -lactamase VIM-2 and extended-spectrum -lactamase GES-7: identification of the In8 integron carrying the blaVIM-2 gene

We have previously reported the isolation of a VIM-2-producing Escherichia coli clinical isolate and the coexistence of blaVIM-2 and blaGES-7 in the same isolate. The aim of this study was to elucidate the genetic environment of these genes. PCR and sequence analysis were used to identify and analyse the blaVIM-containing integron. The location of the blaVIM-2 and blaGES-7 genes was identified by hybridization experiments on I-CeuI-digested genomic DNA after PFGE. Only the blaVIM-2 gene has been found on a class I integron, designated In8 (1474 bp). Hybridization with the blaVIM-2, the blaGES-7 and the 16S-23S rRNA probes confirmed the chromosomal location of both genes. The blaVIM-2 probe hybridized with a 710 kb fragment whereas the blaGES-7 probe hybridized with a 144 kb fragment of genomic DNA of the E. coli isolate. This report provides evidence for the chromosomal location of both blaVIM-2 and blaGES-7 genes carried by an E. coli clinical isolate. In8, containing blaVIM-2, presents an emerging threat of carbapenem resistance among E. coli isolates in Greece.