Anke Heisig

Commonality among Fluoroquinolone-Resistant Sequence Type ST131 Extraintestinal Escherichia coli Isolates from Humans and Companion Animals in Australia

ABSTRACT Escherichia coli sequence type 131 (ST131), an emergent multidrug-resistant extraintestinal pathogen, has spread epidemically among humans and was recently isolated from companion animals. To assess for human-companion animal commonality among ST131 isolates, 214 fluoroquinolone-resistant extraintestinal E. coli isolates (205 from humans, 9 from companion animals) from diagnostic laboratories in Australia, provisionally identified as ST131 by PCR, selectively underwent PCR-based O typing and blaCTX-M-15 detection. A subset then underwent multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) analysis, extended virulence genotyping, antimicrobial susceptibility testing, and fluoroquinolone resistance genotyping. All isolates were O25b positive, except for two O16 isolates and one O157 isolate, which (along with six O25b-positive isolates) were confirmed by MLST to be ST131. Only 12% of isolates (25 human, 1 canine) exhibited blaCTX-M-15. PFGE analysis of 20 randomly selected human and all 9 companion animal isolates showed multiple instances of ≥94% profile similarity across host species; 12 isolates (6 human, 6 companion animal) represented pulsotype 968, the most prevalent ST131 pulsotype in North America (representing 23% of a large ST131 reference collection). Virulence gene and antimicrobial resistance profiles differed minimally, without host species specificity. The analyzed ST131 isolates also exhibited a conserved, host species-independent pattern of chromosomal fluoroquinolone resistance mutations. However, eight (89%) companion animal isolates, versus two (10%) human isolates, possessed the plasmid-borne qnrB gene (P < 0.001). This extensive across-species strain commonality, plus the similarities between Australian and non-Australian ST131 isolates, suggest that ST131 isolates are exchanged between humans and companion animals both within Australia and intercontinentally.

Prominence of an O75 clonal group (clonal complex 14) among non-ST131 fluoroquinolone-resistant Escherichia coli causing extraintestinal infections in humans and dogs in Australia

ABSTRACT Fluoroquinolone (FQ)-resistant extraintestinal pathogenic Escherichia coli (FQr ExPEC) strains from phylogenetic group B2 are undergoing epidemic spread. Isolates belonging to phylogenetic group B2 are generally more virulent than other E. coli isolates; therefore, resistance to FQs among group B2 isolates is concerning. Although clonal expansion of sequence type 131 (ST131) is a major factor, the contribution of additional clonal groups has not been quantified. Group B2 FQr ExPEC isolates from humans (n = 250) and dogs (n = 12) in Australia were screened for ST131, a recently recognized and rapidly emerging multidrug-resistant and virulent clonal group that is important in both human and companion animal medicine. Non-ST131 isolates underwent virulence genotyping, PCR-based O typing, partial multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and FQ resistance mechanism analysis. Of 49 non-ST131 isolates (45 human, 4 canine), 49% (24 human, 2 canine) represented O-type O75 and exhibited conserved virulence genotypes (F10 papA allele, iha, fimH, sat, vat, fyuA, iutA, kpsMII, usp, ompT, malX, K1/K5 capsule) and MLST allele profiles corresponding with clonal complex CC14. Two clusters, each containing canine and human isolates, were identified by PFGE (differentiated by K1 and K5 capsules). Australian FQr O75 isolates exhibited commonality with an historical FQ-susceptible O75 urosepsis isolate (also CC14). The isolation from humans and dogs of highly similar FQr derivatives of the classic O75:K1/K5 (CC14) ExPEC lineage suggests recent acquisition of FQ resistance and potential cross-host-species transfer. This lineage should be targeted with ST131 in future epidemiological investigations of FQr ExPEC.

The antirepressor of phage P1 Isolation and interaction with the C1 repressor of P1 and P7

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in‐frame start codon. Another open reading frame, icd, 5′ upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene‐carrying plasmids we have overproduced and purified Ant½ in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N‐terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N‐terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co‐precipitate with the Ant½ complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1‐inactivating function by a direct protein—protein interaction.

Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor.

The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis. The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene. We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22. Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced. On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.

Regulation of the ban gene containing operon of prophage P1

SummaryA physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac additional gene(s) — crr/ban in the clockwise direction of the circular map of P1.

Detection of a Novel gyrB Mutation Associated With Fluoroquinolone-Nonsusceptible Salmonella enterica serovar Typhimurium Isolated From a Bloodstream Infection in Ghana

A multidrug-resistant Salmonella enterica serovar Typhimurium with reduced susceptibility to ciprofloxacin was isolated from the blood of a hospitalized child in Ghana. DNA sequencing identified a novel gyrB mutation at codon 466 (Glu466Asp). An increase in fluoroquinolone susceptibility after the introduction of a wild-type gyrB(+) allele demonstrated that the gyrB466 mutation had a direct effect on fluoroquinolone susceptibility.

Uncomplicated Urinary Tract Infections and Antibiotic Resistance—Epidemiological and Mechanistic Aspects

Uncomplicated urinary tract infections are typically monobacterial and are predominantly caused by Escherichia coli. Although several effective treatment options are available, the rates of antibiotic resistance in urinary isolates of E. coli have increased during the last decade. Knowledge of the actual local rates of antibiotic resistant pathogens as well as the underlying mechanisms are important factors in addition to the geographical location and the health state of the patient for choosing the most effective antibiotic treatment. Recommended treatment options include trimethoprim alone or in combination with sulfamethoxazol, fluoroquinolones, β-lactams, fosfomycin-trometamol, and nitrofurantoin. Three basic mechanisms of resistance to all antibiotics are known, i.e., target alteration, reduced drug concentration and inactivation of the drug. These mechanisms—alone or in combination—contribute to resistance against the different antibiotic classes. With increasing prevalence, combinations of resistance mechanisms leading to multiple drug resistant (mdr) pathogens are being detected and have been associated with reduced fitness under in vitro situations. However, mdr clones among clinical isolates such as E. coli sequence type 131 (ST131) have successfully adapted in fitness and growth rate and are rapidly spreading as a worldwide predominating clone of extraintestinal pathogenic E. coli.