Investigation of High-Risk ST131 Clone in Extended Spectrum β-Lactamase–Producing Escherichia coli Isolates in Children

Objective Antimicrobial resistance poses a serious threat to children's health. In recent years, high-risk Escherichia coli ST131 has become an important target for global surveillance studies. The E.coli ST131 clone is associated with extended spectrum β-lactamase (ESBL) production, as well as multidrug resistance and treatment failure. Studies on this clone in the pediatric age group are limited. We aim to investigate the rate of high-risk E. coli ST131 clone in ESBL-positive E. coli isolates obtained from pediatric patients. Methods A total of 292 ESBL-positive E. coli isolates from clinical samples of pediatric patients was included in the study. MALDI-TOF MS system was used for bacterial identification. Susceptibility tests were performed using BD Phoenix automated system. ST131 detection was done by MALDI-TOF-MS. Fisher's exact test was used to compare the groups (significance <0.05). Results A total of 292 isolates was analyzed. The high-risk ST131 clone was detected in 117 (40%) of the 292 ESBL-positive isolates. ST131 rates were found to be significantly higher in children under the age of 5 years compared with children over the age of 5 years (49.3 vs. 31.1%, p = 0.0019). Ciprofloxacin resistance was higher in ST131 isolates (45.6 vs. 31.7%; p < 0.05). Conclusion The rate of the ST131 clone was found to be high in the pediatric population. The significantly high rate of resistance to ciprofloxacin, which is not commonly used in the pediatric population, in ST131 isolates reveals the importance of the spread of high-risk clones for the development of resistance.

Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of Escherichia coli

Background SOS response suppression (by RecA inactivation) has been postulated as a therapeutic strategy for potentiating antimicrobials against Enterobacterales. Objectives To evaluate the impact of RecA inactivation on the reversion and evolution of quinolone resistance using a collection of Escherichia coli clinical isolates. Methods Twenty-three E. coli clinical isolates, including isolates belonging to the high-risk clone ST131, were included. SOS response was suppressed by recA inactivation. Susceptibility to fluoroquinolones was determined by broth microdilution, growth curves and killing curves. Evolution of quinolone resistance was evaluated by mutant frequency and mutant prevention concentration (MPC). Results RecA inactivation resulted in 2–16-fold reductions in fluoroquinolone MICs and modified EUCAST clinical category for several isolates, including ST131 clone isolates. Growth curves and time–kill curves showed a clear disadvantage (up to 10 log10 cfu/mL after 24 h) for survival in strains with an inactivated SOS system. For recA-deficient mutants, MPC values decreased 4–8-fold, with values below the maximum serum concentration of ciprofloxacin. RecA inactivation led to a decrease in mutant frequency (≥103-fold) compared with isolates with unmodified SOS responses at ciprofloxacin concentrations of 4×MIC and 1 mg/L. These effects were also observed in ST131 clone isolates. Conclusions While RecA inactivation does not reverse existing resistance, it is a promising strategy for increasing the effectiveness of fluoroquinolones against susceptible clinical isolates, including high-risk clone isolates.

Prevalence of E. coli ST131 among Uropathogenic E. coli Isolates from Iraqi Patients in Wasit Province, Iraq

The emergence of Escherichia coli sequence type 131 (E. coli ST131) clone represents a major challenge to public health globally, since this clone is reported as highly virulent and multidrug-resistant, thus making it successfully disseminated worldwide. In Iraq, there is no previous study dealing with this important clone, so this project was suggested to investigate its presence within uropathogenic E. coli (UPEC) from Iraqi patients in Wasit Province. A total of 112 UPEC isolates from cases of acute urinary tract infection (UTI) were analysed for phylogenetic groups by quadruplex PCR; then, these isolates were investigated for E. coli ST131 clone by both conventional and real-time PCR procedures. The antibiotic susceptibility test was performed by the disk diffusion method. The results revealed that, out of 112 UPEC isolates, 38 (33.9%) belonged to phylogroup B2. For conventional PCR, 92.1% (35/38) of B2 E. coli isolates were positive for E. coli ST131, of which 34 were O25b-ST131 strain and 1 was O16-ST131 strain. However, serogroups O25b and O16 represented 17.1% and 2.8%, respectively. By RT-PCR assay, 15.1% (17/112) and 44.7% (17/38) of total and B2 E. coli isolates were confirmed as being E. coli ST131, respectively. The highest resistance rates of E. coli ST131 isolates were against the β-lactams, while low resistance rates were against amikacin, nitrofurantoin, and gentamicin. Fortunately, all isolates were susceptible to carbapenems. Moreover, 52.9% (9 out of 17) of E. coli ST131 isolates were MDR. In conclusion, the presence of E. coli ST131 among UPEC isolates from Iraqi patients is confirmed with high resistance to most antimicrobials included in this study.

Complex Multilevel Control of Hemolysin Production by Uropathogenic Escherichia coli

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin. IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.

Molecular epidemiological of extended-spectrum β-lactamase producing Escherichia coli isolated in Djibouti

Introduction: While the molecular epidemiology of extended-spectrum-b-lactamase (ESBL)-producing E. coli is well known in Europe due to effective surveillance networks and substantial literature, data for Africa are less available, especially in Djibouti. Methodology: We studied 31 isolates of ESBL-producing E. coli from Djibouti and compared these molecular results with data available in Africa. Results: Susceptibility rates were 3.2% for ceftazidim, 48.4% for piperacillin-tazobactam, 90.3% for amikacine and 16.1% for ofloxacin. No isolate showed resistance to carbapenems or colistin. 30 E. coli (96.8%) were positive to blaCTX-M-15, 1 (3.2%) to blaCTX-M-14 and 10 (32.3%) to narrow-broad-spectrum blaTEM. No blaSHV were detected. Fluoroquinolone resistance analysis showed that 30 ofloxacin-resistant E. coli had the mutation Ser-83->Leu on the gyrA gene. 24 E. coli (77.4%) harboured the plasmid-borne aac(6 ')-Ib-cr gene. No E. coli carried the genes qnrA, qnrB and qepA. 10 isolates (32.3%) belonging to the ST131 clone. The plasmid incompatibility group most widely represented in our collection was IncFIA/IB/II. Conclusions: There is no major difference with African epidemiology. In particular, we notice the international diffusion of specific clonal group ST131.

Rapid and Simple UniversalEscherichia coliGenotyping Method Based on Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Single-Tube Multiplex PCR and Standard Gel Electrophoresis

ABSTRACTWe developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing ofEscherichia coliandShigellaisolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: theE. colireference (ECOR) collection (n = 72), O1:K1 isolates causing neonatal meningitis (n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone (n = 38), Shiga toxin-producingE. coli(STEC) isolates of serogroups O157:H7 (n = 21) and O26 (n = 16, 8 of which belonged to an outbreak), 27Shigellaisolates (22Shigella sonneiisolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27Shigellaisolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing ofE. coli/Shigellaisolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE.IMPORTANCEFast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of allE. coliisolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).