Development of amoxicillin resistance in Escherichia coli after exposure to remnants of a non-related phagemid-containing E. coli:an exploratory study

OBJECTIVE: To determine the effect of exposure to remnants of a phagemid-containing E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates. METHODS: An in vitro model was developed in which a clinical E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development. RESULTS: In 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only (risk difference: 10.0%; 95% CI 5.4-14.6%)) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 (risk difference: 10.5%; 95% CI 6.1-14.9%). In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction. CONCLUSION: Exposure of an E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities

Distinguishing blaKPC -gene-containing IncF plasmids from epidemiologically related and unrelated Enterobacteriaceae based on short- and long-read sequence data

BACKGROUND: Limited information is available on whether blaKPC -containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. This study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids. METHOD: Epidemiologically related isolates, were short- and long-read whole genome sequenced. A hybrid assembly was performed and plasmid sequences were extracted from the assembly graph. Epidemiologically unrelated plasmid sequences were extracted from the GenBank. Pairwise comparisons were performed of epidemiologically related and unrelated plasmids based on SNP differences using snippy, phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser-index). The percentage of pairwise comparisons misclassified as genetically related or as clonally unrelated was determined using different genetic thresholds for genetic relatedness. RESULTS: The ranges in number of SNP differences, Roary phylogenetic distance, and Stoesser-index overlapped between the epidemiologically related and unrelated plasmids. When using a genetic similarity threshold that classified 100% of epidemiologically related plasmid pairs as genetically related, the percentages of plasmids misclassified as epidemiologically related ranged from 6.7% (Roary) to 20.8% (Stoesser-index). DISCUSSION: Although epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic differences, blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids show a high degree of sequence similarity. The phylogenetic distance as determined using Roary showed the highest degree of discriminatory power between the epidemiologically related and unrelated plasmids

Detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicons in Enterobacteriaceae using PlasmidSPAdes assembly of short-read sequence data

Knowledge of the epidemiology of plasmids is essential for understanding the evolution and spread of antimicrobial resistance. PlasmidSPAdes attempts to reconstruct plasmids using short-read sequence data. Accurate detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicon genes is a prerequisite for the use of plasmid assembly tools to investigate the role of plasmids in the spread and evolution of ESBL production in Enterobacteriaceae. This study evaluated the performance of PlasmidSPAdes plasmid assembly for Enterobacteriaceae in terms of detection of ESBL-encoding genes, plasmid replicons and chromosomal wgMLST genes, and assessed the effect of k-mer size. Short-read sequence data for 59 ESBL-producing Enterobacteriaceae were assembled with PlasmidSPAdes using different k-mer sizes (21, 33, 55, 77, 99 and 127). For every k-mer size, the presence of ESBL genes, plasmid replicons and a selection of chromosomal wgMLST genes in the plasmid assembly was determined. Out of 241 plasmid replicons and 66 ESBL genes detected by whole-genome assembly, 213 plasmid replicons [88 %; 95 % confidence interval (CI): 83.9-91.9] and 43 ESBL genes (65 %; 95 % CI: 53.1-75.6) were detected in the plasmid assemblies obtained by PlasmidSPAdes. For most ESBL genes (83.3 %) and plasmid replicons (72.0 %), detection results did not differ between the k-mer sizes used in the plasmid assembly. No optimal k-mer size could be defined for the number of ESBL genes and plasmid replicons detected. For most isolates, the number of chromosomal wgMLST genes detected in the plasmid assemblies decreased with increasing k-mer size. Based on our findings, PlasmidSPAdes is not a suitable plasmid assembly tool for short-read sequence data for ESBL-encoding plasmids of Enterobacteriaceae

A European perspective on nosocomial urinary tract infections II. Report on incidence, clinical characteristics and outcome (ESGINI–04 study)

ObjectivesTo estimate the incidence of nosocomially acquired urinary tract infections (NAUTI) in Europe and provide information on the clinical characteristics, underlying conditions, etiology, management and outcome of patients.Materials and methodsWe collected clinical information from NAUTI patients with a microbiology report on the named study day.ResultsA total of 141 hospitals from 25 European countries participated in the study. Written institutional bladder catheter guidelines were in place in 90.3% of EU hospitals and 55% of non-EU hospitals (P <0.05). The total number of new NAUTI episodes on the day of the study was 298, representing an incidence of 3.55 episodes/1000 patient-days and an estimated prevalence of 10.65/1000. The five most commonly isolated micro-organisms were Escherichia coli, Enterococcus sp., Candida sp., Klebsiella sp. and Pseudomonas aeruginosa. Patients from non-EU countries were younger, with more severe underlying diseases with a higher incidence of obstructive uropathy/lithiasis. Overall, 22.8% of patients had no ‘classic’ UTI-predisposing factors. Catheter-associated UTI (CAUTI) was present in 187 patients (62.8%). A closed drainage system was used in only 78.5% of catheterised patients. The indication for bladder catheterisation was not considered adequate in 7.6% of cases and continuation of bladder catheterisation was considered unnecessary in 31.3%. Opening of the closed drainage system was the most frequent major error in catheter management (16.8%). Antimicrobial treatment was not considered adequate in 19.8% of all cases.ConclusionsThe incidence of NAUTI in a large European population is 3.55/1000 patient-days. There is clearly room for improvement in the area of bladder catheterisation, catheter care and medical management of NAUTI. We recommend that European authorities draw up and implement practical and specific guidelines to reduce the incidence of this infection