Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli.
In the present study, we examine the prevalence of phylogenetic groups, O-serogroups, adhesin genes, antimicrobial resistance, the level of gene expression associated with biofilm formation, and the presence of extended-spectrum beta-lactamase (ESBL) in UPEC strains isolated from both pediatric and adult patients.
In this cross-sectional study, 156 UPEC isolates were collected from UTI patients. ESBL-producing isolates were detected using the double-disc synergy (DDS) method, and biofilm formation was assessed through a microplate assay. The presence of O-serogroups, adhesion factors and resistance genes, including ESBLs and PMQR genes, was detected by PCR, and isolates were categorized into phylogenetic groups using multiplex PCR. Additionally, the quantitative real-time PCR method was also used to determine the expression level of genes related to biofilm.
During the study period, 50.6% (79/156) of the samples were obtained from children, and 49.4% (77/156) were from adults. The highest rate of resistance was to NA (91.7%), while FM (10.9%) had the lowest rate of antibiotic resistance. In addition, 67.9% (106/156) of UPEC isolates were ESBL producers. Most of UPEC isolates belonged to phylogenetic group B2 (37.1%). This study revealed that blaCTX-M and qnrS are widely distributed among UPEC isolates. The mean expression levels of fimA genes were significantly higher in non-biofilm producers than in biofilm producers (p < 0.01).
The high antibiotic resistance rates in this study highlight the significance of local resistance monitoring and investigating underlying mechanisms. Our findings indicate the dominance of phylogroup B2 and group D as the prevailing phylogenetic groups. Consequently, it is imperative to investigate the epidemiological aspects and characterize UPEC isolates across diverse regions and time frames.
Talieh Mostaghimi
，Pournajaf A
，Bijani A
，Mohammadi M
，Rajabnia M
，Halaji M
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High prevalence of plasmid-mediated quinolone resistance in escherichia coli strains producing extended-spectrum beta-lactamases isolated from faeces and urine of pregnant women with acute cystitis.
Escherichia coli is the most common etiological agent of urinary tract infections (UTIs). Meanwhile, plasmid-mediated quinolone resistance (PMQR) is reported in E. coli isolates producing extended-spectrum β-lactamases (ESBLs). Furthermore, the reservoirs and mechanisms of acquisition of uropathogenic Escherichia coli (UPEC) strains are poorly understood. On the other hand, UTIs are common in pregnant women and the treatment challenge is alarming.
In the present study, 54 pregnant women with acute cystitis were included. A total of 108 E. coli isolates, 54 isolates from UTI and 54 isolates from faeces of pregnant women (same host) were collected. In the antimicrobial susceptibility test, the highest rate of antibiotic resistance was to nalidixic acid (77%, 83/108) and the lowest rate was to imipenem (9%, 10/108). Among the isolates, 44% (48/108) were ESBLs producers. A high frequency of PMQR genes was observed in the isolates. The frequency of PMQR genes qnrS, qnrB, aac(6')-Ib-cr, and qnrA was 58% (63/108), 21% (23/108), 9% (10/108), and 4% (4/108), respectively. Meanwhile, PMQR genes were not detected in 24% (20/85) of isolates resistant to nalidixic acid and/or fluoroquinolone, indicating that other mechanisms, i.e. chromosomal mutations, are involved in resistance to quinolones, which were not detected in the present study. In ESBL-producing isolates, the frequency of PMQR genes was higher than that of non-ESBL-producing isolates (81% vs. 53%). Meanwhile, UTI and faeces isolates mainly belonged to phylogenetic group B2 (36/54, 67% and 25/54, 46%, respectively) compared to other phylogenetic groups. In addition, virulence factors and multidrug-resistant (MDR) were mainly associated with phylogenetic group B2. However, predominant clones in faeces were not found in UTIs. Rep-PCR revealed the presence of 85 clones in patients. Among the clones, 40 clones were detected only in faeces (faeces-only), 35 clones only in UTI (UTI-only) and 10 clones in both faeces and UTI (faeces-UTI). We found that out of 10 faeces-UTI clones, 5 clones were present in the host's faeces flora.
This study revealed a high rate of resistance to the quinolone nalidixic acid and a widespread distribution of PMQR genes in MDR E. coli strains producing ESBLs. The strains represented virulence factors and phylogenetic group B2 are closely associated with abundance in UTI and faeces. However, the predominant clones in faeces were not found in UTIs and it is possible that rep-PCR is not sufficiently discriminating clones.
Sohrabi M
，Fathi J
，Mohebi S
，Hashemizadeh Z
，Kholdi S
，Hadadi M
，Keshavarz K
，Darvishvand Z
... -
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Exploring uncatalogued genetic variation in antimicrobial resistance gene families in Escherichia coli: an observational analysis.
Antimicrobial resistance (AMR) in Escherichia coli is a global problem associated with substantial morbidity and mortality. AMR-associated genes are typically annotated based on similarity to variants in a curated reference database, with the implicit assumption that uncatalogued genetic variation within these is phenotypically unimportant. In this study, we evaluated the performance of the AMRFinder tool and, subsequently, the potential for discovering new AMR-associated gene families and characterising variation within existing ones to improve genotype-to-susceptibility phenotype predictions in E coli.
In this cross-sectional study of international genome sequence data, we assembled a global dataset of 9001 E coli sequences from five publicly available data collections predominantly deriving from human bloodstream infections from: Norway, Oxfordshire (UK), Thailand, the UK, and Sweden. 8555 of these sequences had linked antibiotic susceptibility data. Raw reads were assembled using Shovill and AMR genes (relevant to amoxicillin-clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, gentamicin, piperacillin-tazobactam, and trimethoprim) extracted using the National Center for Biotechnology Information AMRFinder tool (using both default and strict [100%] coverage and identity filters). We assessed the predictive value of the presence of these genes for predicting resistance or susceptibility against US Food and Drug Administration thresholds for major and very major errors. Mash was used to calculate the similarity between extracted genes using Jaccard distances. We empirically reclustered extracted gene sequences into AMR-associated gene families (≥70% match) and antibiotic-resistance genes (ARGs; 100% match) and categorised these according to their frequency in the dataset. Accumulation curves were simulated and correlations between gene frequency in the Oxfordshire and other datasets calculated using the Spearman coefficient. Firth regression was used to model the association between the presence of blaTEM-1 variants and amoxicillin-clavulanic acid or piperacillin-tazobactam resistance, adjusted for the presence of other relevant ARGs.
The performance of the AMRFinder database for genotype-to-phenotype predictions using strict 100% identity and coverage thresholds did not meet US Food and Drug Administration thresholds for any of the seven antibiotics evaluated. Relaxing filters to default settings improved sensitivity with a specificity cost. For all antibiotics, most explainable resistance was associated with the presence of a small number of genes. There was a proportion of resistance that could not be explained by known ARGs; this ranged from 75·1% for amoxicillin-clavulanic acid to 3·4% for ciprofloxacin. Only 18 199 (51·5%) of the 35 343 ARGs detected had a 100% identity and coverage match in the AMRFinder database. After empirically reclassifying genes at 100% nucleotide sequence identity, we identified 1042 unique ARGs, of which 126 (12·1%) were present ten times or more, 313 (30·0%) were present between two and nine times, and 603 (57·9%) were present only once. Simulated accumulation curves revealed that discovery of new (100% match) ARGs present more than once in the dataset plateaued relatively quickly, whereas new singleton ARGs were discovered even after many thousands of isolates had been included. We identified a strong correlation (Spearman coefficient 0·76 [95% CI 0·73-0·80], p<0·0001) between the number of times an ARG was observed in Oxfordshire and the number of times it was seen internationally, with ARGs that were observed six times in Oxfordshire always being found elsewhere. Finally, using the example of blaTEM-1, we showed that uncatalogued variation, including synonymous variation, is associated with potentially important phenotypic differences; for example, two common, uncatalogued blaTEM-1 alleles with only synonymous mutations compared with the known reference were associated with reduced resistance to amoxicillin-clavulanic acid (adjusted odds ratio 0·58 [95% CI 0·35-0·95], p=0·031) and piperacillin-tazobactam (0·50 [95% CI 0·29-0·82], p=0·005).
We highlight substantial uncatalogued genetic variation with respect to known ARGs, although a relatively small proportion of these alleles are repeatedly observed in a large international dataset suggesting strong selection pressures. The current approach of using fuzzy matching for ARG detection, ignoring the unknown effects of uncatalogued variation, is unlikely to be acceptable for future clinical deployment. The association of synonymous mutations with potentially important phenotypic differences suggests that relying solely on amino acid-based gene detection to predict resistance is unlikely to be sufficient. Finally, the inability to explain all resistance using existing knowledge highlights the importance of new target gene discovery.
National Institute for Health and Care Research, Wellcome, and UK Medical Research Council.
Lipworth S
，Crook D
，Walker AS
，Peto T
，Stoesser N
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《Lancet Microbe》
ResearchGate
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