The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131.

BACKGROUND: Escherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children's infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran. METHODS: The E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing. RESULTS: Among 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern. CONCLUSIONS: Our results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

Combined Anti-Bacterial Actions of Lincomycin and Freshly Prepared Silver Nanoparticles: Overcoming the Resistance to Antibiotics and Enhancement of the Bioactivity.

Bacterial drug resistance to antibiotics is growing globally at unprecedented levels, and strategies to overcome treatment deficiencies are continuously developing. In our approach, we utilized metal nanoparticles, silver nanoparticles (AgNPs), known for their wide spread and significant anti-bacterial actions, and the high-dose regimen of lincosamide antibiotic, lincomycin, to demonstrate the efficacy of the combined delivery concept in combating the bacterial resistance. The anti-bacterial actions of the AgNPs and the lincomycin as single entities and as part of the combined mixture of the AgNPs-lincomycin showed improved anti-bacterial biological activity in the Bacillus cereus and Proteus mirabilis microorganisms in comparison to the AgNPs and lincomycin alone. The comparison of the anti-biofilm formation tendency, minimum bactericidal concentration (MBC), and minimum inhibitory concentration (MIC) suggested additive effects of the AgNPs and lincomycin combination co-delivery. The AgNPs' MIC at 100 µg/mL and MBC at 100 µg/mL for both Bacillus cereus and Proteus mirabilis, respectively, together with the AgNPs-lincomycin mixture MIC at 100 + 12.5 µg/mL for Bacillus cereus and 50 + 12.5 µg/mL for Proteus mirabilis, confirmed the efficacy of the mixture. The growth curve test showed that the AgNPs required 90 min to kill both bacterial isolates. The freshly prepared and well-characterized AgNPs, important for the antioxidant activity levels of the AgNPs material, showed radical scavenging potential that increased with the increasing concentrations. The DPPH's best activity concentration, 100 µg/mL, which is also the best concentration exhibiting the highest anti-bacterial zone inhibition, was chosen for evaluating the combined effects of the antibiotic, lincomycin, and the AgNPs. Plausible genotoxic effects and the roles of AgNPs were observed through decreased Bla gene expressions in the Bacillus cereus and BlaCTX-M-15 gene expressions in the Proteus mirabilis.

Nationwide Surveillance and Molecular Characterization of Critically Drug-Resistant Gram-Negative Bacteria: Results of the Research University Network Thailand Study.

A large-scale surveillance is an important measure to monitor the regional spread of antimicrobial resistance. We prospectively studied the prevalence and molecular characteristics of clinically important Gram-negative bacilli, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii complex (ABC), and Pseudomonas aeruginosa, from blood, respiratory tract, urine, and sterile sites at 47 hospitals across Thailand. Among 187,619 isolates, 93,810 isolates (50.0%) were critically drug resistant, of which 12,915 isolates (13.8%) were randomly selected for molecular characterization. E. coli was most commonly isolated from all specimens, except the respiratory tract, in which ABC was predominant. Prevalence of extended-spectrum cephalosporin resistance (ESCR) was higher in E. coli (42.5%) than K. pneumoniae (32.0%), but carbapenem-resistant (CR)-K. pneumoniae (17.2%) was 4.5-fold higher than CR-E. coli (3.8%). The majority of ESCR/CR-E. coli and K. pneumoniae isolates carried blaCTX-M (64.6% to 82.1%). blaNDM and blaOXA-48-like were the most prevalent carbapenemase genes in CR-E. coli/CR-K. pneumoniae (74.9%/52.9% and 22.4%/54.1%, respectively). In addition, 12.9%/23.0% of CR-E. coli/CR-K. pneumoniae cocarried blaNDM and blaOXA-48-like. Among ABC isolates, 41.9% were extensively drug resistant (XDR) and 35.7% were multidrug resistant (MDR), while P. aeruginosa showed XDR/MDR at 6.3%/16.5%. A. baumannii was the most common species among ABC isolates. The major carbapenemase gene in MDR-A. baumannii/XDR-A. baumannii was blaOXA-23-like (85.8%/93.0%), which had much higher rates than other ABC species. blaIMP, blaVIM, blaOXA-40-like, and blaOXA-58-like were also detected in ABC at lower rates. The most common carbapenemase gene in MDR/XDR-P. aeruginosa was blaIMP (29.0%/30.6%), followed by blaVIM (9.5%/25.3%). The findings reiterate an alarming situation of drug resistance that requires serious control measures.

Seven-year surveillance of the prevalence of antimicrobial-resistant Escherichia coli isolates, with a focus on ST131 clones, among healthy people in Osaka, Japan.

OBJECTIVES: Escherichia coli (E. coli) is an indicator of antimicrobial resistance, and some strains of E. coli cause infectious diseases. E. coli sequence type 131 (ST131) - a global antimicrobial-resistant pandemic E. coli clone - is frequently detected in clinical specimens. Antimicrobial-resistant bacteria are monitored via national surveillance in clinical settings; however, monitoring information in non-clinical settings is limited. This study elucidated antimicrobial resistance trends of E. coli and dissemination of ST131 among healthy people in non-clinical settings. METHODS: This study collected 517 E. coli isolates from healthy people in Osaka, Japan, between 2013 and 2019. It analysed antimicrobial susceptibility of the isolates and detected the bla and mcr genes in ampicillin-resistant and colistin-resistant isolates, respectively, and the ST131 clone. RESULTS: Antimicrobial resistance rates of the bacteria isolated from healthy people in non-clinical settings were lower than for those in clinical settings. The resistance of the isolates to cefotaxime (4.4%) and ciprofloxacin (13.5%) gradually increased during the study period. In 23 cefotaxime-resistant isolates, the most frequent bla genes belonged to the blaCTX-M-9 group, followed by blaCTX-M-1 goup, blaTEM and blaCMY-2. One mcr-1-harbouring colistin-resistant isolate was detected in 2016. The incidence of the E. coli O25b-ST131 clone was approximately 5% until 2015 and 10% after 2016. CONCLUSION: Both ciprofloxacin resistance and O25b-ST131 clone frequency increased during the study period. Antimicrobial-resistant bacteria gradually spread in healthy people in non-clinical settings; one reason behind this spread was dissemination of global antimicrobial-resistant pandemic clones.

Why cannot a ß-lactamase gene be detected using an efficient molecular diagnostic method?

OBJECTIVE: Fast detection of ß-lactamase (bla) genes can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We have evaluated a further refinement of our fast and accurate molecular method, developed to overcome these limitations, using clinical isolates. METHODS: We have recently developed the efficient large-scale bla detection method (large-scaleblaFinder) that can detect bla gene types including almost all clinically available 1,352 bla genes with perfect specificity and sensitivity. Using this method, we have evaluated a further refinement of this method using clinical isolates provided by International Health Management Associates, Inc. (Schaumburg, Illinois, USA). Results were interpreted in a blinded manner by researchers who did not know any information on bla genes harbored by these isolates. RESULTS: With only one exception, the large-scaleblaFinder detected all bla genes identified by the provider using microarray and multiplex PCR. In one of the Escherichia coli test isolates, a blaDHA-1 gene was detected using the multiplex PCR assay but it was not detected using the large-scaleblaFinder. CONCLUSION: The truncation of a blaDHA-1 gene is an important reason for an efficient molecular diagnostic method (large-scaleblaFinder) not to detect the bla gene.

Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene.

The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum ß-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.

Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin

Abstract Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek® MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla -genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM & blaCTX-M (52.7%), blaTEM & blaSHV (20%), blaTEM & blaCTX-M & blaSHV (12.7%), and blaSHV & blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes.

Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin.

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek(®) MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla-genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM &blaCTX-M (52.7%), blaTEM &blaSHV (20%), blaTEM &blaCTX-M &blaSHV (12.7%), and blaSHV &blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes.

Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review.

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011-14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A ß-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoß sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoß correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus.

Genetic, biochemical characterization and mutagenesis of the chromosomal class A ß-lactamase of Raoultella (formerly Klebsiella) terrigena.

BACKGROUND: Chromosomal class A ß-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena. MATERIALS AND METHODS: The blaTER-1 gene of R. terrigena strain ATCC33257(T) was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing>2mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on ß-lactam resistance was assessed by site-directed mutagenesis experiments. RESULTS: R. terrigena strains ATCC33257(T) and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A ß-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent ß-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth. CONCLUSION: TER is a new class A ß-lactamase belonging to functional group 2b.

Use of R plasmid and bla Gene for Epidemiological Fingerprinting of Clinical Isolates of Enterobacteriaceae resistant to beta- lactam Antibiotics

Clinical isolates of Enterobacteriaceae (189 Klebsiella, 61 Enterobacter, 32 Serratia, 19 E. coli, 7 Proteus, and 3 Citrobacter) from one university hospital were epidemiologically analyzed by using transferable R plasmids resistant beta-lactam antibiotics including broad-spectrum cephalosporins. About 30% of E. cloacae and S. marcescens and about 5% of K. pneumoniae were resistant to one or more broad-spectrum j3-lactam antibiotics including cefotaxim, ceftazidime, aztreonam, or cefoxitin but all isolates of E. aerogenes, K oxytoca, and P. mirabilis were susceptible. Thirty-six conjugative R plasmids including 8 plasmids resistant expanded-spectrum cephalosporins were obtained from multiple resistant K. pneumoniae (19), E. cloacae (9), E. coli (4), and C. freundii (1). Thirty-one plasmids were subjected to R plasmid analysis and classified 20 different plasmid types. Among them 5, 2, and 2 plasmids belong to 3 different types respectively showed identical molecular size, endonuclease fragment pattern by Southem hybridization pattern by TEM-1 probe, pI value by isoelctric focusing, and also identical antibiogram and biotype of wild strains harboring plasmids. But all of plasmids resistant to cefotaxim, ceftazidime, aztreonam or cefoxitin showed different palsmid anlysis patterns. These results indicate that the epidemic strains of 3 clonal types had been present in this hospital and anlysis using transferable R plasmid and bla gene can be used to discriminate multi-resistant clinical isolates of Enterobacteriaceae.