Nitroxoline and its derivatives are potent inhibitors of metallo-ß-lactamases.

Carbapenemases such as metallo-ß-lactamases (MBLs) are spreading among Gram-negative bacterial pathogens. Infections due to these multidrug-resistant bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase producing bacteria include ß-lactamase inhibitor combinations. Nitroxoline is a broad-spectrum antibiotic with restricted indication for urinary tract infections. In this study, we report on nitroxoline as an inhibitor of MBLs. We investigate the structure-activity relationships of nitroxoline derivatives considering in vitro MBL inhibitory potency in a fluorescence based assay using purified recombinant MBLs, NDM-1 and VIM-1. We investigated the most potent nitroxoline derivative in combination with imipenem against clinical isolates as well as transformants producing MBL by broth microdilution and time-kill kinetics. Our findings demonstrate that nitroxoline derivatives are potent MBL inhibitors and in combination with imipenem overcome MBL-mediated carbapenem resistance.

Fecal Carriage and Molecular Characterization of Carbapenemase-Producing Enterobacterales in the Pediatric Population in Qatar.

Whole-genome sequencing was used to characterize carbapenemase-producing Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic. IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study.

Comparison of sCIM and Other Phenotypic Detection Methods for Carbapenemase-Producing Enterobacterales.

Rapid detection and reporting of carbapenemase-producing Enterobacterales (CPE) is one of the top priorities of clinical microbiology laboratories. The Clinical and Laboratory Standards Institute recommends the modified carbapenem inactivation method (mCIM) as the preferred method for this purpose, but it requires a broth incubation process which can be cumbersome. Here, we compared the performance of mCIM with three alternative rapid CPE detection methods against a collection of genetically defined CPE, with most carrying blaIMP, and non-CPE clinical isolates. The sensitivities of mCIM, simplified carbapenem inactivation method (sCIM), Rapidec Carba NP, and NG-Test Carba 5 were 98.0%, 54.9%, 90.2%, and 72.5%, whereas the specificities were 89.5%, 84.2%, 89.5%, and 100%, respectively. Modification of the interpretive criteria of sCIM increased its sensitivity to 88.2% and specificity to 89.5%. The results suggest that mCIM is currently the optimal method for CPE detection in an epidemiological setting where CPE-producing IMP group carbapenemase is predominant. While sCIM is easier to perform, it requires further validation before it can be widely adopted as an alternative to mCIM in the clinical laboratory. IMPORTANCE Simple identification methods for carbapenemase-producing Enterobacterales are required for the clinical laboratory. The simplified carbapenem inactivation method (sCIM) is a carbapenemase detection method that can be performed with less hands-on time than mCIM, but its sensitivity and specificity were suboptimal compared with other phenotypic detection methods when tested against a collection of IMP-producing CPE. Insufficient inactivation of imipenem from inadequate inoculation was suspected as the cause. While sCIM is easier to perform, it requires optimization before it can be widely adopted as an alternative to mCIM in the clinical laboratory.

Rapid Detection and Differentiation of KPC and MBL Carbapenemases among Enterobacterales Isolates by a Modified Combined-Disk Test.

This study was conducted to develop a cheap, rapid, and accurate modified combined-disk test (mCDT) approach to detect and differentiate KPC and MBL carbapenemases among clinical carbapenem-resistant Enterobacterales (CRE) isolates and simultaneously distinguish them from carbapenem-susceptible Enterobacterales (CSE) isolates. A total of 163 CRE and 90 third-generation cephalosporin-resistant Enterobacterales isolates were tested using imipenem and meropenem disks and different concentrations of carbapenemase inhibitors. The optimal sensitivity and specificity for detecting KPC carbapenemase were 97.2% and 100%, respectively. The sensitivity and specificity for detecting MBL carbapenemase were 100% and 100% with imipenem or meropenem and carbapenemase inhibitors within six hours. The inhibitory zone diameter of 18 mm for imipenem or meropenem disks without inhibitor could distinguish CRE from CSE isolates. Therefore, this mCDT approach may be a useful tool in clinical laboratories to detect CRE isolates and differentiate KPC and MBL producers, which is beneficial for patient management and hospital infection prevention and control.

Application of a multiplex immunochromatographic assay for rapid identification of carbapenemases in a clinical microbiology laboratory: performance and turn-around-time evaluation of NG-test Carba 5.

BACKGROUND: Prompt and accurate identification of carbapenemase production is essential for appropriate treatment and infection control. NG-Test Carba 5 (termed herein "Carba 5"; NG Biotech, Guipry, France) is a multiplex immunochromatographic assay for the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) from bacterial isolates. This study aimed to evaluate the diagnostic performance of Carba 5 and its impact on the turn-around-time in a clinical microbiology laboratory. RESULTS: Carba 5 was retrospectively evaluated using 78 carbapenemase producers and 23 non-carbapenemase producers confirmed by PCR and sequencing. The performance and time required for carbapenemase identification were prospectively evaluated using 47 carbapenem resistant Enterobacteriaceae isolates, and the results were compared to those obtained using Xpert Carba-R (Cepheid, Sunnyvale, CA, USA). For the bacterial isolates included in retrospective and prospective evaluation, the Carba 5 assay correctly identified 147 isolates except one isolate with a sensitivity of 99.13% (95% CI 95.25-99.98%) and specificity of 100% (95% CI 89.42-100%). The Carba 5 assay missed one VIM-1 among 13 VIM producers. The assay showed a sensitivity of 92.31% (95% CI 63.97-99.81%) for detecting VIM and 100% for detecting KPC, NDM, OXA-48-like, and IMP. Compared to the Xpert Carba-R assay, Carba 5 exhibited 100% agreement and was more time-efficient (median time 24 min vs. 1 h 11 min). CONCLUSIONS: The Carba 5 assay has potential as an alternative to molecular methods for detecting major carbapenemases from bacterial isolates in a clinical microbiology laboratory. Compared to the Xpert Carba-R, Carba 5 turns out to be more affordable and time-efficient while showing a comparable performance, and may accelerate therapeutic and infection control decisions.

Macrolides: a novel risk factor for carbapenemase-producing Enterobacterales in intensive care units.

INTRODUCTION: Carbapenemase-producing Enterobacterales (CPE) have emerged as a substantial cause of morbi-mortality worldwide, with a prevalence of approximately 5% in areas with high endemicity. However, available data may not be representative of developing countries, such as Ecuador. In this study, the incidence of CPE in Ecuador and risk factors for infection/colonisation were evaluated. METHODOLOGY: A prospective cohort study was performed from February to April 2016 in seven intensive-care units of Guayaquil, Ecuador. Samples were processed according to the Centers for Disease Control and Prevention laboratory protocol and the CHROMagar mSuper CARBA agar method. Resistance to carbapenems was defined according to Clinical and Laboratory Standards Institute breakpoints. A modified carbapenemase inactivation method was used to identify carbapenamase production phenotypically with molecular confirmation by multiplex polymerase chain reaction. RESULTS: In total, 640 patients were enrolled. The incidence of CPE was 36.4% (N = 233). A multivariate analysis indicated that several factors were associated with CPE acquisition, included a long intensive care unit stay (OR 1.05; 95% CI 1.03-1.08; p < 0.01), tracheostomy (OR 3.52; 95% CI 1.90-6.75; p < 0.01), hospitalisation 3 months prior to admission (OR 2.07; 95% CI 1.17-3.71; p < 0.01), vancomycin use (OR 3.31; 95% CI 2.02-5.18; p < 0.01), and macrolide use (OR 3.31; 95% CI 1.43-7.76; p < 0.01). CONCLUSIONS: Macrolide use was a risk factor for CPE acquisition. This association should be evaluated further, especially in developing countries.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Fast and automated detection of common carbapenemase genes using multiplex real-time PCR on the BD MAX™ system.

Fast detection of carbapenemases in Gram-negative bacilli is necessary for accurate antibiotic treatment, prevention of further spreading and surveillance purposes. We analyzed the current occurrence of gene variants and designed two multiplex PCRs with hydrolysis probes. The assay was developed for the BD MAX™ system that combines DNA extraction and PCR in a fully automated procedure providing results within 3 h and was evaluated for detection of carbapenemases from bacterial isolates and directly from rectal swabs. The assay has a theoretic coverage of 97.1% for carbapenemases detected during the last years by the German National Reference Laboratory (NRL). A collection of 151 isolates from the NRL was used and all carbapenemase-positive bacteria (58/58) were identified correctly. The direct-PCR on rectal swabs revealed additional carbapenemase genes in 7 samples that were not identified by the culture-based method used as reference method. The assay allows detection of carbapenemases from clinical isolates and might also help in rapid detection directly from rectal samples.

Genomic and clinical characterisation of multidrug-resistant carbapenemase-producing ST231 and ST16 Klebsiella pneumoniae isolates colonising patients at Siriraj hospital, Bangkok, Thailand from 2015 to 2017.

BACKGROUND: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data. METHODS: The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were "colonised" or "infected" with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes. RESULTS: Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was blaOXA-232, with blaNDM-1 additionally identified in 19 of them. All CPE isolates carrying either blaOXA-232 or blaNDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n = 18) and CPE-colonised patients who developed CPE infections during the study (n = 3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n = 15) and ST231 (n = 14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes. CONCLUSIONS: This study has revealed the genomic features of colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.

High-Speed Quenching Probe-Polymerase Chain Reaction Assay for the Rapid Detection of Carbapenemase-Producing Gene Using GENECUBE: A Fully Automatic Gene Analyzer.

BACKGROUND: The prevalence of carbapenemase-producing organisms (CPOs) globally poses a public health threat; however, detecting carbapenemases is a challenge because of their variety. METHODS: GENECUBE, a fully automated gene analyzer, detects a target gene in a short time and simultaneously detects its single nucleotide polymorphism. We used this property to develop for the first time a rapid assay for detecting CPOs from cultured bacteria using GENECUBE. The original primer-probe sets were used to detect blaKPC, blaIMP, blaVIM, blaNDM, and blaOXA-48-like from 149 CPOs (nine types) and 61 non-CPOs. RESULTS: The sensitivity, specificity, and positive and negative predictions of the GENECUBE assay were 100%. This assay detected carbapenemase single-producers and carbapenemase co-producers with 100% accuracy. The time required for detects of four types of carbapenemase at one run was about 30 min, but it took about 1 h to detect all five types. In addition, this assay performed the rapid detection and classification of blaOXA-48, blaOXA-181, blaOXA-232, and blaOXA-244 simultaneously. CONCLUSIONS: The GENECUBE assay is a promising tool for controlling the spread of CPOs and helping to select accurate and rapid antibiotic therapies.

Evaluation of three molecular carbapenemase tests: Eazyplex SuperBug complete B, Novodiag CarbaR+, and Amplidiag CarbaR+MCR.

BACKGROUND: Carbapenemase-producing Gram-negative bacilli, i.e., Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter, are of increased concern for the public health around the world. There is urgent need for rapid and accurate tests in order to provide correct treatment and to prevent bacterial spread in healthcare settings. METHODS: The aim of this study was to evaluate three commercial multiplex carbapenemase tests with CE-IVD marking: Eazyplex SuperBug complete B (AmplexDiagnostics), Novodiag CarbaR+ (Mobidiag), and Amplidiag CarbaR+MCR (Mobidiag). All these tests recognize KPC, NDM, OXA-48/181 group, VIM, OXA-23 group, and OXA-24/40 group, and Novodiag CarbaR+ and Amplidiag CarbaR+MCR additionally recognize IMP, OXA-51 group (with promoter located within ISAbaI), OXA-58 group, and MCR, and Amplidiag CarbaR+MCR further recognizes GES (carbapenemase-type only). RESULTS: The sensitivities and specificities of these tests with bacterial isolates were 100%. The sensitivity directly from clinical samples was 100%, but the specificity was lower, which is simply explained by the higher sensitivity of the molecular methods compared with culture method. CONCLUSIONS: Overall, these CE-IVD marked tests provide a good alternative in the detection of carbapenemase-producing organisms.

Detection of various beta-Lactamases in Escherichia coli and Klebsiella sp.: A study from Tertiary Care Centre of North India.

Objective: The emergence of carbapenem-resistant Escherichia coli and Klebsiella species is a global threat. We aimed to compare two phenotypic methods and evaluate the genotypic method for the detection of beta-lactamases produced by E. coli and Klebsiella spp. Materials and Methods: One hundred and twenty-six E. coli and Klebsiella isolates were examined for phenotypic production of beta-lactamases by using disc diffusion, combined disc test (CDT) and modified carbapenem inactivation method (mCIM). All strains were also studied for the presence of various genes by polymerase chain reaction. Results: Out of 126 isolates, 96% of the isolates were extended-spectrum ß-lactamase (ESBL) producers based on the presence of various ESBL genes. CDT method showed higher number of total (89%) carbapenemases in comparison to mCIM (81%). Among carbapenemases none of the isolates were Klebsiella pneumoniae carbapenemase producer by CDT, while 69% isolates were metallo-beta-lactamase (MBL) producers. Another method, mCIM/ethylene diamine tetraacetic acid mCIM showed 100% agreement for MBL detection. As regards, AmpC and class D carbapenemases; 0.04% and 16% positivity was detected, respectively, based on CDT method. Molecular analysis revealed 91% of the isolates harbouring carbapenemase genes. blaNDMwas the most common gene detected followed byblaOXA-48. Nine of the blaNDM-positive isolates also possessed blaOXA-48gene. Conclusion: Our finding shows high percentages of ESBL and carbapenemases in E. coli and Klebsiella spp. Among phenotypic methods, CDT seems to be a better choice as prevalence of carbapenemases shows lots of variation in our country. For Class B enzymes, both CDT and mCIM/eCIM can be used in the routine laboratories.

Characterization of a novel carboxylesterase belonging to family VIII hydrolyzing ß-lactam antibiotics from a compost metagenomic library.

A novel esterase, EstCS3, was isolated from a metagenomic library constructed from a compost. The EstCS3, which consists of 409 amino acids with an anticipated molecular mass of 44 kDa, showed high amino acid sequence identities to predicted esterases, serine hydrolases and ß-lactamases from uncultured and cultured bacteria. Phylogenetic analysis suggested that EstCS3 belongs to family VIII of lipolytic enzymes. EstCS3 had catalytic Ser78 residue in the consensus tetrapeptide motif SXXK, which is characteristic of family VIII esterases. Two conserved YXX and W(H or K)XG motifs in an oxyanion hole of family VIII esterases were also present in EstCS3. EstCS3 demonstrated the highest activity toward p-nitrophenyl butyrate (C4) and was stable up to 70 °C with optimal activity at 55 °C. EstCS3 had optimal activity at pH 8 and maintained its stability within pH range of 7-10. EstCS3 had over 70% activity in the presence of 20% (v/v) methanol and DMSO and hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. EstCS3 hydrolyzed ampicillin, cephalothin and cefepime. The properties of EstCS3, including moderate thermostability, stability against organic solvents and activity toward esters of tertiary alcohols, indicated that it has the potential to be used in industrial applications.

Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru. / Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú.

Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

Performance evaluation of automated BD Phoenix NMIC-500 panel for carbapenemase detection in carbapenem-resistant and carbapenem-susceptible Enterobacterales.

Rapid detection of carbapenemases and accurate reporting of carbapenem MICs is critical for appropriate treatment and infection control. We evaluated the BD Phoenix NMIC-500 panel for detection and classification of carbapenemases and antimicrobial susceptibility testing (AST) for carbapenems. A total of 235 isolates were tested; 47 carbapenemase-producing Enterobacterales, 52 non-carbapenemase-producing carbapenem-resistant Enterobacterales (non-CP-CRE), 136 carbapenem-susceptible Enterobacterales (CSE). The sensitivity of carbapenemase-producing organism (CPO) detection was 97.9%, the specificity was 100% for CSE but 32.7% for non-CP-CREs. All the 35 false-positive cases were non-CP-CREs; 23 out of the 35 were determined as untyped carbapenemase producer (CP), nine were mistyped as class B, and three were as class A. The detection rate/correct classification rate for class A, B, and D carbapenemase was 100%/78.6%, 100%/100%, and 80%/60%, respectively. To supplement the low specificity, it is suggested to report carbapenemase-producer (CP) positive results as "strongly suspicious for carbapenem resistance but carbapenemase production needs to be confirmed" and perform the confirmatory test. The EA and CA for ertapenem, imipenem, and meropenem was 99.1%/99.6%, 89.4%/90.6%, and 95.3%/95.7%. In conclusion, the BD Phoenix CPO detect panel provides advantage in that the carbapenemase test is automated and the results can be obtained within 6 h but the low specificity in CREs needs to be improved. In addition, accurate reporting of meropenem MICs will be helpful for clinicians to choose treatment options.

Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú / Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru

RESUMEN Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

ABSTRACT Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Detection and characterization of ESBL-producing Enterobacteriaceae from the gut of healthy chickens, Gallus gallus domesticus in rural Nepal: Dominance of CTX-M-15-non-ST131 Escherichia coli clones.

The surge in the prevalence of drug-resistant bacteria in poultry is a global concern as it may pose an extended threat to humans and animal health. The present study aimed to investigate the colonization proportion of extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae (EPE and CPE, respectively) in the gut of healthy poultry, Gallus gallus domesticus in Kaski district of Western Nepal. Total, 113 pooled rectal swab specimens from 66 private household farms and 47 commercial poultry farms were collected by systematic random sampling from the Kaski district in western Nepal. Out of 113 pooled samples, 19 (28.8%) samples from 66 backyard farms, and 15 (31.9%) from 47 commercial broiler farms were positive for EPE. Of the 38 EPE strains isolated from 34 ESBL positive rectal swabs, 31(81.6%) were identified as Escherichia coli, five as Klebsiella pneumoniae (13.2%), and one each isolate of Enterobacter species and Citrobacter species (2.6%). Based on genotyping, 35/38 examined EPE strains (92.1%) were phylogroup-1 positive, and all these 35 strains (100%) had the CTX-M-15 gene and strains from phylogroup-2, and 9 were of CTX-M-2 and CTX-M-14, respectively. Among 38 ESBL positive isolates, 9 (23.7%) were Ambler class C (Amp C) co-producers, predominant were of DHA, followed by CIT genes. Two (6.5%) E. coli strains of ST131 belonged to clade C, rest 29/31 (93.5%) were non-ST131 E. coli. None of the isolates produced carbapenemase. Twenty isolates (52.6%) were in-vitro biofilm producers. Univariate analysis showed that the odd of ESBL carriage among commercial broilers were 1.160 times (95% CI 0.515, 2.613) higher than organically fed backyard flocks. This is the first study in Nepal, demonstrating the EPE colonization proportion, genotypes, and prevalence of high-risk clone E. coli ST131 among gut flora of healthy poultry. Our data indicated that CTX-M-15 was the most prevalent ESBL enzyme, mainly associated with E. coli belonging to non-ST131clones and the absence of carbapenemases.

The type I-E CRISPR-Cas system influences the acquisition of blaKPC-IncF plasmid in Klebsiella pneumonia.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and blaKPC-IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of blaKPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and blaKPC-IncF plasmids in such isolates. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and blaKPC-positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered blaKPC-IncF plasmids invasion and existence. Notably, most blaKPC-IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of blaKPC in K. pneumoniae populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of blaKPC-IncF plasmids in CG258 K. pneumoniae.

α-Triazolylboronic Acids: A Promising Scaffold for Effective Inhibitors of KPCs.

Boronic acids are known reversible covalent inhibitors of serine ß-lactamases. The selectivity and high potency of specific boronates bearing an amide side chain that mimics the ß-lactam's amide side chain have been advanced in several studies. Herein, we describe a new class of boronic acids in which the amide group is replaced by a bioisostere triazole. The boronic acids were obtained in a two-step synthesis that relies on the solid and versatile copper-catalyzed azide-alkyne cycloaddition (CuAAC) followed by boronate deprotection. All of the compounds show very good inhibition of the Klebsiella pneumoniae carbapenemase KPC-2, with Ki values ranging from 1 nM to 1 µM, and most of them are able to restore cefepime activity against K. pneumoniae harboring blaKPC-2 . In particular, compound 1 e, bearing a sulfonamide substituted by a thiophene ring, proved to be an excellent KPC-2 inhibitor (Ki =30 nM); it restored cefepime susceptibility in KPC-Kpn cells (MIC=0.5 µg/mL) with values similar to that of vaborbactam (Ki =20 nM, MIC in KPC-Kpn 0.5 µg/mL). Our findings suggest that α-triazolylboronates might represent an effective scaffold for the treatment of KPC-mediated infections.

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual, SPR and NMR screening.

NDM-1 can hydrolyze nearly all available ß-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of 15N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.