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245600.9625MgrB Alterations Mediate Colistin Resistance in Klebsiella pneumoniae Isolates from Iran. Colistin is one of the last-resort therapeutic agents to combat multidrug-resistant Gram-negative bacteria (GNB) including Klebsiella pneumoniae. Although it happens rarely, resistance to colistin has been reported for several GNB. A total of 20 colistin resistant (col-R) and three colistin susceptible (col-S) clinical isolates of K. pneumoniae were studied to explore the underlying mechanisms of colistin resistance. The presence of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 genes were examined by PCR. The nucleotide sequences of pmrA, pmrB, phoP, phoQ, and mgrB genes were determined. To evaluate the association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons, transcriptional level of the pmrK and pmrC genes encoding for lipopolysaccharide target modifying enzymes was quantified by RT-qPCR analysis. None of the plasmid encoded resistance genes were detected in the studied isolates. Inactivation of MgrB due to nonsense mutations and insertion of IS elements was observed in 15 col-R isolates (75%). IS elements (IS5-like and IS1-like families) most commonly targeted the coding region and in one case the promoter region of the mgrB. Complementation with wild-type MgrB restored colistin susceptibility in isolates with altered mgrB. All col-R isolates lacked any genetic alterations in the pmrA, phoP, and phoQ genes and substitutions identified in the pmrB were not found to be involved in resistance conferring determined by complementation assay. Colistin resistance linked with upregulation of pmrHFIJKLM and pmrCAB operons with the pmrK and pmrC being overexpressed in 20 and 11 col-R isolates, respectively. Our results demonstrated that MgrB alterations are the major mechanisms contributing to colistin resistance in the tested K. pneumoniae isolates from Iran.201729326662
503510.9620Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods. A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools.201627153928
600620.9618Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae. NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.202333685902
245830.9612Mutation in mgrB is the major colistin resistance mechanism in Klebsiella pneumoniae clinical isolates in Tehran, Iran. Colistin is considered as one of a last resort antimicrobial agent against multidrug-resistant Gram-negative bacteria including Escherichia coli and Klebsiella pneumoniae. However, the recent emergence of colistin resistance (ColR) worldwide that severely restricts therapeutic options is a serious threat to global public health. In this study we have investigated the molecular determinants in ColR K. pneumoniae isolates collected from clinical specimens. A total of 98 E. coli and 195 K. pneumoniae clinical isolates were collected from two hospitals from August 2018 to December 2019 in Tehran, Iran. Colistin susceptibility and minimum inhibitory concentrations (MIC) were determined according to the Clinical and Laboratory Standards Institute by disk diffusion method, and microdilution method, respectively. For isolates with colistin MIC ≥4 μg mL-1, PCR was performed for the detection of mcr-1 to mcr-4 genes. Moreover, nucleotide sequences of mgrB, phoP, phoQ, pmrA, and pmrB genes were determined by sequencing. Finally, the transcriptional level of pmrK and pmrC genes was evaluated by quantitative reverse transcription PCR (RT-qPCR). None of the E. coli isolates were resistant to colistin while 21 out 195 K. pneumoniae isolates were identified as resistant, 19 of which carried mutation in the mgrB gene. Three different mutations were observed in the pmrB gene in 3 K. pneumoniae isolates. None of the ColR isolates showed alternations in pmrA, phoP, and phoQ genes. Furthermore, none of the plasmid-encoding genes were detected. Transcriptional level of the pmrK gene increased in all ColR isolates meanwhile, pmrC overexpression was detected in 16 out 21 (76.19%) isolates. Eventually, all ColR isolates were susceptible to tigecycline. Our results demonstrated that the alternation of mgrB gene is the main mechanism related to colistin resistance among ColR K. pneumoniae isolates in this study.202235113039
245740.9611Prevalence and molecular mechanisms of colistin resistance in Acinetobacter baumannii clinical isolates in Tehran, Iran. Colistin is one of the last remaining active antibiotics against multidrug resistant Gram-negative bacteria. However, several recent studies reported colistin-resistant (ColR) Acinetobacter baumannii from different countries. In the current study, we investigated molecular mechanisms involved in colistin resistance in A. baumannii isolates from different clinical samples. A total of 110 clinical A. baumannii isolates were collected from two hospitals in Tehran. Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute. For the ColR isolates, mutation was detected in pmrA, pmrB, lpxA, lpxC, and lpxD genes using the polymerase chain reaction (PCR) and sequencing. Moreover, the relative expression of the pmrC gene was calculated using quantitative reverse transcription PCR. Three colistin resistant isolates were identified with MIC between 8 and 16 μg/mL and were resistant to all the tested antimicrobial agents. All the three isolates had a mutation in the pmrB, pmrA, lpxA, lpxD, and lpxC genes. Moreover, the overexpression of pmrC gene was observed in all isolates. Our results showed that the upregulation of the PmrAB two component system was the primary mechanism linked to colistin resistance among the studied colistin resistant A. baumannii isolates.202134370684
246050.9610Emergence of cefiderocol resistance during therapy in NDM-5-producing Klebsiella pneumoniae isolates harboring siderophore receptors mutations. Cefiderocol, a siderophore-conjugated cephalosporine, is a promising drug used to treat infection with carbapenem-resistant gram-negative bacteria. Here, we report a case of pneumonia induced by multiple gram-negative pathogens, including a carbapenem-resistant Klebsiella pneumoniae developing cefiderocol resistance within 32 days of cefiderocol therapy. Whole genome sequencing of three consecutive K. pneumoniae isolates revealed that the bacteria were isogenic and were carrying several broad-spectrum β-lactamases (bla(NDM5) and bla(CTX-M-15)). Two isolates with elevated minimum inhibitory concentration against cefiderocol harbored mutations in genes encoding siderophore: one in the cirA gene and one in both the cirA and the fiu genes. The combination of a metallo-β-lactamase background and mutations in siderophore receptors was associated with phenotypic resistance to cefiderocol.202539617206
245360.9610Prevalence and molecular determinants of colistin resistance among commensal Enterobacteriaceae isolated from poultry in northwest of Iran. BACKGROUND: The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria. Here we aimed to determine the prevalence of colistin resistance, among enterobacteria isolated from poultry and the possible underlying colistin resistance mechanisms. METHODS: A collection of 944 cloacal samples were obtained from poultry and screened for colistin resistance. To uncover the molecular mechanism behind colistin resistance, the presence of plasmid encoded colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4 was examined by PCR. The nucleotide sequences of the mgrB, pmrA, pmrB, phoP, phoQ, crrA and crrB genes were determined. The genetic relatedness of the colistin resistant (ColR) isolates was evaluated by Multilocus sequence typing. Three ColR mutants were generated in vitro by repetitive drug exposure. RESULTS: Overall from 931 enteric bacteria isolated from poultry samples obtained from 131 farms, nine ColR bacteria (0.96%) with high level colistin resistance (MICs ≥ 64 mg/L) were detected all being identified as K. pneumoniae. The 9 ColR bacteria originated from different farms and belonged to 7 distinct Sequence types including ST11 (22.2%) and ST726 (22.2%) being the most prevalent STs followed by ST37, ST74, ST485, ST525 and novel sequence type 3380 (11.1% each). mcr-type genes were not detected in any isolate. In 88.8% of the isolates (n = 8), MgrB was inactivated by Insertion of IS elements (IS1-like, IS3-like, IS5-like families, positions + 75, + 113, + 117, + 135) and nonsense mutations at codons 8, 16, 30. All ColR isolates harboured wild type PmrA, PhoP, PhoQ or polymorphic variants of PmrB. Sequence analysis of the CrrB revealed a familiar S195N and 4 novel I27V, T150R, F303S and K325R substitutions. PmrB T93N substitution and mgrB locus deletion were identified in two laboratory induced ColR mutants and one mutant lacked alteration in the studied loci. In one ColR isolate with wild type MgrB an A83V substitution was detected in CrrA. CONCLUSION: It is concluded from our results that colistin resistance in the studied avian K. pneumoniae isolates was mostly linked to alterations identified within the mgrB gene.201930728861
245570.9610Molecular Mechanisms of Colistin Resistance Among Klebsiella Pneumoniae Strains. BACKGROUND: The increasing rate of infections caused by multiple drug resistant gram-negative bacteria has led to resuscitation of colistin. As a result, colistin resistance, mainly among Klebsiella pneumoniae strains has also been increased. The aim of this study was to investigate molecular mechanisms behind colistin resistance. METHODS: Twenty colistin-resistant K. pneumoniae strains isolated from clinical samples of different patients were involved in this study. VITEK2 automated ID/AST system (Biomeriux, France) was used for the identification and also the susceptibility testing for antibiotics other than colistin. Colistin susceptibility was determined by broth microdilution method. To identify the mechanisms of resistance, mutations on mgrB genes, expression levels of pmrA, pmrB, pmrC, pmrD, pmrE, pmrK, phoQ, and phoP genes, and the presence of plasmid mediated colistin resistance genes, mcr-1 and mcr-2 were investigated. RESULTS: As a result of the study, increased expression levels of the pmrA, pmrB, pmrD, pmrK, phoP, and phoQ genes were observed. All colistin resistant strains were found wild type for the mgrB gene which is thought to be esponsible for colistin resistance. Also, no mcr-1 or mcr-2 genes which are the causes of plasmid mediated colistin resistance have been detected in any of the strains. CONCLUSIONS: Among the colistin resistant K. pneumoniae strains included in our study, increased expression Levels of the genes responsible for cell membrane modifications related with colistin resistance were the most common mechanisms.201931307167
140580.9610The threat of carbapenem resistance in Eastern Europe in patients with decompensated cirrhosis admitted to intensive care unit. BACKGROUND: Multidrug-resistant organisms are an increasing concern in patients with decompensated cirrhosis. AIM: We aimed to evaluate the prevalence of infections with carbapenem-resistant Enterobacteriaceae in patients with decompensated cirrhosis. METHODS: Patients with decompensated cirrhosis admitted to ICU were included. The isolated Enterobacteriaceae strains were tested for carbapenemase-producing genes using the Roche LightMix® Modular VIM/IMP/NDM/GES/KPC/OXA48-carbapenemase detection kit. RESULTS: 48 culture-positive infections were registered in 75 patients with acutely decompensated cirrhosis. Thirty patients contracted a second infection. 46% of bacteria isolated at admission and 60% of bacteria responsible for infections identified during ICU-stay were multiresistant. ESBL+ Enterobacteriaceae were predominant at admission, while carbapenem-resistance was dominant in both Enterobacteriaceae and Non-Fermenting-Gram-Negative Bacteria responsible for infections diagnosed during hospitalisation. OXA 48 or KPC type carbapenemases were present in 30% of the analyzed Enterobacteriaceae and in 40% of the phenotypically carbapenem-resistant Klebsiella pneumoniae strains. The length of ICU stay was a risk-factor for a second infection (p=0.04). Previous carbapenem usage was associated with occurence of infections with carbapenem-resistant Gram-negative bacteria during hospitalization (p=0.03). CONCLUSION: The prevalence of infections with carbapenem-resistant Enterobacteriaceae is high in patients with decompensated cirrhosis admitted to ICU. Carbapenemase-producing genes in Enterobacteriaceae in our center are bla(OXA-48) and bla(KPC).202235732546
506690.9608Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli. Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.202438905152
6173100.9606Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae. BACKGROUND: RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants. METHODS: A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression. RESULTS: Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance. CONCLUSION: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.202337331490
2454110.9606Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms. BACKGROUND: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. RESULTS: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. CONCLUSIONS: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.202134798825
1400120.9603Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
5044130.9602Detection of Colistin Resistance in Salmonella enterica Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer. Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.202032582090
1435140.9602Epidemiology, Phenotypic and Genotypic Characterization of Carbapenem-Resistant Gram-Negative Bacteria from a Libyan Hospital. Antimicrobial resistance, particularly resistance to carbapenems, has become one of the major threats to public health. Seventy-two isolates were collected from patients and hospital environment of Ibn Sina Hospital, Sirte, Libya. Antibiotic susceptibility tests, using the disc diffusion method and E-Test strips, were performed to select carbapenem-resistant strains. The colistin (CT) resistance was also tested by determining the minimum inhibitory concentration (MIC). RT-PCR was conducted to identify the presence of carbapenemase encoding genes and plasmid-mediated mcr CT resistance genes. Standard PCR was performed for positive RT-PCR and the chromosome-mediated CT resistance genes (mgrB, pmrA, pmrB, phoP, phoQ). Gram-negative bacteria showed a low susceptibility to carbapenems. Molecular investigations indicated that the metallo-β-lactamase New Delhi metallo-beta-lactamases-1 was the most prevalent (n = 13), followed by Verona integron-encoded metallo-beta-lactamase (VIM) enzyme (VIM-2 [n = 6], VIM-1 [n = 1], and VIM-4 [n = 1]) that mainly detected among Pseudomonas spp. The oxacillinase enzyme OXA-23 was detected among six Acinetobacter baumannii, and OXA-48 was detected among one Citrobacter freundii and three Klebsiella pneumoniae, in which one coharbored the Klebsiella pneumoniae carbapenemase enzyme and showed resistance to CT (MIC = 64 μg/mL) by modification in pmrB genes. In this study, we report for the first time the emergence of Pseudomonas aeruginosa carrying the bla(NDM-1) gene and belonging to sequence type773 in Libya. Our study reported also for the first time CT resistance by mutation in the pmrB gene among Enterobacteriaceae isolates in Libya.202337145891
5037150.9600Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas. Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n=248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla(IMP) genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7×10(4)cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.201121986031
1474160.9599Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria. PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.201728966495
2459170.9598In vitro antimicrobial activity and resistance mechanisms of cefiderocol against clinical carbapenem-resistant gram-negative bacteria. BACKGROUND: The rise of carbapenem-resistant gram-negative bacteria (CRGNB) necessitates new therapeutic options such as cefiderocol. OBJECTIVE: To evaluate the in vitro efficacy of cefiderocol against clinical CRGNB and investigate associated resistance mechanisms. METHODS: A total of 370 CRGNB isolates were analyzed. Minimum inhibitory concentration (MIC) values were determined, and whole genome sequencing, efflux pump inhibition assays, and RT-qPCR were conducted to assess resistance-related mutations, gene loss, and expression changes. RESULTS: Cefiderocol demonstrated potent in vitro activity, with high susceptibility rates in C. freundii (100%), K. pneumoniae (93.3%), and E. hormaechei (92.2%), and notable activity against P. aeruginosa (80.0%) and Escherichia coli (76.8%). Efflux pump inhibition by Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP) significantly reduced MICs in resistant strains. Key resistance mechanisms included β-lactamase gene variants (bla (OXA-66), bla (OXA-23), bla (SHV-12)), mutations in envZ, cirA, nuoC, ampC, and loss or altered expression of iron transporter genes (piuA, pirA, fepA). CONCLUSION: Cefiderocol is highly effective against CRGNB; however, resistance may arise through diverse mechanisms, including efflux pump activity. Continued surveillance of emerging resistance is essential to guide its optimal clinical use.202541113641
1721180.9598Convergence of MCR-8.2 and Chromosome-Mediated Resistance to Colistin and Tigecycline in an NDM-5-Producing ST656 Klebsiella pneumoniae Isolate From a Lung Transplant Patient in China. We characterized the first NDM-5 and MCR-8.2 co-harboring ST656 Klebsiella pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant patient. Complete genome sequences were obtained through Illumina HiSeq sequencing and nanopore sequencing. The acquired resistance genes and mutations in chromosome-encoded genes associated with colistin and tigecycline resistance were analyzed. Comparative genomic analysis was conducted between mcr-8.2-carrying plasmids. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonging to ST656, and harbored plasmid-encoded bla(NDM-5) and mcr-8.2 genes. The bla(NDM-5) gene was located on an IncX3 type plasmid. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8, which had a common ancestor with another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. The MIC of KP32558 for colistin was 256 mg/L. The mcr-8.2 gene and mutations in the two-component system, pmrA and crrB, and the regulator mgrB, had a synergistic effect on the high-level colistin resistance. The truncation in the acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.202235899054
5375190.9596Mechanism of Eravacycline Resistance in Clinical Enterococcus faecalis Isolates From China. Opportunistic infections caused by multidrug-resistant Enterococcus faecalis strains are a significant clinical challenge. Eravacycline (Erava) is a synthetic fluorocycline structurally similar to tigecycline (Tige) that exhibits robust antimicrobial activity against Gram-positive bacteria. This study investigated the in vitro antimicrobial activity and heteroresistance risk of Eravacycline (Erava) in clinical E. faecalis isolates from China along with the mechanism of Erava resistance. A total of 276 non-duplicate E. faecalis isolates were retrospectively collected from a tertiary care hospital in China. Heteroresistance to Erava and the influence of tetracycline (Tet) resistance genes on Erava susceptibility were examined. To clarify the molecular basis for Erava resistance, E. faecalis variants exhibiting Erava-induced resistance were selected under Erava pressure. The relative transcript levels of six candidate genes linked to Erava susceptibility were determined by quantitative reverse-transcription PCR, and their role in Erava resistance and heteroresistance was evaluated by in vitro overexpression experiments. We found that Erava minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates ranged from ≤0.015 to 0.25 mg/l even in strains harboring Tet resistance genes. The detection frequency of Erava heteroresistance in isolates with MICs ≤ 0.06, 0.125, and 0.25 mg/l were 0.43% (1/231), 7.5% (3/40), and 0 (0/5), respectively. No mutations were detected in the 30S ribosomal subunit gene in Erava heteroresistance-derived clones, although mutations in this subunit conferred cross resistance to Tige in Erava-induced resistant E. faecalis. Overexpressing RS00630 (encoding a bone morphogenetic protein family ATP-binding cassette transporter substrate-binding protein) in E. faecalis increased the frequency of Erava and Tige heteroresistance, whereas RS12140, RS06145, and RS06880 overexpression conferred heteroresistance to Tige only. These results indicate that Erava has potent in vitro antimicrobial activity against clinical E. faecalis isolates from China and that Erava heteroresistance can be induced by RS00630 overexpression.202032523563