Carbapenem Resistance Mechanisms Research Articles

Extensively drug-resistant Acinetobacter baumannii co-producing VIM-2 and OXA-23 in intensive care units: Results of a one-day point prevalence in a Tunisian hospital

ObjectiveThe main objective of this study was to identify carbapenem resistance mechanisms among clinical, commensal and environmental carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in 5 Tunisian intensive care units (ICUs). Materials and MethodsCRAB isolates were recovered from different sources: clinical specimens, rectal and environmental swabs. Bacterial identification was carried out using conventional methods and susceptibility testing according to EUCAST recommendations. Evaluation of phenotypic carbapenemase production of was performed using seven different methods, and molecular detection of carbapenemase-coding genes (blaOXA23, blaOXA24, blaOXA58, blaNDM, blaGES, blaOXA48, blaIMP, blaVIM and blaKPC) was done by PCR. The genetic relationships between CRAB isolates were analyzed by pulsed-field gel electrophoresis. ResultsAll in all, 46 CRAB isolates were identified in clinical specimens (n = 26/26), rectal swabs (n = 17/36) and environmental swabs (n = 3/63). Most of them (n = 41/46) were clonally related and found in the different ICUs. All of the CARB isolates harbored blaOXA-51-like and blaOXA-23 genes, while blaVIM-2 gene was detected in 42 isolates (91.3 %). Phenotypic carbapenemase activity was found in all 46 strains, using the CIM-Tris test with 100 % sensitivity for OXA-23 and VIM-carbapenemase detection, thereby justifying its routine use. ConclusionThe emergence and diffusion of clonal CRAB strains inducing high mortality rates in ICUs is a major public health concern. Enhanced infection control practices and mandatory staff education are needed to control the spread of these multidrug-resistant bacteria.

Hybrid IncFIA/FIB/FIC(FII) plasmid co-carrying blaNDM-5 and fosA3 from an Escherichia coli ST117 strain of retail chicken

Carbapenems and fosfomycin are important antibiotics used to treat Enterobacteriaceae-associated infections. This study aimed to characterize the co-resistance and co-dissemination mechanism of carbapenem and fosfomycin resistance in an Escherichia coli ST117 strain isolated from retail chicken meat. Antimicrobial susceptibility testing showed that an E. coli CS18F strain had a multidrug resistance profile, including carbapenem and fosfomycin resistance. The presence of blaNDM-5 and fosA3 genes was confirmed by PCR and Sanger sequencing. The blaNDM-5 and fosA3 genes were successfully transferred to the recipient strain E. coli J53 via conjugation, and the transconjugants had elevated minimum inhibitory concentrations (MICs) for meropenem and fosfomycin. Whole genome sequencing (WGS) of E. coli CS18F revealed that blaNDM-5 and fosA3 were colocalized on an IncFIA/FIB/FIC(FII) type plasmid of 189,141 bp, which was designated as pCS18F-NDM-Fos. A novel structure with five IS26 sequences flanking the multiple drug resistance region (MDRR) was identified, and three copies of IS26 were found to be flanked blaNDM-5, fosA3, dfrA12, aadA2, and sul1. Three types of translocation units (TUs) were identified by PCR, containing either the resistance gene blaNDM-5 and an IS26 sequence, fosA3, and an IS26 sequence, or both, indicating their potential co-transfer via TUs. Thus, this is an unprecedented report of the presence of a plasmid co-carrying blaNDM-5 and fosA3 and TUs potentially mediating their simultaneous transfer.

Carbapenemase- and Colistin Resistant Escherichia coli Strains from Children in China: High Genetic Diversity and First Report of bla NDM-5, bla CTX-M-65, bla OXA-10, bla TEM-1, and mcr-1.1 Genes Co-Occurrence in E. coli ST156.

BackgroundThe emergence of carbapenem and colistin co-resistant Escherichia coli poses a huge challenge to infection control. The purpose of this study was to clarify the mechanism of the carbapenems and colistin co-resistance in E. coli strains.MethodsAntimicrobial susceptibility test was carried out by agar dilution methods and colistin resistance was confirmed by broth microdilution methods. Whole genome sequencing was carried out, and resistance genes, sequence types and virulence genes of carbapenems and colistin co-resistance E. coli isolates were analyzed.ResultsThe results showed that among the 176 carbapenem-resistant Enterobacteriaceae strains, 5 multidrug resistant E. coli strains exhibiting coresistance to carbapenem and colistin. The main mechanism of 5 E. coli strains in this study was generating carbapenem. Four E. coli strains were mcr-positive, while one mcr-negative strain had a new MgrB mutation. The blaNDM-5, blaCTX-M-65, blaOXA-10, blaTEM-1 and mcr-1.1 genes were simultaneously detected in E. coli 20IR1127 strain belonging to ST156 lineage. Other antimicrobial resistance genes encoding aminoglycosides-, sulfonamide-, chloramphenicol-, tetracyclines- and macrolides resistance were also detected.ConclusionThe main mechanisms of carbapenem and colistin resistance were encoded by blaNDM and mcr1.1, meanwhile mgrB mutations also contribute to colistin resistance. To our knowledge, this study is the first to report of E. coli ST156 strain in which the blaNDM-5, blaCTX-M-65, blaOXA-10, blaTEM-1 and mcr1.1 genes coexist. In addition, there is also an E. coli ST457 strain, which carries blaTEM-1, blaNDM-9, blaCTX-M-199 and is positive for mcr1.1 gene.

Proteomics profiling of ertapenem challenged major porin deficient carbapenem-resistant Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent threat to human health. Major outer membrane proteins (OMPs) porin mutation is one important resistance mechanism of CRKP, and may also affect the inhibition activity of β-lactam and β-lactamase inhibitor combinations. The ertapenem-resistant K. pneumoniae strain 2018B120 with major porin mutations was isolated from a clinical patient. Genomic and time-series proteomic analyses were conducted to retrieve the ertapenem-challenged response of 2018B120. The abundance changing of proteins from PTS systems, ABC transporters, the autoinducer 2 (AI-2) quorum sensing system, and antioxidant systems can be observed. Overexpression of alternative porins was also noticed to balance major porins' defection. These findings added a detailed regulation network in bacterial resistance mechanisms and gave new insights into bypass adaptation mechanisms the porin deficient bacteria adopted under carbapenem antibiotics pressure. SignificanceOuter membrane porins deficiency is an important mechanism of carbapenem resistance in K. pneumoniae. Comprehensive genomic and proteomic profiling of an ertapenem-resistant K. pneumoniae strain 2018B120 gives a detailed systematic regulation network in bacterial resistance mechanisms. Overexpression of alternative porins to balance major porins' defection was noticed, giving new insights into bypass adaptation mechanisms of porin deficient bacteria.

Risk factors for mortality in hospitalized patients infected with carbapenem-resistant Pseudomonas aeruginosa in Iran.

Mortality due to carbapenem-resistant Pseudomonas aeruginosa (CRPA) infection has increased worldwide in recent years. The risk factors associated with hospital settings in Iran and the role of strain resistance mechanisms in many studies are unclear. A retrospective study was conducted on consecutive non-repetitive patients with CRPA infections isolated from seven major hospitals from northwest of Iran. We evaluated different risk factors and characteristics of bacteria for the death or survival of patients. In this study, 116 CRPA isolates were obtained from patients admitted to seven hospitals. Forty-one (35.3%) patients were enrolled in the study of mortality risk factors. Significant risk factors associated with mortality included the site of infection, hospitalization in different wards, the use of invasive devices, and the type of carbapenem resistance mechanisms. ICU admission, the use of mechanical ventilation and chest tube and infection with pandrug-resistant strains were the most important factors in increasing mortality due to CRPA infection. These results suggested that the clinicians should emphasize the proper use of antibiotic and invasive procedures.

Plasmid-Borne AFM Alleles in Pseudomonas aeruginosa Clinical Isolates from China.

ABSTRACTCarbapenem-resistant Pseudomonas aeruginosa (CRPA) is a pathogen of global concern due to the fact that therapeutic drugs are limited. Metallo-β-lactamase (MBL)-producing P. aeruginosa has become a critical part of CRPA. Alcaligenes faecalis metallo-β-lactamase (AFM) is a newly identified subclass B1b MBL. In this study, 487 P. aeruginosa strains isolated from patients and the environment in an intensive care unit were screened for AFM alleles. Five AFM-producing strains were identified, including four AFM-2-producing strains (ST262) and one AFM-4-producing strain (ST671). AFM-2-producing strains were isolated from rectal and throat swabs, and AFM-4-producing strains were isolated from the water sink. The blaAFM-2 carrying plasmids belonged to the IncP-2 type, while the blaAFM-4 carrying plasmid pAR19438 was a pSTY-like megaplasmid. Plasmid pAR19438 was acquired blaAFM-4 by the integration of the Tn1403-like transposon. All blaAFM genes were embedded in an ISCR29-blaAFM unit core module flanked by class 1 integrons. The core module of blaAFM-2 was ISCR29-ΔgroL-blaAFM-2-bleMBL-ΔtrpF-ΔISCR, while the core module of blaAFM-4 was ISCR29-ΔgroL-blaAFM-2-bleMBL-ΔtrpF-ISCR-msrB-msrA-yfcG-corA-ΔISCR. The flanking sequences of ISCR29-blaAFM units also differed. The expression of AFM-2 and AFM-4 in DH5α and PAO1 illustrated the same effect for the evaluation of the MICs of β-lactams, except for aztreonam. Identification of AFM-4 underscores that the quick spread and emerging development of mutants of MBLs require continuous surveillance in P. aeruginosa.IMPORTANCE Acquiring metallo-β-lactamase genes is one of the important carbapenem resistance mechanisms of P. aeruginosa. Alcaligenes faecalis metallo-β-lactamase is a newly identified metallo-β-lactamase, the prevalence and genetic context of which need to be explored. In this study, we identified AFM-producing P. aeruginosa strains among clinical isolates and found a new mutant of AFM, AFM-4. The blaAFM-4 carrying plasmid pAR19438 was a pSTY-like megaplasmid, unlike the plasmids encoding other blaAFM alleles. The genetic context of blaAFM-4 was also different. However, AFM-2 and AFM-4 had the same impacts on antibiotic susceptibility. The presence and transmission of AFM alleles in P. aeruginosa pose a challenge to clinical practice.

Molecular Detection of OXA-type Carbapenemases among Acinetobacter baumannii Isolated from Burn Patients and Hospital Environments

Background:Acinetobacter baumanniiis known as one of the major causes of nosocomial infections, especially in intensive care units and burn patients. The emergence of antimicrobial resistance in burn wound bacterial pathogens is a severe health crisis. Detection of carbapenem resistance and genetic elements inA. baumanniiassociated with burn patients and hospital environments play a key role in the control and alerting in clinical settings.Purpose:In this study, the prevalence of OXA-type carbapenemases was investigated inA. baumanniistrains isolated from burn patients and from a hospital environment in Tehran, 2021.Methods:A total of 85 non-duplicateA. baumanniiisolates (53 from various surfaces of the hospital environment and 32 from burn patients) were recovered in the Burns Hospital in Tehran. TheA. baumanniiisolates were screened for antibiotic susceptibility and the presence of the most common OXA-type carbapenemase genes.Results:A. baumanniiwas isolated from 38.5% of hospital patient burn wounds and 22.1% of surfaces, including burn units (15.6%) and intensive care units (52.4%). Antibiotic susceptibility results showed that (100%) of burn patient isolates were resistant to imipenem, while (100%) of ICU isolates and (96.8%) of burn isolates were resistant to imipenem. All clinical isolates were identified as MDR and XDR, whereas all (100%) and 98.1% of environmental isolates were identified as MDR and XDR, respectively. All studiedA. baumanniiisolates carriedblaOXA-51-likegene. Moreover, 50 (94.3%) and 49 (92.5%) of environmental isolates, 32 (100%) and 30 (93.7%) of burn patient isolate harboredblaOXA-23-likeandblaOXA-24/40–likegenes, respectively. None of the isolates carried theblaOXA-58orblaOXA-143genes and all isolates had at least 2 OXA-type carbapenemase genes.Conclusion:Our results suggest that surfaces in the hospital environment, particularly in ICUs, are contaminated with MDR or XDRA. baumanniistrains. They may be considered a potential reservoir for the colonization of hospital patients. In addition, OXA-type carbapenemases, including OXA-23-like and OXA-24/40-like, appear to be one of the major mechanisms of carbapenem resistance in the clinical and environmentalA. baumanniistrains.

Imipenem Induced Transcriptional Modulation of Multidrug Efflux Pumps and Porin Genes in Carbapenem Resistant Escherichia coli Isolates of Animal Origin

Background: The emergence of a multidrug resistance phenotype is most likely outcome of co-existing multiple mechanisms. The underlying molecular mechanism of carbapenem resistance is poorly understood in bacterial isolates of animal origin. The present study investigates the resistance mechanism for reduced susceptibility to carbapenem in E. coli. Methods: The study includes 12 multidrug resistant E.coli isolates showing decreased susceptibility to imipenem. Carbapenemase production was evaluated by the mCarba-NP test. The presence of the carbapenem resistance gene was confirmed by multiplex PCR. Efflux pump activity was accessed by broth microdilution method with or without selective efflux pump inhibitor. We analyzed differential expression of the outer membrane porin genes (ompF, ompC) and efflux pump genes (acrA, acrB) by quantitative real-time PCR analysis. Result: We observed increased expression of acrA and acrB genes in all the E. coli isolates under imipenem stress. A positive correlation (p less than 0.001) was evident between imipenem MIC and acrA and acrB over-expression in these isolates. A significant decrease in ompF and ompC expression was observed in these isolates under imipenem stress. The change in MIC was not correlated with the corresponding OmpC and OmpF gene expression. The results suggest the importance of efflux pump and porins proteins in the emergence of multidrug resistance besides carbapenemase production.

Comparing the activity of novel antibiotic agents against carbapenem-resistant Enterobacterales clinical isolates.

We compared the activity of 8 novel β-lactam and tetracycline-derivative antibiotics against a cohort of clinical carbapenem-resistant Enterobacterales (CRE) isolates and investigated the incremental susceptibility benefit of the addition of an aminoglycoside, fluoroquinolone, or polymyxin to the β-lactam agents to assist with empiric antibiotic decision making. A collection of consecutive CRE clinical isolates from unique patients at 3 US hospitals (2016-2021) was assembled. Broth microdilution was performed to obtain antimicrobial susceptibility testing results. Mechanisms of carbapenem resistance were investigated through short-read and long-read whole-genome sequencing. Of the 603 CRE isolates, 276 (46%) were carbapenemase producing and 327 (54%) were non-carbapenemase producing, respectively. The organisms most frequently identified were Klebsiella pneumoniae (38%), Enterobacter cloacae complex (26%), and Escherichia coli (16%). We obtained the following percent susceptibility to novel β-lactam agents: ceftazidime-avibactam (95%), meropenem-vaborbactam (92%), imipenem-relebactam (84%), and cefiderocol (92%). Aminoglycosides and the polymyxins provided greater incremental coverage as second agents, compared to fluoroquinolones. Amikacin and plazomicin exhibited the greatest additive value. Ceftazidime-avibactam, meropenem-vaborbactam, and cefiderocol were active against 94% of the 220 KPC-producing isolates. Cefiderocol was active against 83% of the 29 NDM-producing isolates. Ceftazidime-avibactam had 100% activity against the 9 OXA-48-like-producing isolates. Tigecycline had the highest activity compared to other tetracyclines against KPC, NDM, or OXA-48-like-producing isolates. Selection among novel agents requires a nuanced understanding of the molecular epidemiology of CRE. This work provides insights into the comparative activity of novel agents and the additive value of a second antibiotic for empiric antibiotic decision making.

Carbapenemase-Encoding Gene Copy Number Estimator (CCNE): a Tool for Carbapenemase Gene Copy Number Estimation.

ABSTRACTCarbapenemase production is one of the leading mechanisms of carbapenem resistance in Gram-negative bacteria. An increase in carbapenemase gene (blaCarb) copies is an important mechanism of carbapenem resistance. No currently available bioinformatics tools allow for reliable detection and reporting of carbapenemase gene copy numbers. Here, we describe the carbapenemase-encoding gene copy number estimator (CCNE), a ready-to-use bioinformatics tool that was developed to estimate blaCarb copy numbers from whole-genome sequencing data. Its performance on Klebsiella pneumoniae carbapenemase gene (blaKPC) copy number estimation was evaluated by simulation and quantitative PCR (qPCR), and the results were compared with available algorithms. CCNE has two components, CCNE-acc and CCNE-fast. CCNE-acc detects blaCarb copy number in a comprehensive and high-accuracy way, while CCNE-fast rapidly screens blaCarb copy numbers. CCNE-acc achieved the best accuracy (100%) and the lowest root mean squared error (RMSE; 0.07) in simulated noise data sets, compared to the assembly-based method (23.4% accuracy, 1.697 RMSE) and the OrthologsBased method (78.9% accuracy, 0.395 RMSE). In the qPCR validation, a high consistency was observed between the blaKPC copy number determined by qPCR and that determined with CCNE. Reverse transcription-qPCR transcriptional analysis of 40 isolates showed that blaKPC expression was positively correlated with the blaKPC copy numbers detected by CCNE (P < 0.001). An association study of 357 KPC-producing K. pneumoniae isolates and their antimicrobial susceptibility identified a significant association between the estimated blaKPC copy number and MICs of imipenem (P < 0.001) and ceftazidime-avibactam (P < 0.001). Overall, CCNE is a useful genomic tool for the analysis of antimicrobial resistance genes copy number; it is available at https://github.com/biojiang/ccne.IMPORTANCE Globally disseminated carbapenem-resistant Enterobacterales is an urgent threat to public health. The most common carbapenem resistance mechanism is the production of carbapenemases. Carbapenemase-producing isolates often exhibit a wide range of carbapenem MICs. Higher carbapenem MICs have been associated with treatment failure. The increase of carbapenemase gene (blaCarb) copy numbers contributes to increased carbapenem MICs. However, blaCarb gene copy number detection is not routinely conducted during a genomic analysis, in part due to the lack of optimal bioinformatics tools. In this study, we describe a ready-to-use tool we developed and designated the carbapenemase-encoding gene copy number estimator (CCNE) that can be used to estimate the blaCarb copy number directly from whole-genome sequencing data, and we extended the data to support the analysis of all known blaCarb genes and some other antimicrobial resistance genes. Furthermore, CCNE can be used to interrogate the correlations between genotypes and susceptibility phenotypes and to improve our understanding of antimicrobial resistance mechanisms.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Isolated from Intensive Care Unit Patients in Jordanian Hospitals.

Acinetobacter baumannii is a common cause of healthcare-associated infections (HAI) worldwide, mostly occurring in intensive care units (ICUs). Extended-spectrum beta lactamases (ESBL)-positive A. baumannii strains have emerged as highly resistant to most currently used antimicrobial agents, including carbapenems. The most common mechanism for carbapenem resistance in this species is β-lactamase-mediated resistance. Carbapenem-hydrolyzing class D oxacillinases are widespread among multidrug-resistant (MDR) A. baumannii strains. The present study was conducted to determine the presence and distribution of blaOXA genes among multidrug-resistant A. baumannii isolated from ICU patients and genes encoding insertion sequence (IS-1) in these isolates. Additionally, the plasmid DNA profiles of these isolates were determined. A total of 120 clinical isolates of A. baumannii from various ICU clinical specimens of four main Jordanian hospitals were collected. Bacterial isolate identification was confirmed by biochemical testing and antibiotic sensitivity was then assessed. PCR amplification and automated sequencing were carried out to detect the presence of blaOXA-51, blaOXA-23, blaOXA-24, and blaOXA-58 genes, and ISAba1 insertion sequence. Out of the 120 A. baumannii isolates, 95% of the isolates were resistant to three or more classes of the antibiotics tested and were identified as MDR. The most frequent resistance of the isolates was against piperacillin (96.7%), cephalosporins (97.5%), and β-lactam/β-lactamase inhibitor combinations antibiotics (95.8%). There were 24 (20%) ESBL-producing isolates. A co-existence of blaOXA-51 gene and ISAba1 in all the 24 ESBL-producing isolates was determined. In addition, in the 24 ESBL-producing isolates, 21 (87.5%) carried blaOXA-51 and blaOXA-23 genes, 1 (4.2%) carried blaOXA-51 and blaOXA-24, but all were negative for the blaOXA-58 gene. Plasmid DNA profile A and profile B were the most common (29%) in ESBL-positive MDR A. baumannii isolates while plasmid DNA profile A was the most common in the ESBL-negative isolates. In conclusion, there was an increase in prevalence of MDR-A. baumannii in ICU wards in Jordanian hospitals, especially those having an ESBL phenotype. Thus, identification of ESBL genes is necessary for the surveillance of their transmission in hospitals.

Molecular characterization of carbapenem-resistant Pseudomonas aeruginosa isolated from four medical centres in Iran.

Understanding the mechanisms of antibiotic resistance is important for designing new therapeutic options and controlling resistant strains. The goal of this study was to look at the molecular epidemiology and mechanisms of resistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from Tabriz, Iran. One hundred and forty P. aeruginosa were isolated and antibiotic susceptibility patterns were determined. Overproduction of AmpC and efflux pumps were discovered using phenotypic techniques. Polymerase chain reaction (PCR) was used to determine the presence of carbapenemase-encoding genes. In addition, the expressions of OprD and efflux pumps were evaluated by the Real-Time PCR. Random amplified polymorphic DNA typing (RAPD) was performed for genotyping. Among 140 P. aeruginosa isolates, 74 (52.8%) were screened as CRPA. Overexpression of efflux systems was observed in 81% of isolates, followed by decreased expression of OprD (62.2%), presence of carbapenemase genes (14.8%), and overproduction of AmpC (13.5%). In most isolates, carbapenem resistance was multifactorial (60.8%). According to our results, the prevalence of CRPA is at alarming levels. Overexpression of efflux systems was the most common mechanism of carbapenem resistance. Most isolates may originate in patients themselves, but cross-infection is possible. Therefore, we suggest a pattern shift in the strategy of CRPA in our setting.

Risk Factors for the Development of Colistin Resistance during Colistin Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

ABSTRACTColistin is one of the last-resort options for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections if novel antibiotics are unavailable, where the development of colistin resistance during treatment represents a major challenge for clinicians. We aimed to investigate the risk factors associated with the development of colistin resistance in patients with CRKP infections following colistin treatment. We conducted a retrospective case-control study of patients with CRKP strains available before and after colistin treatment at a medical center in Taiwan, between October 2016 and November 2020. Cases (n = 35) included patients with an initial colistin-susceptible CRKP (ColS-CRKP) strain and a subsequent colistin-resistant CRKP (ColR-CRKP) strain. Controls (n = 18) included patients with ColS-CRKP as both the initial and subsequent strains. The 30-day mortality rate after the subsequent CRKP isolation was not different between cases and controls (12/35 [34%] versus 5/18 [28%] [P = 0.631]). blaKPC (n = 38) and blaOXA-48 (n = 11) accounted for the major mechanisms of carbapenem resistance. Alterations in mgrB were found in 18/35 (51%) ColR-CRKP strains, and mcr-1 was not detected in any of the strains. More patients received combination therapy in the control group than in the case group (17/18 versus 21/35 [P = 0.008]). The logistic regression model indicated that combination therapy with tigecycline was protective against the acquisition of colistin resistance (odds ratio, 0.17; 95% confidence interval, 0.05 to 0.62 [P = 0.008]). We observed that the inclusion of tigecycline in colistin treatment mitigated the risk of acquiring colistin resistance. These results offer insight into using the combination of tigecycline and colistin for the treatment of CRKP infections in antimicrobial stewardship.IMPORTANCE Treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is challenging due to the limited options of antibiotics. Colistin is one of the last-resort antibiotics if novel antimicrobial agents are not available. It is crucial to identify modifiable clinical factors associated with the emergence of resistance during colistin treatment. Here, we found that the addition of tigecycline to colistin treatment prevented the acquisition of colistin resistance. Colistin-tigecycline combination therapy is therefore considered a hopeful option in antimicrobial stewardship to treat CRKP infections.

Genome-wide identification of carbapenem-resistant Gram-negative bacterial (CR-GNB) isolates retrieved from hospitalized patients in Bihar, India

Carbapenemase-producing clinical isolates are becoming more common over the world, posing a severe public health danger, particularly in developing nations like India. Carbapenem-resistant Gram-negative bacterial (CR-GNB) infection has become a fast-expanding global threat with limited antibiotic choice and significant mortality. This study aimed to highlight the carbapenem-resistance among clinical isolates of hospital admitted patients in Bihar, India. A cross-sectional study was conducted with 101 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. All GNB isolates were tested for their antimicrobial susceptibility using Kirby-Bauer disc diffusion method. Double disc synergy test / modified Hodge test (DDST/MHT) were used to detect carbapenemase production by these isolates. Subsequently, these isolates were evaluated for carbapenem-resistance genes using whole-genome sequencing method. The overall percentage of carbapenem-resistance among GNB was (17/101) 16.8%. The genomic analysis of antimicrobial-resistance (AMR) demonstrates a significantly high prevalence of blaCTX-M followed by blaSHV, blaTEM, blaOXA, and blaNDM β-lactam or carbapenem resistance genes among clinical isolates of GNB. Co-occurrence of blaNDM with other beta-lactamase-encoding genes was found in 70.6% of carbapenemase-producing isolates. Our study highlights the mechanism of carbapenem-resistance to curb the overwhelming threat posed by the emergence of drug-resistance in India.

Co-occurrence of genes encoding carbapenemase, ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E.coli clinical isolates in Tunisia.

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried blaNDM-1 (n = 11), blaOXA-48 (n = 11), blaNDM-1 + blaOXA-48 (n = 1), blaNDM-1 + blaVIM-1 (n = 1), blaOXA-204 (n = 1), along with blaCTX-M , blaOXA , blaTEM , blaCMY , blaDHA and blaSHV genes variants on conjugative plasmid of IncL/M, IncR, IncFIIK , IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.

Evaluation of In Vitro Efficacy of Ceftazidime-Avibactam, Meropenem, and Colistin Single and Binary Combinations Against Carbapenem Resistant Klebsiella pneumoniae Strains Isolated from Various Clinical Specimens

It has been reported that many antibiotics used today, including the carbapenem group, fail to treat Klebsiella pneumoniae infections effectively. Despite many studies in recent years, the definitive treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is still uncertain. In this study, it was aimed to investigate in vitro activities of colistin (COL) and meropenem (MEM), which are frequently used in the treatment of CRKP infections, and ceftazidime-avibactam (CZA), which is recently used in our country, alone or in combination against different CRKP isolates having different carbapenem resistance mechanisms andto analyze whether the presence of colistin resistance, which is an important problem in CRKP strains, influences the drug interaction results. This study was carried out in 42 K.pneumoniae isolates, which were isolated from various clinical samples as an infectious agent in Süleyman Demirel University Faculty of Medicine, Department of Medical Microbiology, Bacteriology Laboratory and whose carbapenem resistance was confirmed by carbapenemase inactivation test. The carbapenemase genes of the isolates were determined by the polymerase chain reaction (PCR) method. Antimicrobial susceptibilities of CRKP strains to CZA, MEM, and COL were determined by the broth microdilution method and in vitro synergy activities of dual combinations of these drugs were evaluated by checkerboard and time-kill methods. Statistical evaluation of categorical data was performed using Fisher's exact test, and p-value of less than 0.05 was considered statistically significant in terms of difference between the groups. Of the 42 CRKP isolates 34 (81%) were only OXA-48 positive, 5 (11.9%) were OXA-48+NDM and 3 (7.1%) were OXA-48+KPC positive. In the checkerboard test, synergy was detected against 97.6% of the isolates both with CZA+MEM and CZA+COL combinations, whereas this rate was 50% with MEM+COL. In the time-kill test, synergy was detected with CZA+MEM and CZA+COL combinations in the OXA-48 positive isolate and OXA-48+KPC positive isolate, while synergy was detected with CZA+COL and MEM+COL combinations in the OXA-48+NDM positive isolate. There was no significant relationship between whether the isolates were resistant to colistin or not and the checkerboard test results of antibiotic combinations (pCZA+MEM= 0.33, pCZA+COL= 0.11, pMEM+COL= 0.61). Results of our study revealed that the most common carbapenemase type in CRKP isolates was OXA-48 in our hospital, and the combinations of CZA with MEM and COL had high potential for synergism against these isolates.

In vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii isolated from the urinary tract

The old antimicrobial nitroxoline is currently repurposed for oral treatment of uncomplicated urinary tract infections (UTIs). To investigate the in vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii (CRAb). From an international collection of previously well-characterized clinical A. baumannii isolates, 34 isolates from urinary tract sources with different carbapenem-resistance mechanisms were selected. Nitroxoline activity was analysed with broth microdilution (BMD), disc diffusion (DD) and within an in vitro biofilm model. MICs of meropenem and imipenem were assessed with BMD. Susceptibility to ciprofloxacin and trimethoprim/sulfamethoxazole was investigated using DD. Escherichia coli ATCC 25922 and A. baumannii NCTC 13304 were used for quality control. All isolates were carbapenem resistant (MIC90 >32 mg/L for meropenem and imipenem) and most isolates were resistant to ciprofloxacin (33/34) and trimethoprim/sulfamethoxazole (31/34). Nitroxoline yielded MIC50/90 values of 2/2 mg/L (MIC range 1-2 mg/L) and inhibition zone diameters ranging from 20 to 26 mm. In contrast, for definite eradication of biofilm-associated CRAb in vitro, higher nitroxoline concentrations (≥16 to ≥128 mg/L) were necessary for all isolates. Nitroxoline showed excellent in vitro activity against a collection of CRAb despite high resistance rates to other antimicrobials for parental and oral therapy of A. baumannii UTI. Currently, nitroxoline is recommended for the treatment of uncomplicated UTI in Germany with a EUCAST breakpoint limited to uncomplicated UTI and E. coli (S ≤16 mg/L). Nitroxoline could be a promising drug for oral treatment of lower UTI caused by CRAb. More data are warranted to correlate these findings with in vivo success rates.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms.MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years.ResultsOur CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment.ConclusionsCollectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Conventional and Real-Time PCR Targeting blaOXA Genes as Reliable Methods for a Rapid Detection of Carbapenem-Resistant Acinetobacter baumannii Clinical Strains.

Multidrug-resistant Acinetobacter baumannii, particularly those producing carbapenemases, are spread worldwide. A reliable detection of carbapenemases is essential to choose the appropriate antimicrobial therapy and, consequently, prevent the dissemination of carbapenem-resistant strains. The aim of this study is to examine the molecular basis of the carbapenem resistance mechanism and estimation of conventional PCR and real-time PCR usefulness for the detection of oxacillinases when compared to phenotypic carbapenemases detection. The following methods were evaluated: the CarbAcineto NP test, Carbapenem Inactivation Method, CPO panels of semiautomated antimicrobial susceptibility testing method on the BD Phoenix™ M50 system, conventional Polymerase Chain Reaction and real-time PCR. The eazyplex® SuperBug complete A assay was used as the reference method. Among the tested strains, 39 (67.2%) carried the blaOXA-40 gene, while the blaOXA-23 gene was noted amongst 19 (32.8%) isolates. The diagnostic sensitivities of the studied assays were as follows: CarbAcineto NP—65.5%; CIM—100%; CPO—100%; conventional PCR—100%; real-time PCR—100%.

Mechanisms of Action of Carbapenem Resistance.

Carbapenem antibiotics are the most effective antimicrobials for the treatment of infections caused by the most resistant bacteria. They belong to the category of β-lactams that include the penicillins, cephalosporins, monobactams and carbapenems. This class of antimicrobials has a broader spectrum of activity than most other beta-lactams antibiotics and are the most effective against Gram-positive and Gram-negative bacteria. All β-lactams antibiotics have a similar molecular structure: the carbapenems together with the β-lactams. This combination gives an extraordinary stability to the molecule against the enzymes inactivating the β-lactams. They are safe to use and therefore widespread use in many countries has given rise to carbapenem resistance which is a major global public health problem. The carbapenem resistance in some species is intrinsic and consists of the capacity to resist the action of antibiotics with several mechanisms: for the absence of a specific target, or an intrinsic difference in the composition of cytoplasmatic membrane or the inability to cross the outer membrane. In addition to intrinsic resistance, bacteria can develop resistance to antibiotics with several mechanisms that can be gathered in three main groups. The first group includes antibiotics with poor penetration into the outer membrane of bacterium or antibiotic efflux. The second includes bacteria that modify the target of the antibiotics through genetic mutations or post-translational modification of the target. The third includes bacteria that act with enzyme-catalyzed modification and this is due to the production of beta-lactamases, that are able to inactivate carbapenems and so called carbapenemases. In this review, we focus on the mode of action of carbapenem and the mechanisms of carbapenem resistance.