Imipenem Minimum Inhibitory Concentrations Research Articles

Investigation of antimicrobial susceptibility and resistance gene prevalence in Capnocytophaga spp. isolated from dogs and cats and characterization of novel class A β-lactamase CST-1.

Capnocytophaga spp., common inhabitants of the animal oral cavity, are zoonotic pathogens transmitted to humans through dog/cat bites and cat scratches. Appropriate antimicrobial therapy is essential for treatment this zoonotic disease because of the rapid deterioration of systemic symptoms at disease onset; however, antimicrobial resistance of animal bite-associated Capnocytophaga spp. has not been fully investigated. We sought to understand the antimicrobial susceptibility and prevalence of resistance genes among Capnocytophaga sp. isolates obtained from dogs and cats. Minimum inhibitory concentrations (MICs) of nine antibiotics for a total of 57 isolates belonging to 6 species (C. canimorsus, C. cynodegmi, C. canis, C. felis, C. stomatis, and C. catalasegens) were assayed using E-test. Resistance genes were detected using polymerase chain reaction, nucleotide sequencing, and whole-genome sequencing. The MICs of penicillin, ceftriaxone, cefepime, clindamycin, minocycline, nalidixic acid, and ciprofloxacin were high for some isolates. The MICs of imipenem and amoxicillin/clavulanic acid were low for all isolates. Known resistance genes blacfxA2, blaOXA-347, emrF, and tetQ were detected using polymerase chain reaction. Mutation in the quinolone resistance-determining region of gyrA was also detected. Cst-1, a previously unreported gene, was identified using whole-genome analysis of two C. stomatis isolates. CST-1 was proposed as a class A, subclass A2, β-lactamase based on amino acid sequence and phylogenetic relationship. In recombination experiments, CST-1 inactivated penicillin and first- and second-generation cephems; however, sulbactam inhibited it. Known and novel resistance genes are prevalent among Capnocytophaga spp. in animal oral cavities. The findings have clinical implications, especially in antimicrobial treatment.

Molecular detection of extended-spectrum β-lactamase- and carbapenemase-producing Klebsiella pneumoniae isolates in southwest Iran.

The global emergence of carbapenem-resistant Klebsiella pneumoniae is considered a significant contemporary concern., as carbapenems are the last resort for treating infections caused by multidrug-resistant Gram-negative bacteria. This study aimed to investigate the carbapenem-resistance genes in extended-spectrum β-lactamase producing K. pneumoniae isolates. Seventy-five non-duplicate clinical K. pneumoniae strains were isolated from urine, blood, sputum, and wound samples. Antimicrobial susceptibility tests for 12 different antibiotics were performed using the disk diffusion method, followed by determining minimum inhibitory concentrations of imipenem and meropenem. Phenotypic detection of extended-spectrum β-lactamase and carbapenemase enzymes was performed by double-disc synergy test and modified Hodge test, respectively. PCR assay further investigated resistant isolates for extended-spectrum β-lactamase and carbapenemase-encoding genes. The highest and lowest resistance rates were observed against ampicillin (93.3%) and tigecycline (9.3%). According to phenotypic tests, 46.7% of isolates were positive for extended-spectrum β-lactamase enzymes and 52.8% for carbapenemase. A total of 11 isolates contained carbapenemase genes, with blaOXA-48 (19.4%; 7/36) being the predominant gene, followed by blaNDM (8.3%; 3/36). The study's findings reveal the alarming prevalence of beta-lactamase enzymes in K. pneumoniae strains. Early detection of carbapenem-resistant isolates and effective infection control measures are necessary to minimise further spread, as carbapenem resistance has become a public health concern.

Molecular epidemiology of Carbapenemase-encoding genes and comparative evaluation of carbapenem MIC with genotypic carbapenem resistance in Klebsiella isolates from neonatal sepsis cases

Objectives: The objective of this study was to determine the molecular epidemiology of Carbapenemase-encoding genes in Klebsiella isolates from neonatal sepsis cases and comparative evaluation of carbapenem minimum inhibitory concentration (MIC) with genotypic carbapenem resistance. Materials and Methods: One hundred cases of neonatal sepsis with blood cultures positive for Klebsiella spp. were included in the study. MIC for imipenem and meropenem was determined by Epsilometer-test. Antimicrobial susceptibility testing (AST) was performed by modified Kirby Bauer disc diffusion method. All the isolates of Klebsiella spp. were tested for the presence of beta-lactamase Klebsiella pneumoniae carbapenemase (blaKPC ), beta-lactamase New Delhi metalloβ-lactamase-1(blaNDM-1), beta-lactamase imipenemase (blaIMP), beta-lactamase Verona imipenemas e (blaVIM) genes by multiplex polymerase chain reaction (PCR) and uniplex PCR for beta-lactamase oxacillinase-48 (blaOXA-48). Comparison of individual antibiotic susceptibility between carbapenemase-encoding gene positive and negative Klebsiella spp. isolates was performed. Statistical analysis: Statistical analysis was done using the Fisher’s exact test. P < 0.05 was considered significant. Results: The prevalence of carbapenemase-encoding genes in Klebsiella spp. was 16%. Most predominant carbapenemase-encoding gene was blaOXA-48 gene (12%) followed by blaNDM-1 gene (6%). Coexpression of both blaOXA-48 and blaNDM-1 was observed in 2% of isolates. All the Klebsiella spp. isolates harboring the carbapenemases gene (100%) had resistant MIC values for Meropenem, whereas, for imipenem, only 75% of isolates had resistant MIC values. Conclusions: Determination of prevalence of carbapenemase-encoding genes is of paramount importance in the development of effective antibiotic policies at various levels.

The importance of meropenem resistance, rather than imipenem resistance, in defining carbapenem-resistant Enterobacterales for public health surveillance: an analysis of national population-based surveillance

BackgroundIn Japan, carbapenem-resistant Enterobacterales (CRE) infections were incorporated into the National Epidemiological Surveillance of Infectious Diseases (NESID) in 2014, necessitating mandatory reporting of all CRE infections cases. Subsequently, pathogen surveillance was initiated in 2017, which involved the collection and analysis of CRE isolates from reported cases to assess carbapenemase gene possession. In this surveillance, CRE is defined as (i) minimum inhibitory concentration (MIC) of meropenem ≥2 mg/L (MEPM criteria) or (ii) MIC of imipenem ≥2 mg/L and MIC of cefmetazole ≥64 mg/L (IPM criteria). This study examined whether the current definition of CRE surveillance captures cases with a clinical and public health burden.MethodsCRE isolates from reported cases were collected from the public health laboratories of local governments, which are responsible for pathogen surveillance. Antimicrobial susceptibility tests were conducted on these isolates to assess compliance with the NESID CRE definition. The NESID data between April 2017 and March 2018 were obtained and analyzed using antimicrobial susceptibility test results.ResultsIn total, 1681 CRE cases were identified during the study period, and pathogen surveillance data were available for 740 (44.0%) cases. Klebsiella aerogenes and Enterobacter cloacae complex were the dominant species, followed by Klebsiella pneumoniae and Escherichia coli. The rate of carbapenemase gene positivity was 26.5% (196/740), and 93.4% (183/196) of these isolates were of the IMP type. Meanwhile, 315 isolates were subjected to antimicrobial susceptibility testing. Among them, 169 (53.7%) fulfilled only the IPM criteria (IPM criteria-only group) which were susceptible to meropenem, while 146 (46.3%) fulfilled the MEPM criteria (MEPM criteria group). The IPM criteria-only group and MEPM criteria group significantly differed in terms of carbapenemase gene positivity (0% vs. 67.8%), multidrug resistance rates (1.2% vs. 65.8%), and mortality rates (1.8% vs 6.9%).ConclusionThe identification of CRE cases based solely on imipenem resistance has had a limited impact on clinical management. Emphasizing resistance to meropenem is crucial in defining CRE, which pose both clinical and public health burden. This emphasis will enable the efficient allocation of limited health and public health resources and preservation of newly developed antimicrobials.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid β-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-β-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Characterization of blaKPC-2 and blaNDM-1 Plasmids of a K. pneumoniae ST11 Outbreak Clone.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.

Development of a Risk Prediction Model of Subsequent Bloodstream Infection After Carbapenem-Resistant Enterobacteriaceae Isolated from Perianal Swabs in Hematological Patients.

Patients with hematological diseases are at high risk of carbapenem-resistant Enterobacteriaceae (CRE) infection, and CRE-related bloodstream infection (BSI) is associated with high mortality risk. Therefore, developing a predictive risk model for subsequent BSI in hematological patients with CRE isolated from perianal swabs could be used to guide preventive strategies. This was a single-center retrospective cohort study at a tertiary blood diseases hospital, including all hematological patients hospitalized from 10 October 2017 to 31 July 2021. We developed a predictive model using multivariable logistic regression and internally validated it using enhanced bootstrap resampling. Of 421 included patients with CRE isolated from perianal swabs, BSI due to CRE occurred in 59. According to the multivariate logistic analysis, age (OR[odds ratio]=1.04, 95% CI[confidence interval]: 1.01-1.06, P=0.004), both meropenem and imipenem minimal inhibitory concentration (MIC) of the isolate from perianal swabs>8ug/mL (OR=5.34, 95% CI: 2.63-11.5, P<0.001), gastrointestinal symptoms (OR=3.67, 95% CI: 1.82-7.58, P<0.001), valley absolute neutrophil count (109/L)>0.025 (OR=0.07, 95% CI: (0.02-0.19, P<0.001) and shaking chills at peak temperature (OR=6.94, 95% CI: (2.60-19.2, P<0.001) were independently associated with CRE BSI within 30 days and included in the prediction model. At a cut-off of prediction probability ≥ 21.5% the model exhibited a sensitivity, specificity, positive predictive value and negative predictive value of 79.7%, 85.6%, 96.27% and 47.47%. The discrimination and calibration of the prediction model were good on the derivation data (C-statistics=0.8898; Brier score=0.079) and enhanced bootstrapped validation dataset (adjusted C-statistics=0.881; adjusted Brier score=0.083). The risk prediction model is freely available as a mobile application at https://liujia1992.shinyapps.io/dynnomapp/. A prediction model based on age, meropenem and imipenem MIC of isolate, gastrointestinal symptoms, valley absolute neutrophil count and shaking chills may be used to better inform interventions in hematological patients with CRE isolated from perianal swabs.

1421. A 5-year Review of Mycobacterium abscessus Susceptibility in a Tertiary Hospital in Thailand

Abstract Background Mycobacterium abscessus is a common rapid growing mycobacteria (RGM). It causes chronic nodular or cavitary lung disease in adults with bronchiectasis or cystic fibrosis, skin and soft tissue infection following penetrating injury or an unsterile skin procedure. It was also a pathogen of disseminated infection in patients with anti-interferon-gamma autoantibodies. The 2018 clinical and laboratory standards institute (CLSI) guideline suggested that amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, meropenem, moxifloxacin, trimethoprim-sulfamethoxazole, tigecycline and tobramycin should be tested against RGM. We aimed to review the antimicrobial susceptibility of this organism in our hospital. Methods We performed a retrospective descriptive review of the minimal inhibitory concentration (MIC) of clinical isolates of M. abscessus from 2013 to 2018. Results We found 267 isolates of M. abscessus. Eighty-four isolates were tested for MICs. Of these, 4 isolates were excluded due to duplication. The remaining 80 isolates were included for analysis. The susceptibility results were as follows. Conclusion To our knowledge, this is the largest report of susceptibility pattern of M. abscessus in Thailand. MICs of cefoxitin and imipenem, which are the recommended intravenous antimicrobials, were high. Most isolates also demonstrated high MICs of doxycycline, linezolid, trimethoprim-sulfamethoxazole, tobramycin, ciprofloxacin and moxifloxacin. In contrast, most of MICs of amikacin and clarithromycin were in susceptible range. These findings may be used to guide the treatment regimen although clinical outcomes of each drug are still to be investigated. Disclosures All Authors: No reported disclosures.

Screening of Carbapenem Resistance Klebsiella pneumoniae and its MIC against Imipenem

Klebsiella pneumoniae is a common opportunistic pathogen causing a wide range of infections; pneumonia, urinary tract infection, bacteremia, and liver abscesses. It infects primarily immunocompromised and immunocompetent individuals. It presents itself as an antibiotic-resistant bacterium, especially for third-generation cephalosporins and carbapenems, creating serious global challenges. Therefore, this cross-sectional study was conducted in B &amp; B Hospital, Lalitpur to screen the distribution of carbapenem resistance K. pneumoniae through ertapenem and to assess the minimum inhibitory concentration of imipenem for screened carbapenem resistance K. pneumoniae. From 3447 different clinical samples collected according to standard guidelines, K. pneumoniae was identified using standard microbiological techniques; staining and a panel of biochemical tests. The antibiotic susceptibility test of the isolates was performed by the Kirby-Bauer disc diffusion method as per CLSI 2018 guidelines. The screening of carbapenem resistance was assessed by using ertapenem disc and the MIC of imipenem for carbapenem resistance and intermediate was performed using an Epsilometer. A total of 85 K. pneumoniae were identified and their antibiotic susceptibility test revealed that ceftriaxone was the least effective antibiotic. The number of MDR, carbapenem-resistant and intermediate isolates was 51, 46, and 3, respectively. The MIC of imipenem through an Epsilometer from ertapenem resistant and intermediate revealed that 31, 5, and 13 isolates were resistant, intermediate, and sensitive respectively. These findings showed the inconsistency in the detection of carbapenem-resistant isolates in routine microbiology laboratories and further support the other tests for the detection of carbapenem resistance as suggested by CLSI.

Prevalence of faecal carriage of Carbapenemase Producing Enterobacteriaceae in healthy Indian subjects from the community

PurposeFaecal carriage of carbapenemase-producing Enterobacterales (CPE) has been extensively investigated in hospitalized patients, but limited data is available on the carriage rate in healthy individuals in India. MethodsA total of 1000 stool samples were screened for CPE from healthy individuals in Chennai (n ​= ​50), Hyderabad (n ​= ​184) and Mumbai (n ​= ​766). Diluted stool samples were cultured on chromID CARBA SMART plates. Growing colonies were screened for CPE by RAPIDEC® CARBA NP Test and minimum inhibitory concentration (MIC) of imipenem by E-Test. PCR was performed for confirmation of CPE genes. ResultsOut of the 1000 stool samples tested, 6.1% were positive for CPE. A total of 64 carbapenem resistant isolates (56 ​E.coli, 4 Klebsiella pneumoniae, 3 Enterobacter cloacae and 1 Citrobacter freundii) were recovered from ChromID CARBA SMART biplate. Carbapenemase production was identified in 57/64 isolates by RAPIDEC® CARBA NP test. PCR analysis showed 28 blaNDM-1 and 33 blaOXA48. Three remaining isolates (2 ​E.coli, 1 ​K.pneumoniae) were negative for the tested carbapenemase genes. Interestingly, out of these 61 PCR positive isolates, 49.1% displayed imipenem MIC within the susceptibility range on the basis of CLSI interpretative criteria. ConclusionsFaecal carriage of CPE among healthy individuals was 6.1%. Comprehensive measures to improve the sanitation scenario and implementation of National AMR action plan are needed to prevent further generation and dissemination of carbapenem resistant Enterobacterales (CRE).

Epidemiology of carbapenem-resistant and carbapenemase-producing Enterobacterales in the Netherlands 2017\u20132019

BackgroundThe Netherlands is currently considered a low endemic country for carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE), experiencing only sporadic hospital outbreaks. This study aims to describe susceptibility to carbapenems and the epidemiology of carbapenemase production in Enterobacterales in the Netherlands in 2017–2019.MethodsThree complementary nationwide surveillance systems are in place to monitor carbapenem susceptibility in the Netherlands. Routine antimicrobial susceptibility test results from medical microbiology laboratories were used to study phenotypic susceptibility of Escherichia coli and Klebsiella pneumoniae. Pathogen surveillance (of all Enterobacterales species) and mandatory notifications were used to describe the characteristics of CPE positive isolates and affected persons.ResultsThe prevalence of isolates with gradient strip test-confirmed elevated meropenem (> 0.25 mg/L) or imipenem (> 1 mg/L) minimum inhibitory concentration (MIC) in the Netherlands was very low in 2017–2019, with percentages of 0.06% in E. coli and 0.49% in K. pneumoniae, and carbapenem resistances of 0.02% and 0.18%, respectively. A total of 895 unique species/carbapenemase-encoding allele combinations of CPE from 764 persons were submitted between 2017 and 2019, with the annual number of submissions increasing slightly each year. Epidemiological data was available for 660 persons. Screening because of presumed colonisation risk was the reason for sampling in 70.0% (462/660) of persons. Hospitalization abroad was the most common risk factor, being identified in 45.9% of persons.ConclusionsCarbapenem resistance of E. coli and K. pneumoniae remains low in the Netherlands. The annual number of CPE isolates slightly increased during the period 2017–2019. Recent hospitalization abroad is the main risk factor for acquisition of CPE.

The using of the polymerase chain reaction for the detection of resistance genes in gram-negative bacteria in routine practice in a pediatric hospital.

Objective - assessment of RT-PCR for the detection of carbapenem-resistance genes in gram-negative bacteria. A total, 499 strains of gram-negative microorganisms isolated in two pediatric hospitals in 2019-2020 were studied. Species identification was performed using MALDI-ToF mass-spectrometry (Bruker Daltonics, Germany). Meropenem and imipenem minimal inhibitory concentration (MIC) was determined by E-test method (BioMerieux, France). The presence of acquired carbapenemase genes of IMP, NDM, VIM, KPC, OXA-48, OXA-23, OXA-40, OXA-58-groups was determined by RT-PCR. Klebsiella pneumoniae (34%), Escherichia coli (4%), Serratia marcescens (6%) and other members of Enterobacterales (6%), also gram-negative non-glucose-fermenting bacteria Acinetobacter baumannii (14%), Pseudomonas aeruginosa (36%) were found among selected strains. Carbapenemase production was found in 385 isolates (77%). The main mechanism determining carbapenem resistance in P. aeruginosa was the production of blaVIM (100%). A. baumanii strains harbored OXA-23 (55%) and OXA-40 (45%) carbapenemases. The major determinant of carbapenem resistance in K. pneumoniae isolates was OXA-48 carbapenemase, detected in 63% strains, 13% of the strains possessed blaNDM-group, 16% isolates had a combination of blaNDM-group and blaOXA-48-like. Carbapenemase of KPC-group was found in 8% K. pneumoniae strains. OXA-48 carbapenemase prevailed (95%) among S. marcescens strains. Most of E. coli isolates harbored metallo-beta-lactamase NDM (89%). Other members of Enterobacterales most often had OXA-48 carbapenemase (57%), 39% of the isolates carried blaNDM-group. In one strain, a combination of blaNDM-group and blaOXA-48-like was discovered. RT-PCR is a fast and reliable method for the detection of acquired carbapenemases and can be recommended for routine use in bacteriological laboratories.

A retrospective study on molecular epidemiology trends of carbapenem resistant Enterobacteriaceae in a teaching hospital in Malaysia.

BackgroundCarbapenem resistant Enterobacteriaceae (CRE) has rapidly disseminated worldwide and has become a global threat to the healthcare system due to its resistance towards “last line” antibiotics. This study aimed to investigate the prevalence of CRE and the resistance mechanism as well as the risk factors associated with in-hospital mortality.MethodsA total of 168 CRE strains isolated from a tertiary teaching hospital from 2014–2015 were included in this study. The presence of carbapenemase genes and minimum inhibitory concentration of imipenem, meropenem and colistin were investigated. All carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains were characterised by PFGE. The risk factors of patients infected by CRE associated with in-hospital mortality were determined statistically.ResultsThe predominant CRE species isolated was K. pneumoniae. The carbapenemases detected were blaOXA-48, blaOXA-232, blaVIM and blaNDM of which blaOXA-48 was the predominant carbapenemase detected among 168 CRE strains. A total of 40 CRE strains harboured two different carbapenemase genes. A total of seven clusters and 48 pulsotypes were identified among 140 CRKp strains. A predominant pulsotype responsible for the transmission from 2014 to 2015 was identified. Univariate statistical analysis identified that the period between CRE isolation and start of appropriate therapy of more than 3 days was statistically associated with in-hospital mortality.

Outbreak of KPC-producing Klebsiella pneumoniae ST15 strains in a Chinese tertiary hospital: resistance and virulence analyses.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major cause of clinical infection. However, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae ST15 strains are occasionally identified and have seldom been reported to cause hospital outbreaks in PR China.Hypothesis/Gap Statement. In this study, we describe nosocomial outbreaks caused by KPC-producing K. pneumoniae ST15 strains in a Chinese tertiary hospital.Aim. To characterize the molecular relationship, resistance and virulence factors of the 32 KPC-producing K. pneumoniae ST15 strains isolated in a Chinese hospital.Methodology. A total of 102 non-repetitive carbapenem-resistant Enterobacteriaceae (CRE) strains were collected from a Chinese tertiary hospital in 2018. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize the clonal relationship of the K. pneumoniae isolates, and the ST15 strains were selected for further study. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Fifteen carbapenem resistance genes, bla KPC genetic structures and 12 virulence factors were detected by PCR. Whole-genome sequencing (WGS) was performed using next-generation sequencing combined with single-molecule real-time sequencing.Results. Thirty-two K. pneumoniae ST15 strains were characterized, and 31 of them presented a PFGE similarity of >92 %, indicating clonal spread. In 81.3 % (26/32) of strains, the imipenem (IPM) and meropenem (MEM) MICs were ≤8 and≤16 µg ml-1, while only 1 isolate (KP18069) exhibited ≥64 µg ml-1 for both agents. The bla KPC-2 gene embedded in the Tn3-Tn4401 chimaera and synonymous mutations of the ompK35 gene were detected in all the strains. However, a nonsense mutation at amino acid position 248 (K248X) of OmpK36 was found in the highly carbapenem-resistant strain KP18069. No virulence gene was detected in any of the ST15 strains. WGS analyses further confirmed the genetic characteristics of the K. pneumoniae KP18069 strain.Conclusion. Nosocomial outbreaks caused by the clonal spread of K. pneumoniae ST15 strains were characterized in a Chinese tertiary hospital, and strict monitoring of highly resistant CRKP is required.

Comparative 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=183h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64µg 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.

Molecular Epidemiology of Enterobacter cloacae Complex Isolates with Reduced Carbapenem Susceptibility Recovered by Blood Culture.

The Enterobacter cloacae complex (ECC) is one of the most common causes of bacteremia and leads to poor clinical outcomes. The aim of this study was to clarify the antimicrobial susceptibility profiles and genetic backgrounds of non-carbapenemase-producing reduced-carbapenem-susceptible (RCS) ECC blood isolates in Japan using agar dilution antimicrobial susceptibility testing, whole-genome sequencing, and quantitative polymerase chain reaction for ampC, ompC, and ompF transcripts. Forty-two ECC blood isolates were categorized into RCS and carbapenem-susceptible groups based on the minimum inhibitory concentration of imipenem. The RCS ECC blood isolates belonged to distinct species and sequence types and produced varying class C β-lactamases. The E. roggenkampii, E. asburiae, and E. bugandensis isolates belonged only to the RCS group. Some E. hormaechei ssp. steigerwaltii isolates from the RCS group exhibited AmpC overexpression caused by amino acid substitutions in AmpD and AmpR along with ompF downregulation. These findings suggest that non-carbapenemase-producing RCS ECC blood isolates are genetically diverse.

Virulence Characteristics of Biofilm-Forming Acinetobacter baumannii in Clinical Isolates Using a Galleria mellonella Model.

Acinetobacter baumannii is a Gram-negative coccobacillus responsible for severe hospital-acquired infections, particularly in intensive care units (ICUs). The current study was designed to characterize the virulence traits of biofilm-forming carbapenem-resistant A. baumannii causing pneumonia in ICU patients using a Galleria mellonella model. Two hundred and thirty patients with hospital-acquired or ventilator-associated pneumonia were included in our study. Among the total isolates, A. baumannii was the most frequently isolated etiological agent in ICU patients with pneumonia (54/165, 32.7%). All A. baumannii isolates were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, while the minimum inhibitory concentrations of imipenem and colistin were estimated using the broth microdilution technique. The biofilm formation activity of the isolates was tested using the microtiter plate technique. Biofilm quantification showed that 61.1% (33/54) of the isolates were strong biofilm producers, while 27.7% (15/54) and 11.1% (6/54) showed moderate or weak biofilm production. By studying the prevalence of carbapenemases-encoding genes among isolates, blaOXA-23-like was positive in 88.9% of the isolates (48/54). The BlaNDM gene was found in 27.7% of the isolates (15/54 isolates). BlaOXA-23-like and blaNDM genes coexisted in 25.9% (14/54 isolates). Bap and blaPER-1 genes, the biofilm-associated genes, coexisted in 5.6% (3/54) of the isolates. For in vivo assessment of A. baumannii pathogenicity, a Galleria mellonella survival assay was used. G. mellonella survival was statistically different between moderate and poor biofilm producers (p < 0.0001). The killing effect of the strong biofilm-producing group was significantly higher than that of the moderate and poor biofilm producers (p < 0.0001 for each comparison). These findings highlight the role of biofilm formation as a powerful virulence factor for carbapenem-resistant A. baumannii that causes pneumonia in the ICU.

IN VITRO ACTIVITIES OF POLYMYXINS GROUP COMBINED WITH IMIPENEM OR MEROPENEM AGAINST CARBAPENEM-RESISTANT PSEUDOMONAS AERUGINOSA

The evaluation of the combined effects between the antibiotics against P. aeruginosa is among the efforts to counteract its antimicrobial resistance. This study aimed to evaluate the possible combined effect of colistin or polymyxin-B with carbapenem antibiotic group against clinical isolates of multi-drug resistant P. aeruginosa. Strains were isolated from biological samples of hospitalized and non-hospitalized patients with any type of infection related to this pathogen. Only carbapenem-resistant strains were included in the study. After determining the minimum inhibitory concentration (MIC) of antibiotics against the strains by broth microdilution test, the checkerboard method was used for evaluation of any possible effect of both colistin or polymyxin-B with imipenem or meropenem. Fifty-nine strains of reduced susceptibility to carbapenems were recovered. It was noted that (45.7%) of strains had an imipenem MIC=1024 µg/ml and (28.8%) had a meropenem MIC=512 µg/ml. The checkerboard technique demonstrated that the ten representative strains showed reductions in their minimal inhibitory concentration (MIC) for at least one of the antibiotics in the combinations evaluated. These results from the in vitro evaluation suggest that combinations of colistin and polymyxin-B may significantly reduce the MICs of the carbapenem antibiotics tested.

Prevalence of Carbapenemase Encoding Genes and Antimicrobial Resistance Pattern of Enterobacteriaceae Isolated from Hospitalized Patients in Khartoum State, Sudan

Carbapenem-resistant Enterobacteriaceae strains have been responsible for an increasing number of hospital-acquired infections globally. This study aimed to determine the prevalence of carbapenemase-producing Enterobacteriaceae and their frequency of antimicrobial-resistant patterns among hospitalized patients across three Khartoum State Teaching Hospitals, Sudan. Materials and Methods. A cross-sectional study was conducted at Khartoum State Teaching Hospitals from April 2018 to October 2019. A total of 384 non-duplicative Enterobacteriaceae strains were isolated from1062 clinical samples obtained from hospitalized patients receiving treatment across three main teaching hospitals. The samples were cultured into a MacConkey agar plate. The Enterobacteriaceae strains were differentiated by specific colony color and again by biochemical test. Antimicrobial susceptibility testing was performed by the disc diffusion method. The minimum inhibitory concentration (MIC) of imipenem and meropenem was performed by the agar dilution method. Multiplex polymerase chain reaction (PCR) was performed to investigate the presence of carbapenemase-encoding genes. Data analysis was carried out using SPSS version 21. Results. Of the 36.2% (384/10.62) nonduplicate of Enterobacteriaceae strains isolated from clinical samples, 122 (31.8%) were carbapenemase-producing Enterobacteriaceae (CPE). Of these isolates, 37 (30.3%) harbored the blaIMP followed by; 29 (23.8%) blaNDM, 21 (17.2%) blaOXA-48, 6 (4.9%) blaGES, 5 (4.1%) were blaKPC, 3(2.5%) blaGIM-1, 2(1.6%) blaVIM and 1 (0.8%) blaSIM-1, while the remaining 19(15.6%) isolates carried combinations carbapenemase blagenes. The most predominant CPE strains were Escherichia coli 40 (32.8%) followed by Klebsiella pneumoniae 24 (19.7%) and Enterobacter aerogenes 14(11.5%). Most of the CPE isolates were isolated from wound swab 40(32.8), sputum 33(27.0), and urine 22(18.0) samples. Furthermost strains showed high resistance rates (>70%) to the antibiotics tested. Resistance to amikacin, tetracycline, co-trimoxazole, and nalidixic acid was 36.9%, 43.4%, 62.3%, and 63.9%, respectively and 82.8 % of CPE strains were susceptible to colistin. The detection of blagenes carbapenemases in CPE strains had a significant effect on both imipenem and meropenem MICs. Conclusion. The most prevalent carbapenemase-producing blagenes among clinical Enterobacteriaceae clinical isolates from the three Khartoum state regions were blaIMP, blaNDM, and blaOXA-48. In contrast, the propensity of the multidrug-resistant profile that has been associated with producing carbapenemase blagenes is alarming. Therefore, it is very important to establish a routine screening of carbapenemase-producing blagenes in clinical isolates to prevent the dissemination of resistant strains among both inpatients and outpatients in hospital settings.

Biosynthesis of Zinc Oxide Nanoparticles from Acacia nilotica (L.) Extract to Overcome Carbapenem-Resistant Klebsiella Pneumoniae.

Recently, concerns have been raised globally about antimicrobial resistance, the prevalence of which has increased significantly. Carbapenem-resistant Klebsiella pneumoniae (KPC) is considered one of the most common resistant bacteria, which has spread to ICUs in Saudi Arabia. This study was established to investigate the antibacterial activity of biosynthesized zinc oxide nanoparticles (ZnO-NPs) against KPC in vitro and in vivo. In this study, we used the aqueous extract of Acacia nilotica (L.) fruits to mediate the synthesis of ZnO-NPs. The nanoparticles produced were characterized by UV-vis spectroscopy, zetasizer and zeta potential analyses, X-ray diffraction (XRD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The antimicrobial activity of ZnO-NPs against KPC was determined via the well diffusion method, and determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the results showed low MIC and MBC when compared with the MIC and MBC of Imipenem and Meropenem antibiotics. The results of in vitro analysis were supported by the results upon applying ZnO-NP ointment to promote wound closure of rats, which showed better wound healing than the results with imipenem ointment. The biosynthesized ZnO-NPs showed good potential for use against bacteria due to their small size, applicability, and low toxicity to human cells.