Modified Carbapenem Inactivation Method Research Articles

Four Types of ST11 Novel Mutations From Increasing Carbapenem-Resistant Klebsiella pneumoniae in Guangdong, 2016-2020.

Objectives: This study aimed to explore changes in carbapenem-resistant Klebsiella pneumoniae (CR-KP) isolates collected in Guangdong over the period of 2016–2020.Methods: Antibacterial susceptibility was quantified through VITEK 2 compact and K-B method. Carbapenemase phenotypes and genotypes were characterized by modified carbapenem inactivation method (mCIM), EDTA-carbapenem inactivation method (eCIM), and polymerase chain reaction (PCR). Molecular characteristics and evolutionary trends were analyzed by multilocus sequence typing and evolutionary tree.Results: Isolates (2,847) of K. pneumoniae were separated in 2016–2020, and the separate rate of CR-KP increased from 5.65 to 9.90% (p = 0.009). The top 3 wards were intensive care unit (ICU) (21.92%), neonatal wards (13.70%), and respiratory wards (12.33%). In 146 CR-KP strains, serine carbapenemase was the main phenotype, and KPC was the main genotype, and 57 contained two resistant genes, and 1 contained three resistant genes. Two polygenic strains were first found: IMP + GES and KPC + NDM + VIM, but all the phenotypes were metalloenzyme, which indicated that metalloenzyme was usually the first choice for CR-KP resistance. In addition, all the ST54 of metalloenzyme type contained IMP, and all the ST45, ST37, and ST76 contained OXA. ST11 was the most prevalent (42.47%); ST11 and its mutants proved the predominant sequence type making up 51.1% of the carbapenemase-producing isolates. A novel type of ST11 mutation, the rpoB was mutated from sequence 1 to sequence 146, was in an independent separate branch on the evolutionary tree and was resistant to all antibacterial agents. The other three mutants, rpoB 1–15, infB 3–148, and infB 3–80, are also resistant to all antibacteria. Of note, all the four mutants produced serine carbapenemase and contained KPC, and indicated that the prevalent strain in China, ST11, has serious consequences and potential outbreaks.Conclusion: The infection rate of CR-KP has increased, and ICU and neonatal wards have become the key infection areas. Producing serine enzyme, the KPC genotype, and ST11 are the predominant CR-KP. Polygenic strains and ST11 mutation made clinical treatment difficult and may become a potential threat.

Investigation of Extended-Spectrum β-Lactamase and Carbapenemase Producing Gram-Negative Bacilli in Rectal Swabs Collected from Neonates and Their Associated Factors in Neonatal Intensive Care Units of Southern Ethiopia.

BackgroundCurrently extended-spectrum β-lactamase (ESβL) and carbapenemase producing gram-negative bacteria are the greatest concern among the neonatal population with very limited therapeutic options. The aim of this study was to assess the prevalence of ESβL and carbapenemase producing gram-negative bacilli, associated factors and antimicrobial resistance patterns among neonates in intensive care units.MethodsAn institutional-based cross-sectional study was conducted from February to June 2021 on 212 neonates in intensive care units. Risk factors data were collected by using a well-designed questionnaire. A rectal swab sample was collected using a sterile cotton swab and inoculated on MacConkey agar. Bacterial isolates were identified using various biochemical tests. ESβL and carbapenemase were first screened by indicator cephalosporins (cefotaxime (30µg) and ceftazidine (30µg)) and carbapenem (meropenem and ertapenem), respectively. ESβL and carbapenemase were confirmed by a double-disk synergy test and modified carbapenem inactivation methods, respectively. SPSS version 21.0 was used for data analysis. A P-value ≤ 0.05 was considered as statistically significant.ResultsThe overall prevalence of ESβL-producing gram-negative bacilli was 72/212 (34%). The predominant ESβL-producing isolate was Klebsiella pneumoniae 23/72 (31.9%) followed by Escherichia coli 17/72 (23.6%). Five (2.4%) carbapenemase-producing gram-negative bacilli were isolated. ESβL-producing isolates showed a high resistance against ampicillin 72/72 (100%), augmentin 69/72 (95.8%) and gentamycin 57/72 (79.2%). The majority 63/72 (87.5%) of isolated ESβL-producing gram-negative bacilli were multi-drug resistant (MDR). Rectal carriage of ESβL by neonates showed a statistically significant association with endotracheal intubation (p = 0.001; AOR = 4.2; 96% CI = (1.8–9.5)), treatment with ampicillin+gentamycin (p = 0.004; AOR = 3.3; 95% CI = (1.5–7.6)) and staying in a neonatal intensive care unit (NICU) between 11 and 20 days (p = 0.042; AOR = 2; 95% CI = (1.0–4.5)).ConclusionA high prevalence of ESβL-producing bacterial isolates was observed for commonly used antibiotics which needs further attention. Therefore, continuous and regular follow-ups of drug resistance patterns is important for the proper treatment and management of ESβL and carbapenemase producing gram-negative bacilli.

Emergence of blaNDM-1 and blaVIM producing Gram-negative bacilliin ventilator-associated pneumonia at AMR Surveillance Regional Reference Laboratory in India.

IntroductionVentilator-associated pneumonia (VAP) may be a life threatening nosocomial infection encountered in intensive care units. Currently the emergence of carbapenem-resistant Gram-negative pathogens has become worrisome threat worldwide.Material and methodsEndotracheal aspirates samples were collected from patients who were under mechanical ventilation for > 48 h. The bacterial isolates were identified by MALDI-TOF-MS and antibiotic susceptibility testing performed. All carbapenem resistant isolates were tested by Modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and EDTA-CIM (eCIM) and PCR were performed to detect blaIMP, blaVIM and blaNDM producing MBL genes.ResultsVAP occurred in 172/353(48.7%), 23.3% had early-onset VAP and 76.7% had late-onset VAP. Males (69.2%) were found to suffer more from VAP. Prior antibiotic therapy, CPI>6, prior surgery and tracheostomy were associated with VAP. The mortality in VAP (58.1%) contrasted with non-VAP (40%). 99/169 (58.6%) Gram-negative isolates were resistant to carbapenems. Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were common pathogens found in late onset VAP, whereas K. pneumoniae, A. baumannii and Staphylococcus aureus were common in early onset VAP. The PCR results detected blaNDM in 37/172(21.5%) and blaVIM in 30/172(17.4%); 15/172(8.7%) isolates carried both genes.ConclusionThe blaNDM-1 and blaVIM genes are the main antibiotic-resistance genes that induce resistance patterns to carbapenems in VAP, highlighting CRE strains of potential public health concern and therapeutic challenge. Diagnostic laboratories in India must get on high caution for early MBL detection as it may limit the wide dispersal of MBL genes.

Application of Modified Carbapenem Inactivation Method and Its Derivative Tests for the Detection of Carbapenemase-Producing Aeromonas.

PurposeInfection and transmission of carbapenem-resistant Aeromonas is a serious threat to public health. Rapid and accurate detection carbapenem-resistant of these organisms is essential for reasonable treatment and infection control. This study aimed to find a simple and effective method to detect carbapenem-resistant phenotype in Aeromonas.MethodsA total of 131 clinical preserved Aeromonas strains were used in this study. The carbapenemase genes were detected by PCR. Modified carbapenem inactivation method (mCIM) in conjunction with EDTA-modified carbapenem inactivation method (eCIM) and simplified carbapenem inactivation method (sCIM) were performed to detect carbapenemases. We also designed a simple method, carbapenem inactivation method using supernatant (CIM-s), to detect the carbapenemase activity in the medium.ResultsOf the 131 Aeromonas strains, 79 contained carbapenemase genes, including 68 blaCphA, 6 blaKPC-2, 2 blaNDM-1 and 3 blaKPC-2+CphA. However, routine antibiotic susceptibility testing could not completely identify carbapenemase-producing Aeromonas. In phenotypic assays, the sensitivity and specificity of mCIM were 100%. The combined mCIM and eCIM could distinguish serine carbapenemase and metallo-β-carbapenemases except co-producing organisms. The sensitivity and specificity of sCIM were 92.4% and 100%, respectively, which could not detect CphA totally. CIM-s results indicate that these carbapenemases could secrete into the medium to perform their hydrolytic activities and had a sensitivity and specificity of 97.5% and 100%, respectively.ConclusionThe combination of mCIM and eCIM can effectively detect and distinguish different types of carbapenemase in Aeromonas, and could be used as an important supplement approach to the antibiotic susceptibility testing.

Occurrence of Multiple, Extensive and Pan Drug-Resistant Pseudomonas aeruginosa and Carbapenemase Production from Presumptive Isolates Stored in a Biobank at Ethiopian Public Health Institute.

PurposePseudomonas aeruginosa is a common cause of nosocomial infections with associated morbidity and mortality because the organism is unresponsive to commonly available antimicrobials. This study was undertaken to determine the multiple drug-resistant (MDR), extensive drug-resistant (XDR) and pan drug-resistant (PDR) phenotype of P. aeruginosa and its carbapenemase production rate from presumptive isolates stored in the biobank at the Ethiopian Public Health Institute (EPHI).MethodsA cross-sectional study was conducted at the EPHI laboratory, Addis Ababa, Ethiopia from March to June 2021. Stored isolates of Pseudomonas spp. which had been characterized by manual identification methods were further processed for species-level identification (ID) and antimicrobial susceptibility testing (AST) using a Becton Dickinson (BD) Phoenix automated system. The isolates were analyzed for carbapenemase enzyme production using the modified Carbapenem Inactivation Method (mCIM). The data analysis was done using SPSS version 20 software.ResultsIn this study, 110 presumptive Pseudomonas isolates from a biobank were re-analyzed, 100 of them were found to be Pseudomonas and among these P. aeruginosa accounted for 98% and P. putida accounted for 2%. The majority of isolates were recovered from wound (46%) specimens followed by ear swabs (18%). The highest level of resistance was observed against ceftazidime (35%) and the lowest level of resistance was observed against amikacin (2%). Twenty-seven isolates were identified as candidates for carbapenemase enzyme production testing, of which only 3/27 (11%) isolates were detected as carbapenemase enzyme producers.ConclusionThis study shows an increasing rate of MDR and XDR isolates and the appearance of PDR in P. aeruginosa strains; this is a serious problem in Ethiopia. The lack of newer anti-pseudomonal antibiotics adds to the problem. In order to alleviate this, infection prevention activities should be promoted, and treatment of bacterial infections should be guided by antibiotic susceptibility test results.

Insights on carbapenem-resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is ubiquitous in nature, and may be a causative agent in severe, life-threatening infections. In >60% of cases, β-lactam antibiotics are used in the therapy of P. aeruginosa infections, therefore the emergence of carbapenem-resistant P. aeruginosa (CRPA) is a significant clinical concern. In this study, phenotypic methods were used to characterize fifty-four (n = 54) P. aeruginosa isolates, which were included based on their suspected non-susceptibility to meropenem. Minimum inhibitory concentrations (MICs) of meropenem, ceftazidime, cefepime, ciprofloxacin, gentamicin, were determined using E-tests, while colistin MICs were determined using broth microdilution. The isolates were subjected to the modified Hodge test (MHT), the modified carbapenem-inactivation method (mCIM) and the imipenem/EDTA combined disk test (CDT). AmpC and efflux pump overexpression was studied using agar plates containing cloxacillin and phenylalanine-arginine β-naphthylamide (PAβN), respectively. Assessment of biofilm-formation was carried out using the crystal violet tube-adherence method. 38.9% of the strains showed meropenem MICs in the resistant range (>8 mg/L). Efflux-pump overexpression and AmpC-hyperproduction was seen in 44.4% and 35.2% of isolates, respectively. 88.8% of the isolates were characterized as strong biofilm-producers. On the other hand, the presence of carbapenemases was suspected in a minority (16.7%) of tested isolates. As safe and effective therapeutic options in carbapenem-resistant Gram-negative infections are severely limited, characterization of these isolates using phenotypic and molecular-based methods is important to provide insights into the epidemiological features of these pathogens.

Insights on carbapenem-resistant Acinetobacter baumannii

Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen, which may be a causative agent in a wide-range of human pathologies. Carbapenems are usually considered the last safe and effective choice of drugs for the treatment of Gram-negative infections. The emergence of carbapenem-resistant A. baumannii (CRAB) is a critical public health issue as they leave clinicians with limited therapeutic options. In this study, phenotypic methods were used to characterize sixty-two (n = 62) A. baumannii isolates, which were included based on their suspected non-susceptibility to meropenem. Minimum inhibitory concentrations (MICs) of meropenem, levofloxacin, gentamicin, sulfamethoxazole/trimethoprim, tigecycline were determined using E-tests, while colistin MICs were determined using broth microdilution. The isolates were subjected to the modified Hodge test (MHT), the modified carbapenem-inactivation method (mCIM) and the imipenem/EDTA combined disk test (CDT). Efflux pump overexpression was studied using agar plates containing phenylalanine-arginine β-naphthylamide (PAβN). Assessment of biofilm-formation was carried out using the crystal violet tube-adherence method. 64.5% of the strains showed meropenem MICs in the resistant range (>8 mg/L), resistance rates were similarly high to the other tested antibiotics. The MHT and mCIM assay were positive in 79.0% and 67.7% of cases, respectively; the presence of an MBL was suggested for 29.0% of isolates. Efflux-pump overexpression was seen in 12.9% of isolates. 54.8% of the isolates were characterized as strong biofilm-producers. Microbiology laboratories have an important role in differentiating the distinct mechanisms by which these pathogens develop the CRAB phenotype, as plasmid-borne carbapenemases are significant from the standpoint of public health microbiology.

Virulence characterization and clonal analysis of uropathogenic Escherichia coli metallo-beta-lactamase-producing isolates

BackgroundUropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL.MethodsA cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR.ResultsAmong 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments.ConclusionMBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1

PurposeCarbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India. MethodsFifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis. ResultsIsolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 ​% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 ​h detected 92.3 ​% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 ​h at cut off 17 ​mm while for NDM-1 CIM was the test of choice at 16 ​mm. ConclusionCIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 ​h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 ​mm was considered adequate for detection of OXA-48-like and 16 ​mm of NDM-1 by mCIM at 4 ​h and CIM respectively.

Carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter species at a large tertiary referral hospital in Lusaka, Zambia

BackgroundPseudomonas aeruginosa (P. aeruginosa) and Acinetobacter species and are among the leading causes of hospital-acquired infections (HAI). Treating patients with HAI caused by these pathogens is usually a challenge due to intrinsic and acquired resistance to the commonly used and affordable antibiotics, which has been heightened by the emergence of metallo-β lactamases thereby exhibiting resistance to carbapenems. Currently, carbapenem resistance represents an increasing problem in the health care settings globally but there is still a paucity of data on carbapenem resistance in our setting. ObjectiveTo detect carbapenem resistance and the susceptibility to other antimicrobial agents in P. aeruginosa and Acinetobacter species isolated from blood, sputum, skin and soft tissue specimens between March, 2018 and June, 2019 at the University Teaching Hospital (UTH), Lusaka, Zambia. MethodThis was a retrospective hospital based study, involving routine blood, sputum, skin and soft tissue samples submitted to the microbiology laboratory. Conventional microbiology methods were used to identify the organisms, while Kirby-Bauer disc diffusion method was used to determine the antimicrobial susceptibility profiles of the isolates. In order to detect carbapenemase production in P. aeruginosa isolates that were resistant to imipenem by disc diffusion, the isolates were subjected to the modified carbapenem inactivation method (mCIM) method. ResultsA total of 384 samples were analyzed, of which 84 P. aeruginosa and 11 Acinetobacter species were isolated. The P. aeruginosa isolates were susceptible to imipenem (94%), piperacillin-tazobactum (95.2%), amikacin (91%), ciprofloxacin (69%), gentamicin (63.1%) and ceftazidime (51.2%). The Acinetobacter species isolates were most susceptible to amikacin (90.9%), imipenem (82.9%) and piperacillin-tazobactum (82.8%), ciprofloxacin, tetracycline and cefepime all at 27.3% each but had the lowest susceptibility at 9.1% to both gentamicin and cefotaxime. Carbapenem resistance in P. aeruginosa was 5/84 (6%) and 2/11 (18.2%) in Acinetobacter species. Carbapenemase production was confirmed in all five P. aeruginosa isolates. ConclusionCarbapenem resistance was low in both organisms demonstrating that imipenem is still an effective treatment choice for invasive infections caused by these organisms in our setting. In order to reduce HAI and improve patient outcome, there is need to strengthen antimicrobial surveillance/stewardship and infection control at the UTH.

Phenotypic/Genotypic Profile of OXA-10-Like-Harboring, Carbapenem-Resistant Pseudomonas aeruginosa: Using Validated Pharmacokinetic/Pharmacodynamic In Vivo Models To Further Evaluate Enzyme Functionality and Clinical Implications.

In vitro MICs and in vivo pharmacodynamics of ceftazidime and cefepime human-simulated regimens (HSR) against modified carbapenem inactivation method (mCIM)-positive Pseudomonas aeruginosa isolates harboring different OXA-10-like subtypes were described. The murine thigh model assessed ceftazidime (2 g every 8 h [q8h] HSR) and cefepime (2 g and 1 g q8h HSR). Phenotypes were similar despite possessing OXA-10-like subtypes with differing spectra. Ceftazidime produced ≥1-log10 killing in all isolates. Cefepime activity was dose dependent and MIC driven. This approach may be useful in assessing the implications of β-lactamase variants.

Resistance Phenotype and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Isolates in Shanghai.

Background: The emergence and wide global spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates are of great concern, and the aim of this study was to investigate drug resistance, molecular epidemiology, and genetic relationship of CRKP isolates from patients in Shanghai, China. Methods: A retrospective study was conducted from April 2018 to July 2019, and a total of 133 CRKP isolates were collected. Antimicrobial susceptibility was determined by VITEK-2 automated microbiology analyzer platform (bioMérieux, France) and the broth microdilution method. Polymerase chain reaction assays were used to investigate the presence of drug resistance genes. A modified carbapenem inactivation method was performed to detect carbapenemases. Multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) were conducted for genetic relatedness of 50 CRKP isolates selected. Results: Among 670 isolates of K. pneumoniae, 133 (19.9%) strains were identified as CRKP, of which, 76.7% (102/133) strains were isolated from intensive care units (ICUs). All the 133 CRKP isolates were found to be carbapenemase-producers and harbor blaKPC-2 gene. No other carbapenemase genes of blaNDM, blaOXA-48, blaVIM, and blaIMP were detected. Furthermore, β-lactamase genes of blaSHV, blaCTX, and blaTEM were the most common resistance-associated genes among these KPC-2 producing isolates. All the 133 CRKP strains displayed >95% of resistance to cephalosporins and carbapenems, except for gentamicin, trimethoprim-sulfamethoxazole, amikacin, tigecycline and colistin, and ceftazidime-avibactam. The most common sequence type was ST11, accounting for 90.0% of the 50 CRKP selected, followed by ST15 (10.0%). PFGE analysis clustered the 50 KPC-2-producing isolates into seven (A-G) distinct clonal clusters at 85% cutoff. Of which, A and G were the two major clusters, accounting for the majority of the strains collected in emergency ICU and neurosurgical ICU. And all the strains of clusters D and E were collected in cardiothoracic surgery ICU, except for one strain collected in one outpatient. Conclusion: The KPC-2-producing K. pneumoniae belonged to ST11 was widely disseminated in ICUs, and active and effective surveillance of infection control strategies was initiated to limit the spread of CRKP strains.

The ERACE-PA Global Surveillance Program: Ceftolozane/tazobactam and Ceftazidime/avibactam in vitro Activity against a Global Collection of Carbapenem-resistant Pseudomonas aeruginosa

The cephalosporin-β-lactamase-inhibitor-combinations, ceftolozane/tazobactam and ceftazidime/avibactam, have revolutionized treatment of carbapenem-resistant Pseudomonas aeruginosa (CR-PA). A contemporary assessment of their in vitro potency against a global CR-PA collection and an assessment of carbapenemase diversity are warranted. Isolates determined as CR-PA by the submitting site were collected from 2019–2021 (17 centers in 12 countries) during the ERACE-PA Global Surveillance Program. Broth microdilution MICs were assessed per CLSI standards for ceftolozane/tazobactam, ceftazidime/avibactam, ceftazidime, and cefepime. Phenotypic carbapenemase testing was conducted (modified carbapenem inactivation method (mCIM)). mCIM positive isolates underwent genotypic carbapenemase testing using the CarbaR, the CarbaR NxG, or whole genome sequencing. The MIC50/90 was reported as well as percent susceptible (CLSI and EUCAST interpretation). Of the 807 isolates, 265 (33%) tested carbapenemase-positive phenotypically. Of these, 228 (86%) were genotypically positive for a carbapenemase with the most common being VIM followed by GES. In the entire cohort of CR-PA, ceftolozane/tazobactam and ceftazidime/avibactam had MIC50/90 values of 2/ > 64 and 4/64 mg/L, respectively. Ceftazidime/avibactam was the most active agent with 72% susceptibility per CLSI compared with 63% for ceftolozane/tazobactam. For comparison, 46% of CR-PA were susceptible to ceftazidime and cefepime. Against carbapenemase-negative isolates, 88 and 91% of isolates were susceptible to ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Ceftolozane/tazobactam and ceftazidime/avibactam remained highly active against carbapenem-resistant P. aeruginosa, particularly in the absence of carbapenemases. The contemporary ERACE-PA Global Program cohort with 33% carbapenemase positivity including diverse enzymology will be useful to assess therapeutic options in these clinically challenging organisms with limited therapies.

HB&L system for rapid phenotypic detection of clinical carbapenem-resistant Enterobacterales isolates

The prevalence of carbapenem-resistant Enterobacterales (CRE) has increased rapidly worldwide in the last two decades. CRE infection poses a huge challenge for today's clinical therapy. Rapid and accurate detection of clinical CRE isolates can avoid inappropriate antimicrobial treatment and reduce mortality. However, existing detection methods are either time consuming, expensive or inaccurate, making them unable to fully meet clinical demands. In this study, the HB&L system was designed to distinguish CRE from carbapenem-susceptible Enterobacterales (CSE), as it can accelerate the growth of bacteria, detect both carbapenemase-producing CRE (CP-CRE) and non-CP-CRE isolates in real time, and provide time-kill curves. The broth microdilution method and PCR and sequencing were used as the reference methods to identify CRE and carbapenemase-producing Enterobacterales (CPE) isolates, respectively. Three methods for detecting CRE isolates, including the Carba NP test, modified carbapenem inactivation method (mCIM) and HB&L system, were evaluated. The accuracy of the HB&L system was extremely high with 100% sensitivity and 96.0% specificity at only 6 h of culture time for detecting CRE. Time-kill curves may provide information on effective treatment options for clinicians. This system is superior to the mCIM (20-24 h detection time; 90.6% sensitivity and 96.6% specificity) and Carba NP test (2 h detection time; 85.2% sensitivity and 98.4% specificity), which are only designed to detect CP-CRE. The HB&L system is promising for wide application for detection of clinical CRE in hospitals.

MCIM test as a reliable assay for the detection of CRE in the Gulf region.

Introduction. Carbapenem resistant Enterobacterales (CRE) are one of the leading causes of systemic and nosocomial infections and are multidrug-resistant organisms producing different carbapenemases. There are many genotypic and phenotypic methods for detecting the carbapenemases; however, there is a limitation for each. Modified carbapenem inactivation method (mCIM) assay is a recent phenotypic method which has been published by the Clinical and Laboratory Standards Institute.Hypothesis / Gap Statement. mCIM assay could provide a reliable method for the detection of carbapenemases in CRE.Aim. Evaluation of the mCIM assay performance for the detection of carbapenemases in Enterobacterales and the identification of the common carbapenemase genes at Eastern Province of Saudi Arabia and Kingdom of Bahrain.Methodology. A collection of 197 non-duplicate carbapenem resistant Enterobacterales clinical isolates, were evaluated with the mCIM test comparing its performance to multiplex PCR. The minimum inhibitory concentration susceptibility testing was done by the Etest method for imipenem, meropenem, and ertapenem.Results. The sensitivity of the mCIM assay was 94 % (95 % CI, (89.3-97.1)). In Saudi Arabia and Bahrain, OXA-48 was the most prevalent carbapenemase gene followed by NDM. Coexistence of multiple carbapenemase genes is reported in eleven cases.Conclusion. These findings indicate that the mCIM test is a reliable and simple assay for detecting the activity of carbapenemase in Enterobacterales, especially in resource-limited laboratories.

Modified CIM test as a useful tool to detect carbapenemase activity among extensively drug-resistant Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii

PurposeTimely detection of carbapenemases is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates. The purpose of this study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant isolates of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. In addition, the efficacy of the modified Hodge test (MHT), Carba NP test, and modified carbapenem inactivation method (mCIM) were compared.MethodsA total of 122 carbapenem-resistant clinical isolates including 77 K. pneumoniae, 39 A. baumannii, and six E. coli were collected from hospitalized patients. Three phenotypic methods, including the MHT, Carba NP test, and mCIM were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was performed to detect carbapenemase-encoding genes.ResultThe sensitivity and specificity of the MHT were 75.0% and 100%, respectively. In addition, Carba NP displayed 80.8% sensitivity and 100% specificity, whereas the sensitivity and specificity were 90.4% and 100% for the mCIM test, respectively. Among carbapenem-resistant isolates, 70, 84, and 87 isolates exhibited positive results according to the MHT, Carba NP test, and mCIM, respectively. PCR indicated the presence of one or more carbapenemase genes in 119 of carbapenem-resistant isolates, with blaKPC and blaVIM being the most commonly encountered. Co-production of ‘KPC and OXA-48’, ‘KPC and VIM’, and ‘KPC and IMP’ was detected in three, nine, and seven isolates, respectively.ConclusionOur results confirm that the mCIM test is a useful tool for the reliable detection of carbapenemase activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources.

Genotypic characterization of carbapenem resistant Enterobacterales in clinical isolates from western Maharashtra

PurposeTo study distribution of carbapenemase genes namely; New Delhi metallo-beta-lactamase (blaNDM), Oxacillinase-48 (blaOXA48), Verona Integron-Encoded Metallo-beta-lactamase (blaVIM) and Imipenemase (blaIMP) in carbapenem resistant Enterobacterales (CRE), isolated from clinical samples. MethodThis cross-sectional study was conducted at a tertiary care hospital of western Maharashtra over six months period. CREs were identified by conventional disc diffusion and modified carbapenem inactivation method (mCIM). A total of 50 consecutive CRE isolates from clinical samples were subjected to home brewed polymerase chain reaction (PCR) for detection of carbapenemases. ResultsOut of the 50 CRE isolates, at least one of the four carbapenemase genes was detected in 49 (98%) isolates. The frequency of distribution of these genes were NDM 90% (n = 45), OXA48 60% (n = 30) and VIM 12% (n = 6). Dual combination of blaNDM and blaOXA48 (50%) was the commonest pattern observed, which was frequently associated with Klebsiella pneumoniae. ConclusionsThe study indicate high prevalence of NDM warranting strict anti-microbial stewardship practices. Surveillance of CRE and resistance mechanism is essential to monitor the trend and take informed decision for appropriate anti-microbial therapy.

Carbapenemase detection by NG-Test CARBA 5-a rapid immunochromatographic assay in carbapenem-resistant Enterobacterales diagnosis.

BackgroundThe global spread of carbapenem-resistant Enterobacterales (CRE) represents a serious public health concern as these organisms are associated with limited treatment options, high mortality rate and rapid transmissibility. The identification of carbapenemase remains a challenge in microbiological laboratories as no single method is perfect when considering cost, carbapenemase coverage, accuracy, handling complexity and TATs together.MethodsNG-Test CARBA 5 assay and modified carbapenem inactivation method in conjunction with EDTA carbapenem inactivation method (mCIM/eCIM) were challenged with a collection of 299 molecularly characterized CRE isolates in China in order to evaluate the performance in detecting five major carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48) among Enterobacterales.ResultsNG-Test CARBA 5 detected all KPC-, NDM-, VIM- and OXA-48-producing isolates perfectly with a weak false-positive signal for NDM in an IMP-4 producer, which makes the specificity for NDM decreases to 99.6%. The overall specificity/sensitivity were 99.9%/100% for NG-Test CARBA 5. mCIM/eCIM achieved high specificity of 100%/100% and sensitivity of 99.6%/97.4%, with one S. marcescens isolate harboring VIM-2 undetected.ConclusionsBoth NG-Test CARBA 5 and mCIM/eCIM showed excellent results in the tested carbapenemase (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48) detection compared with molecular genotypic test. As every assay has its own limitations, suitable methods should be combined for the establishment of the CRE diagnostic pathways.

Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Sequence Type 11 Due to a Mutation in Plasmid-Borne bla kpc-2 to bla kpc-33, in Henan, China.

PurposeCarbapenem-resistant Klebsiella pneumoniae (CRKP) represents a serious problem worldwide. Herein, we describe the evolution of ceftazidime–avibactam (CZA) resistance by sequencing clinical isolates from a patient with CRKP infection undergoing CZA treatment.Patients and MethodsIn this study, six CRKP strains were isolated from sputum and blood samples of a patient with CRKP infection after intracerebral hemorrhage. Two strains were selected for whole-genome analysis.ResultsDrug susceptibility testing showed that the MIC of CZA for CRKP strains isolated after 6 days of CZA treatment was 64-fold higher than that for three CRKP strains isolated before CZA treatment (4 vs >256 μg/mL), whereas the MIC of imipenem and meropenem was 128-fold (>32 vs 0.25 μg/mL) and 16-fold (> 32 vs 2 μg/mL) lower relatively, respectively. Multilocus sequence typing showed that all six CRKP strains isolated from the patient were ST11 and pulsed-field gel electrophoresis confirmed that they were of the same clone. Two strains were selected for whole-genome analysis. The aspartic acid residue at position 179 in the Ω loop was replaced by a tyrosine residue in the resistant strain, and the plasmid carried a blaKPC-2 to blaKPC-33 mutation. The results of the modified carbapenem inactivation method and the carbapenemase inhibitor enhancement and colloidal gold enzyme immunochromatographic assays for blaKPC-33 were negative.ConclusionThis is the first report from Henan to show that treatment with CZA for 6 days can cause mutations and change the phenotype from CZA sensitive to resistant. Therefore, routine testing for drug susceptibility and carbapenemase phenotypes should be conducted during treatment with CZA, and genotype determination is essential.

The prediction values of carbapenemase detection methods and carbapenem susceptibility testing for clinical outcomes of patients with Acinetobacter bacteremia under carbapenem treatment.

Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89-97%) and specificity (81%; 95% CI, 73-88%) for predicting imipenem non-susceptibility. Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.