Common Carbapenemase Research Articles

A Rare Report of Carbapenem Resistant Vibrio fluvialis Isolated from a case of Walled off Necrosis: A Sequelae of Acute Necrotising Pancreatitis

Vibrio fluvialis (V. fluvialis) is commonly found in coastal environments and is an emerging pathogen encountered in diarrhoeal outbreaks and sporadic extraintestinal cases. V. fluvialis infections usually manifest as watery bloody diarrhoea, nausea, vomiting, gastroenteritis and peritonitis. In this case report, an unusual presentation of Multidrug Resistant (MDR) V. fluvialis along with Klebsiella pneumoniae co-infection from a case of Walled Off Necrosis (WON)- A complication of Acute Necrotising Pancreatitis (ANP) is discussed. The patient presented with the acute abdomen and had peripancreatic collection for which Ultrasound (USG) guided 12F pigtail drain placed over the right lower quadrant of the abdomen and conservatively managed. Routine cultures were done and the growth on Sheep Blood Agar (SBA) plate and MacConkey (MAC) agar plates were subjected for automated phenotypic identification by Matrix Assisted Laser Desorption Ionisation Time of Flight-Mass Spectrometry (MALDITOF-MS) and Vitek-2 systems and was identified as V. fluvialis. Also, molecular tests like 16S rRNA Polymerase Chain Reaction (PCR) followed by Sanger’s sequencing was done and the identification was further confirmed. Phenotypic RESIST-5 TRURAPID O.K.N.V.I. commercial lateral flow immunochromatographic assay for detection of clinically relevant and common carbapenemase genes like NDM, OXA-48, VIM, IMP and KPC was done which revealed this V. fluvialis isolate as New Delhi Metallo betalactamase (NDM) type of Carbapenemase producer. Patient was managed with levofloxacin and doxycycline and patient condition improved and was discharged.

Monte Carlo simulation for dosage optimization of the best available therapy for bloodstream infections secondary to carbapenemase-producing Klebsiella pneumoniae in critically ill patients

ObjectivesWe aimed to use Monte Carlo simulation, based on pharmacokinetic/ pharmacodynamic (PK/PD) targets, to investigate and determine the optimal dosage of the available combination therapies for carbapenem-resistant Klebsiella pneumoniae (CRKP) in critically ill patients. MethodsWe collected CRKP clinical isolates from Phramongkutklao Hospital between October 2020 and June 2022. A molecular study of resistant genes was performed using polymerase chain reaction (PCR). Broth microdilution checkerboards were used to evaluate the mono- and synergistic antibiotic activities. Monte Carlo simulation was used to determine the optimal antibiotic regimens, based on the probability of target achievement (PTA) and cumulative fraction of response (CFR). ResultsThe 54 CRKP isolates were resistant to tigecycline (100%), colistin (75.9%), amikacin (70.4%), and gentamicin (63.0%). The most common carbapenemase genotype was blaoxacillinases (OXA)-48-like (42.6%), followed by blaNew Delhi metallo beta lactamase (NDM) (29.6%) and coexistence of blaOXA-48-like and blaNDM (22.2%). Based on the PTA, synergistic and additive activities against CRKP isolates were observed with appropriate dosages of tigecycline–colistin (67.9%), tigecycline–gentamicin (62.2%), and tigecycline–amikacin (51.4%). ConclusionsTigecycline–colistin was the best available combination therapy for critically ill patients with CRKP, especially NDM. When used in combination with tigecycline, a colistin creatinine clearance (CrCl) of <90 mL/min can raise the CFR target and less nephrotoxicity.

Laboratory detection of carbapenemases among Gram-negative organisms.

SUMMARYThe carbapenems remain some of the most effective options available for treating patients with serious infections due to Gram-negative bacteria. Carbapenemases are enzymes that hydrolyze carbapenems and are the primary method driving carbapenem resistance globally. Detection of carbapenemases is required for patient management, the rapid implementation of infection prevention and control (IP&C) protocols, and for epidemiologic purposes. Therefore, clinical and public health microbiology laboratories must be able to detect and report carbapenemases among predominant Gram-negative organisms from both cultured isolates and direct from clinical specimens for treatment and surveillance purposes. There is not a "one size fits all" laboratory approach for the detection of bacteria with carbapenemases, and institutions need to determine what fits best with the goals of their antimicrobial stewardship and IP&C programs. Luckily, there are several options and approaches available for clinical laboratories to choose methods that best suits their individual needs. A laboratory approach to detect carbapenemases among bacterial isolates consists of two steps, namely a screening process (e.g., not susceptible to ertapenem, meropenem, and/or imipenem), followed by a confirmation test (i.e., phenotypic, genotypic or proteomic methods) for the presence of a carbapenemase. Direct from specimen testing for the most common carbapenemases generally involves detection via rapid, molecular approaches. The aim of this article is to provide brief overviews on Gram-negative bacteria carbapenem-resistant definitions, types of carbapenemases, global epidemiology, and then describe in detail the laboratory methods for the detection of carbapenemases among Gram-negative bacteria. We will specifically focus on the Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex.

Antimicrobial susceptibility of enterobacterales causing bloodstream infection in United States medical centres: comparison of aztreonam-avibactam with beta-lactams active against carbapenem-resistant enterobacterales

BackgroundBloodstream infection (BSI) is associated with poor outcomes especially when effective antimicrobial therapy and control of infection source are delayed. As the frequency of Enterobacterales producing metallo-β-lactamases (MBL) and/or OXA-48–like carbapenemases is increasing in some United States (US) medical centres, effective antimicrobials to treat the infections caused by these organisms are urgently needed. Aztreonam-avibactam is under clinical development for treatment of infections caused by Gram-negative bacteria, including MBL producers.ObjectivesTo evaluate the antimicrobial susceptibility of Enterobacterales causing BSI in US medical centres and compare the activity of aztreonam-avibactam with ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, cefiderocol, and other antimicrobials used to treat BSI.Methods4,802 Enterobacterales were consecutively collected (1/patient) from 72 US medical centres in 2020–2022 and susceptibility tested by broth microdilution. Aztreonam-avibactam was tested with avibactam at a fixed concentration of 4 mg/L. A pharmacokinetic/pharmacodynamic susceptible breakpoint of ≤ 8 mg/L was applied for aztreonam-avibactam for comparison. Carbapenem-resistant Enterobacterales (CRE) isolates were tested for β-lactamase–encoding genes using Next-generation sequencing.ResultsAztreonam-avibactam was highly active against Enterobacterales; only 2 isolates showed aztreonam-avibactam MICs > 8 mg/L: 1 meropenem-susceptible E. coli and 1 K. aerogenes (CRE). All carbapenemase producers and 98.0% of CRE were inhibited at an aztreonam-avibactam MIC of ≤ 8 mg/L. CRE susceptibility rates were 81.6% for ceftazidime-avibactam, 65.3% for meropenem-vaborbactam, 61.2% for imipenem-relebactam, and 87.8% for cefiderocol. Aztreonam-avibactam retained activity (MIC, ≤ 8 mg/L) against all (100.0%) meropenem-vaborbactam nonsusceptible (n = 17), 99.5% of imipenem-relebactam nonsusceptible (n = 206), and 90.0% of ceftazidime-avibactam nonsusceptible (n = 10) isolates. The most common carbapenemases were KPC-2/3 (57.1% of CREs), OXA-48–like (16.3%), and NDM (14.3%). A carbapenemase gene was not observed in 12.3% of CREs. Ceftazidime-avibactam and meropenem-vaborbactam were active against 100.0% of KPC producers, but ceftazidime-avibactam showed limited activity against MBL producers and meropenem-vaborbactam showed limited activity against OXA-48–like and MBL producers. The most active non–β-lactam comparators against CRE were gentamicin (49.0% susceptible) and amikacin (44.9% susceptible).ConclusionsAztreonam-avibactam demonstrated potent activity against a large collection of Enterobacterales isolated from patients with BSI in US hospitals, including CRE, MBL producers, and isolates resistant to recently approved β-lactamase inhibitor combinations.

Nosocomial cluster of patients infected with imipenemase-1-producing Enterobacter ludwigii.

Introduction. Imipenemase (IMI) enzymes are an uncommon class A carbapenemases that have been isolated from aquatic environments and, occasionally, from clinical isolates of Enterobacterales.Aim. We describe a cluster of three patients infected by IMI-1 carbapenemase-producing Enterobacter ludwigii (IMI-1-Elud) in a tertiary university hospital in Gran Canaria, Spain.Methodology. Antimicrobial susceptibility was determined using the Vitek2 AST-N355 card and antibiotic gradient strips. The modified carbapenem inactivation method (CIM) test was performed in cases where the ertapenem MIC value was higher than 0.125 mg l-1. The carbapenemase was identified by PCR and DNA microarray and later characterized by whole-genome next-generation sequencing (NGS) with Illumina.Results. Three patients presented thoracic or abdominal infections caused by IMI-1-Elud ST1677 from 14 June 2022 to 14 July 2022. All patients underwent at least one gastroscopy during their admission, and two of them were located in adjoining rooms. Isolates were resistant to carbapenems, colistin and fosfomycin but susceptible to ciprofloxacin. IMI/NMC-A carbapenemase was detected by PCR and hybridization test and confirmed by NGS as IMI-1. All patients underwent at least one gastroscopy, and two of them were in nearby rooms. Patients showed microbiological and clinical improvement following focus drainage and targeted antibiotic treatment with a fluoroquinolone.Conclusions. This study reports the first documented global outbreak of patients infected with IMI-1-Elud. The source appeared to be related to endoscopes. Contact transmission may also have played a role. A screening method such as the modified CIM test is crucial for detecting less common carbapenemases that might not be identified by rapid molecular or immunochromatographic tests, as these often do not include bla IMI genes, which could lead to the undetected dissemination of carbapenemase-producing Enterobacterales. Effective infection source control and targeted treatment are essential for achieving a favourable clinical outcome.

A novel machine-learning aided platform for rapid detection of urine ESBLs and carbapenemases: URECA-LAMP.

Pathogenic gram-negative bacteria frequently carry genes encoding extended-spectrum beta-lactamases (ESBL) and/or carbapenemases. Of great concern are carbapenem resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite the need for rapid AMR diagnostics globally, current molecular detection methods often require expensive equipment and trained personnel. Here, we present a novel machine-learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The platform consists of (i) an affordable device for sample lysis, LAMP amplification, and visual fluorometric detection; (ii) a LAMP screening panel to detect the most common ESBL and carbapenemase genes; and (iii) a smartphone application for automated interpretation of results. Validation studies on clinical isolates and urine samples demonstrated percent positive and negative agreements above 95% for all targets. Accuracy, precision, and recall values of the machine learning model deployed in the smartphone application were all above 92%. Providing a simplified workflow, minimal operation training, and results in less than an hour, this study demonstrated the platform's feasibility for near-patient testing in resource-limited settings.IMPORTANCEExtended-spectrum beta-lactamases (ESBL) and carbapenemases confer resistance to third-generation cephalosporins and carbapenems in pathogenic Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Conventional antimicrobial susceptibility testing is based on phenotypic methods, and results can take several days to be obtained. Current genotypic detection methods can be rapid but require expensive equipment and trained personnel. In this study, we present a novel machine learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The validation of the platform demonstrated percent positive and negative agreements above 95% for all targets. The newly developed platform provided a simplified workflow, minimal technical training, and results in less than an hour. This study demonstrated the platform's feasibility for rapid testing of ESBL and carbapenemases in bacteria and urine specimens.

A 5-year study of bloodstream infections caused by carbapenemase-producing Gram-negative bacilli in southern Spain.

The aim of this study was to evaluate the microbiological epidemiology of carbapenemase-producing Gram-negative bacilli (CPGNB) isolated from blood during a 5-year period. A total of 80 isolates from 78 patients were finally included; fifty-five (70.5%) were men and the mean age was 60 years. Detection of carbapenemase production was performed by immunocromatography (IC) and polymerase chain reaction (PCR). Genotyping was carried-out by pulsedfield gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), and characterization of carbapenemase-producing isolates was performed by whole genome sequencing (WGS). The main microorganisms isolated were K. pneumoniae (29.4%), E. cloacae (28.2%), A. baumannii (17.9%) and P. aeruginosa (15.3%). Overall, the most common carbapenemase in Enterobacterales was OXA-48 group (57.7%). The most common carbapenemase in non-fermenting bacilli was OXA-23 (60.8%). The most common ST in K. pneumoniae producing OXA-48 types was ST45 and in E. cloacae ST114, while in E. cloacae producing VIM types was ST78. In OXA-23 types, the most common clone in A. baumannii was ST2, whereas in P. aeruginosa producing IMP types was ST253. There was an increase in cases recorded in the years of highest incidence and severity of the SARS-CoV-2 pandemic, with a significant number of cases in patients admitted to the ICU. All bacteremias caused by A. baumannii were caused by the same clone, and 12 of the 14 cases caused by A. baumannii were part of outbreaks in the ICU.

Phenotypic antimicrobial resistance associated with different &lt;em&gt;bla&lt;/em&gt; genotypes and their combinations in Enterobacterales

Introduction: Varying combinations of β-lactamase genes (bla) in Enterobacterales manifest different resistant phenotypes.Objectives: This study aimed to identify bla gene types and combinations resulting in variations in phenotypic antimicrobial resistance.Methods: Minimum inhibitory concentrations (MICs) of 75 extended spectrum β-lactamase (ESBL), AmpC β-lactamase and carbapenemase-producing Entero-bacterales isolates from a variety of clinical specimens were determined by Vitek micro-broth dilution. Bla genes were detected by 16-plex tandem PCR panel and Xpert Carba-R assay. BlaCTX-M group-1 and blaOXA-48 were the most common ESBL and carbapenemase genes, respectively.Results: Majority of the isolates (92%, 69/75) had bla gene combinations. Thirty-eight isolates (51%) carried ESBL, AmpC genes and/or narrow spectrum bla genes without any carbapenemase genes. The common bla gene combination in these isolates was blaCTX-M group-1 + blaOXA1 which is known to confer decreased susce-ptibility to piperacillin-tazobactam. Antibiotics with potential activity against ESBL-producing bacteria such as fourth generation cephalosporins, β-lactam/β-lactam-inhibitor combinations, quinolones and gentamicin also showed low rates of susceptibility. Highest susceptibility rates were to meropenem (92%) and amikacin (92%). Thirty-seven isolates (49%) carried carbapenemase genes as well. These isolates showed even higher resistance rates to all antibiotics, except amikacin (92%susceptible). Significantly higher MIC values for cefoxitin, ceftazidime, ceftriaxone, cefepime, meropenem, amikacin, tobramycin and trimethoprim-sulfamethoxazole were seen in isolates carrying blaNDM than blaOXA-48. However, a higher MIC for norfloxacin was seen in isolates with blaOXA-48.Conclusions: In conclusion, this study showed that Enterobacterales with different bla gene combinations manifest variations in phenotypic resistance to both β-lactam and non-β-lactam antibiotics.

Genomic analysis of &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; strains virulence and antibiotic resistance

Recently, Klebsiella pneumoniae strains have become widespread both in community-acquired infectious processes and in nosocomial infections. There are two pathotypes of K. pneumoniae: classical (cKp) and hypervirulent (hvKp). Representatives of any pathotype are prone to acquire and further transmit genetic factors of antibiotic resistance and virulence. This combination accounts for severity of the infectious process. Therefore, information about whether the strain belongs to either pathotype can help in prescribing proper therapy. Since there is no consensus upon hypervirulence marker, we attempted to find the most significant combinations of genetic markers of virulence and antibiotic resistance in K. pneumoniae strains. The study was aimed to conduct a genomic analysis of virulence and antibiotic resistance of K. pneumoniae clinical isolates. Materials and methods. There were examined 85 strains of K. pneumoniae isolated from diverse clinical material samples from patients in large St. Petersburg hospitals. In our work, we used classical bacteriological methods, including determination of the hypermucoviscous type using the “string test”, the mass spectrometric method (MALDI-ToF MS) for identifying bacteria, molecular methods for studying markers of virulence and antibiotic resistance (multilocus sequence typing, genome sequencing of K. pneumoniae strains). Results. Among the studied K. pneumoniae strains, the most common carbapenemase genes were OXA-48 (18.7%) and NDM-1 genes — 17.3% of strains; in 6.7% of strains, NDM-1 and OXA-48 genes were found simultaneously. The percentage of strains with β-lactamase genes CTX-M-15 was 54.7%, OXA-1 — 17.3%, TEM-1D — 13.3%, and in 17.3% of cases the OXA-1 and TEM-1D genes were simultaneously present in bacterial strains. Quinolone resistance genes were found in 68.4% of strains. The most common genes were qnrS1 (40% of strains) and qnrB1 (22.7%). Phenotypic antimicrobial susceptibility testing showed that 23.5% and 64.7% strains were resistant to colistin and carbapenems, respectively. 32.9% K. pneumoniae strains, isolated in patients with phlegmon, pneumonia, sepsis, and peritonitis, had a hypermucoid phenotype. The most common sequence types were: ST395 (24.3%), ST23 (17.6%) and ST512 (9.5%). 8% and 25.3% of strains belonged to capsule types K1 and K2, respectively. The polyketide synthesis locus ybt, which characterizes virulent strains, was detected in 69.3% isolates, and the clb locus was present in 10.7% of strains. In 73.3% and 14.7% strains, the plasmid-associated virulence loci iuc and iro were identified, which encode the biosynthesis of the siderophores aerobactin and salmochelin. We described 44 cases (58.7% of strains) of genotypic convergence of virulence and antibiotic resistance, as shown by simultaneously detected the aerobactin (iuc) locus and β-lactamase or carbapenemase genes. Thus, identification of hypervirulence may provide valuable information for the clinical management of patients with hvKp infections. Therefore, it is is obviously necessary to develop comprehensive diagnostic test for simultaneous screening of multidrug-resistant hypervirulent K. pneumoniae strains.

Modification of carbapenemase inhibition test and comparison of its performance with NG-Test CARBA 5 for detection of carbapenemase-producing enterobacterales.

Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. Carbapenemase genes in 2665 non-duplicate carbapenem-resistant Enterobacterales (CRE) clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4mm. The overall sensitivities of the current CIT, the modified CIT and NG-Test CARBA 5 were 91.4%, 94.9% and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0%vs. 53.3%), though this difference was not statistically significant (p=0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance.

Antimicrobial susceptibility profile of ceftolozane/tazobactam, ceftazidime/avibactam and cefiderocol against carbapenem-resistant Pseudomonas aeruginosa clinical isolates from Türkiye.

Carbapenem resistant Pseudomonas aeruginosa (CR-PA) is escalating worldwide and leaves clinicians few therapeutic options in recent years, β-lactam/β-lactamase inhibitor combinations (ceftolozane-tazobactam, ceftazidime-avibactam) and a new siderophore cephalosporin (cefiderocol) have been approved for the treatment of P. aeruginosa infection and have shown potent activity against isolates defined as carbapenem resistant. The aim of this study was to determine the phenotypic profile of these agents against CR-PA in the emerging setting of carbapenemases. CR-PA clinical isolates were collected from three teaching hospitals in different geographical regions between January 2017-December 2021. All isolates were subjected to phenotypic carbapenemase testing using modified carbapenem inactivation method. MICs were determined by reference broth microdilution and evaluated according to EUCAST standards, while genotypic profiling was determined using PCR methods. 244 CR-PA sourced most frequently from the respiratory tract (32.2%), blood (20.4%) and urine (17.5%) were evaluated. Of all isolates, 32 (13.1%) were phenotypically and 38 (15.6%) were genotypically defined as carbapenemase-positive. The most common carbapenemase was GES (63.1%), followed by VIM (15.8%). The MIC50/90(S%) of ceftazidime/avibactam, ceftolozane/tazobactam and cefiderocol in all CR-PA isolates were 4 and 32 (80%), 1 and > 64 (69%) and 0.25 and 1mg/L (96%), respectively. Cefiderocol was also the most active agent in carbapenemase-positive isolates (90%). While ceftolozane/tazobactam and ceftazidime/avibactam remained highly active against CR-PA devoid of carbapenemases, cefiderocol provided potent in vitro activity irrespective of carbapenemase production. When considering the potential clinical utility of newer agents against CR-PA, regional variations in carbapenemase prevalence must be considered.

Efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli.

Carbapenem-resistant Enterobacterales (CRE) have become a major public health problem worldwide. The aim of this study was to investigate efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates. Susceptibility of imipenem, meropenem, ertapenem, ceftazidime/avibactam and plazomicin was investigated by broth-microdilution method. Major carbapenemases NDM, VIM, IMP, KPC, OXA-48 as well as other β-lactamases namely, TEM, SHV, OXA-1-like, CTX-M, ACC, FOX, MOX, DHA, CIT, EBC, VEB, GES, PER were investigated by PCR. A total of 120 carbapenem-resistant isolates (60 E. coli and 60 K. pneumoniae) were included in this study and blaOXA-48-like was found in 78.33%, blaNDM in 26.66%, blaKPC in 7.5%, blaIMP in 5.83%, and blaVIM in 5%. Among 94 isolates with the blaOXA-48-like gene, 22.3% were resistant to ceftazidime/avibactam and 51.1% were resistant to plazomicin. Of 32 isolates with blaNDM, 31 (96.9%) were resistant to ceftazidime/avibactam and 30 (93.75%) were resistant to plazomicin, and both antibiotics had limited effects against blaNDM carriers (P < 0.001). Of the 12 isolates with blaNDM+OXA-48 combination, 11 (91.7%) were resistant to ceftazidime/avibactam and plazomicin. The effect of both antibiotics was significantly lower in strains with blaNDM+OXA-48 combination (P < 0.005).The most common carbapenemase genes in this study were blaOXA-48-like and blaNDM. Ceftazidime/avibactam demonstrated a good efficacy among OXA-48 producing K. pneumoniae and E. coli, however, plazomicin had a significantly lower antibacterial effect in our study. Both antimicrobial agents should be considered as an option by evaluating combined susceptibility results and gene patterns obtained by regional and global molecular data in the treatment of CRE infections.

Detection of Carbapenemase Production in Klebsiella pneumoniae Isolates by Rapid Carbapenemase Detection Method in an OXA-48 Endemic Region

Background: Carbapenem resistance is one of the major global public health threats caused by the overuse or misuse of carbapenems, which are often the last resort in treating multidrug-resistant infections. Today, timely detection of carbapenemase-producing organisms is critical for infection control. Therefore, rapid and accurate detection of carbapenemases using a cost-effective method with high sensitivity and specificity is essential to guide appropriate treatment and prevent further spread. Objectives: This study aimed to evaluate the sensitivity of the newly developed rapid carbapenemase detection method (rCDM) with isolates whose resistance genes were determined by molecular methods and to compare it with the modified carbapenem inactivation method (mCIM), whose diagnostic performance characteristics are well-defined. Methods: A total of 130 Klebsiella pneumoniae isolates (65 carbapenem-susceptible, 65 carbapenem-resistant) from various clinical specimens were included in the study. Identification and antibiotic susceptibility testing of the isolates was performed using the BD Phoenix™ automated system. Phenotypic carbapenemase detection methods, mCIM and rCDM, were studied for all carbapenem-sensitive and carbapenem-resistant isolates with known resistance genes. Results: Among carbapenem-resistant K. pneumoniae isolates, blaOXA-48 was the most frequently detected resistance gene (78%). Using PCR results as the reference method, the sensitivity of rCDM was 100%. When rCDM and mCIM results were compared, both phenotypic methods were 100% compatible, with no statistically significant difference observed. Conclusions: Rapid carbapenemase detection method is a rapid and reliable phenotypic carbapenemase detection method with high sensitivity. Its ease of use and interpretation make it suitable for use in laboratories with limited resources. Additionally, its excellent performance in detecting common carbapenemase types and high consistency with mCIM support its utility in various laboratory settings without requiring additional materials or technical infrastructure.

Antimicrobial Resistance Profiles of Pseudomonas aeruginosa in the Arabian Gulf Region Over a 12-Year Period (2010-2021).

To evaluate literature from a 12-year period (2010-2021) on the antimicrobial resistance profile of Pseudomonas aeruginosa from the Arabian Gulf countries (Bahrain, Kuwait, Oman, Qatar, Saudi Arabia, and the United Arab Emirates). An electronic literature search was conducted for articles on antimicrobial resistance in P. aeruginosa and associated phenotypes, covering the period of 1st January 2010 to 1st December 2021. Antimicrobial resistance in the Arabian Gulf was highest to meropenem (10.3-45.7%) and lowest to colistin (0.0-0.8%), among the agents tested. Annual data showed that ceftazidime resistance (Kuwait), piperacillin-tazobactam non-susceptibility (Qatar), and aztreonam, imipenem, and meropenem resistance (Saudi Arabia) increased by 12-17%. Multiple mechanisms of carbapenem resistance were identified and multiple clones were detected, including high-risk clones such as ST235. The most common carbapenemases detected were the VIM-type metallo-β-lactamases. Among P. aeruginosa in the Arabian Gulf countries, resistance to meropenem was higher than to the other agents tested, and meropenem resistance increased in Saudi Arabia during the study period. Resistance to colistin, a classic antibiotic used to treat Pseudomonas spp. infections, remained low. The VIM-type β-lactamase genes were dominant. We recommend local and regional antimicrobial resistance surveillance programs to detect the emergence of resistance genes and to monitor antimicrobial resistance trends in P. aeruginosa.

The impact of colistin-based regimens on mortality compared to other antimicrobials in patients with carbapenem-resistant Enterobacterales bacteremia in South African hospitals: a cross-sectional study

BackgroundTreatment of carbapenem-resistant Enterobacterales (CRE) infections in low-resource settings is challenging particularly due to limited treatment options. Colistin is the mainstay drug for treatment; however, nephrotoxicity and neurotoxicity make this drug less desirable. Thus, mortality may be higher among patients treated with alternative antimicrobials that are potentially less efficacious than colistin. We assessed mortality in patients with CRE bacteremia treated with colistin-based therapy compared to colistin-sparing therapy.MethodsWe conducted a cross-sectional study using secondary data from a South African national laboratory-based CRE bacteremia surveillance system from January 2015 to December 2020. Patients hospitalized at surveillance sentinel sites with CRE isolated from blood cultures were included. Multivariable logistic regression modeling, with multiple imputations to account for missing data, was conducted to determine the association between in-hospital mortality and colistin-based therapy versus colistin-sparing therapy.ResultsWe included 1 607 case-patients with a median age of 29 years (interquartile range [IQR], 0–52 years) and 53% (857/1 607) male. Klebsiella pneumoniae caused most of the infections (82%, n=1 247), and the most common carbapenemase genes detected were blaOXA-48-like (61%, n=551), and blaNDM (37%, n=333). The overall in-hospital mortality was 31% (504/1 607). Patients treated with colistin-based combination therapy had a lower case fatality ratio (29% [152/521]) compared to those treated with colistin-sparing therapy 32% [352/1 086]) (p=0.18). In our imputed model, compared to colistin-sparing therapy, colistin-based therapy was associated with similar odds of mortality (adjusted odds ratio [aOR] 1.02; 95% confidence interval [CI] 0.78-1.33, p=0.873).ConclusionIn our resource-limited setting, the mortality risk in patients treated with colistin-based therapy was comparable to that of patients treated with colistin-sparing therapy. Given the challenges with colistin treatment and the increasing resistance to alternative agents, further investigations into the benefit of newer antimicrobials for managing CRE infections are needed.

Molecular epidemiology and risk factors for carbapenem-resistant Enterobacteriaceae infections during 2020-2021 in Northwest China

Severe infection caused by Carbapenem-resistant Enterobacteriaceae (CRE) is a challenge for clinical anti-infective therapy, and clinical intervention to improve control of CRE is of great significance. The study aims to determine the molecular epidemiology and risk factors of CRE infections to provide evidence for effective control of nosocomial infection in patients with CRE. A total of 192 non-repetitive CRE strains were collected from January 2020 to December 2021 in Northwest China. To explore the risk factors of CRE infection by univariate and Logistic regression analysis, 1:1 case-control study was used to select Carbapenem sensitive Enterobacteriaceae (CSE) infection patients at the same period as the control group. Among the 192 CRE strains, the most common isolates included Klebsiella pneumoniae (Kpn) and Enterobacter cloacae (Ecl). The CRE strain showed the lowest rate of resistance to amikacin at 58.3. 185 CRE strains carried carbapenemase resistance genes of concern in this study. KPC-2 (n=94) was the most common carbapenemase, followed by NDM-1 (n=69), NDM-5 (n=22) and IMP-4 (n=5). OXA-48 and VIM were not detected. And KPC-2 was the most common in all strains. Logistic regression analysis implicated days of invasive ventilator-assisted ventilation (OR=1.452; 95 % CI 1.250～1.686), antibiotic combination therapy (OR=2.149; 95 % CI 1.128～4.094), hypoalbuminemia (OR=6.137; 95 % CI 3.161～11.913), history of immunosuppressant use (OR=25.815; 95 % CI 6.821～97.706) and days of hospitalization (OR=1.020; 95 % CI 1.006～1.035) as independent risk factors associated with CRE infection. Age (OR=0.963; 95% CI 0.943～0.984) and history of hormone use (OR=0.119; 95 % CI 0.028～0.504) were protective factors for CRE infection (P < 0.05). The resistance of commonly used antibiotics in clinical is severe, and CRE strains mainly carry KPC-2 and NDM-1. Multiple risk factors for CRE infection and their control can effectively prevent the spread of CRE.

Prevalence of serine carbapenemase genes among multi-resistant strains of Klebsiella pneumoniae isolated from patients in the departments of surgery and intensive care units

The frequency of serine carbapenemases in phenotypically multi-drug resistant K. pneumoniae obtained from patients of surgery and intensive care units in Belarus was assessed. Beta-lactamase genes KPC, OXA-48, OXA-23, and OXA-24 were detected by PCR in 115 phenotypically multi-drug resistant K. pneumoniae. The most common carbapenemase gene was blaOXA-48 (70 %). The genes KPC and blaOXA-24 were identified in 43.6 and 39.1 % isolates, respectively. Single serine carbapenemase gene was detected in 23.5 % strains. On other strains, the molecular analysis determined double or triple carbapenemase gene combinations.

Carbapenemases Produced by Multidrug-Resistant Strains of &lt;i&gt;Klebsiella Pneumoniae&lt;/i&gt; Isolated from Intensive Care Patients

Relevance Klebsiella pneumoniae is one of the main pathogens of nosocomial infections. Hospital strains of this pathogen are characterized by a high frequency of resistance to many antibiotics, including carbapenems. The main mechanism for the formation of resistance to carbapenems is the production of carbapenemases by bacteria. To date, K. pneumoniae is considered one of the main “distributors” of clinically important antibiotic resistance genes.Aim of the study To study the frequency of occurrence of the most common carbapenemase genes in multiresistant K.pneumoniae strains isolated from patients of intensive care units in an emergency hospital.Material and methods 4708 samples of various types of clinical material from patients of 5 intensive care units of the N.V. Sklifosovsky Research Institute for Emergency Medicine were analyzed. Microbiological studies were carried out using standard generally accepted methods. For the purposes of this study, unique sequential K.pneumoniae strains resistant to imipenem and/or meropenem were selected. DNA isolation was carried out using the RIBO-prep kit (Russia). Carbapenemase genes were detected by real-time PCR using the kits of reagents “AmpliSens MDR-MBL-FL” and “AmpliSens MDR-KPC/OXA-48-FL” on a “Rotor Gene” device (Corbett Research, Australia).Results Etiologically significant microorganisms were detected in 64.7% of the studied samples. K. pneumoniae was isolated in a quarter of the samples. 194 unique carbapenem-resistant strains of K.pneumoniae were selected. Of these, 11.3% of the genes of the studied carbapenemases were not detected. In 38.1% of strains, 1 carbapenemase was detected, in 29.9% — two and in 20.6% — three or more. Among the strains with one carbapenemase gene, OXA-48 (19.1%) and CATTLE (13.4%) producers prevailed. Strains producing only NDM betalactamase were found in 5.7% of cases. Isolated allocation of VIM and IMP was not detected. In 34%, metallobectalamases were isolated in combination with serine carbapenemases. The production of serine carbapenemases alone was detected in 48.5% of the strains. Depending on the specialization of the intensive care unit, there are differences in the frequency of detection of serine and metallobetalactamases in strains of carbapenem-resistant Klebsiella.Conclusion K. pneumoniae is the causative agent of nosocomial infections in 25% of cases. In 11.3% of carbapenem-resistant strains, the production of KPC, OXA-48, NDM, VIM and IMP genes was not detected. When developing algorithms for antibacterial therapy, it is necessary to take into account that from 25.7% to 60.6% of K. pneumoniae strains in different intensive care units are the producers of metallobetalactamases.

Development of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes

Carbapenem and colistin are often used as last-resort treatment for Gram-negative multi-drug resistant (MDR) bacteria. Nevertheless, co-resistance of these drugs is threatening the global healthcare system. Rapid and accurate detection of carbapenem and colistin resistant bacteria is critical for adequate antibiotic therapy and infection control, particularly in the context of an outbreak. The presence of blaNDM, blaKPC, blaIMP-1 and blaOXA-48 is responsible for greater than 95% phenotypic resistance in carbapenem-resistant Enterobacteriaceae, while mcr-1 is the most prevalent and well disseminated of all mcr genes in colistin-resistant strains. In this study, we aim to develop a multiplex real time-PCR assay for simultaneous detection of the five genes blaNDM, blaKPC, blaIMP-1, blaOXA-48 and mcr-1. The melting curve-based multiplex real time PCR assay was established with the dissociation temperature range extended from 76°C to 87°C. The whole process is completed within one hour and half, allowing rapid screening of the five genes in cultured bacteria samples with a limit of detection of 10 CFU/ml. The proposed multiplex real-time PCR assay is a robust, reliable and rapid method for the detection of bacterial strains carrying blaOXA-48, blaIMP, blaNDM, blaKPC and mcr-1 gene individually or in cocktail of genes. This assay will be a valuable tool for surveillance and monitoring of MDR bacteria additionally resistant to either carbapenem or colistin or both drugs.

Role of amino acid 159 in carbapenem and temocillin hydrolysis of OXA-933, a novel OXA-48 variant.

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The blaOXA-933 gene was borne on Tn2208, a 2,696-bp composite transposon made of two IS1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the blaOXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the blaOXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it.