In vitro activity, pharmacodynamic profile and dose optimization of biapenem against NDM and OXA-48-like carbapenemase-producing Klebsiella pneumoniae: A multicentre study in Thailand.

BACKGROUND: Biapenem (BIPM) exhibited a less efficient substrate for various metallo-ß-lactamase (MBL) than other carbapenems. OBJECTIVE: We aimed to evaluate in vitro susceptibility data of BIPM and optimal dose based on Monte Carlo simulation to extend treatment options. METHODS: We collected 192 carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates from unique patients among multicentres in Thailand, from June 2019 to March 2023. BIPM disk diffusion and broth-microdilution testing were performed to obtain minimum inhibitory concentration (MIC). Each BIPM regimen was simulated using the Monte Carlo technique to calculate the probability of target attainment (PTA) and the cumulative fraction of response (CFR). RESULTS: The most common genotypes among 192 CRKP isolates were blaOXA-48 (62.3%), blaOXA-48+blaNDM (22.6%) and blaNDM (15.1%). BIPM showed 22.4 and 28.6% susceptible rate when interpreted at clinical breakpoints of 1 and 2 mg/L. The MIC50 and MIC90 of BIPM against CRKP were 8 and 32 mg/L. The BIPM dosing regimens of 300 mg q 6 h infused 6 h and 600 mg q 8 h infused 8 h met the PTA target of %fTime >MIC at 50%, 75% and 100% against isolates MICs of ≤2 mg/L. Based on CFR ≥90%, no BIPM regimens were effective against all the studied CRKP isolates. CONCLUSION: BIPM exhibited a partially susceptible rate among the CRKP isolates in Thailand. The current suggested dose of BIPM with prolonged infusion appears appropriate regimen against CRKP MICs of ≤2 mg/L. However, the empirical use of BIPM for severe CRE infection is not recommended unless the susceptibility has been confirmed.

Nosocomial transmission and rearrangement of large resistance-virulence hybrid plasmids between two bacteremic ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains with low fitness cost.

OBJECTIVES: To characterize nosocomial transmission and rearrangement of the resistance-virulence plasmid between two ST11-K64 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains (JX-CR-hvKP-10 and JX-CR-hvKP-9) with low fitness. METHODS: Phenotypic tests were used to assess the virulence of JX-CR-hvKP-10 and JX-CR-hvKP-9. Whole-genome sequencing was used to analyze JX-CR-hvKP-10 and JX-CR-hvKP-9 chromosomes and plasmids. Fitness and conjugation experiments were also conducted using these two CR-hvKP isolates. RESULTS: Phenotypic tests indicated that both JX-CR-hvKP-10 and JX-CR-hvKP-9 were multidrug-resistant and hypervirulent K. pneumoniae. Whole-genome sequencing and clinical information demonstrated that the super large resistance-virulence fusion plasmid pJX10-1 formed precisely by the fusion of pJX9-1 and pJX9-2 via the nosocomial transmission. Interestingly pJX9-1 itself was also a classic resistance-virulence fusion plasmid by way of the blaKPC-carrying resistance plasmid and pLVPK-like virulence plasmid. Compared with classic K. pneumoniae ATCC700603, fitness analysis revealed no significant difference in growth was observed between JX-CR-hvKP-10 and JX-CR-hvKP-9. CONCLUSION: Nosocomial transmission and rearrangement of a blaKPC-harboring plasmid and a pLVPK-like virulence plasmid with a low fitness cost in ST11 K. pneumoniae enhances drug resistance and virulence simultaneously. Thus, active surveillance of this hybrid plasmid is needed to prevent these efficient resistance-virulence plasmids from disseminating in hospital settings.

Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment.

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in blaKPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in blaKPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored blaKPC-2, blaSHV-182 and blaTEM-1B. Compared with KP135194, KP137060 harbored an additional blaNDM-5 positive plasmid. blaNDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that blaKPC-2- and blaNDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a blaNDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.

The Detection of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary Teaching Hospital in Malaysia and Assessment of Hypermucoviscous as Marker of Hypervirulence.

Background: Hypermucoviscous carbapenem-resistant Klebsiella pneumoniae (hmCRKp) is emerging globally and approaching the worst-case scenario in health care system. Aims: The main objective in this study was to determine the hypermucoviscous characteristics among the carbapenem-resistant K. pneumoniae (CRKp) isolated from a teaching hospital in Malaysia. The association of hypermucoviscous phenotype with the virulence traits and clinical presentations were also investigated. Methods: A retrospective study was conducted in University Malaya Medical Centre (UMMC). The presence of hypermucoviscous K. pneumoniae was identified among a collection of CRKp clinical isolates (first isolate per patient) from 2014 to 2015 using string test. Correlation between clinical and microbial characteristics of the hmCRKp was investigated. Results: A total of nine (7.5%) hmCRKp were detected among 120 CRKp isolates. Majority of the isolates were hospital acquired or health care-associated infections. None of the patients had typical pyogenic liver abscess. All of the hmCRKp isolates harbored carbapenemase genes and were multidrug resistant. K1/K serotype, peg-344, allS, and magA were not identified among hmCRKp isolates, whereas aerobactin siderophore receptor gene (iutA), iroB, rmpA, and rmpA2 were detected. Only three hmCRKp isolates were resistant to serum bactericidal. Conclusions: All the isolates presented inconclusive evidence for the interpretation of hypervirulence. Therefore, more study should be performed in the future to have a better understanding of the virulence mechanisms in correlation with the clinical and microbial determinants.

The use of multi-pronged screening strategy to understand the epidemiology of carbapenemase-producing Enterobacteriaceae in Hong Kong: transition from epidemic to endemic setting.

A multi-pronged carbapenemase-producing Enterobacteriaceae (CPE) screening strategy was implemented in Hong Kong West healthcare network. Of 199,192 fecal specimens from 77,194 patients screening from 1 July 2011 to 30 June 2019, the incidence of CPE per 1000 patient admission significantly increased from 0.01 (2012) to 1.9 (2018) (p<0.01). With appropriate infection control measures, the incidence of nosocomial CPE per 1000 CPE colonization day decreased from 22.34 (2014) to 10.65 (2018) (p=0.0094). Exposure to wet market for purchasing raw pork (p=0.007), beef (p=0.017), chicken (p=0.026), and vegetable (p=0.034) for >3 times per week significantly associated with community acquisition of CPE. Strategic CPE control measures should be implemented in both the hospital and the community.

Assessment of the performance of CHROMagar KPC and Xpert Carba-R assay for the detection of carbapenem-resistant bacteria in rectal swabs: First comparative study from Abu Dhabi, United Arab Emirates.

OBJECTIVES: The objective of this study was to evaluate the performance of CHROMagar™ KPC compared with Xpert® Carba-R assay for the detection of carbapenem-resistant bacterial isolates from rectal swabs. METHODS: Rectal swabs were obtained from patients admitted to Cleveland Clinic Abu Dhabi (United Arab Emirates) over a period of 7 months and were screened for carbapenem resistance by either culture on CHROMagar KPC or carbapenemase production using the Xpert Carba-R molecular method. Further testing for carbapenem susceptibility of isolates recovered from CHROMagar KPC was performed using VITEK®2. RESULTS: A total of 1813 rectal swabs were screened, of which 61 (3.4%) were positive for carbapenem resistance by either one or both methods. Both methods were equally efficient in detecting carbapenem resistance in 37/61 swabs (60.7%), mostly positive for Klebsiella pneumoniae (22 isolates), of which 40.9% (9/22) carried blaOXA-48-like and blaNDM. Xpert Carba-R assay detected 12 additional swabs with negative CHROMagar KPC culture and revealed additional carbapenemase-producing organisms carrying blaOXA-48-like and/or blaNDM. CHROMagar KPC recovered organisms in nine swabs not detected by the genotypic method, 44.4% of which were K. pneumoniae. Three swabs yielded false-positive results (carbapenem-susceptible organisms) by both methods. Sensitivity and specificity were, respectively, 75.4% and 99.8% for CHROMagar KPC and 80% and 99.8% for Xpert Carba-R. CONCLUSION: This comparative study of CHROMagar KPC versus Xpert Carba-R in rectal swabs showed a slightly higher sensitivity for the PCR-based method. Whilst CHROMagar KPC provides a less expensive screening method, Xpert Carba-R may be more accurate and faster.

Direct ß-Lactam Inactivation Method: a New Low-Cost Assay for Rapid Detection of Carbapenemase- or Extended-Spectrum-ß-Lactamase-Producing Enterobacterales Directly from Positive Blood Culture Bottles.

We validate and evaluate a new phenotypic assay, named the direct ß-lactam inactivation method (dBLIM), for the rapid and simultaneous detection of carbapenemase or extended-spectrum-cephalosporinase activity directly from Enterobacterales (EB)-positive blood cultures (BCs). It originates from the carbapenem inactivation method (CIM), an inexpensive and highly sensitive assay for carbapenemase activity detection. dBLIM cutoff values to detect extended-spectrum ß-lactamase (ESBL) and carbapenemase activities resulted in diameters of ≤12 mm for a 5-µg-cefotaxime disk and for a 10-µg-meropenem disk. dBLIM assessment was determined with both aerobic and anaerobic BC bottles spiked with 422 characterized EB strains, classifiable into the following 4 phenotypic groups: (i) ESBL/AmpC-type ß-lactamase (ACBL)/carbapenemase (CARB)-nonproducing (np-ESBL/ACBL/CARB) EB (n = 116), (ii) ESBL-producing EB (n = 111), (iii) AmpC-ß-lactamase-producing EB (n = 33), and (iv) carbapenemase-producing EB (n = 162). No false-positive results were obtained in any of the np-ESBL/ACBL/CARB EB, ESBL, and AmpC groups, demonstrating an overall assay specificity of 100%. There were no significant discrepancies in dBLIM performance between aerobic and anaerobic BCs across all groups, except with VIM-type carbapenemase-expressing EB. Interestingly, among BCs spiked with blaVIM-harboring EB, the sensitivity rates of the assay in anaerobic and aerobic bottles were 53.6% and 100%, respectively. In contrast, dBLIM performance was deemed excellent for the KPC, OXA-48, and NDM carbapenemase producers regardless of the type of bottle being tested, with a sensitivity rate ranging between 99% and 100%. Concerning the detection of the extended-spectrum cephalosporinases of the ESBL-producing and AmpC types, dBLIM sensitivities was 100% and 84 to 87%, respectively. dBLIM may be a cost-effective and highly robust phenotypic screening method for the reliable detection of carbapenemases or extended-spectrum cephalosporinases directly from BCs on the same day of bottle positivity detection.

A multiplex real-time PCR for the direct, fast, economic and simultaneous detection of the carbapenemase genes blaKPC, blaNDM, blaVIM and blaOXA-48.

A novel multiplex real-time PCR was designed to detect the clinically most important carbapenemase genes, blaKPC, blaVIM, blaNDM and blaOXA-48. The multiplex assay was verified testing genomic DNA of 24 carbapenemase-producing strains. It was validated using a blinded panel of 82 carbapenemase-producing and 50 non-producing isolates by direct colony PCR.