Carbapenemase-producing Enterobacterales Isolates Research Articles

Epidemiology and clinical significance of carbapenemases in Australia: a narrative review.

Carbapenemase-producing gram-negative bacteria (CP-GNB) infections threaten public health with high mortality, morbidity and treatment costs. Although frequencies remain low in Australia (total number of CP-GNB infections reported was 907 in 2022), blaIMP-4 has established low levels of endemicity in many states. Imipenemase metallo-β-lactamase types alone accounted for more than half of all carbapenemases in carbapenemase-producing Enterobacterales isolates in Australia, particularly in Enterobacter cloacae complex. New Delhi metallo-β-lactamase constitutes almost 25% of all carbapenemases in Australia and was identified predominantly in Escherichia coli. The OXA-48-like carbapenemases include almost 10% of all carbapenemases and are mainly seen in Klebsiella pneumoniae and E. coli. Although K. pneumoniae carbapenemase-type carbapenemases are rare in Australia, some local outbreaks have occurred. Most carbapenem-resistant (CR) Pseudomonas aeruginosa strains in Australia do not produce carbapenemases. Finally, OXA-23-like carbapenemases are overwhelmingly positive in CR-Acinetobacter baumannii strains in Australia. Treatment of CR-GNB infections challenges physicians. Of 10 new antibiotics active against at least some CR-GNB infections that are approved by the USFood and Drug Administration, just three are approved for use in Australia. In this context, there is still an unmet need for novel antibacterials that can be used for the treatmentof CR-GNB infections in Australia, as well as a pressing requirement for new mechanisms to 'de-link' antibiotic sales from their availability. In this narrative review, we aim to overview the epidemiology and clinical significance of carbapenem resistance in Australia as it pertains to Enterobacterales, P. aeruginosa and A. baumannii.

Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes.

Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Active surveillance of carbapenemase-producing Enterobacterales using genomic sequencing for hospital-based infection control interventions.

Whole-genome sequencing (WGS) is increasingly used to characterize hospital outbreaks of carbapenemase-producing Enterobacterales (CPE). However, access to WGS is variable and testing is often centralized, leading to delays in reporting of results. We describe the utility of a local sequencing service to promptly respond to facility needs over an 8-year period. The study was conducted at Royal Prince Alfred Hospital in Sydney, Australia. All CPE isolated from patient (screening and clinical) and environmental samples from 2015 onward underwent prospective WGS. Results were notified to the infection control unit in real time. When outbreaks were identified, WGS reports were also provided to senior clinicians and the hospital executive administration. Enhanced infection control interventions were refined based on the genomic data. In total, 141 CPE isolates were detected from 123 patients and 5 environmental samples. We identified 9 outbreaks, 4 of which occurred in high-risk wards (intensive care unit and/or solid-organ transplant ward). The largest outbreak involved Enterobacterales containing an NDM gene. WGS detected unexpected links among patients, which led to further investigation of epidemiological data that uncovered the outpatient setting and contaminated equipment as reservoirs for ongoing transmission. Targeted interventions as part of outbreak management halted further transmission. WGS has transitioned from an emerging technology to an integral part of local CPE control strategies. Our results show the value of embedding this technology in routine surveillance, with timely reports generated in clinically relevant timeframes to inform and optimize local control measures for greatest impact.

Predominance of OXA-48 Carbapenemase-Producing Enterobacterales in a Moroccan Hospital.

The emergence of carbapenemase-producing Enterobacterales (CPE) is a major concern that is increasingly reported worldwide. Our study aimed at investigating the resistance of CPE isolates in a Moroccan teaching hospital using phenotypic and genotypic methods. Enterobacterales strains from March to June 2018 were collected from different clinical samples. The Enterobacterales isolates resistant to third-generation cephalosporins (3GC) and/or carbapenems were subjected to the Carba NP test and an immunochromatographic test for phenotypic detection. Detection of extended-spectrum β-lactamases (ESBL) was also performed following standards. Molecular screening of carbapenemases genes (OXA-48, NDM, blaKPC, blaIMP, blaVIM, and blaOXA-24, blaOXA-23, OXA-51, OXA-58) using conventional multiplex PCR assays was also performed on 143 isolates. Enterobacterales represented 52.7% with a proportion of 21.8% of bacteria resistant to 3GC and/or carbapenems. Within 143 isolates MDR to 3GC, K. pneumoniae, E. coli, and E. cloacae represent 53.1%, 40.6%, and 6.3%, respectively. These strains were isolated mainly from urinary samples (74.8%) in patients admitted to emergency and surgical units. 81.1% of strains are producing ESBL and 29% are carbapenemase producers as confirmed by the Carba NP test, immunochromatographic test, and molecular testing. OXA-48 carriers represent 83.3% of these strains, followed by NDM with 16.7%. blaKPC, blaIMP, blaVIM, and blaOXA-24, blaOXA-23, OXA-51, OXA-58 were not detected in any of these bacteria. A high rate of CPE carrying OXA-48 among Enterobacterales resistant to 3GC and/or carbapenems isolates was found. Strict observance of hospital hygiene measures and more rational use of antibiotics are mandatory. Implantation of carbapenemases detection should be encouraged in our hospital settings to estimate the true burden of the CPE.

First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the β-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate.

MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25- > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-μl loopful of bacterial colonies was suspended in 150 μl 0.01 M Na-PBS buffer, pH 7.4, a 10 μg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study.

The increasing multi-drug carbapenem resistance among Enterobacterales are a severe health problem limiting therapeutic options and worsen the prognosis. This study characterizes carbapenemase genes and integrons among uropathogenic carbapenem resistant Enterobacterales (CRE) isolates recovered from Mansoura University Hospitals and evaluates the effect of colistin, fosfomycin and meropenem-vaborbactam on these isolates. A total of 200 Enterobacterales isolates were collected from patients with urinary tract infections. Antimicrobial susceptibility testing was performed by the disc diffusion method. Colistin susceptibility was tested using the broth microdilution method and fosfomycin and meropenem/vaborbactam susceptibility were tested by MIC Test Strips. Carbapenem resistant isolates were screened for carbapenemase activity phenotypically using the modified carbapenem inactivation method and EDTA-modified carbapenem inactivation method and genotypically by multiplex PCR. Integrons class 1 and 2 and fosA gene were assayed by PCR. Data were statistically analyzed using the Statistical Package for Social Sciences (SPSS) version 16. The Chi-square or Fisher's exact test was used to compare groups, as appropriate. Ninety-two Enterobacterales isolates were resistant to meropenem (46%); 52 E. coli and 40 K. pneumoniae strains. All CRE isolates were multi-drug resistant (MDR). Sensitivity of CRE isolates to colistin, fosfomycin and meropenem/vaborbactam were 67.4%, 82.6% and 58.7%, respectively. Carbapenemase genes were detected by multiplex PCR in 69.6% of CRE isolates (Carbapenemase producing Enterobacterales (CPE) mainly blaNDM (37%). CPE isolates were significantly more resistant to meropenem/vaborbactam than non-CPE isolates; 51.6% vs 17.8%, respectively (P = 0.003) especially blaNDM carrying isolates (70.6%). Class 1 integrons and fosA gene were detected in 91.3% and 11.9% of CRE isolates, respectively. This study revealed that about half of the uropathogenic Enterobacterales isolates were MDR CRE. Carbapenemase gene blaNDM was the main gene among CRE isolates. Meropenem/vaborbactam sensitivity was significantly higher on non-CPE than CPE isolates and limited by the predominance of blaNDM .

Implementation of Chromatic Super CAZ/AVI® medium for active surveillance of ceftazidime-avibactam resistance: preventing the loop from becoming a spiral

Acquired resistance towards ceftazidime-avibactam (CAZ-AVI) is increasingly reported. Several mechanisms can be involved, but mutations in the Ω-loop region of β-lactamases are the most described. Herein, we assessed the implementation of Chromatic Super CAZ/AVI® medium in rectal swab surveillance cultures in a geographic area with endemic distribution of KPC-producing Klebsiella pneumoniae. Routine rectal swabs collected from the intensive care unit (ICU) and non-ICU patients were screened for carbapenemase-producing Enterobacterales (CPE), carbapenem-resistant Gram-negative organisms (CR-GN) and CAZ-AVI-resistant organisms by Chromatic CRE and Super CAZ/AVI® media. Among the 1839 patients screened, 146 (7.9%) were found to be colonized by one or more CPE and/or CR-GN isolates during hospitalization. Overall, among colonized patients the most common bacteria encountered were KPC-producing Enterobacterales (n = 60; 41.1%), carbapenem-resistant Pseudomonas aeruginosa (n = 41; 28.1%) and carbapenem-resistant A. baumannii (n = 34; 23.3%). Among patients colonized by KPC-producing Enterobacterales, thirty-five (58.3%) had CAZ-AVI-resistant strains. A 30.5% rate of faecal carriage of CAZ-AVI-resistant KPC-producing K. pneumoniae, substantially higher than that of susceptible isolates (2.8%), was observed in the COVID-19 ICU. Prevalence of faecal carriage of metallo-β-lactamase-producing organisms was low (0.5% and 0.2% for Enterobacterales and P. aeruginosa, respectively). Chromatic Super CAZ/AVI® medium showed 100% sensitivity in detecting CPE or CR-GN isolates resistant to CAZ-AVI regardless of both MIC values and carbapenemase content. Specificity was 86.8%. The Chromatic Super CAZ/AVI® medium might be implemented in rectal swab surveillance cultures for identification of patients carrying CAZ-AVI-resistant organisms to contain the spread of these difficult-to-treat pathogens.

Comparison of carbapenem minimum inhibitory concentrations of Oxacillin-48-like Klebsiella pneumoniae by Sensititre, Vitek 2, MicroScan, and Etest

ObjectivesThe aim of this laboratory-based study was to compare carbapenem MICs yielded by Sensititre, Vitek 2, MicroScan WalkAway plus and Etest for Oxacillin (OXA)-48-like Klebsiella pneumoniae isolates. MethodsAnalysis was performed for categorical agreement for ertapenem, meropenem, and imipenem, and the proportion of isolates with MICs ≤8μg/mL and the MIC50/MIC90 for meropenem and imipenem, from a convenience sample of 82 deduplicated blood culture OXA-48-like K. pneumoniae isolates. ResultsThe proportion of isolates testing susceptible to ertapenem by Etest (19/82, 23.1%) differed from Sensititre/Vitek (0/82) and MicroScan (2/82, 2.4%) (p < 0.001 for all). For meropenem, the proportion of isolates susceptible by Etest (31/82, 37.8%) differed from Sensititre/Vitek (16/82, 19.5%) (p = 0.015). There was variation in the proportion of isolates that tested imipenem susceptible when comparing Sensititre (9/82, 11%) and Vitek (8/82, 9.8%) to MicroScan (27/82, 32.9%), p = 0.001 and p < 0.001, respectively, Sensititre and Vitek to Etest (45/82, 54.9%), p < 0.001 for both, and MicroScan to Etest, p = 0.007. The proportion of isolates with meropenem MICs ≤8μg/mL with Sensititre and Vitek differed significantly from Etest, 58.5% and 85.4%, respectively, p < 0.001. A 2-fold difference between the Sensititre and Vitek meropenem and imipenem MIC at which ≥50% of isolates were inhibited compared to the MicroScan, and a 4-fold difference compared to Etest, was present. ConclusionsSubstantial variability in carbapenem MICs for OXA-48-like carbapenemase-producing Enterobacterales isolates by the four methods was demonstrated. Performance characteristics verification of MIC methods in use for the predominant carbapenemase-producing Enterobacterales type is required by laboratories to optimize the accuracy of carbapenem reporting.

Whole genome sequencing reveals hidden transmission of carbapenemase-producing Enterobacterales

Carbapenemase-producing Enterobacterales (CPE) infection control practices are based on the paradigm that detected carriers in the hospital transmit to other patients who stay in the same ward. The role of plasmid-mediated transmission at population level remains largely unknown. In this retrospective cohort study over 4.7 years involving all multi-disciplinary public hospitals in Singapore, we analysed 779 patients who acquired CPE (1215 CPE isolates) detected by clinical or surveillance cultures. 42.0% met putative clonal transmission criteria, 44.8% met putative plasmid-mediated transmission criteria and 13.2% were unlinked. Only putative clonal transmissions associated with direct ward contact decreased in the second half of the study. Both putative clonal and plasmid-mediated transmission associated with indirect (no temporal overlap in patients’ admission period) ward and hospital contact did not decrease during the study period. Indirect ward and hospital contact were identified as independent risk factors associated with clonal transmission. In conclusion, undetected CPE reservoirs continue to evade hospital infection prevention measures. New measures are needed to address plasmid-mediated transmission, which accounted for 50% of CPE dissemination.

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

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

Clonal spread of carbapenemase-producing Enterobacteriaceae in a region, China

BackgroundThe increasing number of carbapenemase-producing Enterobacterales (CPE) has become a serious problem globally. This study aimed to elucidate their geographically epidemiological characteristics.MethodsResistance genes were identified by polymerase chain reaction (PCR) and sequencing. Bacterial genotyping was studied using multilocus sequence typing (MLST) and wzi typing. The transferability of carbapenemase genes was determined by a broth mating method. The relationships between the rates of antimicrobial consumption and the prevalence of CRE were performed by Pearson's or Spearman's correlation analyses.ResultsA total of 930 phenotypically confirmed carbapenem-resistant Enterobacterales (CRE) isolates collected from 19 hospitals were genotypically characterized. K. pneumoniae (KP) and E. coli isolates were 785 (85.14%) and 96 (10.41%) among 922 CPE isolates. Two major carbapenemase genes blaKPC-2 and blaNDM in CPE isolates accounted for 84.6% (n = 780) and 13.77% (n = 127). ST11 comprised 86.83% (633/729) of KPC-2 KP isolates. Different combinations of extended spectrum-β-lactamase (ESBL) genes of blaSHV, blaCTX, and blaTEM were found in KPC-2 producing KP isolates, and blaCTM-M-14/15, blaSHV-11/12 and blaTEM-1 were common ESBL genotypes. The wzi typing method could further subdivide ST11 KP group into at least five subgroups, among which wzi209 (69.83%, 442/633) was the most frequently isolated, followed by wzi141 (25.28%, 160/633). Conjugation assays showed that high conjugation rates were observed in CPE (15.24%, 32/210) for NDM plasmids, but relatively low (8.1%, 17/210) for KPC-2 plasmids. Different STs, different wzis and temperature could influence plasmid conjugation efficiency. No associations between the rates of antibiotics consumption and CPE prevalence were observed. The number of intra-hospital and inter-hospital transfers of CPE patients increased gradually from 18 (17.82%, 101) and 12 (11.88%, 101) in 2015 to 63 (30.73%, 205) and 51 (24.88%, 205) in 2018 (p = 0.016 and p = 0.008), respectively. Evidence-based measures could effectively reduce the prevalence of ST11-wzi209 clone but failed to control the dissemination of ST11-wzi141 KP clone.ConclusionsClonal spread of CPE, especially KPC-2 ST11 KP was the key factor contributing to the CPE increase in the region. Continued vigilance for the importations should be maintained. Coordinated regional interventions are urgently needed to reduce CPE threat.

Differences in the Distribution of Species, Carbapenemases, Sequence Types, Antimicrobial Heteroresistance and Mortality Rates Between Pediatric and Adult Carbapenemase-Producing Enterobacterales in Bloodstream Infections.

The dissemination of carbapenemase-producing Enterobacterales (CPE) is worrisome given their scarce treatment options. CPE bloodstream infections (BSIs) had a high mortality rate in adults, and there was little data on pediatric CPE-BSIs around the world. We comprehensively explored the differences in the clinical and microbiological characteristics between pediatric and adult CPE-BSIs. Forty-eight pediatric and 78 adult CPE-BSIs cases were collected. All-cause 30 day-mortality in children with CPE-BSIs (14.6%, 7/48) was significantly lower than that in adult patients (42.3%, 33/78, p = 0.001). The subgroup in adults empirically treated with tigecycline as an active drug displayed a significantly higher 30-days crude mortality (63.3%, 19/30) than the subgroup treated without tigecycline (29.2%, 14/48, p = 0.003). K. pneumoniae was the most prevalent species in both the pediatric (45.8%, 22/48) and adult populations (64.1%, 50/78), with discrepant carbapenemase genes in each population: 95.4% (21/22) of the pediatric K. pneumoniae isolates carried blaNDM, while 82.0% (41/50) of the adult strains harbored blaKPC. The ratio of E. coli in children (37.5%) was significantly higher than that in adults (12.8%, p = 0.002). In both populations, the majority of E. coli expressed blaNDM, particularly blaNDM−5. With statistical significance, blaNDM was much more common in children (95.8%, 46/48) than in adults (34.6%, 27/78). The rate of multiple-heteroresistance phenotypes in children was as high as 87.5%, which was much lower in adults (57.1%). Agar dilution checkboard experiment against one pediatric carbapenemase-producing E. coli isolates showed that the combination of amikacin and fosfomycin yielded an additive effect. Overall, K. pneumoniae was the most common CPE-BSIs pathogen in both populations, with NDM-producing K. pneumoniae and KPC-producing ST11 K. pneumoniae being the most prevalent species in children and adults, respectively. E. coli was more prevalent in children than in adults, yet blaNDM−5 was the most common carbapenem-resistant mechanism in E. coli in both populations. The wide range of multiple-heteroresistance combination traits found in different pathogen species from different host populations should provide a good foundation for future combination therapy design. Further investigations from more CPE isolates of various species are needed to evaluate the possible in vitro partial synergy of the amikacin and fosfomycin combination.

A Longitudinal Nine-Year Study of the Molecular Epidemiology of Carbapenemase-Producing Enterobacterales Isolated From a Regional Hospital in Taiwan: Predominance of Carbapenemase KPC-2 and OXA-48.

Enterobacterales clinical isolates are now being resistant to clinically achievable concentrations of most commonly used antibiotics that makes treatment of hospitalized patients very challenging. We hereby determine the molecular characteristics of carbapenemase genes in carbapenem-resistant Enterobacterales (CRE) isolates in Taiwan. A total of 455 CRE isolates were identified between August 2011 to July 2020. Minimum inhibitory concentrations for selected carbapenems were tested using Vitek 2, and carbapenemase genes were determined using polymerase chain reaction in combination with sequencing. Phenotypic detection of carbapenemase was determined by modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) to validate our PCR screening results. Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of carbapenemase-producing Enterobacterales (CPE) isolates, and the transferability of carbapenemase-carrying plasmids was determined by conjugation assays. A slight increase in carbapenem-resistant E. coli (CREC) was observed, however, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) was steady, during 2011–2020. The dominant species among our CRE was K. pneumoniae (270/455, 59.3%), followed by E. coli (81/455, 17.8%), Morganella morganii (32/455, 7.0%), and Enterobacter cloacae (25/455, 5.5%). From 2011 to 2020, the total percentage of CPE increased steadily, accounting for 61.0% of CRE in 2020. Moreover, 122 of 455 CRE isolates (26.8%) were CPE. Among the CPE isolates, the dominant carbapenemase gene was blaOXA–48–like (54/122, 44.3%), and the second most common carbapenemase gene was blaKPC–2 (47/122, 38.5%). The sensitivity and specificity for mCIM to detect carbapenemase in the 455 isolates were both 100% in this study. The PFGE results showed that 39 carbapenemase-producing E. coli and 69 carbapenemase-producing K. pneumoniae isolates carrying blaKPC–2 and/or blaNDM–5 could be classified into 5 and 12 clusters, respectively. In conclusion, our results showed an increase in CPE isolates in Taiwan. Moreover, the distribution of carbapenemase and antimicrobial susceptibility in CPE were associated with PFGE typing.

Resistome and virulome accretion in an NDM-1-producing ST147 sublineage of Klebsiella pneumoniae associated with an outbreak in Tuscany, Italy: a genotypic and phenotypic characterisation

Carbapenemase-producing Enterobacterales (CPE), particularly those producing metallo-β-lactamases, are among the most challenging antibiotic-resistant pathogens, causing outbreaks of difficult-to-treat nosocomial infections worldwide. Since November 2018, an outbreak of New Delhi metallo-β-lactamases-positive CPE (NDM-CPE) has emerged in Tuscany, Italy. In this study, we aimed to investigate the NDM-CPE associated with the outbreak and characterise the responsible Klebsiella pneumoniae clone. We used whole-genome sequencing and bioinformatic analysis to characterise NDM-CPE isolates that caused bloodstream infections in 53 patients at 11 hospitals in Tuscany and that were collected between Jan 1, 2018, and July 5, 2019 (ie, the early phase of the outbreak and preceding months). The CPE isolates characterised in this study were isolated and identified at the species level and as NDM producers by six diagnostic microbiology laboratories that serve the 11 hospitals. We used comparative genomic analysis, antimicrobial susceptibility testing, plasmid conjugal transfer assays, evaluation of virulence potential in the Galleria mellonella infection model, and serum bactericidal assays to further characterise the clone causing the outbreak. The outbreak was sustained by an ST147 K pneumoniae producing NDM-1, which had a complex resistome that mediated resistance to most antimicrobials (except cefiderocol, the aztreonam-avibactam combination, colistin, and fosfomycin). The clone belonged to a sublineage of probably recent evolution, occurred by the sequential acquisition of an integrative and conjugative element encoding the yersiniabactin siderophore, an FIB(pQil)-type multiresistance plasmid carrying blaNDM-1, and a transferable chimeric plasmid, derived from virulence elements of hypervirulent K pneumoniae, carrying several resistance and virulence determinants. Infection of G mellonella larvae revealed a variable virulence potential. The behaviour in serum bactericidal assays was different from typical hypervirulent K pneumoniae strains, with variable grades of serum resistance apparently associated with mutations in specific chromosomal loci (csrD, pal, and ramR). This description of a sublineage of ST147 K pneumoniae with a complex resistome and virulome that is capable of sustaining a large regional outbreak adds to existing research on the evolutionary trajectories within high-risk clones of K pneumoniae. Global surveillance programmes are warranted to track the dissemination of these lineages, and to prevent and control their spread. Italian Ministry of Health and Department of Experimental and Clinical Medicine, University of Florence.

Multicenter surveillance of in vitro activities of cefepime-zidebactam, cefepime-enmetazobactam, omadacycline, eravacycline, and comparator antibiotics against Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex causing bloodstream infection in Taiwan, 2020

ABSTRACT Objectives To determine the in vitro activities of novel and comparator antibiotics against Gram-negative bacteria (GNB) in Taiwan. Methods Isolates of Escherichia coli (n = 335), Klebsiella pneumoniae (n = 316; 144 isolates with hyperviscosity characteristics), Pseudomonas aeruginosa (n = 271), Acinetobacter baumannii complex (n = 187), and non-typhoidal Salmonella species (n = 226), Shigella species (n = 13) from miscellaneous culture sources were collected in 2020 in Taiwan. The MICs of the isolates to test antibiotics were determined using the broth microdilution method. GeneXpert was used to detect genes encoding carbapenemases among the carbapenem-non-susceptible (NS) Enterobacterales isolates. Results The MIC values of the cefepime-enmetazobactam combination against extended-spectrum β-lactamase-producing E. coli and K. pneumoniae isolates (MIC90 ≤ 0.5 mg/L), bla KPC-harboring E. coli isolates (0.25 mg/L; n = 2), and 80% of bla OXA-48-like gene-harboring K. pneumoniae isolates (≤2 mg/L) were low. The MIC ranges of the cefepime-zidebactam against carbapenemase-producing Enterobacterales isolates (irrespective of the carbapenemase type [MIC90 ≤ 4 mg/L]) and carbapenem-NS or ceftolozane-tazobactam-NS P. aeruginosa isolates (MIC90 value, 8 mg/L) were significantly lower than those of the cefepime-enmetazobactam. Conclusions The efficacy of novel antibiotics against important drug-resistant GNB must be monitored and validated during the clinical treatment of patients.

Evaluating NG-Test CARBA 5 Multiplex Immunochromatographic and Cepheid Xpert CARBA-R Assays among Carbapenem-Resistant Enterobacterales Isolates Associated with Bloodstream Infection.

ABSTRACTDecreased susceptibility to carbapenems in Enterobacterales is an emerging concern. Conventional methods with short turnaround times are crucial for therapeutic decisions and infection control. In the current study, we used the Xpert CARBA-R (Cepheid, Sunnyvale, CA, USA) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) assays for carbapenemase detection in 214 carbapenem-resistant Enterobacterales (CRE) blood isolates. We used the modified carbapenem inactivation method, conventional PCR, and sequencing to determine the production of five common carbapenemase families and their subtypes. We performed wzc-genotyping for all CR-Klebsiella pneumoniae (CRKP) and multilocus sequence typing for all carbapenemase-producing CRE isolates to reveal their genetic relatedness. The results showed a sensitivity of 99.8% and a specificity of 100% by the Xpert assay, and a sensitivity of 100% and a specificity of 99% by the NG-Test in detecting carbapenemases of 84 CRKP isolates with only one (VIM-1+IMP-8) failure in both tests. For CR-Escherichia coli, four carbapenemase-producing isolates were detected accurately for their subtypes. The two major clones of carbapenemase-producing CRKP isolates in Taiwan were ST11-K47 producing KPC-2 (n = 47) and ST11-K64 producing OXA-48-like (n = 9). Our results support the use of either test in routine laboratories for the rapid detection of common carbapenemases. Caution should be taken using the Xpert assay in areas with a high prevalence of CRE carrying blaIMP-8.IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) are emerging worldwide, causing nosocomial outbreaks and even community-acquired infections since their appearance 2 decades ago. Our previous national surveillance of CPE isolates in Taiwan identified five carbapenemase families (KPC, OXA, NDM, VIM, and IMP) with the KPC-2 and OXA-48-like types predominant. Timely detection and classification of carbapenemases in CPE may be a useful test to guide optimal therapy and infection control. Genetic detection methods using the Xpert CARBA-R assay and the immunochromatographic assay using the NG-Test CARBA 5 have been validated with the advantage of short turnaround time. Our study demonstrated that the NG and Xpert assays are convenient methods to accurately identify carbapenemases in carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Escherichia coli blood isolates. Detecting IMP variants remains challenging, and the results of Xpert CARBA-R assay should be carefully interpreted.

NDM-1 carbapenemase resistance gene vehicles emergent on distinct plasmid backbones from the IncL/M family.

To assess the genetic contexts surrounding blaNDM-1 genes carried on IncM plasmids harboured by six carbapenemase-producing Enterobacterales (CPE) isolates referred to the UK Health Security Agency's Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit. Between 2014 and 2018, the AMRHAI Reference Unit undertook WGS of CPE isolates using Illumina NGS. Nanopore sequencing was used for selected isolates and publicly available plasmid references were downloaded. Analysis of incRNA, which encodes the antisense RNA regulating plasmidic repA gene expression, was performed and bioinformatics tools were used to analyse whole plasmid sequences. Of 894 NDM-positive isolates of Enterobacterales, 44 NDM-1-positive isolates of five different species (Citrobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca) encoded the IncRNA locus of IncM2 plasmids. Long-read sequencing of six diverse isolates revealed related IncM2, NDM-1-encoding plasmids. Plasmid 'backbone' areas were conserved and contrasted with highly variable resistance regions. Sub-groupings of IncM2 plasmids encoding blaNDM-1 were detected; one sub-group occurred in five different health regions of England in every year. The diversity of NDM-1-encoding resistance gene integrons and transposons and their insertions sites in the plasmids indicated that NDM-1 has been acquired repeatedly by IncM2 variants. The use of sequencing helped inform: (i) a wide geographical distribution of isolates encoding NDM-1 on emergent IncM2 plasmids; (ii) variant plasmids have acquired NDM-1 separately; and (iii) dynamic arrangements and evolution of the resistance elements in this plasmid group. The geographical and temporal distribution of IncM2 plasmids that encode NDM-1 highlights them as a public health threat that requires ongoing monitoring.