A comparative evaluation of five phenotypic methods for identification of carbapenemase-producing Enterobacteriaceae: a modified carbapenemase detection test.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) is urgently needed to prevent their spread in healthcare settings. Here, we have evaluated the performance of the phenotypic methods for detection of carbapenemase production directly from bacterial cultures. A total of 99 clinical and rectal Enterobacteriaceae isolates were included (81 carrying known carbapenemase-encoding genes and 18 without carbapenemase production). All isolates were subjected to the five phenotypic tests including in-house Carba NP (iCarba NP), modified-Carba NP, E-Test MBL, modified Hodge test (MHT), and commercial combination disk test. Test results were read at different time points for iCarba NP and modified-Carba (1 min, 5 min, 15 min, 1 h and 2 h). The sensitivity and specificity of the iCarba NP were 78.87% and 100%, respectively, whereas those of the modified-Carba NP test were 95.06% and 94.44%, respectively. False-negative results were detected in four OXA-48 isolates with the use of modified-Carba NP, whereas one non-carbapenemase isolate had false-positive results. The sensitivity/specificity was 91.30%/100% and 80.25%/83.33% for the E-Test MBL and MHT, respectively. The sensitivity and specificity of the aminophenylboronic acid synergy test were 100% and 97.94%, respectively, whereas those of the dipicolinic acid synergy test were 82.61% and 96.23%, respectively. Rapid, simple, and reliable methods are needed for laboratory detection of CPE isolates to improve the detection and surveillance of these clinically relevant pathogens in an epidemiological context. We conclude that the modified-Carba NP test can be one of the reliable tests for the prediction of carbapenemase-producing bacteria.IMPORTANCEThe emergence of carbapenem resistance among Gram-negative bacteria is a serious global health threat. Here, we investigate the performance of the five phenotypic assays against carbapenemase-producing and carbapenemase-non-producing Enterobacteriaceae. Accurate and rapid detection of CPE isolates is critically required for clinical management and treatment of infections caused by these organisms. Among the five evaluated phenotypic tests, the mCNP test presented the highest sensitivity (95.06%) and, therefore, can be considered the best test to be used as a screening phenotypic methodology.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

Impact of removing ESBL status labelling from culture reports on the use of carbapenems for non-bacteraemic patients diagnosed with ESBL-positive urinary tract infections.

OBJECTIVES: To evaluate carbapenem prescribing rates for initial definitive treatment of urinary tract infections and clinical outcomes before and after removing ESBL status labels on antibiotic susceptibility reports. METHODS: This was a retrospective cohort study of adult patients treated for at least 48 h for an ESBL-producing/ceftriaxone-resistant Enterobacterales urinary tract infection. ESBL status reporting ceased in September 2022 for a network of seven community hospitals within the USA. The primary endpoint was the rate of carbapenem prescribing for initial definitive treatment of urinary tract infections. Secondary endpoints included total days of therapy for initial definitive treatment with carbapenems, clinical cure rates, time to transition to oral antibiotic therapy for initial definitive treatment, rate of guideline-compliant therapy, rate of relapsed infection within 30 days, 30 day readmission rate, and 30 day all-cause in-hospital mortality. RESULTS: Of 3055 patients screened, 199 were included in the pre group and 153 were included in the post group. The rate of carbapenem prescribing for initial definitive treatment was 156 patients (78%) in the pre group, compared with 93 patients (61%) in the post group (P = <0.01). Days of therapy for initial definitive therapy with carbapenem was 620 in the pre group compared with 372 in the post group (P < 0.01). There was no difference between other secondary outcomes. CONCLUSIONS: Removing ESBL status labels from laboratory reports reduced carbapenem use for initial definitive treatment of urinary tract infections from 78% to 61% (P < 0.01) without impacting clinical outcomes.

Prevalence and molecular epidemiology of carbapenemase-producing Enterobacterales isolated from dog and cat faeces submitted to veterinary laboratories in the USA.

AIMS: To estimate the prevalence of carbapenemase-producing Enterobacterales (CPE) carriage among pets using faecal specimens submitted to veterinary diagnostic laboratories throughout the US. A secondary aim was to employ whole-genome sequencing (WGS) to characterize isolates of CPE from companion animals and compare them to publicly available CPE genomes. METHODS AND RESULTS: To estimate the prevalence of CPE in companion animals in the USA, a multicenter surveillance study including 8 different veterinary diagnostic laboratories from across the USA was conducted. Briefly, remnant faecal specimens from dogs and cats were screened using two selective agar plates (CHROMID Carba and MacConkey with 1 mg/L cefotaxime and 0.125 mg/L meropenem) and presumptive CPE isolates screened by the modified carbapenemase inactivation method for carbapenemase production. A total of 2393 specimens were screened and yielded 196 isolates for carbapenemase screening. A total of 5 isolates from 4 dogs and 1 cat at 3 different veterinary diagnostic laboratories were confirmed to produce a carbapenemase (0.21%). Whole-genome sequencing (WGS) revealed two E. coli (ST167) isolates that both produced an NDM-5 carbapenemase, two Enterobacter hormaechei (ST171) isolates that produced an NDM-5 carbapenemase and a KPC-4 carbapenemase respectively and one Klebsiella oxytoca (ST199) that produced an Oxa-48-type carbapenemase. Both E. coli isolates were found to be within at least 22 SNPs of previously characterized canine and human CPE isolates. CONCLUSIONS: This study demonstrates that the prevalence of CPE among companion animals is relatively low (0.21%) but that given the genetic relatedness of animal isolates to human isolates, additional surveillance is needed.

Global epidemiology and antimicrobial resistance of Enterobacterales harbouring genes encoding OXA-48-like carbapenemases: insights from the results of the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme 2018-2021.

OBJECTIVES: The recent emergence of carbapenem-resistant Enterobacterales poses a major and escalating threat to global public health. This study aimed to analyse the global distribution and antimicrobial resistance of Enterobacterales harbouring variant OXA-48-like carbapenemase-related genes. METHODS: Enterobacterales isolates were collected from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme during 2018-2021. Comprehensive antimicrobial susceptibility testing and ß-lactamase gene detection were also conducted, along with statistical analysis of the collected data. RESULTS: Among the 72 244 isolates, 1934 Enterobacterales isolates were identified to harbour blaOXA-48-like genes, predominantly Klebsiella spp. (86.9%). High rates of multidrug resistance were observed, with only ceftazidime/avibactam and tigecycline showing favourable susceptibility. A discrepancy between the genotype and phenotype of carbapenem resistance was evident: 16.8% (233 out of 1384) of the Enterobacterales isolates with blaOXA-48-like genes exhibited susceptibility to meropenem. Specifically, 37.4% (64/95) of Escherichia coli strains with blaOXA-48-like genes displayed meropenem susceptibility, while the corresponding percentages for Klebsiella pneumoniae and Enterobacter cloacae complex were 25.2% (160/1184) and 0% (0/36), respectively (P < 0.05). Geographical analysis revealed that the highest prevalence of blaOXA-48-like genes occurred in Asia, the Middle East and Eastern Europe. The proportion of K. pneumoniae isolates harbouring blaOXA-232 increased from 23.9% in 2018 to 56.0% in 2021. By contrast, the proportion of blaOXA-48 decreased among K. pneumoniae isolates during 2018-2021. CONCLUSIONS: This study underscores the widespread and increasing prevalence of blaOXA-48-like genes in Enterobacterales and emphasizes the need for enhanced surveillance, improved diagnostic methods and tailored antibiotic stewardship to combat the spread of these resistant pathogens.

The formate-hydrogen axis and its impact on the physiology of enterobacterial fermentation.

Formic acid (HCOOH) and dihydrogen (H2) are characteristic products of enterobacterial mixed-acid fermentation, with H2 generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H2, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H2 and CO2 by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H2 production in enterobacteria.

Multidrug-resistance and extended-spectrum beta-lactamase-producing lactose-fermenting enterobacteriaceae in the human-dairy interface in northwest Ethiopia.

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.

Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to ß-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

OBJECTIVES: The blaNDM gene was prevalent among children and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%), and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%) and was dominant in blaNDM-Klebsiella penumoniae (79.8%), blaNDM-Escherichia coli (58.2%), and blaNDM-Enterobacter cloacae (61.0%), respectively. Most (79.0%) clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, P <0.01). CONCLUSION: blaNDM is highly prevalent in CRE among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Rapid detection of plasmid-mediated AmpC-producers by eazyplex® SuperBug AmpC assay compared to whole-genome sequencing.

Current methods for plasmid-mediated AmpC ß-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of Enterobacterales without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated Escherichia coli isolates. The remaining 28 E. coli isolates harboured previously described mutations in the blaEC promoter, leading to the overexpression of chromosomally encoded E. coli specific AmpC ß-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in Enterobacterales like Klebsiella spp. and Proteus spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in E. coli.

Antimicrobial activity of ceftazidime-avibactam against KPC-2-producing Enterobacterales: a cross-combination and dose-escalation titration study with relebactam and vaborbactam.

With the introduction of ceftazidime-avibactam worldwide, the antimicrobial activity of new ß-lactam/ß-lactamase inhibitors (BL/BLIs) needs to be investigated. From January 2020 to June 2023, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were collected. With a broth microdilution test of new BL/BLIs, cross-activity test with nine combinations of BLs and new BLIs and dose-escalation titration test for non-susceptible isolates were conducted to investigate inhibitory activities of new BLIs. A total of 188 isolates was collected and most isolates (186/188, 98.9%) carried the KPC-2 gene exclusively, while two isolates (1.1%) co-harbored NDM-1. Among the 186 KPC-2-producing isolates, 184 (98.9%) were susceptible to ceftazidime-avibactam, 173 (93.0%) to imipenem-relebactam, and 184 (98.9%) to meropenem-vaborbactam. All isolates non-susceptible to imipenem-relebactam or meropenem-vaborbactam became susceptible when avibactam replaced relebactam or vaborbactam, with 7 of 11 (63.6%) imipenem-relebactam non-susceptible isolates and both (100.0%) of the meropenem-vaborbactam non-susceptible isolates. When the minimum inhibitory concentrations (MICs) of BLs were compared using log2 scales, combinations with avibactam showed statistically significant efficacy in lowering MICs compared to relebactam and vaborbactam (all P < 0.05). In the dose-escalation test of new BLIs, increasing dose of all new BLIs corresponded to increased susceptibility to BLs. Ceftazidime-avibactam exhibited excellent susceptibility against KPC-2-producing Enterobacterales unless co-harboring metallo-ß-lactamase. The cross-combination test against non-susceptible isolates suggests that the inhibitory activity of avibactam was superior to those of relebactam or vaborbactam. Increasing the dose of new BLIs produced increased susceptibility to BLs, suggesting that high-concentration regimen need to be developed. IMPORTANCE: This study investigated 188 Klebsiella pneumoniae carbapenemase (KPC)-2-producing Enterobacterales collected from January 2020 to June 2023 in a tertiary care hospital of Korea. Most isolates were susceptible to ceftazidime-avibactam (98.9%) and meropenem-vaborbactam (98.9%), while susceptibility to imipenem-relebactam was lower (93.0%). The cross-combination test using nine combinations of the individual ß-lactams (BLs) and new ß-lactamase inhibitors (BLIs) showed that the inhibitory activity of avibactam was significantly superior to relebactam or vaborbactam when the Log2 MIC of BLs were compared for each combination with BLIs (all P < 0.05). The dose-escalation test of new BLIs demonstrated that increasing doses of new BLIs corresponded to increased susceptibility to BLs. Taken together, this study illustrates the excellent activity of ceftazidime-avibactam against KPC-2-producing Enterobacterales and suggests further investigation into high-concentration regimens for potentially non-susceptible clinical isolates.

MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

PURPOSE: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. METHOD: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in ß-lactamases, and compared with that of the reference double disk synergy test. ß-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several ß-lactamases, and 22 strains that produced other ß-lactamases. RESULTS: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. CONCLUSION: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

A practical approach to screening for carbapenemase-producing Enterobacterales- views of a group of multidisciplinary experts from English hospitals.

INTRODUCTION: Carbapenemase-producing Enterobacterales (CPE) are an important public health threat, with costly operational and economic consequences for NHS Integrated Care Systems and NHS Trusts. UK Health Security Agency guidelines recommend that Trusts use locally developed risk assessments to accurately identify high-risk individuals for screening, and implement the most appropriate method of testing, but this presents many challenges. METHODS: A convenience sample of cross-specialty experts from across England met to discuss the barriers and practical solutions to implementing UK Health Security Agency framework into operational and clinical workflows. The group derived responses to six key questions that are frequently asked about screening for CPE. KEY FINDINGS: Four patient groups were identified for CPE screening: high-risk unplanned admissions, high-risk elective admissions, patients in high-risk units, and known positive contacts. Rapid molecular testing is a preferred screening method for some of these settings, offering faster turnaround times and more accurate results than culture-based testing. It is important to stimulate action now, as several lessons can be learnt from screening during the COVID-19 pandemic, as well as from CPE outbreaks. CONCLUSION: Further decisive and instructive information is needed to establish CPE screening protocols based on local epidemiology and risk factors. Local management should continually evaluate local epidemiology, analysing data and undertaking frequent prevalence studies to understand risks, and prepare resources- such as upscaled screening- to prevent increasing prevalence, clusters or outbreaks. Rapid molecular-based methods will be a crucial part of these considerations, as they can reduce unnecessary isolation and opportunity costs.