Phenotypic and genotypic discrepancies for carbapenemase-producing Citrobacter freundii in multiple isolates from a single patient.

BACKGROUND: Carbapenemase-producing gram-negative organisms continue to be a significant healthcare concern and a therapeutic challenge. Members of the genus Citrobacter have emerged as increasingly multidrug resistant and versatile healthcare-associated pathogens. In this study we investigated five KPC-producing Citrobacter freundii isolates, from the same patient, that presented unusual phenotypic characteristics including false susceptibility to carbapenems detection by culture-based methods. METHODS: The isolates were tested for antimicrobial susceptibility using broth microdilution and disk diffusion. Production of serine carbapenemase was confirmed with the mCIM (modified carbapenem inactivation method) test. Genotypes were determined by PCR and whole genome sequencing analysis. RESULTS: The five isolates were susceptible to meropenem by broth microdilution and presented varying colonial morphologies and levels of susceptibility to carbapenems by multiple phenotypic methods, despite being positive for carbapenemase production by mCIM and positive for blaKPC by PCR. Whole genome sequence analysis showed that three of the five highly related isolates harbor an additional gene cassette, including blaCARB-2, ant(2''), aadA2, dfrA19, catB3, cmlA1, mph(E), msr(E), and qnrA1. The presence of these genes explains the difference in phenotypes observed. CONCLUSION: Failure to detect and completely eradicate the carbapenemase-producing C. freundii in the urine with ertapenem therapy, likely due to the presence of a heterogeneous population, resulted in the phenotypic and genotypic adaptations of the organism as it disseminated to the bloodstream and kidneys. The fact that carbapenemase-producing C. freundii can elude detection by phenotypic methods and can so easily acquire and transfer resistance gene cassettes is of concern.

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

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

Evaluation of the Xpert Carba-R NxG Assay for Detection of Carbapenemase Genes in a Global Challenge Set of Pseudomonas aeruginosa Isolates.

The growing prevalence and diversity of carbapenemase producers among carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates warrants an expansion of detection capabilities. The purpose of this study was to evaluate the performance of the commercially available Xpert Carba-R (Carba-R) and the research-use-only Xpert Carba-R NxG (Carba-R NxG) in a global collection of P. aeruginosa The challenge set included 123 P. aeruginosa clinical isolates from 12 countries. Isolates were previously categorized via PCR or whole-genome sequencing. Carbapenemase classes tested include VIM, IMP, NDM, SPM, KPC, and GES. Non-carbapenemase (non-CP)-harboring isolates were also tested (negative control). Isolates were tested using the Carba-R NxG and the Carba-R tests per the manufacturer's instructions. Carba-R NxG testing was completed by Cepheid (Sunnyvale, CA), blinded to genotype. Both assays gave negative results for all non-CP isolates and positive results for all VIM, NDM, and KPC isolates. An improvement in IMP detection among isolates was observed (100% detection by Carba-R NxG versus 58% by Carba-R). All SPM and GES isolates, targets not present in commercially available Carba-R, were positive by Carba-R NxG. Two isolates harbored both VIM and GES, while a third isolate contained VIM and NDM. The Carba-R NxG identified both targets in all 3 isolates, while the Carba-R was negative for both GES-containing isolates. Overall, the Carba-R NxG successfully categorized 100% of isolates tested compared with 68% for its predecessor. The Carba-R NxG will expand the detection spectrum of the current Carba-R assay to include SPM, GES, and expanded IMP variants, increasing the global utility of the test.

Emergence of Oxacillin Resistance in Stealth Methicillin-Resistant Staphylococcus aureus Due to mecA Sequence Instability.

Staphylococcus aureus strains that possess a mecA gene but are phenotypically susceptible to oxacillin and cefoxitin (OS-MRSA) have been recognized for over a decade and are a challenge for diagnostic laboratories. The mechanisms underlying the discrepancy vary from isolate to isolate. We characterized seven OS-MRSA clinical isolates of six different spa types from six different states by whole-genome sequencing to identify the nucleotide sequence changes leading to the OS-MRSA phenotype. The results demonstrated that oxacillin susceptibility was associated with mutations in regions of nucleotide repeats within mecA Subinhibitory antibiotic exposure selected for secondary mecA mutations that restored oxacillin resistance. Thus, strains of S. aureus that contain mecA but are phenotypically susceptible can become resistant after antibiotic exposure, which may result in treatment failure. OS-MRSA warrant follow-up susceptibility testing to ensure detection of resistant revertants.

Updating Molecular Diagnostics for Detecting Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Isolates in Blood Culture Bottles.

Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test (in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCCmec)-orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa, which produced a false S. aureus-negative result. A control strain of S. aureus that harbored an SCCmec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCCM1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCCmec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCCmec cassettes, 1.6% carried SCCM1, <1% had spa deletions, and <1% contained SCCmec variants other than those with SCCM1 These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay.

Streptococcus agalactiae Strains with Chromosomal Deletions Evade Detection with Molecular Methods.

Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb, the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States.

Changes in molecular epidemiology and antimicrobial resistance profiles of Clostridioides (Clostridium) difficile strains in the United States between 2011 and 2017.

PCR ribotyping and antimicrobial susceptibility testing were used to characterize 940 Clostridioides (Clostridium) difficile isolates collected from 26 U S. hospitals over three time periods from 2011 to 2017. The proportion of ribotype (RT) 027 isolated during the three surveys decreased significantly over time from 31% in 2011-2012, to 22% in 2013-2014, and to 14% in 2015-2017 (p < 0.001 and p = 0.010, respectively), while we observed an increase in prevalence of RT106, that rose from 7% in our first survey to 19% of isolates in our last survey (p < 0.001). In addition, both RT056 and RT002 rose from 3% to 10% (p < 0.001). The proportions of all other ribotypes remained steady over time, and RT014/020 was the third most common strain type in our convenience sample in the final survey. Overall, resistance to moxifloxacin, rifampin, and vancomycin decreased during our studies, mainly due to the decline in RT027 isolates. A decrease in moxifloxacin resistance and an increase in tetracycline resistance were found among RT027 strains isolated in the last survey. Although the proportion of RT027 isolates declined, multidrug resistance among this ribotype continues to be common.

Strain types and antimicrobial resistance patterns of Clostridium difficile isolates from the United States, 2011 to 2013.

We determined the PCR ribotypes and antimicrobial susceptibility patterns of 508 toxigenic Clostridium difficile isolates collected between 2011 and 2013 from 32 U.S. hospitals. Of the 29 PCR ribotypes identified, the 027 strain type was the most common (28.1%), although the rates varied by geographic region. Ribotype 014/020 isolates appear to be emerging. Clindamycin and moxifloxacin resistances (36.8% and 35.8%, respectively) were the most frequent resistance phenotypes observed. Reduced susceptibility to vancomycin was observed in 39.1% of 027 isolates.

Characterization of Beta-Lactamase and Fluoroquinolone Resistance Determinants in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa Isolates from a Tertiary Hospital in Yola, Nigeria.

Infections due to antimicrobial resistant gram-negative bacteria cause significant morbidity and mortality in sub-Saharan Africa. To elucidate the molecular epidemiology of antimicrobial resistance in gram-negative bacteria, we characterized beta-lactam and fluoroquinolone resistance determinants in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates collected from November 2017 to February 2018 (Period 1) and October 2021 to January 2022 (Period 2) in a tertiary medical center in north-eastern Nigeria. Whole genome sequencing (WGS) was used to identify sequence types and resistance determinants in 52 non-duplicate, phenotypically resistant isolates. Antimicrobial susceptibility was determined using broth microdilution and modified Kirby-Bauer disk diffusion methods. Twenty sequence types (STs) were identified among isolates from both periods using WGS, with increased strain diversity observed in Period 2. Common ESBL genes identified included blaCTX-M, blaSHV, and blaTEM in both E. coli and K. pneumoniae. Notably, 50% of the E. coli in Period 2 harbored either blaCTX-M-15 or blaCTX-M-1 4 and phenotypically produced ESBLs. The blaNDM-7 and blaVIM-5 metallo-beta-lactamase genes were dominant in E. coli and P. aeruginosa in Period 1, but in Period 2, only K. pneumoniae contained blaNDM-7, while blaNDM-1 was predominant in P. aeruginosa. The overall rate of fluoroquinolone resistance was 77% in Period 1 but decreased to 47.8% in Period 2. Various plasmid-mediated quinolone resistance (PMQR) genes were identified in both periods, including aac(6')-Ib-cr, oqxA/oqxB, qnrA1, qnrB1, qnrB6, qnrB18, qnrVC1, as well as mutations in the chromosomal gyrA, parC and parE genes. One E. coli isolate in Period 2, which was phenotypically multidrug resistant, had ESBL blaCTX-M-15, the serine carbapenemase, blaOXA-181 and mutations in the gyrA gene. The co-existence of beta-lactam and fluoroquinolone resistance markers observed in this study is consistent with widespread use of these antimicrobial agents in Nigeria. The presence of multidrug resistant isolates is concerning and highlights the importance of continued surveillance to support antimicrobial stewardship programs and curb the spread of antimicrobial resistance.

Characterization of Carbapenemase- and ESBL-Producing Gram-Negative Bacilli Isolated from Patients with Urinary Tract and Bloodstream Infections.

A total of 199 Gram-negative bacterial isolates from urinary tract infections and 162 from bloodstream infections were collected from 12 healthcare systems throughout the United States between May 2021 and August 2022. The isolates, phenotypically non-susceptible to 2nd or 3rd generation cephalosporins or carbapenems, were characterized through antimicrobial susceptibility testing and whole genome sequence analysis to obtain a broad snapshot of beta-lactamase-mediated resistance among these two sample types. Overall, 23 different carbapenemase genes were detected among 13 species (20.5% of isolates). The blaKPC-3 and blaKPC-2 subtypes were the most common carbapenemase genes identified, followed by blaNDM and the co-carriage of two different blaOXA carbapenemases by Acinetobacter baumannii isolates. All carbapenemase-producing A. baumannii isolates were mCIM negative. Extended-spectrum beta-lactamase genes were identified in 66.2% of isolates; blaCTX-M-15 was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of blaKPC-positive organisms were susceptible to ertapenem, imipenem, and meropenem, respectively. The correlation between broth microdilution and disk diffusion results was high for most drugs except cefepime, where the detection of resistance was statistically lower by disk diffusion. Thus, there were gaps in the accuracy of susceptibility testing for some mechanisms of resistance.

Phenotypic and molecular characterization of beta-lactam resistant Multidrug-resistant Enterobacterales isolated from patients attending six hospitals in Northern Nigeria.

Infections caused by multi-drug resistant Enterobacterales (MDR-E) are difficult to treat and cause significant mortality, especially in developing countries. This study characterized the phenotypic and genotypic profiles of 49 randomly selected beta-lactam resistant MDR-E previously isolated from patients being managed in hospitals in Nigeria using whole genome sequencing. The study isolates exhibited 85.5% resistance to 3rd generation cephalosporins and 65.3% resistance to carbapenems. The blaTEM-1B (29, 59.2%), blaCTX-M-15 (38, 77.6%), and blaNDM-1 (17, 51.5%) were the most common penicillinase, ESBL, and carbapenem resistant genes across isolates, respectively. Seventeen (45%) of blaCTX-M-15 was carried on the insertion sequence ISEc9 while blaNDM-1 (11, 64.7%) were associated with ISEc33. None of the 21 plasmids detected were associated with ß-lactamase genes. Higher resistance rates were found in E. coli ST-88 (n = 2) and the high-risk ST-692 (n = 2). For Klebsiella species, the high-risk clones ST-476 (n = 8) and ST-147 (n = 3) predominated and had higher phenotypic resistance rates and higher number of AMR genes. The mechanisms and pattern of antibiotic resistance differ from patterns previously described with isolates harbouring a wide range of AMRGs. The detection of several chromosomally mediated carbapenemases in our study also represents a significant finding that warrants further investigation to better understand its' implications for clinical practice and public health. The selected MDR-Es were found to be pan-susceptible to tigecycline and had very low resistance to fosfomycin, suggesting a potential for these as empiric treatments. A surveillance approach incorporating both conventional laboratory techniques and modern molecular techniques is essential for the comprehensive characterization of the emergence and dissemination of antimicrobial resistance in Enterobacterales infections within Nigeria.

The diagnostic accuracy of the GeneXpert ESBL-ampC prototype assay for rapid PCR-based detection of extended-spectrum beta-lactamase genes directly from urine.

IMPORTANCE: Early identification of complicated urinary tract infections caused by ESBL-producing Enterobacterales has the potential to limit the use of carbapenems to those patients without alternative antibiotic options and avoid the empirical use of carbapenems in patients without ESBL-producing bacteria. The purpose for such a test will differ by setting and ESBL prevalence rates. Countries with low ESBL rates and cephalosporins as empiric treatment (e.g., The Netherlands) will need a rule-in test to decide to use carbapenems, while countries with high ESBL rates and empiric carbapenem treatment will need a rule-out test for ESBLs to de-escalate therapy early. Anyway, such as a test would-at least theoretically-improve patient care and reduce selective pressure for the emergence of carbapenem resistance.

Antimicrobial-resistant strains of Clostridium difficile from North America.

A total of 316 toxigenic Clostridium difficile clinical isolates of known PCR ribotypes from patients in North America were screened for resistance to clindamycin, metronidazole, moxifloxacin, and rifampin. Clindamycin resistance was observed among 16 different ribotypes, with ribotypes 017, 053, and 078 showing the highest proportions of resistance. All isolates were susceptible to metronidazole. Moxifloxacin resistance was present in >90% of PCR-ribotype 027 and 053 isolates but was less common among other ribotypes. Only 7.9% of the C. difficile isolates were resistant to rifampin. Multidrug resistance (clindamycin, moxifloxacin, and rifampin) was present in 27.5% of PCR-ribotype 027 strains but was rare in other ribotypes. These results suggest that antimicrobial resistance in North American isolates of C. difficile varies by strain type and parallels rates of resistance reported from Europe and the Far East.

Characterization of nasal and blood culture isolates of methicillin-resistant Staphylococcus aureus from patients in United States Hospitals.

A total of 299 nares and 194 blood isolates of methicillin-resistant Staphylococcus aureus (MRSA), each recovered from a unique patient, were collected from 23 U.S. hospitals from May 2009 to March 2010. All isolates underwent spa and staphylococcal cassette chromosome mec element (SCCmec) typing and antimicrobial susceptibility testing; a subset of 84 isolates was typed by pulsed-field gel electrophoresis (PFGE) using SmaI. Seventy-six spa types were observed among the isolates. Overall, for nasal isolates, spa type t002-SCCmec type II (USA100) was the most common strain type (37% of isolates), while among blood isolates, spa type t008-SCCmec type IV (USA300) was the most common (39%). However, the proportion of all USA100 and USA300 isolates varied by United States census region. Nasal isolates were more resistant to tobramycin and clindamycin than blood isolates (55.9% and 48.8% of isolates versus 36.6% and 39.7%, respectively; for both, P < 0.05). The USA300 isolates were largely resistant to fluoroquinolones. High-level mupirocin resistance was low among all spa types (<5%). SCCmec types III and VIII, which are rare in the United States, were observed along with several unusual PFGE types, including CMRSA9, EMRSA15, and the PFGE profile associated with sequence type 239 (ST239) isolates. Typing data from this convenience sample suggest that in U.S. hospitalized patients, USA100 isolates of multiple spa types, while still common in the nares, have been replaced by USA300 isolates as the predominant MRSA strain type in positive blood cultures.

Detection of Methicillin-Resistant Staphylococcus aureus Infections Using Molecular Methods.

The application of molecular detection methods for bacterial pathogens has dramatically improved the outcomes of septic patients, including those with methicillin-resistant Staphylococcus aureus (MRSA) infections. Molecular methods can be applied to a variety of clinical specimens including nasal swabs, growth in blood culture bottles, and wounds. While data show that the overall accuracy of molecular tests for MRSA is high, results can be confounded by the presence of multiple staphylococcal species in a specimen, insertions and deletions of DNA in and around the Staphylococcal Cassette Chromosome mec (SCCmec) element, and point mutations in mecA. Herein, we explore the complexities of molecular approaches to MRSA detection and the instances where phenotypic methods should be pursued to resolve discrepancies between genotypic and phenotypic results.

Mechanisms of carbapenemase-mediated resistance among high-risk Pseudomonas aeruginosa lineages in Peru.

OBJECTIVES: Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections globally. High-risk carbapenemase-encoding P. aeruginosa clones are disseminating in many regions. The aim of this study was to learn more about the lineages and mechanisms of resistance of P. aeruginosa circulating in Peru. METHODS: A total of 141 carbapenemase-producing isolates recovered from hospitalized and ambulatory patients in Lima were sequenced and analyzed to infer their lineages through whole-genome sequence typing (wgST) and to identify their antimicrobial resistance genes. RESULTS: wgST identified nine sequence types (STs); ST111 and ST357 were the most frequently encountered (44.0% and 38.3%, respectively), followed by ST179 (8.5%), with the remaining six detected only sporadically. Among ST357 isolates, 96.3% carried the novel blaIMP-93 allele, whereas the remainder harbored blaIMP-74. 74.2% of ST111 isolates co-harbored blaIMP-18 and blaVIM-2, while the rest carried either of these genes individually. All other ST lineages carried a single carbapenemase, which was either blaIMP-16, blaIMP-74, or blaVIM-2. CONCLUSION: Our study shows that the high-risk P. aeruginosa clones ST357, which harbors the novel blaIMP-93, and ST111, which carries blaIMP-18 and blaVIM-2, have apparently become endemic in the region.

Characterization of carbapenem-resistant gram-negative bacterial isolates from Nigeria by whole genome sequencing.

This study characterized the mechanisms of carbapenem resistance in gram-negative bacteria isolated from patients in Yola, Nigeria. Whole genome sequencing (WGS) was performed on 66 isolates previously identified phenotypically as carbapenem-non-susceptible. The patterns of beta-lactamase resistance genes identified were primarily species-specific. However, blaNDM-7 and blaCMY-4 were detected in all Escherichia coli and most Providencia rettgeri isolates; blaNDM-7 was also detected in 1 Enterobacter cloacae. The E. coli and E. cloacae isolates also shared blaOXA-1, while blaOXA-10 was found in all P. rettgeri, one Pseudomonas aeruginosa and 1 E. coli. Except for Stenotrophomonas maltophilia isolates, which only contained blaL1, most species carried multiple beta-lactamase genes, including those encoding extended-spectrum beta-lactamases, AmpC and OXA in addition to a carbapenemase gene. Carbapenemase genes were either class B or class D beta-lactamases. No carbapenemase gene was detected by WGS in 13.6% of isolates.

Characterisation of carbapenem-resistant Gram-negative organisms from clinical specimens in Yola, Nigeria.

OBJECTIVES: This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria. METHODS: Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes. RESULTS: Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-ß-lactamase (blaNDM) gene was detected in 26 isolates and Verona integron-encoded metallo-ß-lactamase (blaVIM) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates. CONCLUSION: While blaNDM and blaVIM accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates.

Mobile genetic elements responsible for discordant Staphylococcus aureus phenotypes and genotypes in the same blood culture bottle.

Approximately 15-20% of the S. aureus genome contains mobile genetic elements that can cause discrepancies between phenotypic and genotypic identification methods. Three blood culture bottles (each from a different patient) that showed discordant results, were shown to contain 2 S. aureus isolates after additional subcultures. One bottle had MRSA and MSSA that by DNA sequence analysis differed only by 31 kb; however, the deletions encompassed parts of SCCmec including mecA and SCCM1. The second bottle contained MRSA and MSSA that differed by 124 kb; the MSSA was missing the entire SCCmec and spa regions. The last bottle contained 2 MRSA, one with ACME II disrupting SCCmec and a 24 bp spa deletion. The deletions in SCCmec and the other elements gave rise to the discrepancies between molecular and the original culture results. Such discrepancies should prompt a search for additional strains in the blood culture bottle.

Does the presence of multiple ß-lactamases in Gram-negative bacilli impact the results of antimicrobial susceptibility tests and extended-spectrum ß-lactamase and carbapenemase confirmation methods?

OBJECTIVES: Many multidrug-resistant Gram-negative bacilli (MDR-GNB) harbour multiple ß-lactamases. The aim of this study was to assess the impact of multiple ß-lactamase carriage on the accuracy of susceptibility tests and extended-spectrum ß-lactamase (ESBL) and carbapenemase confirmation methods. METHODS: A total of 50 MDR-GNB, of which 29 carried multiple ß-lactamases, underwent broth microdilution (BMD) and disk diffusion (DD) testing as well as confirmation tests for ESBLs and carbapenemases. Whole-genome sequencing (WGS) was used for ß-lactamase gene identification. RESULTS: Categorical agreement of BMD and DD testing results ranged from 86.5 to 97.7% for 10 ß-lactam agents. BMD and DD algorithms for ESBL detection were highly variable; 6 of 8 positive strains carried an ESBL plus a carbapenemase or an AmpC enzyme, which may confound antimicrobial selection. The sensitivity and specificity of the modified carbapenem inactivation method (mCIM) were both 100%, whilst mCIM and EDTA-modified carbapenem inactivation method (eCIM) when used together to differentiate serine from metallo-ß-lactamase carriage were both 96%. Xpert® Carba-R results (in vitro diagnostic test) were consistent with WGS results. Predicting phenotypic carbapenem resistance from WGS data overall showed 100% specificity but only 66.7% sensitivity for Enterobacterales isolates that were non-susceptible to imipenem and meropenem. CONCLUSIONS: Multiple ß-lactamases in MDR-GNB does not impact DD results, the utility of mCIM/eCIM tests, or Xpert Carba-R results. However, ESBL algorithms produced inconsistent results and predicting carbapenem resistance from WGS data was problematic in such strains.