Carbapenem Minimum Inhibitory Concentrations Research Articles

Molecular epidemiology of Carbapenemase-encoding genes and comparative evaluation of carbapenem MIC with genotypic carbapenem resistance in Klebsiella isolates from neonatal sepsis cases

Objectives: The objective of this study was to determine the molecular epidemiology of Carbapenemase-encoding genes in Klebsiella isolates from neonatal sepsis cases and comparative evaluation of carbapenem minimum inhibitory concentration (MIC) with genotypic carbapenem resistance. Materials and Methods: One hundred cases of neonatal sepsis with blood cultures positive for Klebsiella spp. were included in the study. MIC for imipenem and meropenem was determined by Epsilometer-test. Antimicrobial susceptibility testing (AST) was performed by modified Kirby Bauer disc diffusion method. All the isolates of Klebsiella spp. were tested for the presence of beta-lactamase Klebsiella pneumoniae carbapenemase (blaKPC ), beta-lactamase New Delhi metalloβ-lactamase-1(blaNDM-1), beta-lactamase imipenemase (blaIMP), beta-lactamase Verona imipenemas e (blaVIM) genes by multiplex polymerase chain reaction (PCR) and uniplex PCR for beta-lactamase oxacillinase-48 (blaOXA-48). Comparison of individual antibiotic susceptibility between carbapenemase-encoding gene positive and negative Klebsiella spp. isolates was performed. Statistical analysis: Statistical analysis was done using the Fisher’s exact test. P < 0.05 was considered significant. Results: The prevalence of carbapenemase-encoding genes in Klebsiella spp. was 16%. Most predominant carbapenemase-encoding gene was blaOXA-48 gene (12%) followed by blaNDM-1 gene (6%). Coexpression of both blaOXA-48 and blaNDM-1 was observed in 2% of isolates. All the Klebsiella spp. isolates harboring the carbapenemases gene (100%) had resistant MIC values for Meropenem, whereas, for imipenem, only 75% of isolates had resistant MIC values. Conclusions: Determination of prevalence of carbapenemase-encoding genes is of paramount importance in the development of effective antibiotic policies at various levels.

Characterization of Verona Integron-Encoded Metallo-β-Lactamase-Type Carbapenemase-Producing Escherichia coli Isolates Collected over a 16-Year Period in Bolzano (Northern Italy).

Multidrug-resistant Escherichia coli, particularly carbapenemase producers, are a major source of concern. This study aims to investigate the long-term epidemiology of Verona integron-encoded metallo-β-lactamase (VIM)-producing E. coli in the health district of Bolzano, Northern Italy, by examining the phenotypic and genotypic characteristics of 26 isolates obtained during 2005-2020. Isolates were identified with matrix-assisted laser desorption/ionization time-of-flight, susceptibility testing was by Vitek 2, Sensititre, and Etest; carbapenemase activity was confirmed by Etest and Carbapenemase Inactivation Method (CIM) test; and the VIM-antigen was identified by the NG-Test CARBA 5. Genome sequencing was performed on an Illumina MiSeq platform. Carbapenem minimum inhibitory concentrations varied across methodologies, and overall category agreement between phenotypic methods was low. All 23 sequenced isolates contained blaVIM-1. Eleven (47.8%) isolates belonged to the clonal lineage ST131, with fimH30 being the most common subclone. In Bolzano ST131-fimH30 was present as early as 2005. While the ST131 clonal lineage predominated for the first 10 years, various clonal lineages were present, especially in subsequent years, indicating the concurrent circulation of multiple clonal lineages. Future efforts should focus on the implementation of surveillance methods, including genomic analysis, as well as the use of updated infection control strategies and antibiotic stewardship programs to prevent the spread of these carbapenem-resistant strains.

Global genomic epidemiology of chromosomally mediated non-enzymatic carbapenem resistance in Acinetobacter baumannii: on the way to predict and modify resistance.

Although carbapenemases are frequently reported in resistant A. baumannii clinical isolates, other chromosomally mediated elements of resistance that are considered essential are frequently underestimated. Having a wide substrate range, multidrug efflux pumps frequently underlie antibiotic treatment failure. Recognizing and exploiting variations in multidrug efflux pumps and penicillin-binding proteins (PBPs) is an essential approach in new antibiotic drug discovery and engineering to meet the growing challenge of multidrug-resistant Gram-negative bacteria. A total of 980 whole genome sequences of A. baumannii were analyzed. Nucleotide sequences for the genes studied were queried against a custom database of FASTA sequences using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) system. The correlation between different variants and carbapenem Minimum Inhibitory Concentrations (MICs) was studied. PROVEAN and I-Mutant predictor suites were used to predict the effect of the studied amino acid substitutions on protein function and protein stability. Both PsiPred and FUpred were used for domain and secondary structure prediction. Phylogenetic reconstruction was performed using SANS serif and then visualized using iTOL and Phandango. Exhibiting the highest detection rate, AdeB codes for an important efflux-pump structural protein. T48V, T584I, and P660Q were important variants identified in the AdeB-predicted multidrug efflux transporter pore domains. These can act as probable targets for designing new efflux-pump inhibitors. Each of AdeC Q239L and AdeS D167N can also act as probable targets for restoring carbapenem susceptibility. Membrane proteins appear to have lower predictive potential than efflux pump-related changes. OprB and OprD changes show a greater effect than OmpA, OmpW, Omp33, and CarO changes on carbapenem susceptibility. Functional and statistical evidence make the variants T636A and S382N at PBP1a good markers for imipenem susceptibility and potential important drug targets that can modify imipenem resistance. In addition, PBP3_370, PBP1a_T636A, and PBP1a_S382N may act as potential drug targets that can be exploited to counteract imipenem resistance. The study presents a comprehensive epidemiologic and statistical analysis of potential membrane proteins and efflux-pump variants related to carbapenem susceptibility in A. baumannii, shedding light on their clinical utility as diagnostic markers and treatment modification targets for more focused studies of candidate elements.

Interplay between OXA-10 β-Lactamase Production and Low Outer-Membrane Permeability in Carbapenem Resistance in Enterobacterales.

The OXA-10 class D β-lactamase has been reported to contribute to carbapenem resistance in non-fermenting Gram-negative bacilli; however, its contribution to carbapenem resistance in Enterobacterales is unknown. In this work, minimum inhibitory concentrations (MICs), whole genome sequencing (WGS), cloning experiments, kinetic assays, molecular modelling studies, and biochemical assays for carbapenemase detection were performed to determine the impact of OXA-10 production on carbapenem resistance in two XDR clinical isolates of Escherichia coli with the carbapenem resistance phenotype (ertapenem resistance). WGS identified the two clinical isolates as belonging to ST57 in close genomic proximity to each other. Additionally, the presence of the blaOXA-10 gene was identified in both isolates, as well as relevant mutations in the genes coding for the OmpC and OmpF porins. Cloning of blaOXA-10 in an E. coli HB4 (OmpC and OmpF-deficient) demonstrated the important contribution of OXA-10 to increased carbapenem MICs when associated with porin deficiency. Kinetic analysis showed that OXA-10 has low carbapenem-hydrolysing activity, but molecular models revealed interactions of this β-lactamase with the carbapenems. OXA-10 was not detected with biochemical tests used in clinical laboratories. In conclusion, the β-lactamase OXA-10 limits the activity of carbapenems in Enterobacterales when combined with low permeability and should be monitored in the future.

Phenotypic and genotypic detection of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Accra, Ghana.

To describe the occurrence of carbapenem resistance among multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae isolated from clinical specimens in Accra using phenotypic and genotypic methods. The study was cross-sectional, involving 144 clinical MDR E. coli and K. pneumoniae isolates recovered from the Central Laboratory of the Korle Bu Teaching Hospital (KBTH). The isolates were re-cultured bacteriologically, identified using standard biochemical tests, and subjected to antibiotic susceptibility testing using the Kirby-Bauer method. Carbapenem resistance was determined based on imipenem, meropenem, and ertapenem zones of inhibition, as well as minimum inhibitory concentrations (MICs). Carbapenemase production was determined phenotypically by modified Hodge test (MHT) and modified carbapenem inactivation method (mCIM), and genotypically with multiplex PCR targeting the blaKPC, blaIMP, blaNDM, blaVIM, and blaOXA-48 genes. Of the 144 MDR isolates, 69.4% were E. coli, and 30.6% were K. pneumoniae. The distribution of antimicrobial resistance rates among them was ampicillin (97.2%), cefuroxime (93.1%), sulfamethoxazole-trimethoprim (86.8%), tetracycline (85.4%), cefotaxime and cefpodoxime (77.1% each), amoxicillin-clavulanate (75%), ceftriaxone (73.6%), ciprofloxacin (70.8%), levofloxacin (66.0%), cefepime (65.3%), ceftazidime (64.6%), gentamicin (48.6), piperacillin-tazobactam (40.3%), cefoxitin (14.6%), amikacin (13.9%), ertapenem and meropenem (5.6% each), and imipenem (2.8%). In total, 5.6% (8/144) of them were carbapenem-resistant (carbapenem MIC range = 0.094-32.0 μg/ml), with 75% (6/8) of these testing positive by the phenotypic tests and 62.5% (5/8) by the genotypic test (of which 80% [4/5] carried blaOXA-48 and 20% (1/5) blaNDM). The blaVIM, blaIMP, and blaKPC genes were not detected. Although the rates of antibiotic resistance among the isolates were high, the prevalence of carbapenemase producers was low. The finding of blaOXA-48 and blaNDM warrants upscaling of antimicrobial resistance surveillance programmes and fortification of infection prevention and control programmes in the country.

145. Harnessing the Inoculum Effect to Diagnose Molecular Mechanisms of Carbapenem Resistance

Abstract Background The global spread of carbapenem-resistant Enterobacterales (CRE) presents a major threat to public health. CRE employ two molecular mechanisms to evade carbapenems: 1) expression carbapenemases (CPases), which efficiently hydrolyze carbapenems or 2) disruption of porins, which reduces carbapenem influx to levels that can be hydrolyzed by certain beta-lactamases. Diagnosing mechanisms underlying carbapenem resistance is important for infection control and to administer appropriate treatments. Methods We measured Meropenem and Ertapenem minimum inhibitory concentrations (MICs) for 103 clinical Enterobacterales isolated from hospitals in Massachusetts and California using broth microdilution assays at 14 inocula spanning four orders of magnitude. They represented 30 isolates encoding CPases and intact porins, 46 isolates with disruptions in one or both of the porins responsible for carbapenem influx (OmpC and OmpF), 25 encoding CPases with disrupted porins, and two controls encoding intact porins and no CPases. Results We observed that the two mechanisms result in distinct profiles; first the carbapenem MICs of CPase-encoding strains show a strong inoculum dependence (Fig 1.A), whereas the MICs of porin deficient strains remain largely constant at all inocula (Fig 1.B). The synergistic action of these two mechanisms leads to high-level resistance that we termed “hyper-CRE” (Fig 1.C). Together these factors explain the level of resistance in nearly all our CRE isolates. To validate the hyper-CRE phenotype, we successfully employed CRISPR-based gene editing to show that knocking out the major porin in CPase-producing strains elevates their carbapenem resistance to hyper-CRE levels. We also determined 18% of our isolates changed susceptibility classification within the Clinical Laboratory and Standards Institute (CLSI) recommended inoculum range. This is worrisome for the treatment of infections with strains that are deemed susceptible via in vitro AST assays but are truly resistant in vivo. Conclusion Overall, our approach has demonstrated that measuring MICs at different inoculum can yield crucial diagnostic information about mechanisms of resistance which has important implications for patient care, infection control, and surveillance of emerging CPases. Disclosures All Authors: No reported disclosures.

Discovery of an Effective Small-Molecule Allosteric Inhibitor of New Delhi Metallo-β-lactamase (NDM).

To identify novel inhibitors of the carbapenemase New Delhi metallo-β-lactamase (NDM) as possible therapeutic compounds, we conducted a high-throughput screen of a 43,358-compound library. One of these compounds, a 2-quinazolinone linked through a diacylhydrazine to a phenyl ring (QDP-1) (IC50 = 7.9 ± 0.5 μM), was characterized as a slow-binding reversible inhibitor (Kiapp = 4 ± 2 μM) with a noncompetitive mode of inhibition in which substrate and inhibitor enhance each other's binding affinity. These studies, along with differential scanning fluorimetry, zinc quantitation, and selectivity studies, support an allosteric mechanism of inhibition. Cotreatment with QDP-1 effectively lowers minimum inhibitory concentrations of carbapenems for a panel of resistant Escherichia coli and Klebsiella pneumoniae clinical isolates expressing NDM-1 but not for those expressing only serine carbapenemases. QDP-1 represents a novel allosteric approach for NDM drug development for potential use alone or with other NDM inhibitors to counter carbapenem resistance in enterobacterales.

1281. An Evaluation of Tebipenem In Vitro Activity Against a Panel of Pseudomonas aeruginosa Isolates with Efflux, AmpC, and OprD Mutations

1281. An Evaluation of Tebipenem <i>In Vitro</i> Activity Against a Panel of <i>Pseudomonas aeruginosa</i> Isolates with Efflux, AmpC, and OprD Mutations

Co-occurrence of Carbapenemase-encoding Genes Among Klebsiella pneumoniae Clinical Isolates: Positive Relationship of bla NDM and bla SIM with Imipenem Resistance

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-KP), known as a significant public health threat, is the most common causative agent of nosocomial and community-acquired infections. Objectives: This study aimed to evaluate resistance to carbapenems and determine the prevalence of carbapenemase genes and multilocus sequence typing (MLST) of K. pneumoniae clinical isolates. Methods: One-hundred K. pneumoniae isolates were evaluated. The minimum inhibitory concentrations (MIC) of imipenem and meropenem were assessed by the broth microdilution method. Multiplex-polymerase chain reaction (PCR) was applied to detect 11 carbapenemase-encoding genes belonging to different classes. The alleles and sequence types (ST) of three isolates were identified by MLST. Results: The MIC of carbapenems for the isolates ranged from 0.062 to 32 µg/mL. Overall, resistance rates to imipenem and meropenem were reported 11% and 34%, respectively. The bla IMP gene was the most abundant (78.4%), followed by bla OXA-48 (48.6%), bla GIM (27%), bla KPC (27%), bla SIM (21.6%), bla BIC (21.6%), bla NDM (16.2%), bla AIM (16.2%), bla VIM (16.2%), bla DIM (8.1%), and bla SPM (8.1%). The co-existence of carbapenemase genes was observed in 81.8% of the isolates. A positive relationship was found between the presence of bla NDM and bla SIM and resistance to imipenem. Multilocus sequence typing results showed three different sequence types, including ST14, ST5188, and ST1861. Conclusions: This study revealed a high prevalence of CR-KP isolates that suggests a high risk of horizontal gene transfer and potential to spread resistance among other strains. Since STs are reported for the first time in Iran, they can be considered as emerging strains.

Carbapenemase detection testing in the era of ceftazidime/avibactam-resistant KPC-producing Enterobacterales: A 2-year experience

ObjectivesThe aim of this study was to investigate the prevalence of ceftazidime/avibactam (CZA) resistance among carbapenemase-producing Enterobacterales (CPE) blood culture isolates as well as the performance of the main carbapenemase phenotypic detection methods to identify KPC variants associated with CZA resistance. MethodsNon-duplicate CPE strains isolated from blood cultures during 2018–2020 were tested for antimicrobial susceptibility. Molecular testing was used to identify carbapenemase-producers. Strains harbouring blaKPC and with a CZA minimum inhibitory concentration (MIC) ≥8 mg/L were investigated by sequencing. Subsequentially, five phenotypic carbapenemase detection methods were evaluated on these strains, namely the modified carbapenem inactivation method (mCIM), Rapidec® Carba NP, the disk diffusion synergy test, NG-Test CARBA® 5 and RESIST-5 O.O.K.N.V. ResultsOverall, the CZA resistance rate was high (13.7%) and remained relevant (5.9%) excluding metallo-β-lactamases-producers. All isolates harbouringblaKPC mutants (n = 8) were associated with reduced carbapenem MICs and negative results by all detection methods based on revelation of enzyme activity. Lateral flow immunoassays failed to detect KPC-31 (n = 4) and KPC-33 (n = 2) but correctly identified KPC-14 (n = 2). Conversely, isolates harbouring wild-type KPC genes (n = 3) were associated with high-level CZA resistance and carbapenem resistance and tested positive by all of the evaluated methods. ConclusionIn the era of CZA-based therapies, molecular blaKPC identification followed by a carbapenem hydrolysis-based phenotypic assay could be the most reasonable diagnostic algorithm to detect all KPC-producers and to identify mutants associated with impaired carbapenemase activity and CZA resistance.

Evaluation of Discrepancies in Carbapenem Minimum Inhibitory Concentrations Obtained at Clinical Laboratories Compared to a Public Health Laboratory

Background: Automated testing instruments (ATIs) are commonly used by clinical microbiology laboratories to perform antimicrobial susceptibility testing (AST), whereas public health laboratories may use established reference methods such as broth microdilution (BMD). We investigated discrepancies in carbapenem minimum inhibitory concentrations (MICs) among Enterobacteriaceae tested by clinical laboratory ATIs and by reference BMD at the CDC. Methods: During 2016–2018, we conducted laboratory- and population-based surveillance for carbapenem-resistant Enterobacteriaceae (CRE) through the CDC Emerging Infections Program (EIP) sites (10 sites by 2018). We defined an incident case as the first isolation of Enterobacter spp (E. cloacae complex or E. aerogenes), Escherichia coli, Klebsiella pneumoniae, K. oxytoca, or K. variicola resistant to doripenem, ertapenem, imipenem, or meropenem from normally sterile sites or urine identified from a resident of the EIP catchment area in a 30-day period. Cases had isolates that were determined to be carbapenem-resistant by clinical laboratory ATI MICs (MicroScan, BD Phoenix, or VITEK 2) or by other methods, using current Clinical and Laboratory Standards Institute (CLSI) criteria. A convenience sample of these isolates was tested by reference BMD at the CDC according to CLSI guidelines. Results: Overall, 1,787 isolates from 112 clinical laboratories were tested by BMD at the CDC. Of these, clinical laboratory ATI MIC results were available for 1,638 (91.7%); 855 (52.2%) from 71 clinical laboratories did not confirm as CRE at the CDC. Nonconfirming isolates were tested on either a MicroScan (235 of 462; 50.9%), BD Phoenix (249 of 411; 60.6%), or VITEK 2 (371 of 765; 48.5%). Lack of confirmation was most common among E. coli (62.2% of E. coli isolates tested) and Enterobacter spp (61.4% of Enterobacter isolates tested) (Fig. 1A), and among isolates testing resistant to ertapenem by the clinical laboratory ATI (52.1%, Fig. 1B). Of the 1,388 isolates resistant to ertapenem in the clinical laboratory, 1,006 (72.5%) were resistant only to ertapenem. Of the 855 nonconfirming isolates, 638 (74.6%) were resistant only to ertapenem based on clinical laboratory ATI MICs. Conclusions: Nonconfirming isolates were widespread across laboratories and ATIs. Lack of confirmation was most common among E. coli and Enterobacter spp. Among nonconfirming isolates, most were resistant only to ertapenem. These findings may suggest that ATIs overcall resistance to ertapenem or that isolate transport and storage conditions affect ertapenem resistance. Further investigation into this lack of confirmation is needed, and CRE case identification in public health surveillance may need to account for this phenomenon.Funding: NoneDisclosures: None

Routinely available antimicrobial susceptibility information can be used to increase the efficiency of screening for carbapenemase-producing Enterobacteriaceae.

Introduction. Increased carbapenem resistance is often caused by carbapenemase production.Aim. The objective of our study was to assess which antibiotic susceptibility patterns, as tested by automated systems, are highly associated with the absence of carbapenemase production in Enterobacteriaceae isolates, and could therefore be used as a screening tool.Methodology. Routine antibiotic susceptibility testing data from 42 medical microbiology laboratories in the Netherlands in the period between January 2011 and June 2017 were obtained from the national antimicrobial resistance surveillance programme. Data on Enterobacteriaceae isolates that had an elevated minimum inhibitory concentration (MIC) for carbapenems (meropenem >0.25 mg l-1 or imipenem >1.0 mg l-1) were selected and subjected to phenotypic or genotypic carbapenemase production testing. Routinely available amoxicillin/clavulanic acid, piperacillin/tazobactam, cefuroxime and ceftriaxone/cefotaxime susceptibilities were studied in relation to carbapenemase production by calculating the negative predictive value.Results. No evidence for carbapenemase-producing Enterobacteriaceae (CPE) was found in 767 of 1007 (76 %) isolates. The negative predictive value was highest for amoxicillin/clavulanic acid (99.6 %) and piperacillin/tazobactam (98.8 %).Conclusion. Enterobacteriaceae isolates with elevated carbapenem MICs that are susceptible to amoxicillin/clavulanic acid or piperacillin/tazobactam are unlikely to be carbapenemase producers. Preselection based on this susceptibility pattern may lead to increased laboratory efficiency and reduction of costs. Whether this is also true for countries with a different distribution of CPE species and types or a higher prevalence of CPE needs to be studied.

Carbapenem-Resistant Klebsiella pneumoniae Outbreak in a Neonatal Intensive Care Unit: Risk Factors for Mortality.

The aim of our study was to determine the factors associated with mortality in neonates with carbapenem-resistant Klebsiella pneumoniae (CRKP). This retrospective, single-center study was conducted in the Neonatal Intensive Care Unit of Harran University Faculty of Medicine between January 2017 and July 2018 who had CRKP growth in their blood, urine or cerebrospinal fluid cultures. The discharged group was designated as the control group (Group 1), whereas the group that faced mortality was classified as the case group (Group 2). The demographic data, clinical findings and laboratory and microbiological results of the two groups were compared to identify risk factors. A total of 58 patients (36 in Group 1 and 22 in Group 2) exhibited CRKP growth during the study period. Low birth weight (p = 0.039), previous antifungal (p = 0.002) or amikacin use (p = 0.040), congenital anomalies (p = 0.002), total parenteral nutrition (TPN) administration (p = 0.002), surgery (p = 0.035), thrombocytopenia (p = 0.007), low platelet mass index (p = 0.011), elevated C-reactive protein (p = 0.004), high carbapenem minimum inhibitory concentration (MIC) (p = 0.029) and high amikacin MIC (p = 0.019) were associated with mortality. In a multivariate regression analysis, previous antifungal use (p = 0.028), congenital anomalies (p = 0.032) and TPN use (p = 0.013) were independent factors in predicting mortality. Previous antifungal use, congenital anomalies and TPN use were found to be independent risk factors for mortality in neonates with CRKP infection.

Phenotypic Variation and Carbapenem Resistance Potential in OXA-499-Producing Acinetobacter pittii.

Acinetobacter pittii is increasingly recognized as a clinically important species. Here, we identified a carbapenem-non-resistant A. pittii clinical isolate, A1254, harboring blaOXA–499, blaOXA–826, and blaADC–221. The blaOXA–499 genetic environment in A1254 was identical to that of another OXA-499-producing, but carbapenem-resistant, A. pittii isolate, YMC2010/8/T346, indicating the existence of phenotypic variation among OXA-499-producing A. pittii strains. Under imipenem-selective pressure, the A1254 isolate developed resistance to carbapenems in 60 generations. Two carbapenem-resistant mutants (CAB009 and CAB010) with mutations in the blaOXA–499 promoter region were isolated from two independently evolved populations (CAB001 and CAB004). The CAB009 mutant, with a mutation at position −14 (A to G), exhibited a four-fold higher carbapenem minimum inhibitory concentration (MIC) and a 4.53 ± 0.19 log2 fold change higher expression level of blaOXA–499 than the ancestor strain, A1254. The other mutant, CAB010, with a mutation at position −42 (G to A), showed a two-fold higher carbapenem MIC and a 1.65 ± 0.25 log2 fold change higher blaOXA–499 expression level than the ancestor strain. The blaOXA–499 gene and its promoter region were amplified from the wild-type strain and two mutant isolates and then individually cloned into the pYMAb2-Hygr vector and expressed in Acinetobacter baumannii ATCC 17978, A. pittii LMG 1035, and A. pittii A1254. All the transformed strains were resistant to carbapenem, irrespective of whether they harbored the initial or an evolved promoter sequence, and transformed strains expressing the promoter from the most resistant mutant, CAB009, showed the highest carbapenem MICs, with values of 32–64 μg/ml for imipenem and 128 μg/ml for meropenem. RNA sequencing was performed to confirm the contribution of blaOXA–499 to the development of carbapenem resistance. Although the CAB009 and CAB010 transcriptional patterns were different, blaOXA–499 was the only differentially expressed gene shared by the two mutants. Our results indicate that carbapenem-non-resistant Acinetobacter spp. strains carrying blaOXA genes have the potential to develop carbapenem resistance and need to be further investigated and monitored to prevent treatment failure due to the development of resistance.

2159. Accurate Carbapenem Susceptibility Testing Within 5–6 Hours

BackgroundPatients infected with multi-drug-resistant (MDR) pathogens may experience long delays to targeted therapies due to the incomplete antimicrobial menus and/or breakpoints tested on current commercial antimicrobial susceptibility testing (AST) systems. Detection of carbapenem resistance poses a challenge to rapid, accurate, minimum inhibitory concentrations (MIC) determinations because some resistant organisms may be inhibited by a carbapenem antibiotic until sufficient carbapenemase production has been achieved and traditional AST platforms must wait to make MIC calls. More accurate carbapenem MICs can be determined by implementing a carbapenemase test alongside rapid AST.MethodsWe demonstrate a novel, proprietary test to detect carbapenemase production that enables rapid MIC testing for carbapenem antibiotics. The test is processed in parallel with the Selux next-generation phenotyping (NGP) AST method, enabling rapid, <6-hour, accurate MIC determinations. The carbapenemase assay utilizes high concentrations of intact bacteria. After 3 hours of incubation, a fluorescent pH indicator is read spectroscopically. The solution pH is lowered by carbapenemase-mediated imipenem degradation and is indicative of enzyme activity.ResultsThis assay accurately identifies carabapenemases across multiple enzyme classes and bacterial species. Figure 1 shows the accuracy and speed of NGP AST at determining MICs for representative isolates from the FDA-CDC antimicrobial resistance bank compared with results from overnight broth microdilution (BMD). To date, over 100 challenge strains of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii have been tested with no very major errors and an average time-to-result of 5.3 hours.ConclusionBy incorporating a rapid, on-board carbapenemase activity assay, the NGP AST platform rapidly delivers accurate carbapenem results. Combined with NGP’s comprehensive antibiotic menus, this platform will therefore ensure prompt delivery of personalized antibiotic therapies for all patients, including those infected with MDROs, and enable streamlined antibiotic stewardship coordination. Disclosures All authors: No reported disclosures.

Emergence and transmission of New Delhi metallo-beta-lactamase-5-producing Escherichia coli Sequence Type 361 in a Tertiary Hospital in South Korea.

BackgroundThe emergence of carbapenem‐resistant Escherichia coli (E coli) is a serious global health threat, but little is known about carbapenemase‐producing E coli in Daejeon, South Korea. The aim of this study was to investigate characteristics of thirteen carbapenem‐resistant E coli isolates in a tertiary hospital.MethodsThirteen non‐duplicate carbapenem‐resistant E coli strains were collected from October 2017 to January 2018. Antimicrobial susceptibility was determined with the E test or disk diffusion method. The carbapenem minimum inhibitory concentrations (MICs) were determined by the agar dilution method. The colistin and tigecycline MICs were determined by broth microdilution. The resistance genes, including carbapenemase genes, were evaluated by polymerase chain reaction, and DNA sequencing was performed to characterize the genes. Pulsed‐field gel electrophoresis and multilocus sequence typing (MLST) were performed to evaluate the clonal relatedness of isolates. The clinical data of patients were retrospectively reviewed.ResultsAll the E coli isolates harbored blaNDM‐5 gene and were resistant to most of the antimicrobial agents, such as carbapenem, cephalosporins, ciprofloxacin, and chloramphenicol, excluding amikacin and colistin. Other resistant genes, such as blaTEM‐1, blaCTX‐M‐15, blaCMY‐2, aac(6')‐Ib‐cr, and qepA, were detected. The E coli isolates harboring bla NDM‐5 belonged to ST361 (n = 11), ST12 (n = 1), ST410 (n = 1), and PFGE types A (n = 11), B (n = 1), and C (n = 1).ConclusionsThis study reports on an outbreak of a predominant epidemic clone, the NDM‐5 producing, multidrug‐resistant E coli ST361 isolate. These findings suggest that we should pay attention to infection control measures to limit the spread of NDM‐5‐producing pathogens.

Molecular Epidemiology of Multidrug-Resistant Klebsiella pneumoniae Isolates in a Brazilian Tertiary Hospital.

Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of blaKPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A blaKPC–2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Genomic characterization of a KPC-23-producing Klebsiella pneumoniae ST258 clinical isolate resistant to ceftazidime-avibactam

ObjectivesWe characterized the first ceftazidime-avibactam-resistant KPC-producing-Klebsiella pneumoniae clinical isolate detected in Greece, before the introduction of ceftazidime-avibactam in clinical practice. MethodsK. pneumoniae KP-90 was isolated from a hospitalized patient in Thessaloniki during a nationwide surveillance study conducted between 2014 and 2016. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing (Ion Torrent TM platform) of the isolate was carried out to identify the acquired resistance genes and mutations that were associated with ceftazidime-avibactam resistance. ResultsThe K. pneumoniae isolate belonged to multilocus sequence type ST258 and harboured blaKPC-23 as the only carbapenemase gene. The isolate had a minimum inhibitory concentration (MIC) of 16 mg/L to ceftazidime-avibactam and was highly resistant to imipenem, meropenem (MICs, 512 mg/L) and ceftazidime (MIC, >1024 mg/L). blaKPC-23 was detected on a Tn4401a transposon, located on a pKPQIL-type plasmid. A non-functional outer membrane protein OmpK35 and an OmpK36 variant that had been previously associated with K. pneumoniae isolates of ST258 were detected. Transformation studies with Escherichia coli TOP10 showed that KPC-23 offered similar carbapenem MICs as KPC-2 and KPC-3. However, KPC-23 conferred a four-fold higher ceftazidime MIC (>1024 mg/L), which in the presence of avibactam was reduced (>7-fold) to 8 mg/L, which is just within the limit of the susceptibility breakpoint. ConclusionsCeftazidime-avibactam resistance in a KPC-23- producing K. pneumoniae clinical isolate was due to increased ceftazidime hydrolysis and was likely enhanced by OmpK35 porin deficiency.

Carbapenem-resistant Acinetobacter baumannii isolates carrying blaOXA genes with upstream ISAba1: First report of a novel OXA subclass from Iran

Carbapenem-resistant Acinetobacter baumannii (CRAB) have emerged as a serious threat to public-health worldwide. This study aimed to determine the antimicrobial susceptibility of A. baumannii isolates in Iran and to investigate oxacillinase-encoding determinants and their association with insertion sequence ISAba1 in CRAB isolates. This study was performed on A. baumannii isolates recovered from patients with burn wound infections during 2013. All isolates were evaluated for antimicrobial susceptibility by the disk diffusion method. Minimum inhibitory concentrations (MICs) of five antibiotics (imipenem, meropenem, polymyxin B, colistin and tigecycline) were determined for all CRAB isolates. PCR was performed to determine the distribution of blaOXA determinants and ISAba1 insertion upstream of each corresponding gene in the CRAB isolates. A total of 65 A. baumannii isolates were recovered during the 1-year period, with CRAB accounting for 63 (96.9%) of isolates. Polymyxin B, colistin and tigecycline were the most effective agents against CRAB isolates, with susceptibility rates of 100%, 87.3% and 65.1%, respectively. The proportion of CRAB isolates carrying oxacillinase determinants was as follow: blaOXA-51-like, 100%; blaOXA-23-like, 74.6%; blaOXA-24/40-like, 47.6%; and blaOXA-235-like, 12.7%. ISAba1, ISAba1-blaOXA-23-like and ISAba1-blaOXA-51-like were detected in 100%, 41.3% and 1.6% of CRAB isolates, respectively. Co-occurrence of blaOXA determinants or inserted ISAba1 upstream of the corresponding genes was associated with increased carbapenem MICs (≥128μg/mL). The emergence of high-level CRAB with blaOXA and ISAba1-blaOXA family in burn patients is a matter of increasing clinical concern, emphasising the need for infection control efforts to limit such problematic bacteria.

FALSE POSITIVE PHENOTYPIC DETECTION OF METALLO-BETA-LACTAMASES IN ACINETOBACTER BAUMANNII.

SUMMARYPhenotypic detection of metallo-β-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.