KPC Isolates Research Articles

Whole-genome sequencing of Klebsiella pneumoniae MDR circulating in a pediatric hospital setting: a comprehensive genome analysis of isolates from Guayaquil, Ecuador

BackgroundKlebsiella pneumoniae is the major cause of nosocomial infections worldwide and is related to a worsening increase in Multidrug-Resistant Bacteria (MDR) and virulence genes that seriously affect immunosuppressed patients, long-stay intensive care patients, elderly individuals, and children. Whole-Genome Sequencing (WGS) has resulted in a useful strategy for characterizing the genomic components of clinically important bacteria, such as K. pneumoniae, enabling them to monitor genetic changes and understand transmission, highlighting the risk of dissemination of resistance and virulence associated genes in hospitals. In this study, we report on WGS 14 clinical isolates of K. pneumoniae from a pediatric hospital biobank of Guayaquil, Ecuador.ResultsThe main findings revealed pronounced genetic heterogeneity among the isolates. Multilocus sequencing type ST45 was the predominant lineage among non-KPC isolates, whereas ST629 was found more frequently among KPC isolates. Phylogenetic analysis suggested local transmission dynamics. Comparative genomic analysis revealed a core set of 3511 conserved genes and an open pangenome in neonatal isolates. The diversity of MLSTs and capsular types, and the high genetic diversity among these isolates indicate high intraspecific variability. In terms of virulence factors, we identified genes associated with adherence, biofilm formation, immune evasion, secretion systems, multidrug efflux pump transporters, and a notably high number of genes related to iron uptake. A large number of these genes were detected in the ST45 isolate, whereas iron uptake yersiniabactin genes were found exclusively in the non-KPC isolates. We observed high resistance to commonly used antibiotics and determined that these isolates exhibited multidrug resistance including β-lactams, aminoglycosides, fluoroquinolones, quinolones, trimetropins, fosfomycin and macrolides; additionally, resistance-associated point mutations and cross-resistance genes were identified in all the isolates. We also report the first K. pneumoniae KPC-3 gene producers in Ecuador.ConclusionsOur WGS results for clinical isolates highlight the importance of MDR in neonatal K. pneumoniae infections and their genetic diversity. WGS will be an imperative strategy for the surveillance of K. pneumoniae in Ecuador, and will contribute to identifying effective treatment strategies for K. pneumoniae infections in critical units in patients at stratified risk.

P28 Evaluation of one in-house and three commercial phenotypic tests for detection of KPC, NDM and OXA-48-like carbapenemase in Klebsiella pneumoniae: is routine detection possible?

Abstract Background Phenotypic characterization of the OXA-48-like carbapenemase XDR and pandrug-resistant (PDR) Klebsiella pneumoniae in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing one in-house and three commercial phenotypic tests for detection of KPC, NDM and OXA-48-like carbapenemases in an attempt to study the feasibility of making detection of OXA-48-like carbapenemases a routine activity of a diagnostic microbiology laboratory. Methods A total of 40 representative non-duplicate XDR and PDR clinical Klebsiella pneumoniae isolates were screened on the basis of susceptibility profile, resistance to piperacillin/tazobactam; susceptible, intermediate, or resistant to third generation cephalosporins; elevated MIC for meropenem. These isolates were then subjected to one in-house test (OXA-48 disc test) and three commercial tests: Xpert® Carba-R (Cepheid), MASTDISCS® Combi Carba Plus (D73C), Immunochromatographic assay (ICT); KPC/IMP/NDM/OXA-48 Combo test (LFIA), Medomics. Results Xpert ®Carba-R identified OXA-48 in 62.5% (n=25) isolates, NDM in 17.5% (n=7), KPC+NDM co-carriage in 15% (n=6) and KPC in one isolate. Both Xpert® Carba-R and ICT were considered as possible gold standards and the outcomes of the other tests were compared with these two. With Xpert® Carba-R as the gold standard, high sensitivity of 96.15% was detected in OXA-48-like isolates, followed by NDM (92.85%) and NDM+KPC (85.7%). The lowest sensitivity was seen with the OXA-48 disc test (48% in OXA-48, 53.8% in NDM) followed by D73C test (64% in OXA-48, 38.46% in NDM). These two tests could not detect co- carriage of NDM and KPC in six isolates, although in two cases one of the two was detected. Specificity was high among all the tests (96.29%–93.33%). With ICT as the gold standard, 100% sensitivity was reported for NDM and KPC isolates, followed by OXA-48 and KPC+NDM (96% and 83.33%, respectively). The specificity was higher with ICT (99%) than Xpert® Carba-R (96.29%) as in detection of co- carriage of NDM and KPC. In terms of cost ICT ($16 per test) was much cheaper than Xpert® Carba-R($52 per test). Conclusions We recommend immunochromatographic assay (ICT) as an excellent tool for detection of carbapenemases (KPC/IMP/NDM/OXA-48). OXA-48 disc test and MASTDISCS® Combi Carba plus (D73C) cannot be considered as good standard since they could not detect co-carriage of NDM and KPC and had lower sensitivity.

Carbapenem-Resistant Klebsiella pneumoniae in COVID-19 Era-Challenges and Solutions.

The COVID-19 era brought about new medical challenges, which, together with nosocomial bacterial infections, resulted in an enormous burden for the healthcare system. One of the most alarming nosocomial threats was carbapenem-resistant Klebsiella pneumoniae (CRKP). Monitoring CRKP incidence and antimicrobial resistance globally and locally is vitally important. In a retrospective study, the incidence of CRKP in the pre-COVID-19 period (2017-2019) and the COVID-19 pandemic (2020-2022) was investigated in the Central Military Hospital in Ružomberok, Slovak Republic. The relative incidence of CRKP significantly increased during the COVID-19 period-by 4.8 times, from 0.18 to 0.76%. At the same time, 47% of CRKP-positive patients also had COVID-19. Twenty-six KPC and sixty-nine NDM-producing isolates were identified. CRKPs isolated in the year 2022 were submitted to whole genome sequencing, and their susceptibility was tested to cefiderocol, ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam, with excellent results. KPC-producing isolates were also highly susceptible to colistin (92%). The NDM isolates revealed lower susceptibility rates, including only 57% colistin susceptibility. ST-307 prevailed in KPC and ST-11 in NDM isolates. Despite the excellent activity of new antimicrobials, rational antibiotic policy must be thoroughly followed, supported by complementary treatments and strict anti-epidemic precautions.

Evaluation of the BD Phoenix CPO Detect Panel for Detection and Classification of Carbapenemase Producing Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) pose a serious public health threat due to their resistance to most antibiotics. Rapid and correct detection of carbapenemase producing organisms (CPOs) can help inform clinician decision making on antibiotic therapy. The BD Phoenix™ CPO detect panel, as part of antimicrobial susceptibility testing (AST), detects carbapenemase activity (P/N) and categorizes CPOs according to Ambler classes. We evaluated a CPO detect panel against 109 carbapenemase producing Enterobacterales (CPE) clinical isolates from Korea. The panel correctly detected carbapenemases production in 98.2% (n = 107/109) isolates and identified 78.8% (n = 26/33) class A, 65.9% (n = 29/44) class B, and 56.3% (n = 18/32) class D carbapenemase producers as harboring their corresponding Ambler classes. Specifically, the panel correctly classified 81.3% (n = 13/16) of K. pneumoniae KPC isolates to class A. However, the panel failed to classify 40.0% (n = 4/10) IMP and 63.6% (n = 7/11) VIM isolates to class B. Despite 27.5% (n = 30/109) CPE not being assigned Ambler classes, all of them tested carbapenemase positive. Our results demonstrate that the CPO detect panel is a sensitive test for detecting CPE and classifying KPC as class A, helping with antibiotics selection, but one-third of CPE remained unclassified for Ambler classes.

The molecular and epidemiological characteristics of carbapenemase-producing Enterobacteriaceae isolated from children in Shanghai, China, 2016-2021.

We isolated the carbapenemase-producing Enterobacteriaceae (CPE) strains from children during 2016-2021 in Shanghai, China and investigated the antimicrobial resistance, molecular and epidemiological features of these isolates. Antimicrobial susceptibility tests were performed to confirm the carbapenem resistance. Carbapenemase production was assessed by the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48), which were further confirmed by PCR amplification and sequencing. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. A total of 320 CPE strains were collected from 2016 to 2021, consisting of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn, 55.0%), Escherichia coli (CP-Eco, 24.5%) and Enterobacter cloacae (CP-Ecl, 20.4%) and others (2, 0.1%). NDM was the primary carbapenemase (67.6%) in children, followed by KPC(26.4%), IMP(5.3%) and OXA-48 (0.6%). The minimum inhibitory concentration (MIC) for imipenem has been increasing from 2016 to 2021. NDM and KPC isolates are high resistant while IMP strains show the lower resistant to imipenem. Invasive infection accounted for 10.7% of CPE-related infections and was mainly caused by CP-Kpn (70.6%). NDM-Kpn was detected in 51.8% of infants (70.8% of neonates), while KPC-Kpn was mainly isolated from non-infants (56.3%∼64.3%). ST11 was the primary clone (64.6%) of KPC-Kpn and presented an increasing trend from 2016 to 2021. NDM is widely prevalent and transfers among CPE strains in children. NDM-Kpn shows the most serious threat to infants, especially to neonates. High-risk clone of ST11 KPC-Kpn should be paid more attention and monitored continuously in children.

Covid a karbapeném-rezistentná Klebsiella pneumoniae v nemocničnom prostredí

The COVID-19 pandemic era brought about new medical challenges, which, together with nosocomial bacterial infections, resulted in an enormous burden for the healthcare system. One of the most alarming nosocomial threats was carbapenem-resistant Klebsiella pneumoniae (CRKP). Monitoring CRKP incidence and antimicrobial resistance globally and locally is vitally important. In a retrospective study, the incidence of CRKP in the pre-COVID-19 period (2017–2019) and the COVID-19 pandemic (2020–2022) was investigated in the central Military Hospital in Ružomberok, Slovak Republic. The relative incidence of CRKP significantly increased during the COVID-19 period—by 4.8 times, from 0.18 to 0.76%. At the same time, 47% of CRKP-positive patients also had COVID-19. Twenty-six KPC and sixty-nine NDM-producing isolates were identified. CRKPs isolated in the year 2022 were submitted to whole genome sequencing, and their susceptibility was tested to cefiderocol, ceftazidime–avibactam, imipenem–relebactam and meropenem–vaborbactam, with excellent results. KPC-producing isolates were also highly susceptible to colistin (92%). The NDM isolates revealed lower susceptibility rates, including only 57% colistin susceptibility. St-307 prevailed in KPC and St-11 in NDM isolates. Despite the excellent activity of new antimicrobials, rational antibiotic policy must be thoroughly followed, supported by complementary treatments and strict anti-epidemic precautions.

In vivo exposure-response relationship of meropenem against metallo-β-lactamase-harbouring Pseudomonas aeruginosa: an assessment using MICs from conventional and zinc-limited broth.

Previous investigations into metallo-β-lactamase (MBL)-harbouring Enterobacterales suggest that susceptibility testing in zinc-limited media may be more appropriate in predicting β-lactam in vivo activity. There are limited data with MBL-harbouring Pseudomonas aeruginosa. Forty-three MBL-harbouring P. aeruginosa isolates (IMP, n = 11; VIM, n = 12; NDM, n = 10; SPM, n = 10) and two P. aeruginosa control isolates (KPC, n = 1; WT, n = 1) were evaluated. Meropenem activity was evaluated in the murine neutropenic thigh model using humanized exposures. Susceptibility testing was conducted in conventional CAMHB, EDTA-supplemented CAMHB (3-300 mg/L EDTA) and Chelex-treated CAMHB (0-1.0 mg/L re-supplemented zinc), resulting in a range of meropenem MIC values for each isolate. A sigmoidal Emax model was fitted to fT>MIC versus change in log10 cfu/thigh to estimate the goodness of fit (R2). Increasing EDTA concentrations or limiting the amount of zinc in broth resulted in several-fold reductions in MIC among the majority of the MBL-harbouring P. aeruginosa while the MICs for the KPC and WT isolates were unchanged. Bacterial killing in vivo was variable, with the range of killing spanning -3.29 to +4.81 log10 change in cfu/thigh. Addition of 30 mg/L EDTA and Chelex-treated CAMHB (with no zinc supplementation) provided broth conditions for susceptibility testing that best predicted in vivo efficacy (R2 > 0.7). Among MBL-harbouring P. aeruginosa, meropenem in vivo efficacy is best represented by the pharmacodynamic profile generated using MICs determined in EDTA-supplemented or zinc-limited broth. In addition to previous data with Enterobacterales, antibiotic susceptibility testing in media that approximates physiological conditions makes it possible to uncover potential and existing therapeutic agents.

Risk factors for bloodstream infection by multidrug-resistant organisms in critically ill patients in a reference trauma hospital

Bloodstream infections (BSI) by multidrug-resistant (MDR) organisms are responsible for significant mortality in critically ill trauma patients. Our objective is to identify the risk factors for BSI by MDR agents and their resistance mechanisms in a trauma reference hospital. During 18 months, all patients admitted in our Intensive Care Unit (ICU) were enrolled in this prospective cohort. We included the first episode of BSI by carbapenem-resistant Gram-negative bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococcus. Demographic and clinical data were compared among patients with and without BSI and variables with P < .05 were tested in a multivariate analysis. We performed PCR for identification of carbapenemase and SCC mec genes and Pulsed-field gel electrophoresis for clonality. Out of 1,528 patients, 302 (19.8%) were trauma and 66 (4.3%) had a MDR-BSI (19.5% were trauma). The multivariate analysis showed that mechanical ventilation (OR3.16; 95% CI 1-8; P=.02), hemodialysis (OR3.16; 95% CI 1-5; P=.0003) and surgery (OR1.76; 95% CI 1-3; P=.04) were independent risk factors for MDR-BSI. The most frequent MDR were Klebsiella pneumoniae (n=26) and MRSA (n=27). Regarding K pneumoniae strains (n=24), 20 (83.8%) harbored bla KPC gene and 1 bla NDM. The majority of KPC isolates belonged to a predominant clone; while the MRSA were polyclonal and SCC mec type II. Mechanical ventilation, surgery and hemodialysis were independent risk factors for MDR-BSI in our cohort, but trauma was not. KPC was the main mechanism of resistance among carbapenem-resistant K pneumoniae that belonged to a predominant clone which could indicate cross-transmission.

1215. Evaluation of the Carba-R NxG Assay in a Global Challenge Set of Pseudomonas aeruginosa

BackgroundCarbapenem-resistant P. aeruginosa (CRPA) is a growing clinical challenge. Carbapenemase production is particularly problematic due to high transmissibility and limited treatment options. Carbapenemase prevalence and diversity are largely driven by geography and thus testing spectrum will dictate utility in certain regions. The purpose of this study was to evaluate the performance of the research-use-only Xpert® Carba-R NxG (Carba-R NxG) and commercially available Xpert® Carba-R (Carba-R) in a global collection of P. aeruginosa.MethodsThe challenge set included 123 clinical P. aeruginosa isolates from 11 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 per manufacturer instructions. Carba-R NxG testing was completed by Cepheid (Sunnyvale, CA) blinded to genotype. Test performances were tabulated for each assay by carbapenemase class.ResultsBoth 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 (Carba-R NxG 100% vs. Carba-R 58% detection). All SPM and GES isolates, targets not present in the current 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. Table 1 provides the test performance of both assays. Overall, the Carba-R NxG successfully categorized 100% of isolates tested compared with 68% for its predecessor.Table 1. Test performance of Carba-R NxG and Commercially Available Carba-R by Carbapenemase Class. ConclusionAs the prevalence and diversity of carbapenemase-producing CRPA continues to expand, the Carba-R offers a rapid and sensitive assay to identify clinically relevant carbapenemase genotypes to inform infection prevention and therapeutic interventions. The Carba-R NxG will expand the current targets including SPM, GES, NMC/IMI, and IMP-subtypes, outside the previous testing spectrum, increasing the global utility of the test.Disclosures Isabella A. Tickler, BS, Cepheid (Employee) Caitlin dela Cruz, BS, Cepheid (Employee) Fred C. Tenover, PhD, Cepheid (Employee) David P. Nicolau, PharmD, Cepheid (Other Financial or Material Support, Consultant, speaker bureau member or has received research support.)Merck & Co., Inc. (Consultant, Grant/Research Support, Speaker’s Bureau)Wockhardt (Grant/Research Support)

EDTA-modified carbapenem inactivation method (eCIM) for detecting IMP Metallo-\u03b2-lactamase\u2013producing Pseudomonas aeruginosa: an assessment of increasing EDTA concentrations

BackgroundPrompt identification of carbapenemase-harboring organisms is valuable in informing therapeutic and infection-control measures. The modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) are inexpensive and easy to interpret phenotypic tests endorsed by the Clinical and Laboratory Standards Institute (CLSI) for the detection of carbapenemase-harboring Enterobacterales. Only mCIM is endorsed by CLSI for detecting carbapenemase-harboring Pseudomonas aeruginosa. eCIM’s ability to delineate serine and metallo-β-lactamases (MBL) could be advantageous in areas prevalent with carbapenemase-harboring P. aeruginosa. A recent assessment of mCIM/eCIM on MBL-harboring P. aeruginosa demonstrated high eCIM sensitivity for NDMs and VIMs but not for IMP-producers. Therefore, this study aimed to determine whether increasing EDTA concentrations would enhance eCIM sensitivity for a collection of IMP-harboring P. aeruginosa isolates.Twenty-six IMP-harboring P. aeruginosa isolates were utilized. For test validation, additional P. aeruginosa isolates harboring NDM (n = 3), VIM (n = 3), KPC (n = 8), wild-type (n = 1), and Enterobacterales isolates harboring IMP (n = 6) and NDM (n = 1) were assessed. The mCIM test was conducted as outlined by CLSI. Simultaneously, the eCIM test was performed with the standard 5 mM EDTA concentration and doubling EDTA concentrations: 10 mM, 20 mM, and 40 mM.ResultsConcentration-dependent improvement was observed among the IMP-harboring P. aeruginosa with eCIM sensitivities at 0, 31, 85, and 100% respectively. Remaining Enterobacterales and P. aeruginosa responded concordantly with their genotype at the standard 5 mM eCIM concentration, with doubling EDTA concentrations providing no greater sensitivity.ConclusionCombination of mCIM and an eCIM with a 40 mM EDTA concentration appropriately capture IMP-harboring P. aeruginosa without sacrificing test utility for other carbapenemase-harboring isolates.

External validation of the INCREMENT-CPE mortality score in a carbapenem-resistant Klebsiella pneumoniae bacteraemia cohort: the prognostic significance of colistin resistance

External validation of the INCREMENT-CPE risk score (ICS) for 30-day all-cause mortality is needed. There is also scarce information about whether colistin resistance influences the prognosis of carbapenem-resistant Klebsiella pneumoniae (CRKp) bacteraemia. In this study, the ability of ICS to predict all-cause mortality in the KAPECOR cohort was calculated using the area under the receiver operating characteristic (AUROC) curve. The association of colistin resistance with mortality was studied. The ICS showed an AUROC curve of 0.77 (95% CI 0.68-0.86). A cut-off of 8 points showed 96.8% sensitivity and 50.7% specificity. Mortality of low-risk patients was not different in those treated with monotherapy versus combination therapy. However, mortality of high-risk patients treated with combination therapy (37.8%) was significantly lower than in those treated with monotherapy (68.4%) (P = 0.008). To study the prognostic significance of colistin resistance, 83 selected cases of bacteraemia due to colistin-susceptible CRKp were obtained from the INCREMENT cohort for comparison. Colistin resistance could not be shown to be associated with higher mortality in either the high-risk ICS group [adjusted odds ratio (aOR) = 1.56, 95% CI 0.69-3.33; P = 0.29] or in 37 ICS-matched pairs (aOR = 1.38, 95% CI 0.55-3.42; P = 0.49), or in a sensitivity analysis including only KPC isolates (aOR = 1.81, 95% CI 0.73-4.57; P = 0.20), but the precision of estimates was low. These results validate ICS for all-cause mortality and to optimise targeted therapy for CRKp bacteraemia. Colistin resistance was not clearly associated with increased mortality.

Nationwide epidemiology of carbapenem resistant Klebsiella pneumoniae isolates from Greek hospitals, with regards to plazomicin and aminoglycoside resistance

BackgroundTo evaluate the in vitro activities of plazomicin and comparator aminoglycosides and elucidate the underlying aminoglycoside resistance mechanisms among carbapenemase-producing K. pneumoniae isolates collected during a nationwide surveillance study in Greek hospitals.MethodsThree hundred single-patient carbapenemase-producing K. pneumoniae isolates were studied, including 200 KPC-, 50 NDM-, 21 VIM-, 14 KPC & VIM-, 12 OXA-48-, two NDM & OXA- and one KPC & OXA-producing isolates. Susceptibility testing was performed by broth microdilution, and minimum inhibitory concentrations (MICs) interpreted per EUCAST breakpoints. Carbapenemase-, aminoglycoside modifying enzyme- and 16S rRNA methylase- encoding genes were detected by PCR.ResultsOf 300 isolates tested, 5.7% were pandrug resistant and 29.3% extensively drug resistant. Plazomicin inhibited 87.0% of the isolates at ≤2 mg/L, with MIC50/MIC90 of 0.5/4 mg/L. Apramycin (a veterinary aminoglycoside) inhibited 86.7% of the isolates at ≤8 mg/L and was the second most active drug after plazomicin, followed by gentamicin (S, 43%; MIC50/MIC90, 4/> 256) and amikacin (S, 18.0%; MIC50/MIC90, 32/128). Twenty-three (7.7%) isolates (16 KPC-, 6 VIM- and one KPC & OXA-48-producers) exhibited MICs ≥64 mg/L for plazomicin, and harbored rmtB (n = 22) or armA (n = 1). AAC(6′)-Іb was the most common aminoglycoside modifying enzyme (84.7%), followed by AAC(3΄)-IIa (25.3%), while those two enzymes were co-produced by 21.4% of the isolates.ConclusionsPlazomicin retains activity against most carbapenemase-producing K. pneumoniae isolated from Greek hospitals, with MICs consistently lower than those of the other aminoglycosides, even in the presence of aminoglycoside modifying enzymes. Dissemination of 16S- rRNA methylases in 8% of the isolates is an unwelcome event that needs strict infection control measures and rigorous stewardship interventions.

Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that causes a range of diseases. The appearance of extended-spectrum β-lactamase -and carbapenemase-producing strains, in addition to the biofilm-forming phenotype, is a major problem in the clinical environment. A total of 33 clinical K. pneumoniae isolates were used in this study. Antimicrobial susceptibilities were assessed by a disc diffusion assay. Biofilm formation was determined by a microtiter plate assay, staining with 1% crystal violet and measuring absorbance after destaining. Moreover, expression of acrA, kdeA, ketM, kpnEF, and kexD efflux associated genes was measured by qRT-PCR. Isolates displayed high resistance to β-lactams such as cefazolin, cefuroxime, ceftriaxone, cefepime, piperacillin-tazobactam, imipenem, and meropenem and decreased resistance to gentamicin, amikacin, ciprofloxacin, and levofloxacin. ESBL-producing isolates formed more biofilm than carbapenemase-producing isolates. The mRNA expression levels in KPC isolates for acrA (2-fold), kdeA (2.7-fold), ketM (2.2-fold), and kpnEF (3.4-fold) were significantly increased compared to ESBL-producing isolates. There was no significant difference in kexD expression level. Under the conditions used here ESBL-producing isolates formed more biofilm than KPC postive isolates; this was associated with virulence determinants which were also transferred by plasmids together with ESBLs enzymes. Moreover, the upregulation of acrA, kdeA, ketM, and kpnEF efflux pumps was seen in carbapenemase-producing isolates demonstrating that high expression of efflux pumps alone could not confer resistance but may act as a physiological determinant such as bacterial pathogenicity and virulence, and cell-to-cell communication for bacteria.

Covert dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) in a successfully controlled outbreak: long- and short-read whole-genome sequencing demonstrate multiple genetic modes of transmission.

BackgroundCarbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety.ObjectivesTo use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak.Materials and methodsEnterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013–14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI.ResultsTwenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQIL-D2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts.ConclusionsWGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts.

Antibiofilm peptides increase the susceptibility of carbapenemase-producing Klebsiella pneumoniae clinical isolates to β-lactam antibiotics.

Multidrug-resistant carbapenemase-producing Klebsiella pneumoniae (KpC) strains are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and β-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of β-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates.

Risk factors for KPC-producing Klebsiella pneumoniae bacteremia

The molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae (KPC) has been largely investigated, but limited clinical information is available. A case-control study was performed to evaluate the risk factors for KPC bacteremia in hospitalized patients. Cases were patients with KPC bacteremia and controls were patients with non-KPC bacteremia. A total of 85 patients were included, 18 (21.2%) were KPC, and 67 (78.8%) were non-KPC (40 [59.7%] of them were extended-spectrum beta-lactamase producers). All KPC isolates were type 2 producers. These isolates belong to five distinct clones. Multivariate analysis showed that age (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.02 – 1.11; p = 0.004), presence of mechanical ventilation (OR, 11.1; 95% CI, 1.92 – 63.3; p = 0.007) and fluoroquinolone exposure during hospitalization (OR, 28.9; 95% CI, 1.85 – 454.6; p = 0.02) were independent risk factors for KPC in patients with K. pneumoniae bacteremia. Factors associated with severity of illness, such as age and mechanical ventilation, seem to be the main risks factors for KPC. Fluoroquinolones use might be a risk factor for KPC bacteremia. Further investigations on risk factors for KPC are warranted.

In vitro pharmacodynamics of simulated pulmonary exposures of tigecycline alone and in combination against Klebsiella pneumoniae isolates producing a KPC carbapenemase.

Multidrug-resistant Klebsiella pneumoniae strains that produce a serine carbapenemase (KPC) are emerging worldwide, with few therapeutic options that retain consistent susceptibility. The objective of this study was to determine the effect of combination therapy with tigecycline versus tigecycline alone against KPC-producing isolates (KPC isolates). An in vitro pharmacodynamic model was used to simulate adult steady-state epithelial lining fluid concentrations of tigecycline (50 mg every 12 h) given alone and in combination with either meropenem (2 g by 3-hour infusion every 8 h) or rifampin (600 mg every 12 h). Five KPC isolates with various phenotypic profiles were exposed over 48 h. Time-kill curves were constructed, and the areas under the bacterial killing and regrowth curves (AUBCs) were calculated. No regimens tested were able to maintain bactericidal reductions in CFU over 48 h. The AUBCs for tigecycline and meropenem monotherapies at 48 h ranged from 375.37 to 388.11 and from 348.62 to 383.83 (CFU-h/ml), respectively. The combination of tigecycline plus meropenem significantly reduced the AUBCs at 24 and 48 h for isolates with tigecycline MICs of ≤ 2 μg/ml and meropenem MICs of ≤ 16 μg/ml (P < 0.001) but added no additional activity when the meropenem MIC was 64 μg/ml (P = 0.5). Rifampin provided no additional reduction in CFU or AUBC over tigecycline alone (P = 0.837). The combination of tigecycline with high-dose, prolonged-infusion meropenem warrants further study as a potential treatment option for these multidrug-resistant organisms.

In VivoEfficacy of Simulated Human Dosing Regimens of Prolonged-Infusion Doripenem against Carbapenemase- ProducingKlebsiella pneumoniae

Carbapenemase-producing Klebsiella pneumoniae (KPC) bacteria are rapidly becoming one of the most detrimental drug-resistant Gram-negative pathogens. Doripenem is the newest FDA-approved carbapenem that has the greatest in vitro potency against a wide range of Gram-negative organisms, including multidrug-resistant organisms. Previous work in an animal model has shown efficacy against Pseudomonas aeruginosa with MICs above the current breakpoints of susceptibility. The purpose of this study is to evaluate the efficacy of 1-g and 2-g dose prolonged infusions of doripenem against KPC isolates in both an immunocompetent and neutropenic murine thigh model. Seven clinical KPC isolates (broth microdilution [BMD] MIC range, 4 to 32 μg/ml; Etest MIC range, 3 to >32 μg/ml) were used. After infection, groups of mice were administered doripenem doses previously shown to simulate the exposures observed in humans after the administration of 1 or 2 g every 8 h as a 4-h infusion. In immunocompromised mice, 1- and 2-g doses of doripenem achieved bacteriostasis against isolates with MICs up to and including 8 μg/ml and 16 μg/ml, respectively. In immunocompetent animals, statistically significant reductions in the number of CFU were observed with overall decreases of approximately 1 log (P < 0.05). While carbapenemase-producing Klebsiella pneumoniae continues to decrease our meager supply of active agents, the ability of doripenem to produce CFU reductions in the presence of white blood cells (WBCs) using humanized exposures suggests the potential utility of this agent in combination against this increasingly problematic pathogen.

Comparison of Meropenem MICs and Susceptibilities for Carbapenemase-Producing Klebsiella pneumoniae Isolates by Various Testing Methods

We describe the levels of agreement between broth microdilution, Etest, Vitek 2, Sensititre, and MicroScan methods to accurately define the meropenem MIC and categorical interpretation of susceptibility against carbapenemase-producing Klebsiella pneumoniae (KPC). A total of 46 clinical K. pneumoniae isolates with KPC genotypes, all modified Hodge test and bla(KPC) positive, collected from two hospitals in NY were included. Results obtained by each method were compared with those from broth microdilution (the reference method), and agreement was assessed based on MICs and Clinical Laboratory Standards Institute (CLSI) interpretative criteria using 2010 susceptibility breakpoints. Based on broth microdilution, 0%, 2.2%, and 97.8% of the KPC isolates were classified as susceptible, intermediate, and resistant to meropenem, respectively. Results from MicroScan demonstrated the most agreement with those from broth microdilution, with 95.6% agreement based on the MIC and 2.2% classified as minor errors, and no major or very major errors. Etest demonstrated 82.6% agreement with broth microdilution MICs, a very major error rate of 2.2%, and a minor error rate of 2.2%. Vitek 2 MIC agreement was 30.4%, with a 23.9% very major error rate and a 39.1% minor error rate. Sensititre demonstrated MIC agreement for 26.1% of isolates, with a 3% very major error rate and a 26.1% minor error rate. Application of FDA breakpoints had little effect on minor error rates but increased very major error rates to 58.7% for Vitek 2 and Sensititre. Meropenem MIC results and categorical interpretations for carbapenemase-producing K. pneumoniae differ by methodology. Confirmation of testing results is encouraged when an accurate MIC is required for antibiotic dosing optimization.

KPC Antibiogram in a large teaching Brazilian Hospital

Background: Describe susceptibility profile of KPC Klebsiella pneumoniae producers recovered in a teaching hospital from São Paulo. Methods: We analyzed 27 isolates of KPC, one per patient, from July 2008 to April 2009. Identification was performed by GNI650 Cards Vitek™ (Biomerieux, Marcy l’Etoile, France); susceptibility test was performed by disk diffusion and the Hodge Test was done according to CLSI-M100S19 recommendations. blaKPC detection was done by PCR with previously described primers and Pulsed Field Gel Electrophoresis (PFGE) was also performed. Minimal Inhibitory Concentration (MIC) for polymyxin B, tigecycline and carbapenens was performed by Etest™ (Biomerieux, Marcy l’Etoile, France). Results: KPC antibiogram showed multiple resistance as expected: 100% to ertapenem, 81,4% to meropenem, 77,7% to imipenem, 93,6% to amikacin; 82,5% to gentamicin, 89% to cefepime and ceftazidime and 100% to ciprofloxacin, sulphametoxazol/trimetropim, piperacillin/tazobactam and to aztreonam. MIC 50/90 were 2 mg/l and 3 mg/l to tigecyclin, and 2 mg/l and 48 mg/l to polymyxin B. PFGE showed that 89% belonged to the same molecular profile. Conclusion: KPC isolates showed very few therapeutically options being tygecyclin the most active drug among those isolates. In our institution treatment with polymyxin B alone has to be carefully monitored until MIC is available. The same molecular profile emphasizes the horizontal spreading of these multiresistant bacteria. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive