OXA-48 Producers Research Articles

In vitro activity of cefepime-enmetazobactam on carbapenem-resistant gram negatives

ObjectivesCefepime-enmetazobactam is a new β-lactam/βlactamase inhibitor combination with broad-spectrum activity against multidrug-resistant Enterobacterales, including extended-spectrum β-lactamase producers. This study evaluated the in vitro activity of cefepime-enmetazobactam towards a collection of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa and Acinetobacter baumannii compared to the other β-lactam/β-lactamase inhibitor combinations. MethodsThe MIC of cefepime, cefepime-enmetazobactam, ceftazidime, ceftazidime-avibactam, meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, and ertapenem were determined by broth microdilution on 2212 CRE, including 2089 carbapenemase producers (1000 OXA-48-like, 49 KPC, 697 NDM, 180 VIM, 1 IMP, 9 IMI, and 158 multiple carbapenemases) and 123 CRE that do not produce carbapenemase received at the French National Reference Centre (from March 1, 2023 to August 31, 2023), 50 P. aeruginosa, and 30 A. baumannii. All strains were fully sequenced. ResultsWe confirmed the absence of inhibitory activity of enmetazobactam towards metallo-β-lactamases. Cefepime-enmetazobactam and ceftazidime-avibactam exhibited a similar susceptibility (96.7% vs. 99.5%, respectively) on OXA-48-producers. Cefepime-enmetazobactam exhibited 66.9% and 63.3% susceptibility for CRE non-EPC and KPC, whereas those rates rose to 96.7%/95.9%, 93.4%/95.9%, and 95.9%/98.0% for ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, respectively. Low MICs (≤0.25 mg/L) were obtained for ceftazidime-avibactam-resistant KPC variants.Cefepime-enmetazobactam did not display a significant added value when compared with cefepime alone on Pseudomonas aeruginosa and Acinetobacter baumannii. DiscussionOXA-48 producers displayed high susceptibility to cefepime-enmetazobactam, which is similar to ceftazidime-avibactam, including for OXA-48 producers that coproduce a ceftazidime hydrolyzing enzyme (extended-spectrum β-lactamases or AmpC). In vivo experiments have to be implemented to confirm if cefepime-enmetazobactam might be a relevant alternative to ceftazidime-avibactam for the treatment of infections caused by OXA-48 producers.

Outbreak caused by multidrug-resistant OXA-48 and NDM-1 producing Klebsiella pneumoniae inthe intensive care unit of a cancer hospital.

We report a nosocomial outbreak caused by a multidrug-resistant carbapenemase-producing Klebsiella pneumoniae (MDRCPKp), that was detected in six patients admitted to the medical intensive care unit between 20th of December 2023 and 15th of January 2024 in Ankara, Turkey. The investigation of this outbreak was started on 29th of December 2023. During the outbreak 11 samples were collected from the six patients with MDRCPKp. Pulsed-field gel electrophoresis (PFGE) was performed to determine the genetic relatedness and clonality of MDRCPKp strains. MDRCPKp was isolated in the tracheal aspiration culture, blood, urine, and screening samples. Five patients with MDRCPKp colonization developed healthcare-associated infection. In one patient MDRCPKp was isolated from tracheal aspirate and the screening cultures were considered as colonization not infection. PFGE analysis revealed that all isolates belonged to the same K. pneumoniae clone. MDRCPKp strain of this outbreak exhibited multidrug resistance and co-produced OXA-48 and NDM-1. This outbreak ended after application of strict infection control measures. An outbreak of MDRCPKp can occur in hospitals, especially in the intensive care units; thus, it should be detected early by infection control teams. A strong collaboration between infection control team and microbiology laboratory is essential to cope with MDR bacterial outbreaks in hospitals.

Efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli.

Carbapenem-resistant Enterobacterales (CRE) have become a major public health problem worldwide. The aim of this study was to investigate efficacy of ceftazidime/avibactam and plazomicin on carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates. Susceptibility of imipenem, meropenem, ertapenem, ceftazidime/avibactam and plazomicin was investigated by broth-microdilution method. Major carbapenemases NDM, VIM, IMP, KPC, OXA-48 as well as other β-lactamases namely, TEM, SHV, OXA-1-like, CTX-M, ACC, FOX, MOX, DHA, CIT, EBC, VEB, GES, PER were investigated by PCR. A total of 120 carbapenem-resistant isolates (60 E. coli and 60 K. pneumoniae) were included in this study and blaOXA-48-like was found in 78.33%, blaNDM in 26.66%, blaKPC in 7.5%, blaIMP in 5.83%, and blaVIM in 5%. Among 94 isolates with the blaOXA-48-like gene, 22.3% were resistant to ceftazidime/avibactam and 51.1% were resistant to plazomicin. Of 32 isolates with blaNDM, 31 (96.9%) were resistant to ceftazidime/avibactam and 30 (93.75%) were resistant to plazomicin, and both antibiotics had limited effects against blaNDM carriers (P < 0.001). Of the 12 isolates with blaNDM+OXA-48 combination, 11 (91.7%) were resistant to ceftazidime/avibactam and plazomicin. The effect of both antibiotics was significantly lower in strains with blaNDM+OXA-48 combination (P < 0.005).The most common carbapenemase genes in this study were blaOXA-48-like and blaNDM. Ceftazidime/avibactam demonstrated a good efficacy among OXA-48 producing K. pneumoniae and E. coli, however, plazomicin had a significantly lower antibacterial effect in our study. Both antimicrobial agents should be considered as an option by evaluating combined susceptibility results and gene patterns obtained by regional and global molecular data in the treatment of CRE infections.

Carbapenemases Produced by Multidrug-Resistant Strains of &lt;i&gt;Klebsiella Pneumoniae&lt;/i&gt; Isolated from Intensive Care Patients

Relevance Klebsiella pneumoniae is one of the main pathogens of nosocomial infections. Hospital strains of this pathogen are characterized by a high frequency of resistance to many antibiotics, including carbapenems. The main mechanism for the formation of resistance to carbapenems is the production of carbapenemases by bacteria. To date, K. pneumoniae is considered one of the main “distributors” of clinically important antibiotic resistance genes.Aim of the study To study the frequency of occurrence of the most common carbapenemase genes in multiresistant K.pneumoniae strains isolated from patients of intensive care units in an emergency hospital.Material and methods 4708 samples of various types of clinical material from patients of 5 intensive care units of the N.V. Sklifosovsky Research Institute for Emergency Medicine were analyzed. Microbiological studies were carried out using standard generally accepted methods. For the purposes of this study, unique sequential K.pneumoniae strains resistant to imipenem and/or meropenem were selected. DNA isolation was carried out using the RIBO-prep kit (Russia). Carbapenemase genes were detected by real-time PCR using the kits of reagents “AmpliSens MDR-MBL-FL” and “AmpliSens MDR-KPC/OXA-48-FL” on a “Rotor Gene” device (Corbett Research, Australia).Results Etiologically significant microorganisms were detected in 64.7% of the studied samples. K. pneumoniae was isolated in a quarter of the samples. 194 unique carbapenem-resistant strains of K.pneumoniae were selected. Of these, 11.3% of the genes of the studied carbapenemases were not detected. In 38.1% of strains, 1 carbapenemase was detected, in 29.9% — two and in 20.6% — three or more. Among the strains with one carbapenemase gene, OXA-48 (19.1%) and CATTLE (13.4%) producers prevailed. Strains producing only NDM betalactamase were found in 5.7% of cases. Isolated allocation of VIM and IMP was not detected. In 34%, metallobectalamases were isolated in combination with serine carbapenemases. The production of serine carbapenemases alone was detected in 48.5% of the strains. Depending on the specialization of the intensive care unit, there are differences in the frequency of detection of serine and metallobetalactamases in strains of carbapenem-resistant Klebsiella.Conclusion K. pneumoniae is the causative agent of nosocomial infections in 25% of cases. In 11.3% of carbapenem-resistant strains, the production of KPC, OXA-48, NDM, VIM and IMP genes was not detected. When developing algorithms for antibacterial therapy, it is necessary to take into account that from 25.7% to 60.6% of K. pneumoniae strains in different intensive care units are the producers of metallobetalactamases.

Usefulness of inclusion of ertapenem and temocillin screening breakpoints in the EUCAST rapid antimicrobial susceptibility testing (RAST) for rapid detection of OXA-48-producing Klebsiella pneumoniae directly from positive blood cultures.

The aims of this study were: (i) to assess the ability of the meropenem screening breakpoint as part of the screening rapid antimicrobial susceptibility testing (sRAST) of EUCAST for the detection of OXA-48 carbapenemase-producing Klebsiella pneumoniae directly from positive blood cultures (BCs); and (ii) to evaluate the inclusion of ertapenem and temocillin discs into the sRAST to enhance the detection of OXA-48-producing isolates. BC bottles were spiked with a total of 117 K. pneumoniae isolates, including 77 previously characterized OXA-48 producers and 40 non-OXA-48 producers. Disc diffusion assays were directly performed from positive BCs with meropenem (10 µg), ertapenem (10 µg) and temocillin (30 µg) discs, and inhibition zones were manually measured after 4, 6 and 8 h of incubation. The screening cut-off values of sRAST were applied to evaluate their capability in detecting OXA-48-producing isolates. Receiver operating characteristic curves were constructed to illustrate the performance efficacy of the disc diffusion assays to detect OXA-48 producers. The meropenem cut-off values of sRAST only detected 90.91% of the OXA-48-producing isolates after 6 and 8 h of incubation. With the proposed cut-off points for ertapenem [<19 mm (4/6 h) and <20 mm (8 h)] and temocillin [<10 mm (4 h) and <11 mm (6/8 h)], all OXA-48-positive isolates were detected without any false-positive results at any reading time. In healthcare settings with a high prevalence of OXA-48 producers, the inclusion of ertapenem and temocillin discs in the sRAST procedure may improve the detection of OXA-48-producing K. pneumoniae isolates directly from positive BCs, providing reliable results after only a 4 h incubation period.

Wastewater from healthcare centers in Burkina Faso is a source of ESBL, AmpC-β-lactamase and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae

BackgroundExtended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC-β-lactamase and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae have spread into the environment worldwide posing a potential public health threat. However, the prevalence data for low- and middle-income countries are still scarce. The aim of this study was to evaluate the presence of ESBL, AmpC-β-lactamase and carbapenemase-producing and multidrug-resistant E. coli and K. pneumoniae in wastewaters from healthcare centers in Burkina Faso.ResultsEighty-four (84) wastewater samples were collected from five healthcare centers and plated on selective ESBL ChromAgar. E. coli and Klebsiella pneumoniae isolates were identified using API20E. ESBL-producing bacteria were detected in 97.6% of the samples and their average concentration per hospital ranged from 1.10 × 105 to 5.23 × 106 CFU/mL. Out of 170 putative ESBL-producing isolates (64% of them were E. coli) and 51 putative AmpC-β-lactamase-producing isolates, 95% and 45% were confirmed, respectively. Carbapenemase production was detected in 10 isolates, of which 6 were NDM producers, 3 were OXA-48 producers and 1 was NDM and OXA-48 producer. All isolates were multidrug resistant and, moreover, all of them were resistant to all tested β-lactams. Resistance to ESBL inhibitors was also common, up to 66% in E. coli and 62% in K. pneumoniae. Amikacin, fosfomycin and nitrofurantoin were the antibiotics to which the least resistance was detected.ConclusionsThis study showed that wastewater from healthcare centers constitutes a reservoir of multidrug-resistant bacteria in Burkina Faso, including carbapenemase producers. Untreated healthcare wastewater entering the environment exposes people and animals to infections caused by these multi-resistant bacteria, which are difficult to treat, especially in the resource-poor settings.

Healthcare Equipment and Personnel Reservoirs of Carbapenem-Resistant Acinetobacter baumannii Epidemic Clones in Intensive Care Units in a Tunisian Hospital.

Carbapenem-resistant Acinetobacter baumannii (CRAB) strains can cause severe and difficult-to-treat infections in patients with compromised general health. CRAB strains disseminate rapidly in nosocomial settings by patient-to-patient contact, through medical devices and inanimate reservoirs. The occurrence of CRAB in patients residing in the intensive care units (ICUs) of the Sahloul University hospital in Sousse, Tunisia is high. The objective of the current study was to determine whether the surfaces of items present in five ICU wards and the medical personnel there operating could serve as reservoirs for CRAB strains. Furthermore, CRAB isolates from patients residing in the ICUs during the sampling campaign were analyzed for genome comparison with isolates from the ICUs environment. Overall, 206 items were screened for CRAB presence and 27 (14%) were contaminated with a CRAB isolate. The items were located in several areas of three ICUs. Eight of the 54 (15%) screened people working in the wards were colonized by CRAB on the hands. Patients residing in the ICUs were infected with CRAB strains sharing extensive genomic similarity with strains recovered in the nosocomial environment. The strains belonged to three sub-clades of the internationally disseminated clone (ST2). A clone emerging in the Mediterranean basin (ST85) was detected as well. The strains were OXA-23 or NDM-1 producers and were also pan-aminoglycoside resistant due to the presence of the armA gene. Hygiene measures are urgent to be implemented in the Sahloul hospital to avoid further spread of difficult-to-treat CRAB strains and preserve health of patients and personnel operating in the ICU wards.

Emergence of NDM in Association with OXA-48 Carbapenemase in Raoultella Terrigena from a Moroccan University Hospital in Casablanca, Morocco

Objective: The purpose of this study was to investigate the emergence of Raoultella terrigena, a rarely found opportunistic pathogen, co-producing NDM and OXA-48 carbapenemases in isolates recovered from various clinical specimens in our institution. Methods: From April 2020 to November 2021, we focused on strains of Enterobacteriaceae with reduced sensitivity to ertapenem giving rise to suspicion of the production of carbapenemases according to the CASFM-EUCAST criteria. These strains were isolated from samples for diagnostic purposes and identified in the microbiology laboratory of our institution. Only non-duplicate clinical and surveillance isolates obtained from patients were included. Results: Out of 159 Enterobacteriaceae suspected, 53 strains of Raoultella terrigena producing carbapenemases were isolated. Among these isolates, 64%(n=34) were NDM producers, 4% (n=2) were OXA-48 producers and 32% (n=17) had both the NDM and OXA-48. The co-existence of NDM and OXA-48 carbapenemases was found more often in Raoultella terrigena than Klebsiella pneumoniae. Among the seventeen isolates of Raoultella terrigena, all were multidrug-resistant. Conclusion: This study reported for the first time the emergence of NDM and OXA-48 carbapenemases co-existence in Raoultella terrigena isolates in our country which is starting to limit treatment options.

In vitro activities of omadacycline, eravacycline, cefiderocol, apramycin, and comparator antibiotics against Acinetobacter baumannii causing bloodstream infections in Greece, 2020–2021: a multicenter study

Resistance of Acinetobacter baumannii to multiple clinically important antimicrobials has increased to very high rates in Greece, rendering most of them obsolete. The aim of this study was to determine the molecular epidemiology and susceptibilities of A. baumannii isolates collected from different hospitals across Greece. Single-patient A. baumannii strains isolated from blood cultures (n = 271), from 19 hospitals, in a 6-month period (November 2020–April 2021) were subjected to minimum inhibitory concentration determination and molecular testing for carbapenemase, 16S rRNA methyltransferase and mcr gene detection and epidemiological evaluation. 98.9% of all isolates produced carbapenemase OXA-23. The vast majority (91.8%) of OXA-23 producers harbored the armA and were assigned mainly (94.3%) to sequence group G1, corresponding to IC II. Apramycin (EBL-1003) was the most active agent inhibiting 100% of the isolates at ≤16 mg/L, followed by cefiderocol which was active against at least 86% of them. Minocycline, colistin and ampicillin-sulbactam exhibited only sparse activity (S <19%), while eravacycline was 8- and 2-fold more active than minocycline and tigecycline respectively, by comparison of their MIC50/90 values. OXA-23-ArmA producing A. baumannii of international clone II appears to be the prevailing epidemiological type of this organism in Greece. Cefiderocol could provide a useful alternative for difficult to treat Gram-negative infections, while apramycin (EBL-1003), the structurally unique aminoglycoside currently in clinical development, may represent a highly promising agent against multi-drug resistant A. baumanni infections, due to its high susceptibility rates and low toxicity.

The Clash of the Titans: COVID-19, Carbapenem-Resistant Enterobacterales, and First mcr-1-Mediated Colistin Resistance in Humans in Romania.

(1) Background: Antibiotic resistance and coronavirus disease-19 (COVID-19) represent a dual challenge in daily clinical practice, inducing a high burden on public health systems. Hence, we aimed to dynamically evaluate the impact of COVID-19 on patients with carbapenem-resistant Enterobacterales (CRE) urinary tract infections (UTIs), as well as the antibiotic resistance trends after the onset of the pandemic. (2) Methods: We conducted a prospective study including patients with CRE UTIs who were enrolled both pre- and during the pandemic from 2019 to 2022. We further performed a standardized and comparative clinical, paraclinical, and microbiological assessment between patients with and without COVID-19. (3) Results: A total of 87 patients with CRE UTIs were included in this study (46 pre-pandemic and 41 during the pandemic, of which 21 had associated Severe Acute Respiratory Syndrome Coronavirus-2 infection). Klebsiella pneumoniae was the main etiological agent of the UTIs, with the majority of strains (82.7%) being carbapenemase producers (mainly OXA-48 producers), while five of the 34 colistin-resistant isolates were harboring the mobile colistin resistance-1 (mcr-1) gene. COVID-19 patients presented a significantly worse outcome with higher rates of intensive care unit (ICU) admissions (66.7% for COVID patients vs. 18.2% for non-COVID patients, p < 0.001), while the fatality rates were also considerably higher among patients with concomitant viral infection (33.3% vs. 12.1%, p < 0.001). Besides COVID-19, additional risk factors associated with increased mortality were urinary catheterization, sepsis with K. pneumoniae, impaired liver and kidney function, and an inappropriate initial empiric antibiotic therapy. (4) Conclusions: COVID-19 showed a pronounced negative impact on patients with CRE UTIs, with significantly longer hospitalizations and higher ICU admissions and mortality rates.

659. In Vitro Activity of Ceftazidime-Avibactam and Comparator Agents against Enterobacterales Collected from Patients with Bloodstream Infections (BSI) as Part of the ATLAS (India) Surveillance Program, 2019-2020

Abstract Background Ceftazidime-avibactam (CAZ-AVI) is indicated for patients with bacteremia that occurs in association complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI) and hospital-acquired pneumonia (HAP). The present report evaluated the in vitro activity of CAZ-AVI and comparators against Enterobacterales clinical isolates collected from BSI as part of the ATLAS (The Antimicrobial Testing Leadership and Surveillance) surveillance program in 2019-2020 from India. Methods A total of 2126 Enterobacterales (493 from blood samples) non-duplicate clinically significant isolates, were collected from 2019-2020. In vitro activity of Ceftazidime-avibactam and its comparators were assessed against Enterobacterales blood isolates. Results Overall susceptibility to the β -lactam/β -lactamase inhibitor (BL-BLI) was 60 -76% for Enterobacterales. with highest susceptibility to Ceftazidime-avibactam. Overall highest susceptibility (97%) was noted for Tigecycline for Enterobacterales(n=476).The susceptibility to colistin was lower(83%) in Enterobacterales (n=409). Among 168 CR-Enterobacterales (CRE), Ceftazidime avibactam showed 29% susceptibility (n=49) while susceptibility to Tigecycline was 96%(n=161) followed by Colistin at 84%(n=141). Molecular analysis for CRE isolates showed 55 % (n=93) to be OXA-48 like producers (with or without NDM) ,68%(n=114)to be NDM producers (with or without OXA- 48) and 30%(n=51) to be co producers of OXA-48 like &amp; NDM. In vitro activity of commonly used antibiotics against Enterobacterales, blood isolates (2019-2020) Conclusion Caz-Avi showed overall good susceptibility against the blood isolates tested hence it may be a valuable therapeutic option for treating BSI caused by MBL-negative Enterobacterales. Tigecycline followed by Colistin showed the highest susceptibilities. Tigecycline attains very low plasma concentrations and is not licensed for use in blood stream infections, while the use of colistin may be limited by the lack of CLSI susceptibility breakpoints and safety concerns. Disclosures Abhisek Routray, PhD, Pfizer India Limited: Advisor/Consultant Akshata Mane, MD, Pfizer India Limited: Advisor/Consultant|Pfizer India Limited: Stocks/Bonds.

Evaluation of In Vitro Activity of Double-Carbapenem Combinations against KPC-2-, OXA-48- and NDM-Producing Escherichia coli and Klebsiella pneumoniae.

Double-carbapenem combinations have shown synergistic potential against carbapenemase-producing Enterobacterales, but data remain inconclusive. This study evaluated the activity of double-carbapenem combinations against 51 clinical KPC-2-, OXA-48-, NDM-1, and NDM-5-producing Escherichia coli and Klebsiella pneumoniae and against constructed E. coli strains harboring genes encoding KPC-2, OXA-48, or NDM-1 in an otherwise isogenic background. Two-drug combinations of ertapenem, meropenem, and doripenem were evaluated in 24 h time-lapse microscopy experiments with a subsequent spot assay and in static time-kill experiments. An enhanced effect in time-lapse microscopy experiments at 24 h and synergy in the spot assay was detected with one or more combinations against 4/14 KPC-2-, 17/17 OXA-48-, 2/17 NDM-, and 1/3 NDM-1+OXA-48-producing clinical isolates. Synergy rates were higher against meropenem- and doripenem-susceptible isolates and against OXA-48 producers. NDM production was associated with significantly lower synergy rates in E. coli. In time-kill experiments with constructed KPC-2-, OXA-48- and NDM-1-producing E. coli, 24 h synergy was not observed; however, synergy at earlier time points was found against the KPC-2- and OXA-48-producing constructs. Our findings indicate that the benefit of double-carbapenem combinations against carbapenemase-producing E. coli and K. pneumoniae is limited, especially against isolates that are resistant to the constituent antibiotics and produce NDM.

Increasing trends of colistin resistance in patients at high-risk of carbapenem-resistant Enterobacteriaceae

Introduction Occurrence of colistin-resistant Enterobacteriaceae in response to the unregulated use of this antibiotic has been documented. This study reports an investigation of colistin resistance rates among carbapenem-resistant enterobacterial clinical isolates. Methods A total of 196 multidrug-resistant Enterobacteriaceae isolates (Klebsiella pneumoniae (n = 100), Escherichia coli (n = 89) and Enterobacter cloacae (n = 7) were selected from Gram-negative isolates over one year. Susceptibility to antimicrobials was determined using Vitek2. Broth microdilution method was used to detect colistin antimicrobial susceptibility. Identification of ESBL and carbapenemases were both done phenotypically and by PCR. Results All the studied isolates showed multidrug-resistant phenotypes with 51.5% resistance to carbapenems (meropenem, imipenem). Very low resistance rates towards tigecycline (n = 9) 4.6% were found. Thirty-nine isolates (19.9%) showed reduced susceptibility to colistin among the MDR isolates. Sixty-four isolates (32.7%) were ESBL producers. Hundred isolates (51%) were carbapenemase producers using Carba NP test. The PCR amplification results revealed that 40 isolates (20%) harboured NDM-1 and 40 isolates contained OXA-48-like gene. Coexistence of both (NDM-1 and OXA-48-like) was observed in nine (4.59%) isolates. A Statistically significant relationship was observed between carbapenem resistance and each of the followings; OXA-48 producers (p= .009), amikacin resistance (p = .000), gentamicin resistance (p = .032), tobramycin resistance (p = .000), and tigecycline resistance (p-value ≤ .001). A statistical significance was detected between ESBL-producing isolates and carbapenem susceptible isolates ESBL producers with p = 0.000. Conclusion An alarming sign is the increasing colistin resistance rates among carbapenem-resistant isolates. Aminoglycosides are still a therapeutic option to decrease the use of colistin and avoid further development of resistance. KEY MESSAGES High rates of colistin resistance among carbapenem-resistant Enterobacteriaceae. The choice of antibiotic is significantly associated with the clinical site of infection. Aminoglycosides are offered choices for treating multiple drug-resistant Enterobacteriaceae to preserve the colistin and carbapenems.

Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Romanian Hospitals Co-Presenting Resistance and Heteroresistance to Colistin.

Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l’Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime–avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem–vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

Outcomes in Oxacillinases β-Lactamases (OXA-48) and New Delhi Metallo-β-Lactamase (NDM-1)-Producing, Carbapenem-Resistant Klebsiella Pneumoniae Isolates Obtained From Bloodstream Infections.

Background: Carbapenemase-producing Klebsiella pneumoniae (CRKP) has become a menace in several intensive care units, which needs to be controlled immediately after being reported by a laboratory. Detection in the laboratory is usually done using phenotypic methods and it is not known whether knowledge of these genes helps in individual patient management. This study aimed to compare the outcomes of oxacillinases β-lactamases (OXA-48) and New Delhi metallo-β-lactamase (NDM-1)-producing CRKP isolates, the two most common carbapenemases reported from India, obtained from patients with bloodstream infections in an ICU in a tertiary care center in North India and to compare the different laboratory methods for their detection.Materials and methods: Klebsiella pneumoniae isolates obtained from the blood culture of patients admitted to various ICUs were subjected to conventional polymerase chain reaction (PCRs) for blaNDM and blaOXA48-like genes. Those positive for any of the genes were tested by the modified carbapenem inactivation method (mCIM) and if found positive were also subjected to ethylenediamine tetraacetic acid (EDTA)-modified carbapenem inactivation method (eCIM). Antibiotic susceptibility tests (AST) were performed and clinical data were recorded.Results: A total of 49 isolates were positive for one or more carbapenemase genes (30 {61.2%} for blaNDM gene only, 13 {26.5%)} for blaOXA48-like gene only, and six {12.2%} for both). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of mCIM were found to be 77.6%, 100%, 100%, and 78.9%, respectively. Statistically significant differences were found in the AST pattern between the isolates with two genes. Increased MIC levels of colistin were observed, though they lay in the sensitive range. Mortality occurred in all six patients who were infected with CRKP harboring both the genes though no significant difference was observed in NDM and OXA-48 producing CRKP isolates.Conclusion: Surveillance of carbapenemase genes in a hospital setting is essential. The possible reasons for the low diagnostic accuracy of mCIM and differences in AST patterns are discussed.

Carbapenem resistance determinants in Klebsiella pneumoniae strains isolated from blood cultures-comparative analysis of molecular and phenotypic methods

Abstract Introduction: This study provides data on carbapenemases identified in carbapenem-resistant Klebsiella pneumoniae (CR-KP) isolated from blood-cultures by the multiplex molecular method. Material and method: Between October 2016 and September 2017, 47 non-duplicate Klebsiella pneumoniae (KP) were isolated from blood cultures, from hospitalized patients in the Regional Institute of Gastroenterology and Hepathology, Cluj-Napoca, Romania. Identification and antimicrobial susceptibility tests (AST) were performed by Vitek 2 Compact. The combination disks test (CDT) was used for phenotypic analysis and the LightCycler® Multiplex DNA assay was used to detect and identify the carbapenemases by the LightCycler®z 480 Instrument. The following targets were chosen: blaKPC, blaNDM, blaGES, blaIMP and blaOXA-48 genes and the Cobas® 4800 software variant 2.2.0 was used for the results interpretation. Results: Taking into consideration the meropenem minimum inhibitory concentration (MIC), 29 KP were susceptible and 18 were not-susceptible (MIC≥0.5 µg ml-1). In the CR-KP group, the CDT identified OXA-48 (10/18) and KPC (7/18) producers. One isolate showed a noninterpretable profile. The multiplex molecular analyses confirmed the carbapenemases production as: 9 CR-KP were KPC and OXA-48 co-producers, 8 were OXA-48 and one was KPC producing strains. In CR-KP group, we found a significant correlation between the CDT and RT-PCR tests results, concerning KPC (p = 0.671). Eight phenotypic results were confirmed by molecular Light-Cycler® Multiplex DNA assay. For CR-KP co-producers (KPC and OXA-48), the CDT could indicate only one carbapenem-hydrolyzing enzyme. Conclusion: This study highlights the CR-KP co-producers (OXA-48 and KPC). OXA-48-like is more frequently encountered in our area than other carbapenemases.

Discovery of Quercetin and Its Analogs as Potent OXA-48 Beta-Lactamase Inhibitors.

Carbapenem resistance in Enterobacteriaceae caused by OXA-48 β-lactamase is a growing global health threat and has rapidly spread in many regions of the world. Developing inhibitors is a promising way to overcome antibiotic resistance. However, there are few options for problematic OXA-48. Here we identified quercetin, fisetin, luteolin, 3′,4′,7-trihydroxyflavone, apigenin, kaempferol, and taxifolin as potent inhibitors of OXA-48 with IC50 values ranging from 0.47 to 4.54 μM. Notably, the structure-activity relationship revealed that the substitute hydroxyl groups in the A and B rings of quercetin and its structural analogs improved the inhibitory effect against OXA-48. Mechanism studies including enzymatic kinetic assay, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) analysis demonstrated that quercetin reversibly inhibited OXA-48 through a noncompetitive mode. Molecular docking suggested that hydroxyl groups at the 3′, 4′ and 7 positions in flavonoids formed hydrogen-bonding interactions with the side chains of Thr209, Ala194, and Gln193 in OXA-48. Quercetin, fisetin, luteolin, and 3′,4′,7-trihydroxyflavone effectively restored the antibacterial efficacy of piperacillin or imipenem against E. coli producing OXA-48, resulting in 2–8-fold reduction in MIC. Moreover, quercetin combined with piperacillin showed antimicrobial efficacy in mice infection model. These studies provide potential lead compounds for the development of β-lactamase inhibitors and in combination with β-lactams to combat OXA-48 producing pathogen.

P09 Activity of newer antibiotics against NDM- and OXA-48-producing organisms

BackgroundSeveral antibiotics active against carbapenemase-producing organisms have recently been licensed for clinical use. These organisms usually have multiple integrative and conjugative elements with different resistance genes. The positioning of these agents for empirical use in sepsis caused by different carbapenemase-producing organisms is important in optimizing the likelihood of appropriate treatment before susceptibility testing is complete. Here we present the activities of meropenem-vaborbactam, imipenem-relebactam, eravacycline and tigecycline against NDM- and OXA-48-producing clinical isolates in a London teaching hospital.MethodsSixty-two previously characterized carbapenemase-producing Gram-negative clinical isolates were studied. These comprised 31 NDM and 31 OXA-48 producers. OXA-48 producers comprised Klebsiella pneumoniae (17), Escherichia coli (10), Enterobacter aerogenes (3) and Serratia marcescens (1). NDM producers comprised K. pneumoniae (11), E. coli (10), Acinetobacter baumannii (3), Klebsiella oxytoca (1), Enterobacter asburiae (1), Enterobacter cloacae (1), Pseudomonas aeruginosa (1), Citrobacter koseri (1), Citrobacter freundii (1) and Leclercia adecarboxylata (1). MICs of meropenem/vaborbactam, imipenem/relebactam, eravacycline and tigecycline were determined using Etests. Interpretation was done using EUCAST breakpoints.Results OXA-48 producers: Eight of 17 K. pneumoniae, 8/10 E. coli and 2/3 E. aerogenes isolates were meropenem/vaborbactam susceptible. Of these, 2 K. pneumoniae, 5 E. coli and 1 E. aerogenes isolates were imipenem/relebactam susceptible. All imipenem/relebactam/susceptible isolates were also meropenem/vaborbactam susceptible. The S. marcescens isolate (1/1) was resistant to both antibiotics. The only isolates demonstrating susceptibility to the glycylcyclines were E. coli. Five of 10 were susceptible to both glycylcyclines. One of 10 was susceptible to one glycylcycline but resistant to the other. NDM producers: Five of 11 K. pneumoniae, 6/10 E. coli and 1/1 K. oxytoca, E. asburiae and C. koseri isolates were susceptible to meropenem/vaborbactam. Of these, two K. pneumoniae and the K. oxytoca (1/1) and E. asburiae (1/1) isolates were imipenem/relebactam susceptible. The L. adecarboxylata isolate (1/1) was meropenem/vaborbactam resistant but imipenem/relebactam susceptible. All P. aeruginosa, C. freundii, A. baumannii and E. cloacae isolates were resistant to both meropenem/vaborbactam and imipenem/relebactam. Seven of 10 E. coli isolates were tigecycline susceptible of which 6 were also eravacycline susceptible. One of 11 K. pneumoniae, 1/1 K. oxytoca and 1/1 L. adecarboxylata isolates were susceptible to both glycylcyclines. All other isolates were resistant to both glycylcyclines.ConclusionsIn both OXA-48 and NDM producers, the activity of the four antibiotics tested was better against E. coli than K. pneumoniae. Meropenem/vaborbactam provided better cover than imipenem/relebactam. Eravacycline and tigecycline showed similar activity to each other, but their activity was inferior compared with the β-lactam combinations, particularly against K. pneumoniae.

An animal model of limitation of gut colonization by carbapenemase-producing Klebsiella pneumoniae using rifaximin

Current knowledge suggests that infection by carbapenem-resistant enterobacteria is preceded by gut colonization. It is hypothesized that colonization is eradicated by non-absorbable antibiotics like rifaximin. We investigated the effect of rifaximin against carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in a mouse model. We studied the in vitro efficacy of rifaximin against 257 CRKP clinical isolates, 188 KPC producers and 69 OXA-48 producers, by minimum inhibitory concentration and time-kill assays. We then developed a model of gut colonization by feeding 30 C57Bl6 mice with 108 cfu of one KPC-KP isolate for 7 days; mice were pre-treated orally with saline, omeprazole or ampicillin. Then, another 60 mice with established KPC-2 gut colonization received orally for 7 consecutive days rifaximin 180 mg/kg dissolved in ethanol and 4% bile or vehicle. On days 0, 3 and 7 stool samples were collected; mice were sacrificed for determination of tissue outgrowth. At a concentration of 1000 μg/ml rifaximin inhibited 84.8% of CRKP isolates. Α 3 × log10 decrease of the starting inoculum was achieved by 100, 250 and 500 μg/ml of rifaximin after 24 h against 25, 55 and 55% of isolates. Pre-treatment with ampicillin was necessary for gut colonization by KPC-KP. Treatment with rifaximin succeeded in reducing KPC-KP load in stool and in the intestine. Rifaximin inhibits at clinically meaningful gut concentrations the majority of CRKP isolates and is efficient against gut colonization by KPC-KP.

First identification of bla NDM-5 producing Escherichia coli from neonates and a HIV infected adult in Tanzania.

Introduction. Carbapenem-resistant members of the family Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice. Gap Statement. There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens. Aim. To describe the resistance mechanisms of carbapenem-resistant Escherichia coli . Methods. Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for faecal carriage of carbapenamases and OXA-48 producing members of the family Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem-resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads. Results. Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried bla NDM-5. Two E. coli from neonates belonged to ST167 and bla NDM-5 co-existed with bla CTX-M-15 and bla OXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belonged to ST2083, and carried bla NDM-5 on an IncX3 type plasmid and bla CMY-42 on an IncI type plasmid. All bla NDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harboured resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3), also on the IncFIA plasmid. All three isolates were susceptible to colistin. Conclusion. This is the first report, to our knowledge, from Tanzania detecting bla NDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania.