NDM-producing Enterobacteriaceae Research Articles

Management and control of an outbreak of NDM-producing Klebsiella pneumoniae in a teaching hospital

Abstract Background Outbreaks caused by New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae are a major public health threat. We report an outbreak caused by NDM-producing Klebsiella pneumoniae (KP) in an Italian teaching hospital and describe the composite management approach that led to a successful containment. Methods On August 12 2022, a 39-year old Ukrainian patient with known colonization (rectal swab) by NDM-producing KP was admitted to the Hematology Unit of the University Hospital of Verona, Italy. Six days after, a second patient tested positive for carbapenem-resistant (CR) KP from rectal swab and 2 additional cases (rectal and pharyngeal swab respectively) were identified in the following 2 weeks. The 4 cases were identical strains. Results A multidisciplinary team coordinated the response actions. Isolation measures were ordered for positive patients, including use of dedicated instruments. Screening at admission and every 7 days was placed for all the ward patients. On-site inspections and microbiological screening of ward surfaces and devices were carried out. Infection prevention and control (IPC) measures were strengthened among personnel. Environmental cleaning was implemented by deep treatment of critical points (e.g. toilets) and reinforcement of routine cleaning. The Risk Manager developed an original checklist based on literature to review the implementation of IPC practices for CR Enterobacteriaceae. No new cases were identified from screening in the following weeks and the Unit returned to standard IPC measures. Given that environmental samples were negative and the cases had overlapping personnel, the hypothesis of contact transmission through caregivers was the most plausible. Reinforcement of training on hand hygiene and transmission-based precautions was planned. Conclusions It was possible to contain the hospital outbreak through rapid and diversified reaction capacity. Continuous IPC education is an essential control measure. Key messages • The spread of NDM-producing Enterobacteriaceae in the hospital setting must be countered with enhanced prevention and, in case of outbreak, rapid control. • Effective containment involves a mix of established measures (screening, hygiene, environmental controls) and innovative approaches (e.g. literature-based checklists).

In Vitro Activities and Inoculum Effects of Cefiderocol and Aztreonam-Avibactam against Metallo-β-Lactamase-Producing Enterobacteriaceae.

Cefiderocol and aztreonam-avibactam (ATM-AVI) both had activity against carbapenem-resistant Gram-negative bacilli, including those that produce metallo-β-lactamases (MBLs). We compared the in vitro activities and inoculum effects of these antibiotics against carbapenemase-producing Enterobacteriaceae (CPE), especially MBL-producing isolates. The MICs of cefiderocol and ATM-AVI were determined using broth microdilution method for a 2016 to 2021 collection of Enterobacteriaceae isolates which produced MBL, KPC, or OXA-48-like carbapenemases. MICs with high bacteria inoculum were also evaluated for susceptible isolates. A total of 195 CPE were tested, including 143 MBL- (74 NDM, 42 IMP, and 27 VIM), 38 KPC-, and 14 OXA-48-like-producing isolates. The susceptible rates of MBL-, KPC-, and OXA-48-like producers to cefiderocol were 86.0%, 92.1%, and 92.9%, respectively, and that to ATM-AVI were 95.8%, 100%, and 100%, respectively. NDM producers displayed lower susceptibility and higher MIC50s/MIC90s of cefiderocol (78.4%, 2/16 mg/L) than IMP (92.9%, 0.375/4 mg/L) and VIM (96.3%, 1/4 mg/L) producers. NDM- and VIM-producing Escherichia coli showed lower susceptibility to ATM-AVI (77.3% and 75.0%, respectively) compared to MBL-CPE of other species (100% susceptible). Inoculum effects for cefiderocol and ATM-AVI were observed among 95.9% and 95.2% of susceptible CPE, respectively. A switch from susceptible to resistant category was observed in 83.6% (143/171) of isolates for cefiderocol and 94.7% (179/189) for ATM-AVI. Our results revealed that NDM-producing Enterobacteriaceae had lower susceptibility to cefiderocol and ATM-AVI. Prominent inoculum effects on both antibiotics were observed for CPE, which suggested a risk of microbiological failure when they were used for CPE infections with high bacteria burden. IMPORTANCE The prevalence of infections caused by carbapenem-resistant Enterobacteriaceae is increasing worldwide. Currently, therapeutic options for metallo-β-lactamase (MBL)-producing Enterobacteriaceae remain limited. We demonstrated that clinical metallo-β-lactamase (MBL)-producing Enterobacteriaceae isolates were highly susceptible to cefiderocol (86.0%) and aztreonam-avibactam (ATM-AVI) (95.8%). However, inoculum effects on cefiderocol and ATM-AVI were observed for over 90% of susceptible carbapenemase-producing Enterobacteriaceae (CPE) isolates. Our findings highlight a potential risk of microbiological failure when using monotherapy with cefiderocol or ATM-AVI to treat severe CPE infection.

Epidemiological characteristics of New Delhi Metallo-β-Lactamase-producing Enterobacteriaceae in the Fourth hospital of Hebei Medical University

The epidemiological characteristics of New Delhi Metallo-β-Lactamase-Producing (NDM) Enterobacteriaceae were analyzed to provide theoretical support for clarifying the distribution characteristics of carbapenem-resistant Enterobacteriaceae (CRE) in the hospital environment and early identification of susceptible patients. From January 2017 to December 2021,42 strains of NDM-producing Enterobacteriaceae were gathered from the Fourth Hospital of Hebei Medical University, primarily Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. The micro broth dilution method combined with the Kirby-Bauer method was used to determine the minimal inhibitory concentrations (MICs) of antibiotics. The carbapenem phenotype was detected by the modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM). Carbapenem genotypes were detected by colloidal gold immunochromatography and real-time fluorescence PCR. The results of antimicrobial susceptibility testing showed that all NDM-producing Enterobacteriaceae were multiple antibiotic resistant, but the sensitivity rate to amikacin was high. Invasive surgery prior to culture, the use of excessive amounts of different antibiotics, the use of glucocorticoids, and ICU hospitalization were clinical characteristics of NDM-producing Enterobacteriaceae infection. Molecular typing of NDM-producing Escherichia coli and Klebsiella pneumoniae was carried out by Multilocus Sequence Typing (MLST), and the phylogenetic trees were constructed. Eight sequence types (STs) and two NDM variants were detected in 11 strains of Klebsiella pneumoniae, primarily ST17, and NDM-1. A total of 8 STs and 4 NDM variants were detected in 16 strains of Escherichia coli, mainly ST410, ST167, and NDM-5. For high-risk patients who have CRE infection, CRE screening should be done as soon as feasible to adopt prompt and efficient intervention measures to prevent outbreaks in the hospital.

NDM Production as a Dominant Feature in Carbapenem-Resistant Enterobacteriaceae Isolates from a Tertiary Care Hospital.

The worldwide spread and increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is of utmost concern and a problem for public health. This resistance is mainly conferred by carbapenemase production. Such strains are a potential source of outbreaks in healthcare settings and are associated with high rates of morbidity and mortality. In this study, we aimed to determine the dominance of NDM-producing Enterobacteriaceae at a teaching hospital in Karachi. A total of 238 Enterobacteriaceae isolates were collected from patients admitted to Jinnah Postgraduate Medical Centre (Unit 4) in Karachi, Pakistan, a tertiary care hospital. Phenotypic and genotypic methods were used for detection of metallo-β-lactamase. Out of 238 isolates, 52 (21.8%) were CRE and 50 isolates were carbapenemase producers, as determined by the CARBA NP test; two isolates were found negative for carbapenemase production by CARB NP and PCR. Four carbapenemase-producing isolates phenotypically appeared negative for metallo-β-lactamase (MBL). Of the 52 CRE isolates, 46 (88.46%) were blaNDM positive. Most of the NDM producers were Klebsiella pneumoniae, followed by Enterobacter cloacae and Escherichia coli. In all the NDM-positive isolates, the blaNDM gene was found on plasmid. These isolates were found negative for the VIM and IPM MBLs. All the CRE and carbapenem-sensitive isolates were sensitive to colistin. It is concluded that the NDM is the main resistance mechanism against carbapenems and is dominant in this region.

Draft Genome Sequence of NDM-Encoding Klebsiella pneumoniae Isolated from Feral Swine.

ABSTRACTNew Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae pose a great threat to public health globally. Most known NDM-producing Enterobacteriaceae are associated with human hospital or community infections. Here, we report the draft genome sequence of an NDM-1-encoding Klebsiella pneumoniae strain isolated from feral swine (Sus scrofa) captured in Florida, USA.

Pharmacodynamics of the Novel Metallo-β-Lactamase Inhibitor ANT2681 in Combination with Meropenem for the Treatment of Infections Caused by NDM-Producing Enterobacteriaceae.

Enterobacteriaceae that produce metallo-β-lactamases (MBLs) are an emerging threat to public health. The metallo-β-lactamase inhibitor (MBLi) ANT2681 inhibits the enzymatic activity of MBLs through interaction with the dinuclear zinc ion cluster present in the active site that is common to these enzymes. ANT2681 is being codeveloped, with meropenem as the partner β-lactam, as a novel combination therapy for infections caused by MBL-producing bacteria. The pharmacokinetics/pharmacodynamics of meropenem-ANT2681 were studied in a murine neutropenic thigh model of NDM-producing Enterobacteriaceae Dose-ranging studies were performed with both meropenem and ANT2681. Dose fractionation experiments were performed to identify the relevant pharmacodynamic index of ANT2681 when coadministered with meropenem. A background of meropenem at 50 mg/kg of body weight every 4 h (q4h) subcutaneously (s.c.) had minimal antibacterial effect. On this background, half-maximal effect was observed with an ANT2681 dose of 89 mg/kg q4h intravenously (i.v.). The dose fractionation study showed that area under the concentration-time curve (AUC) was the relevant pharmacodynamic index for the inhibitor. The magnitude of the meropenem-ANT2681 exposure required to achieve stasis was explored using 5 NDM-producing strains. A 3-dimensional surface fitted to the pharmacodynamic data from the 5 strains suggested that stasis was achieved with an fT > potentiated meropenem MIC of 40% and ANT2681 AUC of 700 mg · h/liter. These data and analyses provide the underpinning evidence for the combined use of meropenem and ANT2681 for clinical infections.

Aggressive Colonization Screening and Infection Control Measures in Containment of NDM-5 Carbapenemase-Producing CRE

Background: In April 2019, Nebraska Public Health Laboratory identified an NDM-producing Enterobacter cloacae from a urine sample from a rehabilitation inpatient who had recently received care in a specialized unit (unit A) of an acute-care hospital (ACH-A). After additional infections occurred at ACH-A, we conducted a public health investigation to contain spread. Methods: A case was defined as isolation of NDM-producing carbapenem-resistant Enterobacteriaceae (CRE) from a patient with history of admission to ACH-A in 2019. We conducted clinical culture surveillance, and we offered colonization screening for carbapenemase-producing organisms to all patients admitted to unit A since February 2019. We assessed healthcare facility infection control practices in ACH-A and epidemiologically linked facilities by visits from the ICAP (Infection Control Assessment and Promotion) Program. The recent medical histories of case patients were reviewed. Isolates were evaluated by whole-genome sequencing (WGS). Results: Through June 2019, 7 cases were identified from 6 case patients: 4 from clinical cultures and 3 from 258 colonization screens including 1 prior unit A patient detected as an outpatient (Fig. 1). Organisms isolated were Klebsiella pneumoniae (n = 5), E. cloacae (n = 1), and Citrobacter freundii (n = 1); 1 patient had both NDM-producing K. pneumoniae and C. freundii. Also, 5 case patients had overlapping stays in unit A during February–May 2019 (Fig. 2); common exposures in unit A included rooms in close proximity, inhabiting the same room at different times and shared caregivers. One case-patient was not admitted to unit A but shared caregivers, equipment, and devices (including a colonoscope) with other case patients while admitted to other ACH-A units. No case patients reported travel outside the United States. Screening at epidemiologically linked facilities and clinical culture surveillance showed no evidence of transmission beyond ACH-A. Infection control assessments at ACH-A revealed deficiencies in hand hygiene, contact precautions adherence, and incomplete cleaning of shared equipment within and used to transport to/from a treatment room in unit A. Following implementation of recommended infection control interventions, no further cases were identified. Finally, 5 K. pneumoniae of ST-273 were related by WGS including carriage of NDM-5 and IncX3 plasmid supporting transmission of this strain. Further analysis is required to relate IncX3 plasmid carriage and potential transmission to other organisms and sequence types identified in this study. Conclusions: We identified a multiorganism outbreak of NDM-5–producing CRE in an ACH specialty care unit. Transmission was controlled through improved infection control practices and extensive colonization screening to identify asymptomatic case-patients. Multiple species with NDM-5 were identified, highlighting the potential role of genotype-based surveillance.Funding: NoneDisclosures: Muhammad Salman Ashraf reports that he is the principal investigator for a study funded by an investigator-initiated research grant.

In Vitro Activity of Cefiderocol Against Multidrug-Resistant Gram-Negative Clinical Isolates

Background: New antimicrobials are being developed as a response to the global threat of multidrug-resistant and panresistant bacterial pathogens. Cefiderocol (FDC; Shionogi & Co) is a novel parenteral siderophore cephalosporin with activity against gram-negative rods. Here, we report on the in vitro activity of FDC against multidrug-resistant gram-negative isolates collected by the CDC, including isolates available through the CDC and FDA Antibiotic Resistance Isolate Bank (AR Isolate Bank). Methods: The challenge set of gram-negative isolates (n = 339), most of which were obtained from the AR isolate bank (n = 258), comprised 188 Enterobacteriaceae (ENT), 72 Pseudomonas aeruginosa (PSA), and 79 Acinetobacter baumannii (ACB). Minimum inhibitory concentrations (MICs) for FDC in iron-depleted cation-adjusted Mueller-Hinton broth were determined using frozen reference broth microdilution panels (IHMA, Schaumburg, IL) according to CLSI guidelines. Isolates displaying nonsusceptibility to FDC (MIC >4 µg/mL) underwent additional testing with β-lactamase inhibitors (FDC with 4 µg/mL avibactam, FDC with 100 µg/ml dipicolinic acid (DPA), and FDC with both 100 µg/mL dipicolinic acid (DPA) and 4 µg/mL avibactam). Results: Cefiderocol MICs ranged from ≤0.03 to >64 µg/mL, and 313 (92.3%) isolates displayed susceptibility to FDC (MIC ≤4 μg/mL). The proportions of susceptible ENT, PSA, and ACB were 93.1%, 94.4%, and 88.6%, respectively. Among isolates harboring Ambler class A, class B, or class D carbapenemases, the proportions of susceptible isolates were 96.5%, 79.5%, and 94.0%, respectively. Overall, 26 (7.7%) isolates were categorized as FDC nonsusceptible (MIC ≥ 8 µg/mL); 65% of these were NDM producers. We selected 23 isolates for testing with β-lactamase inhibitors. The combination FDC-avibactam reduced the MIC to susceptible for all isolates harboring an Ambler class A or D carbapenemase, except for 1 OXA-71–producing ACB and 1 KPC-producing Citrobacter farmeri. The combination FDC-DPA reduced the MIC to susceptible for 9 of 13 (69.2%) NDM-producing and 4 of 4 (100%) OXA-23–producing ACB. By combining FDC with both DPA and avibactam, the MIC was reduced to susceptible (91%) for all but 1 KPC-producing and 1 NDM-producing Enterobacteriaceae isolate. Conclusions: Cefiderocol (FDC) demonstrated potent activity against a diverse collection of multidrug-resistant, gram-negative isolates, including producers of Ambler class A, B, and D carbapenemases. Among the 26 FDC nonsusceptible isolates, 65% were NDM positive. Our data indicate that FDC combined with β-lactamase inhibitors may restore susceptibility in FDC nonsusceptible isolates. Additional studies are needed to understand the underlying mechanism(s) of FDC resistance and to further explore the use of β-lactamase inhibitors in combination with FDC.Funding: NoneDisclosures: None

Transmission of Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae in Georgia, 2018–2019

Background: In April 2019, the Georgia Department of Public Health (DPH) initiated whole-genome sequencing (WGS) on NDM-producing Enterobacteriaceae identified since January 2018. The WGS data analyzed at CDC identified related Klebsiella pneumoniae isolates with hypervirulence markers from 2 patients. Carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) are rarely reported in the United States, but they can to cause serious, highly resistant, invasive infections. We conducted an investigation to identify cases and prevent spread. Methods: We defined a case as NDM-producing K. pneumoniae with ≥4 hypervirulence markers identified by WGS, isolated from any specimen source from a Georgia patient. We reviewed the case patient’s medical history to identify potentially affected facilities. We also performed PCR-based colonization screening and retrospective and prospective laboratory-based surveillance. Finally, we assessed facility infection control practices. Results: Overall, 7 cases from 3 case patients (A, B, and C) were identified (Fig. 1). The index case specimen was collected from case-patient A at ventilator-capable skilled nursing facility 1 (vSNF1) in May 2018. Case-patient A had been hospitalized for 1 month in India before transfer to the United States. Case-patient B’s initial isolate was collected in January 2019 on admission to vSNF2 from a critical access hospital (CAH). The CAH laboratory retrospectively identified case-patient C, who overlapped with case-patient B at the CAH in October 2018. The CAH and the vSNF2 are geographically distant from vSNF1. Case-patients B and C had no known epidemiologic links to case-patient A. Colonization screening occurred at vSNF1 in May 2018, following detection of NDM-producing K. pneumoniae from case-patient A ∼1 year before determining that the isolate carried hypervirulence markers. Among 30 residents screened, 1 had NDM and several had other carbapenemases. Subsequent screening did not identify additional NDM. Colonization screening of 112 vSNF2 residents and 13 CAH patients in 2019 did not reveal additional case patients; case-patient B resided at vSNF2 at the time of screening and remained colonized. At all 3 facilities, the DPH assessed infection control practices, issued recommendations to resolve lapses, and monitored implementation. The DPH sequenced all 27 Georgia NDM–K. pneumoniae isolates identified since January 2018; all were different multilocus sequence types from the CP-hvKP isolates, and none possessed hypervirulence markers. Conclusions: We hypothesize that CP-hvKP was imported by a patient hospitalized in India and spread to 3 Georgia facilities in 2 distinct geographic regions through indirect patient transfers. Although a response to contain NDM at vSNF1 in 2018 likely limited CP-hvKP transmission, WGS identified hvKP and established the relatedness of isolates from distinct regions, thereby directing the DPH’s additional containment activities to halt transmission.Funding: NoneDisclosures: None

Evaluation of the in vitro activity of new polymyxin B analogue SPR206 against clinical MDR, colistin-resistant and tigecycline-resistant Gram-negative bacilli.

SPR206 is a novel polymyxin analogue. Activity against clinical isolates is littledocumented. A collection of 200 MDR, carbapenem-resistant, tigecycline-resistant, colistin-resistant and non-MDR clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Stenotrophomonas maltophilia was obtained from 50 centres across China (2016-17). All isolates were derived from respiratory tract, urine and blood samples. Strains were purposely selected on the basis of phenotypes, genotypes and specimen origins. MICs of SPR206 and other antimicrobials were determined. SPR206 was active against all bacteria tested except colistin-resistant isolates. The MIC50/90 values of SPR206 for colistin-resistant strains were comparable to known polymyxins (16/128 versus 8/128 mg/L). SPR206 exhibited potent activity against colistin-susceptible OXA-producing A. baumannii (MIC50/90 = 0.064/0.125 mg/L), NDM-producing Enterobacteriaceae (MIC50/90 = 0.125/0.25 mg/L) and KPC-2-producing Enterobacteriaceae (MIC50/90 = 0.125/0.5 mg/L). In fact, SPR206 was the most potent agent tested, with 2- to 4-fold lower MICs than colistin and polymyxin B for A. baumannii, P. aeruginosa and Enterobacteriaceae. Additionally, MIC values of SPR206 (MIC50/90 = 0.064/0.125 mg/L) were 16- to 32-fold lower than those of tigecycline (MIC50/90 = 2/2 mg/L) for tigecycline-susceptible carbapenem-resistant A. baumannii. SPR206 showed good in vitro activity against MDR, tigecycline-resistant and non-MDR clinical isolates of Gram-negative pathogens. SPR206 also exhibited superior potency to colistin and polymyxin B, with 2- to 4-fold lower MIC50/90 values.

In vitro activity of the novel β-lactamase inhibitor taniborbactam (VNRX-5133), in combination with cefepime or meropenem, against MDR Gram-negative bacterial isolates from China.

To evaluate in vitro activity of the novel β-lactamase boronate inhibitor taniborbactam (VNRX-5133) combined with cefepime or meropenem against 500 urinary Gram-negative bacilli. Cefepime/taniborbactam and 14 comparators were tested by broth microdilution or agar dilution methods. A total of 450 Enterobacteriaceae and 50 Pseudomonas aeruginosa were selected from 2017 to 2019 based on different β-lactamase-producing or resistance phenotypes. For carbapenem-non-susceptible isolates, the modified carbapenem inactivation method (mCIM), EDTA-CIM (eCIM) and amplification of carbapenemase genes were performed. For NDM-producing isolates and those with cefepime/taniborbactam MICs >8 mg/L, the MICs of meropenem/taniborbactam and/or mutations in PBP3 were investigated. Taniborbactam improved cefepime activity with the same efficiency as avibactam improved ceftazidime activity against 66 KPC-2 producers, 30 non-carbapenemase-producing carbapenem-non-susceptible Enterobacteriaceae and 28 meropenem-susceptible P. aeruginosa. However, cefepime/taniborbactam exhibited more potent activity than ceftazidime/avibactam against 56 ESBL-producing, 61 AmpC-producing, 32 ESBL and AmpC co-producing, 87 NDM-producing and 21 MBL-producing Enterobacteriaceae predicted by phenotypic mCIM and eCIM, 82 Enterobacteriaceae that were susceptible to all tested β-lactams and 22 carbapenem-non-susceptible P. aeruginosa. A four-amino acid 'INYR' or 'YRIN' insertion, with or without a one/two-amino acid mutation in PBP3, may have caused cefepime/taniborbactam MICs >8 mg/L among 96.6% (28/29) of the NDM-5-producing Escherichia coli, which accounted for the majority of isolates with cefepime/taniborbactam MICs >8 mg/L (76.1%, 35/46). Taniborbactam's superior breadth of activity, when paired with cefepime or meropenem, suggests these β-lactam/β-lactamase inhibitor combinations could be promising candidates for treating urinary tract infections caused by ESBL and/or AmpC, KPC or NDM-producing Enterobacteriaceae or P. aeruginosa.

In Vitro Efficacy of Meropenem-Cefmetazole Combination Therapy against New Delhi Metallo-β-lactamase-producing Enterobacteriaceae

BackgroundLimited treatment options complicate management of infections with New Delhi metallo-β-lactamase (NDM)-producing organisms. The efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) was assessed against NDM-producing Enterobacteriaceae. Materials and MethodsTwelve Escherichia coli clinical isolates harbouring blaNDM-1 and a positive control E. coli BAA-2469 harbouring blaNDM-1 were studied. Minimum inhibitory concentrations (MICs) of MEM, ertapenem (ERT) and CMZ were determined by broth microdilution. Checkerboard and time-kill assays were performed to confirm the in vitro efficacy of the MEM/CMZ combination. Scanning electron microscopy, kinetic studies and whole-genome sequence analysis were used to determine the antimicrobial resistance mechanisms. ResultsMICs of MEM, ERT and CMZ in monotherapy ranged from 8 to 32, 16 to 128, and 32 to 512 µg/mL, respectively. In the checkerboard assay, MEM/ERT resulted in no synergy, whereas MEM/CMZ showed a synergistic effect in all the tested isolates. Furthermore, the MIC of MEM in combination decreased by 2- to 8-fold compared with that of MEM alone. The time-kill study revealed a bactericidal effect in 4 of 13 isolates at 24 h. Scanning electron microscopy showed spheroidisation of the bacterial cell in the MEM/CMZ combination; this was not observed in single antibiotic conditions. Kinetic studies indicated CMZ was a better antagonist for NDM-1 than ERT. Whole-genome sequence analysis did not reveal any explainable differences between isolates susceptible and those non-susceptible to combination therapy. ConclusionIn vitro studies showed the potential effectiveness of MEM/CMZ combination therapy against NDM-producing organisms.

Molecular Analysis of the Antibiotic Resistant NDM-1 Gene in Clinical Isolates of Enterobacteriaceae

The emergence of the New Dehli metallo-beta-lactamase (NDM) gene in Enterobacteriaceae is responsible for multidrug resistance responsible for severe infections and serious morbidity in patients. Our study aimed to define the molecular characteristics and antibiogram of the NDM-1 producing Enterobacteriaceae. We isolated 370 individual enterobacteria from the clinical specimens collected from the two tertiary hospitals in Sakaka, Saudi Arabia. Bacterial isolation was performed using standard microbiological techniques and the Phoenix and Microscan WalkAway Plus automated analyzers. Bacterial strains were characterized by phenotypic methods and PCR, and DNA sequencing was used for the molecular characterization of NDM genes. The blaNDM gene was detected among the 68 members of the Enterobacteriaceae including a single case of rarely reported Cedecea lapagei. Of these 68, 43 isolates (63.2%) were blaNDM-1 and 25 (36.8%) were blaNDM variants. A statistically significant relationship between the NDM-1 and Klebsiella pneumoniae (p = 0.004) was seen, and the relationship between the NDM variants was significantly associated with Citrobacter freundii (p = 0.02) and Escherichia coli (p = 0.03). The in vitro minimum inhibitory concentrations (MICs) of NDM-producing Enterobacteriaceae revealed a very high rate of antibiotic resistance against several groups of antibiotics. These bacterial strains were less resistant to two aminoglycosides, gentamicin (39; 57.3%) and amikacin (27; 39.7%), and showed minimum resistance to tigecycline (25; 36.8%). The emergence of a large number of NDM-1 enterobacteria in our study identifies a substantial public concern, both within hospitals and the wider community, and leaves us a narrow choice of therapeutic options: the aminoglycosides, co-trimoxazole, and tigecycline.

Primer aislamiento de enterobacteria productora de carbapenemasa tipo-NDM en un hospital público de Rosario, Argentina

Introducción: La emergencia de enterobacterias productoras de carbapenemasas en el ámbito hospitalario representa un verdadero problema de salud pública mundial. Las carbapenemasas son enzimas que producen resistencia a los antibióticos carbapenémicos, teniendo un directo impacto en la disponibilidad de alternativas terapéuticas. En Argentina, a partir de 2013 han emergido carbapenemasas tipo-NDM (Nueva Delhi Metalo-β-lactamasa, MβL), que constituyen una resistencia emergente a nivel global.
 Objetivo: Reportar el primer aislamiento clínico de enterobacteria portadora de NDM en nuestra institución.
 Materiales y métodos: El aislamiento estudiado fue recuperado de una muestra ósea de un paciente adulto. La identificación bacteriana y los ensayos de susceptibilidad antibiótica se realizaron mediante metodología manual y sistema automatizado Vitek 2C (Biomérieux). La detección y caracterización de carbapenemasas se efectuó por ensayos fenotípicos y moleculares.
 Resultados: Los ensayos revelaron que el aislamiento, tipificado como Citrobacter freundii, es productor de carbapenemasa tipo NDM. Resultó sensible a aztreonam, colistina y fosfomicina. No se detectó fenotípicamente la presencia de beta lactamasas de espectro extendido.
 Discusión: Se reporta el primer aislamiento de enterobacteria productor de MβL tipo-NDM en nuestro nosocomio, siendo multirresistente, con escasas alternativas terapéuticas. Dado que la presencia de este tipo de aislamiento es considerado de alto riesgo, se requiere un monitoreo activo de este mecanismo de resistencia y la instauración de medidas de control adecuadas para hacer frente a la amenaza que suponen.

598. In Vitro Activity of Aztreonam in Combination with Ceftazidime–Avibactam, Amoxicillin–Clavulanate, and Piperacillin–Tazobactam vs. NDM-Producing Escherichia coli and Klebsiella pneumoniae Clinical Isolates

BackgroundThere are limited options available for the treatment of infections caused by Enterobacteriaceae that produce an NDM metallo-β-lactamase. The purpose of this study was to compare the in vitro activity of aztreonam in combination with three different β-lactam/β-lactamase inhibitors (ceftazidime–avibactam, amoxicillin-clavulanate, piperacillin–tazobactam) vs. NDM-positive Enterobacteriaceae clinical isolates.MethodsSeven Escherichia coli and three Klebsiella pneumoniae clinical isolates (all NDM-positive by PCR) were included in this study. The in vitro activities of ceftazidime–avibactam, amoxicillin-clavulanate, piperacillin–tazobactam, and aztreonam were determined by disk diffusion as described by CLSI. For synergy testing, disks containing a β-lactamase inhibitor (ceftazidime–avibactam, amoxicillin-clavulanate, piperacillin tazobactam) were applied to Mueller–Hinton agar plates inoculated with the test organisms, and the plates were incubated for 1 hour. The disks were then removed and aztreonam disks were dropped on the previous disk sites. The plates were then incubated as per standard CLSI recommendations for disk diffusion testing.ResultsAll ten isolates demonstrated phenotypic resistance to aztreonam, amoxicillin-clavulanate, and piperacillin–tazobactam, and eight were resistant to ceftazidime–avibactam (CLSI breakpoints). The zone diameter observed for aztreonam in combination with ceftazidime–avibactam was greater than for either antimicrobial on its own for nine isolates. Seven isolates (70%) had susceptibility to aztreonam restored (zone diameter ≥21 mm) in the presence of avibactam. Aztreonam in combination with amoxicillin-clavulanate demonstrated in increase in zone diameter for all isolates relative to the zone for each antimicrobial alone, but only two (20%) had aztreonam susceptibility restored. Aztreonam susceptibility was not restored for any of the isolates in combination with piperacillin–tazobactam.ConclusionOf the three β-lactam/β-lactamase inhibitor-aztreonam combinations evaluated, ceftazidime–avibactam plus aztreonam demonstrated the greatest in vitro activity vs. NDM-producing Enterobacteriaceae.Disclosures All authors: No reported disclosures.

Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS.

NDM carbapenemases have spread worldwide. However, little information exists about the impact of NDM-producing Enterobacteriaceae in Spain. By WGS, we sought to elucidate the population structure of NDM-like-producing Klebsiella pneumoniae and Escherichia coli in Spain and to determine the plasmids harbouring blaNDM-like genes. High-resolution SNP typing, core-genome MLST and plasmid reconstruction (PlasmidID) were performed on 59 NDM-like-producing K. pneumoniae and 8 NDM-like-producing E. coli isolated over an 8 year period in Spain. Five major epidemic clones of NDM-producing K. pneumoniae caused five important nationwide outbreaks: ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1; in contrast, the spread of NDM-producing E. coli was polyclonal. Three blaNDM types were identified: blaNDM-1, 61.2%; blaNDM-7, 32.8%; and blaNDM-5, 6%. Five K. pneumoniae isolates co-produced other carbapenemases (three blaOXA-48 and two blaVIM-1). The average number of acquired resistance genes was higher in K. pneumoniae than in E. coli. The plasmids encoding blaNDM-like genes belonged to IncFII, IncFIB, IncX3, IncR, IncN and IncC types, of which IncF, IncR and IncC were associated with MDR. The genetic surroundings of blaNDM-like genes showed a highly variable region upstream of ISAba125. In recent years NDM-producing K. pneumoniae and E. coli have emerged in Spain; the spread of a few high-risk K. pneumoniae clones such as ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1 have caused several interregional outbreaks. In contrast, the spread of NDM-producing E. coli has been polyclonal. Plasmid types IncFII, IncFIB, IncX3, IncR, IncN and IncC carried blaNDM, and the same IncX3 plasmid was detected in K. pneumoniae and E. coli.

Assessment of a newly developed immunochromatographic assay for NDM-type metallo-\u03b2-lactamase producing Gram-negative pathogens in Myanmar

BackgroundTo detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-β-lactamases (NDMs) was developed.MethodsThe immunochromatographic assay for New Delhi metallo-β-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing.ResultsOf the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, − 4, − 5 and − 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs.ConclusionsThe immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.

New Delhi Metallo-Beta-Lactamase-Producing Enterobacteriaceae in South Korea Between 2010 and 2015.

This study was carried out to investigate the epidemiological time-course of New Delhi metallo-beta-lactamase- (NDM-) mediated carbapenem resistance in Enterobacteriaceae in South Korea. A total of 146 non-duplicate NDM-producing Enterobacteriaceae recovered between 2010 and 2015 were voluntarily collected from 33 general hospitals and confirmed by PCR. The species were identified by sequences of the 16S rDNA. Antimicrobial susceptibility was determined either by the disk diffusion method or by broth microdilution, and the carbapenem MICs were determined by agar dilution. Then, multilocus sequence typing and PCR-based replicon typing was carried out. Co-carried genes for drug resistance were identified by PCR and sequencing. The entire genomes of eight random selected NDM producers were sequenced. A total of 69 Klebsiella pneumoniae of 12 sequence types (STs), 34 Escherichia coli of 15 STs, 28 Enterobacter spp. (including one Enterobacter aerogenes), nine Citrobacter freundii, four Raoultella spp., and two Klebsiella oxytoca isolates produced either NDM-1 (n = 126), NDM-5 (n = 18), or NDM-7 (n = 2). The isolates co-produced CTX-M-type ESBL (52.1%), AmpCs (27.4%), additional carbapenemases (7.1%), and/or 16S rRNA methyltransferases (4.8%), resulting in multidrug-resistance (47.9%) or extensively drug-resistance (52.1%). Among plasmids harboring blaNDM, IncX3 was predominant (77.4%), followed by the IncFII type (5.8%). Genome analysis revealed inter-species and inter-strain horizontal gene transfer of the plasmid. Both clonal dissemination and plasmid transfer contributed to the wide dissemination of NDM producers in South Korea.

Occurrence of blaNDM Variants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital.

Carbapenem-resistance among enterobacteriaceae has become a global health concern. The objective of this study was to understand NDM producing enterobacteriaceae and their genetic basis of resistance, spreading in neonatal intensive care unit. Carbapenem resistant NDM producing enterobacteriaceae isolates were recovered from rectal swab and blood sample of infants admitted in NICU. These were determined by using Carba-NP test. All isolates were identified using BD PhoenixTM−100 and MICs were determined by broth microdilution method. The blaNDM and associated resistant markers were checked by PCR followed by sequencing. Moreover, ERIC-PCR and genetic environment of blaNDM gene were also performed for the analysis of clonal relationship and genetic surrounding of the strains. We characterized 44 isolates with blaNDM variants in Escherichia coli (45.5%), Klebsiella pneumoniae (40.9%), Citrobacter freundii (4.5%), Citrobacter braakii (2.3%), Klebsiella oxytoca (2.3%), Enterobacter cloacae (2.3%), Enterobacter aerogenes (2.2%) from NICU, showing resistance against all antibiotics except colistin and polymixin B. ISAba125 and bleomycin gene were found surrounding all blaNDM variants, besides class I integron on plasmid. (ERIC)-PCR data revealed non-clonal relatedness among most of the isolates. The transfer of resistant markers was confirmed by conjugation experiment. The PCR-based replicon typing was carried out using DNA of transconjugants. These isolates carried NDM-1 (20.45%), NDM-4 (36.36%), NDM-5 (38.64%), NDM-7 (4.55%), along with OXA, CMY, and SHV variants on conjugative plasmid of IncFIA, IncFIC, IncF, IncK, IncFIB, IncB/O, IncHI1, IncP, IncY, IncFIIA, IncI1, and IncN types. An increased number of carbapenem-resistant NDM producing enterobacteriaceae isolates recovered from NICU which is alarming signal for health workers and policy makers. Hence, it is utmost important to think about infection control measures.

Hospital sewage water: a reservoir for variants of New Delhi metallo-β-lactamase (NDM)- and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have become a threat to public health. Hospital sewage is generally unexplored, having the potential to harbour bacteria causing healthcare-associated infections. Hence, this study was initiated to monitor NDM-producing Enterobacteriaceae in hospital sewage water. A total of 32 isolates with blaNDM variants were detected in hospital sewage water, including 17 Escherichia coli, 8 Citrobacter freundii, 4 Shigella boydii, 2 Citrobacter braakii and 1 Citrobacter farmeri, showing resistance to all antibiotics except colistin. All 32 isolates carried blaNDM (9 blaNDM-1, 11 blaNDM-4, 10 blaNDM-5 and 2 blaNDM-7), 24 isolates carried blaCMY variants (1 blaCMY-2, 3 blaCMY-4, 5 blaCMY-6, 11 blaCMY-42, 2 blaCMY-86 and 2 blaCMY-139), 20 isolates carried blaOXA-type (17 blaOXA-1 and 3 blaOXA-48), 19 isolates carried blaCTX-M and 9 isolates carried ampC on conjugative plasmids of IncFIA, IncFIB, IncFIC, IncP, IncY, IncHI1 and IncI1 types. In E. coli, coexistence of blaNDM-1 with blaCMY-6 and blaCMY-139, of blaNDM-4 with blaCMY-6, blaCMY-42 and blaCMY-86, of blaNDM-5 with blaCMY-6 and blaCMY-42, and of blaNDM-7 with blaCMY-6 was observed. NDM-5-producing S. boydii and NDM-7-producing C. freundii were identified as well as detection of an association of blaNDM-4 and blaOXA-48 in C. braakii and C. farmeri. A class 1 integron was also found on a plasmid. ISAba125 and bleomycin genes were found surrounding all blaNDM variants. The emergence and dissemination of blaNDM variants in hospital sewage water is a matter of concern, creating an endemic scenario leading to the level of an outbreak.