A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae. This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of bla KPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

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Healthcare personnel experiences implementing carbapenem-resistant Enterobacterales infection control measures at a ventilator-capable skilled nursing facility—A qualitative analysis

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2021.265 ◽

2021 ◽

pp. 1-7

Author(s):

Katharina R. Rynkiewich ◽

Jinal Makhija ◽

Mary Carl M. Froilan ◽

Ellen C. Benson ◽

Alice Han ◽

...

Keyword(s):

Infection Control ◽

Multidrug Resistant ◽

Control Measures ◽

Healthcare Personnel ◽

Skilled Nursing ◽

Infection Control Measures ◽

Carbapenem Resistant ◽

Resident Care ◽

Semistructured Interviews ◽

Self Protection

Abstract Objective: Ventilator-capable skilled nursing facilities (vSNFs) are critical to the epidemiology and control of antibiotic-resistant organisms. During an infection prevention intervention to control carbapenem-resistant Enterobacterales (CRE), we conducted a qualitative study to characterize vSNF healthcare personnel beliefs and experiences regarding infection control measures. Design: A qualitative study involving semistructured interviews. Setting: One vSNF in the Chicago, Illinois, metropolitan region. Participants: The study included 17 healthcare personnel representing management, nursing, and nursing assistants. Methods: We used face-to-face, semistructured interviews to measure healthcare personnel experiences with infection control measures at the midpoint of a 2-year quality improvement project. Results: Healthcare personnel characterized their facility as a home-like environment, yet they recognized that it is a setting where germs were ‘invisible’ and potentially ‘threatening.’ Healthcare personnel described elaborate self-protection measures to avoid acquisition or transfer of germs to their own household. Healthcare personnel were motivated to implement infection control measures to protect residents, but many identified structural barriers such as understaffing and time constraints, and some reported persistent preference for soap and water. Conclusions: Healthcare personnel in vSNFs, from management to frontline staff, understood germ theory and the significance of multidrug-resistant organism transmission. However, their ability to implement infection control measures was hampered by resource limitations and mixed beliefs regarding the effectiveness of infection control measures. Self-protection from acquiring multidrug-resistant organisms was a strong motivator for healthcare personnel both outside and inside the workplace, and it could explain variation in adherence to infection control measures such as a higher hand hygiene adherence after resident care than before resident care.

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Molecular and Epidemiological Characterization of IMP-Type Metallo-β-Lactamase-Producing Enterobacter cloacae in a Large Tertiary Care Hospital in Japan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02652-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3441-3450 ◽

Cited By ~ 35

Author(s):

Kayoko Hayakawa ◽

Tohru Miyoshi-Akiyama ◽

Teruo Kirikae ◽

Maki Nagamatsu ◽

Kayo Shimada ◽

...

Keyword(s):

Hospital Mortality ◽

Tertiary Care ◽

Enterobacter Cloacae ◽

Control Measures ◽

Care Hospital ◽

Invasive Procedures ◽

Content Type ◽

Infection Control Measures ◽

Large Tertiary Care ◽

Close Relationship

ABSTRACTIMP-type metallo-β-lactamase enzymes have been reported in different geographical areas and in various Gram-negative bacteria. However, the risk factors and epidemiology pertaining to IMP-type metallo-β-lactamase-producingEnterobacter cloacae(IMP-producingE. cloacae) have not been systematically evaluated. We conducted a retrospective, matched case-control study of patients from whom IMP-producingE. cloacaeisolates were obtained, in addition to performing thorough molecular analyses of the clinically obtained IMP-producingE. cloacaeisolates. Unique cases with IMP-producingE. cloacaeisolation were included. Patients with IMP-producingE. cloacaewere matched to uninfected controls at a ratio of 1 to 3. Fifteen IMP-producingE. cloacaecases were identified, with five of the isolates being obtained from blood, and they were matched to 45 uninfected controls. All (100%) patients from whom IMP-producingE. cloacaeisolates were obtained had indwelling devices at the time of isolation, compared with one (2.2%) uninfected control. Independent predictors for isolation of IMP-producingE. cloacaewere identified as cephalosporin exposure and invasive procedures within 3 months. Although in-hospital mortality rates were similar between cases and controls (14.3% versus 13.3%), the in-hospital mortality of patients with IMP-producingE. cloacae-caused bacteremia was significantly higher (40%) than the rate in controls. IMP-producingE. cloacaeisolates were frequently positive for other resistance determinants. The MICs of meropenem and imipenem were not elevated; 10 (67%) and 12 (80%) of the 15 IMP-producingE. cloacaeisolates had a MIC of ≤1 μg/ml. A phylogenetic tree showed a close relationship among the IMP-producingE. cloacaesamples. Indwelling devices, exposure to cephalosporin, and a history of invasive procedures were associated with isolation of IMP-producingE. cloacae. Screening for carbapenemase production is important in order to apply appropriate clinical management and infection control measures.

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Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States

mBio ◽

10.1128/mbio.02881-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Victor I. Band ◽

Sarah W. Satola ◽

Richard D. Smith ◽

David A. Hufnagel ◽

Chris Bower ◽

...

Keyword(s):

United States ◽

Klebsiella Pneumoniae ◽

Diagnostic Testing ◽

Cladistic Analysis ◽

The United States ◽

Clinical Diagnostics ◽

Infection Model ◽

Content Type ◽

Carbapenem Resistant ◽

Clinical Diagnostic

ABSTRACT Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States. IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

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Spread of Carbapenemase Producing Klebsiella pneumoniae Isolates in Our Hospital: Investigation of Molecular Typing and Clonal Relationship

Türk Mikrobiyoloji Cemiyeti Dergisi ◽

10.5222/tmcd.2021.09226 ◽

2021 ◽

Author(s):

Reyhan Kiş ◽

Ebru Demiray Gündüz ◽

Ayşe Nur Sarı ◽

Zeynep Gülay

Keyword(s):

Intensive Care ◽

Klebsiella Pneumoniae ◽

Carbapenem Resistance ◽

Hospital Staff ◽

Control Measures ◽

Automated System ◽

Microbiology Laboratory ◽

Clonal Relationship ◽

Infection Control Measures ◽

Carbapenem Resistant

Objective: Carbapenem resistance has been reported with increasing frequency among members of Enterobacterales, especially in the last 10 years. Screening and detection of carbapenemase-producing isolates is important in terms of both directing the treatment and preventing its spread. In our study, it was aimed to determine the carbapenemase types and molecular epidemiological relationships of carbapenem resistant Klebsiella pneumoniae isolates, which were isolated sequentially from the samples sent to microbiology laboratory of our hospital. Method: A total of 32 carbapenem-resistant K. pneumoniae isolates of the samples sent to microbiology laboratory between July and September 2014, were included in the study. In addition to classical methods, identification of isolates at species level was made with BD Phoenix ID/AST automated system. Carbapenemase types (blaOXA-48, blaNDM, blaIMP, blaKPC, blaVIM and blaGES) of the isolates were investigated by PCR. The clonal relationship between the isolates was assessed with PFGE. Results: It was noted that 18 isolates were obtained from intensive care units, 9 from inpatient and 5 from outpatient departments. The blaOXA48 gene was found in all isolates while the other carbapenemase genes were not found. It was determined that strains were isolated from 32 patients in our hospital had 12 different PFGE pulsotypes, named as A-L. Among these, the most common ones were B (n=18) and closely related B1 pattern (n=2). The remaining isolates were represented by 11 different types. It was observed that the first isolate with B pulsotype was responsible for the spread of the outbreak from General Intensive Care Unit. Conclusion: It has been thought that the spread of carbapenem- resistant K. pneumoniae isolates in the hospital was probably occurred through the transfer of isolates from patients with gastrointestinal colonization to other patients through hospital staff. Therefore, the spread of the isolates in hospitals can be limited by detecting colonization with active surveillance programs and by applying contact isolation and effective infection control measures.

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Epigenomics, genomics, resistome, mobilome, virulome and evolutionary phylogenomics of carbapenem-resistant Klebsiella pneumoniae clinical strains

Microbial Genomics ◽

10.1099/mgen.0.000474 ◽

2020 ◽

Vol 6 (12) ◽

Author(s):

Katlego Kopotsa ◽

Nontombi M. Mbelle ◽

John Osei Sekyere

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Type I ◽

Bacteriophage Therapy ◽

Content Type ◽

Carbapenem Resistant ◽

Plasmid Replicon ◽

Size Number ◽

Plasmid Size ◽

Epidemiological Trends

Carbapenem-resistant Klebsiella pneumoniae (CRKP) remains a major clinical pathogen and public health threat with few therapeutic options. The mobilome, resistome, methylome, virulome and phylogeography of CRKP in South Africa and globally were characterized. CRKP collected in 2018 were subjected to antimicrobial susceptibility testing, screening by multiplex PCR, genotyping by repetitive element palindromic (REP)-PCR, plasmid size, number, incompatibility and mobility analyses, and PacBio’s SMRT sequencing (n=6). There were 56 multidrug-resistant CRKP, having bla OXA-48-like and bla NDM-1/7 carbapenemases on self-transmissible IncF, A/C, IncL/M and IncX3 plasmids endowed with prophages, traT, resistance islands, and type I and II restriction modification systems (RMS). Plasmids and clades detected in this study were respectively related to globally established/disseminated plasmids clades/clones, evincing transboundary horizontal and vertical dissemination. Reduced susceptibility to colistin occurred in 23 strains. Common clones included ST307, ST607, ST17, ST39 and ST3559. IncFIIk virulent plasmid replicon was present in 56 strains. Whole-genome sequencing of six strains revealed least 41 virulence genes, extensive ompK36 mutations, and four different K- and O-loci types: KL2, KL25, KL27, KL102, O1, O2, O4 and O5. Types I, II and III RMS, conferring m6A (G A TC, G A TGNNNNNNTTG, CA A NNNNNNCATC motifs) and m4C (C C WGG) modifications on chromosomes and plasmids, were found. The nature of plasmid-mediated, clonal and multi-clonal dissemination of blaOXA-48-like and blaNDM-1 mirrors epidemiological trends observed for closely related plasmids and sequence types internationally. Worryingly, the presence of both bla OXA-48 and bla NDM-1 in the same isolates was observed. Plasmid-mediated transmission of RMS, virulome and prophages influence bacterial evolution, epidemiology, pathogenicity and resistance, threatening infection treatment. The influence of RMS on antimicrobial and bacteriophage therapy needs urgent investigation.

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The epidemiology, risk factors and outcomes of Carbapenem-resistant Klebsiella pneumoniae infections in the intensive care unit from 2012 to 2018

10.21203/rs.3.rs-17412/v1 ◽

2020 ◽

Author(s):

Ping Wang ◽

Xiaocui Zou ◽

Boting Zhou ◽

Tao Yin

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Klebsiella Pneumoniae ◽

Control Measures ◽

Icu Patients ◽

Infection Control Measures ◽

Carbapenem Resistant ◽

Incidence And Mortality ◽

Changing Trend ◽

Different Sources

Abstract Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an increasing globally threat for human health, but the trends and clinical characteristics of CRKP infections in the intensive care unit(ICU) remain uninvestigated.Methods: A retrospective study was conducted among ICU patients infected with KP isolates from January 2012 to December 2018. Carbapenem resistant to Klebsiella pneumoniae was defined according to Clinical and Laboratory Standards Institute (CLSI) criteria. The incidence and changing trend of CRKP were determined. CRKP patient sources, specimen types, infection sources and outcomes were investigated. Results: There were 256(40.13%) patients with CRKP and 382(59.87%) patients with CSKP. The incidence of CRKP increased from 2012(11.11%) to 2017(63.48%) and decreased in 2018(51.52%). The proportion of isolates not susceptible to three carbapenems increased from 0 to 98.04%. The rates of CRKP isolated from blood, wound, urine and pleural fluid were higher than that of CSKP. CRKP infections were mainly ICU acquired, rather than input acquired. Conclusion: The incidence of CRKP was high in ICU, but showed a downward trend. Implementation of different infection control measures to different sources of patients, specimen types, and KP infections are necessary. Surveillance data will be needed for ICU patients to decrease the incidence and mortality of CRKP.

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Vaccine Protection against Multidrug-Resistant Klebsiella pneumoniae in a Nonhuman Primate Model of Severe Lower Respiratory Tract Infection

mBio ◽

10.1128/mbio.02994-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 3

Author(s):

Natalia Malachowa ◽

Scott D. Kobayashi ◽

Adeline R. Porter ◽

Brett Freedman ◽

Patrick W. Hanley ◽

...

Keyword(s):

United States ◽

Risk Factors ◽

Health Care ◽

Lower Respiratory Tract ◽

The United States ◽

Multidrug Resistant ◽

Sequence Type ◽

Content Type ◽

Carbapenem Resistant ◽

Cynomolgus Macaques

ABSTRACT Klebsiella pneumoniae is a human gut communal organism and notorious opportunistic pathogen. The relative high burden of asymptomatic colonization by K. pneumoniae is often compounded by multidrug resistance—a potential problem for individuals with significant comorbidities or other risk factors for infection. A carbapenem-resistant K. pneumoniae strain classified as multilocus sequence type 258 (ST258) is widespread in the United States and is usually multidrug resistant. Thus, treatment of ST258 infections is often difficult. Inasmuch as new preventive and/or therapeutic measures are needed for treatment of such infections, we developed an ST258 pneumonia model in cynomolgus macaques and tested the ability of an ST258 capsule polysaccharide type 2 (CPS2) vaccine to moderate disease severity. Compared with sham-vaccinated animals, those vaccinated with ST258 CPS2 had significantly less disease as assessed by radiography 24 h after intrabronchial installation of 108 CFU of ST258. All macaques vaccinated with CPS2 ultimately developed ST258-specific antibodies that significantly enhanced serum bactericidal activity and killing of ST258 by macaque neutrophils ex vivo. Consistent with a protective immune response to CPS2, transcripts encoding inflammatory mediators were increased in infected lung tissues obtained from CPS-vaccinated animals compared with control, sham-vaccinated macaques. Taken together, our data provide support for the idea that vaccination with ST258 CPS can be used to prevent or moderate infections caused by ST258. As with studies performed decades earlier, we propose that this prime-boost vaccination approach can be extended to include multiple capsule types. IMPORTANCE Multidrug-resistant bacteria continue to be a major problem worldwide, especially among individuals with significant comorbidities and other risk factors for infection. K. pneumoniae is among the leading causes of health care-associated infections, and the organism is often resistant to multiple classes of antibiotics. A carbapenem-resistant K. pneumoniae strain known as multilocus sequence type 258 (ST258) is the predominant carbapenem-resistant Enterobacteriaceae in the health care setting in the United States. Infections caused by ST258 are often difficult to treat and new prophylactic measures and therapeutic approaches are needed. To that end, we developed a lower respiratory tract infection model in cynomolgus macaques in which to test the ability of ST258 CPS to protect against severe ST258 infection.

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Fighting Antibiotic-Resistant Klebsiella pneumoniae with “Sweet” Immune Targets

mBio ◽

10.1128/mbio.00874-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 4

Author(s):

Roberto Adamo ◽

Immaculada Margarit

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Therapeutic Approaches ◽

Antibiotic Resistant ◽

Content Type ◽

Carbapenem Resistant ◽

Surface Polysaccharides ◽

Resistant Strains ◽

Murine Monoclonal Antibodies

ABSTRACT Antibiotics and vaccines have greatly impacted human health in the last century by dramatically reducing the morbidity and mortality associated with infectious diseases. The recent challenge posed by the emergence of multidrug-resistant bacteria could possibly be addressed by novel immune prophylactic and therapeutic approaches. Among the newly threatening pathogens, Klebsiella pneumoniae is particularly worrisome in the nosocomial setting, and its surface polysaccharides are regarded as promising antigen candidates. The majority of Klebsiella carbapenem-resistant strains belong to the sequence type 158 (ST258) lineage, with two main clades expressing capsular polysaccharides CPS1 and CPS2. In a recent article, S. D. Kobayashi and colleagues (mBio 9:e00297-18, 2018, https://doi.org/10.1128/mBio.00297-18) show that CPS2-specific IgGs render ST258 clade 2 bacteria more sensitive to human serum and phagocytic killing. E. Diago-Navarro et al. (mBio 9:e00091-18, 2018, https://doi.org/10.1128/mBio.00091-18) generated two murine monoclonal antibodies recognizing distinct glycotopes of CPS2 that presented functional activity against multiple ST258 strains. These complementary studies represent a step toward the control of this dangerous pathogen.

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510. Exposure Investigation Following a Confirmed Case of Candida auris and Multiple Carbapenemase-Producing Carbapenem-Resistant Organisms

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.579 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S247-S247

Author(s):

Frances Nicholson ◽

Melanie Curless ◽

Maggie Schiffhauer ◽

Sean Zhang ◽

Patricia Simner ◽

...

Keyword(s):

Infection Control ◽

Index Patient ◽

The United States ◽

Point Prevalence ◽

Control Measures ◽

Candida Auris ◽

Infection Control Measures ◽

Carbapenem Resistant ◽

Surveillance Cultures ◽

Routine Surveillance

Abstract Background Co-infections of Candida auris and carbapenemase-producing carbapenem-resistant Gram-negative organisms (CP-CRO) are an increasing global concern and rarely seen in the United States. We report the case of a 59-year-old male, with recent hospitalization in India, admitted to our facility with C. auris isolated from urine and axilla/groin specimens and CP-CRO from five body sites. Methods Travel screening in the emergency department identified a patient at high risk for colonization/infection with multidrug-resistant organisms (MDRO). Contact precautions were initiated. Eight CP-CRO isolates were subsequently identified from clinical and routine surveillance cultures from five separate sites. Of the isolates, seven contained one or more carbapenemase-producing genes detected by Xpert Carba-R assay (Cepheid, Sunnyvale, CA) (Table 1). The microbiology laboratory alerted the infection control department of a presumptive positive C. auris from a clinical urine culture from the same patient. Enhanced mitigation strategies were initiated in regards to cleaning and disinfection. An exposure investigation was also conducted using a point prevalence approach. Surveillance cultures were obtained from inpatients currently admitted to the same unit as the index patient. Axilla/groin specimens were collected for C. auris testing, and rectal specimens were collected for CP-CRO gene testing (CRE Real-Time PCR). Results Eighteen patients in addition to the index patient were hospitalized on the acute medicine unit. One patient refused testing for CP-CRO; therefore, 17 patients were tested for CP-CRO, and 18 patients were tested for C. auris. Neither CP-CRO nor C. auris were recovered from any patient. Conclusion A patient co-infected with C. auris and multiple CP-CRO was identified by clinical and routine surveillance cultures at Johns Hopkins Hospital. Travel screening allowed proactive isolation upon presentation. Enhanced infection control measures were implemented and a point prevalence surveillance study was conducted on the general acute care medicine inpatient unit. No transmission of either C. auris or CP-CRO was detected, likely due in part to rapid identification and strict infection control measures. Disclosures All authors: No reported disclosures.

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Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00432-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 7

Author(s):

Melissa D. Barnes ◽

Magdalena A. Taracila ◽

Caryn E. Good ◽

Saralee Bajaksouzian ◽

Laura J. Rojas ◽

...

Keyword(s):

Molecular Modeling ◽

Klebsiella Pneumoniae ◽

The United States ◽

Alternative Mechanism ◽

Enhancer Activity ◽

Content Type ◽

Class A ◽

Carbapenem Resistant ◽

Biochemical Analyses ◽

Molecular Modeling And Docking

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-lactam “enhancer” activity against Enterobacteriaceae. In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Ki app] = 31 ± 3 μM) more efficiently than the K234R variant (Ki app = 270 ± 27 μM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M−1 s−1 for KPC-2 versus 247 ± 25 M−1 s−1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam’s aminoethoxy tail formed unproductive interactions with the K234R variant’s active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae. Moreover, our data suggest that β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

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