Faculty Bibliography

Twenty patients with carbapenem-resistant Enterobacteriaceae (CRE) infections were treated with meropenem-vaborbactam. Thirty-day clinical success and survival rates were 65% (13/20) and 90% (18/20), respectively. Thirty-five percent of patients had microbiologic failures within 90 days. One patient developed a recurrent infection due to meropenem-vaborbactam non-susceptible, ompK36 porin mutant Klebsiellapneumoniae.

Mycobacterium abscessus (Mab) is a highly drug-resistant nontuberculous mycobacteria (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating with a focus on cell wall synthesis proteins (L, D-transpeptidases, Ldt_Mab1-5, and D,D-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase, Bla_Mab Using a "mechanism based" approach, we show that a novel ceftaroline-imipenem combination effectively lowered the minimal inhibitory concentrations (MICs) of Mab isolates (MIC₅₀ ≤ 0.25, MIC₉₀ ≤ 0.5). Ceftaroline and imipenem combined with a β-lactamase inhibitor, relebactam or avibactam, demonstrated only a modest effect on susceptibility, compared to each of the beta-lactams alone. In steady state kinetic assays, Bla_Mab exhibited a lower K_{i app} (K_{i app} = 0.30 ± 0.03 μM, avibactam; 136 ± 14 μM, relebactam) and a faster acylation rate for avibactam (k₂/K = 3.4 ± 0.4 x 10⁵ M^-1s^-1, avibactam; 6 ± 0.6 x 10² M^-1s^-1, relebactam). The k_cat/K_m was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM^-1s^-1) compared to imipenem (0.056 ± 0.006 μM^-1s^-1). Timed mass spectrometry captured complexes of avibactam and Bla_Mab, Ldt_{Mab1, 2, and 4}, and D,D-carboxypeptidase, whereas relebactam bound only Bla_Mab and Ldt_{Mab1 and 2} Interestingly, Ldt_{Mab1, 2, 4 and 5} and D, D-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil to Ldt_{Mab1, 2, 4 and 5} and showed that imipenem bound > 100 fold more avidly than ceftaroline fosamil for Ldt_Mab1 and Ldt_Mab2 (e.g. K_{i app} or K_m LdtMab1 = 0.01 ± 0.01 μM for imipenem vs 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modelling indicates that Ldt_Mab2 readily accommodates imipenem, but the active site must widen to ≥ 8Ã… for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (L, D-transpeptidases and D, D-carboxypeptidase) provides mechanistic rationale for the effectiveness of this dual β-lactam combination in Mab infections.

BACKGROUND:Carbapenem-resistant Enterobacterales (CRE) are a global threat. We aimed to describe the clinical and molecular characteristics of Centers for Disease Control and Prevention (CDC)-defined CRE in the USA. METHODS:CRACKLE-2 is a prospective, multicentre, cohort study. Patients hospitalised in 49 US hospitals, with clinical cultures positive for CDC-defined CRE between April 30, 2016, and Aug 31, 2017, were included. There was no age exclusion. The primary outcome was desirability of outcome ranking (DOOR) at 30 days after index culture. Clinical data and bacteria were collected, and whole genome sequencing was done. This trial is registered with ClinicalTrials.gov, number NCT03646227. FINDINGS/RESULTS:1040 patients with unique isolates were included, 449 (43%) with infection and 591 (57%) with colonisation. The CDC-defined CRE admission rate was 57 per 100 000 admissions (95% CI 45-71). Three subsets of CDC-defined CRE were identified: carbapenemase-producing Enterobacterales (618 [59%] of 1040), non-carbapenemase-producing Enterobacterales (194 [19%]), and unconfirmed CRE (228 [22%]; initially reported as CRE, but susceptible to carbapenems in two central laboratories). Klebsiella pneumoniae carbapenemase-producing clonal group 258 K pneumoniae was the most common carbapenemase-producing Enterobacterales. In 449 patients with CDC-defined CRE infections, DOOR outcomes were not significantly different in patients with carbapenemase-producing Enterobacterales, non-carbapenemase-producing Enterobacterales, and unconfirmed CRE. At 30 days 107 (24%, 95% CI 20-28) of these patients had died. INTERPRETATION/CONCLUSIONS:Among patients with CDC-defined CRE, similar outcomes were observed among three subgroups, including the novel unconfirmed CRE group. CDC-defined CRE represent diverse bacteria, whose spread might not respond to interventions directed to carbapenemase-producing Enterobacterales. FUNDING/BACKGROUND:National Institutes of Health.

The objective of this study was to utilize a co-culture hollow-fiber infection model (HFIM) to characterize the interplay between a small, difficult-to-detect, New Delhi metallo-β-lactamase-producing Klebsiella pneumoniae (NDM-Kp) minor population and a larger K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae population in the presence of KPC-directed antibacterial therapy. Selective plating onto agar with ceftazidime-avibactam was used to track the density of the NDM-Kp population. Susceptibility testing and the Verigene System failed to identify the small initial NDM-Kp population. However, a ceftazidime-avibactam Etest detected resistant colonies that were confirmed to be NDM-Kp. In the HFIM, all of the investigated drug regimens caused regrowth within 24 h and resulted in >10⁹ CFU/mL of NDM-Kp. Our study demonstrates that the HFIM is a powerful tool for studying the population dynamics of multiple pathogens during antimicrobial exposure and also highlights that difficult-to-detect minor populations of drug-resistant bacteria may cause treatment failure without appropriate antibacterial therapy.

Plazomicin was tested against 697 recently acquired carbapenem resistant Klebsiella pneumoniae isolates from the Great Lakes Region of the United States. Plazomicin MIC₅₀ and MIC₉₀ values were 0.25 and 1 mg/L, respectively; 680 isolates were susceptible (97.6%, MIC ≤ 2 mg/L), 9 intermediate (1.3%, MIC 4mg/L), and 8 resistant (1.1%, MIC > 32 mg/L). Resistance was associated with rmtF-, rmtB- or armA-encoded 16S ribosomal RNA methyltransferases, in all but one isolate.

Hypervirulent Klebsiella pneumoniae strains are the major causes of liver abscesses throughout East Asia, and these strains are usually antibiotic susceptible. Recently, multidrug resistant and hypervirulent (MDR-HV) K. pneumoniae strain emerged, arisen by hypervirulent strains acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic MDR strain. In this study, we characterized the clinical and microbiological properties of K. pneumoniae liver abscess (KPLA) caused by MDR-HV strains in Taiwan. Patients with community-onset KPLA were retrospectively identified at Taipei Veterans General Hospital during January 2013 and May 2018. Antimicrobial resistance mechanisms, capsular types, and sequence types were determined. MDR-HV strains and their parental antimicrobial-susceptible strains further underwent whole genome sequencing (WGS) and in vivo mice lethality test. Thirteen MDR-HV strains were identified from a total of 218 KPLA episodes. MDR-HV strains resulted in similar outcomes to antimicrobial-susceptible strains. All MDR-HV strains were traditional hypervirulent clones carrying virulence capsular types. The major resistance mechanisms were the overexpression of efflux pumps and/or the acquisition of ESBL or AmpC β-lactamase genes. WGS showed two hypervirulent strains evolved to MDR phenotype after having a mutation in ramR and acquiring a SHV-12-bearing plasmid, respectively. Both the MDR-HV strains retained high virulence in comparison to their parental strains. The spread of MDR-HV K. pneumoniae strains in the community raises significant public concerns, and measures should be taken to prevent the further acquisition of carbapenemase and other resistance genes among these strains in order to avoid the occurrence of untreatable KPLA.

Tigecycline serves as one of the last-resort antibiotics to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanisms, Tet(X), challenge the clinical efficacy of this class of antibiotics. In this study, we detected one hundred and eighty tet(X)-harboring Acinetobacter isolates (8.9%, n=180) from 2018 samples collected from the avian farms and adjacent environment in China. Eighteen tet(X)-harboring isolates (10.0%) were found to co-carry the carbapenemase gene bla_NDM-1, mostly from waterfowls samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, the tet(X) and bla_NDM-1 were found to co-localize on the same plasmids. Moreover, WGS revealed a novel orthologue of tet(X) in the six tet(X)- and bla_NDM-1-co-harboring isolates. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be co-transferred. Sequence comparison between six tet(X) and bla_NDM-1-co-harboring plasmids showed they shared a highly homologous plasmid backbone, even though they were isolated from different Acinetobacter species (3 A. indicus, 2 A. schindleri, and 1 A. lwoffii) in various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X) and bla_NDM-1-co-harboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis. Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2-98.6) and specificity of 97.9% (95% CI: 96.1-99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5-100) but lower specificity of 91.8% (95% CI: 87.9-95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility.

OBJECTIVES/OBJECTIVE:To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. METHODS:The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. RESULTS:We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. CONCLUSIONS:Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

BACKGROUND:While several studies have assessed the associations between biological factors and tuberculosis (TB) transmission, our understanding of the associations between TB transmission and social and economic factors remains incomplete. We aimed to explore associations between community TB transmission and socio-economic factors within a high TB-HIV burdened setting. METHODS:We conducted a cross-sectional molecular epidemiology study among adult patients attending a routine TB clinic. Demographic and clinical data were extracted from TB registers and clinical folders; social and economic data were collected using interviewer-administered questionnaires; Mycobacterium tuberculosis isolates were genotyped and classified as clustered/non-clustered using IS6110-based Restriction Fragment Length Polymorphism. Composite "social" and "economic" scores were generated from social and economic data. Data were analyzed using StataCorp version 15.0 software. Stratified, bivariable analyses were performed using chi-squared. Wilcoxon signed rank tests; univariable and multivariable logistic regression models were developed to explore associations in the social, economic, traditional and composite TB risk factors with TB transmission. RESULTS:Of the 505 patient Mtb  strains, 348(69%) cases were classified as clustered and 157(31%) were non-clustered. Clustered cases were more likely to have lived longer in the study community, (odds ratio [OR] = 1.05, 95% Confidence interval [C.I]:1.02-1.09, p = 0.006); in the same house (OR = 1.04, C.I: 0.99-1.08, p = 0.06); and had increased household crowding conditions (i.e fewer rooms used for sleeping, OR = 0.45, C.I:0.21-0.95, p = 0.04). Although a higher proportion of clustered cases had a low economic score, no statistically significant association was found between clustering and either the economic score (p = 0.13) or social score (p = 0.26). CONCLUSIONS:We report a novel association between Mtb transmission and prolonged stay within a high burdened community. Transmission was also associated with fewer rooms for sleeping in a household. Increased social interaction and prolonged residence in a high burdened community are important factors linked to Mtb transmission, possibly due to increased probability of higher effective contact rates. The possible importance of degrees of poverty within low socio-economic setting warrants further study.