Faculty Bibliography

BACKGROUND:Multidrug-resistant Enterobacterales (MDR-E), including carbapenem-resistant and third-generation cephalosporin-resistant Enterobacterales (CRE, CefR-E), are major pathogens following solid organ transplantation (SOT). METHODS:We prospectively studied patients who underwent lung, liver and small bowel transplant from February 2015-March 2017. Weekly peri-rectal swabs (up to 100 days post-transplant) were cultured for MDR-E. Whole genome sequencing (WGS) was performed on gastrointestinal (GI) tract-colonizing and disease-causing isolates. RESULTS:Twenty-five percent (40/162) of patients were MDR-E GI-colonized. Klebsiella pneumoniae was the most common CRE and CefR-E. K. pneumoniae carbapenemases and CTX-M were leading causes of CR and CefR, respectively. Thirty-five percent of GI-colonizers developed MDR-E infection versus 2% of non-colonizers (p<0.0001). Attack rate was higher among CRE colonizers than CefR-E colonizers (53% versus 21%, p=0.049). GI-colonization and high body mass index were independent risk factors for MDR-E infection (p≤0.004). Thirty-day mortality among infected patients was 6%. However, 44% of survivors developed recurrent infections; 43% of recurrences were late (285 days-3.9 years post-initial infection). Long-term survival (median: 4.3 years post-transplant) did not differ significantly between MDR-E-infected and non-infected patients (71% versus 77%, p=0.56). WGS phylogenetic analyses revealed that infections were caused by GI-colonizing strains, and suggested unrecognized transmission of novel clonal group-258 sublineage CR-K. pneumoniae and horizontal transfer of resistance genes. CONCLUSIONS:MDR-E GI-colonization was common following SOT, and predisposed patients to infections by colonizing strains. MDR-E infections were associated with low short- and long-term mortality, but recurrences were frequent and often occurred years after initial infections. Findings provide support for MDR-E surveillance in our SOT program.

Mycobacterium abscessus subsp. abscessus is one of the most difficult pathogens to treat and its incidence in disease is increasing. Dual β-lactam combinations act synergistically in vitro, but are not widely employed in practice. A recent study shows that a combination of imipenem and ceftaroline significantly lowers the minimum inhibitory concentration (MIC) of clinical isolates despite both drugs targeting the same peptidoglycan synthesis enzymes. The underlying mechanism of this effect provides a basis for further investigations of dual β-lactam combinations in the treatment of M. abscessus subsp. abscessus eventually leading to a clinical trial. Furthermore, dual β-lactam strategies may be explored for other difficult mycobacterial infections.

BACKGROUND:Levofloxacin prophylaxis is recommended to prevent Gram-negative bloodstream infections (BSIs) in patients with prolonged chemotherapy-induced neutropenia. However, increasing fluoroquinolone resistance may decrease the effectiveness of this approach. METHODS:We assessed the prevalence of colonization with fluoroquinolone-resistant Enterobacterales (FQRE) among patients admitted for hematopoietic cell transplantation (HCT) from November 2016-August 2019 and compared the risk of Gram-negative BSI between FQRE-colonized and non-colonized patients. All patients received levofloxacin prophylaxis during neutropenia. Stool samples were collected upon admission for HCT and weekly thereafter until recovery from neutropenia, and underwent selective culture for FQRE. All isolates were identified and underwent antimicrobial susceptibility testing by broth microdilution. FQRE isolates also underwent whole-genome sequencing. RESULTS:Fifty-four (23%) of 234 patients were colonized with FQRE prior to HCT, including 30 (25%) of 119 allogeneic and 24 (21%) of 115 autologous HCT recipients. Recent antibacterial use was associated with FQRE colonization (P=0.048). Ninety-one percent of colonizing FQRE isolates were Escherichia coli and 29% produced extended-spectrum ß-lactamases. Seventeen (31%) FQRE-colonized patients developed Gram-negative BSI despite levofloxacin prophylaxis, compared to only two (1.1%) of 180 patients who were not colonized with FQRE on admission (P<0.001). Of the 17 Gram-negative BSIs in FQRE-colonized patients, 15 (88%) were caused by FQRE isolates that were genetically identical to the colonizing strain. CONCLUSIONS:Nearly one-third of HCT recipients with pre-transplant FQRE colonization developed Gram-negative BSI while receiving levofloxacin prophylaxis and infections were typically caused by their colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients not initially colonized with FQRE.

Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species that comprises three subspecies; M. abscessus subsp. abscessus,M. abscessus subsp. massiliense, andM. abscessus subsp. bolletii These predominantly environmental microorganisms have emerged as life-threatening chronic pulmonary pathogens in both immunocompetent and immunocompromised patients and their acquisition of macrolide resistance due to the erm(41) gene and mutations in the 23S rrl has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools for the subspeciation of M. abscessus, and the testing for macrolide resistance is often not done. Here we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid and highly accurate method to both distinguish M. abscessus sub-species and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspeciation sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a two-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 hours.

BACKGROUND:Recent studies indicated that the monosubstituted deoxystreptamine aminoglycoside apramycin is a potent antibiotic against a wide range of MDR Gram-negative pathogens. OBJECTIVES/OBJECTIVE:To evaluate the in vitro activity of apramycin against carbapenem-resistant Klebsiella pneumoniae (CRKp) isolates from New York and New Jersey, and to explore mechanisms of apramycin resistance. METHODS:Apramycin MICs were determined by broth microdilution for 155 CRKp bloodstream isolates collected from 2013 to 2018. MLST STs, wzi capsular types and apramycin resistance gene aac(3')-IV were examined by PCR and Sanger sequencing. Selected isolates were further characterized by conjugation experiments and WGS. RESULTS:Apramycin MIC50/90 values were 8 and >128 mg/L for CRKp isolates, which are much higher than previously reported. Twenty-four isolates (15.5%) were apramycin resistant (MIC ≥64 mg/L) and they were all from the K. pneumoniae ST258 background. The 24 apramycin-resistant K. pneumoniae ST258 strains belonged to six different capsular types and 91.7% of them harboured the apramycin resistance gene aac(3')-IV. Sequencing analysis showed that different ST258 capsular type strains shared a common non-conjugative IncR plasmid, co-harbouring aac(3')-IV and blaKPC. A novel IncR and IncX3 cointegrate plasmid, p59494-RX116.1, was also identified in an ST258 strain, demonstrating how apramycin resistance can be spread from a non-conjugative plasmid through cointegration. CONCLUSIONS:We described a high apramycin resistance rate in clinical CRKp isolates in the New York/New Jersey region, mainly among the epidemic K. pneumoniae ST258 strains. The high resistance rate in an epidemic K. pneumoniae clone raises concern regarding the further optimization and development of apramycin and apramycin-like antibiotics.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 9 (ST9) has emerged and disseminated in Asia. It is associated with colonization or infection in both humans and animal hosts; however, the genetic factors underpinning its adaptation to animal and human population remain to be determined. Here, we conducted a genomic analysis of 191 ST9 S. aureus genomes collected from 12 different countries, including 174 genomes retrieved from public databases and 17 sequenced in this study. In silico spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance and virulence gene mining were conducted, and the temporal phylogenetic signal was assessed by Bayesian inference. Our results point toward a human methicillin-susceptible S. aureus (MSSA) origin of ST9 that evolved approximately 2 centuries ago. Three major genetic events occurred during ST9 host shift from human to animals: the loss of the immune evasion cluster genes (scn, chp, and sak), which were reported to contribute to virulence in human infections, the acquisition of the SaPIbov4-like element-encoding vwb gene, which is an animal-specific virulence factor responsible for the clotting of animal plasma, and the acquisition of antibiotic resistance genes, including SCCmec, quinolone resistance-determining region (QRDR) mutations, and a multidrug resistance genetic element (MDR_ST9). Evidence of direct transmission of animal-adapted strains to human hosts also suggest that transmission could potentially reshape the resistance and virulence genetic pool in these isolates. The rapid clonal expansion of MDR ST9 strains in mainland China and Taiwan highlights the increasing need for effective surveillance of antibiotic consumption in animal husbandry to control antimicrobial resistance spread. IMPORTANCE Staphylococcus aureus sequence type 9 (ST9) is the main LA-MRSA clone spreading in the Asian continent. It can colonize and cause mild to severe infections both in animal and humans. Previous work described its genotypic characteristics; however, the molecular history of global spread of ST9 strains remains largely unclear. We conducted a detailed analysis of genomic evolution of global ST9 strains and identified key genetic changes associated with its adaptation to specific hosts. Our results suggest that the ST9 clone originated from human-adapted strains, which lost genes related to the evasion of the immune system. The introduction of ST9 strains in animal populations was aligned with the acquisition of animal-specific virulent factors and mobile elements harboring multiple antimicrobial resistance genes, especially in isolates from mainland China and Taiwan.

Mycobacterium abscessus is an emerging opportunistic human pathogen that naturally resists most major classes of antibiotics, making infections difficult to treat. Thus far, little is known about M. abscessus physiology, pathogenesis, and drug resistance. Genome-wide analyses have comprehensively catalogued genes with essential functions in Mycobacterium tuberculosis and Mycobacterium avium subsp. hominissuis (here, M. avium) but not in M. abscessus. By optimizing transduction conditions, we achieved full saturation of TA insertion sites with Himar1 transposon mutagenesis in the M. abscessus ATCC 19977^T genome, as confirmed by deep sequencing prior to essentiality analyses of annotated genes and other genomic features. The overall densities of inserted TA sites (85.7%), unoccupied TA sites (14.3%), and nonpermissive TA sites (8.1%) were similar to results in M. tuberculosis and M. avium. Of the 4,920 annotated genes, 326 were identified as essential, 269 (83%) of which have mutual homology with essential M. tuberculosis genes, while 39 (12%) are homologous to genes that are not essential in M. tuberculosis and M. avium, and 11 (3.4%) only have homologs in M. avium. Interestingly, 7 (2.1%) essential M. abscessus genes have no homologs in either M. tuberculosis or M. avium, two of which were found in phage-like elements. Most essential genes are involved in DNA replication, RNA transcription and translation, and posttranslational events to synthesize important macromolecules. Some essential genes may be involved in M. abscessus pathogenesis and antibiotics response, including certain essential tRNAs and new short open reading frames. Our findings will help to pave the way for better understanding of M. abscessus and benefit development of novel bactericidal drugs against M. abscessus. IMPORTANCE Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis. Most essential M. abscessus genes have mutual homology with essential M. tuberculosis genes, providing a foundation for leveraging available knowledge from M. tuberculosis to develop more effective drugs and other interventions against M. abscessus. A small number of essential genes unique to M. abscessus deserve further attention to gain insights into what makes M. abscessus different from other mycobacteria. The essential genes and other genomic features such as short open reading frames and noncoding RNA identified here will provide useful information for future study of M. abscessus pathogenicity and new drug development.

AIMS/OBJECTIVE:The aim at this study was to determine the effects of unsaturated fatty acids on clinical plasmids. METHODS AND RESULTS/RESULTS:, respectively, were used to assess the effects on conjugative transfer of a mcr-1-harboring plasmid pCSZ4 (IncX4) in conjugation experiment. The inhibitory mechanisms were analyzed by molecular docking and the gene expression of virB11 was quantitated by qRT-PCR. Target plasmid diversity was carried out by TrwD/VirB11 homology protein sequence prediction analysis. Our results showed that LA and ALA inhibit plasmid pCSZ4 transfer by binding to the amino acid residues (Phe124 and Thr125) of VirB11 with dose-dependent effects. The expression levels of virB11 gene were also significantly inhibited by LA and ALA treatment. Protein homology analysis revealed a wide distribution of TrwD/VirB11-like genes among over 37 classes of plasmids originated from both Gram-negative and Gram-positive bacteria, CONCLUSIONS: This study demonstrates representing a diversity of plasmids that may be potentially inhibited by unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF STUDY/CONCLUSIONS:Our work reported here provides additional support for application of curbing the spread of multiple plasmids by unsaturated fatty acids.

Ceftazidime/Avibactam (CAZ/AVI) is a β-lactam/β-lactamase inhibitor combination with activity against type A and C β-lactamases. Resistance emergence has been seen with multiple mechanisms accounting for the resistance. We performed four experiments in the dynamic Hollow Fiber Infection Model, delineating the linkage between drug exposure and both rate of bacterial kill and resistance emergence by all mechanisms. The P. aeruginosa isolate had an MIC of 1.0 mg/L (CAZ)/4 mg/L (AVI). We demonstrated that Time>4.0 mg/L AVI was linked to rate of bacterial kill. Linkage to resistance emergence/suppression was more complex. In one experiment where CAZ/AVI administration was intermittent/continuous and where AVI was given in unitary steps from 1-8 mg/L, AVI up to 3 mg/L allowed resistance emergence, whereas higher values did not. The threshold value was 3.72 mg/L as a continuous infusion to counterselect resistance (AUC of 89.3 mg*h/L AVI). The mechanism was by a 7 amino acid deletion in the Ω-loop region of the PDC β-lactamase. Further experiments, where CAZ/AVI were both administered intermittently with regimens above and below the AUC of 89.3 mg*hr/L resulted in resistance in the lower exposure groups. Deletion mutants were not identified. Finally, an experiment where paired exposures both as continuous and intermittent infusions were performed, the lower value of 25 mg*hr/L by both profiles allowed selection of deletion mutants. Of the five instances where these mutants were recovered, 4/5 were by the continuous infusion profile. Both continuous infusion administration and low avibactam AUC exposures have a role in selection of this mutation.