T. Nicholas Domitrovic

Unexpected Challenges in Treating Multidrug-Resistant Gram-Negative Bacteria: Resistance to Ceftazidime-Avibactam in Archived Isolates of Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.

Extensively drug-resistant pseudomonas aeruginosa isolates containing blaVIM-2 and elements of Salmonella genomic island 2: a new genetic resistance determinant in Northeast Ohio

ABSTRACT Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-β-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and β-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this regions health care system.

Analysis of β-Lactamase Resistance Determinants in Enterobacteriaceae from Chicago Children: a Multicenter Survey

ABSTRACT Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type β-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR contained plasmid replicon FIIA. We found that β-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further.

Monoclonal Antibody Protects Against Acinetobacter baumannii Infection by Enhancing Bacterial Clearance and Evading Sepsis

Background Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions. Methods We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate. Results C8 targets capsular carbohydrate on the bacterial surface, enhancing opsonophagocytosis. Treating with a single dose of C8 as low as 0.5 μg/mouse (0.0167 mg/kg) markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia models of XDR A. baumannii infection. C8 was also synergistic with colistin, substantially improving survival compared to monotherapy. Treatment with C8 significantly reduced blood bacterial density, cytokine production (tumor necrosis factor α, interleukin [IL] 6, IL-1β, and IL-10), and sepsis biomarkers. Serial in vitro passaging of A. baumannii in the presence of C8 did not cause loss of mAb binding to the bacteria, but did result in emergence of less-virulent mutants that were more susceptible to macrophage uptake. Finally, we developed a highly humanized variant of C8 that retains opsonophagocytic activity in murine and human macrophages and rescued mice from lethal infection. Conclusions We describe a promising and novel mAb as therapy for lethal, XDR A. baumannii infections, and demonstrate that it synergistically improves outcomes in combination with antibiotics.

Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III.

ABSTRACT The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas

ABSTRACT Among Gram-negative bacteria, carbapenem-resistant infections pose a serious and life-threatening challenge. Here, the CRACKLE network reports a sentinel detection and characterization of a carbapenem-resistant Klebsiella pneumoniae ST147 isolate harboring blaNDM-5 and blaOXA-181 from a young man who underwent abdominal surgery in India. blaNDM-5 was located on an IncFII plasmid of ≈90 kb, whereas blaOXA-181 was chromosomally encoded. Resistome and genome analysis demonstrated multiple copies of the transposable element IS26 and a “hot-spot region” in the IncFII plasmid.

A cold hard menace: A contaminated ice machine as a potential source for transmission of carbapenem-resistant Acinetobacter baumannii

Highlights:Carbapenem‐resistant Acinetobacter baumannii isolates were recovered from 3 patients, the hands of a nurse, and an ice machine water outlet and spout.Genetic typing demonstrated that the A. baumannii isolates were genetically related.Contaminated ice machines could serve as a reservoir for dissemination of multidrug‐resistant gram‐negative bacilli. &NA; During an investigation of potential sources of transmission of multidrug‐resistant gram‐negative bacilli on a spinal cord injury unit, we recovered genetically related carbapenem‐resistant Acinetobacter baumannii isolates from the stool of 3 patients, the hands of a nurse, and an ice machine water outlet spout and drain. Our findings suggest that contaminated ice machines could serve as a potential reservoir for dissemination of multidrug‐resistant gram‐negative bacilli.

A Prospective Observational Study of the Epidemiology, Management, and Outcomes of Skin and Soft Tissue Infections Due to Carbapenem-Resistant Enterobacteriaceae

Abstract Background This study was performed to characterize the epidemiology, management, and outcomes of skin and soft tissue infection (SSTI) and colonization due to carbapenem-resistant Enterobacteriaceae (CRE). Methods Patients from the Consortium on Resistance Against Carbapenem in Klebsiella and Other Enterobacteriaceae (CRACKLE-1) from December 24, 2011 to October 1, 2014 with wound cultures positive for CRE were included in the study. Predictors of surgical intervention were analyzed. Molecular typing of isolates was performed using repetitive extragenic palindromic polymerase chain reaction (PCR). Carbapenemase genes were detected using PCR. Results One hundred forty-two patients were included: 62 had SSTI (44%) and 56% were colonized. Mean age was 61 years, and 48% were male: median Charlson score was 3 (interquartile range, 1–5). Forty-eight percent of patients were admitted from long-term care facilities (LTCFs), and 31% were from the community. Two strain types (ST258A and ST258B) were identified (73% of 45 tested). Carbapenemase genes were detected in 40 of 45 isolates (blaKPC-3 [47%], blaKPC-2 [42%]). Sixty-eight patients (48%) underwent surgical intervention, 63% of whom had SSTI. Patients admitted from LTCFs were less likely to undergo surgical intervention (odds ratio [OR], 0.36; 95% confidence interval [CI], 0.18–0.71). In multivariable analysis, among patients with SSTI, those admitted from LTCFs were less likely to undergo debridement (OR, 0.18; 95% CI, 0.04–0.93). Conclusions Patients admitted from LTCFs with CRE SSTI were less likely to undergo surgical intervention. Sixteen percent of the patients died, and approximately 50% of survivors required more intensive care upon discharge. These findings suggest a unique, impactful syndrome within the CRE infection spectrum. Further studies are needed to assess the role of surgical debridement in management of CRE-SSTI, particularly among LTCF residents.

Multidrug Resistant Pseudomonas aeruginosa Causing Prosthetic Valve Endocarditis: A Genetic-Based Chronicle of Evolving Antibiotic Resistance

A rare case of MDR P. aeruginosa causing PVE is analyzed at the genetic level with the goal of understanding acquired resistance mechanisms. The molecular basis of evolving antibiotic resistance gives clinicians insight into the nature of persistent P. aeruginosa infections.

608Can Rapid Molecular Diagnostics Assist in the Choice of b-Lactam Antibiotics? An Analysis of Data from PRIMERS-II of the Antibiotic Resistance Leadership Group (ARLG)

608. Can Rapid Molecular Diagnostics Assist in the Choice of b-Lactam Antibiotics? An Analysis of Data from PRIMERS-II of the Antibiotic Resistance Leadership Group (ARLG) Andrea M. Hujer, BS; Scott Evans, PhD; Hongyu Jiang, PhD; Kristine M. Hujer, BS; Thomas Hall, PhD; Christine Marzan, PhD; Michael Jacobs, MD, PhD; Ranga Sampath, PhD; David J. Ecker, PhD; T. Nicholas Domitrovic, BA; Claudia Manca, PhD; Kalyan Chavda, PhD; Pan Zhang, MD, PhD; Liang Chen, PhD; Carol Hill, PhD; Federico Perez, MD; Barry Kreiswirth, PhD; Vance Fowler, MD; Henry F. Chambers, MD; Robert A. Bonomo, MD; Antimicrobial Resistance Leadership Group; Case Western Reserve University, Cleveland, OH; Center for Biostatistics in AIDS Research, Harvard University, Boston, MA; Biostatistics, Harvard School of Public Health, Boston, MA; Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH; Ibis Biosciences, Carlsbad, CA; Case Western Reserve University/University Hospitals of Cleveland, Cleveland, OH; Ibis Biosciences, Inc., A Division of Abbott, Carlsbad, CA; PHRI UMDNJ, Newark, NJ; Weill Cornell Medical College, New York, NY; PHRI and UMDNJ, Newark, NJ; Duke University Medical Center, Durham, NC; Cleveland VAMC Case Western Reserve University, Cleveland Heights, OH; University of Medicine and Dentistry of NJ, PHRI TB Center, Newark, NJ; University of California, San Francisco General Hospital, San Francisco, CA; Medicine, Pharmacology and Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH