Robert A Bonomo

Case Western Reserve University School of Medicine, Cleveland, Ohio, United States

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Publications (392)1638.69 Total impact

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    ABSTRACT: Patients infected or colonized with carbapenem-resistant Klebsiella pneumoniae (CRKp) are often chronically and acutely ill, which results in substantial mortality that is not related to infection. Therefore, estimating excess mortality due to CRKp infections is challenging. The Consortium on Resistance against Carbapenems in K. pneumoniae (CRACKLE) is a prospective multicenter study. Here, patients in CRACKLE were evaluated at the time of their first CRKp bloodstream infection (BSI), pneumonia, or urinary tract infection (UTI). A control cohort of patients with CRKp urinary colonization without CRKp infection was constructed. Excess hospital mortality was defined as mortality in cases after subtracting mortality in controls. In addition, the adjusted hazard ratios (aHR) for time-to-hospital-mortality censored at 30 days associated with infection as compared to colonization were calculated in Cox proportional hazard models. In the study period, 260 patients with CRKp infections were included in the BSI (90), pneumonia (49), and UTI (121) groups, who were compared to 223 controls. All-cause hospital mortality in controls was 12%. Excess hospital mortality was 27% and 27% in patients with BSI and pneumonia, respectively. Excess hospital mortality was not observed in patients with UTI. In multivariable analyses, BSI and pneumonia as compared to controls was associated with an aHR of 2.59 (95% CI 1.52-4.50, p<0.001) and 3.44 (95% CI 1.80-6.48, p<0.001), respectively. In conclusion, in patients with CRKp infection, pneumonia is associated with the highest excess hospital mortality. Patients with BSI have slightly lower excess hospital mortality rates, whereas excess hospital mortality was not observed in hospitalized patients with UTI.
    No preview · Article · Feb 2016 · Clinical Microbiology and Infection
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    ABSTRACT: Introduction: For the past three decades, carbapenems played a central role in our antibiotic armamentarium, trusted to effectively treat infections caused by drug-resistant bacteria. The utility of this class of antibiotics has been compromised by the emergence of resistance especially among Enterobacteriaceae. Areas covered: We review the current mainstays of pharmacotherapy against infections caused by carbapenem-resistant Enterobacteriaceae (CRE) including tigecycline, aminoglycosides, and rediscovered 'old' antibiotics such as fosfomycin and polymyxins, and discuss their efficacy and potential toxicity. We also summarize the clinical experience treating CRE infections with antibiotic combination therapy. Finally, we review ceftazidime/avibactam and imipenem/relebactam, a new generation of beta-lactamase inhibitors, which may offer alternatives to treat CRE infections. We critically evaluate the published literature, identify relevant clinical trials and review documents submitted to the United States Food and Drug Administration. Expert Opinion: It is essential to define the molecular mechanisms of resistance and to apply insights about pharmacodynamic and pharmacokinetic properties of antibiotics, in order to maximize the impact of old and new therapeutic approaches against infections caused by CRE. A concerted effort is needed to carry out high-quality clinical trials that: i) establish the superiority of combination therapy vs. monotherapy; ii) confirm the role of novel beta-lactam/beta-lactamase inhibitor combinations as therapy against KPC- and OXA-48 producing Enterobacteriaceae; and, iii) evaluate new antibiotics active against CRE as they are introduced into the clinic.
    No preview · Article · Jan 2016 · Expert Opinion on Pharmacotherapy
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    ABSTRACT: Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae as the most problematic pathogens infecting patients in the hospital and community. This broad spectrum resistance to β-lactamas emerges in part via the expression of KPC-2 and SHV-1 β-lactamases, and variants thereof, KPC-2 carbapenemase is particularly worrisome as the genetic determinant encoding this β-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism based inactivators (i.e. clavulanate). In order to develop new β-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54 and 1.87 Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 β-lactamases, respectively, as well as a comparative analysis of the S02030 binding modes including a previously determined S02030 Class C ADC-7 β-lactamase complex. Upon analysis, S02030 is able to inhibit vastly different serine β-lactamases by interacting with the conserved features of these actives sites which includes i ) forming the bond with catalytic serine via the boron atom; ii ) positioning of one of the boronic acid oxygens in the oxyanion hole; and iii ) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, S02030 is able to overcome more distantly located structural differences between the β-lactamases. This unique feature is achieved by re-positionig the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring allowing it to potentially form a unique C-H …O 2.9Å hydrogen bond with S130 in KPC-2.
    No preview · Article · Jan 2016 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). Herein, a series of BATSIs with selectively modified regions (R1, R2 and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a, 1b, 1c, 2a -2e mimicked the side chain of cephalothin whereas for compounds 3a, 3b, 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a -2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C6/C7 of β-lactams, was also changed to the following bioisosteric groups: urea (compound 3b); thiourea (compound 3c); and sulfonamide (compounds 4a and 4b). Among the compounds active against KPC-2 and SHV-1 β-lactamases, nine possessed IC50 values ≤600 nM. The most active compounds contained the thiopheneacetyl group at R1 and carboxy- or hydroxy-substituted aryl group at R2 for the chiral BATSIs. The most active sulfonamido derivative 4b lacked an R2 group. Compound 2b (S02030) was the most active with acylation rates (k2/K) of 1.2 ± 0.2 x 104 M-1s-1 for KPC-2 and 4.7 ± 0.6 x 103 M-1s-1 for SHV-1 and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or -3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A β-lactamase genes (32 → 0.5 mg/L).
    No preview · Article · Jan 2016 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: Carbapenemases have become a significant mechanism for broad-spectrum β-lactam resistance in Enterobacteriaceae and other Gram-negative bacteria such as Pseudomonas and Acinetobacter spp. Intestinal carriage of carbapenemase-producing organisms (CPOs) is an important source of transmission. Isolation of carriers is one strategy that can be used to limit the spread of these bacteria. In this review, we critically examine the clinical performance, advantages, and disadvantages of methods available for the detection of intestinal carriage of CPOs. Culture-based methods (Centers for Disease Control and Prevention [CDC] protocols, chromogenic media, specialized agars, and double-disk synergy tests) for detecting carriage of CPOs are convenient due to their ready availability and low cost, but their limited sensitivity and long turnaround time may not always be optimal for infection control practices. Contemporary nucleic acid amplification techniques (NAATs) such as real-time PCR, hybridization assays, loop-mediated isothermal amplification (LAMP), or a combined culture and NAAT approach may provide fast results and/or added sensitivity and specificity compared with culture-based methods. Infection control practitioners and clinical microbiologists should be aware of the strengths and limitations of available methods to determine the most suitable approach for their medical facility to fit their infection control needs.
    No preview · Article · Jan 2016
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    ABSTRACT: Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime resistant clinical isolates have been described and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded Class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine to alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a non-lipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.
    No preview · Article · Dec 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: We investigate the evolving molecular epidemiology of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan from 2004 to 2006. MBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. bla IMP-1 group was the most predominant (38 isolates, 80%), followed by 3 bla IMP-7, 2 bla IMP-11 group, and 1 bla VIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole bla IMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with bla IMP-11 group or ST235 with bla IMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp. Our data give an important "snapshot" of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.
    Preview · Article · Dec 2015 · BMC Microbiology
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    ABSTRACT: Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase, PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site-saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 → 64 mg/L) when expressed in Escherichia coli . Surprisingly, quantitative immunoblotting shows that steady-state protein levels of the C69F variant are ∼4-fold lower than wild-type PenI (0.76 fg of protein/cell vs. 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis thus suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime, and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. We advance that when ceftazidime is positioned into the active site, the C69F variant is predicted to form an increased number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose “a new twist” for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B . pseudomallei , a pathogen of considerable importance, and suggests that the full repertoire of conformational states of a β-lactamase profoundly effects β-lactam resistance.
    No preview · Article · Nov 2015 · Antimicrobial Agents and Chemotherapy
  • Marisa L Winkler · Robert A Bonomo
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    ABSTRACT: Enzymes are continually evolving in response to environmental pressures. In order to increase enzyme fitness, amino acid substitutions can occur leading to a changing function or an increased stability. These evolutionary drivers determine the activity of an enzyme and its success in future generations in response to changing conditions such as environmental stressors or to improve physiological function allowing continual persistence of the enzyme. With recent warning reports on antibiotic resistance and multi-drug resistant bacterial infections, understanding the evolution of β-lactamase enzymes, which are a large contributor to antibiotic resistance, is increasingly important. Here, we investigated a variant of the SHV β-lactamase identified from a clinical isolate of Escherichia coli in 2011 (SHV-129, G238S-E240K-R275L-N276D) to identify the first instance of a global suppressor substitution in the SHV β-lactamase family. We have used this enzyme to show that several evolutionary principles are conserved in different class A β-lactamases, such as active site mutations reducing stability and requiring compensating suppressor substitutions in order to ensure evolutionary persistence of a given β-lactamase. However, the pathway taken by a given β-lactamase in order to reach its evolutionary peak under a given set of conditions is likely different. We also provide further evidence for a conserved stabilizing substitution among class A β-lactamases, the back to consensus M182T substitution. In addition to expanding the spectrum of β-lactamase activity to include the hydrolysis of cefepime, the amino acid substitutions found in SHV-129 provide the enzyme with an excess of stability, which expands the evolutionary landscape of this enzyme and may result in further evolution to potentially include resistance to carbapenems or β-lactamase inhibitors.
    No preview · Article · Nov 2015 · Molecular Biology and Evolution
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    Full-text · Conference Paper · Oct 2015
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    ABSTRACT: Background: Multi-Drug resistant (MDR) Gram-negative bacilli (GNB) are a growing concern in children. Our long term goals are to describe the impact and define the clinical and molecular epidemiology of this emerging pediatric health threat. Here, we report our molecular findings of ESBL and carbapenemase -containing genotypes in Enterobacteriaceae from children in Chicago. Methods: We conducted a retrospective cohort study of GNB isolates phenotypically identified ESBL or carbapenemase producers, which were recovered from children ages 0-18 years hospitalized between 2011 - 2014 at three Chicago hospitals. We used DNA microarray (Check-Points™) to detect ESBL, plasmid-mediated AmpC (pAmpC) and carbapenemase type beta-lactamase (bla) genes. PCR was performed to assess for plasmid-mediated fluoroquinolone resistance (PMFQR). Repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. Results: The median age was 4.3 years, 55% were female, and 44% were outpatients. Most isolates (68%) were from urine. One hundred ninety-three (of 197) isolates exhibiting ESBL –producing or carbapenemase-producing phenotypes were analyzed genotypically; 207 bla genes were detected. The most common species was E. coli (61%), the most frequent genotype was blaCTX-M-1 (49%); 2.6% were CRE (3 carried blaKPC and 1 carried blaIMP) and PMFQR was found in 52/84 (62%) isolates. Overall, pAmpC (blaACT/MIR and blaCMY) were found in 14.2% (28/197) of all isolates and in 77% (17/22) of Enterobacter spp. The predominant E. coli phylogenetic group was B2 (66%) associated with ST43 (ST131) containing blaCTX-M-1 group (67%), and plasmid replicon types F1A, F11, F1B. The blaKPC harboring K. pneumoniae were non ST258 with replicon I1, A/C. Enterobacter carrying blaACT-MIR contained replicon F11A. Conclusion: ESBL and carbapenemase producing Enterobacteriaceae in children are diverse in origin. Like in adults, the predominant strains responsible for ESBL phenotypes are B2-ST43 clonal groups in E. coli containing blaCTX-M-1 group (containing blaCTX-M-15). In contrast, CRE were rare, and blaKPC bearing K. pneumoniae isolates were non ST258, the predominant strain identified in adults. The finding of an IMP gene suggests introduction of metallo-b-lactamases into our population. Plasmid-mediated AmpC may account for a larger percentage of transferable resistance than previously recognized.
    No preview · Conference Paper · Oct 2015
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    ABSTRACT: This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with bla(NDM), bla(IMP), and bla(OXA-48).
    No preview · Article · Oct 2015 · Journal of Clinical Microbiology
  • Maria F Mojica · Robert A Bonomo · Walter Fast
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    ABSTRACT: Metallo-beta-Lactamases (MBLs) are class B β-lactamases that hydrolyze almost all clinically-available β-lactam antibiotics. MBLs feature the distinctive αβ/βα sandwich fold of the metallo-hydrolase / oxidoreductase superfamily and possess a shallow active-site groove containing one or two divalent zinc ions, flanked by flexible loops. According to sequence identity and zinc ion dependence, MBLs are classified into three subclasses (B1, B2 and B3), of which the B1 subclass enzymes have emerged as the most clinically significant. Differences among the active site architectures, the nature of zinc ligands, and the catalytic mechanisms have limited the development of a common inhibitor. In this review, we will describe the molecular epidemiology and structural studies of the most prominent representatives of class B1 MBLs (NDM-1, IMP-1 and VIM-2) and describe the implications for inhibitor design to counter this growing clinical threat.
    No preview · Article · Oct 2015 · Current drug targets
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    ABSTRACT: CTX-M β-lactamases are one of the fastest growing extended-spectrum β-lactamase (ESBL) families found in Escherichia coli rendering this organism extremely difficult to treat with β-lactam antibiotics . Although they are grouped in class A β-lactamases, the CTX-M family possesses low sequence identity with other enzymes. In addition, they have high hydrolytic activity against oxyimino-cephalosporins, despite having smaller active sites compared to other ESBLs in class A. Similar to most class A enzymes, most of the CTX-M β-lactamases can be inhibited by the clinical inhibitors (clavulanic acid, sulbactam, and tazobactam), but the prevalence of inhibitor resistance is an emerging clinical threat. Thus, the mechanistic details of inhibition pathways are needed for new inhibitor development. Here, we use Raman microscopy to study the CTX-M-9 inactivation reaction with the three commercially available inhibitors and compare these findings to the analysis of the S130G variant. Characterization of the reactions in CTX-M-9 single crystals and solution show the formation of a unique cross-linked species, probably involving Ser70 and Ser130, with subsequent hydrolysis leading to an acrylate species linked to Ser130. In solution, a major population of this species is seen at 25 milliseconds after mixing. Support for this finding comes from the CTX-M-9 S130G variant that reacts with clavulanic acid, sulbactam, and tazobactam in solution, but lacks the characteristic spectroscopic signature for the Ser130-linked species. Understanding the mechanism of inactivation of this clinically important ESBL-type class A lactamase permits us to approach the challenge of inhibitor resistance using knowledge of the bridging species in the inactivation pathway.
    No preview · Article · Sep 2015 · Journal of the American Chemical Society
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    ABSTRACT: Background. Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. Methods. We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. Results. In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%–73% for ceftazidime and 41%–50% for imipenem. Conclusions. RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.
    Full-text · Article · Sep 2015 · Clinical Infectious Diseases
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    ABSTRACT: BACKGROUND The pandemic of carbapenem-resistant Enterobacteriaceae (CRE) was primarily due to clonal spread of bla KPC producing Klebsiella pneumoniae. Thus, thoroughly studied CRE cohorts have consisted mostly of K. pneumoniae. OBJECTIVE To conduct an extensive epidemiologic analysis of carbapenem-resistant Enterobacter spp. (CREn) from 2 endemic and geographically distinct centers. METHODS CREn were investigated at an Israeli center (Assaf Harofeh Medical Center, January 2007 to July 2012) and at a US center (Detroit Medical Center, September 2008 to September 2009). bla KPC genes were queried by polymerase chain reaction. Repetitive extragenic palindromic polymerase chain reaction and pulsed-field gel electrophoresis were used to determine genetic relatedness. RESULTS In this analysis, 68 unique patients with CREn were enrolled. Sixteen isolates (24%) were from wounds, and 33 (48%) represented colonization only. All isolates exhibited a positive Modified Hodge Test, but only 93% (27 of 29) contained bla KPC. Forty-three isolates (63%) were from elderly adults, and 5 (7.4%) were from neonates. Twenty-seven patients died in hospital (40.3% of infected patients). Enterobacter strains consisted of 4 separate clones from Assaf Harofeh Medical Center and of 4 distinct clones from Detroit Medical Center. CONCLUSIONS In this study conducted at 2 distinct CRE endemic regions, there were unique epidemiologic features to CREn: (i) polyclonality, (ii) neonates accounting for more than 7% of cohort, and (iii) high rate of colonization (almost one-half of all cases represented colonization). Since false-positive Modified Hodge Tests in Enterobacter spp. are common, close monitoring of carbapenem resistance mechanisms (particularly carbapenemase production) among Enterobacter spp. is important. Infect. Control Hosp. Epidemiol. 2015;00(0):1-9.
    No preview · Article · Sep 2015 · Infection Control and Hospital Epidemiology
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    ABSTRACT: Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2- Ec ) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33- Ec ) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33- Ec , the patient received cefepime. CMY-33- Ec and CMY-2- Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs . ≤0.5 μg/ml). Identical CMY-2- Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs . 0.25 μg/ml). The k cat / K m or k inact / K I (μM -1 s -1 ) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs . 0.4; ceftazidime: 0.2 vs . not measurable; cefepime: 0.2 vs . not measurable; tazobactam 0.0018 vs . 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2- Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.
    No preview · Article · Sep 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: Background: Infections due to multi-drug resistant (MDR) Gram-negative bacilli (GNB) are a growing problem in children. Our long term goals are to investigate the impact of MDR GNB infections and to define the molecular epidemiology of this emerging health threat. Here, we report our findings concerning PMFQR in children. Methods: A retrospective cohort study of clinical GNB isolates obtained from children ages 0-21 years hospitalized between 2011-14 at three Chicago hospitals was performed. To assess for PMFQR, PCR was used. Repetitive-sequence-based PCR (rep-PCR) was used to determine genetic similarity. PCR and DNA microarray (Check-Points CT101) were used to assess for beta-lactamase genes (bla). Multilocus sequence typing (MLST), PCR and DNA sequencing were performed on representative isolates for bacterial nomenclature and characterization. Results: Of 169 ESBL-producing Enterobacteriaceae isolates with antibiogram data, 85(50.3%) were FQR of which 82 were available for testing. The median age of patients was 4.8 years. The predominant FQR organism was E. coli 65/82 (79%) and predominant bla genotype associated with FQR in Enterobacteriaceae was blaCTX-M-1 group in 51/82 cases (62%). Within ESBL E. coli, FQR was commonly associated with phylogenetic group B2 and/or ST43 (ST131 in Achtman’s MLST scheme) containing blaCTX-M-1 group in 47/63 cases (75%). PMFQR was found in 56/82 (68.3%), of which the aac 6’1b-cr (Y102R and/or D179Y) gene mutation was most common (56%). PMFQR genes oqxA, oqxB, qepA and qnrb were found in 10%, 7%, 9% and 4%, respectively. Several isolates contained >1 bla/PMFQR gene. Conclusions: We define the molecular epidemiology of PMFQR in ESBL-producing Enterobacteriaceae in children. The majority of FQR ESBLs in children are linked with PMFQR mechanisms. The predominant strains responsible are B2-ST43 clonal groups of ESBL-producing E. coli containing blaCTX-M-1 group (containing blaCTX-M-15); however, PMFQR in these organisms is associated with multiple bla types.
    No preview · Conference Paper · Sep 2015
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    ABSTRACT: Antibiotic resistance in bacteria is ever changing and adapting, as once novel β-lactam antibiotics are losing their efficacy primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics including carbapenems and are often co-expressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity is employed. In this study, we elucidate the effect of different amino acid substitutions at position 67 in IMP-type MBLs on their ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were biophysically and biochemically characterized and minimum inhibitory concentrations (MICs) for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed highest kcat/Km. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side-chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor, and by inference, the β-lactam substrates.
    No preview · Article · Sep 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: The increasing rate of antibiotic resistance and slowing discovery of novel antibiotic treatments presents a growing threat to public health. Here, we consider a simple model of evolution in asexually reproducing populations which considers adaptation as a biased random walk on a fitness landscape. This model associates the global properties of the fitness landscape with the algebraic properties of a Markov chain transition matrix and allows us to derive general results on the non-commutativity and irreversibility of natural selection as well as antibiotic cycling strategies. Using this formalism, we analyze 15 empirical fitness landscapes of E. coli under selection by different β-lactam antibiotics and demonstrate that the emergence of resistance to a given antibiotic can be either hindered or promoted by different sequences of drug application. Specifically, we demonstrate that the majority, approximately 70%, of sequential drug treatments with 2-4 drugs promote resistance to the final antibiotic. Further, we derive optimal drug application sequences with which we can probabilistically 'steer' the population through genotype space to avoid the emergence of resistance. This suggests a new strategy in the war against antibiotic-resistant organisms: drug sequencing to shepherd evolution through genotype space to states from which resistance cannot emerge and by which to maximize the chance of successful therapy.
    Full-text · Article · Sep 2015 · PLoS Computational Biology

Publication Stats

12k Citations
1,638.69 Total Impact Points


  • 2007-2015
    • Case Western Reserve University School of Medicine
      • • Department of Molecular Biology and Microbiology
      • • Department of Medicine
      Cleveland, Ohio, United States
    • Emory University
      • Department of Microbiology and Immunology
      Atlanta, Georgia, United States
  • 1996-2015
    • Louis Stokes Cleveland VA Medical Center
      Cleveland, Ohio, United States
  • 1992-2015
    • Case Western Reserve University
      • • Department of Molecular Biology and Microbiology
      • • School of Medicine
      • • Department of Pharmacology
      • • Department of Biochemistry
      • • Division of Infectious Diseases and HIV Medicine
      Cleveland, Ohio, United States
  • 2014
    • University of North Carolina at Chapel Hill
      North Carolina, United States
    • United States Department of Veterans Affairs
      Bedford, Massachusetts, United States
  • 2008-2014
    • San Francisco VA Medical Center
      San Francisco, California, United States
  • 2010-2012
    • Southern Methodist University
      • Department of Chemistry
      Dallas, Texas, United States
  • 1997-2012
    • Cleveland State University
      • Department of Chemistry
      Cleveland, Ohio, United States
  • 2009-2011
    • Mount Sinai School of Medicine
      • Department of Medicine
      Manhattan, NY, United States
    • Saint Edward's University
      Austin, Texas, United States
  • 2006
    • University of Helsinki
      Helsinki, Uusimaa, Finland
  • 2003-2005
    • University of Pittsburgh
      • Division of Infectious Diseases
      Pittsburgh, Pennsylvania, United States
  • 1998-2005
    • University of Connecticut
      • Department of Molecular and Cell Biology
      Сторс, Connecticut, United States
  • 2004
    • Minneapolis Veterans Affairs Hospital
      Minneapolis, Minnesota, United States