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A case-case-control study was conducted to identify independent risk factors for recovery of Escherichia coli strains producing CTX-M-type extended-spectrum β-lactamases (CTX-M E. coli ) within a large Southeastern Michigan medical center. Unique cases with isolation of ESBL-producing E. coli from February 2010 through July 2011 were analyzed by PCR for bla CTX-M , bla TEM , and bla SHV genes. Patients with CTX-M E. coli were compared to patients with E. coli strains not producing CTX-M-type ESBLs (non-CTX-M E. coli ) and uninfected controls. Of 575 patients with ESBL-producing E. coli , 491 (85.4%) isolates contained a CTX-M ESBL gene. A total of 319 (84.6%) patients with CTX-M E. coli (282 [74.8%] CTX-M-15 type) were compared to 58 (15.4%) non-CTX-M E. coli patients and to uninfected controls. Independent risk factors for CTX-M E. coli isolation compared to non-CTX-M E. coli included male gender, impaired consciousness, H2 blocker use, immunosuppression, and exposure to penicillins and/or trimethoprim-sulfamethoxazole. Compared to uninfected controls, independent risk factors for isolation of CTX-M E. coli included presence of a urinary catheter, previous urinary tract infection, exposure to oxyimino-cephalosporins, dependent functional status, non-home residence, and multiple comorbid conditions. Within 48 h of admission, community-acquired CTX-M E. coli ( n = 51 [16%]) and non-CTX-M E coli ( n = 11 [19%]) strains were isolated from patients with no recent health care contacts. CTX-M E. coli strains were more resistant to multiple antibiotics than non-CTX-M E. coli strains. CTX-M-encoding genes, especially bla CTX-M-15 type, represented the most common ESBL determinants from ESBL-producing E. coli , the majority of which were present upon admission. Septic patients with risk factors for isolation of CTX-M E. coli should be empirically treated with appropriate agents. Regional infection control efforts and judicious antibiotic use are needed to control the spread of these organisms. more...

We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures., IMPORTANCE Chemotherapeutic agents, including antibiotics, have been used for more than a century; nevertheless, there are still major gaps in our understanding of how these drugs operate which limit future advances in antibacterial chemotherapy. Although the molecular mechanisms by which antibiotics bind to their target structures are largely known, fundamental questions about how these drugs actually kill and/or inhibit the replication of bacteria remain unanswered and subjects of controversy. We postulate that for the broad class of ribosome-binding bacteriostatic antibiotics, their reducing the number of active (functional) ribosomes per cell provides a sufficient explanation for the abatement of replication and the low rate of decline in densities of viable cells of bacteria exposed to these drugs. Using E. coli K-12 constructs with deletions of from one to six of the seven ribosome-RNA operons and the ribosome-binding bacteriostatic antibiotics tetracycline, chloramphenicol, and azithromycin, we tested this hypothesis. The results of our experiments are consistent with this “numbers game” hypothesis. more...

Persisters are dormant variants that form a subpopulation of cells tolerant to antibiotics. Persisters are largely responsible for the recalcitrance of chronic infections to therapy. In Escherichia coli, one widely accepted model of persister formation holds that stochastic accumulation of ppGpp causes activation of the Lon protease that degrades antitoxins; active toxins then inhibit translation, resulting in dormant, drug-tolerant persisters. We found that various stresses induce toxin-antitoxin (TA) expression but that induction of TAs does not necessarily increase persisters. The 16S rRNA promoter rrnB P1 was proposed to be a persister reporter and an indicator of toxin activation regulated by ppGpp. Using fluorescence-activated cell sorting (FACS), we confirmed the enrichment for persisters in the fraction of rrnB P1-gfp dim cells; however, this is independent of toxin-antitoxins. rrnB P1 is coregulated by ppGpp and ATP. We show that rrnB P1 can report persisters in a relA/spoT deletion background, suggesting that rrnB P1 is a persister marker responding to ATP. Consistent with this finding, decreasing the level of ATP by arsenate treatment causes drug tolerance. Lowering ATP slows translation and prevents the formation of DNA double-strand breaks upon fluoroquinolone treatment. We conclude that variation in ATP levels leads to persister formation by decreasing the activity of antibiotic targets., IMPORTANCE Persisters are a subpopulation of antibiotic-tolerant cells responsible for the recalcitrance of chronic infections. Our current understanding of persister formation is primarily based on studies of E. coli. The activation of toxin-antitoxin systems by ppGpp has become a widely accepted model for persister formation. In this study, we found that stress-induced activation of mRNA interferase-type toxins does not necessarily cause persister formation. We also found that the persister marker rrnB P1 reports persister cells because it detects a drop in cellular ATP levels. Consistent with this, lowering the ATP level decreases antibiotic target activity and, thus, leads to persister formation. We conclude that stochastic variation in ATP is the main mechanism of persister formation. A decrease in ATP provides a satisfactory explanation for the drug tolerance of persisters, since bactericidal antibiotics act by corrupting energy-dependent targets. more...

The ability of microorganisms to develop resistance against agents used to treat infections has resulted in a constant battle between man and microbe since the clinical introduction of antibiotics over 70 years ago. This article summarizes changes in resistance from the perspective of both microbial pathogens and classes of drugs over the past decade. Drugs that inhibit the growth of bacteria, fungi, and viruses are included in this overview. Significant changes have occurred in resistance profiles of some pathogens, particularly multidrug-resistant Gram-negative pathogens, and the effectiveness of certain antimicrobial agents, while little has changed for others. A combination of continued drug development and good stewardship of existing drugs is required to control morbidity and mortality and to try to keep ahead of microbial pathogens. more...

β-Lactam antibiotics have been used clinically since World War II and are considered to be among the safest, most efficacious and most widely prescribed antibiotics for the treatment of bacterial infections. Their therapeutic use began with the introduction of benzylpenicillin (penicillin G) during World War II [1, 2] and continues with the development of newer cephalosporins and carbapenems for antibiotic-resistant infections. These agents act by inhibiting bacterial cell wall synthesis, as a result of their strong covalent binding to essential penicillin binding proteins (PBPs) that catalyze the last steps of cell wall formation in both Gram-positive and Gram-negative bacteria [3, 4]. However, resistance to these agents has been a major concern. The discovery and development of new β-lactams has been driven by the continued selection of resistance mechanisms that result in the loss of efficacy of these agents. more...

Synergy between antibiotics is a strictly defined microbiological phenomenon, requiring two bioactive agents to exhibit enhanced bacterial killing when the two are combined. Because of increasing antibiotic resistance, and few new drugs to treat multidrug-resistant bacteria, combination therapy is often used in the clinical setting. Frequently, these combinations have demonstrated synergistic activity both in vitro and in animal models before being used therapeutically. Antibiotic combinations are more likely to be used in patients with drug-resistant staphylococcal or enterococcal infections, as well as in patients whose diseases are caused by carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa, or Acinetobacter spp. Although well-defined combinations have been approved by regulatory authorities as single agents, such as trimethoprim–sulfamethoxazole or β-lactamase inhibitor combinations, many combinations are used empirically with no clinical data to support their use. Because combination therapy will continue to be used in the absence of supportive clinical data, it will be important in the future to investigate mechanistic principles that may lead to predictive models for successful patient outcomes. more...

The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens., IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem- and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant “superbugs.” more...

β-Lactamases can be named on the basis of molecular characteristics or functional properties. Molecular classes A, B, C, and D define an enzyme according to amino acid sequence and conserved motifs. Functional groups 1, 2, and 3 are used to assign a clinically useful description to a family of enzymes, with subgroups designated according to substrate and inhibitor profiles. In addition, other designations are used to define the functionality of specific subgroups, such as extended-spectrum β-lactamases, or ESBLs, and inhibitor-resistant TEM, or IRT, β-lactamases. None of these systems provides an unambiguous description of this versatile set of enzymes. A proposed classification system involving microbiological, molecular, and biochemical properties is described, based on the traditional classes A, B, C, and D and functional groups 1, 2, and 3 designations. more...

Inactivation of β-lactam antibiotics by β-lactamases in bacterial infections is associated with some of the most serious infectious disease issues that are currently encountered. The evolution of unique β-lactamases has resulted in more than 1,300 distinct enzymes that have been identified in natural clinical isolates. Of these enzymes, the most deleterious β-lactamases are the extended-spectrum β-lactamases, or ESBLs, that hydrolyze most penicillins and cephalosporins, and the carbapenemases that may inactivate all β-lactam classes of drugs. The most prominent ESBLs worldwide are the CTX-M-14 and CTX-M-15 enzymes. Among enzyme families, the TEM and OXA β-lactamases exhibit the greatest number of variants. The broad groups of carbapenemases are particularly treacherous, especially the KPC serine carbapenemases and the NDM family of metallo-β-lactamases, both of which appear in multidrug-resistant Gram-negative pathogens that are often resistant to most classes of antibiotics. Although new β-lactamase inhibitor combinations are being investigated as a means of controlling infections caused by these organisms, additional approaches are sorely needed. more...

Origins of antibiotic drug discovery are frequently traced to 1929 when Alexander Fleming recognized the antibacterial activity of a substance secreted by Penicillium notatum on a contaminated culture plate. However, the subsequent development of penicillin as a therapeutic agent was not realized until the early 1940s, after a consortium of academic and pharmaceutical scientists from England and the United States developed sufficiently advanced fermentation technology to produce high-purity penicillin in large enough quantities for medical supplies. It was at this time that the antibiotic era was truly successfully launched. During the following decade, unprecedented antibiotic research and development emerged in academic laboratories and the pharmaceutical industry, resulting in identification of most of the antibiotic classes currently used therapeutically. This short historical commentary describes some of these early events, beginning with a conference held at the New York Academy of Sciences in 1946, the first conference to focus entirely on the latest science related to the identification and characterization of antibacterial substances produced by microorganisms. more...

RWJ-416457 is a novel investigational oxazolidinone with minimum inhibitory concentrations (MICs) to staphylococci and enterococci that are two- to four-fold lower than those of linezolid. Single-step and serial passage in vitro resistance selection experiments were performed for RWJ-416457 and linezolid with Staphylococcus aureus and Enterococcus faecalis laboratory and clinical isolates. RWJ-416457 selected for resistant mutants in single-step selections at a frequency of ≤1×10(-10), similar to that of linezolid. In serial passage selection experiments, a G2576T transversion in the domain V region of the 23S rRNA gene was the predominant mutation observed for both oxazolidinones, suggesting similar 23S rRNA binding sites. The associated development of increasing oxazolidinone resistance in E. faecalis (four 23S rRNA alleles) required fewer passages than with S. aureus isolates (six 23S rRNA alleles), and resistance was generally proportionate to the number of mutated (G2576T) 23S rRNA alleles. Fold changes in MICs were similar for both compounds, and MICs for RWJ-416457 remained two- to four-fold lower than those of linezolid for mutants selected by either compound. Serial passage of linezolid with S. aureus OC 2878 yielded a novel A2572T 23S rRNA mutation, whilst the final passages of S. aureus OC 10517 with RWJ-416457 resulted in the apparent loss of a mutated (G2576T) allele 6. more...

Resistance to β-lactams and other antibiotics in the Enterobacteriaceae is frequently associated with plasmidic resistance determinants that are easily transferred among species. β-Lactamase-mediated resistance is increasingly associated with plasmid-encoded extended-spectrum β-lactamases (ESBLs) and carbapenemases, specifically the CTX-M family of ESBLs, the KPC family of serine carbapenemases, and the VIM, IMP, and NDM-1 metallo-β-lactamases. Although clonal dispersion of resistant isolates was seen initially, more diverse genetic platforms are being observed as variations of mobile elements are transferred worldwide. These enzymes are now appearing in multiple combinations of ESBLs and carbapenemases, thereby conferring resistance to virtually all β-lactam antibiotics. more...

Single-step Pseudomonas aeruginosa mutants, selected with ceftobiprole, ceftazidime, or cefepime, were generated at frequencies of 10 −6 to −9 at two and four times the MIC. The chromosomal AmpC β-lactamase activity was increased in all ceftazidime-selected mutants. Mutants selected with cefepime either increased AmpC activity or upregulated expression of the mexXY efflux genes. Mutants selected with ceftobiprole did not overexpress AmpC; 90% of these produced elevated levels of mexXY RNA, indicating that increased efflux, not AmpC derepression, is the predominant response to ceftobiprole during first-step mutations in P. aeruginosa . more...

β-Lactam antibiotics are among the most frequently prescribed antibiotics used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of β-lactamases with broad hydrolytic capabilities, especially in multidrug-resistant Gram-negative bacteria.This review describes new β-lactams and β-lactamase inhibitors described in the patent literature primarily between 2007 and 2010, together with supportive meeting abstracts and relevant descriptive literature.Readers will learn which classes of β-lactam antibiotics are being explored as the most promising groups of compounds to counteract resistance in Gram-negative pathogenic bacteria. Somewhat surprisingly, few traditional β-lactam classes such as penicillins or cephalosporins were described in the literature, other than in combinations with other β-lactams or β-lactamase inhibitors that are being developed to inhibit enzymes from all molecular classes.β-Lactam antibiotics are currently being developed as monotherapy by only a few companies. The major emphasis in the past 4 years has been the discovery of novel β-lactamase inhibitors or inhibitor combinations that will allow use of β-lactams against multidrug-resistant bacteria. The use of β-lactams as single agents appears to be a limited option for the future. more...

We compared drugs (imipenem and doripenem), doses (500 mg and 1 g), and infusion times (0.5 and 1.0 [imipenem], 1.0 and 4.0 h [doripenem]) in our hollow-fiber model, examining cell kill and resistance suppression for three isogenic strains of Pseudomonas aeruginosa PAO1. The experiments ran for 10 days. Serial samples were taken for total organism and resistant subpopulation counts. Drug concentrations were determined by high-pressure liquid chromatography-tandem mass spectrometry (LC/MS/MS). Free time above the MIC (time > MIC) was calculated using ADAPT II. Time to resistance emergence was examined with Cox modeling. Cell kill and resistance emergence differences were explained, in the main, by differences in potency (MIC) between doripenem and imipenem. Prolonged infusion increased free drug time > MIC and improved cell kill. For resistance suppression, the 1-g, 4-h infusion was able to completely suppress resistance for the full period of observation for the wild-type isolate. For the mutants, control was ultimately lost, but in all cases, this was the best regimen. Doripenem gave longer free time > MIC than imipenem and, therefore, better cell kill and resistance suppression. For the wild-type organism, the 1-g, 4-h infusion regimen is preferred. For organisms with resistance mutations, larger doses or addition of a second drug should be studied. more...

Induction of mecA by ceftobiprole and oxacillin in 18 methicillin-resistant Staphylococcus aureus clinical isolates with various SCC mec cassettes was examined using reverse transcriptase PCR. The magnitude of mecA induction, 3- to 65-fold for ceftobiprole and 2- to 69-fold for oxacillin, did not correlate with ceftobiprole MICs (≤4 μg/ml), although the 11 isolates with at least 13-fold induction by oxacillin all had oxacillin MICs of ≥256 μg/ml. No correlation between magnitude of induction and SCC mec type was found. more...

In 50 years, the International Conference on Antimicrobial Agents and Chemotherapy—known as ICAAC—has grown from a small, almost entirely U.S.-based meeting to the largest international conference on antimicrobial agents, infectious diseases, and microbiology. One key reason for this success is an enduring focus on scientific excellence and innovation. During those five decades, domestic and international attendance and numbers of scientific presentations has grown steadily, while scientific rigor remained steady. more...

The stability of doripenem to hydrolysis by β-lactamases from molecular classes A to D was compared to the stability for imipenem and meropenem. Doripenem was stable to hydrolysis by extended-spectrum β-lactamases and AmpC type β-lactamases and demonstrated high affinity for the AmpC enzymes. For the serine carbapenemases SME-3 and KPC-2 and metallo-β-lactamases IMP-1 and VIM-2, doripenem hydrolysis was generally 2- to 150-fold slower than imipenem hydrolysis. SPM-1 hydrolyzed meropenem and doripenem fourfold faster than imipenem. more...

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, ≤4 μg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P . aeruginosa resistance to β-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P . aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 μg/ml at baseline but 16 μg/ml posttreatment, unrelated to β-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB , mexCD , and mexEF , increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ . Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB , - CD , and - EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical β-lactam that is a substrate for the MexXY efflux pump in P . aeruginosa . more...

New antibacterial agents to treat infections caused by antibiotic-susceptible and antibiotic-resistant pathogens are in various stages of clinical development. In this review are compounds with demonstrated activity against methicillin-resistant staphylococci including investigational cephalosporins, carbapenems, and a new tetracycline, as well as glycopeptides effective against vancomycin-resistant enterococci (VRE), and fluoroquinolones with improved potency against respiratory pathogens and multidrug-resistant Gram-positive bacteria. Although most recent progress has occurred in the identification of agents for Gram-positive infections, broad-spectrum carbapenems are described for the treatment of multidrug-resistant Gram-negative pathogens. Also discussed are agents with mechanisms of action other than inhibition of protein synthesis, penicillin-binding proteins, and DNA topoisomerases; among these are inhibitors of bacterial fatty acid biosynthesis, peptidoglycan synthesis, and dihydrofolate reductase. more...

In phase 3 clinical trials for ceftobiprole treatment of complicated skin and skin structure infections, 1,219 gram-positive and 276 gram-negative aerobic baseline pathogens were identified. Ceftobiprole inhibited all staphylococcal isolates, including methicillin-resistant strains, at MICs of ≤4 μg/ml. Against Enterobacteriaceae and Pseudomonas aeruginosa isolates, the potency of ceftobiprole was similar to that of cefepime. more...

Background A randomized, double-blind, multicenter trial involving patients with a broad range of complicated skin and skin-structure infections due to either gram-positive or gram-negative bacteria was conducted to compare ceftobiprole monotherapy with treatment with vancomycin plus ceftazidime. Methods Patients were randomized 2:1 to receive ceftobiprole or to receive vancomycin plus ceftazidime. Outcomes were determined at a test-of-cure visit (7-14 days after completion of therapy) and were analyzed for all patients with complicated skin and skin-structure infections, as well as for subgroups, on the basis of major types of infections and severity of disease. Results Among the clinically evaluable and the intent-to-treat populations, clinical cure rates at the test-of-cure visit were similar in the ceftobiprole and comparator treatment arms (clinical cure rate, 90.5% [439 of 485 patients] and 90.2% [220 of 244 patients] in the clinically evaluable population, respectively; 81.9% [448 of 547 patients] and 80.8% [227 of 281 patients] in the intent-to-treat population, respectively). Clinical cure rates in ceftobiprole-treated patients ranged from 86.2% (125 of 145 patients) among those with diabetes who had foot infections to 93.0% (80 of 86 patients) among those with cellulitis. Among patients treated with ceftobiprole, clinical cure rates were similar among patients from whom gram-negative bacteria were isolated (87.9% [109 of 124 patients]) and among patients from whom gram-positive bacteria were isolated (91.8% [292 of 318 patients]) and were not statistically different from the clinical cure rates among comparator-treated patients (89.7% [61 of 68 patients] and 90.3% [149 of 165 patients], respectively). Rates of adverse events and serious adverse events in the 2 treatment groups were similar. Conclusions Ceftobiprole monotherapy is as effective as vancomycin plus ceftazidime for treating patients with a broad range of complicated skin and skin-structure infections and infections due to gram-positive and gram-negative bacteria. more...

Eravacycline is a novel, fully synthetic fluorocycline antibiotic of the tetracycline class being developed for the treatment of complicated urinary tract infections and complicated intra-abdominal infections. Eravacycline has activity against many key Gram-negative pathogens, including Enterobacteriaceae resistant to carbapenems, cephalosporins, fluoroquinolones and β-lactam/β-lactamase inhibitor combinations, including strains that are multidrug-resistant. Carbapenem-resistant Enterobacteriaceae (CRE) isolates from 2010 to 2013 (n=110) were characterized for carbapenemase genes by PCR and sequencing. MICs for eravacycline, tetracycline, tigecycline, amikacin, imipenem, ceftazidime, cefotaxime and levofloxacin were determined in broth microdilution assays. All isolates produced at least one carbapenemase, most frequently KPC-3. Nine isolates produced both a KPC serine carbapenemase and a metallo-β-lactamase, NDM-1 (n=1) or VIM-1 (n=8). The 110 isolates were highly resistant to all the β-lactams tested and to levofloxacin, and had MIC50/MIC90 values in the intermediate range for tetracycline and amikacin. MIC50/MIC90 values for eravacycline were 1/2 μg ml(-1) compared with 2/2 μg ml(-1) for tigecycline. Eravacycline MICs were often twofold lower than for tigecycline, with 64% of the eravacycline MICs2 μg ml(-1) as compared with4% of tigecycline MICs. Overall, eravacycline demonstrated the lowest cumulative MICs against this panel of recent CRE and may have the potential to treat infections caused by CRE. more...

MurF is an essential enzyme of bacterial cell wall biosynthesis. Few MurF inhibitors have been reported, and none have displayed measurable antibacterial activity. Through the use of a MurF binding assay, a series of 8-hydroxyquinolines that bound to the Escherichia coli enzyme and inhibited its activity was identified. To derive additional chemotypes lacking 8-hydroxyquinoline, a known chelating moiety, a pharmacophore model was constructed from the series and used to select compounds for testing in the MurF binding and enzymatic inhibition assays. Whereas the original diverse library yielded 0.01% positive compounds in the binding assay, of which 6% inhibited MurF enzymatic activity, the pharmacophore-selected set yielded 14% positive compounds, of which 37% inhibited the enzyme, suggesting that the model enriched for compounds with affinity to MurF. A 4-phenylpiperidine (4-PP) derivative identified by this process displayed antibacterial activity (MIC of 8 μg/ml against permeable E. coli ) including cell lysis and a 5-log 10 -unit decrease in CFU. Importantly, treatment of E. coli with 4-PP resulted in a 15-fold increase in the amount of the MurF UDP-MurNAc-tripeptide substrate, and a 50% reduction in the amount of the MurF UDP-MurNAc-pentapeptide product, consistent with inhibition of the MurF enzyme within bacterial cells. Thus, 4-PP is the first reported inhibitor of the MurF enzyme that may contribute to antibacterial activity by interfering with cell wall biosynthesis. more...

The interactions of ceftobiprole with purified β-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent β-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 β-lactamase, the class A TEM-1 β-lactamase, and the class C AmpC β-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum β-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressingEnterobacter cloacaeorganisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than β-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the familyEnterobacteriaceae. more...

Ceftobiprole exhibited tight binding to PBP2a in methicillin-resistant Staphylococcus aureus , PBP2x in penicillin-resistant Streptococcus pneumoniae , and PBP3 and other essential penicillin-binding proteins in methicillin-susceptible S. aureus , Escherichia coli , and Pseudomonas aeruginosa . Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria. more...

Ceftobiprole is a broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA) that is undergoing phase III trials for the treatment of complicated skin and skin structure infections and nosocomial pneumonia. The objectives were to describe the pharmacodynamic profiles of ceftobiprole given at 500 mg intravenously (i.v.) every 8 h (q8h) (2-h infusion) and 500 mg i.v. every 12 h (q12h) (1-h infusion) to determine the overall probability of target attainment (PTA) by weighting for the expected distributions of renal function in the populations of interests, to determine the PTA against representative pathogens encountered in clinical trials, and to determine the optimal renal dose adjustment for ceftobiprole at 500 mg i.v. q8h (2-h infusion). Data for a total of 150 subjects in phase I/II trials were analyzed by using the population pharmacokinetic modeling program BigNPOD (nonparametric optimal design). Monte Carlo simulation was performed with the ADAPT II program to estimate the PTA at which the free drug concentrations exceed the MIC for 30 to 60% of the dosing interval (30 to 60% fT > MIC). For ceftobiprole at 500 mg i.v. q12h, the probabilities of achieving 30% and 50% fT > MIC exceeded 90% for MICs ≤2 mg/liter and ≤1 mg/liter, respectively, For ceftobiprole at 500 mg i.v. q8h, the probabilities of achieving 40 and 60% fT > MIC exceeded 90% for MICs ≤4 mg/liter and ≤2 mg/liter, respectively. For ceftobiprole at both 500 mg i.v. q12h and 500 mg i.v. q8h, the probability of achieving a nearly bactericidal effect (50% fT > MIC) exceeded 90% for methicillin-susceptible S. aureus and MRSA. For gram-negative pathogens, the PTA for achieving a nearly maximal bactericidal effect (60% fT > MIC) for ceftobiprole at 500 mg i.v. q8h exceeded 90% for non-AmpC-producing gram-negative organisms. Ceftobiprole at 500 mg i.v. q12h, for patients who had a creatinine clearance rate of ≤50 ml/min, was identified as the most appropriate treatment regimen for patients who require renal dose adjustment for mild to moderate renal impairment. more...

A series of 3-keto-6-O-carbamoyl-11,12-cyclic thiocarbamate erythromycin A derivatives has been synthesized. The best compounds in this series possess potent in vitro antibacterial activity against erythromycin-susceptible and erythromycin-resistant bacteria. more...

RWJ-416457, an investigational pyrrolopyrazolyl-substituted oxazolidinone, inhibited the growth of linezolid-susceptible staphylococci, enterococci, and streptococci at concentrations of ≤4 μg/ml, generally exhibiting two- to fourfold-greater potency than that of linezolid. Time-kill studies demonstrated bacteriostatic effects for both RWJ-416457 and linezolid. more...

Chemobiosynthesis has been used to prepare analogs of erythromycins having unique functional groups at the 15-position. Using diketide thioester feeding to genetically engineered Streptomyces coelicolor, analogs of 6-deoxyerythronolide B were prepared having 15-fluoro, 15-chloro, and 15-azido groups. Bioconversion using a genetically engineered mutant of Saccharopolyspora erythraea was used to produce 15-fluoroerythromycin A and 15-azidoerythromycin A. These new erythromycin analogs provide antibacterial macrolides with unique physicochemical properties and functional groups that allow for selective derivatization. more...

A new series of erythromycin A derivatives, the 6-O-heteroarylcarbamoyl-11,12-lactoketolides, with activity against macrolide-resistant streptococci, are described. Structurally, these macrolide antibiotics are characterized by a heteroaryl side chain attached to the macrolactone core through a carbamate linkage at the C6 position, as well as 11,12-gamma-lactone and 3-keto functionalities. The synthesis and antibacterial activity of this new series of ketolides are discussed. more...

A series of 3-O-acyl-6-O-carbamoyl erythromycin A derivatives has been synthesized. Several functional groups were identified as the optimal C3-substituents, and the best compounds in this series possess potent in vitro antibacterial activity against erythromycin-susceptible and erythromycin-resistant bacteria. more...

OBJECTIVES Prevalence of single quinolone-resistance determining region (QRDR) mutations in Streptococcus pneumoniae was studied from nine institutions over 5 years to track the incidence of single QRDR mutations. METHODS All 1106 levofloxacin-susceptible pneumococci (MICs < or = 2.0 mg/L) identified from 1112 total isolates (99.5% susceptibility) in TRUST 3 (1999), TRUST 5 (2001) and TRUST 7 (2003) surveillance studies from the same nine hospitals in nine states were screened for QRDR mutations. Using pyrosequencing, the strains were screened for mutations corresponding to hot spots Asp-78, Ser-79 and Asp-83 in ParC; Asp-80, Ser-81 and Glu-85 in GyrA; Asp-435 in ParE and Asp-435 in GyrB. DNA sequencing of QRDRs was performed to confirm mutations. RESULTS No QRDR mutations were found in any of the isolates with levofloxacin MICs < or = 0.5 mg/L and no gyrA or gyrB QRDR mutations were found in any of the screened isolates (MICs < or = 2 mg/L). Four single-step QRDR mutants with the following amino acid substitutions were found: ParE Asp-435 to Asn (isolated in 1999 in Colorado); ParC Asp-83 to Asn (isolated in 2001 in Kentucky); ParC Ser-79 to Phe (isolated in 2003 in Indiana) and ParC Ser-79 to Tyr (isolated in 2003 in California). These non-clonal strains had levofloxacin MICs of 1 mg/L and were non-susceptible to ciprofloxacin (MIC 2-4 mg/L). CONCLUSIONS Overall prevalence of single QRDR mutations in levofloxacin-susceptible S. pneumoniae with MICs of < or = 2 mg/L was 0.4% (4/1106) and has remained more...

MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc- l -Ala-γ- d -Glu- meso -diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc- l -Ala-γ- d -Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic l -Ala-γ- d -Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing l -Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 μM was identified. more...

Expanded options for treatments directed against pathogens that can be used for bioterrorism are urgently needed. Treatment regimens directed against such pathogens can be identified only by using data derived from in vitro and animal studies. It is crucial that these studies reliably predict the efficacy of proposed treatments in humans. The objective of this study was to identify a levofloxacin treatment regimen that will serve as an effective therapy for Bacillus anthracis infections and postexposure prophylaxis. An in vitro hollow-fiber infection model that replicates the pharmacokinetic profile of levofloxacin observed in humans (half-life [ t 1/2 ], 7.5 h) or in animals, such as the mouse or the rhesus monkey ( t 1/2 , ∼2 h), was used to evaluate a proposed indication for levofloxacin (500 mg once daily) for the treatment of Bacillus anthracis infections. The results obtained with the in vitro model served as the basis for the doses and the dose schedules that were evaluated in the mouse inhalational anthrax model. The effects of levofloxacin and ciprofloxacin treatment were compared to those of no treatment (untreated controls). The main outcome measure in the in vitro hollow-fiber infection model was a persistent reduction of culture density (≥4 log 10 reduction) and prevention of the emergence of levofloxacin-resistant organisms. In the mouse inhalational anthrax model the main outcome measure was survival. The results indicated that levofloxacin given once daily with simulated human pharmacokinetics effectively sterilized Bacillus anthracis cultures. By using a simulated animal pharmacokinetic profile, a once-daily dosing regimen that provided a human-equivalent exposure failed to sterilize the cultures. Dosing regimens that “partially humanized” levofloxacin exposures within the constraints of animal pharmacokinetics reproduced the antimicrobial efficacy seen with human pharmacokinetics. In a mouse inhalational anthrax model, once-daily dosing was significantly inferior (survival end point) to regimens of dosing every 12 h or every 6 h with identical total daily levofloxacin doses. These results demonstrate the predictive value of the in vitro hollow-fiber infection model with respect to the success or the failure of treatment regimens in animals. Furthermore, the model permits the evaluation of treatment regimens that “humanize” antibiotic exposures in animal models, enhancing the confidence with which animal models may be used to reliably predict the efficacies of proposed antibiotic treatments in humans in situations (e.g., the release of pathogens as agents of bioterrorism or emerging infectious diseases) where human trials cannot be performed. A treatment regimen effective in rhesus monkeys was identified. more...

Why Do Some Plants Have Larger leaves than other plants? Why do the overall shapes of different kinds of leaves vary? How does one determine the area of a peculiarly shaped leaf? Plant biologists are extremely interested in these questions. A biologist might wonder, for instance, which of two plant species carries out the most photosynthesis. Since the amount of light a plant can absorb for use in photosynthesis is related to its area, the biologist might investigate the photosynthetic capacity of two species by comparing the amount of chlorophyll in leaf pieces of the same area. more...

Novel C6-carbamate ketolides with C2-fluorination and C9-oximation have been synthesized. The best compounds in this series displayed MIC values of 0.03–0.12 μg/mL against streptococci containing erm and mef resistance determinants and 2–4 μg/mL against Haemophilus influenzae . Several compounds also showed measurable activity against erm (B)-containing enterococci with MIC values of 2–8 μg/mL. In vivo activity was adversely affected by fluorination, possibly as a result of increased serum protein binding. more...

Respiratory tract infections, including community-acquired pneumonia (CAP), acute exacerbations of chronic bronchitis (AECB), acute bacterial sinusitis (ABS), and pharyngitis, are a frequent cause of illness and, for CAP, a substantial cause of mortality (3, 22). Streptococcus pneumoniae and Haemophilus influenzae are responsible for a majority of the cases of CAP, AECB, and ABS, while Streptococcus pyogenes is the primary causal agent for pharyngitis (5, 23). Macrolides efficacious against S. pneumoniae and H. influenzae are first-line agents for the treatment of CAP (10, 16, 28) and are second-line agents for the treatment of AECB (1). With ABS (2, 26) and pharyngitis (5, 25), macrolides are an important treatment option where intolerance to penicillins is a concern. Macrolide resistance in pneumococci has been increasing since the early 1980s, with worldwide resistance levels of clinical isolates currently at approximately 25% in some studies (19, 34). In the United States, macrolide nonsusceptibility varies by region from 17 to 36% (34), while nonsusceptibility outside the United States ranges from 1.5% in the Czech Republic to 80% in Hong Kong (19). Clinical macrolide resistance results primarily from the expression of the erm(B) dimethylase or the mef(A) efflux gene. The proportion of macrolide-resistant strains expressing the erm or mef phenotypes differs by region and by country. In the United States, resistance results from mef(A) in approximately 64% of resistant isolates, from erm(B) in approximately 23% of cases, and a combination of erm(B) and mef(A) in 12% of resistant isolates (14). Outside the United States, the distribution of resistance varies from 0% mef(A) and 100% erm(B) in Belgium to 83% mef(A) and 17% erm(B) in Argentina. Worldwide, erm(B) is the predominant mechanism of resistance, accounting for 56% of macrolide-resistant strains, while mef(A) accounts for 35%; strains with both mechanisms comprise most of the remainder (14). Other clinically relevant but less frequent mechanisms of macrolide resistance include mutations in the L4 and L22 ribosomal proteins and mutations in domain V of the 23S rRNA (approximately 2% of macrolide-resistant pneumococci) (11, 14, 30). With the proportion of penicillin-nonsusceptible pneumococci concomitantly reaching 37% in the United States and ranging from 4% (The Netherlands) to 74% (Hong Kong) outside the United States (19), the need for new agents to treat respiratory tract infections is increasingly important. Recent efforts to identify new agents with activity against macrolide-resistant respiratory tract pathogens resulted in the development of the ketolide telithromycin (32), a member of a new class of antibiotics structurally related to macrolides. Telithromycin, with demonstrated clinical efficacy against H. influenzae, macrolide-susceptible and -resistant strains of S. pneumoniae, and S. pyogenes (8, 23, 36), is being used for the treatment of CAP, AECB, ABS, and tonsillitis/pharyngitis. To date, most attempts to produce new ketolides have utilized traditional macrolide cores such as erythromycin A as starting material in synthetic schemes. In contrast, in the studies outlined in this report, novel ketolides were synthesized from unique macrolides produced biosynthetically from genetically altered actinomycete strains (9, 20); these unnatural macrolide core molecules were produced as part of a program to yield novel ketolides with potent in vitro and in vivo activities. This is the first publication describing the in vitro antibacterial activities of ketolides derived from bioengineered macrolide core molecules. Herein, the in vitro activities of two naphthyridine-containing ketolides derived from 15-methylerythromycin A are described. (Portions of the data were previously presented [D. Abbanat, G. Webb, G. Ashley, B. Foleno, H. Fu, Y. Li, P. Licari, M. Macielag, D. Montenegro, E. Wira, and K. Bush, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-1663, 2002].) more...

To the Editor—With great sadness, we mark the passing of our cherished colleague and friend, John P. Quinn, MD (Figure ​(Figure1).1). John leaves behind a beloved wife (Dr Maria Virginia Villegas), a daughter (Veronica), 3 sons (John Jr., Robert, and Christopher), and 2 grandchildren. We in the field of infectious diseases will miss his friendship, his smile, his humor, his embrace, and his warmth. His absence will be felt profoundly because it occurs at a time when talent and passion dedicated to the study of drug-resistant organisms are most urgently needed. Figure 1. John P. Quinn in the heights of Machu Picchu, Peru, 2010. Picture provided by Dr Maria Virginia Villegas. John fiercely embodied the qualities we all strive to achieve. He was a gifted clinician and a dedicated and insightful researcher who strove to translate the lessons learned in the laboratory to the patient's bedside. John's contributions led to the increasing recognition of the crisis of antibiotic resistance in gram-negative bacteria. He leaves a great void in our field. The eldest among 8 brothers, John grew up in Chicago's South Shore neighborhood (the “South Side”). He pursued an academic career at his city's venerable institutions, including Rush Medical College, Loyola University, Michael Reese Hospital, University of Illinois at Chicago, and Cook County Hospital, eventually attaining the rank of Professor of Medicine and Vice-Chairman. Like a true son of Chicago, all his accomplishments were earned the “hard way.” He worked evenings and weekends to support his medical school education as he raised a young family. Very early in his career, John established himself as a pioneer who led the way in the field. Soon after imipenem/cilastatin was introduced in 1985, John presciently reported therapeutic failures in the treatment of Pseudomonas aeruginosa. Moreover, he detailed how penetration of imipenem through the outer membrane of P. aeruginosa was prevented by changes in the porin protein OprD2 [1, 2]. Later, he also studied carbapenem-resistant Serratia marcescens isolates from across the United States that harbored SME, a serine carbapenemase [3]. With these sentinel discoveries, John heralded the emergence of resistance to carbapenems decades before the current era of “nightmare bacteria.” John also observed how resistance during treatment with ceftazidime can emerge in Enterobacter cloacae; here, he examined the role of β-lactamase induction [4]. Also, he was quick to grasp the significance of the unusual susceptibility pattern of 2 isolates of Klebsiella pneumoniae that demonstrated resistance to ceftazidime. He identified TEM-10 as the mechanism of resistance, which, together with the subsequent report of TEM-26, were 2 of the first occurrences of plasmid-mediated extended-spectrum β-lactamase (ESBL) in North America [5, 6]. Since then, he advocated for the judicious use of expanded-spectrum cephalosporins to limit selective pressure and the spread of ESBLs. After these findings, John earned for himself the moniker of “ESBLman,” which he later adopted as his email address. Working with others, John demonstrated the importance of detecting β-lactamase–mediated resistance to avoid therapeutic failures, and recognized that facilities such as nursing homes can be “reservoirs of resistance”—notions that continue to enjoy currency in the contemporary epidemic of carbapenem-resistant organisms [7, 8]. John also chronicled the eroding reliability of fluoroquinolones in intensive care units due to the emergence of resistance associated with the increased use of this class of drugs [9]. From his vantage point in Chicago, John described the first nosocomial outbreak of P. aeruginosa producing VIM-2 metallo-β-lactamase (MBL) in the United States, revealing that this country is vulnerable to MBLs [10]. He warned about the threat posed by mobile genetic elements containing MBLs, especially because they may not be readily detected. The need for increased vigilance to curb the spread of MBLs is attested by the recent importation of New Delhi metallo-β-lactamase-1 (NDM-1) into the United States. John leveraged his expertise and reputation to establish collaborations with scientists from around the world. For instance, at an early stage, he and investigators at the Peking Union Medical College documented the predominance of CTX-M–type enzymes in China, a key piece in the global epidemiology of ESBLs [11]. More recently, John authored a definite review on carbapenemase-mediated resistance in K. pneumoniae together with scientists from 14 different countries and 5 continents [12]. The most meaningful of these partnerships was with Dr Villegas from CIDEIM (Centro Internacional de Entrenamiento e Investigaciones Medicas) in Cali, Colombia. Together, they formed a scientific collaboration that extended for more than a decade and led to important insights into the emergence of antibiotic resistance in South America and its significance in the global context [13–16]. At the same time, John and Dr Villegas created research opportunities, scientific capacity, and awareness in antimicrobial resistance throughout the entire region. At the culmination of his academic career, John joined the pharmaceutical industry in order to seek practical solutions to the crisis caused by drug-resistant bacteria. As a physician, John saw firsthand the impact that these pathogens wrought; he also recognized that, contrary to the past, new molecules were not being added to the therapeutic armamentarium. He believed that the discovery and development of new antibacterial agents was a “moral obligation.” Working for Pfizer and later for AstraZeneca, he was recognized as someone who could build bridges between scientists and clinicians and play a critical role in the development of new anti-infectives. To Pfizer's and AstraZeneca's credit, each recognized John's intellectual depth, scientific credentials, and clinical talents. John's vision was to make a difference in the treatment of bacterial infections and to persevere in seeking fundamental answers regarding mechanisms of antibiotic resistance. This was the sentiment he unequivocally voiced in the public television program “Frontline,” broadcast 4 days after his death [17]. Clear lessons emerge from examining John's life and career, and these constitute his legacy: his keen observations and prepared mind, his interest in forming and sustaining collaborations, his global outlook and reach, his ability to inspire and support young investigators, and the resolution of his beliefs and courage to conduct himself accordingly. Keeping faithful to these lessons will be key in our society's efforts to preserve the dividend of antibiotics to global health. John had a lasting impact on our field, and we must ensure that his lessons survive him by passing them to future generations. John will always be in our hearts and minds, and we only hope to emulate him by moving forward with fortitude and conviction. A few days before his death, he told one of us, with a smile on his face, “I am ready to go”; he knew his legacy will continue. more...

At least 18 antibacterial agents are currently undergoing clinical trials for the treatment of infections caused by susceptible and resistant bacteria. The beta-lactam class includes new parenteral carbapenems and cephalosporins with varying spectra of activities. The glycopeptides are antibiotics with in vitro activity primarily against Gram-positive bacteria, including multi-resistant strains. Three quinolones are being investigated for use against a variety of Gram-positive and respiratory Gram-negative organisms. Several other classes of antibacterial agents currently in clinical trials are represented by a glycolipodepsipeptide, a dihydrofolate reductase inhibitor, an oxazolidinone, two peptide antibiotics, a glycylcycline, and a peptide deformylase inhibitor, a member of a new antibacterial class. more...

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum β-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing β-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no β-lactamases gave consistent MIC results with inocula of 10 5 and 10 6 CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total β-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing. more...

We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32μ g/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla SHV and bla TEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The bla KPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination. more...

The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1–120 μM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC 50 value of 0.10 μM, virus isolated at passages 3 and 15 displayed EC 50 values of 10 and >50 μM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC 50 values of 8.4±0.4 and 127±16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K i value of 0.066±0.002 nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K i value of 4.69±0.44 nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5 A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance. more...