Staphylococcus aureus Strain Newman D2C Contains Mutations in Major Regulatory Pathways That Cripple Its Pathogenesis.

Staphylococcus aureus is a major human pathogen that imposes a great burden on the health care system. In the development of antistaphylococcal modalities intended to reduce the burden of staphylococcal disease, it is imperative to select appropriate models of S. aureus strains when assessing the efficacy of novel agents. Here, using whole-genome sequencing, we reveal that the commonly used strain Newman D2C from the American Type Culture Collection (ATCC) contains mutations that render the strain essentially avirulent. Importantly, Newman D2C is often inaccurately referred to as simply "Newman" in many publications, leading investigators to believe it is the well-described pathogenic strain Newman. This study reveals that Newman D2C carries a stop mutation in the open reading frame of the virulence gene regulator, agrA In addition, Newman D2C carries a single-nucleotide polymorphism (SNP) in the global virulence regulator gene saeR that results in loss of protein function. This loss of function is highlighted by complementation studies, where the saeR allele from Newman D2C is incapable of restoring functionality to an saeR-null mutant. Additional functional assessment was achieved through the use of biochemical assays for protein secretion, ex vivo intoxications of human immune cells, and in vivo infections. Altogether, our study highlights the importance of judiciously screening for genetic changes in model S. aureus strains when assessing pathogenesis or the efficacy of novel agents. Moreover, we have identified a novel SNP in the virulence regulator gene saeR that directly affects the ability of the protein product to activate S. aureus virulence pathways.IMPORTANCEStaphylococcus aureus is a human pathogen that imposes an enormous burden on health care systems worldwide. This bacterium is capable of evoking a multitude of disease states that can range from self-limiting skin infections to life-threatening bacteremia. To combat these infections, numerous investigations are under way to develop therapeutics capable of thwarting the deadly effects of the bacterium. To generate successful treatments, it is of paramount importance that investigators use suitable models for examining the efficacy of the drugs under study. Here, we demonstrate that a strain of S. aureus commonly used for drug efficacy studies is severely mutated and displays markedly reduced pathogenicity. As such, the organism is an inappropriate model for disease studies.

Efficacy of a new fluoroquinolone, JNJ-Q2, in murine models of Staphylococcus aureus and Streptococcus pneumoniae skin, respiratory, and systemic infections.

The in vivo efficacy of JNJ-Q2, a new broad-spectrum fluoroquinolone (FQ), was evaluated in a murine septicemia model with methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) and in a Streptococcus pneumoniae lower respiratory tract infection model. JNJ-Q2 and comparators were also evaluated in an acute murine skin infection model using a community-acquired MRSA strain and in an established skin infection (ESI) model using a hospital-acquired strain, for which the selection of resistant mutants was also determined. JNJ-Q2 demonstrated activity in the MSSA septicemia model that was comparable to that moxifloxacin (JNJ-Q2 50% effective dose [ED(50)], 0.2 mg/kg of body weight administered subcutaneously [s.c.] and 2 mg/kg administered orally [p.o.]) and activity in the MRSA septicemia model that was superior to that of vancomycin (JNJ-Q2 ED(50), 1.6 mg/kg administered s.c.). In an S. pneumoniae lower respiratory tract infection model, JNJ-Q2 displayed activity (ED(50), 1.9 mg/kg administered s.c. and 7.4 mg/kg administered p.o.) that was comparable to that of gemifloxacin and superior to that of moxifloxacin. In both MRSA skin infection models, treatment with JNJ-Q2 resulted in dose-dependent reductions in bacterial titers in the skin, with the response to JNJ-Q2 at each dose exceeding the responses of the comparators ciprofloxacin, moxifloxacin, linezolid, and vancomycin. Additionally, in the ESI model, JNJ-Q2 showed a low or nondetectable propensity for ciprofloxacin resistance selection, in contrast to the selection of ciprofloxacin-resistant mutants observed for both ciprofloxacin and moxifloxacin. JNJ-Q2 demonstrated activity that was comparable or superior to the activity of fluoroquinolone or antistaphylococcal comparators in several local and systemic skin infection models performed with both S. aureus and S. pneumoniae and is currently being evaluated in phase II human clinical trials.

Antistaphylococcal activities of the new fluoroquinolone JNJ-Q2.

The new broad-spectrum fluoroquinolone JNJ-Q2 displays in vitro activity against Gram-negative and Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant MRSA isolates. Tested with isogenic methicillin-susceptible S. aureus (MSSA) and MRSA strains bearing quinolone-resistant target mutations, JNJ-Q2 displayed MICs ≤ 0.12 µg/ml, values 16- to 32-fold lower than those determined for moxifloxacin. Overexpression of the NorA efflux pump did not impact JNJ-Q2 MICs. Inhibition of S. aureus DNA gyrase and DNA topoisomerase IV enzymes demonstrated that JNJ-Q2 was more potent than comparators against wild-type enzymes and enzymes carrying quinolone-resistant amino acid substitutions, and JNJ-Q2 displayed equipotent activity against both enzymes. In serial-passage studies comparing resistance selection in parallel MRSA cultures by ciprofloxacin and JNJ-Q2, ciprofloxacin readily selected for mutants displaying MIC values of 128 to 512 µg/ml, which were observed within 18 to 24 days of passage. In contrast, cultures passaged in the presence of JNJ-Q2 displayed MICs ≤ 1 µg/ml for a minimum of 27 days of serial passage. A mutant displaying a JNJ-Q2 MIC of 4 µg/ml was not observed until after 33 days of passage. Mutant characterization revealed that ciprofloxacin-passaged cultures with MICs of 256 to 512 µg/ml carried only 2 or 3 quinolone resistance-determining region (QRDR) mutations. Cultures passaged with JNJ-Q2 selection for up to 51 days displayed MICs of 1 to 64 µg/ml and carried between 4 and 9 target mutations. Established in vitro biofilms of wild-type or ciprofloxacin-resistant MRSA exposed to JNJ-Q2 displayed greater decreases in bacterial counts (7 days of exposure produced 4.5 to >7 log(10) CFU decreases) than biofilms exposed to ciprofloxacin, moxifloxacin, rifampin, or vancomycin.

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007-09.

BACKGROUND: Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. METHODS: Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. RESULTS: Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-ß-lactamases were present in three P. aeruginosa and one A. baumannii isolates. CONCLUSIONS: Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-ß-lactamases were found in <0.1% of isolates.

In vitro antibacterial activities of JNJ-Q2, a new broad-spectrum fluoroquinolone.

JNJ-Q2, a novel fluorinated 4-quinolone, was evaluated for its antibacterial potency by broth and agar microdilution MIC methods in studies focused on skin and respiratory tract pathogens, including strains exhibiting contemporary fluoroquinolone resistance phenotypes. Against a set of 118 recent clinical isolates of Streptococcus pneumoniae, including fluoroquinolone-resistant variants bearing multiple DNA topoisomerase target mutations, an MIC(90) value for JNJ-Q2 of 0.12 microg/ml was determined, indicating that it was 32-fold more potent than moxifloxacin. Against a collection of 345 recently collected methicillin-resistant Staphylococcus aureus (MRSA) isolates, including 256 ciprofloxacin-resistant strains, the JNJ-Q2 MIC(90) value was 0.25 microg/ml, similarly indicating that it was 32-fold more potent than moxifloxacin. The activities of JNJ-Q2 against Gram-negative pathogens were generally comparable to those of moxifloxacin. In further studies, JNJ-Q2 exhibited bactericidal activities at 2x and 4x MIC levels against clinical isolates of S. pneumoniae and MRSA with various fluoroquinolone susceptibilities, and its activities were enhanced over those of moxifloxacin. In these studies, the activity exhibited against strains bearing gyrA, parC, or gyrA plus parC mutations was indicative of the relatively balanced (equipotent) activity of JNJ-Q2 against the DNA topoisomerase target enzymes. Finally, determination of the relative rates or frequencies of the spontaneous development of resistance to JNJ-Q2 at 2x and 4x MICs in S. pneumoniae, MRSA, and Escherichia coli were indicative of a lower potential for resistance development than that for current fluoroquinolones. In conclusion, JNJ-Q2 exhibits a range of antibacterial activities in vitro that is supportive of its further evaluation as a potential new agent for the treatment of skin and respiratory tract infections.

Effect of MexXY overexpression on ceftobiprole susceptibility in Pseudomonas aeruginosa.

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, < or =4 microg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to beta-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 microg/ml at baseline but 16 microg/ml posttreatment, unrelated to beta-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 beta-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.

Transcriptional profiling and drug discovery.

The availability of whole-genome nucleotide sequence data from an ever-growing list of microbial genomes, including complete genomes for multiple strains within a species, presents an opportunity to overcome the challenges presented by antimicrobial drug resistance. The development of DNA microarrays provides a unique tool to understand pathogenic microbial genomes from a global perspective. Genome-wide expression profiles can facilitate the characterization both of the mechanisms of action and of the mechanisms of resistance to antimicrobial agents. Expression data have also been used to initiate the characterization of genes of unknown function, potentially leading to the identification of novel drug targets. Initial studies using DNA microarrays to analyse the host and pathogen responses to infection have also been performed, impacting our understanding of pathogenesis and the strategies taken to combat infectious diseases.

Activities of carbapenem and comparator agents against contemporary US Pseudomonas aeruginosa isolates from the CAPITAL surveillance program.

Antimicrobial susceptibilities of contemporary Pseudomonas aeruginosa clinical isolates were determined from the CAPITAL 2010 surveillance program. Isolates were collected from 100 sites throughout the USA and Puerto Rico, and included isolates representing a range of patient demographics and infection types. A total of 2722 isolates were tested for susceptibility to a broad spectrum of agents, with susceptibilities ranging from 98.8% for colistin to 74% for levofloxacin. Doripenem was the most active carbapenem agent, with 88.6% of isolates susceptible, in comparison with 78.1% and 84.6% for imipenem and meropenem, respectively. Lower respiratory tract isolates and isolates from the intensive care unit setting were the least susceptible overall. Resistance rates were typically highest in lower respiratory tract isolates, with the exception of urinary tract isolates, which displayed the highest resistance for levofloxacin. Overall, multidrug-resistant isolates comprised 14.8% of the total sample population.

Tetrahydroindazole inhibitors of bacterial type II topoisomerases. Part 2: SAR development and potency against multidrug-resistant strains.

We have previously reported a novel class of tetrahydroindazoles that display potency against a variety of Gram-positive and Gram-negative bacteria, potentially via interaction with type II bacterial topoisomerases. Herein are reported SAR investigations of this new series. Several compounds possessing broad-spectrum potency were prepared. Further, these compounds exhibit activity against multidrug-resistant Gram-positive microorganisms equivalent to that against susceptible strains.

Novel pyrazole derivatives as potent inhibitors of type II topoisomerases. Part 1: synthesis and preliminary SAR analysis.

In an attempt to search for a new class of antibacterial agents, we have discovered a series of pyrazole analogs that possess good antibacterial activity for Gram-positive and Gram-negative organisms via inhibition of type II bacterial topoisomerases. We have investigated the structure-activity relationships of this series, with an emphasis on the length and conformation of the linker. This work led to the identification of tetrahydroindazole analogs, such as compound 1, as the most potent class of compounds.

Comparison of the changes in global gene expression of Escherichia coli induced by four bactericidal agents.

DNA microarrays provide a global view of the physiological state of the cell by parallel analysis of the expression levels of all the genes in an organism. The effects of four bactericidal agents on the expression pattern of Escherichia coli MG1655 were assessed. Compounds were chosen on the basis of their different mechanisms of action and included inhibitors of DNA replication and recombination, translation, transcription and cell wall biosynthesis. The addition of rifampin resulted in increased expression of the target, rpoB, as well as several genes involved in nucleotide salvage and purine biosynthesis. The addition of ampicillin resulted in overall changes in gene expression that showed some similarity to changes induced by rifampin. The addition of the antibiotics kanamycin or norfloxacin resulted in the induction of unique gene expression signatures: a heat shock response to kanamycin and an SOS response to norfloxacin. Several genes of unknown function showed expression profiles similar to the genes associated with the SOS or the heat shock response. Thus, these profiles define families of genes with similar expression phenotypes that can be tested for related function.

Simultaneous analysis of host and pathogen interactions during an in vivo infection reveals local induction of host acute phase response proteins, a novel bacterial stress response, and evidence of a host-imposed metal ion limited environment.

A fundamental goal in the study of infections is to understand the dynamic interplay between host and pathogen; however, direct in vivo interrogation of this disease process via transcriptional profiling has been lacking. Here we describe the development and application of novel bacterial RNA amplification technology to simultaneously identify key elements of both host and pathogen responses in a murine infection model. On the bacterial side, we found induction of an unusual pattern of stress response genes, a response to host-induced metal ion limitation, and a failure to achieve stationary phase in vivo. On the mammalian side, we observed the surprising induction of several genes encoding acute phase response proteins including hepcidin, haptoglobin, complement C3 and metallothionein 1 at the site of infection, as well as other mediators of innate immunity. Thus, our results reveal host-pathogen cross-talk not predicted by previous in vitro analyses and provide the framework to eavesdrop on a broad array of host-pathogen interactions in vivo. As described here, the comprehensive examination of host-pathogen interactions during an infection is critical to the discovery of novel approaches for intervention not predicted by current models.