Identification of bile acid and fatty acid species as candidate rapidly bactericidal agents for topical treatment of gonorrhoea.

BACKGROUND: Novel therapeutic strategies are urgently needed for Neisseria gonorrhoeae, given its increasing antimicrobial resistance. Treatment of oropharyngeal N. gonorrhoeae infections has proven particularly challenging, with most reported treatment failures of the first-line drug ceftriaxone occurring at this site and lower cure rates in recent trials of new antibiotics reported for oropharyngeal infections compared with other sites of infection. However, the accessibility of the oropharynx to topical therapeutics provides an opportunity for intervention. Local delivery of a therapeutic at a high concentration would enable the use of non-traditional antimicrobial candidates, including biological molecules that exploit underlying chemical sensitivities of N. gonorrhoeae but lack the potency or pharmacokinetic profiles required for effective systemic administration. METHODS: Two classes of molecules that are thought to limit gonococcal viability in vivo, bile acids and short- and medium-chain fatty acids, were examined for rapid bactericidal activity. RESULTS: The bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), but not other bile acid species, exerted extremely rapid bactericidal properties against N. gonorrhoeae, reducing viability more than 100 000-fold after 1 min. The short-chain fatty acids formic acid and hexanoic acid shared this rapid bactericidal activity. All four molecules are effective against a phylogenetically diverse panel of N. gonorrhoeae strains, including clinical isolates with upregulated efflux pumps and resistance alleles to the most widely used classes of existing antimicrobials. DCA and CDCA are both approved therapeutics for non-infectious indications and are well-tolerated by cultured epithelial cells. CONCLUSIONS: DCA and CDCA are attractive candidates for further development as anti-gonococcal agents.

Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach.

Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.

Gain-of-Function Analysis Reveals Important Virulence Roles for the Yersinia pestis Type III Secretion System Effectors YopJ, YopT, and YpkA.

Virulence of Yersinia pestis in mammals requires the type III secretion system, which delivers seven effector proteins into the cytoplasm of host cells to undermine immune responses. All seven of these effectors are conserved across Y. pestis strains, but three, YopJ, YopT, and YpkA, are apparently dispensable for virulence. Some degree of functional redundancy between effector proteins would explain both observations. Here, we use a combinatorial genetic approach to define the minimal subset of effectors required for full virulence in mice following subcutaneous infection. We found that a Y. pestis strain lacking YopJ, YopT, and YpkA is attenuated for virulence in mice and that addition of any one of these effectors to this strain increases lethality significantly. YopJ, YopT, and YpkA likely contribute to virulence via distinct mechanisms. YopJ is uniquely able to cause macrophage cell death in vitro and to suppress accumulation of inflammatory cells to foci of bacterial growth in deep tissue, whereas YopT and YpkA cannot. The synthetic phenotypes that emerge when YopJ, YopT, and YpkA are removed in combination provide evidence that each effector enhances Y. pestis virulence and that YopT and YpkA act through a mechanism distinct from that of YopJ.

Reversion From Methicillin Susceptibility to Methicillin Resistance in Staphylococcus aureus During Treatment of Bacteremia.

Approximately 3% of Staphylococcus aureus strains that, according to results of conventional phenotypic methods, are highly susceptible to methicillin-like antibiotics also have polymerase chain reaction (PCR) results positive for mecA. The genetic nature of these mecA-positive methicillin-susceptible S. aureus (MSSA) strains has not been investigated. We report the first clearly defined case of reversion from methicillin susceptibility to methicillin resistance among mecA-positive MSSA within a patient during antibiotic therapy. We describe the mechanism of reversion for this strain and for a second clinical isolate that reverts at a similar frequency. The rates of reversion are of the same order of magnitude as spontaneous resistance to drugs like rifampicin. When mecA is detected by PCR in the clinical laboratory, current guidelines recommend that these strains be reported as resistant. Because combination therapy using both a ß-lactam and a second antibiotic suppressing the small revertant population may be superior to alternatives such as vancomycin, the benefits of distinguishing between mecA-positive MSSA and MRSA in clinical reports should be evaluated.

An updated molecular diagnostic for surveillance of tetM in Neisseria gonorrhoeae.

Doxycycline post-exposure prophylaxis (doxy-PEP) for sexually transmitted bacterial infections reduces the risk of syphilis and chlamydia, but effectiveness against gonorrhea is variable, likely attributable to varying resistance rates. As doxy-PEP is incorporated into clinical practice, an urgent unanswered question is whether increased doxycycline use will drive tetracycline-class resistance in Neisseria gonorrhoeae. Here, we report an updated RT-PCR molecular diagnostic to detect the tetM gene that confers high-level tetracycline resistance in N. gonorrhoeae.

Yersinia pestis escapes entrapment in thrombi by targeting platelet function.

BACKGROUND: Platelets are classically recognized for their role in hemostasis and thrombosis. Recent work has demonstrated that platelets can also execute a variety of immune functions. The dual prothrombotic and immunological roles of platelets suggest that they may pose a barrier to the replication or dissemination of extracellular bacteria. However, some bloodborne pathogens, such as the plague bacterium Yersinia pestis, routinely achieve high vascular titers that are necessary for pathogen transmission. OBJECTIVES: It is not currently known how or if pathogens circumvent platelet barriers to bacterial dissemination and replication. We sought to determine whether extracellular bloodborne bacterial pathogens actively interfere with platelet function, using Y  pestis as a model system. METHODS: The interactions and morphological changes of human platelets with various genetically modified Y pestis strains were examined using aggregation assays, immunofluorescence, and scanning electron microscopy. RESULTS: Yersinia pestis directly destabilized platelet thrombi, preventing bacterial entrapment in fibrin/platelet clots. This activity was dependent on two well-characterized bacterial virulence factors: the Y pestis plasminogen activator Pla, which stimulates host-mediated fibrinolysis, and the bacterial type III secretion system (T3SS), which delivers bacterial proteins into the cytoplasm of targeted host cells to reduce or prevent effective immunological responses. Platelets intoxicated by the Y pestis T3SS were unable to respond to prothrombotic stimuli, and T3SS expression decreased the formation of neutrophil extracellular traps in platelet thrombi. CONCLUSIONS: These findings are the first demonstration of a bacterial pathogen using its T3SS and an endogenous protease to manipulate platelet function and to escape entrapment in platelet thrombi.

RNA polymerase mutations cause cephalosporin resistance in clinical Neisseria gonorrhoeae isolates.

Increasing Neisseria gonorrhoeae resistance to ceftriaxone, the last antibiotic recommended for empiric gonorrhea treatment, poses an urgent public health threat. However, the genetic basis of reduced susceptibility to ceftriaxone is not completely understood: while most ceftriaxone resistance in clinical isolates is caused by target site mutations in penA, some isolates lack these mutations. We show that penA-independent ceftriaxone resistance has evolved multiple times through distinct mutations in rpoB and rpoD. We identify five mutations in these genes that each increase resistance to ceftriaxone, including one mutation that arose independently in two lineages, and show that clinical isolates from multiple lineages are a single nucleotide change from ceftriaxone resistance. These RNA polymerase mutations cause large-scale transcriptional changes without altering susceptibility to other antibiotics, reducing growth rate, or deranging cell morphology. These results underscore the unexpected diversity of pathways to resistance and the importance of continued surveillance for novel resistance mutations.

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

Genome-wide mutant fitness profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue.

UNLABELLED: Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes-encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)-both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes. IMPORTANCE: Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because most growth studies of pathogenic bacteria are conducted on laboratory media that do not reflect conditions prevailing in infected animal tissues. Improving our knowledge of this aspect of bacterial biology is important as a potential pathway to the development of novel therapeutics. Yersinia pestis, the plague bacterium, is highly virulent due to its rapid dissemination and growth in deep tissues, making it a good model for discovering bacterial adaptations that promote rapid growth during infection. Using Tn-seq, a genome-wide fitness profiling technique, we identified several functions required for fitness of Y. pestis in vivo that were not previously known to be important. Most of these functions are needed to acquire or synthesize nutrients. Interference with these critical nutrient acquisition pathways may be an effective strategy for designing novel antibiotics and vaccines.