Antibiotic resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer 1. Since new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination 2. Here, using carbapenemresistant Enterobacteriaceae (CRE) clinical isolates, we revealed the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics 3-6 in which only a subset of cells are phenotypically resistant 7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics ("multiple heteroresistance"). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance

Beyond defense against foreign DNA, the CRISPR-Cas9 system of pathogenic Francisella 23 novicida represses expression of an endogenous immunostimulatory lipoprotein and is essential for virulence. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 has a highly specific regulon of four genes which must be repressed for bacterial virulence. Regulation occurs through a PAM-dependent interaction of Cas9 with its endogenous DNA targets, directed by a non-canonical small RNA (scaRNA) duplexed with tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage. This highlights the evolution of the scaRNA to direct transcriptional interference via interaction with endogenous DNA without lethally targeting the chromosome.We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-Cas9 machinery can also be licensed to direct cleavage of target DNA. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens.

Summary The nosocomial pathogen Acinetobacter baumannii is a growing threat to public health due to its increasing resistance to antibiotics including the last-line polymyxin, colistin. Heteroresistance to colistin has been described in A. baumannii, wherein a resistant subpopulation of cells co-existing with a majority susceptible subpopulation actively grows in the presence of antibiotic and can cause treatment failure. The shortcomings of diagnostic tests in detecting colistin heteroresistance are especially worrisome as they may lead to clinicians unknowingly prescribing an ineffective antibiotic, leading to increased patient morbidity and mortality. Several techniques can be used to detect heteroresistance and the purpose of this chapter is to outline effective methods for identifying, quantifying and analyzing heteroresistance to colistin in A. baumannii. We will highlight the advantages and disadvantages of techniques including population analysis profile (PAP), Etest, and disc diffusion, as well as additional methods to distinguish heteroresistance from other forms of resistance. While the scope of this chapter will focus on colistin heteroresistance in A. baumannii, these techniques can be adapted for the study of heteroresistance to other antibiotics and in other bacteria with slight modifications.