Bacterial populations are heterogeneous, which in many cases can provide a selective advantage during changes in environmental conditions. In some instances, heterogeneity exists at the genetic level, in which significant allelic variation occurs within a population seeded by a single cell. In other cases, heterogeneity exists due to phenotypic differences within a clonal, genetically identical population. A variety of mechanisms can drive this latter strategy. Stochastic fluctuations can drive differential gene expression, but heterogeneity in gene expression can also be driven by environmental changes sensed by individual cells residing in distinct locales. Utilizing multiple single cell approaches, workers have started to uncover the extent of heterogeneity within bacterial populations. This review will first describe several examples of phenotypic and genetic heterogeneity, and then discuss many single cell approaches that have recently been applied to define heterogeneity within bacterial populations.

We created a 2013 combination antibiogram of healthcare-associated urinary tract infection. The 2013 antibiogram-determined regimen was evaluated in a 2014 cohort who had received empirical therapy. The regimen was statistically more likely to represent adequate treatment than actual prescriptions. A customized antibiogram may guide empirical therapy for specific patients. Infect Control Hosp Epidemiol 2016;37:1101-1104.

OBJECTIVE To offer antimicrobial stewardship to a long-term acute care hospital using telemedicine. METHODS We conducted an uninterrupted time-series analysis to measure the impact of antimicrobial stewardship on hospital-acquired Clostridium difficile infection (CDI) rates and antimicrobial use. Simple linear regression was used to analyze changes in antimicrobial use; Poisson regression was used to estimate the incidence rate ratio in CDI rates. The preimplementation period was April 1, 2010-March 31, 2011; the postimplementation period was April 1, 2011-March 31, 2014. RESULTS During the preimplementation period, total antimicrobial usage was 266 defined daily doses (DDD)/1,000 patient-days (PD); it rose 4.54 (95% CI, -0.19 to 9.28) per month then significantly decreased from preimplementation to postimplementation (-6.58 DDD/1,000 PD [95% CI, -11.48 to -1.67]; P=.01). The same trend was observed for antibiotics against methicillin-resistant Staphylococcus aureus (-2.97 DDD/1,000 PD per month [95% CI, -5.65 to -0.30]; P=.03). There was a decrease in usage of anti-CDI antibiotics by 50.4 DDD/1,000 PD per month (95% CI, -71.4 to -29.2; P<.001) at program implementation that was maintained afterwards. Anti-Pseudomonas antibiotics increased after implementation (30.6 DDD/1,000 PD per month [95% CI, 4.9-56.3]; P=.02) but with ongoing education this trend reversed. Intervention was associated with a decrease in hospital-acquired CDI (incidence rate ratio, 0.57 [95% CI, 0.35-0.92]; P=.02). CONCLUSION Antimicrobial stewardship using an electronic medical record via remote access led to a significant decrease in antibacterial usage and a decrease in CDI rates.

Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

Bacterial pathogens express virulence-specific transcriptional programs that allow tissue colonization. Although phenotypic variation has been noted in the context of antibiotic exposure, no direct evidence exists for heterogeneity in virulence-specific transcriptional programs within tissues. In a mouse model of Yersinia pseudotuberculosis infection, we show that at least three subpopulations of bacteria develop within a single tissue site in response to distinct host signals. Bacteria growing on the exterior of spleen microcolonies responded to soluble signals and induced the nitric oxide (NO)-detoxifying gene, hmp. Hmp effectively eliminated NO diffusion and protected the interior bacterial population from exposure to NO-derived inducing signals. A third subpopulation, constituting the most peripherally localized bacteria, directly contacted neutrophils and transcriptionally upregulated a virulence factor. These studies demonstrate that growth within tissues results in transcriptional specialization within a single focus of microbial replication, facilitating directed pathogen counterattack against the host response.

During tuberculosis (TB), some Mycobacterium tuberculosis bacilli persist in the presence of an active immunity and antibiotics that are used to treat the disease. Herein, by using the Cornell model of TB persistence, we further explored our recent finding that suggested that M. tuberculosis can escape therapy by residing in the bone marrow (BM) mesenchymal stem cells. We initially showed that M. tuberculosis rapidly disseminates to the mouse BM after aerosol exposure and maintained a stable burden for at least 220 days. In contrast, in the lungs, the M. tuberculosis burden peaked at 28 days and subsequently declined approximately 10-fold. More important, treatment of the mice with the antibiotics rifampicin and isoniazid, as expected, resulted in effective clearance of M. tuberculosis from the lungs and spleen. In contrast, M. tuberculosis persisted, albeit at low numbers, in the BM of antibiotic-treated mice. Moreover, most viable M. tuberculosis was recovered from the bone marrow CD271(+)CD45(-)-enriched cell fraction, and only few viable bacteria could be isolated from the CD271(-)CD45(+) cell fraction. These results clearly show that BM mesenchymal stem cells provide an antibiotic-protective niche for M. tuberculosis and suggest that unraveling the mechanisms underlying this phenomenon will enhance our understanding of M. tuberculosis persistence in treated TB patients.