Pre-existing heterogeneity facilitates development of heteroresistance upon gene acquisition.

Antibiotic resistance causes 1.27 million global deaths annually and is predicted to worsen. Heteroresistance is a form of resistance in which only a minor and unstable subpopulation of cells of a bacterial isolate are resistant to a given antibiotic, and are therefore often undetected by clinical diagnostics. These infrequent and undetected resistant cells can be selected during antibiotic therapy, expand in number, and cause unexplained treatment failures. A major question is how heteroresistance evolves. Here, studying the antibiotic fosfomycin, we report that heteroresistance can develop from a pre-existing state of phenotypic heterogeneity in which an isolate harbors a subpopulation with increased minimum inhibitory concentration (MIC), but below the clinical resistance breakpoint. We call this phenomenon heterosusceptibility and demonstrate that acquisition of a resistance gene, fosA, increases the MIC of the subpopulation beyond the breakpoint, making the isolate heteroresistant. Conversely, deletion of fosA from a heteroresistant isolate led to reduction of the MIC of the resistant subpopulation without a loss of heterogeneity, thus generating heterosusceptibility. A survey of 103 carbapenem-resistant Enterobacterales (CRE) revealed that the Escherichia sp. isolates lacked the fosA gene and uniformly exhibited fosfomycin heterosusceptibility, whereas the Klebsiella and Enterobacter encoded the fosA gene and were almost exclusively heteroresistant. Furthermore, some isolates exhibited heterosusceptibility to other antibiotics, demonstrating that this is a widespread phenomenon. These results highlight a mechanism for the evolution of heteroresistance and suggest that surveillance for heterosusceptibility may facilitate the prediction of impending heteroresistance before it evolves.

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States.

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

Antibiotic-Selected Gene Amplification Heightens Metal Resistance.

The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment.IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.

Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.

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 cancer1. Because 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 combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal 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 antibiotics3-6 in which only a subset of cells are phenotypically resistant7. 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 were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.

Thiol Starvation Induces Redox-Mediated Dysregulation of Escherichia coli Biofilm Components.

A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

The Biology of the Escherichia coli Extracellular Matrix.

Escherichia coli is one of the world's best-characterized organisms, because it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere: in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix (ECM), primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces and resistance to desiccation, the host immune system, and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this article, which concludes with insights into the future of E. coli biofilm research and potential therapies.

The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

The bacterial extracellular matrix encases cells and protects them from host-related and environmental insults. The Escherichia coli master biofilm regulator CsgD is required for the production of the matrix components curli and cellulose. CsgD activates the diguanylate cyclase AdrA, which in turn stimulates cellulose production through cyclic di-GMP (c-di-GMP). Here, we identified and characterized a CsgD- and AdrA-independent cellulose production pathway that was maximally active when cultures were grown under reducing conditions or when the disulfide bonding system (DSB) was compromised. The CsgD-independent cellulose activation pathway was dependent on a second diguanylate cyclase, called YfiN. c-di-GMP production by YfiN was repressed by the periplasmic protein YfiR, and deletion of yfiR promoted CsgD-independent cellulose production. Conversely, when YfiR was overexpressed, cellulose production was decreased. Finally, we found that YfiR was oxidized by DsbA and that intraprotein YfiR disulfide bonds stabilized YfiR in the periplasm. Altogether, we showed that reducing conditions and mutations in the DSB system caused hyperactivation of YfiN and subsequent CsgD-independent cellulose production.

Modulation of curli assembly and pellicle biofilm formation by chemical and protein chaperones.

Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by "chemical chaperones."

Iron induces bimodal population development by Escherichia coli.

Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air-biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H(2)O(2) toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

Experimental manipulation of the microbial functional amyloid called curli.

Curli are proteinaceous fibrous structures produced on the surface of many gram-negative bacteria. As a major constituent of the extracellular matrix, curli mediate interactions between the bacteria and its environment, and as such, curli play a critical role in biofilm formation. Curli fibers share biophysical properties with a growing number of remarkably stable and ordered protein aggregates called amyloid. Here we describe experimental methods to study the biogenesis and assembly of curli by exploiting their amyloid properties. We also present methods to analyze curli-mediated biofilm formation. These approaches are straightforward and can easily be adapted to study other bacterially produced amyloids.