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Enhanced Survival of Rifampin- and Streptomycin-Resistant Escherichia coli Inside Macrophages.

ABSTRACT: The evolution of multiple-antibiotic-resistant bacteria is an increasing global problem. Even though mutations causing resistance usually incur a fitness cost in the absence of antibiotics, the magnitude of such costs varies across environments and genomic backgrounds. We studied how the combination of mutations that confer resistance to rifampin (Rif(r)) and streptomycin (Str(r)) affects the fitness of Escherichia coli when it interacts with cells from the immune system, i.e., macrophages (M?s). We found that 13 Rif(r) Str(r) doubly resistant genotypes, of the 16 tested, show a survival advantage inside M?s, indicating that double resistance can be highly beneficial in this environment. Our results suggest that there are multiple paths to acquire multiple-drug resistance in this context, i.e., if a clone carrying Rif(r) allele H526 or S531 acquires a second mutation conferring Str(r), the resulting double mutant has a high probability of showing increased survival inside M?s. On the other hand, we found two cases of sign epistasis between mutations, leading to a significant decrease in bacterial survival. Remarkably, infection of M?s with one of these combinations, K88R+H526Y, resulted in an altered pattern of gene expression in the infected M?s. This indicates that the fitness effects of resistance may depend on the pattern of gene expression of infected host cells. Notwithstanding the benefits of resistance found inside M?s, the Rif(r) Str(r) mutants have massive fitness costs when the bacteria divide outside M?s, indicating that the maintenance of double resistance may depend on the time spent within and outside phagocytic cells.

SUBMITTER: Durao P

PROVIDER: S-EPMC4914683 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Enhanced Survival of Rifampin- and Streptomycin-Resistant Escherichia coli Inside Macrophages.

Durão Paulo P Gülereşi Daniela D Proença João J Gordo Isabel I

Antimicrobial agents and chemotherapy 20160620 7

The evolution of multiple-antibiotic-resistant bacteria is an increasing global problem. Even though mutations causing resistance usually incur a fitness cost in the absence of antibiotics, the magnitude of such costs varies across environments and genomic backgrounds. We studied how the combination of mutations that confer resistance to rifampin (Rif(r)) and streptomycin (Str(r)) affects the fitness of Escherichia coli when it interacts with cells from the immune system, i.e., macrophages (Mϕs) ...[more]

PMID: 27161646

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Project description:Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats.

Project description:UnlabelledTreatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as "colonization resistance." However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment.ImportanceOur intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers "colonization resistance" through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

Project description:The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the P hlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.

Dataset Information

Enhanced Survival of Rifampin- and Streptomycin-Resistant Escherichia coli Inside Macrophages.

Publications

Enhanced Survival of Rifampin- and Streptomycin-Resistant Escherichia coli Inside Macrophages.

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