Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, https://doi.org/10.1128/AEM. . Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY. The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.KEYWORDS acid, Escherichia coli, experimental evolution, GABA, low pH, RNA polymerase, decarboxylase, fnr E nteric bacteria need to survive a wide range of pH values throughout the human intestinal tract. The pH of the intestinal tract ranges from 1.5 to 3.5 in the stomach, increases to 4 to 7 in the duodenum, reaches 7 to 9 in the jejunum, and decreases to pH 5 to 6 in the cecum (1). On a microscopic scale, extreme gradients of pH occur across the intestinal epithelium where enterobacteria adhere. To cope with such conditions, Escherichia coli maintains a cytoplasmic pH homeostasis...

Blastocystis is a gastrointestinal protist frequently reported in humans and animals worldwide. Wildlife populations, including deer, may serve as reservoirs of parasitic diseases for both humans and domestic animals, either through direct contact or through contamination of food or water resources. However, no studies of the occurrence and subtype distribution of Blastocystis in wildlife populations have been conducted in the United States. PCR and next generation amplicon sequencing were used to determine the occurrence and subtypes of Blastocystis in white-tailed deer (Odocoileus virginianus). Blastocystis was common, with 88.8% (71/80) of samples found to be positive. Twelve subtypes were identified, ten previously reported (ST1, ST3, ST4, ST10, ST14, ST21, and ST23–ST26) and two novel subtypes (ST30 and ST31). To confirm the validity of ST30 and ST31, MinION sequencing was used to obtain full-length SSU rRNA gene sequences, and phylogenetic and pairwise distance analyses were performed. ST10, ST14, and ST24 were the most commonly observed subtypes. Potentially zoonotic subtypes ST1, ST3, or ST4 were present in 8.5% of Blastocystis-positives. Mixed subtype infections were common (90.1% of Blastocystis-positives). This study is the first to subtype Blastocystis in white-tailed deer. White-tailed deer were found to be commonly infected/colonized with a wide diversity of subtypes, including two novel subtypes, zoonotic subtypes, and subtypes frequently reported in domestic animals. More studies in wildlife are needed to better understand their role in the transmission of Blastocystis.

Benzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution of Escherichia coli K-12. Transcriptomes of E. coli isolates evolved with benzoate showed the reversal of benzoate-dependent regulation, including the downregulation of multidrug efflux pump genes, the gene for the Gad acid resistance regulon, the nitrate reductase genes narHJ, and the gene for the acid-consuming hydrogenase Hyd-3. However, the benzoate-evolved strains had increased expression of OmpF and other large-hole porins that admit fermentable substrates and antibiotics. Candidate genes identified from benzoate-evolved strains were tested for their roles in benzoate tolerance and in chloramphenicol sensitivity. Benzoate or salicylate tolerance was increased by deletion of the Gad activator ariR or of the acid fitness island from slp to the end of the gadX gene encoding Gad regulators and the multidrug pump genes mdtEF. Benzoate tolerance was also increased by deletion of multidrug component gene emrA, RpoS posttranscriptional regulator gene cspC, adenosine deaminase gene add, hydrogenase gene hyc (Hyd-3), and the RNA chaperone/DNA-binding regulator gene hfq. Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit gene rpoA, the Mar activator gene rob, and hfq. Deletion of lipopolysaccharide biosynthetic kinase gene rfaY decreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such as aroF (encoding tyrosine biosynthesis) and apt (encoding adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for the loss of multidrug efflux pumps and of hydrogenases that generate a futile cycle of PMF and upregulates porins that admit fermentable nutrients and antibiotics. IMPORTANCE Benzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentrations, benzoic acid conducts a proton across the membrane, depleting the proton motive force. In the absence of antibiotics, benzoate exposure selects against proton-driven multidrug efflux pumps and upregulates porins that admit fermentable substrates but that also allow the entry of antibiotics. Thus, evolution with benzoate and related molecules, such as salicylates, requires a trade-off for antibiotic sensitivity, a trade-off that could help define a stable gut microbiome. Benzoate and salicylate are naturally occurring plant signal molecules that may modulate the microbiomes of plants and animal digestive tracts so as to favor fermenters and exclude drug-resistant pathogens.

Blastocystis is a common intestinal protist in humans and animals worldwide. Wild and domestic animals are thought to be reservoirs of Blastocystis subtypes that also infect humans. There are limited studies on the prevalence and subtype distribution of Blastocystis in horses. In this study, 185 fecal samples were collected from horses (1 month to 17 years of age) in four regions of Colombia (Sabana de Bogotá, Costa Atlántica, Llanos Orientales, and Bogotá D.C.). Blastocystis presence and subtypes were determined by PCR and next generation amplicon sequencing. Eighty-one (43.8%) horses were positive for Blastocystis, with positive horses in all four regions. Molecular characterization identified 12 Blastocystis subtypes, 10 known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26), and 2 novel subtypes (ST33 and ST34). The validity of the novel subtypes was confirmed via phylogenetic and pairwise distance analyses of the full-length SSU rRNA gene sequences. Mixed subtype infections were common (55.6% of Blastocystis-positive horses). ST10 was the most prevalent subtype, present in 82.8% of Blastocystis-positive horses. Potentially zoonotic subtypes were identified in 88.9% of the Blastocystis-positive horses. This constitutes the most comprehensive study of Blastocystis in horses. Our findings indicate that horses harbor potentially zoonotic subtypes and could contribute to the transmission of Blastocystis to humans.