Division of Blastocystis ST10 into three new subtypes: ST42-ST44.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full-length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long-read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full-length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote.

Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk.

Blastocystis is a common intestinal protist with a global distribution in humans and many other animals. Yet, the status of Blastocystis as a pathogen, the risk factors associated with its transmission, and its zoonotic potential remain ill-defined. Here, we explored subtype (ST) diversity and potential risk factors for Blastocystis infection in 98 children from Apulo, Colombia. Samples were screened for Blastocystis via PCR, and ST identification was performed through next-generation amplicon sequencing (NGS). Associations between the presence of Blastocystis and individual STs and sociodemographic variables were assessed via logistic regression analyses. Seventy-one samples (72.4%) were Blastocystis-positive, and NGS revealed the presence of five STs (ST1-ST5). ST1, ST2, and ST3 were common and observed in nearly equal proportions (~ 40%), while samples with ST4 (1.4%) and ST5 (5.6%) were comparatively rare. The presence of mixed STs in the same sample was also common (28.2%). Comparisons among children within the same household identified that shared ST profiles were common, but diversity within family units was also observed. Logistic regression analyses returned significant associations between the presence of Blastocystis, individual subtypes, or mixed subtypes for several variables. Intriguingly, the presence of animals was one of the most common significant associations. Taken together, these data represent an important step forward in understanding both the potential routes and risk factors that may influence Blastocystis transmission and will be useful in shaping future studies which seek to clarify the relationships between STs, pathogenicity, and zoonotic transmission.

Diversity of Blastocystis Subtypes in Horses in Colombia and Identification of Two New Subtypes.

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.

Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing.

Giardia duodenalis is one of the most common parasitic causes of gastrointestinal illness in humans worldwide with widespread infections in mammalian hosts. It frequently infects cattle, producing a high number of cysts. Cattle can harbor both host-adapted assemblage E and human pathogenic assemblages A and B. Previous studies have demonstrated that conventional molecular methods lack the sensitivity required for detecting mixed infections and that the occurrence of mixed infections in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2539 pre-weaned dairy calves from the United Stated were screened for the presence of G. duodenalis using molecular tools. Next generation amplicon sequencing (NGS) was then performed for a subset of around 30% of positive samples (n = 314) and the ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared. The overall prevalence of G. duodenalis in pre-weaned dairy calves in the sample using PCR was high (1013/2539; 39.9%). Molecular genotyping identified only assemblage A and E, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both Sanger and NGS, 9 samples (2.9%) were identified as mixed A/E infections by Sanger while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared with Sanger sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than was hypothesized using values from the literature. This underestimation could be present in the wider cattle population as well, though further exploration would be needed to verify that claim. These findings highlight the advantages of NGS application in molecular epidemiological studies of Giardia. To better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages it is necessary to uncover mixed assemblage infections.

Diarrhoea-causing enteric protist species in intensively and extensively raised pigs (Sus scrofa domesticus) in Southern Spain. Part I: Prevalence and genetic diversity.

Numerous protist species are shared between humans and pigs. Among those, Giardia duodenalis, Cryptosporidium spp. and Balantioides coli have a clear public and animal health significance. For others such as Enterocytozoon bieneusi and Blastocystis sp., their impact on animal health has not been fully established. Little information is currently available on the molecular diversity of these protists in swine populations. To fill this gap, we molecularly assessed G. duodenalis, Cryptosporidium spp., B. coli, Blastocystis sp. and E. bieneusi in faecal samples from Iberian and Large White pigs raised under different (intensive and/or extensive) management systems in southern Spain. A total of 151 extensively raised Iberian pigs, 140 intensively raised Iberian pigs, and 184 intensively raised Large White pigs were investigated. Blastocystis sp. was the agent most prevalently found (47.8%), followed by B. coli (45.5%), G. duodenalis (10.7%), E. bieneusi (6.9%), and Cryptosporidium spp. (5.5%). Blastocystis sp. was significantly less prevalent in intensively raised Iberian pigs (22.9%) than in their extensively raised counterparts (51.0%) or in intensively raised Large White pigs (64.1%). A significantly higher prevalence was found for G. duodenalis, Cryptosporidium spp., and E. bieneusi in Large White pigs than Iberian pigs. Balantioides coli was similarly distributed (40.0-51.1%) in all three investigated swine populations. Sequence analyses revealed the presence of G. duodenalis assemblage E, two Cryptosporidium species (Cryptosporidium scrofarum and Cryptosporidium suis), B. coli (genotypes A and B), Blastocystis sp. (ST1, ST3, and ST5), and E. bieneusi (EbpA, EbpC, EbpD, O, and a novel genotype named PigSpEb2). Novel genotype PigSpEb2 was found alone or in combination with EbpA. Data suggest a widespread exposure to protist enteroparasites in domestic pig populations irrespectively of breed and raising management system. Many of the species/genotypes identified have a zoonotic potential and might represent a public health concern.

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2-37.4%) and 0.6% (2/336, 95% CI: 0.0-1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2-8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.

Wide Genetic Diversity of Blastocystis in White-Tailed Deer (Odocoileus virginianus) from Maryland, USA.

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.

An Illumina MiSeq-Based Amplicon Sequencing Method for the Detection of Mixed Parasite Infections Using the Blastocystis SSU rRNA Gene as an Example.

Parasite mixed infections remain a relatively unexplored field in part due to the difficulties of unraveling complex mixtures of parasite DNA using classical methods of sequencing. Next-generation amplicon sequencing (NGS) is a powerful tool for exploring mixed infections of multiple genetic variants of the same parasite in clinical, environmental (water or soil), or food samples. Here, we provide a method for NGS-based detection of mixed parasite infections which uses the Blastocystis SSU rRNA gene as an example and includes steps for parasite concentration, DNA extraction, sequencing library preparation, and bioinformatic analysis.

Pomegranate peel extract reduced colonic damage and bacterial translocation in a mouse model of infectious colitis induced by Citrobacter rodentium.

The pomegranate fruit peel is a rich source of polyphenols including punicalins, punicalagins, and ellagic acids, but is considered an agricultural waste product. Pomegranate derived products have been reported to have a wide variety of health promoting benefits including antibacterial properties in vitro but there is limited evidence of their antibacterial properties in vivo. The purpose of this study was to test the in vivo antibacterial properties of a pomegranate peel extract (PPX) containing punicalin, punicalagin, and ellagic acid. C3H/He mice were orally pre-treated with water or PPX prior to infection with the mouse bacterial pathogen, Citrobacter rodentium (Cr) that mimics many aspects of human enteropathogenic Escherichia coli infections. Fecal excretion of Cr was monitored and mice were euthanized on day 12 post-infection to assess Cr colonization of the colon and spleen, histological changes, and gene expression. PPX-treatment reduced Cr infection induced weight loss and mortality that was observed in water-treated infected mice. However, Cr colonization of the colon and clearance was unaffected by PPX-treatment. Consistent with this, PPX treatment did not alter the potent Th1/Th17 pro-inflammatory response elicited by Cr infection. Significant colonization of the spleen was only seen in water-treated infected mice and was inversely correlated with the dose of PPX administered. PPX treatment decreased the extent of Cr-induced colon damage that correlated with decreased mortality and reduced colonization of the spleen. Thus, a pomegranate peel extract contains bioactive compounds that mitigate the deleterious effects of an in vivo infection with the model enteropathogenic bacteria, Cr.

Pomegranate peel extract alters the microbiome in mice and dysbiosis caused by Citrobacter rodentium infection.

Treatment of mice with a pomegranate peel extract (PPX) decreased the pathogenicity of Citrobacter rodentium (Cr) infections. Here, we investigate the effects of PPX on the microbiome of uninfected or Cr-infected C3H/HeNCr mice by 16S rRNA gene sequencing. Mice were treated with water or PPX for 14 days, feces were collected, and then, the mice were infected with Cr and feces collected again at day 6 postinfection. DNA was isolated from the fecal samples and subjected to 16S rRNA gene sequencing to determine the microbial composition. Differences in the composition of the microbiome were observed for untreated and PPX-treated mice with PPX mice having decreased diversity. PPX treatment decreased the Firmicutes/Bacteroidetes ratio by increasing Bacteroidetes and decreasing Firmicutes levels. The decrease in Firmicutes was driven by a large reduction in Lactobacillus. PPX treatment increased the abundance of Proteobacteria and Verrucomicrobiae and decreased Actinobacteria. The relative abundance of Cr reached 22% in water-treated but only 5% in PPX-treated infected mice. These results suggest that consumption of pomegranate polyphenols altered the microbiome, making it more resistant to displacement by infection with Cr, indicating that pomegranate polyphenols may mitigate the pathogenic effects of food-borne bacterial pathogens.

Inverted Regulation of Multidrug Efflux Pumps, Acid Resistance, and Porins in Benzoate-Evolved Escherichia coli K-12.

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.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

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, 2015, https://doi.org/10.1128/AEM.03494-14). 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 H2 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.