Environmental contamination with clostridioides (Clostridium) difficile in Vietnam

AIMS: To investigate the prevalence, molecular type, and antimicrobial susceptibility of Clostridioides difficile in the environment in Vietnam, where little is known about C. difficile. METHODS AND RESULTS: Samples of pig faeces, soils from pig farms, potatoes, and the hospital environment were cultured for C. difficile. Isolates were identified and typed by polymerase chain reaction (PCR) ribotyping. The overall prevalence of C. difficile contamination was 24.5% (68/278). Clostridioides difficile was detected mainly in soils from pig farms and hospital soils, with 70%-100% prevalence. Clostridioides difficile was isolated from 3.4% of pig faecal samples and 5% of potato surfaces. The four most prevalent ribotypes (RTs) were RTs 001, 009, 038, and QX574. All isolates were susceptible to metronidazole, fidaxomicin, vancomycin, and amoxicillin/clavulanate, while resistance to erythromycin, tetracycline, and moxifloxacin was common in toxigenic strains. Clostridioides difficile RTs 001A+B+CDT- and 038A-B-CDT- were predominantly multidrug resistant. CONCLUSIONS: Environmental sources of C. difficile are important to consider in the epidemiology of C. difficile infection in Vietnam, however, contaminated soils are likely to be the most important source of C. difficile. This poses additional challenges to controlling infections in healthcare settings

HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice

Body weight and urine volume of WT and Vpr Tg mice.

(A) Four days after the low salt diet the mice were weighed. n = 7–10. n.s., not significant, Student’s t test. (B) Mouse were placed in metabolic cages and overnight urine was collected before and after the mice were placed on low salt diet. n = 9. n.s., not significant, ANOVA, Bonferroni correction. (TIF)</p

Primers for qRT-PCR in mouse kidneys and hDCT cells.

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Primary antibodies used in this study.

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Expression of NCC, NaKa, and MR in kidney homogenates from wild-type (WT) and Vpr transgenic (Tg) mice.

(A) Protein expression of total NCC was markedly suppressed in kidneys from Tg mice compared with WT littermates. n = 6. **P t test. (B) Protein expressions of NaKa and MR were similar in WT and Tg mice. NaKa, Na+/K+-ATPase. n = 3. n.s., not significant, Student’s t test. (C) Immunohistochemical analysis of NCC distribution in kidney cortex sections demonstrated that NCC is located in the apical cellular regions of the renal distal convoluted tubule, with similar distribution in both WT and Tg mice. NCC immunostained tubules were scanned and NCC protein expression was found to be lower in Tg than WT mice both before and after salt depletion. n = 3–4. n.s., not significant; **P P < 0.001, ANOVA, Bonferroni correction.</p

Suppression of MR transcriptional activity by Vpr in human DCT cells.

(A) Human DCT cells were treated with or without soluble Vpr (sVpr, 100ng/ml) for 24-hr followed by aldosterone treatment for the time indicated. Protein expression of NCC and MR were determined by immunoblotting. (B) The density of NCC was quantified by densitometry. The aldosterone-enhanced NCC protein expression, with a peak at 3-hr treatment, was abolished by sVpr (100ng/ml). n = 3. *P P C) Human DCT cells were treated with sVpr, aldosterone, or eplerenone (10μM), individually or in combination as indicated. Expression of SLC12A3 mRNA was determined by qRT-PCR and normalized to β-actin mRNA. The aldosterone-induced increase in SLC12A3 mRNA expression was attenuated by Vpr, but eplerenone had no further effect on SLC12A3 mRNA expression. n = 4–6. **P P (TIF)</p