Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 µg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 µg/mL. This level is comparatively lower than 9.2 µg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

Susceptible trichostrongyloid species mask presence of benzimidazole-resistant Haemonchus contortus in cattle.

BACKGROUND: Benzimidazole (BZ) anthelmintics are widely used to control infections with parasitic nematodes, but BZ resistance is an emerging threat among several nematode species infecting humans and animals. In Sudan, BZ-resistant Haemonchus contortus populations were recently reported in goats in South Darfur State. The objective of this study was to collect data regarding the situation of BZ resistance in cattle parasitic nematodes in South Darfur using phenotypic and molecular approaches, besides providing some epidemiological data on nematodes in cattle. METHODS: The faecal egg count reduction test and the egg hatch test (EHT) were used to evaluate benzimidazole efficacy in cattle nematodes in five South Darfur study areas: Beleil, Kass, Nyala, Rehed Al-Birdi and Tulus. Genomic DNA was extracted from pools of third-stage larvae (L3) (n = 40) during trials, before and after treatment, and pools of adult male Haemonchus spp. (n = 18) from abattoirs. The polymorphisms F167Y, E198A and F200Y in isotype 1 ß-tubulin genes of H. contortus and H. placei were analysed using Sanger and pyrosequencing. RESULTS: Prevalence of gastro-intestinal helminths in cattle was 71% (313/443). Reduced albendazole faecal egg count reduction efficacy was detected in three study areas: Nyala (93.7%), Rehed Al-Birdi (89.7%) and Tulus (88.2%). In the EHT, EC50 values of these study areas ranged between 0.032 and 0.037 µg/ml thiabendazole. Genus-specific PCRs detected the genera Haemonchus, Trichostrongylus and Cooperia in L3 samples collected after albendazole treatment. Sanger sequencing followed by pyrosequencing assays did not detect elevated frequencies of known BZ resistance-associated alleles in codon F167Y, E198A and F200Y in isotype 1 ß-tubulin gene of H. placei (≤ 11.38%). However, polymorphisms were detected in H. contortus and in samples with mixed infections with H. contortus and H. placei at codon 198, including E198L (16/58), E198V (2/58) and potentially E198Stop (1/58). All pooled L3 samples post-albendazole treatment (n = 13) were identified as H. contortus with an E198L substitution at codon 198. CONCLUSIONS: To the knowledge of the authors, this is the first report of reduced albendazole efficacy in cattle in Sudan and is the first study describing an E198L substitution in phenotypically BZ-resistant nematodes collected from cattle.

New codon 198 ß-tubulin polymorphisms in highly benzimidazole resistant Haemonchus contortus from goats in three different states in Sudan.

BACKGROUND: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. METHODS: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 ß-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. RESULTS: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. CONCLUSIONS: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

Epidemiology of strongyle nematode infections and first report of benzimidazole resistance in Haemonchus contortus in goats in South Darfur State, Sudan.

BACKGROUND: Since pastoralists in South Darfur, Sudan, had complained about lack of albendazole (ABZ) efficacy to control nematodes in goats, the frequency of infection with gastrointestinal helminths was studied before in vivo faecal egg count reduction tests (FECRT) were conducted using ABZ orally either at the dose recommended for sheep, 5 mg/kg body weight (bw) or at 10 mg/kg bw. Experiments included goats naturally infected with gastrointestinal nematodes or experimentally infected with local Haemonchus contortus isolates. Three study areas (Nyala, Beleil and Kass) were visited in autumn or winter. RESULTS: Out of 478 screened goats, 82.4% were infected with gastrointestinal helminths and 82% were shedding eggs of strongyle nematodes with 90% of the strongyle larvae representing Haemonchus spp. A FECRT using naturally infected goats (n = 225: 71 untreated, 104 and 50 treated with 5 and 10 mg ABZ/kg bw, respectively) detected reduced ABZ efficacy in Nyala and Kass. Paired and unpaired FECRT calculations detected reductions of 72-92% with samples taken at 8 days post treatment with 5 mg ABZ/kg bw and of 85-94% with 10 mg ABZ/kg bw. The FECRT based on day 14 post treatment samples showed reductions of 69-77% with 5 mg/kg and of 75-87% with 10 mg ABZ/kg bw. In Beleil, ABZ efficacy was 95%. In the egg hatch test EC50 values for Nyala and Kass ranged from 0.12-0.24 µg thiabendazole/ml, corresponding to benzimidazole resistant phenotypes. Only Haemonchus spp. larvae were present after treatments in coprocultures. When the efficacy was evaluated experimentally using isolates of H. contortus from Nyala and Kass, the 5 mg ABZ/kg dose revealed reductions of 76-78% on day 8 and of 62-70% on day 14 with the unpaired method. Using 10 mg ABZ/kg, the FECR was still only 77-82%. CONCLUSIONS: Both, in vivo and in vitro methods detected resistant H. contortus populations in goats from South Darfur State. The time point 14 days post treatment was more sensitive for detection of ABZ resistance than 8 days post treatment. This is the first report on the occurrence of anthelmintic resistance in Sudan confirming that anthelmintic resistance selection is occurring in African subsistence farming systems.