Application of culture, PCR, and PacBio sequencing for determination of microbial composition of milk from subclinical mastitis dairy cows of smallholder farms.

Mastitis is a cow disease usually signalized by irritation, swelling, and soreness of the udder. It is characterized by physical, chemical, and biological changes in the udder and milk. The aim of this study was to detect and characterize pathogens causing subclinical mastitis (SCM) from the milk of dairy cows of small-scale farmers through culture and molecular techniques. Milk was collected from 32 cows belonging to 8 small-scale farmers around Harrismith District, South Africa. The results showed that screening of SCM by California mastitis test and somatic cell counts (SCC) was 21.87 and 25%, respectively. Culture methods revealed the presence of Staphylococcus aureus at 93% followed by Streptococci spp. and Escherichia coli at 36.4 and 13.3%, respectively. The PCR could only detect E. coli, while single-molecule real-time sequencing showed a total of 2 phyla, 5 families, 7 genera, and 131 species. Clostridiaceae was the most abundant family, while Romboutsia was the most abundant genus followed by Turicibacter spp. The present study has documented the occurrence of SCM causing pathogens in milk collected from cows of small-scale farmers in Harrismith, indicating that SCM may be present at higher levels than expected.

In Vitro Antifungal Resistance of Candida auris Isolates from Bloodstream Infections, South Africa.

Candida auris is a multidrug-resistant fungal pathogen that is endemic in South African hospitals. We tested bloodstream C. auris isolates that were submitted to a reference laboratory for national laboratory-based surveillance for candidemia in 2016 and 2017. We confirmed the species identification by phenotypic/molecular methods. We tested susceptibility to amphotericin B, anidulafungin, caspofungin, micafungin, itraconazole, posaconazole, voriconazole, fluconazole, and flucytosine using broth microdilution and Etest methods. We interpreted MICs using tentative breakpoints. We sequenced the genomes of a subset of isolates and compared them to the C. auris B8441 reference strain. Of 400 C. auris isolates, 361 (90%) were resistant to at least one antifungal agent, 339 (94%) to fluconazole alone (MICs of ≥32 µg/ml), 19 (6%) to fluconazole and amphotericin B (MICs of ≥2 µg/ml), and 1 (0.3%) to amphotericin B alone. Two (0.5%) isolates from a single patient were pan-resistant (resistant to fluconazole, amphotericin B, and echinocandins). Of 92 isolates selected for whole-genome sequencing, 77 clustered in clade III, including the pan-resistant isolates, 13 in clade I, and 2 in clade IV. Eighty-four of the isolates (91%) were resistant to at least one antifungal agent; both resistant and susceptible isolates had mutations. The common substitutions identified across the different clades were VF125AL, Y132F, K177R, N335S, and E343D in ERG11; N647T in MRR1; A651P, A657V, and S195G in TAC1b; S639P in FKS1HP1; and S58T in ERG3. Most South African C. auris isolates were resistant to azoles, although resistance to polyenes and echinocandins was less common. We observed mutations in resistance genes even in phenotypically susceptible isolates.

Whole-Genome Sequences of Listeria monocytogenes Sequence Type 6 Isolates Associated with a Large Foodborne Outbreak in South Africa, 2017 to 2018.

We report whole-genome sequences for 10 Listeria monocytogenes sequence type 6 isolates associated with a large listeriosis outbreak in South Africa, which occurred over the period of 2017 to 2018. The possibility of listeriosis spreading beyond South Africa's borders as a result of exported contaminated food products prompted us to make the genome sequences publicly available.

Identification and characterization of Lactobacillus florum strains isolated from South African grape and wine samples.

A total of 213 strains of lactic acid bacteria were examined in this study. Among these, 30 strains previously isolated from South African grape and wine samples remained unidentified. The identification of these isolates was performed by BLAST and phylogenetic analyses of 16S rDNA gene sequences, which indicated that the isolates belonged to Lactobacillus florum. In this work, we also designed a discriminative species-specific primer FLOR targeting the 16S rDNA gene of Lb. florum. The validity and specificity of this primer was confirmed. Of particular interest in this study was to further evaluate the identified strains for the presence of genes encoding enzymes of oenological relevance. Reference strains included three flower-associated Lb. florum (F9-1(T), F9-2 and F17) and two Lactobacillus lindneri (AWRI B530 and DSM 20691) strains. Lb. lindneri strains were incorporated as being the closest relatives of Lb. florum. PCR detection results revealed that all Lb. florum strains and Lb. lindneri AWRI B530 (grape isolate) possessed the majority of the tested genes relative to DSM 20691 (beer isolate); these enzyme-encoding genes included malolactic enzyme, peptidases (PepC, PepI, PepN), citrate lyase (α- and ß-subunits), phenolic acid decarboxylase and arginine deiminase pathway enzymes (arginine deiminase and ornithine transcarbamylase). Sequence verification of PCR-generated fragments was performed by sequencing. The sequence data were used to construct the phylogenetic trees, which indicated that our Lb. florum isolates cluster with other Lb. florum strains of flower origin but rather distinct from other LAB species, with Lb. lindneri being the next closest species.