The Emergence of Colistin- and Imipenem-Associated Multidrug Resistance in Escherichia coli Isolates from Retail Meat.

To determine the prevalence of Escherichia coli and their drug resistance profiles in fresh pork sold at two retail outlets (open-air market and closed retail stores) in Alice, South Africa. Retail meat samples (n = 176) collected from four shops (two from open-air markets and two from closed stores) were analyzed by conventional biochemical and PCR-based molecular confirmatory tests. The confirmed isolates were profiled for antimicrobial susceptibility to a panel of 12 commercial antibiotics: tetracycline, ampicillin, sulphamethoxazole trimethoprim, erythromycin, gentamycin, colistin sulphate, cefotaxime, chloramphenicol, norfloxacin, ciprofloxacin, cefuroxime, and imipenem. Colistin, ampicillin, and erythromycin resistance genes were profiled with the gene-specific primers. Multidrug resistance (MDR) and the association of imipenem and colistin in the MDR profile were determined. A total of 68 (39.08%) E. coli isolates were confirmed by PCR analysis. Resistance was most common to erythromycin (100%), followed by cefotaxime (95.58%), ampicillin (88.23%), cefuroxime (88.23%), trimethoprim-sulphamethoxazole (88.23%), and tetracycline (60.29%). Overall, 27/68 (39.70%) were MDR (≥ 3antibiotics classes). MDR E. coli isolates associated with imipenem resistance (50.00%) and colistin resistance (33.82%) were detected. The resistance genes were detected among the isolates though not in all the phenotypically resistant isolates. The detection of colistin resistance among MDR E. coli isolates from retail meat is troubling as the drug is a last resort antibiotic. Overall, the epidemiological implications of the findings are of public health importance.

Emergence of Theileria species in ticks from free-ranging domestic animals in Raymond Mhlaba local municipality, South Africa.

Ticks infestation and diseases associated with it, are becoming a major life threatening concern to wildlife, domesticated animals and human health in general. Besides causing skin damage, ticks infestations have become a growing burden in food security and transmission of multiple pathogens. There is paucity of data on the occurrence of etiologic agents of tick-borne diseases in the Eastern Cape Province South Africa. We therefore carried out a molecular surveillance on Babesia and Theileria species in ticks obtained from livestock in Raymond Mhlaba District Municipality of the Province. A total of 962 ticks were collected and were morphologically identified and processed for DNA extraction using commercial DNA extraction kit. The extracted DNA samples were used to molecular identification of the ticks, and also to assess the occurrence of the Babesia and Theileria spp by PCR using genus specific primers. Positive amplicons obtained were sequenced, processed and characterised using appropriate bioinformatics tools. The molecular and morphological identifications of ticks obtained from the domestic animals in the study areas revealed that they belong to three different genera namely: Haemophalis, Rhipicephalus, and Amblyomma in ascending order of their abundance. Furthermore, the DNA of Theileria spp. was detected from 10 out of 962 ticks screened, with an overall infection of about 1% for Rhipicephalus spp., while none of the ticks was positive for Babesia spp. The phylogenetic analysis of the 10 theilerial sequences showed that nine (9) clustered distinctly within the T. orientalis complex clade, while only one (1) sequence formed a cluster with reference sequences of T. velifera. The findings from this study therefore expand the knowledge on recent emergence of Theileria spp. in livestock reared in the study area. This calls for an urgent effort in curbing the further spread of the pathogens in the area and beyond.

Genomic Profiling for Piroplasms in Feeding Ixodid Ticks in the Eastern Cape, South Africa.

Importation of tick-infected animals and the uncontrollable migration of birds and wild animals across borders can lead to geographical expansion and redistribution of ticks and pathogen vectors, thus leading to the emergence and re-emergence of tick-borne diseases in humans and animals. Comparatively, little is known about the occurrence of piroplasms in ixodid ticks in the Eastern Cape, South Africa, thus necessitating this study, which is aimed at detecting piroplasms (Theileria and Babesia) from feeding tick samples collected from cattle, sheep, and goats in selected sites in the Eastern Cape, South Africa. A total of 1200 feeding ixodid ticks collected from farm animals at selected homesteads were first subjected to molecular identification using mitochondrial 12S ribosomal RNA (rRNA) gene by PCR and were further tested for the presence of piroplasms through amplification of the 18S rRNA gene via nested-PCR followed by sequencing of the PCR products. The results indicated that 853 (71.1%) corresponded to the genus Rhipicephalus, 335 (27.9%) corresponded to genus Amblyomma, and 12 (1%) corresponded to genus Haemaphysalis. Amblyomma hebraeum and Rhipicephalus appendiculatus were the most common identified ticks from this study. The 18S rRNA nested-PCR revealed that 44 (3.7%) samples were confirmed positive for Theileria. A homology search for the generated sequences revealed a high percentage identity of 98-98.9% similarity to T. buffeli, T. orientalis, and T. sergenti in the GenBank. Based on the results obtained herein, we conclude that there is a big diversity of Theileria species; therefore, we suggest that this research should cover more geographical areas in order to reveal the true prevalence of this pathogen in the studied area because this will be a great step in the possible prevention of an outbreak that could have devastating effects on livestock production and human health in both the studied areas and South Africa at large.

Rickettsia africae an Agent of African Tick Bite Fever in Ticks Collected from Domestic Animals in Eastern Cape, South Africa.

Background: Ticks transmit a plethora of pathogens of zoonotic implications. Their distribution, diversity and the pathogens they transmit differ from one ecological location to another. Rickettsia africae is the agent of African tick bite fever found in South Africa, a zoonotic infection that is frequently reported among travelers who have visited many sub-Saharan African countries where the pathogen is prevalent. Methods: Ticks were collected from domestic animals in Raymond Nkandla Municipality, Eastern Cape, South Africa. The ticks were identified morphologically prior to DNA extraction followed by molecular identification of randomly selected ticks from the morphologically delineated groups. To assess for the presence of tick-borne pathogens belonging to Rickettsia spp. by PCR (polymerase chain reaction), we used specific primer pairs targeting the gltA, ompA and ompB genes. The selected amplified ticks, all positive ompB and forty three ompA amplicons were sequenced in a commercial sequencing facility. The obtained nucleotide sequences were edited and subjected to BLASTn for homology search and phylogenetic analyses were performed with MEGA 7 Version for genetic relationships with curated reference sequences in GenBank. Results: A total of 953 ticks collected in the study were delineated into three genera consisting of Amblyomma, Rhipicephalus and Hyalomma in decreasing order of abundance. The presence of rickettsial DNA was detected in 60/953 (6.3%) from the three genera of ticks screened. Genetic analyses of the DNA sequences obtained showed that they have phylogenetic relationship to members of the spotted fever group rickettsiae with R. africae, being the predominant SFGR (spotted fever group rickettsiae) detected in the screened ticks. Conclusion: This report shows that R. africae is the predominant spotted fever group rickettsiae in ticks collected from domestic animals in the study area and the human health impacts are not known.

Phylogenetic profiling for zoonotic Ehrlichia spp. from ixodid ticks in the Eastern Cape, South Africa.

Ticks are obligate hematophagous parasite of vertebrate that transmit a range of pathogenic microorganisms that can cause diseases in livestock and humans. The range of tick-borne disease causative agents infecting domestic animals and humans has recently increased. Several significant zoonotic tick-borne diseases such as ehrlichiosis among others are on the increase worldwide. This study was designed to investigate the occurrence of zoonotic Ehrlichia spp. from samples collected from livestock in selected communities in the Eastern Cape Province, South Africa. Tick samples were manually collected from domesticated animals in selected homesteads. The ticks were morphologically identified to species and tested for Ehrlichia infection via polymerase chain reaction (PCR), using genus-specific disulphide bond formation protein (dsbA) gene primers. This was followed by sequence analysis of amplicons and phylogeny. Of the 1,200 ticks collected, Amblyomma hebraeum was most prevalent (n = 335; 27.9%), followed by Rhipicephalus appendiculatus (n = 274; 22.8%), Rhipicephalus decoloratus; (n = 224; 18.7%) and Rhipicephalus eversti eversti (n = 200, 16.7%). Ehrlichia DNA was detected in 19/1,200 (1.6%) of the screened DNA samples. A homology search of the generated sequences revealed a high percentage of identity between 95% and 98% with other homologous dsbA gene sequences of other Ehrlichia species in GenBank. Phylogenetic analysis showed that the obtained sequences clustered unambiguously with other Ehrlichia sequences from different geographical regions of the world. We concluded that Ehrlichial pathogens are vectored by the ticks collected from domesticated animals in the study areas, thus suggesting concern for public health, as some of the recovered pathogens are zoonotic in nature and could pose serious public health risk through human exposure to tick bites.

Increasing diversity of swine parvoviruses and their epidemiology in African pigs.

Detection of infectious viral agents has been on the increase globally with the advent and usage of more sensitive and selective novel molecular techniques in the epidemiological study of viral diseases of economic importance to the swine industry. The observation is not different for the pig-infecting member of the subfamily Parvovirinae in the family Parvoviridae as the application of novel molecular methods like metagenomics has brought about the detection of many other novel members of the group. Surprisingly, the list keeps increasing day by day with some of them possessing zoonotic potentials. In the last one decade, not less than ten novel swine-infecting viruses have been added to the subfamily, and ceaseless efforts have been in top gear to determine the occurrence and prevalence of the old and new swine parvoviruses in herds of pig-producing countries worldwide. The story, however, is on the contrary on the African continent as there is presently a dearth of information on surveillance initiatives of the viruses among swine herds of pig-producing countries in the region. Timely detection and characterization of the viral pathogens is highly imperative for the implementation of effective control and prevention of its spread. This review therefore presents a concise overview on the epidemiology of novel porcine parvoviruses globally and also provides up-to-date highlights on the reported cases of the viral agents in the African sub-region.

Prevalence of porcine parvoviruses in some South African swine herds with background of porcine circovirus type 2 infection.

The classical porcine parvovirus is an important pathogen of reproductive disorders in pigs with a confirmed history of global distribution. The detection of many novel porcine parvoviruses has however been on the increase for the past few years, but there is a dearth of information on the occurrence and prevalence of these viruses in South Africa. Molecular detection of some known parvoviruses, namely porcine parvoviruses (PPVs) - 1, 2, 3 and 4, porcine bocavirus-like virus (PBo-likeV) and porcine bocaviruses (PBoV1/2), was carried out from 110 randomly selected archived swine samples collected in the year 2015 and 2016. Samples were drawn from previously screened and confirmed porcine circovirus type 2 (PCV2) infected farms, with farm-level occurrence ranged from 5.6 to 60%. The findings showed that all the screened parvoviruses were present as follows: PPV1 (29.1%), PPV2 (21.8%), PPV3 (5.5%), PPV4 (43.6%), PBo-likeV (21.8%) and PBoV1/2 (44.6%). The frequency of double infections of the viruses was as high as 18.2% of PPV2/PPV4 and PPV4/PBoVs; while 17.3% and 7.3% of the screened samples showed multiple infections of the three and four viruses respectively. Further phylogenetic analyses of partial PPV1, 2 and PBoV1/2 sequences showed two major clades for each of the viruses. This study reports the first epidemiological survey and molecular characterisation of the classical and emerging porcine parvoviruses in South African swine herds. It also gives insights into the diversity and distribution of these viral pathogens within the herds of the study area and confirms their co-infection potentials with PCV2.

Genetic characterization and diversity of porcine circovirus type 2 in non-vaccinated South African swine herds.

The porcine circovirus type 2 (PCV2) is a swine infectious viral pathogen of great significance in global swine herds. It was recently detected at another Province of South Africa sequel to the first detection of North American-like strain (PCV2a) at Gauteng about two decades ago, but there is a dearth of information about the genomic features and diversity of the viral strains in circulation within the country and the entire sub-Saharan Africa region. To date, only one complete genome of the virus from South Africa is available on global data base. This current effort is therefore geared towards the full-genome characterization of the circulating PCV2 strains in the pigs of Eastern Cape Province. With the use of conventional polymerase chain reaction method, fifteen complete PCV2 genomes were successfully amplified, sequenced and assembled from field samples obtained from non-vaccinated pigs in the region. Neighbor Joining and Maximum Likelihood phylogenetic analyses of the ORF2 gene and full genomes unanimously showed that most of the assembled genomes (11) belong to genotype PCV2b. Furthermore, three of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (i.e. PCV2d) strains from the USA, China and South Korea. The last sequence, however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2-IM2), recently identified in a global PCV2 strains phylogenetic analysis. This study reports the first complete genome sequences of PCV2b, PCV2d and PCV2-IM2 in pigs from South Africa, and it gives a possible insight into the genetic characteristics and variability of the viral strains presently in circulation within the country. It further emphasizes the need for more stringent measures in curtailing the introduction and spread of transboundary swine pathogens in the country and entire Southern African region.

Molecular Characterization and Antimicrobial Resistance Pattern of Escherichia coli Recovered from Wastewater Treatment Plants in Eastern Cape South Africa.

Wastewater treatment plants (WWTPs) are designed to eliminate organic matter and pathogens but most WWTPs discharges antimicrobial resistance pathogens into aquatic milieu. The study aimed to examine the antibiotics resistant patterns and the presence of some resistance genes among E. coli isolates from WWTPs effluents. Water were collected from WWTPs final effluents, filtered through nitrocellulose membrane and the filter papers were placed on chromogenic agar plates, incubated for 24 h at 37 °C. Presumptive E. coli isolates (173) were obtained from the culture method. From the presumptive E. coli isolates screened by polymerase chain reaction (PCR), 111 isolates were positive and the positive isolates were further screened for six diarrheagenic E. coli pathotypes (EPEC, ETEC, EHEC, DAEC, EIEC, and EAEC) and from the pathotypes screened, nine isolates harboured daaE gene. The phenotypic susceptibility patterns of the 111 isolates to 12 antibiotics were determined by Kirby-Bauer disk diffusion technique. All the isolates were resistant to erythromycin and clindamycin. From the resistance genes screened, 31 isolates harboured mcr-1 gene and nine isolates harboured ermA gene. The study reveals that water samples recovered from the final effluents of WWTPs may likely be one of the major sources of antibiotic-resistant in Escherichia coli.

Molecular detection of Porcine circovirus type 2 in swine herds of Eastern Cape Province South Africa.

BACKGROUND: Porcine circovirus type 2 (PCV2) remains the main causative viral pathogen of porcine circovirus-associated diseases (PCVAD) of great economic importance in pig industry globally. This present study aims at determining the occurrence of the viral pathogen in swine herds of the Province. RESULTS: The data obtained revealed that 15.93% of the screened samples (54/339) from the swine herds of the studied areas were positive for PCV2; while the severity of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6 to 60% in the studied farms. The majority (15 out of 17 = 88%) of the analyzed sequences were found clustering with other PCV2b strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genogroup, which is presently another fast-spreading genotype with observable higher virulence in global swine herds. CONCLUSION: This is the first report of PCV2 in swine herds of the Province and the first detection of PCV2b and PCV2d in South African swine herds. It follows the first reported case of PCV2a in an outbreak of porcine multisystemic wasting syndrome (PMWS) in Gauteng Province, South Africa more than one decade ago. This finding confirmed the presence of this all-important viral pathogen in pigs of the region; which could result in a serious outbreak of PCVAD and huge economic loss at the instances of triggering factors if no appropriate measures are taken to effectively curb its spread.

Multidrug-Resistant Salmonella Isolates from Swine in the Eastern Cape Province, South Africa.

The exposure of farm animals to antimicrobials for treatment, prophylaxis, or growth promotion can select for resistant bacteria that can be transmitted to humans, and Salmonella as an important zoonotic pathogen can act as a potential reservoir of antimicrobial resistance determinants. We assessed the antibiogram profiles of Salmonella species isolated from pig herds in two commercial farms in South Africa. Two hundred fifty-eight presumptive Salmonella isolates were recovered from the fecal samples of 500 adult pigs. Specific primers targeting Salmonella serogroups A, B, C1, C2, and D were used to determine the prevalence of different serogroups. Only serogroup A (n = 48) was detected, while others were not. Antimicrobial susceptibility of the confirmed Salmonella serogroup A isolates was performed by using the disk diffusion method against a panel of 18 antibiotics. All the 48 isolates were resistant to tetracycline and oxytetracycline, while 75% were resistant to ampicillin, sulphamethoxazole-trimethoprim, nalidixic acid, and streptomycin. All the isolates exhibited multidrug resistance, with the predominant phenotype being against 11 antibiotics, and multiple antibiotic resistance index ranged between 0.3 and 0.6. The incidence of genes encoding resistance against ampicillin (ampC), tetracycline (tetA), and streptomycin (strA) were 54, 61, and 44%, respectively. We conclude that healthy pigs are potential reservoirs of multidrug-resistant Salmonella that could be transmitted to humans through the food chain and, hence, a significant public health threat.

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in two commercial swine farms in the Eastern Cape Province, South Africa.

Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli.

Antibiotic susceptibilities of enterococcus species isolated from hospital and domestic wastewater effluents in alice, eastern cape province of South Africa.

BACKGROUND: Antimicrobial resistance in microorganisms are on the increase worldwide and are responsible for substantial cases of therapeutic failures. Resistance of species of Enterococcus to antibiotics is linked to their ability to acquire and disseminate antimicrobial resistance determinants in nature, and wastewater treatment plants (WWTPs) are considered to be one of the main reservoirs of such antibiotic resistant bacteria. We therefore determined the antimicrobial resistance and virulence profiles of some common Enterococcus spp that are known to be associated with human infections that were recovered from hospital wastewater and final effluent of the receiving wastewater treatment plant in Alice, Eastern Cape. METHODS: Wastewater samples were simultaneously collected from two sites (Victoria hospital and final effluents of a municipal WWTP) in Alice at about one to two weeks interval during the months of July and August 2014. Samples were screened for the isolation of enterococci using standard microbiological methods. The isolates were profiled molecularly after targeted generic identification and speciation for the presence of virulence and antibiotic resistance genes. RESULTS: Out of 66 presumptive isolates, 62 were confirmed to belong to the Enterococcus genusof which 30 were identified to be E. faecalis and 15 E. durans. The remaining isolates were not identified by the primers used in the screening procedure. Out of the six virulence genes that were targeted only three of them; ace, efaA, and gelE were detected. There was a very high phenotypic multiple resistance among the isolates and these were confirmed by genetic analyses. CONCLUSIONS: Analyses of the results obtained indicated that hospital wastewater may be one of the sources of antibiotic resistant bacteria to the receiving WWTP. Also, findings revealed that the final effluent discharged into the environment was contaminated with multi-resistant enterococci species thus posing a health hazard to the receiving aquatic environment as these could eventually be transmitted to humans and animals that are exposed to it.

Molecular epidemiology of HIV in two highly endemic areas of northeastern South Africa.

There is paucity of data on the genetic landscape of HIV-1 viruses circulating in the Limpopo Province of northeastern South Africa. Here, we examine the genetic diversity of viruses from Bela-Bela and Musina, two towns with high HIV prevalence. Between June 2007 and March 2008, blood samples were collected from antiretroviral-drug-naïve individuals. Viruses were analyzed for genetic subtypes and drug resistance mutations. All of the viruses in these samples were shown by phylogenetic analysis based on gag p17, gag p24, reverse transcriptase, protease and envelope C2-C3 gene regions to belong to HIV-1 subtype C. Two of 44 reverse transcriptase sequences (4.5%) contained N rather than the consensus K at position 103. The K103N mutation is normally associated with resistance to NNRTIs. No major mutations were observed in the protease gene. However, several polymorphisms and amino acid changes normally considered to be minor drug resistance mutations were observed in the protease sequences. These results suggest that HIV-1 subtype C remains the predominant variant responsible for the epidemic in northeastern South Africa and that the prevalence of drug-resistant viruses among the naïve population is low.