Browsing by Author "Amoako, Daniel Gyamfi."

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Molecular and genomic analysis of clinical multidrug-resistant coagulase-negative staphylococci from the uMgungundlovu District in the KwaZulu-Natal Province, South Africa.
(2020) Asante, Jonathan.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.; Abia, Akebe Luther King.
Coagulase-negative staphylococci (CoNS) are among the most commonly recovered bacteria in clinical specimens. They are usually colonisers (commensals) of the skin and nasal passages and considered contaminants of microbial cultures. However, they have been recognised as emerging pathogens, frequently causing opportunistic infections. The frequent use of indwelling medical devices and long-term hospitalisation present an increased risk of exposure to CoNS, resulting in infections usually caused by multidrug-resistant pathogens. Few studies focus on CoNS, including characterisation of their mechanisms of resistance, virulence, and persistence. Therefore, this study describes the molecular and genomic profiles of clinical CoNS from public sector hospitals in the uMgungundlovu District in KwaZulu-Natal, South Africa. Eighty-nine clinical CoNS isolates collected from three hospitals within the uMgungundlovu District between October 2019 and February 2020, constituted the sample. Isolates were speciated using the Vitek 2 system. Antibiotic susceptibility testing was done against a panel of 20 antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines using the Kirby-Bauer disk-diffusion method and minimum inhibitory concentration (MIC) was determined using the broth microdilution method for penicillin G, cefoxitin, ceftaroline, ciprofloxacin, moxifloxacin, azithromycin, erythromycin, gentamicin, amikacin, chloramphenicol, tetracycline, doxycycline, teicoplanin, tigecycline, linezolid, clindamycin, rifampicin, sulphamethoxazole/trimethoprim, nitrofurantoin and vancomycin. PCR was used to detect the presence of the mecA gene to confirm phenotypic methicillin resistance. Based on their resistance profiles, a sub-sample of isolates were subjected to wholegenome sequencing (Illumina MiSeq) to ascertain the resistome, virulome, mobilome, clonality and phylogenomic relationships using bioinformatic tools. The SPAdes software was used for the assembly of the raw reads. ResFinder 4.1 and CARD were used to identify antibiotic resistance genes in the isolates, while the virulence factor database (VFDB), Center for Genomic Epidemiology‘s MLST 2.0 server and MobileElementFinder v1.0.3 were used to identify virulence genes, sequence types and mobile genetic elements, respectively. Mutations in fluoroquinolone and rifampicin resistance genes were identified by manual curation using BLASTn alignment which was also used to determine the genetic environment of the resistance genes.S. epidermidis was the most abundant CoNS species isolated. Phenotypic methicillinresistance was detected in 76.4% (n=68) of isolates, 92.6% (n=63) of which were genotypically confirmed by PCR. Multidrug resistance (MDR) was observed in 76.4% (n=68) of isolates, with 51 antibiograms observed. The resistance genes mecA, blaZ, erm(A), erm(B), erm(C), msr(A), aac(6')-aph(2'') and fosB, among others, were detected and corroborated the observed phenotypes. Molecular mechanisms of resistance to tigecycline, teicoplanin, linezolid and nitrofurantoin were not detected even though some isolates were resistant to them. There was no association between ARG type and hospital/department. The ica operon known to facilitate biofilm formation was detected in 7/16 isolates sequenced. Known and putatively novel mutations in the gyrA, parC, parE and rpoB genes were also detected for fluoroquinolone- and rifampicin-resistant isolates. Prediction of isolates’ pathogenicity towards human hosts yielded a high average probability score (Pscore ≈ 0.936), which, together with the several virulence genes detected (including atl, ebh, clfA, ebp, icaA, icaB,icaC), support their pathogenic potential to humans. Seven MLST types were found, while the community-acquired SCCmec type IV was the most common SCCmec type detected. Mobile genetic elements (MGEs) haboured by isolates included plasmid replicon Rep10 and insertion sequence IS256. Defense systems such as arginine catabolic mobile element (type I and III), CRISPR system (16), and the restriction-modification system (type II) were detected. Genetic analysis showed that resistance genes were frequently bracketed by MGEs such as transposons (such as Tn554) and insertion sequences (such as IS257 and IS1182) that facilitated their mobility. Phylogenetic studies showed that the distribution of genes did not coincide with the phylogenetic clades. Despite the relatedness of isolates (clades A and B), there is still considerable variation within individual strains that can facilitate adaptation to local environments. The isolates exhibited several permutations and combinations of ARGs, virulence genes and MGEs, pointing to a complex milieu of mobilized antibiotic resistance and pathogenic characteristics in clonal and multiclonal strains. The study necessitates surveillance of CoNS as emerging pathogens.
Molecular characterization of antibiotic-resistant Staphylococcus aureus in an intensive pig production system in KwaZulu-Natal, South Africa.
(2021) Sineke, Ncomeka.; Amoako, Daniel Gyamfi.; Abia, Akebe Luther King.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.
The increase in antibiotic resistance in food animals and food of animal origin has been attributed to the extensive use of antibiotics during animal husbandry giving rise to multidrug-resistant bacteria. Staphylococcus aureus is a major threat in veterinary medicine, the agricultural sector and public health because of its zoonotic potential. Despite significant research on S. aureus in food animals in other parts of the world, in-depth studies outside healthcare facilities are limited in South Africa. This study characterized the molecular epidemiology of antibiotic resistant S. aureus from farm-to-fork in an intensive pig production chain in the uMgungundlovu district, Kwa-Zulu Natal, South Africa. A total of 333 samples collected along a pig production chain on the farm (faecal, litter and slurry samples) during transport (truck samples) and at the abattoir (caeca, carcass swabs, carcass rinsate and retail meat samples) were investigated for the presence S. aureus using selective media and biochemical tests. Confirmation was done by using PCR targeting the nucA gene. Antibiotic susceptibility patterns were investigated by the Kirby Bauer disk diffusion according to CLSI guidelines against the WHO-AGISAR recommended panel of antibiotics. Selected resistance and virulence genes were detected using PCR. REPPCR was used to evaluate the molecular relatedness of isolates across the pig production chain. Of the 333 samples, 141 (43%) yielded staphylococci isolates. After molecular confirmation, 97(69%) isolates were confirmed S. aureus and 44(31%) as other staphylococcal species. Isolates displayed resistance to erythromycin (85%), clindamycin (85%), penicillin-G (81%), tetracycline (79%), doxycycline (77%), vancomycin (69%), ampicillin (61%), trimethoprim/sulfamethoxazole (57%), rifampicin (57%), teicoplanin (52%), linezolid (51%), chloramphenicol (51%), nitrofurantoin (47%), moxifloxacin (33%), cefoxitin (20%), ciprofloxacin (15%), tigecycline (10%), levofloxacin (8%), gentamicin (8%), and amikacin (2%). Multidrug resistance (MDR) was recorded in 84% (80/97) of isolates with 56 different antibiograms. Resistance genes ermC, blaZ, tetK, tetM, msrA, aac’6, mecA were evident in 82%, 73%, 58%, 28%, 15%, 5%, and 53% respectively and not all resistance phenotypes were genotypically confirmed. The hla (39%), hld (23%), seb (3%), sed (2%), etb (1%), LukS/F-PV (30%) and tst (11%) virulence genes encoding hemolysin, cytotoxins, staphylococcal enterotoxins (sea and seb), exfoliative toxins, PVL pore-forming toxin and toxic shock syndrome toxin-1 were detected. Genetic fingerprinting revealed the diversity of MRSA isolates in the pig production chain with the major REP-types constituting isolates from different sources within the farm, suggesting transmission within the farm environment with no evidence of transmission across the production chain. This study highlights the phenotypic and genotypic diversity of the virulence and resistance profiles of S. aureus isolated across the pig production chain. Resistance to antibiotics used as growth promoters was evident and the high prevalence of MDR isolates with elevated MAR index values >0.2, specifically at farm level indicates exposure to environments of high antibiotic use, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.
Molecular characterization of multi-drug resistant (MDR) gram-negative bacterial pathogens from environments, patients and staff in a teaching hospital in Ghana.
(2023) Yeboah, Esther Eyram Asare.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alexander.; Agyepong, Nicholas.; Abia, Akebe Luther King.; Amoako, Daniel Gyamfi.; Mbanga, Joshua.
Multidrug resistant Gram-negative bacteria (MDR GNB) are implicated in serious infections both of community and nosocomial origin and may be disseminated in the hospital in the absence of efficient infection prevention and control (IPC) practices. The prevalence and risk factors for rectal colonization of MDR GNB among patients, the carriage of MDR GNB on healthcare workers’ (HCWs’) hands and the contamination patients’ environments with MDR GNB were investigated in a teaching hospital in Ghana. In this prospective study, conducted between April 2021 to July 2021, the phenotypic profiles of the MDR GNB isolates were determined using the VITEK 2 system. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis of associated data. The resistome, virulome, mobilome and genetic relatedness of MDR extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and ESBL-producing or carbapenem resistant Klebsiella pneumoniae isolates from patients and their environment were also determined using whole genome sequencing performed on the Nextseq 550 (2 x 150 bp) and bioinformatics analysis. A total of 585 samples were collected from patients, HCWs’ hands and the hospital environment within the study period. The prevalence of MDR GNB rectal colonization among patients was 50.62% on admission and 44.44% after 48 hours. MDR GNB, frequently E. coli and K. pneumoniae were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR,95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 hours of admission while age (21-30 years) (p-value =0.022, OR, 95% CI =0.103(0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. Rectal carriage and acquisition of ESBL-producing E. coli among patients was 13.65% and 11.32% respectively. blaTEM-1B and blaCTX-M-15 were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Multiple virulence genes, predominantly, terC were detected in the ESBL E. coli isolates. Sequence types (STs) were diverse and included one novel ST (ST13846) present in two isolates. Phylogenetic analysis grouped the ESBL E. coli isolates into four main clusters. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. Of the ten selected MDR K. pneumoniae isolates, the β-lactamase gene, blaCTX-M-15 was observed in six isolates. Mutations were found in both ompK36 and ompK37 in all isolates (both carriage isolates and isolates from hospital environments). Genes encoding resistance to fluoroquinolone (qnrB), aminoglycosides (aadA1, aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib , aph(6)-Id) sulphamethoxazole/trimethoprim (sul1, sul2, dfrA14, dfrA15) were also detected. The K. pneumoniae isolates belonged to seventeen different STs with ST39 most commonly observed and common to both carriage isolates and isolates from hospital environments. A myriad of virulence genes, including irp1, irp2, iutA, gndA, ompA, fes, fep, mrkD and fimH were detected in both carriage and isolates from the hospital environment. IncFIB was the most abundant plasmid replicon occurring in nine (four carriage isolates and five isolates from hospital environments). ESBL-producing K. pneumoniae isolates appeared to be introduced into the hospital from the community. The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, the contamination of hospital environments and the circulation of ESBL-producing E. coli and K. pneumoniae isolates with diverse genomic characteristics, highlights the need for patient screening, and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals. The observed clonal relatedness among isolates from patients and the hospital environment, as well as between different patients, suggests a possible transmission within and between sources, hence infection prevention and control practices need to be enhanced to prevent the dissemination and transmission of these resistant strains in the hospital. This study further highlights the usefulness of whole genome sequencing as an effective tool in AMR surveillance.
Molecular characterization of resistance and virulence in Methicillin Resistant Staphylococcus Aureus (MRSA) from the private sector in KwaZulu-Natal, South Africa.
(2016) Amoako, Daniel Gyamfi.; Esaack, Sabiha Yasuf; Bester, Linda Antionette.
Abstract available in PDF file.
Molecular epidemiology of antibiotic resistant Campylobacter spp. from farm-to-fork in an intensive pig production system in Kwazulu-Natal, South Africa.
(2021) Sithole, Viwe.; Amoako, Daniel Gyamfi.; Essack, Sabiha Yusuf.; Abia, Akebe Luther King.; Bester, Linda Antionette.
Background: Campylobacter spp. are among the leading foodborne pathogens, causing Campylobacteriosis, a zoonotic infection that results in bacterial gastroenteritis and diarrhea disease in animals and humans. The emergence and transmission of antibiotic resistance and virulence in Campylobacter spp. is increasingly reported. We investigated the molecular epidemiology of antibiotic resistant Campylobacter spp. isolated across the farm-to-fork-continuum in an intensive pig production system in the uMgungundlovu District, Kwazulu-Natal, South Africa. Methodology: Following ethical approval, samples were collected over a period of sixteen weeks from selected critical points (farm, transport, abattoir and retail) using a farm-to-fork sampling approach according to WHO-AGISAR guidelines. Overall, 520 samples were investigated for the presence of Campylobacter spp. which were putatively identified using selective media with identity and speciation confirmed by polymerase chain reaction (PCR) of specific genes. Resistance profiles were ascertained by the Kirby-Bauer disk diffusion method according to EUCAST and/or CLSI guidelines. Selected antibiotic resistance and virulence genes were identified using PCR and DNA sequencing. Clonal relatedness among the isolates was determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Results: Altogether, 378/520 (72.7%) samples were positive for Campylobacter spp. with C. coli as the most predominant (73.3%), followed by C. jejuni (17.7%) with 9.0% classified as “other”. Relatively high levels of resistance were observed in C. coli and C. jejuni to erythromycin (89% and 99%), streptomycin (87% and 93%), tetracycline (82% and 96%), ampicillin (69% and 85%), and ciprofloxacin (53% and 67%) respectively. The lowest percentage resistance observed was for gentamicin (12%) for both C. coli and C. jejuni, and nalidixic acid (28% and 27%) for C. coli and C. jejuni respectively. Multi-drug resistance (MDR) was noted among 330/378 (87.3%) isolates. The antibiotic resistance genes observed were the tetO (74.6%), the blaOXA-61 (2.9%) and cmeB (11.1%) accounting for the resistance to tetracycline and ampicillin while the membrane efflux pump could confer resistance to ampicillin, tetracycline, ciprofloxacin, and erythromycin. All C. coli and C. jejuni isolates (21) with the gyrA gene exhibited mutation at the Thr-86-Ile region in the quinolone-resistancedetermining region (QRDR) and all C. coli and C. jejuni isolates (18) exhibiting erythromycin resistance showed common transitional mutations A2075G and A2074C in the 23S rRNA gene. Of the virulence genes tested, ciaB, dnaJ, pldA, cdtA, cdtB, cdtC and cadF were detected in 48.6%, 61.1 %, 17.4%, 67.4%, 19.3%, 51% and 5% of all Campylobacter isolates respectively. The ERIC-PCR banding patterns revealed that isolates along the continuum were highly diverse with isolates from the same sampling points belonging to the same major ERIC-types. Conclusion: We showed relatively high levels of resistance to antibiotics commonly used in intensive pig production in South Africa with some evidence, albeit minimal, of transmission across the farm-tofork continuum. This together with the virulence profiles present in Campylobacter spp. presents a challenge to food safety and a potential risk to human health. This is further exacerbated by the reduction in antibiotic treatment options necessitating routine surveillance and monitoring together with antibiotic stewardship, comprehensive biosecurity, and good animal husbandry in intensive pig production.
Molecular epidemiology of antibiotic resistant salmonella spp. from farm to fork in an intensive pig production system in KwaZulu-Natal South Africa.
(2021) Tshakane, Nozipho Pamela.; Essack, Sabiha Yusuf.; Abia Akebe, Luther King.; Amoako, Daniel Gyamfi.
Antibiotic resistance (ABR) is a worldwide challenge, and, if not resolved, can be a danger to humans, animals and the ecosystem. The inappropriate use and misuse of antibiotics in food animal production creates selection pressure for the development of bacterial resistance. We investigated the molecular epidemiology, antibiotic resistance and virulence of Salmonella spp. from farm-to-fork in an intensive pig production system in KwaZulu-Natal. A herd of pigs was followed from birth to slaughter over a period of 4 months. Following ethical approval, a total of 408 samples were collected, which consisted of feces, litter, slurry, hand and nasal swabs from occupationally exposed workers, carcass swabs and rinsate, caecal samples and pork for retail purposes. Salmonella was putatively identified using selective media, i.e., Brilliance Salmonella Agar and Salmonella Shigella Agar (SS agar). Identification to species and sub-species level was confirmed by polymerase chain reaction (PCR), where the invA gene was used to confirm Salmonella spp. and the iroB gene for Salmonella enterica. Isolates were subjected to antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method against a panel of 14 antibiotics. Isolates were screened for selected virulence genes, misL, spiC, orfL, sopB, pipD, hilA and stn, conferring intracellular survival (misL), type III secretion system (spiC), adhesion and autotransporter (orfL), type III secreted effector protein (sopB), type III secreted effector associated with SPI-1 system (pipD), host cell invasion (hilA), and enterotoxin production (stn) by PCR. Genetic relatedness of the isolates was determined by ERIC-PCR. A total of 399 putative Salmonella spp. were detected by selective media, of which 49% (n= 197) were confirmed by the presence of the invA gene and 45% (n=179) were identified as Salmonella enterica by the presence of the iroB gene. The largest number of Salmonella were isolated from retail meat samples. Antibiotic susceptibility testing showed 10% (n=19) resistance to cefoxitin, 8% (n=16) to amoxicillin and 0.5% (n=1) to gentamicin and chloramphenicol. The isolates carried the hilA (91%), stn (91%), misL (89%), pipD (88%), spiC (87%), orfL (85%) and sopB (72%) virulence genes. The isolates were clonally diverse with 26 ERIC-types and four major ERIC-type groups. The large number of isolates in retail meat samples, their virulence, and, to a lesser extent their antibiotic resistance profiles poses a challenge to the food safety system and requires a comprehensive understanding of molecular epidemiology of the organism so that it’s incidence spread can be reduced and better controlled from the primary source within the food chain.
Molecular epidemiology of antibiotic-resistant Enterococcus spp. from farm-to-fork in intensive pig production in KwaZulu-Natal, South Africa.
(2021) Badul, Sasha.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
Background: Substantial antibiotic use and high population densities in intensive farming systems results in the emergence and spread of antibiotic-resistant commensals and pathogens. This study investigated the molecular epidemiology of antibiotic resistance (ABR) and virulence in Enterococcus spp. from pigs in an intensive food production continuum from farm-to-fork in the uMgungundlovu district, Kwa-Zulu Natal. Methods: A total of 174 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) were subjected to the quantification and putative identification of Enterococcus spp. using the IDEXX Enterolert® method and selective media, respectively. Up to three presumptive enterococcal colonies were picked per sampling point for molecular confirmation by real-time PCR, targeting the genus- and species-specific (tuf and sodA) genes, respectively. Antibiotic resistance profiles were determined by the Kirby-Bauer disk diffusion method against a panel of antibiotics for Enterococcus spp. recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. Results: A total of 284 isolates constituted the final sample. Real-time PCR confirmed 79.2% of the isolates as E. faecalis, 6.7% as E. faecium, 2.5% as E. casseliflavus, 0.4% as E. gallinarum, and 11.2% as other Enterococcus spp. Antibiotic susceptibility testing revealed resistance to sulfamethoxazole-trimethoprim (78.8%), tetracycline (76.9%), erythromycin (68.1%), streptomycin (62.6%), chloramphenicol (27.0%), ciprofloxacin (8.5%), gentamicin (8.1%), and levofloxacin (5.6%) but no vancomycin, teicoplanin, tigecycline or linezolid resistance was detected. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. A total of 78% of enterococcal isolates were MDR. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3’)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most commonly detected virulence genes were: gelE, efaAfs, and cpd in 89.1%, 78.5%, and 77.1% of isolates conferring autolysin and biofilm formation capabilities, cell adhesion, and conjugative plasmid accumulation, respectively. Clonality evaluated by REP-PCR revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, largely consisting of isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. There was minimal evidence of clonal transmission across the continuum. Conclusion: Multi-drug resistant Enterococcus spp. were isolated along the farm-to-fork continuum. Isolates harboured a diversity of antibiotic resistance and virulence genes in different combinations forming reservoirs for the potential transfer of these genes from pigs to occupationally exposed workers and consumers via direct contact with animals and animal products/food, respectively. The results highlight the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.
Molecular epidemiology of antibiotic-resistant Escherichia coli and Enterococcus spp. from agricultural soil fertilized with chicken litter in uMgungundlovu district, KwaZulu-Natal Province, South Africa.
(2021) Fatoba, Dorcas Oladayo.; Abia, Akebe Luther King.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.
The application of animal manure contaminated with antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) represents a major route by which antibiotic resistance is transmitted into the soil environment. The introduction and persistence of ARB in agricultural soil may pose a risk to public health via the consumption or handling of contaminated farm produce. Understanding the impact of animal manure application on the agricultural soil resistome and the risk it poses on public health is critical. However, such information is limited in South Africa as most antibiotic resistance research focuses on humans and food animals. This study, therefore, describes the prevalence and the genomic profiles of antibiotic-resistant Escherichia coli and Enterococcus spp. isolated from agricultural soil fertilized with chicken litter and the chicken litter. A total of 237 samples were examined and included soil before litter application, the litter-amended soil, and the chicken litter. Isolation and quantification of Escherichia coli and Enterococci were carried out using the Colilert® -18 / Quantiti-Tray® 2000 system and the Enterolert® -18® Quanti-Tray®/2000 system, respectively. The antibiotic susceptibility profiles of the isolates was determined using the Kirby-Bauer disk diffusion method. Whole-genome sequencing (WGS) and bioinformatics tools were used to determine the resistome, virulome, mobilome, clonal lineages, and phylogenies of the isolates circulating between the soil and the chicken litter. The application of chicken litter to the soil statistically significantly increased Enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil. A total of 835 enterococci (680 from soil and 155 from litter) isolates recovered from the samples was dominated by E. casseliflavus (56%), followed by E. faecalis (22%), E. faecium (8%), E. gallinarum (2%) and other Enterococcus spp 102 (12%). Overall, 55.8% (466/835) of the enterococci isolates were resistant to one or more antibiotics with the highest rate in the litter-amended soil (68.9%, 321/466), followed by chicken litter (19.9%, 93/466) and the least in the soil samples collected before the litter amendment (11.2%; 52/466). The enterococci isolates were mostly resistant to tetracycline (33%), erythromycin (25%), and trimethoprim-sulfamethoxazole (23%), among others, intimating the high usage of these antibiotics in poultry farms in South Africa. Additionally, multidrug resistance (MDR) was recorded in 27.8% (130/466) of the enterococci isolates with MAR indices ranging from 0.13 (resistance to two antibiotics) to 0.44 (resistance to seven antibiotics). A total of 63 different resistance patterns were recorded in the MDR enterococci isolates. Notably, enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil were reduced to levels comparable to the unamended soil at 50 and 28 days after soil amendment respectively. The whole-genome analysis of the few selected enterococci isolates revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). Several resistance genes that confer resistance to aminoglycosides (aac(6’)-Ii, aac(6’)-Iih, ant(6)-Ia, aph(3’)-III, ant(9)-Ia), macrolide-lincosamide-streptogramin AB (MLSAB) [erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)], trimethoprim-sulfamethoxazole (dfrE, and dfrG), tetracycline (tet(M), tet(L), and tet(S)), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat) were detected in the isolates. The bioinformatics analysis further revealed that the chicken litter amendment increased the number and diversity of ARGs in the soil, resulting in increased detection of tetracycline resistance genes (tet(M), tet(L)), and the macrolide resistance gene erm(B) and appearance of some ARGs (ant(6)-Ia, aph(3’)-III, lnu(G), dfrG)) that were not detected in the unamended soil. ARGs were mostly associated with diverse insertion sequences (ISs) (IS982, ISL3, IS6, IS5, IS3, IS256, IS30) and/or transposons (Tn3, Tn916, Tn6009) on plasmids or chromosome. The tet(M) and erm(B) were also co-located on Tn916-like transposons (Tn644, Tn645, and Tn659) in the three sample groups. Some of the isolates also harboured virulence genes that encoded adherence/biofilm formation (ebpA, ebpB, ebpC), anti-phagocytosis (elrA), and bacterial sex pheromones (Ccf10, cOB1, cad, and camE). Phylogenomic analysis showed that few isolates from litter-amended soil clustered with the chicken litter isolates. The isolates from this study also clustered with clinical and animal isolates from South Africa (Pretoria, Pietermaritzburg), Angola, and Tunisia. There was also an increase (albeit statistically insignificant) in E. coli count and the number of antibiotic-resistant E. coli in the soil following chicken litter amendment. A total of 126 E. coli was recovered from the soil and chicken litter samples. In total, 76% (96/126) of the E. coli isolates displayed resistance to at least one antibiotic, with the highest prevalence in the litter-amended soil (71.9%, 69/96) and the least (1%, 1/96) in soil samples collected before the litter amendment. The E. coli isolates displayed a high percentage resistance to tetracycline (78.1%), chloramphenicol (63.5%), ampicillin (58.3%), trimethoprim-sulfamethoxazole (39.6%), cefotaxime (30.2%), ceftriaxone (26.0%), and cephalexin (20.8%). Lower percentages of XVI resistance to cefepime (11.5%), amoxicillin-clavulanic acid (11.5%), cefoxitin (10.4%), nalidixic acid (9.4%), amikacin (6.3%), ciprofloxacin (4.2%), imipenem (3.1%), tigecycline (3.1%), and gentamicin (3.1%) were also recorded in the isolates. All the isolates were completely susceptible to meropenem and ceftazidime. Approximately 54% (52/96) of the resistant isolates were MDR, and the MAR indices of the isolates ranged between 0.11 (resistance to two antibiotics) and 0.56 (resistance to ten antibiotics). Overall, 38.5% (37/96) of all the resistant isolates had a MARI > 0.2, with the highest rate (51.4%) in the litter-amended soil and the least in the soil before litter amendment (2.7%). Twenty-one multidrug resistance patterns were observed among the isolates. These results show that the soil resistome was augmented by chicken litter application. Agricultural soil and chicken litter are rich reservoirs of multidrug-resistant E. coli and Enterococcus spp. that could threaten public health through contamination of food products and the surrounding water bodies. There is therefore a need for urgent and stringent measures to mitigate the spread of antibiotic resistance in the environment via prudent use of antibiotics in food animal production and treatment of animal manure before its application onto agricultural soil.
Molecular epidemiology of carbapenem-resistant Enterobacterales colonization in an intensive care unit.
(2021) Madni, Osama.; Essack, Sabiha Yusuf.; Rout, Joan Allison.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
Background: Due to the high association with mortality and morbidity, carbapenem-resistant Enterobacterales (CRE) in general, and carbapenem-resistant Klebsiella pneumoniae, in particular, have been listed as high-priority pathogens by the World Health Organization (WHO) for the research and development of new antibiotics. Concomitant resistance to multiple antibiotics of different classes, impedes efficient clinical management of CRE infections. We characterized carbapenemase-producing K. pneumoniae (CPKP) isolates from sequential rectal screening of patients in a single intensive care unit (ICU) in a public hospital in the uMgungundlovu District of Kwazulu-Natal, South Africa, collected over one month. Method: Ninety-seven rectal swabs collected from consenting adult patients (n=31) on day 1, 3, 7 and weekly thereafter were screened for carbapenemase-production using Chrome-ID selective media. Fourteen CPKP were subjected to speciation and antibiotic susceptibility testing using the VITEK 2® automated system and their clonality was ascertained by ERIC/PCR. A sub-sample of eight isolates from five patients underwent whole genome sequencing (WGS) on the Illumina MiSeq platform followed by bioinformatics analysis to delineate the resistome, virulome, mobilome, clonality and phylogeography. Results: All isolates (100%) were resistant to ertapenem and meropenem and 71.4% (n=10) were resistant to imipenem. All isolates harbored the blaOXA-181 carbapenemase (100%, n=8) and also carried other β-lactamase genes such as OXA-1, CTX-M-15, TEM-1B and SHV-1. IncF, IncX3, and Col plasmid replicons groups and class I integrons (ln191 and ln27) were detected. All isolates belonged to the same sequence type ST307 and capsular serotypes (K102, O2v2) and several were associated with a single bed located in the ICU. All but one isolate carried the same plasmid multilocus sequence type [K7:A-:B-] and the same virulence repertoire was identified reflecting the epidemiological relationships between isolates. BlaOXA-181 were presumably located on a multi-replicon plasmid similar to that of E. coli p010_B-OXA181, and isolates were aligned with several South African and international clades, demonstrating horizontal and vertical transboundary distribution. Conclusion: OXA-181-producing K. pneumoniae belonging to ST307 was found to be potentially endemic in the hospital ICU environment of a public hospital in KwaZulu-Natal South Africa. The presence of a myriad of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in different permutations and combinations presents challenges to clinical management and infection prevention and control measures. This necessitates a CRE screening programme and strict infection prevention and control measures to detect and eliminate this endemic clone.