Medical Microbiology

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Enterococcus sp. contamination surveillance in different levels of healthcare in eThekwini District, KwaZulu-Natal (KZN) South Africa.
(2021) Shobo, Christiana Omowunmi.; Bester, Linda Antionette.; Essack, Sabiha Yusuf.
Hospital-acquired infections (HAIs) have been identified as long-standing setbacks affecting hospitals' quality of health care. While one of the major challenges related to HAIs is controlling cross-transmission, the role and significance of the inanimate hospital environment chain of transmission are yet to be unequivocally elucidated. Therefore, this study investigated the functional profile and diverseness of bacteria from various inanimate environmental sources, from two different wards in public hospitals at various healthcare levels in eThekwini District, KwaZulu-Natal, South Africa. True to the study focus on investigating the dissemination of bacteria from equipment within the hospital, the study further used Enterococcus as well-known HAI as target bacteria and described the molecular and genomic profiles of this specie isolated from the hospital environments. Samples were collected for a period of three months (September – November 2017) from the four levels of healthcare in eThekwini district, KwaZulu-Natal. The intensive care unit and peadiatic ward were employed in this study. An overall of 620 swabs were collected from areas frequently touched by healthcare workers (HCWs) and patients. These sites include the occupied bed linen, unoccupied bed linen, drip stands, patient files, ward phones, ventilators, nurses' tables, blood pressure apparatus, sinks, linen room door handle and mops. Swabs were placed in Amies transport medium and transported in a cooler box to the laboratory facility to be processed within four hours. The collected swabs (n=620) were pooled and incubated in tryptone soya broth containing 6.5% NaCl at 36.5oC for 24 hrs and subsequently plated on enterococci chromogenic media. The microbial diversity and functional profiles from the sites were identified using 16S rRNA metagenomics. Positive colonies were sub-cultured on bile esculin azide agar, and screened using standard microbiological methods, including haemolytic, oxidase and catalase, and API. Identifications were confirmed with polymerase chain reaction (PCR) with the added genus-specific tuf-gene and species-specific sodA-gene. Antibiotic resistance patterns in the Enterococcus spp. isolates were determined by the Kirby-Bauer disk diffusion method against 14 antibiotics as recommended by the Clinical and Laboratory Standard Institute (CLSI) guidelines. Thirty-seven samples from E. faecalis showed intermediate Resistance to vancomycin and were further analyzed using molecular tools viz. whole-genome sequencing (WGS) and bioinformatics analyses. This enabled determining the resistome, mobile genetic elements (MGEs), and clonal lineages circulating across the sites, wards, and hospitals. Metagenomics identified a total of 288 species, 190 genera, 105 families, 50 orders, 29 classes and 11 phyla from the samples analyzed. The dominant functional metabolic pathways implicated in causing human infection discovered were the signal transduction mechanisms, citrate cycle (TCA), transcription-factor bisphenol degradation, tyrosine metabolism. A total of 295 Enterococcus spp. isolates were recovered from the hospitals` environmental sites, 83% (n=245) were identified as Enterococcus faecium, 13% (n=38) as Enterococcus faecalis, 2% (n=6) Enterococcus gallinarum and another 2% (n=6) Enterococcus casseliflavus. Notably, the pediatric wards had the highest isolation rate compared to ICU, 64% and 36%, respectively. Overall, the sites with the highest isolation rate were occupied beds and mops (to clean ward floors) with 14.9% (n=44) each. The tertiary hospital were the most affected. The most prominent MDR antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for WGS analysis of the E. faecalis samples confirmed that the tet(M) and erm(C) genes were the prevalent antibiotic resistance genes found in hospitals. The isolates harboured mobile genetic elements consisting of plasmids (n =11) and prophages (n=14), predominantly clonally specific. The 37 isolates analyzed consisted of 15 clonal lineages with six major sequence types (ST). Phylogenomic analysis showed that major lineages were mostly conserved within specific hospital environments. This study highlighted the inanimate hospital environment as a possible source of opportunistic nosocomial pathogens using Enterococcus as an illustrative example and emphasized the urgent necessity to optimize infection prevention and control measures to intercept/moderate the spread of bacteria in the hospital environments.
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 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 Escherichia coli from intensively-produced poultry in a farm-to-fork continuum in KwaZulu-Natal, South Africa.
(2020) McIver, Katherine Susan.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.; Abia, Akebe Luther King.
The increased use of antibiotics in intensively produced food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. There is a risk of transfer of this resistance to humans and as such a public health risk. The aim of this study was to investigate the molecular epidemiology of antibiotic resistant Escherichia coli from intensively produced poultry in the uMgungundlovu district of Kwa-Zulu Natal, South Africa. This was a longitudinal descriptive study with the aim to determine the epidemiology of antibiotic resistance of E.coli from hatching through to the final retail product from an intensive poultry farm house. The farm reported the use of zinc bacitracin and Salinomycin included in the feed, but no therapeutic antibiotics used in this batch of chickens. However, the following antibiotics were used on the farm in the previous 12 months: Doxycycline, Sulfadiazine and Trimethoprim, Enrofloxacin, Ceva olaquindox 10%, Avilamycin, Tylosin 10% and Kitasamycin tartate. During the first five weeks, ten samples from litter and faeces were collected. During transfer from the house to abattoir ten swabs from transport trucks and transport crates were taken. At the abattoir ten samples from carcass wash were collected. After slaughter and dressing ten caecums, whole chickens, thighs and necks were collected. Again, during house washing, ten samples were collected. E.coli was putatively identified using Eosin Methylene Blue agar followed by Sorbitol MacConkey agar and confirmed by identification of the uidA gene by polymerase chain reaction. Susceptibility to a panel of antibiotics recommended by the World Health Organization Advisory Group on the Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) was ascertained by the Kirby-Bauer disk diffusion method for 20 antibiotics according to CLSI guidelines. Realtime PCR was used to test for resistance genes tetA, tetB, qnrB, qnrS, aac(6)-lb-cr, sul1, sul2, sul3, blaSHV, blaCTX-M, blaTEM conferring resistance to tetracyclines, quinolones, sulphonamides and cephalosporin antibiotics. Clonal similarities were investigated using ERIC-PCR. A total of 266 E.coli isolates constituted the sample size with a non-susceptibility profile of ampicillin 48.1%, tetracycline 27.4%, nalidixic acid 20.3%, trimethoprim-sulphamethoxazole 13.9%, chloramphenicol 11.7%, cefalexin 4.5%, ciprofloxacin 4.1%, amoxycillin-clavulanic acid 3.4%, gentamicin 1.9%, cefoxitin 1.1%, cefepime 1.1%, cefotaxime 1.1%, amikacin 1.1%, ceftriaxone 0.8% and azithromycin 0.8%. Isolates were fully susceptible to ceftazidime, imipenem, meropenem and tigecycline. Of the 266 isolates 6.4% were multidrug resistant (resistant to one or more antibiotics in three or more distinct antibiotic classes). The most frequently observed resistance genes were blaCTX-M (100%), sul1(80%), tetA(77%), tetB(71%). Using ERIC-PCR the isolates were grouped into 27 clusters with a 75% similarity. Eight clusters comprised of isolates from only one sample. xiv There was an increase in MDR and resistance genes over the farm to fork continuum with lowest and highest levels seen in transport and waste-water samples respectively. ERIC-PCR did not indicate the transmission of clones across the farm-to-fork continuum. There instead appeared to be de novo or evolution of resistance genes or the introduction of plasmids over the time period. As the only antimicrobials used in this flock were salinomycin and zinc bacitracin it is postulated that the resistance observed could be attributed to the co-selection of resistance genes and/or horizontal gene transfer from the environment, insects, chicken food and workers. Overall resistance levels were low over the six weeks of the study, MDR and the prevalence of resistance genes increased over time. The diverse clonality shown by the ERIC PCR results did not support the transmission of clones across the farm-tofork continuum but indicated a de novo evolution of resistance genes and/or the loss or gain of plasmids over the time period.
Molecular epidemiology of antibiotic-resistant enterococcus spp. from farm to food-production chain in intensive poultry production in KwaZulu-Natal, South Africa.
(2019) Molechan, Chantal.; Essack, Sabiha Yusuf.; Akebe, Luther King Abia.; Bester, Linda Antionette.
Extensive antibiotic use in intensively-farmed poultry exerts selection pressure for the emergence of multidrug-resistant pathogens. The aim of this study was to determine the antibiotic resistance and virulence profiles of Enterococcus spp. along the farm to food-production chain continuum in an intensive poultry system in the uMgungundlovu District in Kwazulu-Natal, South Africa. A total of 187 samples along the poultry farm to food-production chain continuum (litter, faeces, transport, holding, abattoir and retail meat) were evaluated for the presence of Enterococcus spp. Molecular confirmation by PCR, targetting the genus- (tuf) and species-specific (sodA) genes was undertaken. Susceptibility profiles were assessed by Kirby-Bauer disk diffusion against the WHO-AGISAR recommended panel of antibiotics for Enterococcus spp. using CLSI guidelines. Antibiotic resistance and virulence genes were detected using real-time PCR. Genetic relatedness between isolates across the continuum was evaluated by REP-PCR. Of 134 isolates identified across the continuum, with a prevalence of 72%, molecular speciation confirmed the isolates as E. faecalis (36%), E. faecium (31%), E. gallinarum (2%) and other Enterococcus spp. (31%). Resistance to tetracycline (80%), erythromycin (71%), nitrofurantoin (17%), ampicillin (15%), streptomycin (15%), chloramphenicol (11%), ciprofloxacin (5%), tigecycline (4%), gentamicin (4%), teicoplanin (3%) was observed among Enterococcus spp. but no vancomycin resistance (0%). E. faecium displayed 24% resistance, and 21% were of intermediate susceptibility to quinupristin-dalfopristin. Twenty-one percent (21%) of E. faecalis and 100% of E. gallinarum, also showed intermediate susceptibility to vancomycin. Forty-three percent (43%) of E. faecium were multidrug-resistant (MDR) (resistant to 1 or more antibiotics in 3 or more antibiotic classes). The most frequently observed antibiotic resistance genes, associated with the phenotypic profiles, were tetM (76%) and ermB (67%) with a smaller percentage noted for aph(3’)-IIIa (12%) and vanC1 (1%). Virulence genes efaAFs (100%), cpd (96%) and gelE (81%) were more frequently detected in E. faecalis. The cell wall adhesin (efaAFm) was more common in E. faecium (100%) and other Enterococcus spp. (71%). Clonality evaluated by REP-PCR revealed that isolates along the continuum are highly diverse with major REP-types often consisting of isolates from the same sampling point in the continuum.
Molecular surveillance and dissemination of Klebsiella pneumoniae on frequently encountered surfaces in South African public hospitals.
(2021) Malinga, Nongcebo Zuzile Zekhethelo.; Zishiri, Oliver Tendayi.; Bester, Linda Antionette.
Hospital equipment and surfaces can harbour Klebsiella pneumoniae. In the absence of effective cleaning, anyone who encounters these surfaces can unknowingly spread this opportunistic pathogen throughout the hospital. This study aimed to investigate the prevalence of K. pneumoniae on inanimate surfaces and evaluate the genetic diversity, antibiotic resistance and virulence profile of the recovered isolates. Overall, 777 swab samples were collected from four different South African public hospitals classified as central (A), tertiary (B), regional (C) and district (D). These samples were taken from 11 predetermined surfaces present in three different wards: the intensive care unit (ICU), paediatric and general. K. pneumoniae was identified using polymerase chain reaction (PCR) followed by antibiotic susceptibility testing using disk diffusion. Extended-spectrum β-lactamases (ESBL) producers were characterised using the combination disc method. Six resistance and three virulence genes were screened using PCR. The genetic diversity of the isolates was examined using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Collectively, 75 (10%) K. pneumoniae isolates were recovered from the collected samples. The isolates recovered were equally abundant in tertiary hospital B and district hospital D. The recovery of K. pneumoniae was highest in the paediatric ward. Six sites harboured K. pneumoniae wherein the occupied beds were the most heavily contaminated. Thirty (40%) isolates were identified as ESBL producers and detected in high quantities in tertiary hospital B and the ICU. The ESBLs were mostly classified as multidrug-resistant (MDR), displaying higher resistance levels to the antibiotics screened than non-ESBLs. Majority of the ESBLs harboured the blaCTX-M group one resistance gene, which was significantly (p<0.05) associated with the aminoglycoside [aac(3')-II and aac(6')-Ib] and fluoroquinolone genes (qnrB) screened. The prevalence of virulence genes was high, mrkD (95%), wabG (93%) and entB (92%). ERIC-PCR demonstrated that clonally related isolates were recovered from different sites within the same hospital suggesting bacterial transmission. This study demonstrated that K. pneumoniae could contaminate diverse surfaces, and the persistence allowed for dissemination within the public hospital environment. The study's findings highlighted the importance of regularly monitoring hospital surfaces and emphasised on the need to strengthen current infection prevention and control (IPC) measures in hospitals to reduce the spread of bacteria.

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