Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana.

BACKGROUND: ESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital. MATERIALS AND METHODS: Twenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny. RESULTS: The prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The ß-lactamase genes, blaTEM-1B (10 isolates) and blaCTX-M-15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX-M-15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. 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. CONCLUSION: We identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

Quantitative microbial risk assessment for waterborne pathogens in a wastewater treatment plant and its receiving surface water body.

BACKGROUND: Access to safe water for drinking and domestic activities remains a challenge in emerging economies like South Africa, forcing resource-limited communities to use microbiologically polluted river water for personal and household purposes, posing a public health risk. This study quantified bacterial contamination and the potential health hazards that wastewater treatment plant (WWTP) workers and communities may face after exposure to waterborne pathogenic bacteria in a WWTP and its associated surface water, respectively. RESULTS: Escherichia coli (Colilert®-18/ Quanti-Tray® 2000) and enterococci (Enterolert®/ Quanti-Tray® 2000) were quantified and definitively identified by real-time polymerase chain reaction targeting the uidA and tuf genes, respectively. An approximate beta-Poisson dose-response model was used to estimate the probability of infection (Pi) with pathogenic E. coli. Mean E. coli concentration ranged from 2.60E+ 02/100 mL to 4.84E+ 06/100 mL; enterococci ranged from 2.60E+ 02/100 mL to 3.19E+ 06/100 mL across all sampled sites. Of the 580 E. coli isolates obtained from this study, 89.1% were intestinal, and 7.6% were extraintestinal pathogenic E. coli. The 579 enterococci obtained were 50.4% E. faecalis (50.4%), 31.4% E. faecium, 3.5%, E. casseliflavus and 0.7% E. gallinarum. The community health risk stemming from the use of the water for recreational and domestic purposes revealed a greater health risk (Pi) from the ingestion of 1 mL of river water from upstream (range, 55.1-92.9%) than downstream (range, 26.8-65.3%) sites. The occupational risk of infection with pathogenic E. coli for workers resulting from a once-off unintentional consumption of 1 mL of water was 0% (effluent) and 23.8% (raw influent). Multiple weekly exposures of 1 mL over a year could result in a Pi of 1.2 and 100% for the effluent and influent, respectively. CONCLUSION: Our findings reveal that there is a potentially high risk of infection for WWTP workers and communities that use river water upstream and downstream of the investigated WWTP.

Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009-2017.

BACKGROUND: Typhoid fever remains a major public health problem in Zimbabwe with recurrent outbreaks reported since 2009. To provide guidance on appropriate treatment choice in order to minimise the morbidity and mortality of typhoid fever and prevent large scale outbreaks, we investigated the antimicrobial susceptibility patterns, prevalence of Salmonella enterica serotype Typhi (S. Typhi) H58 haplotype and molecular subtypes of S. Typhi from outbreak strains isolated from 2009 to 2017 in Zimbabwe and compared these to isolates from neighbouring African countries. METHODS: Antimicrobial susceptibility testing was performed on all isolates using the disk diffusion, and E-Test, and results were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (2017). S. Typhi H58 haplotype screening was performed on 161 (58.3%) isolates. Pulsed-field gel electrophoresis (PFGE) was performed on 91 selected isolates across timelines using antibiotic susceptibility results and geographical distribution (2009 to 2016). RESULTS: Between 2009 and 2017, 16,398 suspected cases and 550 confirmed cases of typhoid fever were notified in Zimbabwe. A total of 276 (44.6%) of the culture-confirmed S. Typhi isolates were analysed and 243 isolates (88.0%) were resistant to two or more first line drugs (ciprofloxacin, ampicillin and chloramphenicol) for typhoid. The most common resistance was to ampicillin-chloramphenicol (172 isolates; 62.3%). Increasing ciprofloxacin resistance was observed from 2012 to 2017 (4.2 to 22.0%). Out of 161 screened isolates, 150 (93.2%) were haplotype H58. Twelve PFGE patterns were observed among the 91 isolates analysed, suggesting some diversity exists among strains circulating in Zimbabwe. PFGE analysis of 2013, 2014 and 2016 isolates revealed a common strain with an indistinguishable PFGE pattern (100% similarity) and indistinguishable from PFGE patterns previously identified in strains isolated from South Africa, Zambia and Tanzania. CONCLUSIONS: Resistance to first line antimicrobials used for typhoid fever is emerging in Zimbabwe and the multidrug resistant S. Typhi H58 haplotype is widespread. A predominant PFGE clone circulating in Zimbabwe, South Africa, Zambia and Tanzania, argues for cross-border cooperation in the control of this disease.

Incidence and molecular characterisation of lumpy skin disease virus in Zimbabwe using the P32 gene.

Between January, 2013 and December, 2014, there was a lumpy skin disease (LSD) outbreak that affected cattle in different localities of Zimbabwe. The outbreak resulted in severe economic losses to the livestock industry. A retrospective study was conducted by examining stored veterinary records of the LSD outbreak at the Central Veterinary Laboratory (CVL) in Harare, Zimbabwe. Over the 2-year period, a total of 10,038 cases and 880 deaths (8.77 %) were recorded. LSD was reported from all regions of the country, with the highest incidence occurring in Mashonaland West (30.95 %) and Midlands province (14.59 %). The frequency of reported outbreaks was highest in March and April, with the lowest reported cases occurring in November. A total of 25 representative specimens (skin biopsies) were collected from nodular skin lesions of infected cattle, and after viral DNA isolation, the P32 gene was successfully amplified, by using PCR, in 88 % (22/25) of all assayed specimens. Out of the 22 samples that showed amplification, 16 (73 %) were selected for DNA sequencing, and from these, 13 sequences were submitted to GenBank and assigned accession numbers: KX033494, KX033495, KX033496, KX033497, KXO33498, KX033499, KX033500, KX033501, KX033502, KX033503, KX033504, KX033505 and KX033506. Phylogenetic analyses of the 13 sequences was done by using MEGA 7 and showed that the viruses formed two major clusters implying that at least two strains of LSDV are in circulation in Zimbabwe. This study provides the first report on the incidence and molecular characterisation of LSDV in Zimbabwe.

Genomic Characterization of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Implicated in Bloodstream Infections, KwaZulu-Natal, South Africa: A Pilot Study.

Staphylococcus aureus is an opportunistic pathogen and a leading cause of bloodstream infections, with its capacity to acquire antibiotic resistance genes posing significant treatment challenges. This pilot study characterizes the genomic profiles of S. aureus isolates from patients with bloodstream infections in KwaZulu-Natal, South Africa, to gain insights into their resistance mechanisms, virulence factors, and clonal and phylogenetic relationships. Six multidrug-resistant (MDR) S. aureus isolates, comprising three methicillin-resistant S. aureus (MRSA) and three methicillin-susceptible S. aureus (MSSA), underwent whole genome sequencing and bioinformatics analysis. These isolates carried a range of resistance genes, including blaZ, aac(6')-aph(2″), ant(9)-Ia, ant(6)-Ia, and fosB. The mecA gene, which confers methicillin resistance, was detected only in MRSA strains. The isolates exhibited six distinct spa types (t9475, t355, t045, t1265, t1257, and t7888) and varied in virulence gene profiles. Panton-Valentine leukocidin (Luk-PV) was found in one MSSA isolate. Two SCCmec types, IVd(2B) and I(1B), were identified, and the isolates were classified into four multilocus sequence types (MLSTs), with ST5 (n = 3) being the most common. These sequence types clustered into two clonal complexes, CC5 and CC8. Notably, two MRSA clones were identified: ST5-CC5-t045-SCCmec_I(1B) and the human-associated endemic clone ST612-CC8-t1257-SCCmec_IVd(2B). Phylogenomic analysis revealed clustering by MLST, indicating strong genetic relationships within clonal complexes. These findings highlight the value of genomic surveillance in guiding targeted interventions to reduce treatment failures and mortality.

Antimicrobial resistance of Escherichia coli isolated from chickens with colibacillosis in and around Harare, Zimbabwe.

Colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC), can lead to great economic losses in the poultry industry. The aim of this study was to determine the prevalence of antibiotic resistance and antibiotic resistance patterns in APEC in Zimbabwe. From 503 chickens diagnosed with colibacillosis, 103 E. coli isolates were obtained. Isolation and identification of E. coli were carried out using microscopy and biochemical tests. The disc diffusion method was used to determine antibiotic susceptibility of the isolates to 8 commercial antibiotics. Many isolates exhibited resistance to more than one antibiotic. Antibiogram profiles indicated maximum resistance to tetracycline (100%), bacitracin (100%), and cloxacillin (100%) and a high prevalence of resistance to ampicillin (94.1%). However; there were high prevalences of sensitivity to ciprofloxacin (100%) and gentamycin (97.1%). The isolates showed moderate rates of sensitivity to chloramphenicol and neomycin. All isolates in this study showed multidrug resistance because they were all resistant to 3 or more antibiotics. Seven multidrug resistance patterns were observed. The most common pattern (resistance to ampicillin, bacitracin, cloxacillin, and tetracycline) was exhibited by 30 isolates. Our findings show that there is emerging drug resistance in APEC associated with colibacillosis in Zimbabwe. The observed high level of multidrug resistance could hamper the treatment of colibacillosis in Zimbabwe.

Antibiotic resistance, pathotypes, and pathogen-host interactions in Escherichia coli from hospital wastewater in Bulawayo, Zimbabwe.

This study aimed to characterise E. coli strains isolated from hospital wastewater effluent in Bulawayo, Zimbabwe, using both molecular and cytological approaches. Wastewater samples were aseptically collected from the sewerage mains of a major public referral hospital in Bulawayo province weekly for one month. A total of 94 isolates were isolated and confirmed as E. coli through biotyping and PCR targeting the uidA housekeeping gene. A total of 7 genes (eagg, eaeA, stx, flicH7, ipaH, lt, and st genes) coding for virulence in diarrheagenic E. coli were targeted. Antibiotic susceptibility of E. coli was determined against a panel of 12 antibiotics through the disk diffusion assay. The infectivity status of the observed pathotypes was investigated using HeLa cells through adherence, invasion, and intracellular assay. None of the 94 isolates tested positive for the ipaH and flicH7genes. However, 48 (53.3%) isolates were enterotoxigenic E. coli (ETEC) (lt gene positive), 2 (2.13%) isolates were enteroaggregative E. coli (EAEC) (eagg gene), and 1 (1.06%) isolate was enterohaemorrhagic E. coli (EHEC) (stx and eaeA). A high level of sensitivity was observed in E. coli against ertapenem (98.9%), and Azithromycin (75.5%). The highest resistance was against ampicillin (92.6%) and sulphamethoxazole-trimethoprim (90.4%). Seventy-nine (84%) E. coli isolates exhibited multidrug resistance. The infectivity study results indicated that environmentally isolated pathotypes were as infective as the clinically isolated pathotypes for all three parameters. No adherent cells were observed using ETEC, and no cells were observed in the intracellular survival assay using EAEC. This study revealed that hospital wastewater is a hotspot for pathogenic E. coli and that the environmentally isolated pathotypes maintained their ability to colonise and infect mammalian cells.

Metal Ions and their Effects on Antimicrobial Resistance Development in Wastewater.

Antimicrobial resistance (AMR) is a global health challenge and there is increasing recognition of the role of the environment, particularly wastewater, in the development and spread of AMR. Although trace metals are common contaminants in wastewater, the quantitative effects of trace metals on AMR in wastewater settings remain understudied. We experimentally determined the interactions between common antibiotic residues and metal ions found in wastewater and investigated their effects on the development of antibiotic resistance in Escherichia coli over time. These data were then used to expand on a previously developed computational model of antibiotic resistance development in continuous flow settings to incorporate the effects of trace metals acting in combination with multiple antibiotic residues. We found that the common metal ions, copper and iron, interact with both ciprofloxacin and doxycycline at wastewater relevant concentrations. This can significantly affect resistance development due to antibiotic chelation of the metal ions causing a reduction in the antibiotics' bioactivity. Furthermore, modeling the effect of these interactions in wastewater systems showed the potential for metal ions in wastewater to significantly increase the development of antibiotic resistant E. coli populations. These results demonstrate the need to quantitatively understand the effects of trace metal-antibiotic interactions on AMR development in wastewater.

Multidrug-resistant Gram-negative bacterial colonization in patients, carriage by healthcare workers and contamination of hospital environments in Ghana.

BACKGROUND: Patients already colonized with multidrug-resistant (MDR) Gram-negative bacteria (GNB) on admission to critical care units may be an important source of transmission of these bacteria in hospitals. We sought to determine the prevalence of MDR GNB colonization in patients, staff and the ward environment and to assess the risk factors for colonization of patients in wards. METHODS: The study was conducted from April 2021 to July 2021 in a teaching hospital in Ghana. MDR GNB were isolated from rectal, and hand swabs were taken from patients on admission and after 48 h. Swabs from HCW's hands and the ward environment were also taken. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis. RESULTS: MDR GNB rectal colonization rate among patients was 50.62% on admission and 44.44% after 48 h. MDR GNB 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 h of admission. 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. CONCLUSION: The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW's hands, and the contamination of hospital environments highlights the need for patient screening and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals.

Mobile genetic elements-mediated Enterobacterales-associated carbapenemase antibiotic resistance genes propagation between the environment and humans: A One Health South African study.

We, (1) studied carbapenem-resistant Enterobacterales (CRE) in the environment, humans, and animals, within the same geographical area and, (2) delineated the isolates' resistome, mobilome, virulome, and phylogeny. Following ethical approval, 587 samples (humans = 230, pigs = 345, and water = 12) were collected and cultured on CRE selective media. Confirmatory identification and antibiotic susceptibility testing were performed using the VITEK 2 automated platform. The resistomes, virulomes, mobilomes, and phylogenies were ascertained by whole genome sequencing. Nineteen (3.2%), i.e., 15/19 humans and 4/19 environmental, but no pig, CRE were obtained. CREs included Klebsiella pneumoniae 9/19 (47%), Enterobacter hormaechei 6/19 (32%), Klebsiella quasipneumoniae 2/19 (11%), a novel ST498 Citrobacter freundii 1/19 (5%) and Serratia marcescens 1/19 (5%). Eleven isolates were extensively drug-resistant; eight were multidrug-resistant. Sixteen CRE harbored the blaOXA-181, blaOXA-48, blaOXA-484, blaNDM-1, and blaGES-5 genes. Multiple species/clones carried blaOXA-48 and blaNDM-1 carbapenemase-encoding genes with respective mobile genetic elements (MGEs). The IncFIB(K) plasmid replicon was found in most human K. pneumoniae strains (7/9) and all environmental K. quasipneumoniae isolates; most K. pneumoniae produced OXA-181 (5/9). The (Col440I) plasmid replicon, identified in 11 (26.82%) isolates, mainly E. hormaechei (n = 6), predominated both sectors. Most ß-lactamase-encoding genes were associated with class 1 integrons IntI1, insertion sequences (IS) (IS91, IS5075, IS30, IS3000, IS3, IS19, ISKpn19, IS5075) and transposons (Tn3). The IncL/M(pMU407) and IncL/M(pOXA48) plasmid replicons were found exclusively in K. pneumoniae; all but one of these strains produced OXA-181. Also, the Klebsiella spp. harbored 80 virulence genes. Phylogenomic clustered identified isolates with other carbapenemase-producing K. pneumoniae, E. hormaechei, S. marcescens, and C. freundii from different South African sources (animals, environment, and humans). We delineated the resistome, mobilome, virulome, and phylogeny of carbapenemase-producing Enterobacterales in humans and environment, highlighting antibiotic resistance genes propagation via MGEs across sectors, emphasizing a One Health approach to AMR.

Molecular Epidemiological Characterisation of ESBL- and Plasmid-Mediated AmpC-Producing Escherichia coli and Klebsiella pneumoniae at Kamuzu Central Hospital, Lilongwe, Malawi.

The global rise in infections caused by multidrug resistant (MDR) Enterobacterales poses a public health problem. We have performed a molecular epidemiological characterisation of representative plasmid-mediated AmpC (pAmpC) and ESBL-positive clinical isolates of Escherichia coli (n = 38) and Klebsiella pneumoniae (n = 17) from a tertiary hospital in Malawi collected in 2017. BlaCTX-M-15 was the most prevalent ESBL-determinant in E. coli (n = 30/38) and K. pneumoniae (n = 17/17), whereas blaCMY-2 was detected in nearly all AmpC-phenotype E. coli (n = 15/17). Whole genome sequencing revealed dominant globally disseminated E. coli sequence types (STs); ST410 (n = 16), ST131 (n = 7), and ST617 (n = 6). The ST distribution in K. pneumoniae was more diverse but included ST101 (n = 2), ST14 (n = 2), and ST340 (n = 2), all considered high-risk MDR clones. The isolates expressed an MDR profile, including resistance against commonly used antibiotics, such as fluoroquinolones, aminoglycosides, and/or trimethoprim-sulfamethoxazole, and harboured corresponding resistance determinants. Clonal analyses of the major STs of E. coli revealed closely related genetic clusters within ST410, ST131, and ST617 supporting within-hospital transmission between patients and/or via a common reservoir. The overall findings add to the limited knowledge on the molecular epidemiology of MDR E. coli and K. pneumoniae in Malawi and may help health policy makers to identify areas to target when addressing this major threat of antibiotic resistance.

Longitudinal Surveillance of Antibiotic Resistance in Escherichia coli and Enterococcus spp. from a Wastewater Treatment Plant and Its Associated Waters in KwaZulu-Natal, South Africa.

We assessed the prevalence, distribution, and antibiotic resistance patterns of Escherichia coli and Enterococcus spp. isolated from raw and treated wastewater of a major wastewater treatment plant (WWTP) in KwaZulu-Natal, South Africa and the receiving river water upstream and downstream from the WWTP discharge point. Escherichia coli and enterococci were isolated and counted using the Colilert®-18 Quanti-Tray® 2000 and Enterolert®-18 Quanti-Tray 2000 systems, respectively. A total of 580 quantitative PCR-confirmed E. coli and 579 enterococci were randomly chosen from positive samples and tested for in vitro antibiotic susceptibility using the disk diffusion assay against 20 and 16 antibiotics, respectively. The removal success of the bacterial species through the treatment procedure at the WWTP was expressed as log removal values (LRVs). Most E. coli were susceptible to meropenem (94.8%) and piperacillin-tazobactam (92.9%), with most Enterococcus susceptible to ampicillin (97.8%) and vancomycin (96.7%). In total, 376 (64.8%) E. coli and 468 (80.8%) Enterococcus isolates showed multidrug resistance (MDR). A total of 42.4% (246/580) E. coli and 65.1% (377/579) enterococci isolates had multiple antibiotic resistance indices >0.2. The LRV for E. coli ranged from 2.97 to 3.99, and for enterococci the range was observed from 1.83 to 3.98. A high proportion of MDR E. coli and enterococci were present at all sampled sites, indicating insufficient removal during wastewater treatment. There is a need to appraise the public health risks associated with bacterial contamination of environmental waters arising from such WWTPs to protect the health of users of the receiving water bodies.

Genomic Insights of Multidrug-Resistant Escherichia coli From Wastewater Sources and Their Association With Clinical Pathogens in South Africa.

There is limited information on the comparative genomic diversity of antibiotic-resistant Escherichia coli from wastewater. We sought to characterize environmental E. coli isolates belonging to various pathotypes obtained from a wastewater treatment plant (WWTP) and its receiving waters using whole-genome sequencing (WGS) and an array of bioinformatics tools to elucidate the resistomes, virulomes, mobilomes, clonality, and phylogenies. Twelve multidrug-resistant (MDR) diarrheagenic E. coli isolates were obtained from the final effluent of a WWTP, and the receiving river upstream and downstream of the WWTP were sequenced on an Illumina MiSeq machine. The multilocus sequence typing (MLST) analysis revealed that multiple sequence types (STs), the most common of which was ST69 (n = 4) and ST10 (n = 2), followed by singletons belonging to ST372, ST101, ST569, ST218, and ST200. One isolate was assigned to a novel ST ST11351. A total of 66.7% isolates were positive for ß-lactamase genes with 58.3% harboring the bla TEM1B gene and a single isolate the blaCTX-M-14 and blaCTX-M-55 extended-spectrum ß-lactamase (ESBL) genes. One isolate was positive for the mcr-9 mobilized colistin resistance gene. Most antibiotic resistance genes (ARGs) were associated with mobile genetic support: class 1 integrons (In22, In54, In191, and In369), insertion sequences (ISs), and/or transposons (Tn402 or Tn21). A total of 31 virulence genes were identified across the study isolates, including those responsible for adhesion (lpfA, iha, and aggR), immunity (air, gad, and iss), and toxins (senB, vat, astA, and sat). The virulence genes were mostly associated with IS (IS1, IS3, IS91, IS66, IS630, and IS481) or prophages. Co-resistance to heavy metal/biocide, antibiotics were evident in several isolates. The phylogenomic analysis with South African E. coli isolates from different sources (animals, birds, and humans) revealed that isolates from this study mostly clustered with clinical isolates. Phylogenetics linked with metadata revealed that isolates did not cluster according to source but according to ST. The occurrence of pathogenic and MDR isolates in the WWTP effluent and the associated river is a public health concern.

Genomic Analysis of Enterococcus spp. Isolated From a Wastewater Treatment Plant and Its Associated Waters in Umgungundlovu District, South Africa.

We investigated the antibiotic resistome, mobilome, virulome, and phylogenomic lineages of Enterococcus spp. obtained from a wastewater treatment plant and its associated waters using whole-genome sequencing (WGS) and bioinformatics tools. The whole genomes of Enterococcus isolates including Enterococcus faecalis (n = 4), Enterococcus faecium (n = 5), Enterococcus hirae (n = 2), and Enterococcus durans (n = 1) with similar resistance patterns from different sampling sites and time points were sequenced on an Illumina MiSeq machine. Multilocus sequence typing (MLST) analysis revealed two E. faecalis isolates that had a common sequence type ST179; the rest had unique sequence types ST841, and ST300. The E. faecium genomes belonged to 3 sequence types, ST94 (n = 2), ST361 (n = 2), and ST1096 (n = 1). Detected resistance genes included those encoding tetracycline [tet(S), tet(M), and tet(L)], and macrolides [msr(C), msr(D), erm(B), and mef(A)] resistance. Antibiotic resistance genes were associated with insertion sequences (IS6, ISL3, and IS982), and transposons (Tn3 and Tn6000). The tet(M) resistance gene was consistently found associated with a conjugative transposon protein (TcpC). A total of 20 different virulence genes were identified in E. faecalis and E. faecium including those encoding for sex pheromones (cCF10, cOB1, cad, and came), adhesion (ace, SrtA, ebpA, ebpC, and efaAfs), and cell invasion (hylA and hylB). Several virulence genes were associated with the insertion sequence IS256. No virulence genes were detected in E. hirae and E. durans. Phylogenetic analysis revealed that all Enterococcus spp. isolates were more closely related to animal and environmental isolates than clinical isolates. Enterococcus spp. with a diverse range of resistance and virulence genes as well as associated mobile genetic elements (MGEs) exist in the wastewater environment in South Africa.

Molecular characterization and antibiotic resistance patterns of avian fecal Escherichia coli from turkeys, geese, and ducks.

BACKGROUND AND AIM: Avian fecal Escherichia coli (AFEC) are considered to be the natural reservoir of pathogenic strains in extraintestinal infections as such characterization of AFEC gives insight into the spread of the potential pathogenic lineage. The aim of the study was to investigate the reservoirs of avian pathogenic E. coli (APEC) from fecal samples of healthy ducks, geese, and turkeys by determining the antibiotic resistance patterns of AFEC isolates from turkeys, geese and ducks and characterization of the isolates using virulence genes, plasmid profiles, and phylogenetic grouping. MATERIALS AND METHODS: The disc diffusion method was used to determine antibiotic resistance of 100 AFEC isolates from turkeys (9), geese (29), and ducks (62) to 8 antibiotics. Molecular characterization of the isolates was done by multiplex polymerase chain reaction to investigate the presence of 12 virulence genes, plasmid profiling, and phylogenetic grouping based on the 16S rRNA sequences. RESULTS: Antibiogram profiles indicated maximum resistance to cloxacillin (100%) and bacitracin (100%) for all AFEC isolates and high sensitivity to ciprofloxacin; however, all isolates exhibited multi-drug resistance. The AFEC isolates from turkeys (6) and geese (12) did not contain virulence genes. The frz (3.7%), sitD (29.6%), and fimH (92.5%) were detected in the duck isolates. None of the isolates had the KpsM, iutA, vat, sitA, hlyF, pstB, ompT, uvrY, and sopB genes. Plasmid profiling gave four plasmid profiles with the plasmids ranging from 1.5 to 55 kb. Phylogenetic analysis of 16S rRNA sequences revealed similarities between AFEC isolates from the different poultry species, as the isolates did not cluster according to avian species. CONCLUSION: AFEC isolates are potential reservoirs of APEC as they contain some of the virulence genes associated with APEC. Multidrug resistance is high in AFEC isolated from healthy birds. This is a public health concern.

Virulence gene profiles of avian pathogenic Escherichia coli isolated from chickens with colibacillosis in Bulawayo, Zimbabwe.

Colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC), is one of the main causes of economic losses in the poultry industry worldwide. This study was carried out in order to determine the APEC-associated virulence genes contained by E. coli isolates causing colibacillosis in chickens. A total of 45 E. coli isolates were obtained from the diagnostics and research branch of the Central Veterinary Laboratories, Bulawayo, Zimbabwe. These isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. The presence of the iutA, hlyF, ompT, frz, sitD, fimH, kpsM, sitA, sopB, uvrY, pstB and vat genes were investigated by multiplex polymerase chain reaction (PCR) assay. Of the 45 isolates, 93% were positive for the presence of at least one virulence gene. The three most prevalent virulence genes were iutA (80%), fimH (33.3%) and hlyF (24.4%). The kpsM, pstB and ompT genes had the lowest prevalence, having been detected in only 2.2% of the isolates. All 12 virulence genes studied were detected in the 45 APEC isolates. Virulence gene profiles were constructed for each APEC isolate from the multiplex data. The APEC isolates were profiled as 62.2% fitting profile A, 31.1% profile B and 6.7% profile C. None of the isolates had more than seven virulence genes. Virulence profiles of Zimbabwean APEC isolates are different from those previously reported. Zimbabwean APEC isolates appear to be less pathogenic and may rely on environmental factors and stress in hosts to establish infection.

Molecular Identification of Mycobacterium Species of Public Health Importance in Cattle in Zimbabwe by 16S rRNA Gene Sequencing.

Mycobacterium species are naturally found in the environment as well as in domestic animals such as cattle. So far, more than 150 species of Mycobacterium, some of which are pathogenic, have been identified. Laboratory isolation, detection and identification of Mycobacterium species are therefore critical if human and animal infections are to be controlled. The objective of this study was to identify Mycobacterium species isolated in cattle in Zimbabwe using 16S ribosomal RNA gene amplification and sequencing. A total of 134 cow dung samples were collected throughout Zimbabwe and mycobacteria were isolated by culture. Only 49 culture isolates that were found to be acid-fast bacilli positive by Ziehl-Neelsen staining. The 16S rRNA gene was successfully amplified by PCR in 41 (84%) of the samples. There was no amplification in 8 (16%) of the samples. Out of the 41 samples that showed amplification, 26 (63%) had strong PCR bands and were selected for DNA sequencing. Analysis of the DNA sequences showed that 7 (27%) belonged to Mycobacterium neoaurum, 6 (23%) belonged to Mycobacterium fortuitum, 3 (12%) to Mycobacterium goodii, 2 (1%) to Mycobacterium arupense, 2 (1%) to Mycobacterium peregrinum or M. septicum and 1 isolate (0.04%) to Mycobacterium elephantis. There were 5 (19%) isolates that were non-mycobacteria and identified as Gordonia terrae, a close relative of Mycobacterium. The study therefore provided a molecular basis for detection and identification of Mycobacterium species in animals and humans.

Evolution of antimicrobial resistance of Salmonella enteritidis (1972-2005).

With the extensive use of antibiotics in livestock production, surveillance revealed an increase in Salmonella resistance to the commonly used antimicrobials in veterinary and public health. This serious threat to health care is further exacerbated by the limited epidemiological information about the common zoonotic agent, Salmonella enteritidis, required to determine antibiotic therapy. The aim was to characterise the antimicrobial resistance patterns of S. enteritidis isolates across different timelines (1972-2005) with accompanying genetic changes being investigated. Thirty-seven stored S. enteritidis isolates were collected from the Central Veterinary Laboratory, Harare, with antimicrobial susceptibility determined against eight antibiotics. Plasmids were isolated to analyse any genetic variation. An overall significant increase in resistance (p < 0.05) to nalidixic acid (0% - 10%), ampicillin (14.3% - 50%), tetracycline (14.3% - 30%) and erythromycin (71.4% - 100%) was observed across the timeline. However, the highest rates of susceptibility were maintained for gentamicin, sulphamethoxazole-trimethoprim, kanamycin and chloramphenicol. We report an increase in multidrug resistance (MDR) of 14.2% - 50% with an increase in resistotypes and plasmid profiles across the timeline. Eleven plasmid profiles were obtained in the 37 isolates studied with a minority of isolates (21.6%, 8/37) harbouring a 54 kb plasmid, commonly serovar-specific. A concerning increase in antimicrobial resistance to commonly administered drugs was observed across the timeline. The surge in MDR is of great concern and implies the need for consistent antimicrobial stewardship. No correlation was observed between the plasmid and antibiotic profiles.

Prevalence and drug resistance in bacteria of the urinary tract infections in Bulawayo province, Zimbabwe.

OBJECTIVE: To obtain data on the prevalence of antibiotic resistance in bacteria isolated from patients with suspected urinary tract infection in Bulawayo province, Zimbabwe. METHOD: Over a period of one year, 257 urine samples were analyzed for bacteria by standard procedures. Antimicrobial susceptibility testing of isolated bacteria was done using the disk diffusion method. RESULTS: The isolated bacteria were Escherichia coli (40.3%), coagulase negative Staphylococcus (16.1%), Klebsiella spp (11.2%), Staphylococcus aureus (8%), Group A Streptococcus (8%) and Klebsiella oxytoca (8%). Antibiotic susceptibility testing was done using the disc diffusion method on Mueller-Hinton agar. It revealed a high resistance to ampicillin (84.5%) and cotrimoxazole (68.5%) among the Gram negative bacilli. Gram positive cocci showed resistance to Nalidixic acid (81%) and cotrimoxazole (69%). E. coli was susceptible to most of the drugs but 84% of the strains were resistant to ampicillin, and 68% to cotrimoxazole. All isolates were sensitive to Nicene. CONCLUSION: The high levels of ampicillin and cotrimoxazole resistance in E. coli and other enterobactericiae suggest the need to perform urinalysis and antibiotic susceptibility testing in all patients. Nicene should be considered as the first line therapy for all age groups. It is important for physicians to know susceptibility data for UTIs in order to optimize the use of empirical therapy.