Investigation of carbapenem-resistant Enterobacterales isolates at a tertiary laboratory in Pretoria, South Africa.

This study aimed to investigate phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales isolates (CRE) at a tertiary laboratory in South Africa. A total of 99 CRE isolates were collected between 2019 and 2021. Carbapenemase production was tested using modified carbapenem inhibitory method. Colistin susceptibility testing was performed using the ComASP™ Colistin broth microdilution method. Conventional PCR assays were conducted for detection of mcr-1 gene and common carbapenemase genes (blaVIM, blaNDM, blaIMP, blaKPC, blaOXA-23, blaOXA-51 and blaOXA-48). Rep-PCR assay was conducted to determine the genetic relatedness of the study isolates. Majority of the isolates were Klebsiella pneumoniae (83%). Carbapenem resistant K. pneumoniae cluster was observed from ICU and surgical ward samples. Colistin resistance was observed in 13% (12/93) of the isolates namely, in 11 K. pneumoniae and one Enterobacter cloacae. The blaOXA-48 (65%) was the most prevalent gene detected followed by blaNDM (25%) and blaVIM (22%). Several K. pneumoniae isolates concomitantly carried multiple carbapenemase genes with one isolate carry up to 5 five genes blaVIM, blaNDM, blaOXA-48, blaOXA-23 and blaOXA-51. The mcr-1 gene was not detected in the isolates. Rep-PCR assay showed that most isolates matched cluster A (50%). The high prevalence of blaOXA-48, blaNDM and emerging colistin resistant isolates is of concern for patient management at this institution and needs close monitoring. Rep-PCR is a valuable tool in establishing infection clusters in resource-limited settings.

Virulence and phylogenetic analysis of enteric pathogenic Escherichia coli isolated from children with diarrhoea in South Africa.

BACKGROUND: Diarrhoeagenic Escherichia coli pose a significant risk to human health. As such, determining the source(s) of these bacteria when isolated from patients with diarrhoea is an important step in disease prevention. OBJECTIVES: To identify the presence of genes coding for virulence and phylogroups among E. coli isolated from children hospitalized due to diarrhoea in Limpopo Province, South Africa. METHODS: E. coli isolates were identified using the VITEK-2 automated system. An 11-gene multiplex polymerase chain reaction (PCR) was used to differentiate five pathogenic types of E. coli: enteroaggregative (EAEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The Clermont quadruplex PCR method was used to identify phylogroups of isolates. RESULTS: From the 133 isolates tested, 79 were confirmed as E. coli. Of these, 19.0% (15/79) were commensals and 81.0% (64/79) were positive for at least one pathotype, of which ETEC was predominant (16.5%, 13/79), followed by EAEC (10.1%, 8/79), EPEC (7.6%, 6/79) and EHEC (2.5%, 2/79). Hybrid pathotypes were also detected and EAEC/ETEC was predominant (25.3%, 20/79). Phylogroup B2 was predominant (30.4%, 24/79), followed by phylogroup B1 (22.8%, 18/79), and phylogroups C and E (both 12.7%, 10/79). Just over 6% (5/79) of isolates were non-typable. CONCLUSION: There was a high distribution of diarrhoeagenic E. coli associated with different phylogroups among children living in Limpopo Province, South Africa. This emphasizes the importance of future monitoring of virulence and phylogroup distribution of E. coli isolates in this province in particular and in South Africa as a whole.

Occurrence of Hybrid Diarrhoeagenic Escherichia coli Associated with Multidrug Resistance in Environmental Water, Johannesburg, South Africa.

This study was undertaken to determine the virulence and antibiotic resistance profiles of diarrhoeagenic Escherichia coli (DEC) in environmental waters of Johannesburg, South Africa. Samples were collected and cultured on selective media. An 11-plex PCR assay was used to differentiate five DEC, namely: enteroaggregative (EAEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The antibiotic resistance profile of isolates was determined using the VITEK®-2 automated system. The virulence profiles of 170 E. coli tested showed that 40% (68/170) were commensals and 60% (102/170) were pathogenic. EPEC had a prevalence of 19.2% (32/170), followed by ETEC 11.4% (19/170), EAEC 6% (10/170) and EHEC 3% (5/170). Hybrid DEC carrying a combination of simultaneously two and three pathogenic types was detected in twenty-eight and nine isolates, respectively. The antibiotic susceptibility testing showed isolates with multidrug resistance, including cefuroxime (100%), ceftazidime (86%), cefotaxime (81%) and cefepime (79%). This study highlighted the widespread occurrence of DEC and antibiotic resistance strains in the aquatic ecosystem of Johannesburg. The presence of hybrid pathotypes detected in this study is alarming and might lead to more severe diseases. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant strains in this area.

Molecular characteristics and genotypic diversity of enterohaemorrhagic Escherichia coli O157:H7 isolates in Gauteng region, South Africa.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is one of the major foodborne and waterborne pathogens causing severe diseases and outbreaks worldwide. There is scarcity of EHEC O157:H7 data in South Africa. This study was carried out to determine the molecular characteristics and genotypic diversity of EHEC O157:H7 isolates in the Gauteng region, South Africa. Samples were cultured on selective chromogenic media. Antibiotic susceptibility profile of isolates was determined using the VITEK®-2 automated system. Isolates were characterised using multiplex PCR assays and the genetic diversity was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 520 samples of which 270 environmental water samples and 250 stool specimens were collected and analysed. Overall, EHEC O157:H7 was recovered from 2.3% (12/520) of samples collected. Environmental water samples and clinical stool specimens showed a prevalence of 4.07% (11/270) and 0.4% (1/250) respectively. Antibiotic susceptibility profile varied from isolates with full susceptibility to isolates with resistance to multiple antibiotics. Most resistance was detected to the penicillins, specifically ampicillin (7/12), amoxicillin (3/12) and piperacillin/Tazobactam (3/12) followed by one of the folate inhibitors, trimethoprim (3/12) and the carbapenems, imipenem and meropenem (2/12) each. Three isolates harboured a combination of Shiga-toxins (Stx)-2, intimin (eae) and enterohaemolysin (hlyA) genes, while two isolates harboured the Stx-1, Stx-2 and hlyA genes. The PFGE performed showed that EHEC O157:H7 isolates were genetically diverse, with two minor pulsotypes and eight singletons. The MLST analysis identified three sequence types (STs) (ST10, ST11 and ST1204) that have been previously reported associated with outbreaks. The STs identified in this study pose a potential public health risk to consumers of untreated environmental water and closed human contacts. There is necessity to enhance surveillance in reducing the propagation of this bacterium which is a public health problem.

Antigenic distribution of Streptococcus agalactiae isolates from pregnant women at Garankuwa hospital - South Africa.

INTRODUCTION: Streptococcus agalactiae (group B streptococcus; GBS) is globally recognised as one of the leading causes of neonatal sepsis and meningitis. It also causes adverse pregnancy outcomes such as stillbirth and miscarriages. Incidence of invasive disease is increasing in non-pregnant adults with underlying medical conditions (e.g., diabetes mellitus). Epidemiological studies of GBS infections are based on capsular serotyping. Genotyping of the surface anchored protein genes is also becoming an important tool for GBS studies. Currently ten different GBS serotypes have been identified. This study was performed to determine the prevalence of GBS capsular types (CTs) and surface anchored protein genes in isolates from colonized pregnant women attending antenatal clinic, at Dr George Mukhari Academic Hospital, Garankuwa, Pretoria, South Africa. METHODS: The samples were collected over 11 months and cultured on selective media. GBS was identified using different morphological and biochemical tests. Capsular typing was done using latex agglutination test and conventional PCR. Multiplex PCR with specific primers was used to detect the surface anchored protein genes. RESULTS: Of the 413 pregnant women recruited, 128 (30.9%) were colonized with GBS. The capsular polysaccharide (CPS) typing test showed that CPS type III (29.7%) was the most prevalent capsular type followed by CPS type Ia (25.8%), II (15.6%), IV (8.6%), V (10.9%) and Ib (8.6%); 0.7% of the isolates were nontypeable. Multiplex PCR revealed that the surface proteins genes were possessed by all the capsular types: rib (44.5%), bca (24.7%), alp2/3 (17.9%), epsilon (8.6%) and alp4 (4.7%). CONCLUSION: The common capsular types found in this study are Ia, III, and II. The most common protein genes identified were rib and bca, and the distribution of the surface protein genes among the isolates of different capsular types showed similar trends to the distribution reported from previous studies.

Antibiotic resistance of Streptococcus agalactiae isolated from pregnant women in Garankuwa, South Africa.

BACKGROUND: This study was undertaken to determine the susceptibility profile and the mechanism of antibiotic resistance in Group B streptococcus (GBS) isolates detected in vaginal and rectal swabs from pregnant women attending Dr George Mukhari Academic Hospital, a University Teaching Hospital in Pretoria, South Africa. METHODS: The samples were collected over an 11-month period, cultured on selective media (colistin and nalidixic acid agar and Todd-Hewitt broth), and GBS positively identified by using different morphological and biochemical tests. The susceptibility testing was done using the Kirby-Bauer and E test methods according to CLSI guidelines 2012. The D test method was used for the detection of inducible clindamycin resistance. Multiplex PCR with specific primers was used to detect different genes coding for resistance. RESULTS: Out of 413 samples collected, 128 (30.9%) were positive with GBS. The susceptibility testing revealed that 100% of isolates were sensitive to penicillin, ampicillin, vancomycin and high level gentamicin. Erythromycin and clindamycin resistance was 21.1 and 17.2%, respectively, in which 69% had harboured constitutive macrolide, lincosamide and streptogramin B (MLS(B)), 17.4% had inducible MLS(B). The M and L phenotypes were present in 6.8% each. The methylation of target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was also distributed among the isolates. CONCLUSIONS: The study reaffirmed the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it identified the challenges of resistance to macrolides and lincosamides used as alternative drugs for individuals allergic to penicillin. More GBS treatment options for penicillin allergic patients need to be researched on.