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.

Antibacterial Activities of Monsonia Angustifolia and Momordica Balsamina Linn Extracts against Carbapenem-Resistant Acinetobacter Baumannii.

Carbapenemase-producing Acinetobacter baumannii (A. baumannii) is resistant to most of the available antibiotics and poses serious therapeutic challenges. The study investigated Monsonia angustifolia (M. angustifolia) and Momordica balsamina Linn (M. balsamina Linn) extracts for antibacterial activity against a clinical isolate of carbapenemase-producing A. baumannii using the Kirby Bauer disc diffusion and TLC coupled with bioautography. MIC determination experiments were conducted on a molecularly characterized A. baumannii isolate identified using VITEK2. Positive PCR detection of blaOXA-51 and blaOXA-23 confirmed isolate identity and the presence of a carbapenemase-encoding gene. Antibacterial activity was observed with the methanolic extract of M. balsamina Linn with a MIC of 0.5 mg/mL. Compounds with Rf values of 0.05; 0.17; 0.39 obtained from M. angustifolia hexane extract; compounds with Rf values of 0.58; 0.78; 0.36; 0.48; 0.5; 0.56; 0.67; 0.9 obtained from M. angustifolia dichloromethane extract; compounds with Rf values of 0.11; 0.56; 0.24; 0.37 obtained from M. angustifolia acetone extract and compounds with Rf values of 0.11; 0.27 obtained from M. angustifolia methanol extract demonstrated a level of antibacterial activity. M. angustifolia and M. balsamina Linn plant extracts have a clinically significant antibacterial activity against a carbapenemase-producing A. baumannii strain.

Phytochemical Screening, Antioxidant and Antibacterial Properties of Extracts of Viscum continuum E. Mey. Ex Sprague, a South African Mistletoe.

Viscum continuum E. Mey. Ex Sprague is a woody evergreen semi-parasitic shrub that grows on the branches of other trees. It is used by African traditional healers for post-stroke management. This study reports on the qualitative phytochemical screening and the antioxidant and antimicrobial activities of Viscum continuum's acetone, methanol, hexane and dichloromethane extracts. Standard protocols for the phytochemical screening of extracts were employed. TLC bio-autography was used for qualitative antioxidants analysis. Assays: 2,2-diphenyl-1-picrylhydrazyl, H2O2 free-radical scavenging and ferric chloride reducing power were carried out for quantitative antioxidant analysis. The antimicrobial potential of extracts was screened using disc diffusion, bio-autography and broth micro-dilution. The results indicate the presence of alkaloids, phenolics and tannins in all extracts. Acetone and methanol revealed significant amount of saponins, phenolics, tannins and terpenoids. The extracts exhibited significant antioxidant potential on TLC with positive compound bands at an Rf range of 0.05-0.89. DPPH, H2O2 and the reduction of Fe3+ to Fe2+ assays indicated that methanol extract has a strong antioxidant potential, followed by acetone, DCM and lastly hexane. The extracts of Viscum continuum show the potential to be antibacterial agents. It can be concluded that Viscum continuum extracts contain phytochemicals which are capable of mitigating against chronic health conditions such as cancer, stroke and stress-related and infectious diseases.

Whole-Genome Sequencing of a Colistin-Resistant Acinetobacter baumannii Strain Isolated at a Tertiary Health Facility in Pretoria, South Africa.

BACKGROUND: Acinetobacter baumannii's (A. baumannii) growing resistance to all available antibiotics is of concern. The study describes a colistin-resistant A. baumannii isolated at a clinical facility from a tracheal aspirate sample. Furthermore, it determines the isolates' niche establishment ability within the tertiary health facility. METHODS: An antimicrobial susceptibility test, conventional PCR, quantitative real-time PCR, phenotypic evaluation of the efflux pump, and whole-genome sequencing and analysis were performed on the isolate. RESULTS: The antimicrobial susceptibility pattern revealed a resistance to piperacillin/tazobactam, ceftazidime, cefepime, cefotaxime/ceftriaxone, imipenem, meropenem, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tigecycline, and colistin. A broth microdilution test confirmed the colistin resistance. Conventional PCR and quantitative real-time PCR investigations revealed the presence of adeB, adeR, and adeS, while mcr-1 was not detected. A MIC of 0.38 µg/mL and 0.25 µg/mL was recorded before and after exposure to an AdeABC efflux pump inhibitor. The whole-genome sequence analysis of antimicrobial resistance-associated genes detected beta-lactam: blaOXA-66; blaOXA-23; blaADC-25; blaADC-73; blaA1; blaA2, and blaMBL; aminoglycoside: aph(6)-Id; aph(3″)-Ib; ant(3″)-IIa and armA) and a colistin resistance-associated gene lpsB. The whole-genome sequence virulence analysis revealed a biofilm formation system and cell-cell adhesion-associated genes: bap, bfmR, bfmS, csuA, csuA/B, csuB, csuC, csuD, csuE, pgaA, pgaB, pgaC, and pgaD; and quorum sensing-associated genes: abaI and abaR and iron acquisition system associated genes: barA, barB, basA, basB, basC, basD, basF, basG, basH, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, and entE. A sequence type classification based on the Pasteur scheme revealed that the isolate belongs to sequence type ST2. CONCLUSIONS: The mosaic of the virulence factors coupled with the resistance-associated genes and the phenotypic resistance profile highlights the risk that this strain is at this South African tertiary health facility.

Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa.

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of bla OXA-51 . Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were bla OXA-51 -positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

Investigating multi-drug resistant Acinetobacter baumannii isolates at a tertiary hospital in Pretoria, South Africa.

PURPOSE: Antimicrobial resistance is now globally recognised amongst the greatest threat to human health. Acinetobacter baumannii's' (A. baumannii) clinical significance has been driven by its ability to obtain and transmit antimicrobial resistance factors. In South Africa, A. baumannii is a leading cause of healthcare associated infections (HAI). In this study, we investigated the genetic determinants of multi-drug resistant A. baumannii (MDRAB) at a teaching hospital in Pretoria, South Africa. METHODS: One hundred non repetitive isolates of A. baumannii were collected for the study. Antimicrobial susceptibility testing was performed using the VITEK2 system. The prevalence of antibiotic resistance associated genes and AdeABC efflux pump system were investigated using conventional PCR. Genetic relatedness of isolates was determined using rep-PCR. RESULTS: Seventy (70) of 100 isolates collected were confirmed multi-drug resistant and were blaOXA51positive. Phenotypically, the isolates where resistant to almost all tested antibiotics. One isolate showed intermediate susceptibility to tigecycline while all were susceptible to colistin. Oxacillinase gene blaOXA-23 was the most detected at 99% and only 1% was positive for blaOXA-40. For Metallo-betalactamases (MBL), blaVIMwas the most frequently detected at 86% and blaSIM-1 at 3% was the least detected. Fifty-six isolates had the required gene combination for an active efflux pump. The most prevalent clone was clone A at 69% of the isolates. Colistin and tigecycline are the most effective against investigated isolates. CONCLUSION: The major genotypic determinant for drug resistances is oxacillinases blaOXA-23. The study reports for the first time, blaOXA-40 and blaSIM-1 detection in A. baumannii in South Africa.