Usha Govinden

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria

The minimal antibiotic options for carbapenemase‐producing Gram‐negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI‐TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem‐resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase‐producing isolates. MALDI‐TOF MS and/or molecular methods like microarray, loop‐mediated isothermal amplification and real‐time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.

Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods.

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase‐producing Gram‐negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans‐complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT‐PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC‐FGH‐IJK, mexAB‐XY‐oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr‐1 gene conferring resistance to colistin was identified via WGS, trans‐complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI‐TOF MS, micro‐array and real‐time multiplex PCR hold much promise for the future as new detection tools.

An investigation of the antimicrobial and anti-inflammatory activities of crocodile oil.

ETHNOPHARMACOLOGICAL RELEVANCE Crocodile oil has been used by traditional practitioners world-wide to treat microbial infections and inflammatory conditions. However, the scientific rationale behind its use is not completely understood. This study provides an updated fatty acid profile and novel scientific evidence of the antimicrobial and anti-inflammatory properties of crocodile oil, obtained from the Nile crocodile (Crocodylus niloticus), justifying its use by traditional healers. MATERIALS AND METHODS The fatty acid content of the oil was determined by gas chromatography and the major fatty acids were identified. A microplate method was used to assess activity of the oil against Staphylococcus aureus, Klebsiella pneumoniae and Candida albicans. The anti-inflammatory activity of the oil was assessed by oral administration and topical application, utilising a mouse model of acute croton oil-induced contact dermatitis. RESULTS Sixteen fatty acids were identified with oleic, palmitic and linoleic acid being the major components of the oil. The optimal activity of the oil against the bacteria and fungus was obtained with 15% and 6% (w/v) oil respectively. No significant selectivity was observed against the bacterial species, but Candida albicans was more susceptible. The anti-inflammatory assays showed optimal activity at 3h after the oral administration of oil (60.8±5.5%) and at 12h after topical application (57.5±5.9%). This suggested a short duration of action when the oil was orally administered, and a longer duration of action when it was topically applied. CONCLUSIONS Subsequent studies may be directed towards the investigation of the mechanisms of action of the antimicrobial and anti-inflammatory activities of crocodile oil and its fatty acids.

The Molecular Epidemiology and Genetic Environment of Carbapenemases Detected in Africa.

Research articles describing carbapenemases and their genetic environments in Gram-negative bacteria were reviewed to determine the molecular epidemiology of carbapenemases in Africa. The emergence of resistance to the carbapenems, the last resort antibiotic for difficult to treat bacterial infections, affords clinicians few therapeutic options, with a resulting increase in morbidities, mortalities, and healthcare costs. However, the molecular epidemiology of carbapenemases throughout Africa is less described. Research articles and conference proceedings describing the genetic environment and molecular epidemiology of carbapenemases in Africa were retrieved from Google Scholar, Scifinder, Pubmed, Web of Science, and Science Direct databases. Predominant carbapenemase genes so far described in Africa include the blaOXA-48 type, blaIMP, blaVIM, and blaNDM in Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter spp., and Escherichia coli carried on various plasmid types and sizes, transposons, and integrons. Class D and class B carbapenemases, mainly prevalent in A. baumannii, K. pneumoniae, E. cloacae, Citrobacter spp., and E. coli were the commonest carbapenemases. Carbapenemases are mainly reported in North and South Africa as under-resourced laboratories, lack of awareness and funding preclude the detection and reporting of carbapenemase-mediated resistance. Consequently, the true molecular epidemiology of carbapenemases and their genetic environment in Africa is still unknown.

Complexity and diversity of beta-lactamase expression in inhibitor-resistant Escherichia coli from public hospitals in KwaZulu-Natal, South Africa

β-lactamase profles of 38 inhibitor-resistant Escherichia coli isolates obtained from public hospitals in KwaZulu-Natal, selected on the basis of their resistance profles to one/more of amoxicillin/clavulanate, ampicillin/sulbactam and piperacillin/tazobactam were analysed. Isolates were subjected to iso-electric focusing, plasmid profle determination, PCR of the different β-lactamase genes and sequencing thereof to detect the possible mechanism/s of inhibitor-resistance. A range of β-lactamases including two inhibitor-resistant TEM β-lactamases (TEM-145 and TEM-146), a plasmid-mediated AmpC-type β-lactamase (CMY-20), OXA-1, TEM-55, SHV-2, CTX-M1 and TEM-1 was detected. Diverse β-lactamase genes and/or enzyme combinations, and plasmid profles inferred extensive mobilisation of resistance genes. Inhibitor resistance could be attributed to a range of mechanisms including but not limited to inhibitor-resistant TEM β-lactamases, hyper-production of TEM-1, hyper-production of chromosomal AmpC and OXA β-lactamases.

CMY-20, a novel AmpC-type β-lactamase from South African clinical Escherichia coli isolates

In this study, we report the presence of a novel plasmid-mediated AmpC-type beta-lactamase that was isolated from 3 clinical Escherichia coli isolates at a tertiary teaching hospital in Durban, South Africa. The nucleotide sequence of the genes encoding this novel beta-lactamase was found to be >94% identical to the nucleotide sequences of the plasmid-mediated AmpC-type beta-lactamases originating from Citrobacter freundii. This enzyme differed from CMY-2 by 3 amino acid substitutions and was designated CMY-20.

Molecular characterisation of multidrug-resistant Pseudomonas aeruginosa from a private hospital in Durban, South Africa

AbstractBackground: Multi-drug resistant Pseudomonas aeruginosa pose a clinical challenge globally. This study delineated the molecular mechanisms of resistance to β-lactam antibiotics in multidrug-resistant P. aeruginosa isolated from a single private hospital in Durban, South Africa and ascertained clonality with regard to the isolates carrying β-lactamase genes.Methods: Seventeen P. aeruginosa isolates recovered from sputum, urine, catheter tips, pus swabs, nasal swabs and endotracheal aspirates underwent MIC determination, and phenotypic screening using the Double Disk Synergy Test (DDST) and Modified Hodge Test (MHT) to identify putative extended-spectrum β-lactamases (ESBLs), metallo-β-lactamases and other carbapenemases. Selected β-lactamase encoding genes were genotypically confirmed by PCR and sequencing. REP-PCR was conducted to determine the clonal relatedness of the 11 isolates carrying β-lactamase genes.Results: Sixteen isolates (94%) were resistant to aztreonam and piperacillin, 15 isolates ...

Mechanisms of antibiotic resistance in Haemophilus parainfluenzae

Haemophilus parainfluenzae is part of the HACEK group of fastidious bacteria commonly implicated in endocarditis and bacteremia. Previously considered as a normal respiratory, oral and sometimes genitourinary commensal, it has been recognised as a pathogen that can cause life-threatening infections in both immunocompromised and healthy individuals. It has also been reported as a bacterium that can harbor transferable antibiotic resistance genes. This paper presents a literature review on the molecular mechanisms of resistance of H. parainfluenzae to commonly prescribed antibiotics and discusses areas for further research.

Emerging fluoroquinolone and ketolide resistance in Haemophilus parainfluenzae in South Africa

Dear Editor, Fluoroquinolones, macrolides and ketolides remain effective antibiotics for the treatment of infections caused by Haemophilus influenzae and Haemophilus parainfluenzae (Haemophilus species) especially the beta lactam-resistant isolates [1,2]. Fluoroquinolone and macrolide resistance in Haemophilus species, although low, has been reported [2,3]. A Chinese study, conducted from 2006 to 2008, reported 9% to 24% resistance to various fluoroquinolones and 5.9% to 8.8% to a number of macrolides in H. parainfluenzae isolates collected from Acute Exacerbation of Chronic Obstructive Pulmonary Diseases (AECOPD) patients [4]. In comparison, a ten-year retrospective study (2004-2014) in a hospital in Germany revealed no ciprofloxacin resistance in Haemophilus species implicated in pneumonia but a high resistance rate to ampicillin (24.4 %), erythromycin (38.3 %), piperacillin (20.8 %), cefuroxime (8.5 %), ampicillinsulbactam (7.3 %), piperacillin-sulbactam (4.3 %), piperacillin-tazobactam (2.5 %), cefotaxime (2.5 %), and levofloxacin (1.6 %) [3]. There is a dearth of information on the antibiotic susceptibility of Haemophilus species (especially H. parainfluenzae) in South Africa. There is very limited information from the public and private sectors, as both of these species are neither routinely cultured, nor included in the few institution-specific surveillance projects in public health institutions. Therefore, the aim of this study is to describe the resistance trends to selected antibiotics in H. influenzae and H. parainfluenzae isolates from 2012 to 2015 using retrospective susceptibility data from a private sector laboratory in South Africa.