Anou M. Somboro

NOTA: a potent metallo-β-lactamase inhibitor

Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa; Antimicrobial Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa; Biomedical Resource Unit, Westville Campus, University of KwaZulu-Natal, Durban, South Africa; Department of Virology, National Health Laboratory Service/University of KwaZulu-Natal, c/o Inkosi Albert Luthuli Central Hospital, Durban, South Africa; Science for Life Laboratory, Drug Discovery and Development Platform, and Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

Tissue distribution of pretomanid in rat brain via mass spectrometry imaging

Abstract 1. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) combines the sensitivity and selectivity of mass spectrometry with spatial analysis to provide a new dimension for histological analyses of the distribution of drugs in tissue. Pretomanid is a pro-drug belonging to a class of antibiotics known as nitroimidizoles, which have been proven to be active under hypoxic conditions and to the best of our knowledge there have been no studies investigating the distribution and localisation of this class of compounds in the brain using MALDI MSI. 2. Herein, we report on the distribution of pretomanid in the healthy rat brain after intraperitoneal administration (20 mg/kg) using MALDI MSI. Our findings showed that the drug localises in specific compartments of the rat brain viz. the corpus callosum, a dense network of neurons connecting left and right cerebral hemispheres. 3. This study proves that MALDI MSI technique has great potential for mapping the pretomanid distribution in uninfected tissue samples, without the need for molecular labelling.

MALDI MSI and LC‐MS/MS: Towards preclinical determination of the neurotoxic potential of fluoroquinolones

Fluoroquinolones are broad-spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague-Dawley rats, via Multicenter matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a sensitive LC-MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright

Synthesis, in vitro evaluation, and 68Ga-radiolabeling of CDP1 toward PET/CT imaging of bacterial infection

Bacterial infections are a major concern in the human health sector due to poor diagnosis and development of multidrug‐resistant strains. PET/CT provides a means for the non‐invasive detection and localization of the infectious foci; however, the radiotracers available are either cumbersome to prepare or their exact contribution toward the imaging is not yet established. Human antimicrobial peptides are of interest for development as PET radiotracers as they are an integral component of the immune system, non‐immunogenic toward the recipient, and show selectivity toward pathogens such as bacteria. Herein we report on the potential of LL37, a human cathelicidin antimicrobial peptide, as a radiotracer for bacterial imaging. Bifunctional chelator 1,4,7‐triazacyclononane,1‐glutaric acid‐4,7‐acetic acid was utilized to functionalize the antimicrobial peptide, which in turn was capable of chelating gallium. The synthesized natGa‐CDP1 showed bacterial selectivity and low affinity toward hepatic cells, which are favorable characteristics for further preclinical application.

Antibiotic resistance profiles of Campylobacter species in the South Africa private health care sector

INTRODUCTION There is a dearth of surveillance data on clinical Campylobacter in South Africa, particularly in the private healthcare environment. We investigated the prevalence of resistance to first-line antibiotics used to treat campylobacterioses in clinical Campylobacter isolates from a private pathology laboratory. METHODOLOGY Identification of the Campylobacter specific genes were confirmed by PCR. Minimum inhibitory concentrations were determined using the broth micro-dilution method against macrolides (erythromycin, azithromycin), fluoroquinolones (ciprofloxacin, gatifloxacin) and tetracycline. RESULTS Seventy-two Campylobacter isolates were identified by PCR, with 54 (75%) being classified as C. jejuni and 18 (25%) as C. coli. Of these, 11 (20.4%) C. jejuni and six (33.3%) C. coli strains were resistant to ciprofloxacin and three (7.41%) C. jejuni and three (16.7%) C. coli strains were resistant to gatifloxacin. The number of C. jejuni strains resistant to erythromycin and azithromycin was 17 (31.5%) and 36 (50%) respectively, while the resistance of C. coli strains to erythromycin and azithromycin were seven (38.9%) and 14 (77.8%) respectively. Resistance to tetracycline was detected in 10 (55.6%) C. coli and 14 (25.9%) C. jejuni strains. CONCLUSION In the light of these resistant profiles, the lack of a South African Campylobacter surveillance program is of concern. Relatively high prevalence of resistance in clinical isolates of C. jejuni and C. coli to the fluoroquinolones, macrolides and tetracycline used in first line treatment is of great concern. The efficacy treating human campylobacteriosis should thus be revisited.

Plasmid-mediated resistance and virulence mechanisms in the private health sector in KwaZulu-Natal, South Africa: An investigation of methicillin resistant Staphylococcus aureus (MRSA) clinical isolates collected during a three month period

OBJECTIVES Due to the lack of information on the plasmid content of MRSA strains in South Africa (SA), this study investigated the resistance and virulence mechanisms of 27 clinical isolates from the private health care sector over a period of 3 months. METHODS Plasmids were extracted and the presence of MRSA confirmed by the presence of mecA. The isolates were subjected to antimicrobial susceptibility testing and molecular characterization of common resistance encoding genes and frequently encountered virulence factors by PCR using plasmid DNA as the template. The genetic relatedness between the isolates was determined by pulsed field gel electrophoresis (PFGE). RESULTS All isolates were plasmid positive, and displayed ampillicin, ciprofloxacin, gentamicin, rifampicin, tetracycline, erythromycin, and clindamycin resistance. They were all fully susceptible to daptomycin, linezolid, vancomycin, tigecycline and fusidic acid. Multidrug resistance (MDR) was found in 74.1% (20/27) of the MRSA isolates. The frequency of the resistance and virulence genes ranged from 100% to 0%. PFGE analysis revealed 10 pulsotypes, designated A-J, which showed correlation with resistance profile of the isolates in each group. Of note, 85.2% (23/27) of the isolates clustered into six major PFGE types giving an indication of similar circulating MRSA clones. CONCLUSIONS This study highlights the genetic diversity and resistance mechanisms in MRSA strains from the private health sector in SA hence the need for implementing effective infection control programs.

Diversity and proliferation of metallo-β-lactamases: a clarion call for clinically effective metallo-β-lactamase inhibitors.

The worldwide proliferation of life-threatening metallo-β-lactamase (MBL)-producing Gram-negative bacteria is a serious concern to public health. MBLs are compromising the therapeutic efficacies of β-lactams, particularly carbapenems, which are last-resort antibiotics indicated for various multidrug-resistant bacterial infections. ABSTRACT The worldwide proliferation of life-threatening metallo-β-lactamase (MBL)-producing Gram-negative bacteria is a serious concern to public health. MBLs are compromising the therapeutic efficacies of β-lactams, particularly carbapenems, which are last-resort antibiotics indicated for various multidrug-resistant bacterial infections. Inhibition of enzymes mediating antibiotic resistance in bacteria is one of the major promising means for overcoming bacterial resistance. Compounds having potential MBL-inhibitory activity have been reported, but none are currently under clinical trials. The need for developing safe and efficient MBL inhibitors (MBLIs) is obvious, particularly with the continuous spread of MBLs worldwide. In this review, the emergence and escalation of MBLs in Gram-negative bacteria are discussed. The relationships between different class B β-lactamases identified up to 2017 are represented by a phylogenetic tree and summarized. In addition, approved and/or clinical-phase serine β-lactamase inhibitors are recapitulated to reflect the successful advances made in developing class A β-lactamase inhibitors. Reported MBLIs, their inhibitory properties, and their purported modes of inhibition are delineated. Insights into structural variations of MBLs and the challenges involved in developing potent MBLIs are also elucidated and discussed. Currently, natural products and MBL-resistant β-lactam analogues are the most promising agents that can become clinically efficient MBLIs. A deeper comprehension of the mechanisms of action and activity spectra of the various MBLs and their inhibitors will serve as a bedrock for further investigations that can result in clinically useful MBLIs to curb this global menace.

Evaluation of MALDI Biotyping for Rapid Subspecies Identification of Carbapenemase-Producing Bacteria via Protein Profiling

The method of direct mass spectrometry profiling is reliable and reproducible for the rapid identification of clinical isolates of bacteria and fungi. This is the first study evaluating the approach of MALDI-TOF mass spectrometry profiling for rapid identification of carbapenemase-resistant enterobacteriaceae (CRE). Proof of concept was achieved by the discrimination of CRE using MALDI Biotyper MS based on the protein. This profiling appears promising by the visual observation of consist- ent unique peaks, albeit low intensity, that could be picked up from the mean spectra (MSP) method. The Biotyper MSP creation and identification methods needed to be optimized to provide significantly improved differences in scores to allow for subspe- cies identification with and without carbapenemases. These spectra were subjected to visual peak picking and in all cases; there were pertinent differences in the presence or absence of potential biomarker peaks to differentiate isolates. We also evaluated this method for potential discrimination between different carbapenemases bacteria, utilizing the same strategy. Based on our data and pending further investigation in other CREs, MALDI-TOF MS has potential as a diagnostic tool for the rapid identification of even closely related carbapenemases but would require a paradigm shift in which Biotyper suppliers enable more flexible software control of mass spectral profiling methods.

Evaluation of 1, 4, 7-Triazacyclononane (TACN) as a potential Metallo-B-Lactamase inhibitor in Enterobacteriaceae: Restoring the Activity of B-lactams

Metallo-β-lactamase producing Enterobacteriaceae are of grave clinical concern particularly as there are no Metallo-β-lactamase (MBL) inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1, 4, 7-triazacyclononane (TACN) for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), serum effect, fractional inhibitory concentrations index and time-kill kinetics were performed using broth microdilution techniques according to the Clinical Laboratory Standard Institute (CSLI) guidelines. Enzyme kinetic parameters and cytotoxicity effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing to 0.03 mg/L in the presence of TACN. TACN-Meropenem combinations showed bactericidal effects with MIC/MBC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be non-cytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active site pockets. This was supported by its inhibition constant Ki = 0.044 µM and inactivation constant kinact= 0.0406 (min-1) demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. Importance Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported as critical in the World Health Organization’s Priority Pathogens List for the Research and Development of New Antibiotics. CRE produce enzymes such as Metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the carbapenem. This study demonstrated that TACN inhibits MBLs efficiently and holds promises as a potential MBLs inhibitor to help curb the global health threat posed by MBL-producing CREs.Metallo-B-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern particularly as there are no MBL inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available B-lactams is thus imperative. We investigated a zinc-chelating moiety, 1, 4, 7-triazacyclononane (TACN) for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae (CRE). Minimum inhibitory concentrations, minimum bactericidal concentrations, serum effect, fractional inhibitory concentrations index and time-kill kinetics were performed using broth micro-dilution techniques according to the CLSI guidelines. Enzyme kinetic parameters and cytotoxicity effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem (MEM) regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing to 0.03 mg/L in the presence of TACN. TACN-MEM combinations showed bactericidal effect with MIC/MBC ratio of less than or equal to 4 and synergistic activity was observed. Human serum effects on the MICs were insignificant and TACN was found to be non-cytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibit MBLs by targeting their catalytic active site pockets. This was supported by its inhibition constant Ki = 0.044 uM and inactivation constant kinact = 0.0406 (min -1 ) demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. Importance of study: Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a huge public health concern and have been reported as critical in the World Health Organization Priority Pathogens List for the Research and Development of New Antibiotics. CRE produce enzymes such as Metallo-B-lactamases (MBLs), which inactivate B-lactam antibiotics. Combination therapies involving a B-lactam antibiotic and a B-lactamase inhibitor remain a major treatment option for infections caused by B-lactamase-producing organisms. Currently, no MBL inhibitor-B-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes remain a way forward to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the carbapenem. This study demonstrated that TACN inhibits MBLs efficiently and holds promise as a potential MBLs inhibitor to help curb the global health threat posed by MBL-producing CREs.

In Vitro Potentiation of Carbapenems with Tannic Acid Against Carbapenemase Producing Enterobacteriaceae: Exploring Natural Products as Potential Carbapenemase Inhibitors

We hypothesized and confirmed that tannic acid (TA) reverses carbapenem resistance by inhibiting carbapenemases in class A and B carbapenemase‐producing Enterobacteriaceae.