Presented by: Kelley Reynolds-Clausen Eskom RT&D Date: 31 July 2017 The concomitant development of antibiotic resistance and biocidal resistance to Isothiazalone and the effect on bacterial species diversity. Presented by: Kelley Reynolds-Clausen Eskom RT&D Date: 31 July 2017

Introduction Antimicrobial products used in industries  biocides ---- those used for human or animal health  Antibiotics. Biocides generally a greater range – target many sites but antibiotics very specific. Antibiotics more strictly controlled but biocides used without monitoring. Abuse and overuse has lead to increased antimicrobial resistance in particularly aquatic bacteria  more expensive treatments. Considered resistant if: Bacterial species not killed/inhibited at commonly used concentrations. Bacterial species not killed where other species are. When bacteria not affected by concentrations which act on other cells in culture.

Introduction Antibiotics are effective against pathogens but also damage host population. Several lab scale studies show development of antibiotic resistance in biofilms and a link between biocide and antibiotic resistance (Høiby et al, 2010). Link between biocide exposure and antibiotic resistance (Levy, 2000). Resistance increases with increasing concentration Cross-resistance due to similar mechanisms which work for both agents – more likely in Gram -. Impermeability Multi-drug efflux pumps Over expression of operons Plasmid transfer Quorum sensing Alteration of the target site. Use of biocides in cooling water systems well documented  biocide resistance in cooling water may be resistant to antibiotics.

Introduction Biofilm cells 100 – 1000x more resistant to antimicrobials than planktonic counterparts. Possible slow or incomplete penetration of antimicrobial into biofilm. Antibiotics cannot penetrate biofilm. Inactivated by biofilm. Absorbed into biofilm matrix. Altered chemical environment in biofilm. Nutrient gradient within biofilm. Anaerobic regions limit oxygen availability. Waste products change pH Osmotic changes Sub-populations in biofilm develop protected spore-like state. Shown in new biofilms where to thin to act as protection 1% of population always remains. Attempt to determine whether similar reaction in planktonic phase.

Biolog Phenotype Microarray® (PM) Simple technology measures bacterial cell respiration in specific substrates. Set of 20 x 96-well microtitre plates 200 assays for C-source 400 assays for N-source 100 assays for P and S-sources 100 assays for biosynthetic pathways 100 assays on ion effects and osmolarity 100 assays of pH effects and control with deaminases and decarboxylases 1000 assays for chemical sensitivity. In chemical assays – 240 chemicals each at 4 concentrations Toxic to bacteria or interfere in cellular pathways. Common antibiotics included. Includes inorganics (Na, K, Fe, Cu, Co, Zn, Mn, Cl-, SO32-, PO42-, NO2, NO3). Sample loaded into wells with tetrazolium dye  reduction due to respiration forms a purple colour – measured colorometrically on Omnilog Reader.

Materials and Methods 500 litre cooling water system simulator. Biocidal resistance developed by sequential addition increasing concentration Sub-effective Isothiazalone Day 1 – 4 mg/l Day 3 – 6 mg/l Day 5 – 10 mg/l Day 8 – 16 mg/l Day 9 – 20 mg/l normal dosage Samples collected Day 1, time 0; Day 9 , pre-dosing, 1, 2, 4, 6, 24 and 48 hr post dosing. Conduct aerobic plate counts  when lowest count = highest resistance. And when counts recovered. Samples analysed on Biolog Ecoplates for substrate utilisation, DGGE molecular analysis and Biolog PM plates. Only reporting on antibiotic resistance development.

Results and discussion 3 samples Day 1 – pre dosing – least resistant Day 9, time 24 hours post dosing – most resistant Day 9, time 48 hours post dosing – recovering population

Results and discussion - Molecular DGGE to determine number of species per sample.

Results and discussion – Biolog PM Plates Kinetic data compared for 3 samples. Kinetic outputs related to bacterial cell growth through increased respiration. Software compares 3 different sample times Antibiotic resistance development induced with biocidal resistance in following: Rifamycin Aztreonam Ethionamide

Results and discussion – Biolog PM Plates Rifamycin Antibiotics produced by Amycolatopsis rifamycinia or artificially. Effective against mycobacteria Used to treat HIV-related TB and leprosy. Based on inhibition of DNA-dependant RNA synthesis. All bacterial populations can use Rifamycin As concentration increases so does resistance. Rifamycin not growth inhibitor but growth substrate.

Results and discussion – Biolog PM Plates Azetreonam Beta-lactam class – selective against aerobic Gram negative bacteria only. Effective against Enterobacteriacace and 90% of Pseudomonas. Some staphylococcal species, Aeromonas hydrophila, Citrobacter diversus, Enterobacter agglomerans, Haemophilus and Streptococcus pyrogenes have increased resistance. Used to treat urinary/respiratory tract infections, pneumonia and bronchitis Inhibits cell wall synthesis.

Results and discussion – Biolog PM Plates Azetreonam – cont Resistant population (red) more able to utilise  increased growth with concentration Sample 1 and 3 remain sensitive. Problematic if drug used without other antibiotics. Treatment always recommended in conjunction with co- antibiotics. People with HIV-related pneumonia will be at risk in this instance.

Results and discussion – Biolog PM Plates Ethionamide Pro-drug – administered and must be metabolised to active form. Activated by enzyme ethA which binds to NAD+ to for adduct that inhibits InhA Disrupts mycolic acid pathway. Used in conjunction with other anti TB drugs to treat multi-drug resistant TB. All sample metabolise ethionamide in the increasing concentrations. Sample 1 is able to utilise ethionamide slightly better at higher concentrations. Samples 2 and 3 remain resistant over the trial period. Individual in contact with resistant population will be unresponsive to treatment.

Conclusions Development of biocidal resistance reduces bacterial species diversity and total plate count. Overall trend shows population diversity and counts begin high, then drop to a minimum followed by gradual recovery. (Nutrients from lysed cells) Bacterial counts and substrate both decrease significantly between samples 1 and 2 with a marginal increase in sample 3. DGGE mirrors substrate utilisation from Biolog Ecoplates – low change from sample 2 to 3 seems likely to increase. Bacterial population dynamics change from high species diversity and a high number of individuals in each species  a high species diversity and a low number of individuals. Biolog PM plates insight into biocidally resistant bacterial populations. In this study the antibiotics all act as substrates for the resident population. Rifamycin is most highly utilised antibiotic while Aztreonum the least. Bacterial populations in cooling water resistant to Isothiazalone biocide may develop antibiotic resistance to the studied antibiotics.

Conduct study with other biocides and other cooling water sources. Next steps Conduct study with other biocides and other cooling water sources. Consider NextGen molecular species identification. Consider the effect of pH and temperature on the results.

Thank-you! Questions?