Molecular analysis and Photodynamic inactivation of multidrug-resistant strains of Acinetobacter baumannii from clinical, abattoir and aquatic samples: Public Health Impact

Title: Molecular analysis and Photodynamic inactivation of multidrug-resistant strains of Acinetobacter baumannii from clinical, abattoir and aquatic samples: Public Health Impact
Creator: ANANE YAW ADJEI
Description: Acinetobacter baumannii is an important hospital-acquired pathogen, frequently associated with morbidity and mortality in immunocompromised patients causing a wide range of clinical complications, such as pneumonia, septicaemia, urinary tract infection, surgical site infection, wound infection, and meningitis. Much of the interest in A. baumannii has been attributed to its genetic plasticity, with its ability to acquire and express resistance in plasmids and chromosome particularly to carbapenems, the drug of choice to treat Acinetobacter infections. Carbapenem resistance in A. baumannii is a growing public health concern and represents a serious problem for treatment of the infection. This situation is not restricted to only clinical settings but also occurs in the extra-hospital settings where these areas can serve as reservoirs for multidrug-resistant (MDR) A. baumannii. The increasing limited access of therapeutic agents against A. baumannii infections and the development of MDR have led to the necessity for alternative strategies to control infections caused by MDR pathogens. Antimicrobial Photodynamic Inactivation (APDI) is a promising treatment modality in the field of biocide research that in the future may have widespread applications, replacing conventional antibiotics. The overall aim of this study was to determine the efficacy of APDI against MDR A. baumannii isolated from clinical, aquatic and abattoir settings, to investigate carbapenem resistance mechanisms and the genetic relationship among isolates circulating in our settings in Mthatha, Eastern Cape, South Africa. The thesis consists of seven chapters. With the exception of chapters 1, 2 and 7, each chapter is designed as a publishable unit. iv The 1 st chapter gives a comprehensive account of the background to the study and the literature review. Here the history of classification, morphology, natural habitat, laboratory diagnosis, infections, epidemiology, virulence and pathogenicity, antimicrobial resistance, A. baumannii in extra-hospital environment (abattoir and aquatic environment), treatment options of A. baumannii and the mechanism of action of APDI were reviewed. Chapter 2 described the techniques and general methods used for sampling, identification, antimicrobial susceptibility testing, PCR extraction and amplification and sequencing processes involved in this work. It covers the general experimental section of chapters 3, 4, 5 and 6. Chapter 3 evaluated the prevalence of antibiotic resistance and frequency of carbapenemase-encoding genes as well as the roles of insertion sequence one (ISAba1) and class 1 integrons (intI1) in MDR A. baumannii clinical isolates obtained from the National Health Laboratory Service (NHLS) at Nelson Mandela Academic Hospital (NMAH). Between August 2016 to July 2017, 100 non-duplicate MDR A. baumannii strains were recovered from various clinical samples. Identification and antimicrobial susceptibility tests of the isolates were performed using the VITEK® 2 automated systems with Gram-negative identification cards and the MicroScan autoSCAN-4 system Gram-negative ID type 2 panel. Minimum inhibitory concentration to imipenem, meropenem and colistin was rechecked by E-test (BioMérieux, France). Colistin susceptibility testing was confirmed using broth microdilution method according to the CLSI and by ComASP™ Colistin according to the manufacturer’s recommendations. Suspected colonies were verified using v Acinetobacter specific primer set Ac436f and Ac676r to amplify the 16S rRNA gene and identification was further confirmed by PCR amplification of the intrinsic blaOXA51-like. The detection of oxacillinase carbapenemase genes (blaOXA-23-like, blaOXA-24-like, blaOXA-51-like and blaOXA-58-like) and metallo-β-lactamase (MBL) genes (blaIMP-1, blaVIM, blaNDM-1 and blaSIM) as well as blaAmpC was carried out by real-time PCR assay using Roche LightCycler 2.0. The occurrence of the activating ISAba1 element upstream of the blaOXA-23-like and blaOXA-51-like genes was also carried out in a PCR assay. Isolates were also assayed for intI1. All strains were resistant to penicillins and cephalosporins except cefepime (87%). Resistant rates against other tested antibiotics were as follows: piperacillin/tazobactam (100%), ampicillin/sulbactam (100%), levofloxacin (91%), ciprofloxacin (89%), tobramycin (87%), gentamicin (84%), meropenem (83%), imipenem (81%), tetracycline (64%), amikacin (50%) and tigecycline (48%). All strains were fully susceptible to colistin. The screening of carbapenemase-encoding genes showed that all strains were found to harbour at least one of the OXA-type carbapenemases. The intrinsic blaOXA-51-like gene was detected in all isolates, 70% of the strains had blaOXA-23-like, 5% had blaOXA-24-like and 8% had blaOXA-58-like genes. For the MBLs, 4% isolates were positive for blaIMP-1, 3% strains were positive for blaVIM and 2% positive for blaNDM-1. Genes encoding blaSIM and blaAmpC enzymes were absent in all the strains. Co-occurrence of blaOXA-23-like and blaIMP-1 or blaOXA-58-like was detected in 1% and 2% MDR A. baumannii strains respectively. A key feature of this study was the co-harbouring of blaOXA-23-like, blaOXA-58-like and blaIMP-1 detected in 2% of the strains and this is the first report in the Eastern Cape province of South Africa. The ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like were detected in 15% and 40% vi isolates respectively whilst intI1 was detected in 80% isolates. Our data revealed a high antibiotic resistance to carbapenems and provide direct evidence of the spread of carbapenemase-encoding genes especially blaOXA-23-like in our local settings. Also the high presence of ISAba1 element upstream of the blaOXA-51-like and blaOXA-23-like increases expression of these carbapenemases and presents an emerging threat in our study setting. The study has also revealed specific insights into the interplay of different mechanisms causing rapid increase in antibiotic resistance in A. baumannii in our local setting. To prevent the development of carbapenem-resistant A. baumannii (CRAB), routine implementation of simple and cost-effective screening methods to detect carbapenemase production in our hospitals is crucial in enhancing infection control practices and finally establishing antimicrobial stewardship programs. Chapter 4 investigated the prevalence of MDR A. baumannii strains in extra-hospital settings, to study its susceptibility to antibiotics and to determine the frequency of carbapenemase-encoding genes. Various samples were obtained from Umzikantu Red Meat Abattoir and Mthatha Dam. Isolates were presumptively identified by direct plating on chromogenic CHROMagar Acinetobacter with supplement Ref. CR102 which allows the growth of CRAB isolates. Species identification and antimicrobial susceptibility testing were carried out using MicroScan autoSCAN-4 automated System. Confirmation of A. baumannii isolates were carried out by PCR analysis of the presence of inherent blaOXA-51-like genes. The presence of the carbapenemase-encoding genes was assessed. Results for 48 and 52 MDR A. baumannii isolated recovered from the abattoir and dam respectively revealed a high resistance rate to some frequently used antimicrobials such as piperacillin [abattoir vii (84%), aquatic (73%)], ceftazidime [abattoir (84%), aquatic (83%)], ciprofloxacin [abattoir (71%), aquatic (70%)]. The percentage of resistant strains to trimethoprim/sulfamethoxazole, amikacin, gentamycin, cefotaxime, and cefepime in abattoir and aquatic isolates were (100%, 92%), (41%, 42%), (81%, 75%), (81%, 79%) and (72%, 72%) respectively. High resistance of (75%, 73%) and (74%, 71%) to imipenem and meropenem were observed in abattoir and aquatic strains respectively. The detection of carbapenemase-encoding genes showed that all MDR A. baumannii strain were positive for blaOXA-51-like. The blaOXA-23-like was detected in 32 (66.7%) of the abattoir strains whereas 11 (21.2%) of the aquatic strains had blaOXA23-like. The blaOXA-58-like was detected in 7 (14.6%) and 4 (7.7%) of abattoir and aquatic isolates respectively. No strain had blaOXA-24-like, blaIMP-type, blaVIM-type, blaNDM-1, blaSIM, and blaAmpC genes among both groups of strains. All the strains lacked ISAba1/blaOXA-23-like and ISAba1/blaOXA-51-like as well as intI1. A high level of multiple antibiotic resistance indexes ranging from 0.22-0.51 indicates that the strains emerged from high-risk sources, in line with conventional standards. In conclusion, the presence of MDR A. baumannii in the extra-hospital environment such as abattoir and aquatic environments may be a threat to public health considering that this may provide an extra-hospital reservoir for the spread of these opportunistic pathogen into both community and hospital settings. Chapter 5 was designed to investigate the genetic relationship among CRAB strains obtained from clinical and extra-hospital sources using the Pasteur MLST scheme. A phylogenetic analysis was performed on 33 representative proportions of A. baumannii strains from clinical, abattoir and aquatic sources. Multi-locus sequence typing was performed using seven fragments of housekeeping genes cpn60, fusA, viii gltA, pyrG, recA, rplB and rpoB provided by Pasteur Institute MLST. Five existing sequence types (STs) were identified in the clinical strains by eBURST algorithm. The most dominant ST for clinical strains was ST2 accounting for 68.8 % (n = 11), followed by ST25 12.5% (n = 2) and 3 STs (ST1, ST85 and ST215) represented by a single strain (singletons) were identified. Extra-hospital isolates had two existing STs, ST1 and ST2 identified in three and fourteen strains respectively. Clonal relation analysis revealed that ST1, ST2, ST25, ST85 and ST215 belonged to the CC1, CC2, CC25, CC85 and CC215 lineage respectively. Phylogenetic tree analysis show that most of the extra-hospital strains were related to each other and also did cluster among the clinical strains. Resistance to carbapenems was related to blaOXA-23-like (all strains), blaOXA-58-like (2 strains) and blaOXA-24-like (1 strain). This study highlights the wide dissemination of highly related blaOXA-23-like producing CRAB belonging to the international clonal complex 2 in Mthatha, South Africa. Thus, appropriate infection control measures are recommended in order to control this clone. As a whole, phylogenetic analysis provided a global view of the molecular epidemiology of A. baumannii in Mthatha, South Africa. In chapter 6, in vitro APDI effect mediated by Protoporphyrin IX (PpIX) and methylene blue (MB) as photosensitizers and with halogen lamp as a light source on A. baumannii biofilm was evaluated. Three MDR A. baumannii strains recovered from clinical, abattoir and aquatic samples were used. The irradiation source has a maximum light dose of 30 Jcm-2 . We studied the APDI according to low or high concentration of MB and PpIX along with exposure time. After treatment, the antibacterial effect of the APDI was assessed by counting colony-forming units per millilitre (CFU/mL). Antimicrobial photodynamic inactivation test groups (L+P1+ and ix L+P2+) showed significant reduction of CFUs compared to the control group (L−P−). The log survival fraction of the treated groups as a percentage of that of L−P− at a concentration of 20 µM is as follows: L+P1+ (5×10-6%), L+P2+ (2×10- 6%); L−P− (100%). (L+P2+ P = 0.0001). The most effective dye was MB which showed higher bacterial reduction of 7.0 log10 whilst PpIX yielded 6.0 log10 CFU/mL at a concentration of 20 µM and light dose of 30 Jcm-2 . Results showed that the efficacy of APDI is dependent on PpIX and MB concentration and light dose. Similar APDI effectiveness was achieved regardless of the origin and minimum inhibitory concentration of isolate indicating that regardless of the origin and resistant rate of isolate, similar APDI effectiveness could be reached. Lastly, general conclusions and recommendations on various parts of findings were captured in chapter 7. The study provides a comprehensive data and information on antimicrobial drug resistance, genes coding for resistance and phylogenetic profiles of local MDR A. baumannii strains and highlights the importance of surveillance. This is essential for epidemiological purposes and specifically in situation requiring empirical treatment. Also in vitro APDI could be considered as an alternative approach to traditional antibiotic therapy if there is an optimized interplay between combinations of different types of photosensitisers and their concentrations, exposure time and light dose. Further research studies employing whole genome sequencing and next generation sequencing, massively parallel or deep technologies are required to understand greater detail of these many unique findings
Date: 2020
Type: PhD Manuscripts
Identifier: http://hdl.handle.net/11260/2080
Identifier: vital:40816
Identifier: DOCTOR OF PHILOSOPHY (PhD) HEALTH SCIENCES (MEDICAL MICROBIOLOGY)
Format: Pdf
Language: English
Rights: Walter Sisulu University

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