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MICROBIAL ASSESSMENT AND ANTIMICROBIAL SUSPECEPTABILITY TEST OF BACTERIA ISOLATED FROM AIR SAMPLES AROUND DUMP SITES IN LOKOJA
TABLE OF CONTENT
Title page- – – – – – – – – i
Approval page – – – – – – – -ii
Dedication – – – – – – – – -iii
Acknowledgement – – – – – – – -iv
Abstract – – – – – – – – – -v
Table of content – – – – – – – -vi
CHAPTER ONE
INTRODUCTION – – – – – – – -1
1.0 Background of the study – – – – -1
1.1 Statement of the problem – – – – -5
1.2 Purpose of the study – – – – – -6
1.3 Significance of the study – – – – -8
1.4 Research questions – – – – – -9
1.5 Scope of the study – – – – – – -10
CHAPTER TWO
LITERATURE REVIEW – – – – – – -11
CHAPTER THREE
Research methodology – – – – – – -39
Design of study – – – – – – – -40
CHAPTER FOUR
Presentation, analysis and interpretation of data – -48
CHAPTER FIVE
Summary of findings – – – – – – -60
Conclusion – – – – – – – – -61
Recommendations – – – – – – – -62
Suggestions for further research – – – – -64
References – – – – – – – – -65
Appendix I – – – – — – – – -68
Questionnaire. – – – – – – – -69
Abstract
Historically, medical practitioners and veterinarians selected antimicrobials to treat bacterial infectious diseases based primarily on past clinical experiences. However, with the increase in bacterial resistance to traditionally used antimicrobials, it has become more difficult for clinicians to empirically select an appropriate antimicrobial agent. As a result, in-vitro antimicrobial susceptibility testing (AST) of the relevant bacterial pathogens, from properly collected specimens, should use validated methods. Thus, AST is an important component of prudent antimicrobial use guidelines in animal husbandry worldwide and veterinarians in all countries should have these data available for informed decision-making. Although a variety of methods exist, the goal of in-vitro antimicrobial susceptibility testing is to provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the infected host. This type of information aids the clinician in selecting the appropriate antimicrobial agent, aids in developing antimicrobial use policy, and provides data for epidemiological surveillance. Such epidemiological surveillance data provide a base to choose the appropriate empirical treatment (first-line therapy) and to detect the emergence and/or the dissemination of resistant bacterial strains or resistance determinants in different bacterial species. The selection of a particular AST method is based on many factors such as validation data, practicality, flexibility, automation, cost, reproducibility, accuracy, and individual preference. The use of genotypic approaches for detection of antimicrobial resistance genes has also been promoted as a way to increase the speed and accuracy of susceptibility testing. Numerous DNAbased assays are being developed to detect bacterial antibiotic resistance at the genetic level. These methods, when used in conjunction with phenotypic analysis, offer the promise of increased sensitivity, specificity, and speed in the detection of specific known resistance genes and can be used in tandem with traditional laboratory AST methods.
CHAPTER ONE
INTRODUCTION
1.0 Background of the study
The majority of infectious diseases are bacterial in origin. With the discovery of laboratory methods to grow these microorganisms using appropriate growth medium known as “culture,” determining the sensitivity and resistance of specific pathogens to a wide range of antimicrobial agents becomes necessary so that healthcare providers can immediately institute proper treatment regimens to their patients.[ Lagier, Edouard, Pagnier, Mediannikov et al., 2015]
Antimicrobial susceptibility testing (AST) is a laboratory procedure performed by medical technologists (clinical laboratory scientists) to identify which antimicrobial regimen is specifically effective for individual patients. On a larger scale, it aids in the evaluation of treatment services provided by hospitals, clinics and national programs for control and prevention of infectious diseases. Recently, researchers have to implement continuous surveillance activities for resistance patterns due to the mutations in bacterial DNA.[ Sawatzky, Liu, Dillon, Allen, Lefebvre et al., 2015]
Clinical laboratories currently employ several methods depending on the laboratory test menu that they provide. These approaches include the disk diffusion and minimum inhibitory concentration (MIC) methods. Commercial systems also became available across health centers and hospital facilities, utilizing both phenotypic and genotypic characterization of bacterial resistance. While routine antimicrobial susceptibility testing for gram-positive (e.g., Staphylococcus aureus) and gram-negative bacteria (e.g., Pseudomonas aeruginosa) are commonly available in peripheral laboratories, drug susceptibility testing (DST) for Mycobacterium tuberculosis are usually carried out within more complex facilities like reference laboratories. Despite the differences in the techniques for susceptibility tests, all laboratories must be critical on each step of the sampling and testing process so that test results are obtainable with consistently high levels of accuracy and reliability.
- Statement of the problem
Susceptibility testing for antimicrobials is necessary for patients who raise suspicion of infection with specific pathogens based on disease manifestation and clinical correlation. Special susceptibility tests via commercial systems may not always require bacterial colonies from culture because they can detect resistance to certain antimicrobial drugs by employing molecular techniques for detecting resistant genes. An example would be the Xpert MTB/Rif assay which determines sensitivity or resistance to rifampicin directly from sputum specimens.[ Xie, Chakravorty, Armstrong, Hall et al., 2017]
Antibacterial agents are then used to detect sensitivity or resistance from bacteria. Although the purpose of this research is primarily towards microbial assessment and antimicrobial suspeceptability test of bacteria isolated from air samples around dump sites in lokoja, it is important to note that antifungal susceptibility tests also exist for addressing fungal infection (e.g., Candida, Aspergillus spp.). Furthermore, antiviral susceptibility tests are also available (e.g., influenza) via molecular technologies including sequencing analysis such as Sanger and pyrosequencing methods.[ Santos, Barros & Hamdan,2006]
1.2 Purpose of the study
A unique impact of AST to patient management is the identification of the specific diagnosis, and additionally, targeting the particular etiologic agent causing the disease. No two patients can be managed similarly, especially if they have the same signs and symptoms (disease manifestation) but with different treatment regimens because the same causative organism can have different resistance patterns. For example, two patients may present with an ordinary strain of Staphylococcus aureus vs. methicillin-resistant Staphylococcus aureus (MRSA); and another example would be patients with drug-susceptible (DS-TB) and drug-resistant tuberculosis (DR-TB).
Once antimicrobial susceptibility results become available, treatment regimens for each patient can be developed by healthcare providers. Prescribed medications of appropriate antibiotics need individualization for each patient diagnosed with an infectious disease. Moreover, resistance from primary drugs will require a higher level of antimicrobial stewardship, including prudent use of second-line drugs.
1.3 Significance of the study
Coagulase-negative staphylococci were previously dismissed as contaminants and were found with in dump site and occur mostly in hospitalized patients, individuals suffering from nosocomial infections and infections arising from the use of catheter or other intra-uterine devices however, it has been shown that CoNS from fermented foods found around dump sites in lokoja also exhibit virulent traits [Lagier, Edouard, Pagnier, Mediannikov et al., 2015] . The major challenge of CoNS-related infections has been the difficulty in therapy due to antimicrobial resistance. Antimicrobial agents used in therapy and as feed supplements to promote growth in food animals may increase the spread of drug-resistant bacteria. Such bacteria may contaminate milk or meat and are subsequently found in fermented food made of such raw material [Sawatzky, Liu, Dillon, Allen, Lefeb etsl., 2015] . The levels of antibiotic resistant infections in the developing world have increased steadily in the last few decades as a result of combination of microbial characteristics and the selective pressure of antimicrobial use [Graham, Dixon, Hughes et al., 2015] . Microbial mechanisms of overcoming the activities of antimicrobial agents include the production of structure-altering or inactivating enzymes (e.g. beta-lactamase or amino glycoside-modifying enzymes), alteration of penicillin-binding proteins or other cell-wall target sites, altered DNA gyrase targets, permeability mutations, active efflux and ribosomal modification [Coorevits, Boelens & Claeys, 2015] . Multidrug-resistant bacteria in both the hospital and community environment are important concern to the clinician, as it is the major cause of failure in the treatment of infectious diseases, increased morbidity, and mortality and the evolution of new pathogens [Kassim, Omuse, Premji & Revathi, 2016] .
Penicillin was initially the drug of choice for treatment of infections caused by Staphylococcus however, penicillin resistance in CoNS became very high since 1968 [Zaman, Hussain, Nye et al., 2017] . Nowadays, resistance is around 91% in clinical strains [Ventola, 2015] . Two mechanisms confer penicillin resistance in staphylococci; the first and the most important is the production of β-lactamase which inactivates penicillin by the hydrolysis of its β-lactam ring. The second is primarily associated with human isolates and confers resistance due to a penicillin-binding protein, PBP2a, encoded by mecA [Sawatzky, Liu, Dillon, Allen et al., 2015] . The blaZ has also been identified as the cause of penicillin resistance among coagulase-negative staphylococci (CoNS) suggesting that blaZ is one of the main mechanism of penicillin resistance in staphylococci [Sawatzky, Liu, Dillon, Allen et al., 2015] . Methicillin resistance in Staphylococcus is caused by the expression of PBP2a encoded by the mecA gene [Ventola, 2015] . Resistance of staphylococci to methicillin and all β-lactam antibiotics is associated with the low affinity of a penicillin-binding protein, PBP2a, which is not present in susceptible staphylococci [Rather, Kim, Bajpai & Park et al., 2017] . This protein is encoded by the mecA gene, which is located in the mec region in which the DNA is of foreign origin [Rather, Kim, Bajpai & Park et al., 2017] . There is evidence of horizontal transfer of SCC cassette between staphylococcal species [Kassim, Omuse, Premji & Revathi, 2016] which implies that CoNS could serve as a reservoir for the spread of resistance genes. Transfer of resistance genes between CoNS and S. aureus has been reported thus indicating that CoNS may act as a resistance gene reservoir for S. aureus. It is thus possible that the different species of staphylococci that are present in the same microenvironment, for example on the skin of dairy cows can exchange mecA and blaZ, if the appropriate bacterial factors are met [[ Sawatzky, Liu, Dillon, Allen, Lefebvre et al., 2015] .
In this study, microbial assessment and antimicrobial suspeceptability test of bacteria isolated from air samples around dump sites in lokoja associated with antibiotic resistance against 9 antibiotics among 255 strains of coagulase-negative staphylococci of fermented food were investigated using disc diffusion technique according to the CLSI guidelines. The antibiotic resistant phenotypes were confirmed molecularly by the detection of mecA genes and cefoxitin screening test.
1.4 Research questions
- What are the microbial from air samples around dump sites in lokoja?
- What are antimicrobial suspeceptability test of bacteria isolated from air samples around dump sites in lokoja?
- what is the effect of the bacteria on the health of the people living around the dump site in lokoja?
