Methicillin resistance staphylococcus aureus: refers to a group of Gram-positive bacteria that are genetically distinct from other strains of Staphylococcus aureus.

Antimicrobial susceptibility pattern: Is the test done to assure the susceptibility to drug of choice for particular infection or detect resistance in individual bacterial isolates.

Clinical specimen: Are biological materials derived from patients used for laboratory analysis to assist differential diagnosis or staging of a disease process.

Multi-drug resistance is antimicrobial resistance shown by a species of microorganism to at least one antimicrobial drug in two or more antimicrobial classes.

Gram-positive bacteria are bacteria that give a positive result in the Gram stain test.

ABBREVIATION

AST - Antimicrobial Susceptibility test

CLSI - Clinical and Laboratory Standards Institute

CPL - Central Pathology Laboratory

GPC -Gram Positive Cocci

HA-MRSA -Health-acquired Staphylococcus aureus

MDR -Multi-Drug resistance

MIC -Minimal Inhibitory Concentration

MNH - Muhimbili National Hospital

MRSA -Methicillin -resistant Staphylococcus aureus

MUHAS -Muhimbili University of Health and Allied Sciences

ABSTRACT

Background

The discovery of antibiotics has created a turning point in medications to pathogenic infections but unfortunately, each discovery was consistently followed by the emergence of resistance. The rise of multi-drug resistant bacteria has generated a great challenge to treat infections caused by bacteria with the available antibiotics. Multi-drug resistant bacteria results in increased morbidity and mortality rates along with treatment cost and hospitalization stay.

Aim To determine the prevalence and antimicrobial susceptibility pattern of multi-drug resistant Gram-positive isolates in clinical specimen at microbiology laboratory MNH.

Methodology This will be laboratory based cross sectional study. The study will be conducted from April to July 2021 at Central pathology laboratory (MNH). The significant isolates of Gram-positive isolates from various clinical specimen particularly methicillin resistance Staphylococcus aureus detection will be performed by using cefoxitin disk (30µg) Kirby-Bauer disc diffusion methods and the antibiotic susceptibility profile by using microdilution method to determine MIC will be studied to detect MDR by following Clinical and laboratory standard institute guidelines.

Budget The total budget will be Tshs. 838,000/=.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background

Multidrug resistance is defined as acquired resistance to at least one agent in three or more antimicrobial classes (1). MDR can be classified into primary, secondary/ acquired and clinical resistance, whereas primary resistance occurs when the organism has never encountered the drug of interest in a particular host, secondary/ acquired arises in an organism after an exposure to the drug and clinical resistance is the situation in which the infecting organism is inhibited by a concentration of an antimicrobial agent that is associated with a high like-hood of therapeutic failure or reappearance of infections within an organism due to impaired host immune function (2–4).

Apart from Gram negative bacteria, multi-drug resistance is now common to Gram positive bacteria particularly Staphylococci aureus, Coagulase negative Staphylococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecium, and these microorganisms are the major causative agents of community-acquired infections, community-acquired pneumonia, skin and soft tissue infections which results into problem such as significant morbidity, mortality, prolonged hospitalization and healthcare costs (5–8). Multidrug resistance in Gram positive bacteria may be generated by one of two mechanisms. First, these bacteria may accumulate multiple genes, each coding for resistance to a single drug, within a single cell. This accumulation occurs typically on resistance (R) plasmids. Second, multidrug resistance may also occur by the increased expression of genes that code for multidrug efflux pumps, extruding a wide range of drugs (9).

Multidrug resistance has disseminated globally and become a leading cause of bacterial infections in both health-care and community settings. Globally Multidrug resistance Gram positive (MDR GPB) prevalence is ranging between 6% and 40% which has been shown to vary greatly with geographical location where by developing countries were reported to have high rate than developed countries (10–12). MDR GPB may be due to prolonged use of antibiotic treatment, self-prescription of antibiotic medication, inappropriate antibiotic therapy, chronic lung disease, chronic liver disease and cerebral disease, recent hospitalization, longer hospitalization stay, immunosuppression, incomplete treatment, tube feeding, endotracheal tracheostomy and mechanical ventilation, (13).

1.2 Problem statement

Antimicrobial resistance is associated with high mortality rates and high medical costs and has a significant impact on the effectiveness of antimicrobial agents. MDR provokes obstruction in disease control by intensifying the possibility of spreading of resistant pathogens, thus, declining efficacy of treatment and, hence, resulting in prolonged time of infection in patient. The cost of treatment is also increased due to MDR as the pathogens have become resistant to commercially available drugs, which has triggered the useof more expensive therapies.

lack of confirmatory gold standard methods for antimicrobial susceptibility test in our setting results most of our setting relied only on Disc diffusion method for susceptibility testing of different bacteria isolates .According to this method performed limits the spectrum use of routine available drugs since it is the qualitative measured of activity of antimicrobial since its based on zone size of inhibition of bacteria and kirby bauer results are observed on three categorical values, whether sensitive, intermediate or resistance, interpretation depends on zone size of inhibition. Due to considerable increase in resistance to the routinely available antibiotic among the Gram postive isolates in our tertiary , there is need of having confirmatory test that will provide estimate concentration of the tested antimicrobial agent and quantitatively measure antimicrobial activity against bacteria in vitro. Many studies have reported that MIC to be the gold standard method for determining antimicrobial susceptibility of organisms , so can be used as a confirmatory test to confirm unusual resistance obtained from other methods . Considering that there is no any further antimicrobial test performed for resistant pathogen in our setting as a routine confirmatory test, it is important to determine one and thus results the need to conduct this study on MDR Gram postive.

1.3 Conceptual framework

Rounded Rectangle: CLINICAL FACTORS
- Immunosuppression
- ward admitted
- Incomplete treatment
- Prolonged use of medication
- Overuse of medication

Rounded Rectangle: SOCIAL FACTORS
- sex
- Patient residence
- age Oval: MDR Rounded Rectangle: DETECTION
- By using Kirby Bauer method
- MIC method

1.4 Rationale of the study

Through obtained data from this study will help clinians to perform continuous and regular detection for MDR Gram positive and for early detection which will help in generating early intervention programs to control transmission.

MIC values obtained will help clinician to improve treatment option against these MDR Gram positive infections, and provide evidence-based data on the importance of incorporating MIC testing as a confirmatory test for resistance pathogen in our laboratories.

1.5 Research questions

1. What is the prevalence of MDR Gram positive isolates in the clinical specimen at MNH?

2. What is the proportional of MRSA among Staphylococcus aureus in clinical specimen at MNH?

3. What is the most multi-drug resistant Gram-positive bacteria?

4. What are the MIC values of MDR Gram positive organism of routine used antibiotics at CLP?

5. What are the MIC values of MRSA of routine used antibiotics at CPL?

6. What are the factors associated with MDR Gram-positive bacteria?

1.6 Objectives

1.6.1 Broad objective

To determine prevalence and antimicrobial susceptibility pattern of MDR Gram positive isolates in clinical specimen at Microbiology laboratory (Muhimbili National Hospital).

1.6.2 Specific objectives

1. To determine prevalence of MDR Gram positive isolates in clinical specimen at Microbiology laboratory MNH.

2. To determine antimicrobial susceptibility pattern of Gram positive in clinical specimen at Microbiology laboratory MNH.

3. To determine MIC values of MDR Gram-positive isolates to commonly used antibiotics at CPL.

4. To determine MIC value of MRSA to commonly used antibiotic at CPL.

CHAPTER TWO

2.0 Literature review

2.1 Multidrug resistance

Multidrug resistance is defined as acquired resistance to at least one agent in three or more antimicrobial classes(1). MDR can be classified (Figure 1) as primary, secondary/acquired and clinical resistance. Primary resistance occurs when the organism has never encountered the drug of interest in a particular host and secondary resistance arises in an organism after an exposure to the drug(2,3). This secondary class of MDR may further be classified as intrinsic resistance and extensive resistance whereas intrinsic resistance refers to the insensitivity of all microorganisms of a single species to certain common first-line drugs, which are used to treat diseases based on the clinical evidence of the patient and extensive resistance defines the ability of organisms to withstand the inhibitory effects of at least one or two most effective antimicrobial drugs. Clinical Resistance is defined by the situation in which the infecting organism is inhibited by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic failure or reappearance of infections within an organism due to impaired host immune function. In other words, the pathogen is inhibited by an antimicrobial concentration that is higher than could be safely achieved with normal dosing(2,3,14).

Fig 1: Classification of MDR

2.2 Multidrug resistance in Gram positive bacteria

Some Gram positive bacteria have ability to resist different antibiotics used clinically for treatment particularly Staphylococci aureus, Coagulase negative Staphylococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecium that have been reported as MDR Gram positive bacteria(5,6,8,10).These bacteria use different mechanisms to resist common available antibiotics as explained below

1.0

2.0

2.1

2.2

2.2.1 Mechanism for multidrug resistance Gram positive bacteria

Multidrug resistance in Gram positive bacteria may be generated by one of two mechanisms. First, these bacteria may accumulate multiple genes, each coding for resistance to a single drug, within a single cell. This accumulation occurs typically on resistance (R) plasmids. Second, multidrug resistance may also occur by the increased expression of genes that code for multidrug efflux pumps, extruding a wide range of drug(9).

2.2.1.1 Mutational Alteration of the Target Protein

This mechanism of resistance arises through mutations in Gram positive bacteria that make the target protein less susceptible to the agent. Example of resistance attributable to target modification is that conferred by the erm gene, which is usually plasmid coded and produces the methylation of adenine at position 2058 of the 50S rRNA, causing resistance to macrolides (erythromycin and many others), lincosamide, and streptogramin of group B, the MLS phenotype(9).

2.3 Magnitude of MRSA

Methicillin-resistant Staphylococcus aureus is a leading cause of health acquired infections in resource limited countries like Tanzania. Globally, MRSA has increasingly became a problem in health care facilities as well as communities and documented to have exceeded 20% in all WHO regions and above 80% in some regions(15). In Africa, MRSA prevalence intra-country and inter-country has been reported to be heterogeneous, the WHO reported in some parts of Africa 80% of S. aureus infections are resistant to methicillin, results treatment with standard antibiotics to be ineffective(16). According to the reports from developing countries including Tanzania have shown as higher as more than 30 % of isolates from cases of health acquire infections being MRSA. And these infections by MRSA cause increasing of morbidity, mortality and cost due to increased durations of hospitalization, not only that but also use of expensive antimicrobial agents for managing infections (17).

A study done in Nigeria based on skin, wound, ear, throat and nose swabs as clinical specimen, 28 isolates were found to be S. aureus and were subjected to range of available antibiotic agents like: erythromycin, gentamycin, chloramphenicol, levofloxacin and streptomycin to evaluate their susceptibility. Susceptibility results showed that amoxicillin and ampiclox were more resisted by isolates, while ciprofloxacin, levofloxacin were more effective against isolates. Multiple antibiotic resistance index has shown to be 85.7% of the confirmed test isolates were multi drug resistant (were showing resistance to three or more classes of antibiotics). But only 14.3% of isolates showed resistance to only two classes of antibiotics. Moreover MDR has been reported to cause surgical site infection among post-operative patients at regional hospital in Uganda about 65.63% of Gram positive isolates were multi-drug resistance and enterococci species were resistant to ampicillin (18).

2.4 Risk factors of Multi-drug resistant bacteria

A study conducted in China to investigate risk factors for MDR bacteria causes lower respiratory tract infections and India for infections then they suggested that prior antibiotic treatment, inappropriate antibiotic therapy, both acute and chronic illness, cerebral disease, immune suppression, nutritional factors, colonization, severe illness , recent hospitalization, longer hospitalization stay, and use of devices such as endotracheal tracheostomy and mechanical ventilation, tube feeding and intravenous catheter urinary catheters were independent risk factors for MDR bacteria(13,19).

CHAPTER THREE

3.0 METHODOLOGY

3.1 Study design

This will be laboratory based cross-sectional study conducted at Microbiology laboratory Central Pathology Laboratory MNH.

3.2 Study setting

The study will be conducted at Central Pathology Laboratory at Muhimbili Upanga, CPL is the largest laboratory which serve in the largest tertiary hospital in Tanzania, serving approximately 6million people from Dar es Salaam, it provides services to approximately 1200 inpatients per week and approximately 1200 outpatients per day. CPL is also a training facility for the Muhimbili University of Health and Allied Sciences and is the main clinical diagnostic referral laboratory which is well equipped and is the one among accredited laboratory in the country practiced under 15189 ISO standard.

3.3 Study population

Samples of patients who attended /admitted at MNH with significant bacterial isolates obtained from all samples in CPL microbiology laboratory.

3.4 Study duration

The study will be conducted for four months starting from April to July 2021.

3.5 Selection criteria

3.0

3.1

3.2

3.3

3.4

3.5

3.5.1 Inclusion criteria

All pure clinically significant Gram-positive isolates from the clinical specimen.

3.5.2 Exclusion criteria

All contaminated Gram-positive isolates from the cultured plates .

3.6 Sample size determination

Sample size will be determined by using formula below,

n = Z² p(100-p),

d²

Where by

Z= standard deviation of the normal distribution = 1.96(confidence level at 95%)

P= prevalence14.3% of MDR (18).

D= Tolerable error 5%

N=188 for MDR

3.7 Sampling technique

Convenient sampling will be used to recruit the available isolates for this study. Every day new clinical identified isolates of Gram-positive isolates will be collected until the required sample size is for MDR Gram positive bacteria are attained.

3.8 Variable of the study

3.6

3.7

3.8

3.8.1 Independent variable

Independent factor for this study will be social demographic (age, sex, ward admitted, residence) and clinical history (incomplete treatment, prolonged use of medication, overuse of antibiotics).

3.8.2 Dependent variables

Dependent variables MDR Gram positive organism isolates

3.8.3 Data collection

A checklist will be used for collecting both demographic and laboratory data. The isolates will be collected from all clinical specimen processed at microbiology (Blood, Urine, body fluid Pus, Swabs and other clinical specimen) after being cultured and identified, the isolates will be collected using Tryptic Soy Broth (TSB) (Oxoid Ltd) containing 20% glycerol and temporarily stored at − 20 °C in the laboratory. Then these isolates will be tested for antimicrobial susceptibility pattern and those MRSA will screened for MDR, Social demographic data which is age, sex, ward admitted physical address, and clinical information such as history of illness, immune status, type of specimen will be collected from laboratory request form and laboratory information system (GIVA) during data collection.

3.9 LABORATORY PROCEDURES

3.9.1 Detection of MRSA

Methicillin Resistant Staphylococcus aureus (MRSA) will be determined by the Kirby-Bauer disc diffusion method using cefoxitin disk (30μg), a suspension of microorganisms will be prepared and equilibrated to match 0.5 McFarland’s then inoculated to MHA followed by adding 30μg cefoxitin disk and incubated at 37^oC Where by S. aureus isolates with zone of inhibition <21mm phenotypically will be confirmed as MRSA positive, following CLSI guidelines.

3.9.2 Antimicrobial susceptibility test

In this method, the inoculums will be adjusted to the turbidity of a 0.5 McFarland standard and swabbed onto the surface of a Muller-Hinton agar plate. Antimicrobial disks that will be used for S. aureus include cefoxitin 30ug, erythromycin 15ug, tetracycline30ug, ciprofloxacin 5ug and gentamycin 10ug will be used. After putting the disks onto the inoculated plates, the plates will be incubated at 37∘C aerobically for 24 hours. All susceptibility results will be interpreted according to the Clinical and Laboratory Standards Institute (CLSI).

MDR will be defined as resistance to at least one antibiotic in two or more antimicrobial classes.

MIC DETERMINATION

Minimum Inhibitory Concentration will be performed by broth dilution method, the stocks solution of antibiotic will be made from powder by using formula below

1000 x V x C = W

where by serial doubling dilution of antibiotic will be made by using micropipette by taking 1µL of prepared solution of antibiotic and dispensed into first tube containing broth medium mix well and take 1µLfrom first tube and dispensed to the second tube repeat the same until the last negative control tube, then 1ml of tested organism will be added to the set of tubes except the last remain as negative control, and incubating overnight at 350C

Finally, the growth examination will be carried out by looking the turbidity of culture media. The MIC results will be interpreted using the CLSI recommended guidelines, Gentamycin will be used for MRSA.

3.10 Data management and analysis

Socio-demographic, clinical information and laboratory results of isolates will be cross-checked and coded before being entered into computer software. Data will be edited, cleaned, entered and analysed using statistical package for social science (SPSS) version 23.0.

3.11 Ethical consideration

Ethical clearance will be obtained from Research and Publications Committee of the Muhimbili University of Health and Allied Sciences (MUHAS). Permission to conduct the study will be obtained from CPL administration.

3.12 Limitation of the study

According to the biochemical test used at our setting can restrict identification of some useful Gram positive organisms and also clinical information concerning patient may be not reported by the clinicians then some information will be missed in the information system.

3.13 Dissemination of research findings

After collection and analysis of data, a research report will be written and submitted to the supervisor in Microbiology and Immunology department at MUHAS.

3.14 Budget

Item category	Item name	Quantity/Unit	Price
Consumable	Examination gloves	1 box	10,000/=
Consumable	Sterile swab	1 pack	30,000/=
Media	Huller Hinton agar	1 500mg/bottle	223,000/=
	Gentamycin powder	1gm@5	125,000/=
	Tryptic soya broth	1 bottle	280,000/=
Disks	Antibiotics		150,000/=
Stationary	Research Proposal and report		20,000/=
Total			838,000/=

3.15 Work flow

Work	January	February	March	April	May	June	July	August	September
Proposal Development
Proposal submission
Proposal presentation
Data collection
Data analysis
Research report
Research presentation

REFERENCES

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5. Pedroso SH, Sandes SH, Filho RA, Nunes AC, Serufo JC, Farias LM, Carvalho MA, Bomfim MR, Santos SG. Coagulase-negative staphylococci isolated from human bloodstream infections showed multidrug resistance profile. Microb Drug Resist. 2018 Jun 1;24(5):635-47.

6. Pedroso SH, Sandes SH, Filho RA, Nunes AC, Serufo JC, Farias LM, Carvalho MA, Bomfim MR, Santos SG. Coagulase-negative staphylococci isolated from human bloodstream infections showed multidrug resistance profile. Microb Drug Resist. 2018 Jun 1;24(5):635-47.

7. Mohammed Kalgo H, Syawani Jasni A, Rohani Abdul Hadi S, Huda Umar N, Nur Adila Hamzah S, Awang Hamat R. Extremely low prevalence of erythromycin-resistant Streptococcus pyogenes isolates and their molecular characteristics by M protein gene and multilocus sequence typing methods. Jundishapur J Microbiol. 2018 May 31;11(5).

8. Koulenti D, Xu E, Yin Sum Mok I, Song A, Karageorgopoulos DE, Armaganidis A, Lipman J, Tsiodras S. Novel antibiotics for multidrug-resistant Gram-positive microorganisms. Microorganisms. 2019 Aug;7(8):270.9.

9. Al-Zoubi MS, Al-Tayyar IA, Hussein E, Al Jabali A, Khudairat S. Antimicrobial susceptibility pattern of Staphylococcus aureus isolated from clinical specimens in Northern area of Jordan Iran J Microbiol. 2015 Oct;7(5):265.

10. Lim CJ, Cheng AC, Kennon J, Spelman D, Hale D, Melican G, Sidjabat HE, Paterson DL, Kong DC, Peleg AY. Prevalence of multidrug-resistant organisms and risk factors for carriage in long-term care facilities: a nested case–control study. J Antimicrob Chemothe. 2014 Jul 1;69(7):1972-80.

11. Raju S, Foundation E, Patil SA, Sciences N, Kelmani C. Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples. 2009;(January).

12. Chen G, Xu K, Sun F, Sun Y, Kong Z, Fang B. Risk Factors of Multidrug-Resistant Bacteria in Lower Respiratory Tract Infections: A Systematic Review and Meta-Analysis. Can J Infect Dis Med Microbiol. 2020 Jun 30;2020.

13. Marks SM, Flood J, Seaworth B, Hirsch-Moverman Y, Armstrong L, Mase S, Salcedo K, Oh P, Graviss EA, Colson PW, Armitige L. Treatment practices, outcomes, and costs of multidrug-resistant and extensively drug-resistant tuberculosis, United States, 2005–2007. Emerg Infect Dis2014 May;20(5):812.

14. Coombs GW, Daley DA, Thin Lee Y, Pearson JC, Robinson JO, Nimmo GR, Collignon P, Howden BP, Bell JM, Turnidge JD. Australian group on antimicrobial resistance Australian Staphylococcus aureus sepsis outcome programme annual report, 2014. Commun Dis Intell Q Rep. 2016 Jun 30;40(2):E244-54

15. Garoy EY, Gebreab YB, Achila OO, Tekeste DG, Kesete R, Ghirmay R, Kiflay R, Tesfu T. Methicillin-resistant Staphylococcus aureus (MRSA): prevalence and antimicrobial sensitivity pattern among patients—a multicenter study in Asmara, Eritrea. Can J Infect Dis Med Microbiol2019 Jan 1;2019.

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APPENDECES

DATA COLLECTION FORM

SECTION A DEMOGRAPHICS

1. ID: ……………………………………………………….

2. Sex: ………………………..

3. Age: …………………..

4. Ward admitted…………….

5. Patient residence ………………..

SECTION B. CLINICAL INFORMATION

1. Type of specimen: ……………………………………

2. Patient clinical history……………………..

SECTION C. LABORATORY RESULTS

1. Isolated pathogen: GPC

2. Identification of isolated pathogen: …………………………………

3. AST results

Antimicrobial Susceptibility test for GPC
Class of Antibiotics	Antibiotic Agent	Zone of inhibition (mm)	Interpretation (S, I, R)	MIC Value

PREVALENCE AND ANTIMICROBIAL SUSPECTIBILIY PATTERN OF MULTI DRUG RESISTANT GRAM-POSITIVE ISOLATES IN CLINICAL SPECIMEN AT MICROBIOLOGY LABORATORY MUHIMBILI NATIONAL HOSPITAL.

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