Ali Proposal

DNA plasmids profile of antibiotic- resistant enteric bacteria in HIV-infected individuals in Jos, Nigeria.

Ali M.A. and Lar, P.M.

ABSTRACT

The research is being proposed to determine the antibiotic-resistance and Plasmid profile of enteric bacteria in HIV-infected/AIDS patients attending some hospitals in Jos, Plateau State. Nigeria made some improvement in the control of HIV/AIDS as the country was ranked number eight among 12 countries in the world that have recorded a decline in the new Human Immune-deficiency Virus, (HIV) in 2012. It is now however said to carry the second highest HIV/AIDS burden in the world, with 3.4 million Nigerians living with the virus in 2014. This figure represents 4.1 national prevalence rate and places Nigeria among countries that most do more in the control of the disease as also recently advocated by the country director of UNAIDS. Opportunistic infections caused by enteric bacteria remains a serious problem in Nigeria due to poor socio-economic standards and hygiene. This is particularly a burden in the management of people with HIV/AIDS due to their inability to control the infections owing to immunosuppression. Even where medical services are available, the increasing multi-drug resistant nature of the causative agents makes the infection difficult to treat, leading to increased morbidity and mortality among this population. The primary objective of the study is to define the enteric bacteria associated with opportunistic infections in HIV- infected individuals and compare them with those of HIV seronegative patients attending some Hospitals in Jos metropolis, Plateau State, North-central Nigeria. The antibiotic-resistance profile of the associated enteric bacteria will also be determined and their plasmids characterized. Three hundred and twenty-five (325) patients with gastrointestinal disturbances from Jos University Teaching Hospital (JUTH) and Faith Alive Foundation Hospital (FAFH) will be enrolled for the study after ethical approval and subsequent informed consents of the participants. Their stools will be processed at the Microbiology laboratory of University of Jos for enteric bacterial agents. Fresh stool specimens will be collected and processed within 4 hours of collection. Enteric bacteria will be isolated and identified using selective and differential media. These will be confirmed through various biochemical tests as per standard protocol. Clostridium difficile toxin A and B will be detected using Clostridium difficile Toxin A + B Antigen Detection Microwell ELISA (IVD Research Inc., Carlsbad, CA 92008 USA). Following isolation and identification of enteric bacteria, their susceptibility profile will be determined using the Kirby Bauer disc diffusion method. Drug resistant bacteria will be further tested for ability to produce ß-lactamases using rapid iodometric method. The extended spectrum ß-lactamases will be detected by double disc synergy test (DDST) on the basis of substrate specificity using Muller Hinton agar. Agarose gel electrophoresis will be used for the extraction and characterisation of plasmid DNA. Data collected will be analysed using appropriate statistical software and results presented using bars, charts and graphs as the case may be. Results will be discussed along other literature from previous researches. Recommendations for further studies will be made based on findings and conclusions drawn.

NIH-FORM BIOSKETCH

PRINCIPAL INVESTIGATOR: Ali, Murna Ahmed

BIOSKETCH

NAME

Ali, Murna Ahmed

POSITION TITLE

Lecturer 1

eRA COMMONS USER NAME (credential, e.g., agency login)

alima

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION

DEGREE

(if applicable)

MM/YY

FIELD OF STUDY

i. Bauchi Collage of Arts and Sciences

ii. Abbubakar Tafawa Balewa University, Bauchi.

iii. University of Jos, Jos.

iv. Abbubakar Tafawa Balewa University Bauchi, Bauchi

IJMB

B.Sc.

M.Sc.

Ph.D.On-going

1986-1988

1988-1994

1999-2002

A-Level

MCB Tech.

App. Par. And Ent Medical Mirobiology

i. Personal Statement

The goal of the proposed research is to investigate the enteric bacteria associated with HIV- infected individuals and compare them with those of HIV seronegative patients attending some Hospitals in Jos metropolis, Plateau State, North-central Nigeria. We also hope to determine the drug resistance profile of the associated enteric bacteria, while the DNA plasmids of their resistant strains will be profiled. I believe I have the capacity and motivation to undertake this research. I have a broad background in Medical Microbiology from the undergraduate to the postgraduate level. My PhD which is currently on-going, is also in Medical Microbiology with emphasis on HIV-infected pregnant women. We have done some works on enteric bacteria and gastrointestinal parasites among different groups of people, some of which have already been published in peer-review journals. I have also been involved in collaborations with many other researchers which have expanded my research capacity and ability for a realistic research plan, time-line and budget. The current application builds logically into my prior works on susceptibility studies among HIV-infected individuals and my mentor has an excellent background in this too.

ii, Positions and Honors

Positions and Employment

Tutor, School of Midwifery Bauchi 1996 - 2002

Tutor, School of Nursing Education, Bauchi, Bauchi State. 2002 - 2004

Assistant Lecturer, Department of Microbiology, University of Jos. 2004 -2008

Lecturer II. Department of Microbiology, University of Jos. 2008 - 2011

Lecturer 1 2013 – date

Other experience and professional membership:

1. Member, Nigeria Society for Parasitology

2. Member, National association of women in academics (NAWACS)

3 Member, Nigerian Society for Microbiology

iii. Selected relevant peer-reviewed publications

Anejo-okopi, AI, Okwori AEJ, Eze MI Onaji AI Ali, M, Adekwu A, Ejiji IS (2015).

Prevalence and antibiotic resistance pattern of urinary tract bacterial infections among symptomatic patients attending University of Maiduguri Teaching Hospital, North Feast Nigeria. European Journal of Advanced Research in Biological and Life Sciences 3(3), 31-41.

Zumbes H.J., M.M. Dashen, G.A. Ayanbimpe, H. Zakari, M.A. Ali, and A.J. Olachi. (2012).

Isolation and identification of Dermatophytes from barbershop clippers in Jos Metropolis, Plateau State, Nigeria. Biological and Environmental Sciences Journal for the Tropics. 9(2):188-190.

Ali, M. A., Ajang, Y. A., Agabi, Y. A., Zakari, H. and Zumbes, H. (2011).Prevalence,

Knowledge of and Management Practices against Malaria in a Rural Community in Alkaleri Local Government Area of Bauchi State, Nigeria. African Journal of Natural Sciences, 13:15-19.

Ali, M.A., Musa, O., Ali, A.D. and Chollom, P.F. (2011). Incidence of Enteric Bacteria From

some Domestic Water Sources in a Rural Community in Bauchi State. Journal of Pure and Applied Sciences,13(1):28-32

Mawak, J.D., Chollom, P.F., Dashen, M.M., Ali, M.A. and Choji, T.E. (2010). Virulence.

Markers of Staphyllococcus aureus isolated from food sold in Jos, Nigeria. International Journal of Pure and Applied Sciences, 3(5),102-105.

Ali, M. A., Ali, A. D. and Ohaeri, H. I. (2007). Bacteriological and physico-chemical some

water supplies at the senior staff quaters,University of Jos, Plateau state. Nigeria. Research Journal of science.13(1 & 2): 59 – 65.

Ali, M.A., Ali, A.D, and Iroemeh, C.E. (2009). Phytochemicals and Antibacterial activity of

the Stem bark of Casuarinas equisetifolia. Science Forum; Journal of Pure and Applied Sciences 12(1):7-11

iv. On-going research support

2013 Tertiary Education Trust Fund (TETFUND) Research grant.

C. RESEARCH BUDGET

	RESEARCH BUDGET
S/NO	ITEM	QUANTITY	COST	TOTAL (#)
A	Direct/Non-Personnel cost
1	Frosted slides (100 pieces)	5	800 x 5	4000.00
2	Antibiotic discs-oxoid			20000
3	wooden slide rack	4	1500 x 4	6,000.00
4	Media and Bio chemicals
a.	EMB agar	500g	14,500	14,500
b.	BCA	500g	16,000	16,000
c.	Salmonella- Shigella agar	500g	13,000	13,000
d.	Muller Hinton agar	500g	16,000	16,000
e.	Nutrient agar	500g	8,500	8,500
f.	Nutrient broth	500g	13,000	13,000
h.	TCBS agar	500g	35,000	35,000
5	Bio chemicals
a.	Urease agar	500g	17,500	17,500
b.	Simeon Citrate agar	500g	18,000	18,000
c.	Pepton water with tripton	500g	13,000	13,000
d.	Tripled Sugar iron Water	500g	17,500	17,500
e.	Lysine agar	500g	25,000	25,000
f.	Glucose	500g	13,000	13,000
g.	Lactose	500g	13,000	13,000
6	Reagents
a.	Gram stain
i.	Chrystal Violet powder	1	1,200	1,200
ii.	Safaranin Powder	1	1,200	1,200
iii.	Acetone	2.5litres	8,500	8,500
b..	Ethanol	2.5litres	8,500	8,500
c.	Glycerol	2.5litres	20,000	20,000
e.	Oxidase reagent		12,000	12,000
f.	Kovac’s reagent		34,000	34,500
g.	V.P reagents		25,000	25,000
h.	Starch		1,000	1,000
7	Glass wares
a.	Test Tubes	20	100	2,000
b.	Durham Tubes	50	100	5,000
c.	Bijou Bottles	50	50	2,500
d.	McCartney Bottles	20	200	4,000
e.	Glass Petri Dishes	3sets	2,000	60,000
8	Disposable hand gloves	2	1000 x 2	2,000
9	Agarose gel electrophoresis kit for plasmid DNA	4	$458.33×4x#350	#641,662,
10	Primers	2	10,000x2	20,000
11	Clostridium difficile toxin A and B antigen detection kit/Cryptosporidium detection kit	4	$449.25x4x350	628,950.
12	Swab	3	1000 x 3	3,000.00
13	Disinfectants		3500	3,500.00
13
	Total			1,747512
B	Personnel cost
1	Principal investigator	200,000x1	200,000	200,000.00
2	Co-investigator	100,000	100,000	100,000.00
3	Technical assistants;	3	60,000x3	180,000
4	Printing and photocopying		50,000	50,000.00
5	Data analysis		70,000	70,000.00
6	Publications		100,000	150,000.00
7	Bench/Lab/Equipment utilization cost		50,000	50,000.00
8	Conferences		120,000	200,000

	TOTAL			870,000

	GRAND TOTAL			2,617,512.

Budget justification

More than 90% of the budget is to be spent on direct/non-personnel cost. This is especially due to the molecular analysis involved. ELISA kits will be ordered from the UK with high exchange rates of Naira to dollar. Laboratory studies of this kind may not give the desired results without molecular analysis.

Role of investigators

The Principal investigator will direct the course of research, and take full responsibility of all monies provided. She will undertake the full conduct of research activities, including securing an ethical permission and informed consent of participants, sample processing and laboratory investigations, as well as data management. The research assistant will provide aid in other lab procedures including sample collection and disposition, sterilization of glass wares and other equipments, etc. The Mentor will provide the needed guide/supervision of research work, to ensure quality investigations and reporting of research findings.

Project narrative

The research grant will enhance our opportunity for conducting future research and acquiring other funds. This is because of the exposure and experience to be acquired at the end of the work. We would be able to mentor other junior faculties in the future, thereby building a strong research network that would meet global standards and competitiveness. Our capacity to attract funds for the University of Jos through research activities and collaborations with other scientists around the world would be enhanced, leading to an upgrade in the status of the institution. We are also better able to utilize finances that may be associated with such researches.

Research plan

Specific aims

The specific aims of the research are as follows:

To isolate and identify the enteric bacterial agents associated with HIV-infected patients attending JUTH and FAFH Jos, Plateau state.
To determine the antibiotic-resistant profile of enteric bacteria associated with HIV-infected individuals in the study population.
To identify the ESßLs producing bacteria among the antibiotic-resistant strains of enteric bacteria.
To profile the DNA plasmids of resistant bacteria..

Background and significance of study

Human Immunodeficiency Virus (HIV) is a lentivirus (slowly replicating retrovirus) that causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive (Douek et al., 2009). Although there has been dramatic reductions in the incidence of opportunistic infections such as diarrhoea among HIV-infected individuals following the introduction of highly active antiretroviral therapy (HAART) world-wide(Wilcox 2014, Siddqui et. al., 2007), these infections remain highly endemic in the African sub-region(including Nigeria) due to poor socio-economic conditions and illiteracy. Consequently, even where HAART is available, HIV-infected individuals still fall victims of opportunistic infections due to non-compliance, inability to access proper medical services owing to ignorance and traditional believes. The situation is further compounded with the emergence of multi-drug resistance of bacterial agents to commonly used antibiotics in the region as reported by several workers (Tyodugh et al., 2013, Habib et al., 2003, Olowe et.al., 2007, Atata et al., 2013, Umaru et.al., 2015, Anejo-Okopi et al., 2015).The plasmid mediated antibiotic resistance facilitate easy spread between species most likely to occur in the gastrointestinal tract (Kruse and Sorum, 1994; Silva et al., 2006). Plasmids encoded ESßLs often show co-resistance to diverse antibiotics including aminoglycosides, sulfonamides and quinolones. Therefore infections caused by such strains may be difficult to treat due to limited choices of antibiotics.

Although widespread resistance to antimicrobial agents have been reported from different parts of the region, there is a dearth of information on the resistance profile of enteric bacteria associated with HIV-infected individuals, as most studies focused on non-infected populations (Beena et.al., 2009, Troy, 2014, Atata et al., 2013, Ojo and Oluyege, 2014, Umaru et.al., 2015, Anejo-Okopi et al., 2015). Patterns of enteric infections in developing countries like Nigeria, (where there is poor hygiene and low socio-economic standards with higher rates of common infections), may differ in several important ways from patterns of developed countries. This study aims at identifying enteric bacteria associated with opportunistic infections in HIV-infected individuals in Jos and determining the plasmid profile of their antibiotic-resistant strains. This can guide antibiotic therapy especially when resource limitations make exact diagnosis of the etiologic agents in HIV-associated opportunistic infections difficult. We hypothesize that the enteric bacterial agents associated with HIV infection are highly resistant to many antimicrobial agents, and that their plasmid resistance profile are different from those of HIV-uninfected.

Experimental design and methods

This is a cross-sectional study aimed at identifying the bacterial agents associated with opportunistic infections in HIV-infected persons and profiling the DNA Plasmids of the resistant strains. The research seeks to find if the Plasmids profile of resistant bacteria associated with HIV-infected persons are similar to those of their HIV-uninfected counterparts in the study population. Study participants will include HIV-infected persons with gastrointestinal complains (study group), as well as HIV-uninfected persons attending Jos University Teaching Hospital(JUTH) and Faith Alive Hospital(FAFH) Jos. Persons who had received antibiotics treatment for gastrointestinal disorders within the past 14 days will be excluded. The study group will be drawn from the HIV clinics while the control group will be drawn from the out patients Department(OPD) of the Hospitals. Demographic and other information will be collected using standard questionnaires,

Sample size of 325 for the study, was arrived at, using the following formula ;

N=t² X P(1-P) M²

Where

N = Sample size, t² = confidence level at 95% confidence limit = standard value = 1.96,

P = Prevalence of opportunistic bacteria among HIV – infected persons from similar studies = 26%

M = Margin of error at 5% (standard value = 0.05)

N = 1.96² x 0.55 (1 – 0.0.55) = 380.16 + 10% attrition (38) =380.16+38 = 418.16 0.05²

(Source=http://www.ifad.org/gender/tools/hfs/anthropometry/ant 3.htm.)

Stool samples will be collected from 325 HIV-infected people with gastrointestinal disturbances, and attending the HIV Clinics of JUTH and FAFH in Jos, Plateau State. This will form the study group, while another 100 samples to be collected from HIV-uninfected persons from the general out-patient Department of the same hospitals will form the control group. These will be processed at the Microbiology Laboratory of University of Jos. Standard procedures will be used for the isolation and identification of enteric bacteria from stool samples. This will involve Culturing on MacConkey agar, Deoxycholate citrate agar and Cysteine Lactose electrolyte efficient (CLED) media and incubated at 36.5oC over night. Biochemical tests such as oxidase test, indole test, motility test, citrate, urease and sugar fermentations will be carried out on suspected isolates as described by Appleton et al., (2009), so as to ascertain the identity of enteric bacteria isolated.

Clostridium difficile toxin A and B, will be detected if present, using the Clostridium difficile toxin A and B antigen detection kit, (IVD Research Inc., Carlsbad, CA 92008 USA). Assays will be analysed visually and/or spectrophotometrically (OD 450 nm/650 nm >0.05).

The antibiotic susceptibility profile of bacteria will be investigated using the disc diffusion method as described by Bauer et al. (1996). These will be further tested for their ability to produce ß-lactamases using rapid iodometric method as described by Catlin (1975) and adopted by Ahmad and Aqil (2007). The extended spectrum ß-lactamases (ESßLs) will be detected by double disc synergy test (DDST) as described by Ahmad and Aqil,(2007). Agarose gel electrophoresis. will be used for DNA plasmid profiling. Qualitative and quantitative data collected will be analysed using SPSS version 2010 software, while STATA software will be used for the comparative analysis of proportions. The 95% confidence limit and probability (P value) of 0.05 will be used to determine level of significance of associations. Prevalence of enteric bacterial agents will be compared using chi-square test, while graphs will be used to present antibiotic resistance and plasmid profiles of organisms.

Reference

Anejo-Okopi, A.J, Okwori A.E.J, Eze M.I, Onaji A.I, Ali, M, Adekwu A, Ejiji I.S. (2015). Prevalence and Antibiotic Resistance Pattern of urinary tract bacterial infections among symptomatic patients attending university of maiduguri teaching hospital, north east nigeria. European Journal of Advanced Research in Biological and Life Sciences 3 (3): 31-41

Ahmad and Aqil (2007). Ahmad I, Aqil F (2007) In vitro efficacy of bioactive extracts of 15 medicinal plants against ESßL producing multidrug resistant bacteria. Microbiology Research 162:264-275.

Atata, R.F. Ibrahim, Y.K.E. Giwa, A. Akanbi , A.A . (2013).Antibiotics resistance profile of bacterial isolates from surgical site and hospital environment in a University teaching hospital in Nigeria. Journal of Medicine and Medical Sciences Vol. 4(4) pp. 181-187.

Bauer et al. (1996). Baur AW, Kirby WMM, Sherris JC, Turch M (1966) Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Patholology 45:494-496.

Beena U, Bineeta K, and Preena B. (2009). Enteric Pathogens in HIV/AIDS from a Tertiary Care Hospital. Indian Jounal of Community Meicine; 34(3): 237–242.

Carcamo C, Hooton T, Wener MH, Weiss NS, Gilman R, Arevalo J, et al. (2005. ). Etiologies and manifestations of persistent diarrhea in adults with HIV-1 infection: a case-control study in Lima, Peru. J Infect Dis.;191:11–9.

Catlin (1975) Catlin BW (1975).Iodometric detection of Haemophilus influenzae _-lactamase: rapid resumptive test for ampicillin resistance. Antimicrobial Agent Chemotherapy 7:265-270.

Douek D.C., Roederer M., Koup R.A. (2009). Emerging concept in the immune-pathogenesis of AIDS. Annual Review in medicine. 60:471-484

Habib AG1, Nwokedi EE, Ihesiulor UI, Mohammed A, Habib ZG. (2003). Widespread antibiotic resistance in savannah Nigeria. African Journal of Medical Science; 32(3):303-5.

Ibrahim, A. K., Ikeh, E. I., Malu, A. O., Okeke, E. N. & Damen, J. G. (2006). Intestinal Parasitosis In Human Immunodeficiency Virus (HIV) Infected Adults with Chronic Diarrhoea At Jos University Teaching Hospital, Nigeria. The Internet Journal of Parasitic Diseases. 2: 1559-4629.

Ojo- B. O. and Oluyege A.O(2014). Antibiotics Resistance of Bacteria Associated with Pneumonia in HIV/AIDS Patients in Nigeria. American Journal of Infectious Diseases and Microbiology; 2 (6), pp 138-144.

Olowe O.A., Eniola K.I.T., Olowe R.A., Olayemi A.B. (2007). Antimicrobial Susceptibility and Betalactamase detection of MRSA in Osogbo. SW Nigeria. Nature and Science, 5(3), pp. 44-48.

Siddiqui U. Bini EJ. Chandarana K, et al. (2007). Prevalence and impact of diarrhea on health-related quality of life in HIV-infected patients in the era of highly active antiretroviral therapy. Journal of Clinical Gastroenterol.;41:484–490. [PubMed]

Silva J, Castillo G, Callejas L, López H, Olmos J (2006) Frequency of transferable multiple antibiotic resistance amongst coliform bacteria isolated from a treated sewage effluent in Antofagasta, Chile. Electronic Journal of Biotechnology. 9:533-540.

Troy Brown, RN, (2014). Antibiotic Resistance a Serious Threat to Global Public Health. Medscape Medical News April 30, 2014.

Tyodugh E.D., Emanghe U.U, Godwin T. Jombo G.T., and Ella B. Abraham E.B.(2012). Contributions of Escherichia coli to Diarrhoea among HIV/AIDS Patients at a Hospital inTropical West Africa . British Microbiology Research Journal 2(3): 175-186.

Umaru, G. A., Adamu, Z.1, Ishaya, D.1, Abubakar, Y. U., Hussaini, A.1, Umar, M.1, Adamu, S.G. and Adamu, N. B. (2015). Prevalence and antimicrobial resistance pattern of Escherichia coli in drinking waters in Jalingo Metropolis, Taraba State, North-Eastern Nigeria. Microbiology Research International 3(1), pp. 8-13,

UNAIDS (2014) 'Epidemiology Fact Sheet on HIV and AIDS in Nigeria. http://www.unaids.org/en/media/unaids/contentassets/documents/factsheet/2014/20

Wilcox C.M., Wanke C.A., Bartlett J.G., Mitty J., (2014), HIV and antibiotic resistance; Evaluation of the HIV-infected patient with diarrhoea. Up to date 2016. 5Pp

Participants will be asked to voluntarily sign an informed consent form after proper education on the objectives of the research. Although there may be no direct financial benefits to participants, study subjects will have all their investigations done free of charge and outcome of research will be used to better their management and that of others in their category. Also, no names of participants will be used in compiling results, instead, patients’ identities will be coded and data obtained will be stored in a computer to be purchased for that purpose.Plan for protection of human subjects

Inclusion of women, minorities and children

There will be no discrimination of potential study participants either based on racial, ethnic or relationship inclination, or even vulnerability. Participants will be enrolled systematically based on eligibility and informed consent only.

MENTOR : LAR M.P.