Background

Antimicrobial resistance (AMR) is a global problem that is limiting treatment choices. As a

result, it has a detrimental influence on a community's health, healthcare costs, and gross

domestic product. Infections caused by resistant microorganisms are becoming more common

around the world (Naylor NR, et al. 2018). AMR is one of the greatest global risks in the

spectrum of infectious diseases, according to a 2014 World Health Organization (WHO)

assessment. According to studies from around the world, antibiotic stock efficiency is falling,

and bacterial resistance to all first-line and last-resort antibiotics is increasing. Antibiotic

resistance has clinical, economic, and societal consequences (Aftab F, et al. 2016). (Tanwar J, et

al. 2014). Hospital patients become infected during their admission, stay in the inpatient ward,

and stay in the intensive care unit. (Entola CL, 2015).

AMR has a huge impact; when new bacterial strains emerge, it will have an influence on

consumer income, employment savings, healthcare delivery, and gross domestic product. (Mazel

D, et al. 2012). Microorganisms naturally develop antibiotic resistance, which cannot be

attributed to the ongoing exposure of microbes to chemicals in the environment. Resistance

strategies in bacteria include enzyme synthesis, antimicrobial agent enzymatic inactivation,

target site changes, loss of outer membrane proteins, spontaneous mutation, and DNA transfer.

(Sherman & Cappuccino, 2002). Infections with drug-resistant bacteria have a substantial impact

on not only public health but also societal economic stability around the world. Microbial

diseases are responsible for at least 25% of the world's 60 million annual fatalities. Despite great

gains in infection control methods, clinical infections caused by drug-resistant organisms

continue to be a major source of morbidity and mortality in hospitalized patients and in the
community, impacting industrialized, middle-income, and sub-Saharan African countries. (Stone

GS et al., 2017).

The most common organisms isolated from infections in clinical and community settings are

Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P.

aeruginosa), Proteus, Staphylococcus aureus, and Streptococcus pneumoniae (S. pneumoniae).

(Shih JD, et al., 2017). They are also the most dangerous antibiotic-resistant bacteria right now.

(Schiavetti B et al., 2018). Extended spectrum beta-lactamase (ESBLs) are enzymes that impart

broad penicillin, cephalosporin, and monobactam resistance but not carbapenem resistance. The

majority of ESBLs are members of the TEM-1, TMH-2, and SHV-1 families of enzymes.

Enterobacteriaceae, primarily E. coli, K. pneumoniae, and Klebsiella oxytoca, produce these

enzymes. Carbapenem resistance is caused primarily by one of two mechanisms: reduced

 intracelllular concentration or drug hydrolysis. Carbapenem resistance may be influenced by pen

icillin binding proteins. Drug resistance is a major concern in low-income nations because to the

high incidence of diseases, inappropriate use of antibiotics, over-the-counter drug availability,

and restrictions in the availability of antimicrobial susceptibility testing. (Pokharel S et al. 2019)

As a result, this study focuses on the isolation of aerobic and anaerobic bacteria and the

assessment of their antibiotic resistance profiles from various body site infections in patients at

the 37 Military Hospital in Accra, Ghana.

Problem statement

There are many infectious diseases caused by Gram- Negative lactose and non-lactose fermenter

bacteria in an individual that become expose to these microorganisms due to poor hygiene,
excessive use of antibiotics, social hygiene, catheterization and prolonged hospitalization.

Wadhwa, et al. (2016). The scope of this study is to identify Gram negative lactose and non-

lactose fermenting bacteria and their antimicrobial susceptibility at 37 Military hospital in the

Grater Accra region.

Hypothesis

Lactose and non-lactose fermenters cause urinary tract infections (UTI’s), infectious wounds,

ventilator associated pneumonia, and blood stream infections in humans.

Lactose fermenter are predominant as compared to non- lactose fermenters and majority of

which is from urine sample.

Some non-lactose fermenters would be resistant to antimicrobial agent such as vancomycin and

cotrimoxazole due to the various mechanisms they undergo.

Aim

The aim of this research focuses on the isolation of aerobic and anaerobic bacteria and their

antibiotic resistance profiles from different body site infections that occurred among patients 37

Military Hospital, Accra.

Specific objectives

There are clinical, economic, and societal consequences of antibiotic resistance.

Isolating and identifying pathogenic microorganisms and the diagnosis, management, and

treatment of infectious diseases.

Significance

To aid in identifying some risk factors contributing to the spread of resistant bacteria in humans.

To help in identifying pathogenic microorganisms and to assist in the diagnosis, management,

and treatment of infectious diseases.

Research design

A prospective cross-sectional study would be used to collect samples from 37 military hospital

and information regarding patients' history, time of sample collection, and safety measures

during the sample collection would be gathered.

Materials

Equipments

Slides

Distilled water/saline

Cotton swab

Cover slips

Petri plates

Inoculating loop and needle

Burson burner
Autoclave

Oven

Microscope

Media (Nutrient agar powder, MaCconkey agar powder, CLED agar powder, Blood agar

powder, Chocolate agar powder, Urea broth powder, Peptone water powder, Citrate agar powder,

Triple Iron sugar agar powder)

Reagents for Gram staining

Gram’s iodine

Crystal violet

70% Ethanol

Neutral red

Reagent for biochemical reactions

Indole

Urea

Citrate

Triple iron sugar (TSI)

Hydrogen peroxide

Kovac’s reagent

Litmus paper

Methodology

Source of sample collection

A cross-sectional study would be carried out on 100 clinically suspected cases collected from 37

Military hospital. Urine, sputum and aspirate samples would be aseptically collected into sterile

plan tubes while the other samples would be taken with sterile swab sticks and capped in into its

tubes respectively. All sample would be packed aseptically into zipped bags and would be

received at the microbiology laboratory at G2 medical laboratory service for culture and

sensitivity testing procedures to assess the quantity of antibiotic resistance bacteria. Samples

collected would include urine, blood, aspirate, sputum, urethral swab, high vaginal swab, stool,

eye swab, nasal swab and wound swab. Permission to undertake the study at G2 medical

laboratory would also be taken from the Laboratory’s management.

Isolation of Bacterial Isolates and Identification

Samples would be cultured according to the sample type plate i.e., urine sample on CLED and

sputum, aspirate on blood agar, chocolate agar and MacConkey agar. And other samples would

be plated on blood, chocolate and MacConkey agar. Urethral swab and High vaginal swab

platted on chocolate agar and smears would be prepared form all sample for gram staining after

24 hours at 37℃ incubation of samples. The colonies on all the plates would be observed for
colony morphology, haemolytic, utilization of lactose and Gram’s reaction under a compound

light microscope. Afterwards, biochemical tests would be performed for all the bacteria strains

isolated following all the standard operational procedures aseptically. (York, et al, 2004).

Antimicrobial Susceptibility Testing

Using the disc diffusion method, an antimicrobial susceptibility test would be performed for all

the bacteria strains from well-isolated colonies from an 18- to 24-hr agar plate in broth or saline

(0.85%) to achieve a turbidity matching a 0.5 McFarland Standard. A sterile cotton swab would

be dipped into the suspension and pressed firmly on the inside of the tube to remove excess

liquid from the isolates, and it would then be inoculated onto a Mueller Hinton agar plate by

streaking the entire surface of the plate in a quadrat direction each time. This will enhance the

even distribution of the bacteria suspension on the agar plate. The plate is then allowed to dry,

covered with the lid, for about 10 to 15 minutes, after which the disc is applied to the surface just

at the center of the agar plate inoculated with the test organism, pressed firmly to obtain a surface

contact with the agar plate, and incubated in an inverted position for 18–24 hours at 37°C. The

plates would be observed after the incubation period to determine the zone that is sensitive (S),

resistant (R) and intermediate (I) for the isolates by measuring the diameters of the zone of

inhibition to the nearest millimeter and compared to those from standard antibiotics. (Acar &

Goldstein, 1996.).

Data analysis

The number of positive cases would be selected and analyzed in percentages and graphical

representation (such as pie chart and bar chart), using categorical variables such as age and
gender. Data would be analyzed, using a two-tailed p value of less than 0.05 [ANOVA (P<0.05)]

would be considered as statistically significant with Statistical Package for the Social Sciences

(SPSS) 20.0 for Windows.

Expected results

1. Lactose fermenter will be predominant as compared to non- lactose fermenters and

majority of which is from urine sample.

2. The most common isolate will be is Escherichia coli

3. Staphylococcus aureus would be predominant isolated bacteria from the clinical

specimens.

4. Men will be more infected than women.

References

Albrecht SJ, Fishman NO, Kitchen J, et al. (2006). Reemergence of gram-negative health

care-associated bloodstream infections. Arch Intern Med ;166(12):1289-94.

Alekshun, MN., & Levy, SB. (2007). Molecular mechanisms of antibacterial multidrug

resistance. Cell;128(6):1037-50.

Astal Z., Sharif, FA., Abdallah, SA., & Fahd, MI. (2004). Extended spectrum beta lactamases

in E. coli isolated from community-acquired urinary tract infectionsin the Gaza strips

Palestine. Ann Saudi Med.; 24:55-7.

Cappuccino, JG., & Sherman, N. (2002). Techniques for isolation of pure culture. In:

Microbiology: A Laboratory Manual. 4th ed. Singapore: Pearson Education Inc.

Kaur, GJ., & Arora, DS. (2009). Antibacterial and phytochemical screening of

Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi. BMC

Complement Altern Med. 6; 9:30.

Mehrgan, H. & Rahbar, M. (2008). Prevalence of extended-spectrum beta lactamase-

producing Escherichia coli in a tertiary care hospital in Tehran, Iran. Int.

Int J Antimicrob Agents.;31(2):147-51.

Mofenson LM1, Brady MT, Danner SP, Dominguez KL, Hazra R, Handelsman E, et al.

(2009). Guidelines for the prevention and treatment of opportunistic infections

Recommendations from CDC,

the national institutes of health, the HIV medicine association of the infectious

society of America, the Pediatric Infectious Disease Society, and the American

Academy of Pediatrics. MMWR Recomm Rep;58(RR-11):1-166. Noori, M.,

Karimi, A., Fallah, F. (2014). High prevalence of metallo-beta Lactamase

producing Acinetobacter baumannii isolated from two hospitals of Tehran, Iran.

Arch Pediatr Infect Dis;2(3): Article ID: e15439.

Rao, GG. (1998). Risk factors for the spread of antibiotic-resistant bacteria. Drugs

55(3):323-30.

Wadhwa, R., Sharma, Y., Upadhyay, R., Bala, K. (2016). Nosocomial infection by non-

fermenting gram-negative bacilli in tertiary care hospital: Screening and cure

Int J Pharm Pharm Sci;8(3):274-7.

York, M.K., Schreckenberger, P.C., and Miller, J.M., (2004). Identification of gram- negative

Bacteria, in Clinical Microbiology Procedures handbook, 2nd, ed., Isenberg, H.D.,

Ed., ASM Press, Washington, DC, Vol. 1, Section 3.18.2

BUDGETS AND JUSTIFICATION

Total
Unit Cost Unit
Cost
Item No. Description
(GHS) Quantity
(GHS)

1 Petri dishes 10.00 20 200.00

2 Durham’s tubes 10.00 20 200.00

3 Microscope 100.00 1 100.00

4 Various reagents 40.00 1x4 160.00

5 Various Media 100.00 1x4 400.00

6 Transportation 50.00 about 3 trips 60.00

7 Susceptibility disc 50.00 3 150.00

8 Lab assistant 150.00 1 150.00

9 Institutional Charges     300.00

10 Miscellaneous     100.00

  Total     1820.00

JUSTIFICATION

1. Petri dishes and media would be needed for culturing microbes and their subsequent

identification and quantification.

2. Microscope and reagents would be used to observe microscopic organisms for their

identification and quantification.

 3. Durham tubes would be needed for preparing bacteria suspension and for biochemical

testing.

4. Antimicrobial susceptibility disc would be needed for sensitivity testing.

TIME LINE

Time (weekly)
Activity
123456789

Sample collection and preparation                

Specific objectives 1                  

Specific objectives 2                  

Data analysis and report submission