Molecular Identification and Antibiotic Sensitivity

Microbiology Research Journal International 24(3): 1-11, 2018; Article no.MRJI.42155 ISSN: 2456-7043 (Past name: British Microbiology Research Journal, Past ISSN: 2231-0886, NLM ID: 101608140)

Molecular Identification and Antibiotic Sensitivity Pattern of Bacteria Associated with Decomposed Domestic Food Wastes from Akure Metropolis Oluwabusayo M. Ologun1*, Bolatito Boboye1 and Oluwole O. Owoyemi1 1

Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure, Ondo State, Nigeria. Authors’ contributions

This work was carried out in collaboration between all authors. Author OMO designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors OMO and BB managed the analyses of the study. Author OOO managed the literature searches. All authors read and approved the final manuscript. Article Information DOI: 10.9734/MRJI/2018/42155 Editor(s): (1) Dr. Giuseppe Blaiotta, Professor, Department of Agriculture, Division of “Grape and Wine Sciences”, University of Naples Federico II, Via Universita' 100 – Palazzo Mascabruno 80055 Portici, Italy. Reviewers: (1) Ruhul Kuddus, Utah Valley University, USA. (2) Maciej Tadeusz Strzemski, Medical University of Lublin, Poland. Complete Peer review History: http://www.sciencedomain.org/review-history/25300

Original Research Article

Received 17th April 2018 th Accepted 24 June 2018 Published 27th June 2018

ABSTRACT Aim: This research was designed to assess the molecular identities and antibiotic sensitivity pattern of the bacteria isolated from decomposed domestic food wastes in Akure metropolis. Methodology: Fifteen bacteria were obtained from the Department of Microbiology, Federal University of Technology Akure. The DNA molecules of the bacterial isolates were extracted using bacterial DNA Mini-Prep Kit. The DNA extracted was amplified and sequenced using universal bacterial primers and ABI Prism DNA sequencer respectively prior their nucleotides blast. The antibiotic sensitivity test of the bacterial isolates was carried out using plate assay. Results: The identified bacterial isolates including Bacillus megaterium, Clostridium difficile, B. thuringiensis, B. sphaericus, B. mycoides, B. cereus, Pseudomonas asplenii, Paenibacillus macerans, Lactobacillus jensenii, B. badius, B. licheniformis, B. subtilis and L. delbrueckii retained their original name after the molecular identification with the exception of C. humiferium and B. pumilus that changed to C. pseudotuberculosis and B. cereus respectively while five bacteria showed no result at the sequencing stage. The molecular techniques revealed the strain name of _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected], [email protected];

Ologun et al.; MRJI, 24(3): 1-11, 2018; Article no.MRJI.42155

the bacteria. L. delbrueckii was resistant to amoxicillin, chloramphenicol, cefriazone while susceptible to erythromycin with a zone of inhibition (59.77±0.66 mm). C. difficile was susceptible to gentamycin with a zone of inhibition (13.93±0.47 mm). Conclusion: From this study, the bacterial isolates (C. pseudotuberculosis, B. cereus, B. licheniformis, B. subtilis, and B. cereus) demonstrated multidrug resistant property. Therefore, this could constitute a serious health threat to the people living in the environment where the wastes are dumped indiscriminately.

Keywords: Bacteria isolates; decomposed; domestic; DNA molecules; food wastes; molecular identity. from infected handlers, transfer of pathogenic microorganisms from unhealthy slaughter animals and most importantly the unhygienic disposal of wastes [7]. The different kinds of microorganisms were reported to be associated with these wastes include Bacillus species, Lactobacillus spp., Corynecbacterium spp., Clostridium spp. and Pseudomonas spp. Most of these microorganisms are harmful to humans and the environment.

1. INTRODUCTION Waste is any substance, solution mixture or article for which no direct use is envisaged but which can be reprocessed, dumped, eliminated by incineration or other methods of disposal [1]. The worldwide urbanisation trends characterised by continuing inward migration into cities have resulted in rapid changes in socio-economic, cultural and environmental conditions [2]. This growth, though important for economic progress, has enormous environmental implications if not managed appropriately. Uncontrolled growth has caused various challenges. One of the huge challenges that face city municipalities is the disposal of increasing quantities of wastes from private households, commercial and industrial enterprises [3]. As the population grows and ensuring food security increases, solid waste productions equally increase rapidly. Pollution resulting from increasing population is a serious problem if the wastes are not properly disposed of and managed. The resulting environmental impact from these wastes can be disastrous. Over 5.2 million people, which include 4 million children were reported to have died yearly from waste-related diseases [4].

The unknown microbial quality and quantity of wastes being discharged into the environment is considered as a source of resistant pathogens. There is an alarming frequency of multidrug resistance of bacterial isolates from food borne infections and this has serious adverse health implications. The widespread emergence of antibiotics resistance among pathogenic microorganisms has become a severe challenge in clinical therapy [8]. Multidrug resistance frequencies of microorganisms in foods contaminants are on the increase [9]. Thus, the apparent increase of the occurrence of antibiotics resistance among microorganisms from foods wastes and its possible implications require adequate surveillance to detect and proffer solutions to the emergence of antimicrobial resistance mechanisms. In order to successfully do this, there is the need to know the correct names of bacteria associated with food wastes to be able to control them well with appropriate antimicrobial agents. Therefore, this study was carried out to assess the molecular identities and antibiotic sensitivity patterns of the bacteria previously isolated from decomposed domestic food wastes obtained in Akure.

The problems of the municipal solid wastes (MSW) are particularly acute in developing countries where the socio-economic changes are not supported by improvements in waste management technologies [5]. Wastes generation has led to the blockage of drainage in watercourses. This causes practical negative effects on the environment in particular pollution by emissions into surface water and groundwater and the resulting risk to human health and property [6]. Restaurants in developing and underdeveloped countries dispose of their generated wastes without any treatment. Wastes accumulate different kinds of microorganism that are sourced directly or indirectly from contact with contaminated kitchen equipment, human transmission of faecally contaminated hands

2. MATERIALS AND METHODS 2.1 Test Bacterial Isolates Fifteen bacteria previously decomposed domestic food

2

isolated from waste (DFW)


samples were collected from the Microbiology Department, the Federal University of Technology Akure (FUTA). The test bacteria were tentatively named as Corynebacterium humiferium, Bacillus megaterium, B. pumilus, Clostridium difficile, B. thuringiensis, B sphaericus, B. mycoides, B. cereus, Pseudomonas asplenii, Paenibacillus macerans, Lactobacillus jensenii, B. lentus, B. licheniformis, B. subtilis and Lactobacillus delbrueckii. The DFW were collected from various locations in Akure Metropolis and were decomposed in the Department of Microbiology, The Federal University of Technology Akure, Nigeria.

The forward primer was AGAGTTTGATCCT GGCTCAG while the reverse primer consisted of GGTTACCTTGTT ACGACTT. The PCR mix (25 uL) contained 15uL of master mix (10x assay buffer, dNTP's, Taq polymerase and MgCl2), 1 uL of forward primer,1uL of reverse primer, 2 µL of template DNA and 6 µL of distilled water. The mixture was vortexed to mix and transferred to a thermo cycler (Corbett Research Australia). Thermal cycling was programmed for the o following conditions: An initialization step at 94 C o for 4 min followed by 30 cycles of 94 C for 1 min, 52oC for 1 min, 72oC for 1 min, final extension at o o 72 C or 5 min and holding temperature at 10 C for 5 min [12].

2.2 Confirmation of the Purity of the Test Bacteria

2.3.3 Purification of PCR products

Microscopic characteristics of the bacteria were determined to ascertain the purity of each test bacterium. Each bacterium was streaked onto nutrient agar in separate Petri plate and o incubated at 37 C for 24 hr. Thereafter, Gram staining, spore staining and microscopic examination was carried out. The Pure bacteria were subsequently streaked onto separate slants of nutrient agar and incubated for 37oC for 24 o hours. They were stored at 4 C until required [10].

Each PCR product was purified before the sequencing using 2M Sodium Acetate wash technique. Agarose gel electrophoresis of the purified PCR product was conducted at 100V, 158mA, 16W for 1 hour, visualized with UV light illumination and photographed with a digital imaging system (Alpha Imager 2000, Alpha Innotech, San Leandro CA). 2.3.4 Sequencing of amplified DNA fragments Sequences of each amplified purified PCR product were determined with a Dye Terminator Sequencing Kit (Applied Biosystems) and an ABI Prism DNA sequencer (Applied Biosystems 3500).

2.3 Molecular Identification of Bacteria 2.3.1 Extraction of bacterial deoxyribonucleic acid (DNA)

The gene sequences of each bacterium were compared with known gene sequences in the GeneBank database. Blasting was done and construction of phylogenetic tree were also done using the Basic Local Alignment Search Tool (BLAST) sequence technique. Phylogenetic Tree was constructed by pasting the set of sequences of each bacterium in the space provided under the one click mode at www.phylogeny.fr website and let the software make decisions by clicking submit.

Extraction of DNA molecules was carried out according to the procedure of Zymo Bacterial DNA Mini-Prep Kit made by Research Corporation in United State of America (USA) using two hundred microlitres of overnight culture of each bacterium. The extracted genomic DNA was stored at 4oC. The quality and quantity of the extracted nucleic acids were determined using a Nanodrop machine set at 260nm and 280nm. The ratio was calculated to determine the purity of the DNA. The concentration of DNA in each sample was calculated with this formula: 50 μg/mL × OD260 × dilution factor [11].

2.3.5 Purity and concentrations extracted bacterial DNA

of

the

The DNA of the test bacteria were purified from proteins and cytoplasmic materials. The ratios of 260/280 nm of the nucleic acids ranged from 1.64 to 2.0 with Corynecbacterium humiferium and Bacillus pumilus having the highest and lowest ratios respectively.

2.3.2 Polymerase chain reaction (PCR) for amplification of bacterial DNA Polymerase Chain Reaction (PCR) was carried out with universal bacterial primers to amplify the portion of the extracted DNA encoding 16SrRNA. 3


2.3.6 The PCR DNA of the bacteria

3. RESULTS

The electrophoresed agarose gel purified amplified DNA molecules of the test bacteria in 1% agarose gel as observed under the UV transilluminator are shown in Plate 1. Most of the DNA molecules of the bacteria showed very bright and strong bands with the size of each DNA fragment at 1500 bp.

3.1 Purity of the Test Bacteria The microscopic morphology of the test bacteria used in this project is presented in Table 1. Both the Gram and spore staining reactions showed that each of the bacteria collected was of single cell shape and Gram reaction. Hence, they were pure. The cells shape for each bacterium was cylindrical form.

2.3.7 Sequences of the 16srdna fragments from the test bacteria

3.2 Purity of the Extracted Bacterial DNA The ten amplified DNA molecules that were sequenced showed varying sizes of nucleotides ranging from 103 to 1742bp. The DNA molecule from probable named Bacillus badius had the lowest number of nucleotides while Corynebacterium humiferium contained the highest number of bases.

The DNA of the test bacteria were purified from proteins and other nucleic acids material. The ratio of 260/280 nm of the nucleic acids ranged from 1.64 to 2.0 with Corynecbacterium humiferium having the lowest ratio. Clostridium difficile, Lactobacillus jensenii, Bacillus sphaericus, Bacillus pumilus, Pseudomonas asplenii and Paenibacillus macerans had the highest ratio of 2.00.

2.4 Antibiotic Sensitivity Test The antibiotic sensitivity test was carried out in order to compare the sensitivity of the bacteria to the different commercially available antibiotics according to the method described by Turner et al. [13]. The microorganisms were seeded separately aseptically by transferring 1mL each of the standardized broth culture of test bacterium into sterile Petri dish. Sterilized Mueller Hinton agar (15 mL) was poured into the plate. The plate was swirled carefully for even distribution of cells and allowed to gel. With the aid of sterile forceps the antibiotic discs were placed firmly on the solidified agar and incubated o for 24 hours at 37 C. After incubation, clear area around each of the discs was measured in millimetres with a metre ruler. This represents the zone of inhibition. Unseeded agar with antibiotics served as the control.

3.3 The PCR DNA of the Bacteria The purified amplified DNA molecules of the test bacteria that were electrophoresed in 1% (w/v) agarose gel and observed under the UV- transilluminator are shown in Plate 1. Most of the DNA molecules of the bacteria showed very bright and strong bands with the sizes of the DNA fragments being 1500 bp.

3.4 Sequences of the DNA fragments from the Test Bacteria Only ten out of the fifteen amplified DNA molecules that were subjected to sequencing showed nucleotides. The remaining five DNA molecules did not show any nucleotide. These include: Clostridium difficile, Lactobacillus jensenii, Bacillus sphaericus, Pseudomanas asplenii and Paenibacillus macerans. Thus showed no result in the sequencing.

2.5 Statistical Analysis Numerical data obtained from the antibiotic sensitivity test and minimum inhibitory concentrations (MIC) of the antibiotic were presented as mean ± STD (standard deviation from the mean). The significance difference (P