Polymerase chain reaction amplification of 16 S rDNA from three nosocomial bacterial isolates in Kaduna State , Northern Nigeria

1 Department of Biological Science, Faculty of Science, Federal University Gashua, P.M.B 1005, Yobe State, Nigeria. 2 Department of Biological Science, Faculty of Science, Nigerian Defence Academy, Kaduna State, Nigeria. 3 Department of Microbiology and Biotechnology, Faculty of Science, Federal University Dutse, Jigawa State, Nigeria. 4 Department of Pure and Applied Biology, Faculty of Pure and Applied Sciences, Ladoke Akintola University of Technology, P. M. B. 4000 Ogbomoso, Oyo State, Nigeria.


INTRODUCTION
Nosocomial infection is one of the most common complications within health care facilities, certain studies have reported outbreaks resulting from contaminated hospital environments, it is called nosocomial infection if it develops 72 h after admission to the hospital (Akihiro et al., 2017).These infections are more dangerous than other infections because they are caused by bacteria with a high resistance to antibiotics.
Health care facilities provide an environment conducive to exposure and transmission of bacteria; infections caused by these bacteria are an important cause of increased morbidity, mortality and health care costs worldwide (Daniel and Michael, 2015).Nowadays, although modern antibiotics have improved; still sometimes the treatment is difficult and causes morbidity and mortality to patients.Many outbreaks of nosocomial infections have come from reservoirs of pathogens in the inanimate hospital surfaces.The contribution of the environment surfaces remains an important factor in nosocomial infection.It has been reported that majority of the patient acquire this infection while staying in hospital (Ananthan et al., 2011).
The major causative agents in the Western world comprise Gram-positive Staphylococcus aureus, Enterococcus spp., Streptococcus spp., Gram-negative Escherichia coli, Enterobacter spp., Proteus mirabilis, Klebsiella spp.In Africa, P. aeruginosa are in increasing proportions due to intensive care treatment (Souza et al., 2015).Pseudomonas sp. are considered opportunistic pathogens that causes opportunistic infection, they are commonly found in nature (soils, water, plants and animals) and water treatment systems, thus demonstrating their adaptation to environments with low nutrient concentration and over a large temperature range (between 4 and 42°C) (Frickmann et al., 2013).Conventional examination of a bacterial infection mainly relies on culture-based techniques.These cultivations usually yield diagnostic results in days or in some cases up to a week after sampling.Furthermore, cultivation of bacteria is not always successful under laboratory conditions (Kerremans et al., 2008;Yoshimura et al., 2011).Such failures may occur due to unsuitable culturing conditions and methods for the bacterial species under study.Alternatively, the particular patient under investigation may have received antimicrobial therapy before sampling.
Molecular methods based on nucleic acid amplification and hybridization aim to circumvent these problems and hasten diagnostic procedures.In such methods, the pathogen is simultaneously detected and identified, which results in more rapid diagnosis than those obtained by conventional culturing methods and obviates the need for additional culture tests (Akihiro et al., 2017).The purpose of this research was to use molecular techniques as alternative methods for strains level identification of nosocomial bacterial found in indoor air of wards, fomites and surgical tools of three prominent hospitals namely, Ahmadu Bello University Teaching Hospital Zaria, Hajiya Gambo Sawaba Hospital Zaria City and Barau Dikko Richard et al. 207 Teaching Hospital Kaduna, Northern Nigeria.

Synopsis of the study location
Kaduna with location Coordinates 10°20'N 7°45'E, and 10.333°N 7.750°E usually referred to as Kaduna state to distinguish it from the city of Kaduna, is a state in Northwest Nigeria.It is the capital of Kaduna with a population of over 6, 066,562 (2006 Census).The selected hospitals are the three major government hospitals in the state and the justification for selection of these hospitals was based on high patronization.

Isolation and molecular characterization of bacteria
Bacteria were isolated from indoor air of wards, fomites and surgical tools of Ahmadu Bello University Teaching Hospital Zaria, Hajiya Gambo Sawaba Hospital Zaria City and Barau Dikko Teaching Hospital Kaduna using standard methods described by Kerremans et al. (2008) and Cheesbrough (2006).The bacteria from bed sheets, pillow cases, nurses' desk and surgical tools were isolated using sterile swab sticks while those from the air in surgical wards and main wards were isolated using exposed plate technique.

Media preparation
All the media used in this research work (nutrient agar, nutrient broth, mannitol salt agar and blood agar) were prepared according to manufacturer's instructions.

Sample collection
Sampling sterile swab sticks were immediately introduced into nutrient broth and taken to Kaduna State University Microbiology Laboratory for incubation and further bacteriological analysis.In the exposed plate technique, the agar plates were opened and exposed to the indoor air of the wards for about 5 min.After 5 min of exposure the plates were covered again and taken to Microbiology laboratory for incubation.Both the agar plates and the broth were incubated at 37°C for 24 h.After incubation, the bacterial colonies that showed positive growth were sub-cultured, gram stained and viewed under the microscope using oil immersion x100 objectives lens (Kerremans et al., 2008).

Biochemical characterization and identification of the nosocomial isolate
Bacteria isolates extracted were characterized and identified after studying their Gram reaction as well as cell micro morphology.
Other tests performed were spore formation, motility and catalase production, citrate utilization, fermentative utilization of glucose, indole production, methyl red-Voges Proskauer reaction, urease and coagulate production, starch hydrolysis, production of H2S from triple sugar iron (TSI) agar and sugar fermentation.The test were carried out according to the methods described (Cheesbrough, 2006;Adeoye, 2007;Agwung-Fobellah and Kemajou, 2007 and Kolhatkar, 2008).Microbial identification was performed using the keys provided in Barrow and Feltham (1993) and Bergey's Manual of Determinative Bacteriology (1994).

Molecular characterization of species of bacteria
The bacteria species characterized using biochemical methods were subjected to strain level identification using molecular approach.These involve extracting the DNA using standard protocols, amplifying the DNA using PCR, sequencing the gene of interest using next generation sequencing (NGS).

DNA extraction using phenol chloroform method
DNA was extracted using phenol chloroform standard method as described by Psifidi et al. (2010).200 μl of the bacterial cells were added in a 1.5 ml Eppendorf tube, 400 μl of lysis buffer (Tris-HCL) and 10 μl of proteinase k was added to the 1.5 ml tube.The tube was vortexed and placed on heat block at 65°C for 1 h while vortexing at interval.400 μl of phenol chloroform was added and vortexed briefly, it was centrifuged for 10 min at 13,000 rpm to separate the phases.The upper layer was carefully removed with a micro pipette, 400 μl of chloroform was added and vortexed, and was then centrifuged for 5 min at 13,000 rpm.The upper layer was carefully removed, 1000 μl of absolute ethanol and 40 μl of 3 M sodium acetate and mixed by inverting the tube several times.The tubes containing pure DNA were incubated at -20°C overnight.The tubes were centrifuged for 5 min at 14,000 rpm using cool centrifuge, the upper layer was carefully removed using micro pipette, 400 μl of 70% ethanol was added to the precipitate, the tubes were centrifuged for 5 min at 14,000 rpm using cold centrifuge, the upper layer was carefully removed using micro pipette in order to remove all traces of ethanol, the DNA was then allowed to dry by leaving the tubes open for 20 min at room temperature and 50 μl of sterile water was added, vortexed and kept at -20°C.

PCR amplification of 16S rDNA using conventional PCR
The extracted DNA from different species of bacteria was quantified using Nano drop.Thereafter, the quantified genomic DNA was placed in a tube containing master mix and primer and the tube was spined for 30 s and introduced into a thermocycler.The thermocycler was operated based on initial denaturation (95°C for 5 min), 25 cycles of denaturation (94°C for 1 min), annealing (52°C for 1 min), extension (72°C for 1 min) and final extension (72°C for 7 min) according to Psifidi et al. (2010).16S rDNA forward sequence 5 1 GGACTACGGGTATCTAAT 3 1 and reverse sequence 3 1 AGAGTTTGATCCAGG 5 1 after the PCR in the thermocycler, the amplified PCR products of expected size 789 bp were confirmed by agarose gel electrophoresis system.The amplified PCR products was run in 1.5% agarose gel electrophoresis stained with ethidium bromide and was viewed under ultra violet (UV) light (Smith et al., 2003;Black and Foarde, 2007).

Sequencing of the gene
The amplified genes of interest were documented, cut and freeze dried.The freeze dried samples of DNA were sent to Macrogen U.S.A laboratory for sequencing.The genomic sequence data was appropriately analyzed using bioinformatics tools, sequence identification was performed using NCBI Basic Local Alignment Search Tool (BLAST) algorithm, similar sequences were downloaded and aligned while phylogenetic tree was drawn with MEGA 7 software (Kumar et al., 2016).

RESULTS
A total number of 66 bacteria were isolated from three hospitals in Kaduna State namely; Ahmadu Bello University Teaching Hospital, Zaria, Hajiya Gambo Sawaba Hospital, Zaria City and Barau Dikko Teaching Hospital, Kaduna (Table 1).Twenty-two (22) samples were collected from each of the hospitals for preliminary antibiotic resistance potency.Observation shows that only 20 of the bacterial isolates were recorded as multi drugs resistant which includes six (6) from Ahmadu Bello University Teaching Hospital, Zaria, eight (8) from Hajiya Gambo Sawaba Hospital, Zaria City and six (6) from Barau Dikko Teaching Hospital, Kaduna.The morphological and biochemical characterization of nosocomial bacterial isolates from hospital environments was presented in Table 1.Investigation revealed that S. aureus occurred as the highest predominant bacterial isolate with the percentage occurrence of 10 (50%) followed by P. aeruginosa with 4 (20%) and Bacillus sp. had 2 (10%), Corynebacterium sp., Klebsiella pneumoniae, Neisseria sp. and Staphylococcus epidermidis all recorded 1 (5%) appearances (Table 1).

DISCUSSION
A number of morphological and biochemical parameters have been used to facilitate the determining of the identities of nosocomial bacteria and other hospital acquired infections (Ateba and Mbewe, 2011).Even though selective and differential media has been used to aid the identification of the bacterial species, yet the sensitivity of these protocols might not be very reproducible between laboratories.Generally, selective and differential media rely on some structural or metabolic property of the species that is preferentially selected.It is highly recommended that they should be combined with confirmatory biochemical and morphological tests.
Table 1 show the morphological and biochemical characteristics of the 20 isolated multidrug resistant nosocomial bacterial (MDRNB).The present study (morphological observation) revealed the cell shapes of 40.00% of the overall isolated microorganisms possessed rod shape under the microscope while 60.00% were found to be cocci in shapes.This is a good characteristic of some organisms but not enough to categorize, hence, all isolates were subjected to grams staining test.About 25.00% of the isolated microorganism from analyzed samples stained pink to red, indicating them to be Gram negative organisms, while 75.00% of the overall isolates stained blue to purple by retaining the crystal violet dye indicating them to be Gram positive organisms (Bergey et al., 1994).

PCR amplification of 16S rDNA from the three highest prevailing nosocomial bacterial isolates in Kaduna State
Organisms of concern in this study are P. aeruginosa, S. aureus and Neisseria sp. Figure 1 shows the amplified 16S rDNA gene with band size of 789 bp bands from Neisseria sp., P. aeruginosa and S. aureus obtained from some selected hospitals in Kaduna State.To test whether we can discriminate between these isolates, Neisseria sp., P. aeruginosa and S. aureus strains were examined for their electrophoretic mobility patterns in PCR amplification (Figure 1).Although the observed differences were small, PCR amplification was capable of distinguishing all the isolates from one and other (Lee et al., 2009).For the Pseudomonas species, the observed electrophoretic mobility patterns showed light differences between Staphylococcus and Neisseria sp.(Figure 1).Because of the closed mobility relativity of all these on electrophoresis gel, these data suggest speciesspecific patterns for identification.To evaluate the applicability of PCR amplification as a general tool for the identification of bacteria, the amplified 16S rDNA products from the three nosocomial bacterial isolates were sequenced according to Cole et al. (2009) and the result obtained is shown in Figure 2. Out of the three bacterial isolates analyzed, only one which is P. aeruginosa gave a DNA sequence.P. aeruginosa was the bacterial isolates sequenced and it showed 100% similarity  having the id query: 86603, when using BLAST National Center for Biotechnology Information (NCBI).The red bullet indicates the sequenced P. aeruginosa (Figure 3).In general, patterns were specific at either the genus level or the species level.However, we opined that in order to overcome lane-tolane and gel-to-gel differences, PCR resolution was improved by the addition of an internal DNA marker for each sample lane.This makes it feasible to precisely compare relative migration times.This research has been able to show that PCR is a promising fast method for the identification of microorganisms.Also, the use of PCR with universal primers and Single Strand Conformational Polymorphism (SSCP) patterns as an identification method can generally be applied to a wide range of nosocomial bacteria without the need of a large panel of probes.
In order to intensify our research, we added an option for molecular identification of methicillin resistant S. species by including the methicillin resistance gene mecA in the assay.The identification was based on PCR amplification as shown in Figure 4. mecA gene was amplified in S. aureus which showed no visible band size.The presence of coagulase negative staphylococcal DNA other than that associated with S. epidermidis was then detected by genus-specific probes.

Conclusion
Molecular and genotypic characterization of bacteria is advantageous when compared to biochemical and phenotypic methods of characterization and identification of nosocomial bacterial.The latter require a prolonged cultivation period for the suspected bacteria and pure bacterial cultures for various biochemical assays.It is therefore concluded that broad-range PCR amplification with subsequent hybridization on a microarray is a rapid diagnostic tool in identifying causative agents of bacterial infections in various specimens from normally sterile site of the hospital environment to medical and surgical tool surfaces.

Figure 3 .
Figure 3. Phylogenetic tree of isolate with query id: 86603 Made by MEGA 7.0 showing percentage similarity of coding regions.

Table 1 .
Morphological and biochemical features of the selected examined Multi-Drugs Resistant (MDRI) isolate.