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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 7  |  Issue : 2  |  Page : 45-49

Isolation, Characterization, and Antibiotic Resistance Profile of Staphylococci from the Indoor Air of the Students' Halls of Residence at the Obafemi Awolowo University, Ile Ife, Nigeria


Department of Microbiology, Obafemi Awolowo University, Ile Ife, Nigeria

Date of Submission21-Jan-2020
Date of Decision09-May-2020
Date of Acceptance30-Sep-2020
Date of Web Publication31-Dec-2020

Correspondence Address:
Mr. Kayode Gabriel Adeyemi
Department of Microbiology, Obafemi Awolowo University, Ile Ife
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/BMRJ.BMRJ_1_20

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  Abstract 


Background: It has become a public health concern to evaluate the safety of the only readily consumable and unfiltered part of the biosphere, the air most especially from the densely populated region like the students' hostel. Aim and Objectives: This study was conducted to investigate the colony-forming unit per unit volume of staphylococci obtained from the indoor air of students' halls of residence, their biochemical characteristics, and antibiotic resistance profile. Materials and Methods: The identification of isolates was based on the standard methods, such as colonial morphology, Gram stain reaction, catalase, and deoxyribonuclease tests, while antibiotic susceptibility profile was on standard disk diffusion method using the commercially available antibiotic discs (Abtek, Habdiscs) such as ciprofloxacin 5 μg/mL, gentamicin 10 μg/mL, and tetracycline 30 μg/mL. Results: A total of 26 staphylococci isolates were obtained from which none (0%), 4 (15%), and 10 (37%) of the isolates were resistant to ciprofloxacin, gentamicin, and tetracycline, respectively. Conclusion: It is observed that the year of discovery of these antibiotics has a nonlinear correlation with the recorded rate of resistance of the staphylococci to the antibiotics, which predicts an efficacy duration window of 104 years from the date of development. This finding predicts a future pattern of the antimicrobial resistance, which may pose a severe public health problem if antimicrobial stewardship measures are not put in place.

Keywords: Air microbes, antibiotics, prediction, resistance, staphylococci


How to cite this article:
Adeyemi KG, Ezekwem FK, Babawale VO. Isolation, Characterization, and Antibiotic Resistance Profile of Staphylococci from the Indoor Air of the Students' Halls of Residence at the Obafemi Awolowo University, Ile Ife, Nigeria. Biomed Res J 2020;7:45-9

How to cite this URL:
Adeyemi KG, Ezekwem FK, Babawale VO. Isolation, Characterization, and Antibiotic Resistance Profile of Staphylococci from the Indoor Air of the Students' Halls of Residence at the Obafemi Awolowo University, Ile Ife, Nigeria. Biomed Res J [serial online] 2020 [cited 2021 Jun 14];7:45-9. Available from: https://www.brjnmims.org/text.asp?2020/7/2/45/305765




  Introduction Top


The air, often referred to as the earth blanket, is the closest of the atmospheric elements to human. Thus, the investigation of aeromicrobes plays a vital role in public health. This inevitable and renewable resource is also called the atmosphere, which hosts a diverse array of microorganisms, which include fungi spores, virus particles, and viable bacteria,[1] which can pose a challenge to the health of the people living in dense areas such as the university's hostels. It also contains other inanimate aerosols including liquids and solids of different kinds.[2] Although the atmosphere is about 560 km (348 miles) from the surface of the earth and different molecules and particles are embedded in it,[3] some of the components of the atmosphere may not be seen with unaided vision. The air also is made up of gases such as methane, nitrous oxide, carbon monoxide, ozone, and fluorocarbon in concentrations ranging from a few μmol/mol to fmol/mol.[3] Due to the action of the wind, altitude, and location, the percentage composition of air is found to vary slightly from place to place.[4]

One serious threat to public health is the unconscious exposure to infectious agents from a seemingly harmless environment like the natural air. A high number of communicable diseases in the world are from the aerosol origin, in which etiological agents are passed via sneezing, coughing, or talking from an infected individual or air current from the reservoir of such etiologic agents.[5] Apart from the known airborne diseases such as the common cold, influenza, and whooping cough, the progression in the rate of airborne staphylococci infection has also been a threat to public health.[6]

This study aims to evaluate the level of antimicrobial resistance that had been developed by the staphylococci isolated from the indoor air concerning the commonly used antibiotics in clinical settings and proper administration. Also, to estimate the total colony forming unit of Staphylococci, mannitol-fermenting and halophilic bacterial in the indoor air of the selected study location.


  Materials and Methods Top


Materials

Sampling location

Samples were collected from the indoor air of the reading rooms from nine students' hostels of the Obafemi Awolowo University, Ile Ife, Nigeria. The hostels included Akintola Hall (7.5161°N, 4.5199°E), Alumni Hall (7.5217°N, 4.5172°E), Angola Hall (7.5217°N, 4.5123°E), Awolowo Hall (7.5206°N, 4.5146°E), Education Trust Fund Hall (7.5180°N, 4.5146°E), Adekunle Fajuyi Hall (7.5181°N, 4.5183°E), Postgraduate Hall (7.5220°N, 4.5166°E), Moremi Hall (7.5202°N, 4.5189°E), and Mozambique Hall (7.5229°N, 4.5144°E). The average atmospheric parameters of the study locations as reported by the Ile Ife Air Quality Index and Nigeria Air Pollution, AirVisual, 2017 were humidity of 75%, wind speed of 12 km/h, and atmospheric pressure of 1016 mb.

Sampling condition and technique

Indoor airborne staphylococci and other halophilic bacterial isolates were collected using the impingement method as described by Omeliansky;[7] in this method, five sterile mannitol salt agar plates were placed on a 1-m high platform and exposed at strategic loci within the reading rooms of each of the students' hostel on a 1-m high platform for 1 h. The collection of samples was done between the period of 09:00 and 14:00, Nigerian local time on each sampling day. This collection was done for a period of 6 weeks (December 6, 2016, to January 18, 2017). After the exposure of the plates, they were incubated at 37°C for 48 h at the laboratory of the Department of Microbiology.

Estimation of total halophilic bacteria isolates

The Omeliansky's formula written below was used to estimate the total number of halophilic bacteria colony-forming unit per unit cubic meter per minute (CFU/m3/min) in the reading rooms.



where A is the total number of colonies per  Petri dish More Details; T is the time of exposure in minutes; 60 min was constant for the period of the study; and S is the surface area of the Petri dish in cm2. This was 59.47 cm2 for all the Petri dishes used. Therefore, CFU/m3/min = 14.012 A. This value was used for each of the hostels per plate.

Estimation of presumptive staphylococci

On incubation, all the colonies that appeared distinct, tiny, yellowish or whitish, and round were counted and recorded as presumptive staphylococci.[8] They were transferred aseptically to a sterile nutrient broth after counting and estimation with the Omeliansky's formula.

Identification and biochemical tests

The following biochemical tests were carried out to characterize and identify the presumed staphylococci isolates. Isolates that were Gram positive, irregularly arranged in cluster, and utilized mannitol and catalase-positive were presumed to be staphylococci, while deoxyribonuclease (DNase) test was used for further characterization.

Gram staining

Samples were Gram stained according to the standard method.[9] Slides that produced purple smear, cocci-shaped cells, and irregular cluster arrangement of colonies were selected for further testing, while further investigation on those that do not meet any of these criteria was discontinued.

Catalase test

Selected colonies from the Gram staining were subjected to catalase test using standard methods.[10] A sterile inoculating loop was used to collect small amount from the 24-h-old colony and placed onto a sterile microscope slide. A drop of 3% H2O2 was applied onto the organisms on the microscope slide with the aid of a dropper. The slide was immediately covered with its lid as effervescence was observed immediately for positive samples, while test that appeared otherwise were recorded as negative. The gas evolved in a positive test is the oxygen gas released from the reaction between catalase enzyme from the bacterial and the hydrogen peroxide in the reagent as represented in this equation:



Deoxyribonuclease test

The selected colonies were tested for their ability to produce DNase enzyme using standard methods.[11] The surface of the DNase agar plates used was dried. To maximize resources, a plate was lightly divided into four quadrants using sterile loop. Colonies were picked and inoculated in a straight streak onto each quadrant of the DNase agar plate. On incubation of the plate at 37°C for 24 h, the surface was flooded with 1 N hydrochloric acid. Excess hydrochloric acid was decanted, while the plates were examined within 5 min against a dark background. Isolates that tested positive for DNase turn the agar colorless due to the release of methyl green, while isolates that do not lead to any change on the agar were negative.

Antibiotic susceptibility test

The disk diffusion method was used for the antibiotic susceptibility testing which involves the use of 5 μg/mL ciprofloxacin, 30 μg/mL tetracycline, and 10 μg/mL gentamicin (Habdiscs ABTEK Biological Limited, Liverpool). The response of each strain to the antibiotic was specific and can be used as an epidemiological antibiotic typing tool of the strains. For susceptible strain, the antibiotic disk caused an inhibition of bacteria growth to such a diameter that satisfies the NCCLS guidelines. The diameter of the zone of growth inhibition revealed the level of susceptibility of the isolate to the antibiotics in reference to the Clinical Laboratory Standards Institute (CLSI) guidelines.[12]

Standardization of the pure culture was done by picking a distinct colony of the isolate with the aid of sterile inoculating needle into a sterile physiological saline solution. The suspension was standardized to 0.5 McFarland turbidity standard by the use of colorimeter at a wavelength of 590 nm. Sterile swab stick was dipped into the standardized inoculum with the head soaked, but the excess liquid was removed by squeezing the cotton head of the stick against the inner wall of the tube. This moderately wet swab stick was uniformly swabbed on the surface of a sterile Mueller–Hinton agar (MHA) plate by rotating the plates at 60° intervals until the surface was fully and evenly inoculated. Antibiotic discs were allowed to stay on the inoculated MHA agar plate for 3 min for adherence, followed by 24 h incubation at 37°C. After this incubation period, the diameter of the clear zone around the disk was measured in millimeters and recorded. The results obtained were then interpreted using the CLSI standard (2019).


  Results Top


The total CFU/m3/min recorded from each hostel on the mixed culture was in the range of 126–4204 CFU/m3/min, with Akintola Hall having the lowest and ETF Hall having the highest as shown in [Table 1]. Following the isolation and biochemical testing, a total number of 26 staphylococci isolates were recovered from the collected samples. All were catalase positive, 46% were DNase positive, while 54% were DNase negative, as shown in [Table 2].
Table 1: The total colony-forming unit per unit cubic meter per minute

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Table 2: Morphological and biochemical characteristics of selected staphylococci isolates

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Antibiotic susceptibility profile

After a clear interpretation of the antibiotic susceptibility result, 0%, 15.4%, and 38.5% of the tested isolates were found to be resistant to ciprofloxacin, gentamicin, and tetracycline, respectively. About 3.8% of the isolates were resistant to both gentamicin and tetracycline, as shown in [Table 3].
Table 3: Antibiotics profile of the isolated staphylococci

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  Discussion Top


Negligence to antibiotics stewardship will lead to resultant increase in resistance rate among both clinical and environmental isolates, thus modelling a prediction of future resistance rate is feasible.[13] The accuracy of such prediction may be undoubted due to high mutation rates of the bacteria. The information obtained from this study had been helpful in evaluating the resistant pattern of the airborne isolates to the selected antibiotics, and in predicting future antibiotics resistance rate of the aerobic staphylococci isolated from the students' hostels. The result of this study revealed that the occurrence of diversity in the number of microorganisms in the air at different locations is in support of the study carried out by Samuel and Abaynee.[14] Each of the susceptibility results obtained in this study, as shown in [Figure 1], is supported by previous independent works. For instance, the 93.4% susceptibility to ciprofloxacin of the Staphylococcus sp. isolated by Taddesse et al.[15] was in support of the findings in this study, from which 100% susceptibility was recorded. The overcrowding nature, unhygienic use of the public buildings, and proximity of the reading rooms to the public restroom of the Education Trust Fund Hall might be some of the factors that predispose the hall to higher microbial contamination. Furthermore, from the study carried out by Bhatt et al., 2014,[16] the recorded 85% susceptibility of staphylococci isolates to gentamicin from this study was supported with the 83% susceptibility obtained by the study. It is also noticed that the result of the 62% susceptibility of the staphylococci isolates to tetracycline in this study is also in line with the results obtained by Foozieh et al., 2018,[20]who recorded 67% susceptibility.
Figure 1: Antibiotics susceptibility profile of staphylococci isolates

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In reference to the year of development of each of the antibiotics, which were 1981,[17] 1963,[18] and 1945,[19] for ciprofloxacin, gentamicin, and tetracycline, respectively, nonlinear regression was used to derive a correlation between these ages with the rate of resistance of the isolates to the antibiotics, as shown in [Figure 2]. This statistical tool gave the best R-square value when compared with both linear and exponential relationships, which were 0.9803 and 0.945, respectively, as compared to an R-square of 1.00 of the nonlinear relationship, as shown in [Figure 2].
Figure 2: Regression and correlation analysis between resistance pattern and antibiotics age since the first use

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The result of this study revealed that there are Staphylococci in the indoor air, implying an easy portal of entrance to the users of the facility, and these isolates had developed different responses to the action of the classes of antibiotics used in the study. There is therefore, an expected evolutionary trend leading that may lead to the emergence of novel strains with more significant resistance potential resulted due to prolong exposure to the particular antibiotics. Although several mathematical models have been developed to relate the resistance pattern to rate of the consumption of antibiotics, models that predict the efficacy window in terms of antibiotics age are limited. Such resistance–consumption models used available databases at a country or state level, and they include the World Health Organization's Global Antimicrobial Resistance Surveillance System, the Center for Disease Dynamics, Economics and Policy Resistance Map, and the European Centre for Disease Prevention and Control's European Antimicrobial Resistance Surveillance Network.[21]

According to cdc.gov, 2020,[22] methicillin resistance Staphylococcus aureus Scientific Name Search  is rated as a serious threat to the public health with more than 300,000 estimated hospitalized cases and more than 10,000 death in 2017

Resistance of any bacteria including staphylococci to ciprofloxacin had been reported to follow two main pathways. First are the point mutations in the genes that encode the topoisomerase IV subunits and DNA gyrase enzymes, respectively, i.e., the grlA/grlB and gyrA/gyrB genes.[23] A 100% susceptibility of the isolates to ciprofloxacin therefore suggested that the staphylococci isolates recovered from the indoor air in this study probably had not developed evasion mechanism to protect their DNA topoisomerase or/and gyrase enzymes from the action of the antibiotics. The enzymes are the primary and secondary targets of ciprofloxacin.[24] The ciprofloxacin is more recent than the other two antibiotics used.[25] The antibiotics, which is a second -generation fluoroquinolones was developed in 1987, though a 15.5% resistant of Staphylococci from wound swab was reported by Yilmaz and Aslantas as against 0% resistant recorded in this study. This reflects diversity in antibiotics resistance in reference to geographical and sample source.[26] There is also the possibility of inability of the membrane-associated NorA protein of the susceptible isolates to expel the fluoroquinolones through their cell membranes.[27] The concept of age–resistance correlation is supported by the results obtained in this study as the same isolates displayed the greatest degree of resistance to the oldest antibiotics class used for this study. The integrity of gentamicin and tetracycline antibiotics was doubtful through the high resistance they yielded in this study. The establishment of resistance suggested that the resistant staphylococci isolates had been able to proceed with the attachment of the aminoacyl-tRNA to the RNA-ribosome translation complex despite the presence of tetracycline. We can, therefore, use the formula to predict the propensity of the loss of antibiotics efficacy toward the strains, as shown in [Table 4].
Table 4: Antibiotics years since first use, isolates resistance rate, and predicted year of total loss of efficacy to the isolates

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This information led to an exponential relationship of:

Resistance rate (%), R = 1500 (7Y2 − 243Y − 332), where Y is the number of years since first use of the antibiotics, which was obtained from the relationship of:

Years (Y) = −0.0133R2 + 1.4669R + 36 as shown in [Figure 2]. Provided all conditions remain the same.

The outcome of this study opens up a research question as to what degree of antibiotic resistant should be expected over a period of years. Further, the establishment of the efficacy of ciprofloxacin to the isolated strains was renewed. The acceptance of the above-proposed relationship is subject to further evidence.


  Conclusion Top


The indoor air of the hostels contains both DNase-positive and DNase-negative staphylococci in the ratio of 7:6. The high range of bacteria count and the presence of antibiotics resistance staphylococci recovered from these hostels could therefore be a potential risk to the health of the students and an indication of air contamination. The degree of health risk posed by these isolates in the hostel can be evaluated by further experiment on the antibiotics resistance strains, which may entail the molecular characterization of the antibiotic resistance plasmid the bacteria possessed. This study has been able to evaluate the safety of the indoor air of the students' hostel, which is a form of preventive measure and channel to unveil the possibility of a future outbreak of high-level antibiotic-resistant staphylococci. Considering likely adaptation of microorganisms to yet-to-made antibiotics, scientists should be encouraged to search for a novel supplement to aid available antimicrobial therapies which can inhibit genetic reassortment and consequentially prevent development of detrimental mutation such as development, or transfer of resistant gene or plasmid. In references to the study location, immediate intervention is needed to be undertaken to maintain hygiene, most notably in the Education Trust Fund Hall, while further molecular probing is needed to be carried out on the resistant strains to identify possible plasmid-borne antibiotic resistance genes. To prevent further risks, it is expedient for the university management to increase the number of reading rooms to reduce overcrowding and improve the ventilation system within the rooms.

Acknowledgment

We acknowledge the complete support of Professor Adebayo Shittu of the Obafemi Awolowo University, Department of Microbiology for supervising this work, and giving us the permission to make use of his laboratory and publish this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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