Mjms2009-0068v.pmd

Maced J Med Sci electronic publication ahead of print,
Ugwu et al. Antibiotic Resistance Patterns of Staphylococcus aureus published on November 03, 2009 as doi:10.3889/MJMS.1857-5773.2009.0068
Macedonian Journal of Medical Sciences. 2009 Dec 15; 2(4):XXX-XXX.
doi:10.3889/MJMS.1857-5773.2009.0068 OPENACCESS
Basic Research
Antibiotic Resistance Patterns of Staphylococcus aureus
Isolated from Nostrils of Healthy Human Subjects in a
Southeastern Nigeria Locality
Malachy C Ugwu, Damian C Odimegwu*, Emmanuel C Ibezim, and Charles O Esimone Division of Pharmaceutical Microbiology, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University ofNigeria, Nsukka, 410001, Enugu State, Nigeria Abstract
Key words:
Background. Antibiotic resistant bacteria have been a source of ever-increasing therapeutic
Staphylococcus aureus; multi-drug resist- problem with profound health and socioeconomic pressures. Hence, continued surveillance for bacteria susceptibility pattern is useful to determine the existing and future challenges of Correspondence:
Aim. This study was carried out to evaluate the resistance patterns of some community
isolates of Staphylococcus aureus to some conventional antibiotics within a locality in Department of Pharmaceutics, Faculty ofPharmaceutical Sciences, University of Material and methods. Preliminary characterizations of the plasmid DNA were also carried
Nigeria, Nsukka, 410001,Enugu State, Nigeria Results. Here we show the results obtained, the resistances of the isolates to the antibiotics
were recorded in the following order: Flucloxacillin > Cotrimoxazole > Cephalexin > Chloram- phenicol > Tetracycline > Minocycline ~ Clindamycin > Gentamicin ~ Erythromycin > Amoxicillin- Clavulanic acid ~ Cloxacillin > Amoxicillin. The MIC results showed that the isolates were highly susceptible to Clindamycin but resistant to Tetracycline. Clindamycin was equally shown toexert a relatively higher bactericidal activity among five other antibiotics tested. Preliminarycharacterization of eight (8) isolated bacteria plasmids from eight resistant bacteria isolatesgave mobilities (distances travelled) ranging from 3- 5 mm. These plasmids may be responsiblefor their observed high level of drug resistance. Early antibiotics susceptibility surveillanceexercises therefore helps ascertain and generate a good framework for effective manage-ment and control of resistant and multiple-drug resistant strains.
Conclusion. The outcome of such surveillance exercises would both find usefulness in
shaping the existing antibiotics prescription policies in order to achieve therapeutic endpoint
and also help to slow down or prevent the emergence of multiple drug-resistant strains.
Introduction
infections, and pneumonia (2-4). Staphylococcal in- Staphylococcus aureus is the cause of a wide fection leads to a worsening of some already existing range of pyogenic infections, though also a commensal superficial infections. Infection ranges from such su- of human skin and nares. It has emerged over the past perficial infection to deep infection as septicaemia, several decades as a leading cause of hospital – and making S. aureus an important subject of consistent community – acquired infections (1). S. aureus has studies (5). Infection rate from S. aureus is high and the been found to be the most frequently isolated pathogen recent increased recognition of community acquired causing bloodstream infections, skin and soft tissue infections has important clinical and pharmacologicalimplications for the health care provider (6). In recent Maced J Med Sci. 2009 Dec 15; 2(4):XXX-XXX.
years, many isolates of S. aureus have evolved resist- 40C until used for further studies. All study activities ance to both synthetic and traditional antimicrobial were conducted at the Pharmaceutical Microbiology chemotherapy and their prevalence outside the hospi- unit of the Department of Pharmaceutics, University of tal is of potential epidemiological threat (7-8). Resist- ance to commonly available and affordable antibioticsposes a major concern in the management of bacterial infections, especially in resource poor countries (9).
The culture media used in the study include, Imprudent practices in the use of antibiotics in human Nutrient broth (Oxoid, England), Mannitol salt agar medicine and for prophylaxis in animal husbandry (Oxoid, England) Nutrient agar (Fluka Spain) and contribute significantly to the emergence of multidrug Peptone water. Gram Staining reagents, buffer solu- resistant (MDR) strains. In several studies worldwide, tion, Tris-ethylenediamine tetra- acetic acid sodium Staphylococcus aureus from normal flora seem to sulfate (TENS), sodium acetate, Ethidium bromide constitute an important reserviour of antimicrobial and Bromo – phenol blue were all analar grade rea- resistance gene (10) which can be transferred to other microbial pathogens thus propagating the resistancetraits among microbial populations. The prevalence of antibiotic – resistant Staphylococci at various skinsites in both healthy and hospitalized patients has The following antibiotics used were obtained received considerable attention because of the role of from ABTEK, India: Amoxicillin – Clavulanic acid these organisms as nosocomial pathogens especially (AUG) 30 μg, Amoxicillin (AMX) 25 μg, Erythromycin in immune-compromised host. Thus surveillance stud- (ERY) 5 μg, Gentamicin (GEN) 10 μg, Cotrimoxazole ies and monitoring of antibiotic resistance in Staphylo- (COT) 25 μg, chloramphenicol (CHL) 30 μg, Cephalexin coccus aureus isolated from the nostril of human (CLX) 30 μg, Clindamycin (DAL) 2 μg, Flucloxacillin subjects is clearly important as data obtained from (FLX) 5 μg and Minocycline 30 μg. The following drugs these exercises may be used to devise mechanisms were also used: Gentamicin (80 mg/ml) (Gentalek) for the appropriate use of antibiotics in chemothera- Yugoslavia, Clindamycin (150 mg) (Dalacin CTM) Pfizer peutics as well as help to stem the emergence and USA, Flucloxacillin (Floxapen 250 mg) Beecham Eng- subsequent spread of drug resistance among bacteria land, Tetracycline (Tetraclin® 250 mg) Greenfield populations. Moreover, beneficial retrospective stud- ies on multi-drug resistance must put the availableconventional antibiotics in the area into consideration.
Based on this we embarked on this study to Antibiotic sensitivity of the isolates was deter- determine the resistance patterns of Staphylococcus mined using previously established procedure (13).
aureus to conventional antibiotics. This paper therefore Briefly, the isolates were cultured in nutrient broth at reports the prevalence of community-acquired MDR S. 370C for 24 h. Two (2) loopfuls of the suspension of each aureus in Nsukka metropolis, Southeastern Nigeria.
isolate were inoculated into 20 ml of sterile molten agarin 10 cm diameter Petri dishes and mixed. The plateswere allowed to set and the antibiotic Sensitivity disc Materials and Methods
(ABTEK, India) containing Amoxicllin – Clavulanate(AUG) 30 μg, Amoxicillin (AMX) 25 μg, Erythromycin (ERY) 5 μg, Gentamicin (GEN) 10 μg, Cotrimoxazole (COT) 25 μg, chloramphenicol (CHL) 30 μg, Cephalexin Community strains of Staphylococcus aureus (CLX) 30 μg, Clindamycin (DAL) 2 μg, Flucloxacillin were isolated from nostrils of 100 healthy human (FLX) 5 μg and Minocycline 30 μg.were aseptically subjects within Nsukka metropolis, Enugu State (hav- placed on their surfaces. The plates were incubated at ing obtained their informed consent, and ethical ap- 370C for 24 h and the resultant inhibition zone diam- proval) using sterile swab sticks. The population com- eters (IZDs) were measured and recorded.
prised of 60 female and 40 male undergraduate stu-dents, all aged between 18 and 26 years. Samples Determination of minimum inhibitory were collected between July and August 2008 while isolation and identification of the bacterial isolateswere performed according to standard bacteriological The antibiotics, Gentamicin, Clindamycin, Tet- techniques previously established (11-12). Thereafter racycline, Cephalexin and Flucloxacillin were used for all the S. aureus isolates were stored in agar slants at this assay. Standard protocols employing agar dilution Ugwu et al. Antibiotic Resistance Patterns of Staphylococcus aureus method were used for this assay (13). Briefly, stock poured into a gel tray. This was allowed for 20 mins to solution of each antibiotic was made with distilled solidity and the comb was carefully removed from the water. Five serial dilutions (2-fold) of each stock solu- gel. The gel carrier was removed from the pouring tray tion were done. Exactly 1 ml from each serial dilution and was placed in the gel electrophoresis box. A 250 was incorporated into 10 ml of molten nutrient agar and ml TBE was used to fill the electrophoresis box until the allowed to solidify. Each of the solidified plate was divided into nine sections and labeled. One loopful ofeach suspension of the test organisms was streaked Electrophoresis of the DNA Samples. Using on the plates according to their numbering. The MIC of micropipette, a 50 μL sample of DNA and 3 μL of each antibiotic for each organism was recorded after loading dye (ethidium bromide) were added together overnight incubation at 370C as the lowest concentra- and this was carefully mixed together by pipetting the tion yielding no growth or a barely visible haze.
solutions up and down (16). Each sample was loadedcarefully into the gel wells, one sample per well and this was placed on the gel box at the negative charge end of the electrophoresis machine. Buffered water wasadded which sealed the agarose containing the sam- This is an extension of the MIC Procedure, ple DNA and acts as electrolyte by moving the current since the agar plates showing no growth in the MIC as well as the sample DNA towards the positive end for tests were used for this test. Discs were cut from each 2 hrs with a voltage of 63 V. Thereafter the agarose agar plate and transferred into corresponding container containing the sample DNA was removed and allowed of fresh nutrient medium (13) and incubated at 370C for to drain off. With the aid of UV light, UV certified safety 48 h. Microbial growth or death were ascertained via glasses and camera, a picture showing size and turbidity of the medium. The minimal concentration of movement of the sample DNA was taken to determine the antibiotic that produced total cell death is the MBC.
the mobility in millimeter using a known sample stand-ard (16, 17).
Plasmid Profile Studies Using Agarose gel ElectrophoresisExtraction of Plasmid DNA. Previously estab- lished protocols were employed for this study (14-16).
Samples were collected from the 100 human Selected resistant isolates were grown in a 5 ml double subjects within the Nsukka community thus repre- strength Mueller Hinton broth for 72 h at 370C. The 72 senting a collection of wild type strains of Staphyloco- h grown cultures were centrifuged in a micro centrifuge ccus aureus available within the assessed commu- for 10 mins at 10,000 rpm to obtain pellets. Thesupernatant was gently decanted and the cell pelletswere vortexed for 5 min. Thereafter, 300 μg of Tris EDTA (TE) buffer and 150 μL of 3.0 M sodium aqueous acetate was added at pH 5.2 and was vortexed for 3mins to lyse the bacteria cell pellet. The samples werecentrifuged again for 2 min in a microcentrifuge (Biofuge,Biotra Bio-trade Hecrus Sepatech Co. Ltd USA) andthe supernatant was transferred to a fresh tube, mixedwell with 0.9 ml of 100% ethanol which had been pre-cooled to – 200C to precipitate the bacteria DNA. It wascentrifuged again for 2 min and the supernatant wasdiscarded. The pellet was rinsed twice with 1 ml of 70%ethanol and was dried under vacuum for 2 – 3 mins,after which it was resuspended in 20 - 40 μL of TE buffer Preparation of Gel. A 1.0 g quantity of agarose was dissolved in 100 ml of Tris Borate EDTA buffer Figure 1: Antibiotic susceptibility rates among the S. aureus (TBE) to form 1.0% gel. The agarose solution was isolates. KEY: AUG = Amoxicillin – Clavulanic acid, AMX = allowed to cool to a temperature of about 400C. There- Amoxicillin, TET = Tetracycline, ERY = Erythromycin, COT = after ethidium bromide was added and the mixture Cotrimoxazole, CHL = Chloramphenicol, CLX = Cephalexin, CXC = Cloxacillin, DAL = Clindamycin, FLX = Flucloxacillin, MIN Maced J Med Sci. 2009 Dec 15; 2(4):XXX-XXX.
nity. Fifty-three (53) isolates of Staphylococcus aureus favourable activity of Clindamycin can be seen to were recovered from the human subjects. They were all reoccur here. This is followed by the microbiocidal effects recorded by Flucloxacillin, Cephalexin, Gen-tamicin, and Tetracycline.
Figure 1 shows the antibiotic percentage resist- ance profile among the tested isolates. From the Table 3: Profile of plasmids isolated from drug-resistant
Figure, the resistances of the isolates of Staphyloco- S. aureus strains.
ccus aureus to the antibiotics were in the followingorder: Flucloxacillin > Cotrimoxazole > Cloxacillin >Chloramphenicol > Tetracycline > Minocycline ~Clindamycin > Gentamicin ~ Erythromycin>Amoxicillin- Clavulanic acid ~ Cloxacillin > Amoxicillin.
Thus, the highest resistance (> 60%) was recorded forFlucloxacillin while the least resistance (< 20%) wasrecorded for Amoxicillin. It is interesting to note herethat while very high resistance (least susceptibility)was displayed for Flucoxacillin, a penicillin, the leastresistance (highest susceptibility) was displayed byAmoxicillin which is another penicillin.
Preliminary characterization of the resistance plasmids isolated from the resistant bacteria strains isshown in Table 3. Mobility values recorded ranges from Table 1: Results of Minimum Inhibitory Concentration
(MIC) in μg/ml + SEM.
Discussion
Bacteria isolates were recovered from the nos- trils of humans. These strains should be expected todisplay the typical community-type acquired geneticsusceptibility traits of the S. aureus microbial speciessince they are non-hospital strains and are relatively Table 1 shows the minimum inhibitory concen- unexposed to wide array of antimicrobial agents asso- tration (MIC) profile of the various isolates to the ciated with the hospital practice and environments. It inhibitory activities of some representative antibiotics is interesting to note (Figure 1) that while very high standards. The very least MICs values of 3.125 μg/ml resistance (least susceptibility) was displayed for were recorded by Clindamycin against isolates 13, 34, Flucoxacillin, a penicillin, the least resistance (highest 45, and 79 respectively. Moderate MICs were recorded susceptibility) was displayed by Amoxicillin which is by Flucloxacillin, Cephalexin, and Gentamicin in the another penicillin. Penicillins are known to exert their aforementioned order, while relatively more isolates antimicrobial effect by inhibition of the synthesis of (isolates 3, 13, 31, 40, 70, and 79 respectively) peptidoglycan, which is a heteropolymeric component recorded highest MICs of 100 μg/ml for Tetracycline of the cell wall, which provides a rigid mechanical thus representing a lower susceptibility outcome.
stability by virtue of its highly cross-linked lattice wallstructure (18-20), and the result of this inhibition is loss Table 2: Results of Minimal biocidal concentration MBC
of bacteria cell rigidity and subsequent rupture or lysis (μg/ml) + SEM.
of the bacteria cells (18). Hence it is very plausible toenvisage quite uniform pattern of susceptibility by thetest microorganisms to the members of the penicillinsfamily, albeit with only slightly varying differences.
Moreover, the inherent weakness associated with thisantimicrobial class is resident in their β-lactam chemi- cal ring nucleus which has been subject to attack byβ-lactamase enzymes produced by certain microor- ganisms including some S. aureus strains (20, 21).
The result of the Minimum Bactericidal Concen- tration (MBC) is presented in Table 2. Again, the more Therefore, if the observed reduced susceptibility Ugwu et al. Antibiotic Resistance Patterns of Staphylococcus aureus of the S. aureus strains to Flucloxacillin is due to the inhibition of bacteria cell wall synthesis were more chemical disruptive activities of possible β-lactamase effective against the S. aureus strains except for the enzymes produced by the S. aureus strains, then, why unusually high resistances recorded for Flucloxacillin this trend does not seem to be replicated with regards and Cloxacillin. This anomaly as have been explained to Amoxicillin does appear to be very clear. This from the foregoing may be related to a combination of observation is further heightened when you consider permeability/absorption factors and inherent degradative the relatively poor activity of Amoxicillin-Clavulanic enzyme-antibiotics SAR-resolved antimicrobial prop- acid in comparison with Amoxicillin used alone.
erty of the antibiotics associated with the Penicillins/ Clavulanic acid present in the Amoxicillin-Clavulanic β-lactam group. Nonetheless, this generally observed acid complex is meant to afford protection to the β- advantage of the Amoxicillin, Amoxicillin-Clavulanic lactam chemical ring nucleus present in the Amoxicillin, acid (Augmentin), and Cephalexin over the other anti- and this protection should be expected to enhance the microbial agents will be expected to find usefulness in activity of Amoxicillin. Hence the Amoxicillin-Clavulanic clinical practice requiring the use of antibiotics in the acid complex should demonstrate clearly significantly management of infections and epidemics caused by higher susceptibility rates over the Amoxicillin alone.
S. aureus strains, and this again underscore the needto always carry out a pre-treatment antimicrobial Instead, a reverse trend is rather recorded from susceptibility testing before embarking on antibiotics the study thus suggesting that other mechanism(s) treatment of infections in clinical settings.
may be responsible for these inconsistencies. Onelikely explanation for this phenomenon may be related Blind treatment of infections with chemothera- to permeability and absorption factors governing anti- peutic agents should be discouraged since this could biotic transfer across the microbial cells. It is quite lead to treatment failure with the possible risk of possible that the Amoxicillin-Clavulanic acid complex, morbidity and mortality, as well as, a waste of eco- which is a larger molecule than Amoxicillin, may nomic resources. MIC results of antimicrobial agents experience greater difficulty in permeability and overall normally represent useful pre-clinical quantitative ana- transport across the microbial cell wall/membrane lytical parameter that finds prospective application in barrier. Thus only relatively limited quantity may be pre-clinical and clinical settings. Apart from the prac- available to exert an antimicrobial effect since antibiot- tical utility of MIC values as a means of cutoff points ics must first penetrate the bacteria cells before they demarcating between microbial species and strains on can be mobilized to produce their antimicrobial effect.
the basis of the antimicrobial susceptibility rate, the Secondly, varied and disproportionate structural hin- possession of lower MICs by an antimicrobial agent is drances introduced by molecular structural differences quite suggestive of a higher inherent antimicrobial among these antibiotics may serve to modulate the property (22). Additionally, MIC values must synchro- compulsory pre-activity structure-activity-relationship nize with pharmacokinetic plasma and tissue distribu- (SAR) between the β-lactamase enzymes and these tion of the antibiotic to ensure that adequate amounts antibiotics thereby rationalizing the overall degradative of the antibiotic are made readily available at the sites effect of these enzymes, and the consequent activities of infection. It is therefore expected that lower MIC values would enhance this outcome as well as help tolimit the clinical occurrence of unwanted drug side Again, from the percentage resistance profile effects since smaller but effective doses of the antibi- results, the three (3) best agents showing relatively otics could then be administered to patients in accord- good susceptibility profile (Amoxicillin, Amoxicillin- ance with pre-determined frequencies.
Clavulanic acid (Augmentin), and Cephalexin) are allbactericidal agents that produce their antimicrobial Consequently, considering the MIC results gen- effect through inhibition of bacteria cell wall synthesis.
erated (Table 1), Clindamycin (followed by Flucloxacillin, The other agents (Chloramphenicol, Tetracycline, Cephalexin, Gentamicin, and Tetracycline) seem to Clindamycin, Minocycline, Gentamycin and Erythro- present as the agent of choice for a general non- mycin) all show quite moderate susceptibilityresistance specific clinical treatment of infections caused by the profile (< 40% > 20%) and they are known to exert their isolated S. aureus strains within the examined Nsukka antimicrobial activities through other means of inhibi- community. This scheme may be extrapolated for tion of bacteria protein synthesis (18). There appears other neighbouring communities within the Enugu therefore, a seeming correlation between the overall State axis due to demographic relatedness of these recorded antimicrobial activity and mode of action of localities. However, since the MIC results also showed the antibiotics used. It would generally appear from the some wide bacteria-strain-specific variations, we would results of this study that antimicrobial agents acting by suggest that antibiotic treatments options should be Maced J Med Sci. 2009 Dec 15; 2(4):XXX-XXX.
rather tailored to confront the specific S. aureus strain carried out for different communities and geographical involved on the basis of their favourable susceptibility settings since the occurrence of antibiotic resistant profile to the specific antibiotic to be used as deter- strains can clearly vary across different environmental mined by a proper laboratory analytical procedure.
settings depending on a host of various factors preva- Given the Minimum Bactericidal Concentration (MBC) lent in such environments that influence selection of (Table 2), although all the antibiotics caused bacteria development of antibiotic resistant strains.
cell death at concentration approaching 100 μg/ml this however is quite far from the actual serum concentra- In a conclusion, the high level of resistance tion usually encountered at the doses employed in among the isolates was found to be common with clinical practice, and even though it is true that that the commonly used antibiotics. The MIC and MBC results prognosis of chemotherapeutic treatment of bacteria showed that among the five antibiotics used, infections is a combination of the antimicrobial prop- Clindamycin had the best antibacterial activity. The erty of the antibiotics used and the overall immunologi- presence of plasmid DNA in the eight most resistant cal dynamics occurring within the host; the MBC isolates may be responsible for their observed high results obtained still point to a very low susceptibility antibiotic resistance. It is therefore recommended that of a large proportion of the bacteria isolates to the a good antibiotics use policy put in place as well as antibiotics employed in the test. This development ethical and rational prescription practices by clini- raises a cause for genuine concern in the future of cians, and every healthcare personnel involved in the infectious diseases control of bacteria origin.
use of antibiotics in clinical and non-clinical settingswould help control and prevent the emergence of MDR Agarose gel electrophoresis was employed for the molecular characterization of the isolated plasmidsfrom the bacteria strains. Thus, the relative profiles ofthe DNA fragments and plasmids were characterized References
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