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ISSN: 2572-4134
Journal of Food & Industrial Microbiology
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Antimicrobial Susceptibility Pattern of Staphylococcus aureus Isolates from Orthopaedic Patients in Abuth, Zaria

Obajuluwa AF1*, Olayinka BO1, Adeshina GO1, Igwe JC2and Onaolapo JA1

1Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria, Nigeria

2National Biotechnology Development Agency, Abuja

*Corresponding Author:
Obajuluwa AF
Department of Pharmaceutics and Pharmaceutical Microbiology
Ahmadu Bello University, Zaria, Nigeria
Tel: +234 803 800 2812
E-mail: funkeyomi6874@gmail.com

Received date September 30, 2015; Accepted date January 18, 2016; Published date January 22, 2016

Citation: Obajuluwa AF, Olayinka BO, Adeshina GO, Igwe JC, Onaolapo JA (2016) Antimicrobial Susceptibility Pattern of Staphylococcus aureus Isolates from Orthopaedic Patients in Abuth, Zaria. J Food Ind Microbiol 2: 106. doi: 10.4172/2572-4134.1000106

Copyright: ©2016 Obajuluwa AF, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

As Staphylococcus aureus remain the predominant microbial flora of the human respiratory tract and skin; it also account for the most human integumental infections and life-threatening systemic diseases especially in orthopaedic surgical site infections (SSIs). This study evaluates the antibiotics susceptibility pattern of Staphylococcus aureus isolates from orthopaedic patients to various antimicrobial agents used in the treatment of surgical sites infection in Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. A total of 100 clinical swab samples of surgical sites were collected from orthopaedic patients in ABUTH, Zaria, Nigeria out of which 39 were identified as Staphylococcus aureus using API STAPH identification kit. Disc agar diffusion method was used for the antibiotics susceptibility test while nitrocefin microplate assay was used to test for beta lactamase production. Our findings showed that 97.4% of the isolates were susceptible to both vancomycin and gentamicin followed by ciprofloxacin (94.9%) and pefloxacin (84.6%). The isolates were highly resistant to ampicilin (94.9%), ceftriaxone (79.5%), cefoxitin (64.1%) and amoxicillin-clavulanic acid (59%) which is beta-lactam antibiotics. Further evaluation showed that 64% of the isolates produced beta-lactamase, while 36% do not. We conclude that the Staphylococcus aureus isolates from orthopaedic patients in ABUTH, Zaria were highly resistant to beta lactam antibiotics used in this study.

Keywords

Staphylococcus aureus ; Orthopaedic patients; Antibiotic susceptibility; ABUTH; Zaria

Introduction

Staphylococcus aureus is commonly carried on the skin or in the nose of healthy individuals. It is an important pathogen in human infections causing illness ranging from minor skin infections and abscesses to life-threatening diseases such as pneumonia, meningitis, endocarditis, toxic shock syndrome and septicaemia which may be rapidly fatal [1]. Bacterial resistance to antibiotics has been recognized since the first drugs were introduced for clinical use. Penicillin was first introduced in 1941, when less than 1% of Staphylococcus aureus strains were resistant to its action. By 1947, 38% of hospital strains had acquired resistance and currently over 90% of Staphylococcus aureus isolates are resistant to penicillin. Increasing resistance to antibiotics is a consequence of selective pressure [2]. In orthopaedics, S. aureus has been implicated in surgical site infection, painful infection of joint fluid known as septic or infective arthritis, post-operative infection, implant devices, infection following trauma, chronic osteomyelitis subsequent to an open fracture, meningitis following skull fracture. This study was aimed at determining the antibiotic susceptibility pattern of the S. aureus isolates from orthopaedic patients in a tertiary hospital in North-western Nigeria.

Methodology

Sample collection

One hundred clinical samples were collected aseptically from the wound, skin and bed of orthopaedic patients in Ahmadu Bello University Teaching Hospital Zaria, Nigeria over a period of 5 months. Ethical approval and patients’ consent were obtained.

Identification of S. aureus isolates

API STAPH identification kit (bioMerieux, Inc, Durham, USA) was used to identify the S. aureus isolates. The procedures were carried out according to the manufacturer’s instructions.

Antibiotic susceptibility test

Disk diffusion tests was performed for each of the isolates previously identified as S. aureus follow the method recommended by the Clinical Laboratory Standard Institute [3]. List of antibiotics used are: Cefoxitin 30 μg, Ceftriaxone 30 μg, Vancomycin 30 μg, Ampicillin 10 μg, Gentamicin 10 μg, Pefloxacin 5 μg, Ciprofloxacin 5 μg, Amoxicillin-clavulanic acid 30 μg, Erythromycin 15 μg and Clindamycin 2 μg (Oxoid Ltd. Basingstoke, London).

Test for β-lactamase production (Nitrocefin test)

Enzyme extracts of the S. aureus isolates were prepared as described by Caddick [4] with modification. Microplate Nitrocefin assay was carried out as follows: 1 mg lyophilized Nitrocefin powder (Oxoid, UK) was reconstituted in 1.9 ml of 0.1 M phosphate buffer, pH 7 supplied by the manufacturer. The reconstituted nitrocefin was further diluted 1 in 10 with PBS to give 50 μg/ml solution. The disrupted cell preparations were used immediately by dispensing 50 μL of preparation into separate wells of a 96 well plate. 50 μL of diluted nitrocefin solution was added into each of the wells and incubated at 37°C for 10 minutes. In the presence of β-lactamase, the chromogenic nitrocefin substrate changes colour from yellow to pink/red.

Determination of multiple antibiotics resistance (MAR) index

The Multiple Antibiotic Resistance (MAR) index was determined for each isolate by dividing the number of antibiotics to which the organisms is resistant to by the total number of antibiotics tested [5-7].

Results

A total number of 100 samples were collected and the distribution of S. aureus from the sample sites is presented in Table 1.

Source No of sample S.aureus No (%)
Wound 22  6(27.3)
Skin 43 16(37.2)
Bed 35 17(48.6)
Total 100 39(39.0)

Table 1: Distribution of S. aureus isolates.

Antibiotic susceptibility pattern of isolates

A high percentage (97.4%) of isolates were susceptible to both vancomycin and gentamicin followed by ciprofloxacin (94.9%) and pefloxacin (84.6%). The level of resistance of the S. aureus isolates to antibiotics is as follows: ampicilin (94.9%), ceftriaxone (79.5%), cefoxitin (64.1%) and amoxicillin-clavulanic acid (59%) (Table 2).

Antibiotics Sensitive Intermediate Resistant
No of sample (%) n=39
Ampicillin 10µg 2 (5.1) 0 37 (94.9)
Ceftriaxone 30µg 8(20.5) 13 (33.3) 18 (46.2)
Cefoxitn 30µg 14 (35.9) 0 25 (64.1)
Amoxicillin-clavulanic acid 30µg 16 (41.0) 0 23(59.0)
Clindamycin 2µg 12(30.8) 13 (33.3) 14 (35.9)
Erythromycin 15µg 19 (48.7) 10 (25.6) 10 (25.6)
Pefloxacin 5µg 33 (84.6) 0 6 (15.4)
Vancomycin 30µg 38 (97.4) 0 1 (2.6)
Ciprofloxacin 5µg 37 (94.9) 2 (5.1) 0
Gentamicin 10µg 38 (97.4) 0 1 (2.6)

Table 2: Percentage susceptibility of S. aureus isolates.

Beta lactamase test and their resistant pattern to antibiotic

The result of the beta lactamase test is shown in Figure 1.

food-industrial-microbiology-Beta-lactamase-production

Figure 1: Beta lactamase production by S. aureus isolates.

Resistant Pattern of beta lactamase producing S. aureus

The resistant patterns of beta lactamase producing S. aureus isolates to antibiotics are presented in Figure 2. There it was observed that these beta lactamase producing isolates were generally resistant to ampicillin (96%), ceftriaxone (88%), cefoxitin (76%) and amoxicillinclavulanate (68%) which are beta lactam antibiotics.

Multiple antibiotic resistant (MAR) index

MAR S. aureus isolates (n= 39)
0.1 4(10.3)
0.2 3 (7.7)
0.25 2 (5.1)
0.3 4(10.3)
0.4 5(12.8)
0.5 9(23.1)
0.6 8(20.5)
0.7 2(5.1)
0.8 1(2.6)
0.9 1(2.6)

Table 3: Result of multiple antibiotic resistant (MAR) index for S. aureussolatesantibiotics resistance pattern of S.Aureus.

food-industrial-microbiology-Resistance-pattern

Figure 2: Resistance pattern of beta-lactamase producing S. aureus to antibiotics.

The result of the MAR index of the S. aureus showed that 32(82.1%) of the resistant S. aureus isolates had MAR index greater than 0.2, details are shown in Table 3.

Antibiotics resistance pattern of S. aureus

This result showed that 50% (19) of the isolated S. aureus were multidrug resistant (MDR) and 28% (11) were extensively drug resistant (XDR). The isolates were observed to show a concurrent pattern of resistance to Cephalosporine, Macrolide and Betalactame/ Betalactame inhibitors (21%) while 10% (4) showed resistance to Cephalosporine, Betalactame/Betalactame inhibitors and Fluoroquinolones.

Discussion

S. aureus is known to be one of the causes of nosocomial infection [8-10], the isolation of S. aureus from the patients’ beddings in this study is in support of this. The majority of nosocomial infection is caused by the patient’s own endogenous microbial flora present upon admission to the hospital [11]. The 97.4% susceptibility to gentamicin observed in this study is consistent with previous reports from Africa [12,13] Gentamicin is an example of aminoglycoside whose mode of action is to inhibit protein synthesis by inhibiting 30 S ribosome of the organism [14]. High susceptibity to vancomycin was observed in this study. Vancomycin is a glycopeptide which inhibits peptidoglycan synthesis in the bacteria, it is a known drug of choice in the treatment of methicillin resistant S. aureus [15].

Ciprofloxacin is a quilonone which acts by inhibition of enzymes involved in DNA replication and function, the 94.9% susceptibility of S. aureus observed in this study is in conformity with previous reports [16-18]. The little level of resistance to ciprofloxacin observed in this study might be connected with the increasing rate of availability of different cheap brands of generic ciprofloxacin in the market which might have probably led to the misuse of it.

The S. aureus isolates in this study were generally resistant to penicillins as was shown by the resistance pattern of the beta lactamase producing S. aureus isolates in Figure 2. Beta lactamase hydrolyses the amide bond of the β-lactam ring resulting in an inactive compound. Some researchers found penicillins to have the highest rate of resistance to clinical samples especially S. aureus . [19,20]. Penicillins inhibit bacterial cell wall synthesis [21], S. aureus develop resistance to penicillins by the production of beta lactamases and by permmeabilty barrier of the cell surface [22].

The high percentage of the S. aureus having MAR index greater than 0.2 (Table 3) suggests that the isolates originated from a high risk source of contamination where antibiotics are often used [6,7]. It also indicates that a large proportion of the bacterial isolates have been exposed to several antibiotics. The high incidence of multi drug resistance observed in this study (Table 4) could be attributed to a combination of microbial characteristics such as selective pressure on antimicrobial usage, societal and technological changes that enhance the transmission of drug resistant organisms [23]. Other reasons could be due to increase in irrational consumption rate of antibiotics, transmission of resistant isolates between people, self-medication, non-compliance with medication and sales of substandard drug.

S/N Isolates Antibiotics Resistant Pattern CART LR
1 W4 AMP BT NIL
2 W7a FOX, AMP, CRO, ERY, DA, AMC CEP, BT, MAC, LIN MDR
3 W7b FOX, AMP, CRO, AMC CEP, BT XDR
4 W20 FOX, AMP, CRO, AMC CEP, BT XDR
5 W39 FOX, AMP, CRO, VA CEP, BT, MAC, GL MDR
6 W51 FOX, AMP, DA, AMC CEP, BT, LIN MDR
7 S1 DA LIN NIL
8 S2 FOX, AMP, AMC, ERY, DA CEP, BT, MAC, LIN MDR
9 S8 AMP BT NIL
10 S12 FOX, AMP, CRO, AMC, VA CEP, BT, GL MDR
11 S20 FOX, AMP, CRO, AMC CEP, BT XDR
12 S23 AMP, ERY, DA BT, MAC, LIN MDR
13 S24 FOX, AMP, CRO, AMC, ERY, DA CEP, BT,MAC, LIN MDR
14 S25 AMP, DA BT, LIN XDR
15 S27 FOX, AMP, CRO, AMC, VA CEP, BT, GL MDR
16 S41 FOX, AMP, CRO, AMC CEP, BT XDR
17 S46 FOX, AMP, CRO, AMC, VA, PER CEP, BT, GL, FLU MDR
18 S47 DA LIN NIL
19 S51 FOX, AMP, AMC CEP, BT XDR
20 S55 FOX, AMP, PER, AMC CEP, BT, FLU MDR
21 S58 AMP BT NIL
22 S72 AMP, DA CEP, BT,MAC,LIN MDR
23 B1 AMP, CRO, ERY, DA CEP, BT,MAC, LIN MDR
24 B7 AMP BT NIL
25 B8 AMP BT NIL
26 B13 FOX, AMP, CRO, AMC, VA CEP, BT, GL MDR
27 B16 FOX, AMP, AMC, ERY CEP, BT, MAC MDR
28 B20 FOX, AMP, CRO CEP, BT XDR
29 B26 FOX, AMP, CRO, AMC CEP, BT XDR
30 B35 FOX, AMP, CRO, AMC CEP, BT XDR
31 B47 FOX, AMP, CRO, AMC CEP, BT XDR
32 B49 FOX, AMP, AMC CEP, BT XDR
33 B50 AMP, ERY, DA BT, MAC, LIN MDR
34 B55 FOX, AMP, AMC, PEF CEP, BT, FLU MDR
35 B60 AMP, ERY, DA BT, MAC, LIN MDR
36 B62 AMP BT NIL
37 B69 FOX, AMP, CRO, AMC, ERY, DA CEP, BT, MAC, LIN MDR
38 B77 FOX, AMP, CRO, PEF CEP, BT, FLU MDR
Key: Lincosamides (LIN), Cephalosporine (CEP), Macrolide (MAC), Betalactame/ Betalactame Inhibitors (BT), Glycopeptide (GL), Fluoroquinolone (FLU), Class of antibiotics resistant to (CART), Level of resistance (LR), Cefoxitin (FOX) Vancomycin (VA), Ampicillin (AMP), Ceftriaxone (CRO), Ciprofloxacin (CIP), Gentamicin (CN), Pefloxacin (PEF), Erythromycin (ERY), Amoxicillin-clavulanic acid (AMC), Clindamycin (DA)

Table 4: Antibiotic resistance pattern of the isolated S. aureus.

Conclusion

High susceptibility S. aureus isolates to gentamicin and ciprofloxacin was observed in this study, this is an indication that these antibiotics can be used for empirical treatment of infections from orthopaedic patients in this hospital since vancomycin is not readily available in this community. The S. aureus isolates were highly resistant to beta lactam antibiotics. The abuse of beta-lactam antibiotics and other classes of antibiotics in our community should therefore be controlled.

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