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ABSTRCT

 

Introduction: Burn wound infection is a potential risk factor for high number of mortality in these patients. This study was conducted to determine the prevalence and analyze antibiotic susceptibility patterns of Methicillin Resistant Staphylococcus aureus isolated from Burn Unit of Allied Hospital Faisalabad, Pakistan

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Methodology: A cross sectional study was designed in which 70 burn wound swabs were collected. Isolation of organism was done by culturing on blood, MacConkey and Mannitol salt agar media. Further gram staining and biochemical characterization was done to identify isolates. Recommended antibiotics of CLSI were used to identify resistance pattern of S. aureus.

Results:  Total 95 isolate obtained from 70 burn wound swabs. It was observed that the major cause of burn injury was flame burn (n = 44) 63%. There was high prevalence of Pseudomonas aeruginosa (36%) following Staphylococcus aureus strains (26%), Gramryp1  positive rod (15%), Kleibsiella spp (14%), Streptococci (4%), Coagulase negative Staphylococcus (3%) and Neisseria spp (2%). Staphylococcus aureus strains (n = 25) comprising of 11 (44 %) methicillin resistant and 14 (56 %)  methicillin sensitive. Multidrug resistant pattern of S.aureus showed resistant predominantly against clindamycin 68% followed by erythromycin, methicillin, levofloxacin, moxifloxacin and vancomycin (56%, 44%, 36%, 16% and 4%) respectively.

Conclusion: A potentially alarming prevalence and multidrug resistance pattern found in MRSA isolates indicates major health concerns in Faisalabad. Therefore attention of health authorities required.

Key words: Methicillin resistance Staphylococcus aureus, Multi drug resistance, Antibiotics, Burn wound infection

 

 

 

 

 

 

 

 

 

 

 

Introduction

Burns are one of the most common and devastating forms of trauma and injury 1. Among the deaths happened due to injuries, burn injury is predominantly common 2. Wounds that result after burn are considered as the favorable sites for bacterial multiplication 3. In most of the burnt patients if the total body surface area (TBSA) is more than 40%  burnt there are higher risk of sepsis that cause death 4. Therefore, specialized management is necessary in order to control morbidity and mortality among burn patients 5.

A high mortality rate has been reported due to burn injuries and rate of injuries are predominantly high in low income countries. Reasons include less facilities to control fire accidents, less health facilities, lack of specialized care, poor management to control the complication arise in burn patients 6. Another  reason of  high mortality rate is  nosocomial infection, almost 75% deaths occur because of bacterial infection and the most common pathogens characterized from burn wounds are Staphylococcus aureus, Pseudomonas aeruginosa, Kleibsiella, Streptococcus pyogenes and various others 7 8.

The persistence of S. aureus as a nosocomial pathogen is a concern all over the world 9. It is the second commonest organism causing nosocomial infection after Pseudomonas 10 11.Despite there are widespread antibiotics are present to cure bacterial infection but at the same time emergence of drug resistance species is a problem 12. Resistance for antimicrobial drugs is one of the global public health concern arises due to over use or misuse of antimicrobial compounds, poor compliance, lack of medical education, self-medication, poor choices of antibiotics at first line treatment and many more 13.  S. aureus is one of the bacterial specie acquiring the resistance against antibiotics day by day. Methicillin resistance aureus is an emerging problem in the field of medical microbiology  and presence of drug resistance specie at the site of burn injury made it  difficult to cure infection 14.

So this study was designed to isolate and characterize S. aureus from burn patients from a tertiary care hospital of Faislabad, Pakistan and to report the prevalence and sensitivity pattern of S. aureus to help in better management of these patients with burn wound infections.

 

 

Methods

Study Design

An observationalryp2  study was designed in which burn wound swab samples from 70 patients were recruited from burn unit, Allied Hospital Faisalabad. This study was done after taking permission from University Institute of Medical Lab Technology (UIMLT) and Ethical Review Committee (ERC) Punjab Medical College, Faisalabad (Ref No: 767/2017). Before taking swab samples from the patients a written or informed consent was taken from patients or relatives of patients.

Sample Collection

Direct swabs from the burn wound were taken aseptically and recommended guidelines of Clinical Laboratory Standard Institute (CLSI) were followed. Samples were transported immediately to the Microbiology section of the main Pathology Laboratory of Allied Hospital Faisalabad with no delay to avoid any contamination.

Isolation and characterization

Samples were cultured on  Blood ,  MacConkey and  Mannitol salt agar medium and incubated at 35-37°C for 18-24 hours to get growth and qualitative and semi quantitative analysis on basis of colony characteristics and morphology 15.

The characteristic colonies of S.aureus were further confirmedryp3  by gram staining, catalase, coagulase and DNase test. For each isolate API strip test were used for confirmation.

Anti-biogramryp4  Assay

The Kirby-Bauer disc diffusion method was used to evaluate the antimicrobial susceptibility pattern of the S. aureus isolates against CLSI recommended antibiotics (Recommended two antibiotics from each group) List of antibiotics used in this study are given in table 1.

Results

Demographic characteristics of burn patients:

A total of 70 burn patients, admitted in Burn Centre, Allied Hospital Faisalabad (AHF), Faisalabad, were recruited and mostly were male patient 41 (59 %) and  29 (41%) were female. The mean age of these patients was 20 year ranging from 1 to 65 year. Etiology of burn patients included in this study revealed 44 (63%) patients suffered with flame burn injury, 16 (23%) with scaled, 4 (6%) with electrical, 3 (4%) with chemical and 3 (4%) had road traffic accident burn injury. Of these 70 patients, 21 (30%) had 1-20% of their Total Body Surface Area (TBSA) burnt, 32 (46%) had 21-40% TBSA, 11 (16%) had 41-60% TBSA and 6 (8%) patients had more than 60% of their TBSA burnt. It was observed that 94% of total patients were culture positive for bacterial growth. Results are presented in table 2.

Isolation and characterization of bacterial strains from burn patients

A total of 95 strains were isolated on bacterial culture from 66 culture positive burn patients.It was observed that 32 isolates were gram positive cocci, 14 were gram positive bacilli, 47 were gram negative bacilli and only 2 were gram negative cocci (Table 3).

On biochemical characterization it was observed that out of 32 gram positive cocci strains, 28 were catalase positive and on further screening 25 strains were coagulase positive and 3 were coagulase negative.

Out of 28 isolated Staphylococcus spp. 25 were confirmed S. aureus based on their cultural and biochemical characteristics. It was observed that Pseudomonas aeruginosa is the predominate organism (36%) among all others. Isolated organisms from these selected burn patients are presented in table no 4.

Antimicrobial sensitivity pattern of S. aureus:

On the basis of antimicrobial sensitivity pattern obtained on Muller Hinton agar it was observed that 44% of S. aureus isolates from burn patients were resistant toward methicillin however 56% were sensitive for this antibiotic. Surprisingly 4% were also resistant against Vancomycin, 36% against levofloxacin and 16% against Moxifloxacin (Table 5). 

When antimicrobial sensitivity pattern was compared between different groups of antibiotic as recommended by CLSI, it was observed that most of the isolates (18) were resistant to both drugs (Clindamycin in combination with Erythromycin) belongs to group “A” used in this research work. Only one isolate was sensitive for both antibiotics of group “A”. For group “B” antibiotics (Methicillin in combination with Vancomycin) 3 were resistant and 12 were sensitive with both of these drugs. For group “C” antibiotics (Levofloxacin in combination with Moxifloxacin) 8 were resistant and 8 were sensitive for both drugs.

Multidrug resistant pattern was observed when combinations of antibiotics of different groups were compared. Three isolates were resistant and one was sensitive for four antibiotics, 2 of group “A” (Clindamycin and Erythromycin) and 2 of group “B” (Methicillin and Vancomycin). For the combination of group “A” and “C” it was observed that 7 isolates were resistant and only 1 isolate was sensitive against all four antibiotics. Maximum antimicrobial sensitivity was observed for group “B” in combination with group “C”. However among all isolates of S. aureus only 1 isolate was sensitive for all 6 antibiotics, 2 of each group “A, B and C” used in this research and 3 isolates were resistant for all of these antibiotics. Results of antimicrobial sensitivity against different combination of antibiotics groups have been presented in figure 1.

Discussion:

This study was designed to investigate the prevalence of . aureus in burn patients, it was also recorded how much methicillin resistant S.aureus is prevalent in these burn patients. This study suggests high prevalence of methicillin resistant S. aureus among burn patients.

Our results indicate that burn injuries are common in male. Aghakhanietryp5  al in 2011 also reported gender difference in burn patients 16 17. In this study flame burn injury was the most prevalent among all other etiological reasons. Flame on scaled is the most common type reported by others 18.

Development of infection in burn patients is profoundly high i.e. 94% of the patients had culture positive results. This indicates presence of any microorganism at site of wound which progressed after burn as reported by previous studies (study) 19. Mixed microbial flora as reported in our studies replicates previous studies. However, Staphylococcus aureus was reported as the common isolates unlike Pseudomonas aeruginosa found in this study. 3, 20. In this study staphylococcusryp6  is the second larger isolate. S. aureus is considered as a significant pathogen among burn patient across the world 21. Various studies from Pakistan have reported different prevalence rate of MRSA in burn patients. A multicenter study have reported a prevalence of 61% MRSA  in Lahore 22, 57% in Karachi 23, 46% in Rawalpindi and Islamabad 24, 36% in Peshawar 25 and 26% in Quetta 6. A similar kind of study in Faisalabad reported 40% MRSA from burn patients 12 which closely matches to our findings i.e. 44%  MRSA.

Interestingly it was observed that all of the MRSA strains isolated in this study were from the mixed bacterial flora that was obtained from the single wound swab culture. Presence of MRSA-the MDR at site of infection impose problems in control of infection. Apart from Methicillin resistance S. aureus (MRSA), Vancomycin resistance S. aureusryp7  (VRSA) was also observed in these samples that is an alarming situation for local health care bodiesryp8 .

For the different groups of antibiotics result of this study suggest multi drug resistance pattern for S. aureus. For primaryryp9  group (A) resistance pattern was much high and interestingly for group C those are considered supplement antibiotics resistance pattern was also high, that indicate the misuse of antibiotics in our local population. However, among all three groups the best combination therapy achieved from Vancomycin. As there is less literature that report prevelance of MRSA among burn patientsryp10  so this data is significant for health care bodies. However due to financial constrains we were unable to do molecular characterization of isolates and to identify mec gene.

Conclusionryp11 : A high prevalence of methicillin resistance S.aureus has been observed among burn patients. The reasons of this elevated pattern of drug resistance of Staphylococcus spp. might be continued use of broad spectrum antibiotics, lack of medical awareness, self-medication and non-adherence to hospital guidelines. We should control the misuse of antibiotics and promotion of medical awareness to cope this alarming increasing rate of antibiotic resistance.

Acknowledgement:

Burn Unit, Allied hospital Faisalabad

ReferencesMGDG12 

1              Association AB (2000) Burn incidence and treatment in the US: 2000 fact sheet.

2.             Association AB (2011) Burn incidence and treatment in the United States: 2011 fact sheet. Chicago: American Burn Association.

3.             Atiyeh BS, Gunn SW, and  Hayek SN (2005) State of the art in burn treatment. World J Surg 29(2):131-148.

4.             Kooistra?Smid M, Nieuwenhuis M, Van Belkum A, Verbrugh H (2009) The role of nasal carriage in Staphylococcus aureus burn wound colonization. Pathogens and Disease 57(1):1-13..

5.             Hussain M, Basit A, Khan A, Rahim K, Javed A, Junaid A, unir S, Niazi R, Sohail M, Hussain T (2013) Antimicrobial sensitivity pattern of methicillin resistant Staphylococcus aureus isolated from hospitals of Kohat district. Pak J Inf Mol Biol 1(1):13-6.

6.             Sharma B (2007) Infection in patients with severe burns: causes and prevention thereof. Infect. Dis. Clin. North Am 21(3):745-759.

7.             Church D, Elsayed S, Reid O, Winston B, Lindsay R (2006) Burn wound infections.  ?Clin. Microbiol. Rev 19(2):403-34

8.             Onwubiko, N.E. and N.M. Sadiq (2011) Antibiotic sensitivity pattern of Staphylococcus aureus from clinical isolates in a tertiary health institution in Kano, Northwestern Nigeria. Pan African Medical Journal 8(1).

9.             Cheesbrough M (2006) District laboratory practice in tropical countries. Cambridge university press.

10.           Dang, K.B., Detection and quantification of staphylococcus aureus enterotoxin B in food product using isotopic dilution techniques and mass spectrometry. 2013.

11.           Rasool MH, Yousaf R, Siddique AB, Saqalein M, Khurshid M (2016) Isolation, Characterization, and Antibacterial Activity of Bacteriophages Against Methicillin-Resistant Staphylococcus aureus in Pakistan. Jundishapur J Microbiol 9(10).

12.           Bhatia R, Narain JP. The growing challenge of antimicrobial resistance in the South-East Asia Region-Are we losing the battle? (2010) Indian J Med Res 132(5):482.

13.           Kazakova SV, Hageman JC, Matava M, Srinivasan A, Phelan L, Garfinkel B, Boo T, McAllister S, Anderson J, Jensen B (2005) A clone of methicillin-resistant Staphylococcus aureus among professional football players. N Engl J Med 352(5):468-75.

14.           Gargis AS, Kalman L, Berry MW, Bick DP, Dimmock DP, Hambuch T, Lu F, Lyon E, Voelkerding KV, Zehnbauer BA (2012) Assuring the quality of next-generation sequencing in clinical laboratory practice. Nature biotechnology 30(11):1033-6.

15.           Aghakhani N, Nia HS, Soleimani MA, Bahrami N, Rahbar N, Fattahi Y, Beheshti Z (2011) Prevalence burn injuries and risk factors in persons older the 15 years in Urmia burn center in Iran. Caspian J Intern Med 2(2):240.

16.           Krishnamoorthy V, Ramaiah R, Bhananker SM. (2012) Pediatric burn injuries. Int J Crit Illn Inj Sci 2(3):128.

17.           Brusselaers N, Monstrey S, Vogelaers D, Hoste E, Blot S (2010) Severe burn injury in Europe: a systematic review of the incidence, etiology, morbidity, and mortality. Crit. Care 14(5):R188.

18.           Alaghehbandan R, Azimi L, Lari AR (2012) Nosocomial infections among burn patients in Teheran, Iran: a decade later. Ann Burns Fire Disasters 25(1):3.

19.           Komolafe O, James J, Kalongolera L, Makoka M (2003) Bacteriology of burns at the Queen Elizabeth Central Hospital, Blantyre, Malawi. Burns 29(3):235-8.

20.           Alebachew T, Yismaw G, Derabe A, Sisay Z (2012) Staphylococcus aureus burn wound infection among patients attending Yekatit 12 hospital burn unit, Addis Ababa, Ethiopia. Ethiop J Health Sci. 22(3).

21.           Namvar AE, Afshar M, Asghari B, Lari AR (2014) Characterisation of SCCmec elements in methicillin-resistant Staphylococcus aureus isolated from burn patients. Burns 40(4):708-12.

22.           Hafiz S, Hafiz A, Ali L, Chughtai A, Memon B, Ahmed A, Hussain S, Sarwar G, Mughal T, Awan A (2002) Methicillin resistant Staphylococcus aureus: a multicentre study. J Pak Med Assoc 52(7):312-4

23.           Ahmed A, S. Qureshi, and N. Ansari (2000) Prevalence and antibiotic sensitivity pattern of methicillin resistant Staphylococcus aureus (MRSA) infection in a tertiary care hospital. in Karachi: Liaquat National Hospital Symposium.

24.           Qureshi A, Rafi S, Quresh S, Ali A (2004) The current susceptibility patterns of methicillin resistant Staphylococcus aureus to conventional anti-staphylococcus antimicrobials at Rawalpindi. Pak J Med Sci 20:361-364

25.           Shafiq R, Shahina M, Ahmad J, Syed H, Malala R (2011) Incidence of Methicillin reistant Staphylococcus in Peshawar. J Ayub Med College Abbotabad 23(1).

 

 

 

Caption of tables:

Table 1: Antibiotics against S. aureus used in this study

Table 2: Demographic Characteristics of Burn Patients (n=70)

Table 3: Characterization of isolates on the basis of Gram staining (n= 95)

Table 4: Organisms isolated from Burn Patients (n=95)

Table 5: Antimicrobial Sensitivity Pattern of S. aureus (n= 25)

Legends of Figure:

Figure 1: Multidrug resistance pattern of S. aureus isolated from burn wounds

 

 ryp1were these not characterized?

 ryp2In abstract, cross sectional was mentioned

 ryp3Can give reference of Bergey’s manual.

 ryp4You can add few lines about the preparation of bacterial inoculum of which bacterial lawn was prepared.

Also about the designation of isolates as resistant, sensitive, or intermediate based on zone of inhibition. 

 ryp5Reference style

 ryp6Or S. aureus?

 ryp7Can give any reference where MRSA and VRSA both are found in bacterial isolates?

 ryp8Any reference?

 ryp9Primary group?

 ryp10Can also say that sample size is small but results are of importance

 ryp11It is just a suggestion

Do you have patient details ? you can check / relate gender and age too with MDR. I have not seen any such relations. Just came in my mind.

 MGDG12* References in the references section are not properly formatted. Examples are provided in the Author Guidelines, in the aforementioned manuscript template, and all the previously published articles. To easily gather and properly fit references into your manuscript, you may want to use Zotero, which is free software available at www.zotero.org.

 

* Please note that the “et al” formula is not allowed in the references section according with the Journal’s style for bibliography. Please use all authors names.

 

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