This study was conducted in the Department of Microbiology, and Burns and Plastic Surgery Department of Vardhman Mahavir Medical College and Safdarjang hospital, New Delhi, India, over a period of 2 years. The present study included 60 burn patients who were admitted to casualties, ICUs and wards of the hospital (group I). The patients who were included were within age range of 10-60 years, whose 20-60% body surface area (BSA) was involved in burns. Blood samples were collected from the same patients if septicaemia (fever, tachycardia, etc.) was evident. Five hundred and twenty five samples were collected from these 60 patients at different intervals- day 1, day 3 and day 7 of their admissions to the hospital. The study was approved by ethical committee of Safdarjang hospital.

A total of 101 environmental samples were collected, which included 51 hand, throat, and nasal swabs obtained from 17 medical staff (three from each) who were working in burn unit; and 50 samples from hospital environment (group II), which were taken from surgical instruments, dressings, suction devices, floors, door walls, beds, commodes, sinks, and antiseptic solutions.

The samples were cultured on Pseudomonas Isolation Agar (PIA), Mac Conkey’s Agar, and Blood Agar and the plates were incubated overnight at 37°C. P. aeruginosa was identified by its colony characteristics, pigment production, grape like odour, oxidase positivity, motility, gram staining (as gram negative bacilli), ability of reducing nitrates to nitrites, non-fermentative character, along with its ability to decarboxylate arginine, liquefy gelatin and to grow at 42°C [5]. Other bacteria which were isolated were also processed and identified by standard microbiological techniques [5].

Antibiotic sensitivity patterns of these isolates were studied by using Kirby Bauer Disc Diffusion method on Mueller –Hinton agar, by following CLSI 2011 Guidelines [6], by using Hi-media antibiotic discs. 11 antibiotics were tested, which included amikacin (30mcg), netilmicin (30mcg), gentamicin (10mcg), ceftazidime (30mcg), aztreonam (30mcg), ciprofloxacin (5mcg), piperacillin (100mcg), piperacillin + tazobactam (100/10mcg), imipenem (10mcg), meropenem (10mcg) and colistin (10mcg). Strains which had the same types of resistance patterns (antibiotype) were considered to be from the same clone. Pseudomonas aeruginosa ATCC 27853 strain was used for quality control in the study. In our work, MDR P. aeruginosa was detected as a bacterium which was resistant to three or more anti-Pseudomonal anti-microbial classes (piperacillin + tazobactam, imipenem, ceftazidime and gentamicin) [4].

The results were statistically analyzed by Chi squire tests and p-values of less than 0.05 were considered as significant.

The mean age of the patients who were included in the study was 29.7 years.The incidence rate of P. aeruginosa was highest (33.93%) in the age group of 21-30 years, as has been shown in [Table/Fig-1]. Culture positivity rate, including all isolates from patients, was determined to be 29.5% (155 strains out of 525 samples). Of the total 155 bacterial strains obtained from patients, P. aeruginosa accounted for 36.12% of the bacterial cultures, followed by Klebsiella sp. and Acinetobacter sp., which accounted for 21.29% and 16.77% of the cultures respectively. Others like Coagulase negative Staphylococci, Staphylococcus aureus, Escherichia coli and Proteus sp. accounted for the rest of the bacterial cultures, as has been described in detail in [Table/Fig-2].

Fifty six P. aeruginosa strains were isolated from inpatients, two were isolated from environmental samples (one from sink and other from door wall) and no P. aeruginosa strains were isolated from hospital staff. Of the 56 strains which were obtained from inpatients, five strains were isolated on day 1,19 strains were isolated after 48 hours of admission and 32 strains were isolated on day 7 of admission of the patients.

Antibiogram results have been described in detail in [Table/Fig-3] and they demonstrated that 58-81% strains were resistant to aminoglycosides (amikacin, netilmicin, and gentamicin), 41-70% were resistant to beta lactams-piperacillin, ceftazidime, and aztreonam, 34.5% were resistant to piperacillin-tazobactam, 12.06% were resistant to ciprofloxacin and that 13-19% were resistant to carbapenems (imipenem, meropenem). All strains were found to be sensitive to colistin (100%).

[Table/Fig-4] describes the Antibiotyping of the P. aeruginosa strains obtained from patients and environment. Antibiotyping was done by studying the antibiotic susceptibilities of the strains and by allotting them under different groups (Type 1-8), based on their patterns of resistance. Type 2 was the most common antibiotype which showed resistance to 7 antibiotics, and which accounted for 29.31 % of the strains. In the present study, 36.2% (21/58) P. aeruginosa strains were MDR and they were categorized into antibiotypes 1, 3 and 5. Among these 21 MDR isolates, 19 were clinical isolates and two were environmental strains.

There has been a rapid emergence of MDR P. aeruginosa in recent times, which is an important concern for clinicians who treat these infections. In the present study, 66.07% (40 /60) patients were found to be infected with P. aeruginosa during their stay in hospital, from among which 56 strains were isolated. Two P. aeruginosa isolates were obtained from environmental samples.

Among the beta-lactams, P. aeruginosa showed highest resistance to ceftazidime (70.68%). However, it was more sensitive to other beta-lactams i.e., piperacillin+ tazobactam, imipenem and meropenem sensitive (65.5%, 72.4% and 79.3% respectively), as has been described in [Table/Fig-3]. Colistin, although it was used only as a salvage drug, showed 100% susceptibility to all the strains. It has to be noted, that according to Srinivasan et al., P. aeruginosa was resistant to beta lactams viz. cephalothin, carbeniciilin, ceftazidime (100%), and cephalexin (98%) respectively [7]. According to the study of Saha et al., it is most sensitive to beta lactams - imipenem (98.72%), followed by aztreonam (33.44%) and ceftazidime (38.32%) [8]. Studies done by Kaushik et al., [9], Singh et al., [10], Taneja et al., [11], Agnihotri et al., [12] and Ganesamoni et al., [13], which were done in Indian context, showed resistance of Pseudomonas spp. in the range of 13.9 - 90% to amikacin, in the range of 4 - 90% to ceftazidime,, in the range of 50 - 77.7% to gentamicin and in the range of 41 - 95.1% to ciprofloxacin, which reflected high resistance profile of this nosocomial pathogen.

In the present study, MDR rate (resistance to three or more of anti Pseudomonal antimicrobials i.e. piperacillin + tazobactam, imipenem, ceftazidime and gentamicin) was determined to be 36.2% (21/58). A study done by Unan et al., [14] in Turkey reported rates of MDR, which were as high as 60%, whereas study done by Sabir et al., in Pakistan detected lower rates of MDR (22.08%) [15]. However, the rates of our study are comparable to a study done in Egypt, where Gad et al., [16] observed 36% MDR P. aeruginosa.

Typing of nosocomial isolates is essential for determining the epidemiology of nosocomial infections and for designing rational pathogen control methods. Antibiotyping was very informative in our work [Table/Fig-4]. Eight different resistance patterns were identified among 58 P. aeruginosa isolates. MDR was categorized in antibiotypes 1, 3 and 5. The two environmental P. aeruginosa isolates were also MDR and they belonged to antibiotype 1(from sink) and antibiotype 5 (from wall). Antibiogram is a sensitive phenotypic marker; however, it has the disadvantage of being non-reproducible in many instances, due to the exchange of R factor among isolates (Ramprasad et al., 2010) [17].

P. aeruginosa has gradually become a major cause of nosocomial infections which occur in burn patients and which requires immediate and effective implementation of infection control strategies, to combat its spread. Environmental sources may play a significant role in spread of MDR among hospitalized patients and it has been corroborated with evidence of two MDR strains which were isolated from hospital environment. In our study, two MDR strains were isolated from hospital environment and these had similar antibiotypes as of those strains which were isolated from patients. Antibiotyping has proved to be effective in determining the sources of nosocomial infections in immunocompromised patients like burn patients, and it can guide the infection control team to take measures for regulating the spread of this pathogen. Molecular characterization of the isolates was not done in the present study, due to financial constraints, which remains a limitation of this study.

In current times, antibiotics with antiPseudomonal activity which are available, include the aminoglycosides, ticarcillin, ureidopenicillins, ceftazidime, cefepime, aztreonam, the carbapenems, and ciprofloxacin. Combination treatments are generally recommended for suspected Pseudomonas infections. It has been reported that the choice of a carbapenem, cefepime, or piperacillin+ tazobactam, in combination with amikacin or tobramycin, in current times, appears to provide the widest potential antimicrobial activity against MDR P.aeruginosa [16]. Interestingly, our study also revealed that carbapenems, piperacillin + tazobactam, ciprofloxacin and gentamicin combinations were very effective in providing reasonable therapeutic options.

The lack of any new compounds in the near future indicates that national and local surveillance efforts are essential, to provide clinicians with correct information for choosing right antimicrobial therapy. Rigorous monitoring for MDR among Pseudomonas isolates is very important, because outbreaks caused by strains which are resistant to potentially useful agents, including carbapenems, have been reported elsewhere [17–19].

In conclusion, restriction of ‘selected antibiotic usage’ and/or infection control policies must be tailored for each institution, to combat the rapid emergence of MDR P. aeruginosa in burn patients. The lack of newer antimicrobial agents with activities against P. aeruginosa, makes periodic studies on the antimicrobial resistance patterns very important.