Antimicrobial resistance pattern of Pseudomonas aeruginosa isolated from various clinical samples in a tertiary care hospital, South Odisha, India

Saudi Journal for Health Sciences - Vol 3, Issue 1, Jan-Apr 2014 15

INTRODUCTION

Pseudomonas aeruginosa is an aerobic, motile, Gram-negative rod and is leading cause of opportunistic nosocomial infections. It is responsible for 10% of all hospital-acquired infections.[1] It has been implicated in diverse infections such as pneumonia, urinary-tract infection, skin and soft-tissue infections, in severe burns and in infections among

immunocompromised individuals. Prior to use of antibiotics, history of P. aeruginosa infection or colonization within the previous year, length of hospital stay, being admitted as in-patient or in the intensive care unit (ICU), mechanical ventilation, malignant disease and history of chronic obstructive pulmonary disease have all been identified as an independent risk factors for multidrug-resistant (MDR) P. aeruginosa infection.[2-4]

Infections caused by P. aeruginosa are often severe, life-threatening and are difficult to treat because of limited susceptibility to antimicrobial agents and high frequency of emergence of antibiotic resistance during therapy.[5] The antibiotic resistance mechanisms include the acquisition of extended-spectrum β-lactamases, carbapenemases, aminoglycoside-modifying enzymes and 16S ribosomal ribonucleic acid methylases. Mutational changes causing the up-regulation of multidrug efflux

Antimicrobial resistance pattern of Pseudomonas aeruginosa isolated from various clinical samples in a tertiary care hospital, South Odisha, IndiaMuktikesh Dash, Sanghamitra Padhi, Moningi Venkat Narasimham, Swetalona Pattnaik

Departments of Microbiology, Maharaja Krishna Chandra Gajapati Medical College and Hospital, Berhampur University, Ganjam, Berhampur, Odisha, India

Original Article

Address for correspondence: Dr. Muktikesh Dash,Department of Microbiology, Maharaja Krushna Chandra Gajapati Medical Collage and Hospital, Ganjam, Berhampur - 760 004, Odisha, India. E-mail: [email protected]

Background: Pseudomonas aeruginosa is an aerobic, motile, Gram-negative rod which is responsible for 10% of all hospital-acquired infections. Objectives: This study was conducted to determine the frequency, risk factors and antibiotic resistance pattern of P. aeruginosa isolated from various clinical samples. Materials and Methods: Present retrospective hospital record based cross-sectional study included a total of 6280 clinical samples collected from patients at a tertiary care hospital, South Odisha, India from January 2011 to December 2012. Samples were processed and identifi ed by standard protocol. The P. aeruginosa was tested for antibiotic resistance by Kirby-Bauer disc diffusion method (according to Clinical and Laboratory Standards Institute guidelines). Results: From 6280 clinical samples, 3378 (53.8%) samples yielded signifi cant growth and 327 samples were positive for (9.7%, 327/3378) P. aeruginosa (6.8% of nosocomial and 2.9% of community-acquired infections). Maximum 221 (67.6%) isolates were obtained from pus/swab, followed by urine 15% and blood (4.9%). Elderly, in-patients and invasive procedures were found to be signifi cant risk factors in the setup investigated (P < 0.05). Out of 327 isolates, 277 (84.7%) isolates were multidrug-resistant, 99 (35.7%, 99/277) isolates were extensively drug-resistant. No pandrug-resistant isolate was obtained. Majority of isolates were sensitive to imipenem, meropenem and piperacillin/tazobactam, showed the least resistance rate of 6.4%, 8% and 11.3% respectively. Conclusion: This hospital based epidemiological data will help to implement better infection control strategies and improve the knowledge of antibiotic resistance patterns among clinicians. Thus, there is a need for periodical antimicrobial surveillance to monitor the resistance patterns in local hospitals.

Key words: Antibiotics, extensively drug-resistant, multidrug-resistant, pandrug-resistant, Pseudomonas aeruginosa

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Dash, et al.: Antimicrobial resistance pattern of Pseudomonas aeruginosa

16 Saudi Journal for Health Sciences - Vol 3,Issue 1, Jan-Apr 2014

pumps, derepression of ampC, modification of antimicrobial targets and changes in the outer membrane permeability barrier are also described. Moreover, the propensity of P. aeruginosa to exist in vivo and in the environment as slow-growing organism embedded in its extracellular matrix adds to its resistance mechanisms. Thus, emergence of MDR P. aeruginosa is of clinical concern and the pandrug-resistant (PDR) isolates, treatable only with colistin, are on the rise.[6]

Therefore, there is a need to conduct area-specific monitoring studies of P. aeruginosa for its resistance patterns and hence the data generated would help clinicians and policy makers to provide correct empirical treatment for patients. Keeping these facts in view, the present study was undertaken to find out the prevalence of drug resistance and antibiotic susceptibility patterns of pathogenic P. aeruginosa isolated from various clinical specimens in a tertiary care hospital.

MATERIALS AND METHODS

Study area, population and methodologyDescriptive cross-sectional study was carried out from January 2011 to December 2012 in the Department of Clinical Microbiology at a tertiary care hospital, South Odisha, India. This 600 bedded hospital has three ICUs, one emergency ward, six medical and surgical wards and out-patients departments. A total of 6280 clinical samples such as pus/swab, urine, sputum, blood, cerebrospinal fluid, pleural fluid, peritoneal fluid, tissue biopsies and bronchial lavage were collected from patients and transferred to the laboratory without delay for further processing. The retrospective evaluation of patient’s age, sex, admission into the hospital, duration of stay and special invasive procedure conducted was carried out on the basis of the case record histories. A health-care-associated infection or nosocomial infection is defined as a localized or systemic condition resulting from an adverse reaction to the presence of infectious agent (s) or its toxin (s) that was not present on admission to the hospital. An infection is considered as nosocomial if all the elements of a Center of Disease Control and Prevention site-specific infection criterion were first present together on or after the 3rd hospital day (day of hospital admission is day 1).[7] Patients from whom P. aeruginosa was isolated in the absence of clinical disease suggesting colonization was not included in this study. The study was conducted after approval from Institutional Ethical Committee.

Sample processing and antibiogramIn the laboratory all collected samples were cultured aerobically on Blood agar and MacConkey agar plates at 37°C

for 24 h. Blood specimen was cultured in Trypticase Soy Broth and sub-cultured in Blood agar and MacConkey agar plates. Suspected colonies of P. aeruginosa were identified using colonial morphology, motility testing, Grams reaction and biochemical tests. Definitive identification of P. aeruginosa included identifying the production of the blue green pigment pyocyanin and its ability to grow at 42°C.[8]

All detected P. aeruginosa isolates were tested for antimicrobial susceptibility test by the standard Kirby-Bauer disc diffusion method according to Bauer et al.,[9] The test organism was picked up with a sterile loop, suspended in peptone water and incubated at 37°C for 2 h. The turbidity of the suspension was adjusted to 0.5 McFarland’s standard (1.5 × 108 CFU/mL). It was then spread on the surface of Mueller-Hinton agar (MHA) plate using sterile cotton swab. The following standard antibiotic disks were placed on MHA plate: Ciprofloxacin (5 mcg/disk), gentamicin (10 mcg/disk), amikacin (30 mcg/disk), ceftazidime (30 mcg/disk), piperacillin/tazobactam (100/10 mcg/disk), imipenem (10 mcg/disk), meropenem (10 mcg/disk), levofloxacin (5 mcg/disk), cefepime (30 mcg/disk) and piperacillin (100 mcg/disk). The plate was incubated at 37°C overnight. The zone of inhibition were measured and interpreted according to Clinical and Laboratory Standards Institute guidelines.[10] All media and antibiotic disks were procured from Himedia Labs., Mumbai, India. In addition, the antibiotic potency of the disk was standardized against the reference strains of Escherichia coli ATCC 25922 (β-lactamase negative) as a positive control and reference strain of P. aeruginosa ATCC 27853 as a negative control.

MDR P. aeruginosa is defined as non-susceptibility to at least one agent in three or more antipseudomonal categories i.e., aminoglycosides, carbapenems, cephalosporins, fluoroquinolones and penicillin/β-lactamase antibiotics. Extensively drug-resistant (XDR) P. aeruginosa is defined as isolates, which remain susceptible to only one or two antipseudomonal categories. Similarly, PDR isolate is non-susceptible to all agents in all antipseudomonal categories (i.e. no agents tested as susceptible for that isolate).[11] Thus, a P. aeruginosa isolate that was characterized as XDR also included as MDR.

Statistical analysisThe data were analyzed for mean, median and standard deviation and P value (Chi-square with Yates’ correction and Fisher’s exact test) by using GraphPad QuickCalcs statistical software Inc., 2236 Avenida de la Playa La Jolla, CA 92037 USA. Statistical significance was defined when P < 0.05.

RESULTS

During the study period from January 2011 to December 2012, a total of 6280 clinical samples were aerobically cultured, out of which 3378 (53.8%) yielded significant growth and the rest 2902 (46.2%) samples were either sterile or showed non-significant growth. From 3378 growth positive samples, a total of 327 (9.7%) P. aeruginosa were isolated. From 327 isolates, majority 229 isolates (70%) were detected from in-patients admitted in the hospital and rest 98 isolates (30%) were isolated from out-patients (community-acquired infection). 221 (67.6%) of P. aeruginosa were isolated from pus/swab samples followed by urine 49 (15%) and blood 16 (4.9%) samples [Table 1].






The mean age of patients infected with P. aeruginosa was 38.6 years old (median 39, standard deviation ± 19.6, 95% confidence intervals 34.7 to 41.8, minimum 3 and maximum 90 years old). The gender (male: female) ratio was 1.4:1, which was not significantly associated (P = 0.13). P. aeruginosa infection was significantly observed among in-patients, elderly (≥55 years), associated with and had under gone any invasive procedure (P < 0.001) [Table 2].

Table 3 shows the frequency of MDR among P. aeruginosa. Out of 327 isolates, 50 (15.3%) isolates were sensitive to all antibiotics tested and 277 (84.7%) isolates were MDR. From 277 MDR P. aeruginosa isolates, 99 (35.7%) isolates were XDR. No isolate was found to be PDR.

In our study, P. aeruginosa was highly resistant to ceftazidime 77.7%, cefepime 64.8%, piperacillin 45%, ciprofloxacin 38.9%, levofloxacin 36.1%, gentamicin 37.3% and amikacin 30%. P. aeruginosa was least resistant to imipenem 6.4%, followed by meropenem 8% and piperacillin/tazobactam 11.3%, which can be considered as effective drugs in this present review [Table 4].

DISCUSSION

P. aeruginosa is the most common non-fermenting bacterium isolated from clinical specimens and presents a serious therapeutic challenge for the treatment of both community-acquired and nosocomial infections. Identification and selection of appropriate antibiotic to initiate therapy is essential to optimizing the clinical outcome.[12] The main objective of the present study was to investigate the epidemiological data of P. aeruginosa isolated from various clinical specimens and to determine the antimicrobial resistance patterns against routinely used antipseudomonal antibiotics.

In our study, from 3378 clinical isolates, 327 P. aeruginosa isolates were obtained, with a prevalence rate of 9.7%. Similar prevalence rate of 9.3% was reported by Srinivas et al. in Andhra Pradesh, India.[13] In comparison, higher prevalence rate of 32.1% and 20.3% was reported by Rajat et al. and Javiya et al. in Gujarat, India respectively.[14,15] Low prevalence of 2.1% was obtained by Okon et al. in Northeastern Nigeria.[16] This varied prevalence of P. aeruginosa in different places may attributed to the type of clinical specimens received for examination, studied population, type of hospitals and geographical locations.

In this present study, majority 221 (67.6%) of P. aeruginosa isolates were recovered from pus/swab followed by urine 15% and sputum 9.5%. Similar findings were obtained by several authors.[13,14,17] Our study revealed P. aeruginosa infection was significantly associated among hospitalized, elderly (≥55 years) and had received any type of invasive procedure such as catheterization, intubation or ventilation (P < 0.001). P. aeruginosa is rarely seen as a member of human normal

flora. However, colonization rates may exceed 50% during hospitalization, especially among impaired immunity patients

Table 1: Distribution of P. aeruginosa isolated from various clinical samples in a tertiary care hospital South Odisha, India

Type of sample

Total no. tested

Total no. (%) showed signifi cant

growth

No. (%) of P. aeruginosa

isolated (% out of signifi cant growth)

Pus/swab 1689 1317 (78) 221 (16.8)

Urine 2443 1118 (45.8) 49 (4.4)

Blood 808 425 (52.6) 16 (3.8)

Sputum 792 374 (47.2) 31 (8.3)

Body fl uids* 411 112 (27.3) 8 (7.1)

Others** 137 32 (23.4) 2 (6.3)

Total 6280 3378 (53.8) 327 (9.7)

*Body fl uids: Includes cerebrospinal fl uid, pleural fl uid and peritoneal fl uid, **Others: Includes tracheal aspirate, bronchial lavage. P. aeruginosa: Pseudomonas aeruginosa

Table 2: Demographic profi les for P. aeruginosa infection among patients attended tertiary care hospital, South Odisha, India (n=6280)

Demographic profi les

Total no. of

patients (n=6280)

No. of P. aeruginosa

isolated (n=327)

Chi-square P value

Attended hospital as

In-patients 3217 229 43.29 <0.001

Out-patients 3063 98

Age (years)

≥55 887 69 11.66 <0.001

<55 5393 258

Invasive procedure conducted (catheterization, intubation, ventilation)

779 66 16.17 <0.001

None 5501 261

Gender

Male 3354 189 2.23 0.13

Female 2926 138

P. aeruginosa: Pseudomonas aeruginosa

Table 3: Frequency of multidrug resistance P. aeruginosa (n=277)

Parameter Resistance to one or several categories of antibiotics (%)

Total

*No. of categories of antibiotics

1 2 3 4 5

**No. (%) isolates of P. aeruginosa

97 (35) 81 (29.2) 77 (27.8) 22 (8) 0 277 (100)

*Category 1: Cephalosporins, 2: Fluoroquinolones, 3: Aminoglycosides, 4: Carbapenems and penicillin/β-lactamase, 5: Resistant to all antibiotics, P. aeruginosa: Pseudomonas aeruginosa. **Fifty isolates of P. aeruginosa were sensitive to all antibiotics tested





18 Saudi Journal for Health Sciences - Vol 3,Issue 1, Jan-Apr 2014

who have experienced mechanical ventilation, tracheostomy, catheters, surgery or severe burns.[18-20]

Our study revealed, 277 (84.7%, 277/327) P. aeruginosa isolates were MDR, out of which 99 (35.7%, 99/277) were XDR and no PDR isolate was obtained. Similar MDR rate of 71% was reported by Mohanasoundaram et al. in Tamil Nadu, India.[17] Gill et al. in Rawalpindi, Pakistan had studied 180 P. aeruginosa isolates, which were obtained from different clinical specimens. Out of these, 22.7% were MDR, while 11% and 4.3% were XDR and PDR, respectively.[21] The high percentage of MDR strains were isolated from different clinical specimens is worrisome for the future. Accurate laboratory detection, control of patient-to-patient transmission and prudent use of antibiotics are cornerstones in containment of drug resistant.

Study showed majority (77.7%) of isolates were resistant to ceftazidime, followed by cefepime 64.8%, piperacillin 45%, ciprofloxacin 38.9%, levofloxacin 36.1%, gentamicin 37.3% and amikacin 30%. Similar resistant pattern against P. aeruginosa was reported in different studies conducted in India.[15,17,22] Imipenem, meropenem and piperacillin/tazobactam were most effective drugs observed in this study, showed resistant rates of 6.4%, 8% and 11.3% respectively. Low resistance rate of P. aeruginosa to carbapenems and piperacillin/tazobactam may be due to its recent introduction for use in our hospital. Higher cost of these drugs is also responsible for its restricted use.

The established fact that the irrational and inappropriate use of antibiotics is responsible for the development of resistance against P. aeruginosa to routinely used antipseudomonal antibiotics. Therefore, there is a need to emphasize the prudent use of antibiotics and strictly adhere to the concept of “reserve drugs” to minimize the misuse of available antimicrobials.[22,23] In addition, regular laboratory detection and antimicrobial susceptibility surveillance P. aeruginosa is essential for local monitoring of resistance pattern. Thus hospitals, as the primary incubators of antimicrobial-resistant pathogens, carry the highest responsibility for proper stewardship of our existing antimicrobial resources.

CONCLUSION

Our study revealed that 84.7% of P. aeruginosa were MDR and most of these isolates were susceptible to imipenem, meropenem and piperacillin/tazobactam. Elderly, in-patients and associated with invasive procedure were found to be risk factors in the setup investigated. To avoid resistance antibiotics should be used judiciously and empirical antibiotic therapy should be determined for each hospital according to the antimicrobial surveillance of that center. Therefore, state and national level antimicrobial policy and guidelines should be introduced to preserve the effectiveness of antibiotics and for better management of the patient. Furthermore, drug like carbapenem should be considered as reserve drug for treatment of severe nosocomial P. aeruginosa infections.

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Table 4: Frequency of antibiotic resistance in P. aeruginosa (n=327)

Antibiotic Disk content (mcg/disk) Sensitivity pattern of P. aeruginosa in number (%)Resistant Intermediate Sensitive

Ceftazidime 30 254 (77.7) 6 (1.8) 67 (20.5)

Cefepime 30 212 (64.8) 3 (1) 112 (34.2)

Ciprofl oxacin 5 127 (38.9) 5 (1.5) 195 (59.6)

Levofl oxacin 5 118 (36.1) 4 (1.2) 205 (62.7)

Gentamicin 10 122 (37.3) 3 (1) 202 (61.7)

Amikacin 30 98 (30) 6 (1.8) 223 (68.2)

Piperacillin 100 147 (45) 5 (1.5) 175 (53.5)

Piperacillin/tazobactam 100/10 37 (11.3) 3 (1) 287 (87.7)

Imipenem 10 21 (6.4) 7 (2.1) 299 (91.5)

Meropenem 10 26 (8) 8 (2.4) 293 (89.6)

P. aeruginosa: Pseudomonas aeruginosa






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How to cite this article: Dash M, Padhi S, Narasimham MV, Pattnaik S. Antimicrobial resistance pattern of Pseudomonas aeruginosa isolated from various clinical samples in a tertiary care hospital, South Odisha, India. Saudi J Health Sci 2014;3:15-9.

Source of Support: Nil, Confl ict of Interest: None declared.




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Antimicrobial resistance pattern of Pseudomonas aeruginosa isolated from various clinical samples in a tertiary care hospital, South Odisha, India