Farooq Ali  ( Department of Microbiology Hazara University Mansehra, KPK Pakistan )
Zeeshan Niaz  ( Department of Microbiology, Hazara University Mansehra, KPK Pakistan )
Pir Tariq Shah  ( Department of Microbiology Hazara University Mansehra, KPK Pakistan )
Qismat Shakeela  ( Department of Microbiology Hazara University Mansehra, KPK Pakistan )
Bibi Uzma  ( Department of Pathology, Ayub Medical College Abbottabad, KPK, Pakistan. )
Shehzad Ahmed  ( Department of Microbiology, Hazara University, Mansehra, KPK, Pakistan. )

Objective: To investigate the frequency rate and sensitivity pattern of extended-spectrum beta-lactamase and metallobeta- lactamase producing Pseudomonas aeruginosa isolated from major hospitals.

Methods: The cross-sectional study was conducted in the Microbiology section of the Pathology Department of Ayub Medical College, Abbottabad, Pakistan, from September 2017 to April 2018, and comprised clinical samples collected from different medical wards of major hospitals in the study area. For the selective growth of Pseudomonas aeruginosa, Cetrimide agar was used, and different antibiotics were evaluated for the sensitivity pattern following Kirby-Bauer diffusion method. Pseudomonas aeruginosa producing extended-spectrum beta lactamase and metallo-beta-lactamase were identified through double disk synergy test and imipenem ethylenediaminetetraacetic acid tests respectively. Patient’s demographic and medical history was noted on a proforma. Data was analysed using SPSS 22.0.

Results: Of the 242 samples screened, 46 (19%) were positive for Pseudomonas aeruginosa. These samples were highly sensitive to levofloxacin, amikacin, imipenem, meropenem and ciprofloxacin (p<0.05). Of the positive cases, 11 (23.91%) were detected for extended-spectrum beta-lactamase production, while 3 (6.52%) samples were detected for metallo-beta-lactamase production.

Conclusion: Pseudomonas aeruginosa samples were widely resistant to most antibiotics, but were sensitive for some antibiotics which may be recommended by physicians when treating Pseudomonas aeruginosa infection.

Keywords: ESBL, MBL, Susceptibility, DDST, Imipenem-EDTA test. (JPMA 70: 1979; 2020)

DOI: https://doi.org/10.5455/JPMA.19089

 

Introduction

 

Pseudomonas aeruginosa (P. aeruginosa) is a rod-shaped, motile, oxidase- and catalase-producing gram-negative bacterium, which is known for incendiary bacterial infection in humans and animals. This opportunistic pathogen is linked with diverse infections such as urinary tract infection (UTI), surgical and nosocomial infections.^1,2 As an opportunistic pathogen, it infects two-third of the hospitalised population which makes it the most aggressive bacterial pathogen. It is the leading gramnegative pathogen in health centres with about 60% mortality rate. Moreover, it complicates almost 90% cystic fibrosis deaths in the community.³ Gasserd discovered and isolated this gram-negative bacillus for the first time from the green pus in 1882. The unique style and diverse nutritional profile of P. aeruginosa permit it to colonise everywhere.⁴ Routinely, this gramnegative rod survives in hospital environment and its reservoir for infection and colonisation cannot be traced.⁵ Wounds of burn patients are colonised by different strains of P. aeruginosa. The frequency of the infections in these patients ranges 22-73%.⁶ Multi-drug resistance (MDR) is a big health challenge linked with P. aeruginosa and this pathogen resists a wide range of antibiotics. The susceptibility pattern of P. aeruginosa is limited to a number of antimicrobial agents because of its genetic resistance to a wide range of antibiotics,⁷ and emerging resistance to the previously used sensitive antibiotics.⁸ Resistance to antibiotics in case of hospital infections are associated with higher costs and adverse effects.⁹ Extended spectrum beta lactamase (ESBL)-producers are the bacterial strains that hydrolyse and emerge resistant to ceftazidime, ceftriaxone, cefotaxime (3G cephalosporin).¹⁰ Studies have reported the frequency rate of ESBL-producing pseudomonas species ranging 22-36%.^11,12 A ventilator-based prevalence rate of pneumonia due to P. aeruginosa was reported as 15.6%, in which Asia contributed 16%, United States 13.5%, Latin America 13.8% and Europe 19.4%.¹³ A study reported patient-to-patient transmission of P. aeruginosa in Vancouver, Canada, in 174 isolates; 157 genetic types were recognized as P. aeruginosa.¹⁴ The current study was planned to assess the occurrence rate, susceptibility pattern and the prevalence rate of ESBLand metallo-beta-lactamase (MBL)-producing P. aeruginosa in indoor and outdoor patients at different hospitals.

 

Materials and Methods

 

The cross-sectional study was conducted in the Microbiology section of the Pathology Department of Ayub Medical College, Abbottabad, Pakistan, from September 2017 to April 2018, and comprised clinical samples collected from different medical wards of major hospitals in the study area. After approval from the institutional bioethical committee of Hazara University, Mansehra, Pakistan. A total of 242 different clinical samples were collected from the patients of different medical wards, like ear, nose, throat (ENT) the burns unit act of the hospitals, and the sample included ear swabs, pus, urine, wounds, body fluids, blood and sputum. Each patient was briefed about the purpose of the study and was also ensured for confidentiality of individual result along with his/her consent. The samples were collected from the patients in the presence of a medical officer and were inoculated on suitable culture media, such as cystine–lactose–electrolytedeficient (CLED) agar, blood agar, MacConkey agar, nutrient agar, and after overnight incubation at 37oC, the culture media were examined for the colonies of gram-negative rod P. aeruginosa. Cetrimide agar was used for selective growth. The colonies were also identified based on staining character, growth pigmentation, colonial morphology and other biochemical tests for P. aeruginosa as per standard lab identification methods.^15,16 For antibacterial susceptibility patterns, tryptic soy broth (CM129-Oxoid) was prepared and was poured 4-5ml into each capped tubes. These tubes after sterilisation by autoclaving were left overnight at 35oC prior to inoculation of samples. Turbidity of inoculum was standardised to 0.5 McFarland index according to Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁷ Routinely used different antibiotics (commercially available, Oxoid) were used to determine the susceptibility pattern by following disc diffusion method.¹⁸ Minimum inhibitory concentration (MIC) of each antibiotic to P. aeruginosawas found, and the break points were standardised in line with literature.¹⁹ For the detection of ESBL-producing P. aeruginosa, all bacterial isolates were screened as prescribed by a study.²⁰ All the stored bacterial isolates at -20oC were refreshed on nutrient agar to find out ESBL via synergy disc diffusion method. ESBL-producing P. aeruginosa were identified using double disk synergy test (DDST). Three discs of thirdgeneration (3G) cephalosporine, ceftriaxone (CRO), cefotaxime (CTX) and ceftazidime (CAZ) 30μg each were placed on Mueller Hinton Agar (MHA) at a distance of 25mm centre-centre from amoxicillin-clavulanic acid (AMC- 20/10μg).²⁰ Enhancement in zone of inhibition of any in the 3G cephalosporine towards AMC indicated the presence of ESBL-producing P. aeruginosa. MBL-producing P. aeruginosa were identified using imipenem-ethylenediaminetetraacetic acid (IMP-EDTA) method. Two IMP-10μg discs and 0.5M EDTA were used. A 0.5M EDTA was prepared by dissolving 186.1g of EDTA (disodium EDTA.2H2O) in one litre of distilled water and potential of hydrogen (pH) was adjusted up to 8.0. IMPresistant culture was inoculated and two 10μg IMP discs were placed on MHA medium and appropriate amount of half-Mole EDTA was added to one of them to obtain the desired concentration of 1000μg. The culture plates were examined for the synergistic effect of IMP and chelator agent EDTA after 16 hours of incubation at 37oC.²¹ Data was analysed using SPSS 22.0. Two-way analysis of variance (ANOVA) and chi-square test were applied as appropriate. P<0.05 was considered significant.

 

Results

 

Of the 242 samples screened, 119(49%) came from males and 123 (51%) from females. The samples included ear swabs 114 (47%), blood 14(5.8%), body fluid 3 (1.2%), pus 54(22.3%), sputum 5 (2.1%), urine 49 (20.2%) and wound 3(1.2%). The mean age of each type of sample along gender lines were noted (Figure 1), and significance was checked in terms of both and gender age (Table 1).

Overall, 46(19%) samples were positive for P. aeruginosa; 27 (58.7%) from males and 19 (41.3%) from females (Table 2).

Also, 32 (69.57%) of the positive samples related to indoor patients and 14 (30.43%) to outdoor patients. Of the positive cases, 11 (23.91%) were detected for ESBL production, and 3 (6.52%) for MBL production (Figure 2).

The highest number of positive cases of P. aeruginosawere identified in ear swab specimens, followed by pus and urine specimens, while no cases were identified positive in body fluid and sputum specimens (Figure 3).

Susceptibility patterns of P. aeruginosa to different classes of antibiotics were checked (Table 3) and the same was done for ESBL- and MBL-producers (Tables 4-5) as well as in relation to indoor and outdoor patients (Table 6).

 

Discussion

 

Bacterial species are commonly known for spreading various infections in both humans and animals. These species can survive in the environment of common healthcare facilities. Some of these, especially P. aeruginosa and Enterobacter, can survive even in toxic conditions. These properties make them able to become resistant to all the drugs available in an environment.²² The current study had more infected samples coming from the male population compared to females which is the opposite of earlier findings.²³ The positive rate of P. aeruginosawas high in pus, secretion and urine samples as compared to sputum, blood etc. This finding is in line with an earlier study.²⁴ In terms of susceptibility pattern in the present study, the clinical isolates of P. aeruginosa showed high susceptibility to imipenem, meropenem, ciprofloxacin, levofloxacin and amikacin. An earlier study highlighted the same susceptibility pattern for the P. aeruginosa.²⁵ But another study disagreed with the efficacy of ciprofloxacin for P. aeruginosa. It is known that bacterial susceptibility pattern is geographically different.²⁶ Commonly, cephalosporins are recommended in general practice for the treatment of infections caused by P. aeruginosa.^27,28 Ceftriaxone (13.04%) and ceftazidime (30.43%) were less effective against P. aeruginosa in the current study. Results for the 3G cephalosporin are affirmed by a study.²⁹ Susceptibility pattern of the cephalosporin reported in India had the same hypothesis for P. aeruginosa.³⁰ In the current study, carbapenem had higher frequency rate of susceptibility against P. aeruginosa species. Imipenem exhibited 68.8% susceptibility rate to indoor and 57.1% to outdoor isolates, while meropenem showed 50% and 35.7% efficacy respectively. These results are supported by a study.³¹ Aminoglycosides are recommended for the treatment of gram-negative bacteria,³² and the current study also found that non β- lactams isolates showed good susceptibility to gentamycin (19.57%) and amikacin (39.13%). Our results are in accordance with an earlier study.³³ Other studies confirmed that amikacin was more effective against P. aeruginosa infections in comparison with gentamycin.^30,34,35 From the results obtained in our study, it appears that gentamycin was widely prescribed in health practices and misused in the community without any consultation with physicians. A total of 6.52% MBL-positive cases were detected in the current study among all the positive cases for P. aeruginosa. These were mostly sensitive to levofloxacin, cefotaxime and ceftazidime, while 100% resistant to imipenem and carbapenem. The findings are in line with a reported study.³⁶ Also, ESBL-positive samples were 23.91% and exhibited high resistance to gentamycin, imipenem and meropenem while remaining sensitive to levofloxacin, ceftazidime and ceftriaxone. These results match earlier findings.^27,31,34 However, other antibiotics, such as amikacin, aztreonam and tazobactam / piperacillin have shown less resistance.²⁴ The present study used two drugs-based tests for the detection of ESBL- and MBL-producing P. aeruginosawhich is in agreement with earlier studies.^21,23 The findings of the current study indicate that there were a few loopholes in hospital and medical setups, and improving these can significantly reduce or eradicate not only the spread, but also the emergence of MDR strains of P. aeruginosa. In order for that to happen, the overall hygienic conditions at hospitals need to be standardised, proper diagnosis and treatment regime must be adopted where antibiotic susceptibility must be detected before a prescription is handed out. The patients must be made aware of the pitfalls of irrational use of antibiotics with incomplete dosage.

 

Conclusion

 

P. aeruginosa samples were found to be widely resistant to most antibiotics, but were sensitive for some others which may be prescribed by physicians when treating P. aeruginosa infections.

 

Disclaimer: None.

Conflict of interests: None.

Source of Funding: None.

 

References

 

1. Kiska D, Gilligan P. Pseudomonas. Manual of clinical microbiology. Washington DC: ASM Press; 2003, pp 719-28.

2. Sekiguchi JI, Asagi T, Miyoshi-Akiyama T, Kasai A, Mizuguchi Y, Araake M, et al. Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan. J Clin Microbiol 2007; 45: 979-89.

3. Fick R. Pseudomonas aeruginosa, the opportunist : pathogenesis and disease. London, Tokyo: CRC Press; 1993.

4. Favero M, Carson L, Bond W, Petersen N. Pseudomonas aeruginosa: growth in distilled water from hospitals. Science 1971; 173: 836-8.

5. Malani PN. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. JAMA 2010; 304: 2067-71.

6. Japoni A, Farshad S, Alborzi A. Pseudomonas aeruginosa: Burn infection, treatment and antibacterial resistance. Iranian Red Crescent Med J 2009; 11: 244-53.

7. Ruimy R, Genauzeau E, Barnabe C, Beaulieu A, Tibayrenc M, Andremont A. Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infection and Immunity 2001; 69: 584-8.

8. Akanji B, Ajele J, Onasanya A, Oyelakin O. Genetic fingerprinting of Pseudomonas aeruginosa involved in nosocomial infection as revealed by RAPD-PCR markers. Biotechnology 2011; 10: 70-7.

9. Afunwa RA, Odimegwu DC, Iroha RI, Esimone CO. Antimicrobial resistance status and prevalence rates of extended spectrum beta-lactamase producers isolated from a mixed human population. Bosn J Basic Med Sci 2011; 11: 91-6.

10. Peirano G, Pitout JD. Molecular epidemiology of Escherichia coli producing CTX-M β-lactamases: the worldwide emergence of clone ST131 O25: H4. Int J Antimicrob Agents 2010; 35: 316-21.

11. Bell JM, Chitsaz M, Turnidge JD, Barton M, Walters LJ, Jones RN. Prevalence and significance of a negative extended-spectrum β-lactamase (ESBL) confirmation test result after a positive ESBL screening test result for isolates of Escherichia coli and Klebsiella pneumoniae: results from the SENTRY Asia-Pacific surveillance program. J Clin Microbiol 2007; 45: 1478-82.

12. Ullah F, Malik SA, Ahmed J. Antimicrobial susceptibility and ESBL prevalence in Pseudomonas aeruginosa isolated from burn patients in the North West of Pakistan. Burns 2009; 35: 1020-5.

13. Kollef MH, Chastre J, Fagon JY, François B, Niederman MS, Rello J, et al. Global prospective epidemiologic and surveillance study of ventilator- associated pneumonia due to Pseudomonas aeruginosa. Crit Care Med 2014; 42: 2178-87.

14. Speert DP, Campbell ME, Henry DA, Milner R, Taha F, Gravelle A, et al. Epidemiology of Pseudomonas aeruginosa in cystic fibrosis in British Columbia, Canada. Am J Resp Crit Care Med 2002; 166: 988-93.

15. Cheesbrough M. District laboratory practice in tropical countries: Cambridge university press; 2006.

16. Cowan ST, Steel KJ. Cowan and Steel's manual for the identification of medical bacteria: Cambridge university press; 2004.

17. Weinstein MP, Patel JB, Bobenchik AM, Campaeu S, Cullen SK, Galas MF, et al. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute (CLSI). 29th ed. 2019.

18. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966; 45: 493-6.

19. Patel JB, Weinstein MP, Eliopoulos GM, Jenkins SG, Lewis JS, Limbago B, et al. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute (CLSI). 27th ed. 2017.

20. Hawser S, Bouchillon S, Lascols C, Hackel M, Hoban D, Badal R, et al. Susceptibility of European Escherichia coli clinical isolates from intraabdominal infections, extended-spectrum β-lactamase occurrence, resistance distribution, and molecular characterization of ertapenem- resistant isolates (SMART 2008–2009). Clin Microbiol Infect 2012; 18: 253-9.

21. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem- EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002; 40: 3798-801.

22. Alp E, Güven M, Yıldız O, Aygen B, Voss A, Doganay M. Incidence, risk factors and mortality of nosocomial pneumonia in intensive care units: a prospective study. Ann Clin Microbiol Antimicrob 2004; 3: 17.

23. Ahmad M, Hassan M, Khalid A, Tariq I, Asad MHHB, Samad A, et al. Prevalence of Extended Spectrum β-Lactamase and Antimicrobial Susceptibility Pattern of Clinical Isolates of Pseudomonas from Patients of Khyber Pakhtunkhwa, Pakistan. BioMed Res Int 2016; 2016: 6068429.

24. Bashir D, Thokar MA, Fomda BA, Bashir G, Zahoor D, Ahmad S, et al. Detection of metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa at a tertiary care hospital in Kashmir. Afr J Microbiol Res 2011; 5: 164-72.

25. Shahcheraghi F, Badmasti F, Feizabadi MM. Molecular characterization of class 1 integrons in MDR Pseudomonas aeruginosa isolated from clinical settings in Iran, Tehran. FEMS Immunol Med Microbiol 2010; 58: 421-5.

26. Messadi AA, Lamia T, Kamel B, Salima O, Monia M, Saida BR. Association between antibiotic use and changes in susceptibility patterns of Pseudomonas aeruginosa in an intensive care burn unit: a 5-year study, 2000–2004. Burns 2008; 34: 1098-102.

27. Cavallo J, Fabre R, Leblanc F, Nicolas-Chanoine M, Thabaut A. Antibiotic susceptibility and mechanisms of β-lactam resistance in 1310 strains of Pseudomonas aeruginosa: a French multicentre study (1996). J Antimicrobial Chemotherapy 2000; 46: 133-6.

28. Gales A, Jones R, Turnidge J, Rennie R, Ramphal R. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001; 32 : S146-S55.

29. Strateva T, Ouzounova-Raykova V, Markova B, Todorova A, Marteva- Proevska Y, Mitov I. Widespread detection of VEB-1-type extendedspectrum beta-lactamases among nosocomial ceftazidime-resistant Pseudomonas aeruginosa isolates in Sofia, Bulgaria. J Chemotherapy 2007; 19: 140-5.

30. Sasirekha B, Manasa R, Ramya P, Sneha R. Frequency and antimicrobial sensitivity pattern of extended spectrum β-lactamases producing E. coli and Klebsiella pneumoniae isolated in a tertiary care hospital. Al Ameen J Med Sci 2010; 3: 265-71.

31. Turner PJ. Meropenem and imipenem activity against Pseudomonas aeruginosa isolates from the MYSTIC Program. Diagn Microbiol Infect Dis 2006; 56: 341-4.

32. Chen H, Yuan M, Livermore D. Mechanisms of resistance to β lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993. J Med Microbiol 1995; 43: 300-9.

33. Cavallo J, Hocquet D, Plesiat P, Fabre R, Roussel-Delvallez M. Susceptibility of Pseudomonas aeruginosa to antimicrobials: a 2004 French multicentre hospital study. J Antimicrobial Chemotherapy 2007; 59: 1021-4.

34. Haque R, Salam M. Detection of ESBL producing nosocomial gram negative bacteria from a tertiary care hospital in Bangladesh. Pak J Med Sci 2010; 26: 887-91.

35. Ullah F, Malik SA, Ahmed J. Antimicrobial susceptibility pattern and ESBL prevalence in Klebsiella pneumoniae from urinary tract infections in the North-West of Pakistan. Afr J Microbiol Res 2009; 3: 676- 80.

36. Nisha KC, Singh S, Ghosh S. A Study of Metallo beta lactamase Producing Pseudomonas aeruginosa in Patients attending D.Y. Patil Hospital and Research Centre, Kolhapur. IOSR J Dental Med Sci 2016; 15: 51-5.