Epidemiology and high incidence of Metallo-β-lactamase and AmpC-β-lactamases in nosocomial Pseudomonas aeruginosa

Document Type : Original Article


1 Institute of Molecular Biology & Biotechnology (IMBB), The University of Lahore, Defence road campus, Lahore, Pakistan

2 Department of Pathology, Combined Military Hospital, Lahore Cantt, Pakistan

3 Department of Respiratory Medicine, Tallaght University Hospital, Dublin, Ireland

4 M. Islam Medical College, Gujranwala, Pakistan

5 Al-Aleem Centre for Advanced Studies and Research, Gulab Devi Educational Complex, Lahore, Pakistan


Objective(s): Isolates producing Metallo-β-lactamase (MBL) have a significant impact on therapeutic and diagnostic layouts, plus their increased frequency has been reported globally. Determination of incidence of clinical isolates of Pseudomonas aeruginosa that are capable of producing MBL and AmpC-β-lactamases making them resistant to imipenem and cefoxitin.
Materials and Methods: Out of 1159 collected samples of urine, wound swabs, blood, tissue, and pus, the isolation rate of P. aeruginosa in the period of March 2020 to February 2021 was 22.0% (255/1159). Bacterial strains that were resistant towards imipenem were further processed for detecting the β-lactamase group of genes followed by statistical analysis of risk factors done based on clinical sample, gender, plus department of sample collection.
Results: The percentage of resistance against imipenem was found to be 53%. Out of 135 strains, phenotypic tests revealed MBLs incidence to be 61.5% by combination disc test and 81.5% by Modified Hodge test (MHT). Frequencies of blaIMP-1, blaVIM, blaSHV, blaTEM, and blaOXA genes were calculated to be 13%, 15%, 32%, 43%, and 21%, respectively. Co-expressions of blaMBLs (blaVIM and blaIMP-1) plus blaESBL (blaSHV, blaOXA, blaTEM) were detected using simplex and multiplex PCR. blaTEM, blaSHV, and blaOXA co-existed in 7.5% of clinical isolates. 5.5% of the isolates exhibited simultaneous expression of MBL/ESBL genes. 15% of the isolates resistant to cefoxitin were positive for the blaAmpC gene (17/114).
Conclusion: This is a pioneer report from Pakistan that concomitantly presents expression of blaVIM and blaIMP-1 with blaTEM, blaOXA, blaSHV, and blaAmpC in isolates of P. aeruginosa.


1. Baran I, Aksu N. Phenotypic and genotypic characteristics of carbapenemresistant Enterobacteriaceae in a tertiary-level reference hospital in Turkey. Ann Clin Microbiol Antimicrob 2016; 15: 20-Last page.
2. Khalifa HO, Soliman AM, Ahmed AM, Shimamoto T, Hara T, Ikeda M, et al. High carbapenem resistance in clinical Gram-negative pathogens isolated in Egypt. Microb Drug Resist 2017; 23: 838–844.
3. Mirsalehian A, Kalantar-Neyestanaki D, Taherikalani M, Jabalameli F, Emaneini M. Determination of carbapenem resistance mechanism in clinical isolates of Pseudomonas aeruginosa isolated from burn patients, in Tehran, Iran. J Epidemiol Glob Health 2017; 7: 155-159. 
4. Xu Y, Gu B, Huang M, Liu H, Xu T, Xia W, Wang T. Epidemiology of carbapenem resistant Enterobacteriaceae (CRE) during 2000-2012 in Asia. J Thorac Dis 2015; 3: 376-385.
5. Satlin MJ, Chen L, Patel G, Gomez-Simmonds  A, Weston G, Kim AC et al. Multicenter clinical and molecular epidemiological analysis of bacteremia due to carbapenemresistant enterobacteriaceae (CRE) in the CRE epicenter of the united states. Antimicrob agents chemother 2017; 61:e02349-16.
6. Wang J-T, Wu U-I, Lauderdale T-LY, Chen M-C, Li S-Y, Hsu L-Y, et al. Carbapenem-nonsusceptible Enterobacteriaceae in Taiwan. PLoS One 2015; 10: e0121668. 
7. Ye Y, Xu L, Han Y, Chen Z, Liu C, Ming L. Mechanism for carbapenem resistance of clinical Enterobacteriaceae isolates. Exp Ther Med 2018; 15: 1143-1149.
8. Dalmolin TV, Bianchini BV, Rossi GG, Ramos AC, Gales AC, Trindade PA, Campos MMA. Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae. Braz J Microbiol 2017; 48: 493-498.
9. Mai M. Zafer, Mohamed H. Al-Agamy, Hadir A. El-Mahallawy, Magdy A. Amin, mohammed seif El-Din ashour. Antimicrobial resistance pattern and their beta-lactamase encoding genes among Pseudomonas aeruginosa strains isolated from cancer patients. Biomed Res Int 2014; 2014 :101635.
10. Iraz M, Özad Düzgün A, Sandallı C, Doymaz MZ, Akkoyunlu Y, Saral A, Peleg et al. Distribution of β-lactamase genes among carbapenem-resistant Klebsiella pneumoniae strains isolated from patients in Turkey. Ann Lab Med 2015; 35: 595-601.
11. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53: 5046-5054.
12. Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995; 39: 1211-1233.
13. Walsh TR. Clinically significant carbapenemases: an update. Curr Opin Infect Dis 2008; 21: 367-371.
14. Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 2002; 8: 321-31.
15. Haruta S, Yamaguchi H, Yamamoto ET, Eriguchi Y, Nukaga M, O’Hara K, et al. Functional analysis of the active site of a metallo-beta-lactamase proliferating in Japan. Antimicrob Agents Chemother 2000; 44: 2304-2309.
16. Ito H, Arakawa Y, Ohsuka S, Wacharotayankun R, Kato N, Ohta M. Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrob Agents Chemother 1995; 39: 824-829.
17. Hawkey PM, Xiong J, Ye H, Li H, M’Zali FH. Occurrence of a new metallo-beta-lactamase IMP-4 carried on a conjugative plasmid in Citrobacter youngae from the People’s Republic of China. FEMS Microbiol Lett 2001; 194: 53-57.
18. Hawkey PM. Multidrug-resistant Gram-negative bacteria: A product of globalization. J Hosp Infect 2015; 89: 241-277.
19. Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr 2016; 4:1-37.
20. Scoulica EV, Neonakis IK, Gikas AI, Tselentis YJ. Spread of bla(VIM-1)-producing Escherichia coli in a university hospital in Greece. Genetic analysis of the integron carrying the bla(VIM-1) metallo-beta-lactamase gene. Diagn Microbiol Infect Dis 2004; 48: 167-172.
21. Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamases: A last frontier for β-lactams? Lancet Infect Dis 2011; 11: 381-393.
22. Kazmierczak KM, Rabine S, Hackel M, McLaughlin RE, Biedenbach DJ, Bouchillon SK, et al. Multiyear, multinational survey of the incidence and global distribution of metallo-β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother 2015; 60: 1067-1078.
23. Rolain JM, Parola P, Cornaglia G. New Delhi metallo-beta-lactamase (NDM-1): towards a new pandemia? Clin Microbiol Infect 2010; 16: 1699-1701.
24. Berrazeg M, Diene S, Medjahed L, Parola P, Drissi M, Raoult D, et al. New Delhi Metallo-beta-lactamase around the world: an eReview using Google Maps. Euro Surveill 2014; 19: 20809.
25. Pfeifer Y, Wilharm G, Zander E, Wichelhaus TA, Göttig S, Hunfeld KP, et al. Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007. J Antimicrob Chemother 2011; 66: 1998-2001.
26. Bonnin RA, Poirel L, Naas T, Pirs M, Seme K, Schrenzel J, et al. Dissemination of New Delhi metallo-β-lactamase-1-producing Acinetobacter baumannii in Europe. Clin Microbiol Infect 2012; 18: E362-365.
27. Yasushi Nakazawa, Ryoko Ii, Taku Tamura, Shinji Kawano, Yasushi Nakazawa, Ryoko Ii, et al. A case of NDM-1-producing Acinetobacter baumannii transferred from India to Japan. J Infect Chemother 2013; 19: 330-332.
28. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 2010; 10: 597-602.
29. Litzow JM, Gill CJ, Mantaring JB, Fox MP, MacLeod WB, Mendoza M, et al. High frequency of multidrug-resistant Gram-negative rods in 2 neonatal intensive care units in the Philippines. Infect Control Hosp Epidemiol. 2009; 30: 543-549.
30. Le NK, Hf W, Vu PD, Khu DTK, Le HT, Hoang BTN, et al. High prevalence of hospital-acquired infections caused by Gram-negative carbapenem resistant strains in Vietnamese pediatric ICUs: A multi-centre point prevalence survey. Medicine (Baltimore) 2016; 95: e4099.
31. Bhat V, Gupta S, Kelkar R, Biswas S, Khattry N, Moiyadi A, et al. Bacteriological profile and antibiotic susceptibility patterns of clinical isolates in a tertiary care cancer center. Indian J Med Paediatr Oncol 2016; 37: 20-24.
32. Walther-Rasmussen J, Høiby N. OXA-type carbapenemases. J Antimicrob Chemother 2006; 57: 373-383.
33. Bonnin RA, Poirel L, Nordmann P. AbaR-type transposon structures in Acinetobacter baumannii. J Antimicrob Chemother 2012; 67: 234-236.
31. Khan MA, Siddiqui BK, Shamim A, Yosuf MA, Ahmed U, Zakiullah N, et al. Emerging bacterial resistance patterns in febrile neutropenic patients: experience at a tertiary care hospital in Pakistan. J Pak Med Assoc 2004; 54: 357-360.
32. Irfan S, Turton JF, Mehraj J, Siddiqui SZ, Haider S, Zafar A, et al. Molecular and epidemiological characterisation of clinical isolates of carbapenem-resistant Acinetobacter baumannii from public and private sector intensive care units in Karachi, Pakistan. J Hosp Infect 2011; 78: 143-148.
33. Khan F, Khan A, Kazmi SU. Prevalence and susceptibility pattern of multi drug resistant clinical isolates of Pseudomonas aeruginosa in Karachi. Pak J Med Sci 2014; 30: 951-954.
34. Kaleem F, Usman J, Hassan A, Khan A. Frequency and susceptibility pattern of metallo-beta-lactamase producers in a hospital in Pakistan. J Infect Dev Ctries 2010; 4: 810-813.
35. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966; 45: 493-496.
36. Wadekar, MD., Anuradha K., Venkatesha D. Phenotypic detection of ESBL and MBL in clinical isolates of Enterobacteriaceae. Into J Current Red Aca Rev 2013; 1: 89-95.
37. Kumar S, Mehra S. Performance of modified Hodge test and combined disc test for detection of Carbapenemases in clinical isolates of Enterobacteriaceae. Int J Curr Microbiol App Sci 2015; 4: 255–561.
38. Abrar S, Vajeeha A, Ul-Ain N, Riaz S. Distribution of CTX-M group I and group III β-lactamases produced by Escherichia coli and klebsiella pneumoniae in Lahore, Pakistan. Microb Pathog 2017; 103: 8-12.
39. Colom K, Pérez J, Alonso R, Fernández-Aranguiz A, Lariño E, Cisterna. Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA–1 genes in Enterobacteriaceae FEMS Microbiol. Lett 2003; 223: 147-151.
40. Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K, et al. PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by Gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003; 41: 5407-5413.
41. Galani I, Souli M, Chryssouli Z, Katsala D, Giamarellou H. First identification of an Escherichia coli clinical isolate producing both metallo-beta-lactamase VIM-2 and extended-spectrum beta-lactamase IBC-1. Clin Microbiol Infect 2004; 10: 757-760.
42. Laxminarayan R, Bhutta ZA. Antimicrobial resistance —a threat to neonate survival. Lancet Glob Health 2016; 4: e676 –677.
43. Ramalingam S, Seetharaman S, Murugesan A, Manoharan M. Carbapenem re-sistance in a rural part of southern India: Escherichia coli versus Klebsiella spp. Indian J Med Res 2016; 144:781-783.
44. Hu FP, Guo Y, Zhu DM, Wang F, Jiang XF, Xu YC, et al. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014. Clin Microbiol Infect 2016; 22 Suppl 1: S9-14.
45. Ilyas M, Khurram M, Ahmad I, Ahmad S, et al. Frequency, susceptibility and co-existence of MBL, ESBL & AmpC positive Pseudomonas aeruginosa in tertiary care hospitals of Peshawar, KPK, Pakistan.  J Pure Appl Microbiol 2015; 9: 981-988.
46. Al-Charrakh AH, Al-Awadi SJ, Mohammed AS. Detection of metallo-β-Lactamase producing Pseudomonas aeruginosa isolated from public and private hospitals in Baghdad, Iraq. Acta Med Iran 2016; 54: 107-113.
47. Ameen N, Memon Z, Shaheen S, Fatima G, Ahmed F. imipenem resistant Pseudomonas aeruginosa: The fall of the final quarterback. Pak J Med Sci 2015; 31: 561-565.
48. 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 –172.
49. Church D, Elsayed S, Reid O, Winston B, Lindsay R. Burn wound infections. Clin Microbiol Rev 2006; 19: 403-434.
50. AL-Aali KY. Microbial profile of burn wound infections in burn patients, Taif. Saudi Arabia. Arch. Clin. Microbiol 2016; 7:1-9.
51. Safari M, Mozaffari Nejad AS, Bahador A, Jafari R, Alikhani MY. Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU). Saudi J Biol Sci 2015; 22: 424-429.
52. Tomás M, Doumith M, Warner M, Turton JF, Beceiro A, Bou G, et al. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2010; 54: 2219-2224.
53. Qureshi MA, Bhatnagar RK. Molecular analysis of metallo -Lactamase in multi drug resistant Pseudomonas aeruginosa among the clinical isolates. SM J Infect Dis 2016; 1:1004: 1-4.
54. Rafiee R, Eftekhar F, Tabatabaei SA, Tehrani DM. Prevalence of extendedspectrum and Metallo -lactamase production in AmpC -lactamase producing Pseudomonas aeruginosa isolates from burns in Jundishapur. J Microbiol 2014; 7: e16436.
55. Hadjadj L, Syed MA, Abbasi SA, Rolain JM, Jamil B. Diversity of carbapenem resistance mechanisms in clinical Gram-negative bacteria in pakistan. Microb Drug Resist 2020; 27: 760-767.