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Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections

  • Applied Microbial and Cell Physiology
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Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Infections caused by carbapenem-resistant Pseudomonas aeruginosa are life-threatening due to its synergistic resistance mechanisms resulting in the ineffectiveness of the used antimicrobials. This study aimed to characterize P. aeruginosa isolates for antimicrobial susceptibility, biofilm formation virulence genes, and molecular mechanisms responsible for resistance against various antimicrobials. Out of 700 samples, 91 isolates were confirmed as P. aeruginosa which were further classified into 19 non-multidrug-resistant (non-MDR), 7 multidrug-resistant (MDR), 19 extensively drug-resistant (XDR), and 8 pan drug-resistant (PDR) pulsotypes based on standard Kirby Bauer disc diffusion test and pulse field gel electrophoresis. In M9 minimal media, strong biofilms were formed by the XDR and PDR pulsotypes as compared to the non-MDR pulsotypes. The virulence genes, responsible for the worsening of wounds including LasB, plcH, toxA, and exoU, were detected among all MDR, XDR, and PDR pulsotypes. Carbapenemase activity was phenotypically detected in 45% pulsotypes and the responsible genes were found as blaGES (100%), blaVIM (58%), blaIMP (4%), and blaNDM (4%). Real-time polymerase chain reaction showed the concomitant use of multiple mechanisms such as oprD under-expression, enhanced efflux pump activity, and ampC overexpression in the resistant isolates. Polymyxin is found as the only class left with more than 80% susceptibility among the isolates which is an alarming situation suggesting appropriate measures to be taken including alternative therapies.

Key points

• Multidrug-resistant P. aeruginosa isolates formed stronger biofilms in minimal media.

• Only polymyxin antimicrobial was found effective against MDR P. aeruginosa isolates.

• Under-expression of oprD and overexpression of ampC were found in resistant isolates.

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References

  • Akhtar J, Saleem S, Shahzad N, Waheed A, Jameel I, Rasheed F, Jahan S (2018) Prevalence of metallo-β-lactamase IMP and VIM producing gram negative bacteria in different hospitals of Lahore, Pakistan. Pak J Zool 50(6).

  • Alipour N, Karagoz A, Taner A, Gaeini N, Alipour N, Zeytin H, Yildiz F, Durmaz R (2017) Outbreak of hospital infection from biofilm-embedded pan drug-resistant Pseudomonas aeroginosa, due to a contaminated bronchoscope. J Prev Med 2(2):1

    Article  Google Scholar 

  • Ameen N, Memon Z, Shaheen S, Fatima G, Ahmed F (2015) Imipenem resistant Pseudomonas aeruginosa: the fall of the final quarterback. PaK J Med Sci 31(3):561

    PubMed  PubMed Central  Google Scholar 

  • Amiel E, Lovewell RR, Toole GA, Hogan DA, Berwin B (2010) Pseudomonas aeruginosa evasion of phagocytosis Is mediated by loss of swimming motility and is independent of flagellum expression. Infect Immun 78(7):2937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Asadpour L (2018) Antimicrobial resistance, biofilm-forming ability and virulence potential of Pseudomonas aeruginosa isolated from burn patients in northern Iran. J Glob Antimicrob Resist 13:214–220

    Article  PubMed  Google Scholar 

  • Awan AB, Schiebel J, Böhm A, Nitschke J, Sarwar Y, Schierack P, Ali A (2019) Association of biofilm formation and cytotoxic potential with multidrug resistance in clinical isolates of Pseudomonas aeruginosa. EXCLI J 18:79

    PubMed  PubMed Central  Google Scholar 

  • Azimi L, Namvar AE, Lari AR, Jamali S (2016) Comparison of efflux pump involvement in antibiotic resistance among Pseudomonas aeruginosa isolates of burn and non-burn patients. Archives of Pediatric Infectious Diseases 4(3).

  • Braun SD, Jamil B, Syed MA, Abbasi SA, Weiß D, Slickers P, Monecke S, Engelmann I, Ehricht R (2018) Prevalence of carbapenemase-producing organisms at the Kidney Center of Rawalpindi (Pakistan) and evaluation of an advanced molecular microarray-based carbapenemase assay. Future Microbiol 13(11):1225–1246

    Article  CAS  PubMed  Google Scholar 

  • Cepas V, López Y, Muñoz E, Rolo D, Ardanuy C, Martí S, Xercavins M, Horcajada JP, Bosch J, Soto SM (2019) Relationship between biofilm formation and antimicrobial resistance in gram-negative bacteria. Microb Drug Resist 25(1):72–79

    Article  CAS  PubMed  Google Scholar 

  • Chaudhry A (2017) Allied tops list of ‘overcrowded’ hospitals. Dawn. https://www.dawn.com/news/1348643. Accessed 10 June 2021.

  • Chuang SK, Vrla GD, Fröhlich KS, Gitai Z (2019) Surface association sensitizes Pseudomonas aeruginosa to quorum sensing. Nat Commun 10(1):1–10

    Article  CAS  Google Scholar 

  • CLSI. Performance standards for antimicrobial susceptibility testing, CLSI document M100-S27. Clinical and Laboratory Standards Institute Wayne; 2017.

  • Cornaglia G, Giamarellou H, Rossolini GM (2011) Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect Dis 11(5):381–393

    Article  CAS  PubMed  Google Scholar 

  • Debalke D, Birhan M, Kinubeh A, Yayeh M (2018) Assessments of antibacterial effects of aqueous-ethanolic extracts of Sida rhombifolia’s aerial part. Sci World J 2018:8429809

    Article  Google Scholar 

  • El Zowalaty ME, Al Thani AA, Webster TJ, El Zowalaty AE, Schweizer HP, Nasrallah GK, Marei HE, Ashour HM (2015) Pseudomonas aeruginosa: arsenal of resistance mechanisms, decades of changing resistance profiles, and future antimicrobial therapies. Future Microbiol 10(10):1683–1706

    Article  PubMed  Google Scholar 

  • Elshamy AA, Aboshanab KM (2020) A review on bacterial resistance to carbapenems: epidemiology, detection and treatment options. Future sci OA 6(3):FSO438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Falagas ME, Bliziotis IA (2007) Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? Int J Antimicrob Agents 29(6):630–636

    Article  CAS  PubMed  Google Scholar 

  • Farooq L, Memon Z, Ismail MO, Sadiq S (2019) Frequency and antibiogram of multi-drug resistant Pseudomonas aeruginosa in a Tertiary Care Hospital of Pakistan. PaK J Med Sci 35(6):1622–1626

    Article  PubMed  PubMed Central  Google Scholar 

  • Filgona J, Banerjee T, Anupurba S (2015) Role of efflux pumps inhibitor in decreasing antibiotic resistance of Klebsiella pneumoniae in a tertiary hospital in North India. J Infect Dev Ctries 9(08):815–820

    Article  CAS  PubMed  Google Scholar 

  • Fournier D, Richardot C, Müller E, Robert-Nicoud M, Llanes C, Plésiat P, Jeannot K (2013) Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa. J Antimicrob Chemother 68(8):1772–1780

    Article  CAS  PubMed  Google Scholar 

  • Freschi L, Vincent AT, Jeukens J, Emond-Rheault J-G, Kukavica-Ibrulj I, Dupont M-J, Charette SJ, Boyle B, Levesque RC (2018) The Pseudomonas aeruginosa pan-genome provides new insights on its population structure, horizontal gene transfer, and pathogenicity. Genome Biol Evol 11(1):109–120

    Article  PubMed Central  Google Scholar 

  • Gheorghe I, Czobor I, Chifiriuc MC, Borcan E, Ghiţă C, Banu O, Lazăr V, Mihăescu G, Mihăilescu DF, Zhiyong Z (2014) Molecular screening of carbapenemase-producing Gram-negative strains in Romanian intensive care units during a one year survey. J Med Microbiol 63(10):1303–1310

    Article  PubMed  Google Scholar 

  • Giamarellou H, Antoniadou A (2001) Antipseudomonal antibiotics. Med Clin North Am 85(1):19–42

    Article  CAS  PubMed  Google Scholar 

  • Gill MM, Usman J, Kaleem F, Hassan A, Khalid A, Anjum R, Fahim Q (2011) Frequency and antibiogram of multi-drug resistant Pseudomonas aeruginosa. J Coll Physicians Surg Pak 21(9):531–534

    PubMed  Google Scholar 

  • Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, Moran GJ, Nicolle LE, Raz R, Schaeffer A (2011) International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 52(5):e103–e120

    Article  PubMed  Google Scholar 

  • Habibi A, Honarmand R (2015) Profile of virulence factors in the multi-drug resistant Pseudomonas aeruginosa strains of human urinary tract infections (UTI). ​Iran Red Crescent Med J 17(12).

  • Hancock REW, Speert DP (2000) Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 3(4):247–255

    Article  CAS  PubMed  Google Scholar 

  • Jordan K, Dalmasso M (2015) Pulse field gel electrophoresis: methods and protocols, 1st edn. Humana Press, New York

    Book  Google Scholar 

  • Kanj SS, Sexton DJ (2019) Principles of antimicrobial therapy of Pseudomonas aeruginosa infections. UpToDate Inc. https://www.uptodate.com/contents/principles-of-antimicrobial-therapy-of-pseudomonas-aeruginosa-infections. Accessed 10 June 2021.

  • Karakonstantis S, Kritsotakis EI, Gikas A (2019) Pandrug-resistant Gram-negative bacteria: a systematic review of current epidemiology, prognosis and treatment options. J Antimicrob Chemother 75(2):271–282

    Google Scholar 

  • Kilmury SL, Burrows LL (2018) The Pseudomonas aeruginosa PilSR two-component system regulates both twitching and swimming motilities. Mbio 9(4):e01310-e1318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar D, Sivakumar S, Sharmila P, Sivasubramani K (2019) ERIC-PCR: a molecular typing tool for genotyping multi drug resistant Pseudomonas aeruginosa isolated from the pus samples. J Drug Deliv Ther 9(4-s):1007–11

    Google Scholar 

  • Lob SH, Karlowsky JA, Young K, Motyl MR, Hawser S, Kothari ND, Gueny ME, Sahm DF (2019) Activity of imipenem/relebactam against MDR Pseudomonas aeruginosa in Europe: SMART 2015–17. J Antimicrob Chemother 74(8):2284–2288

    Article  CAS  PubMed  Google Scholar 

  • Mansoor K, Tanvir SB, Shariq A, Shahnawaz S, Ahmed S (2016) Prevalence and Antimicrobial susceptibility pattern of mono therapy and combination therapy of cefepime in Pseudomonas aeruginosa isolates of patients from a tertiary care hospital in Karachi, Pakistan. Int J Community Med Public Health 6(3).

  • Mcdonnell A (2018) Superbugs: are we returning to an era where bacteria are a major killer without a cure? http://eprints.lse.ac.uk/89967/. Accessed 10 June 2021.

  • Nathwani D, Varghese D, Stephens J, Ansari W, Martin S, Charbonneau C (2019) Value of hospital antimicrobial stewardship programs [ASPs]: a systematic review. Antimicrob Resist Infect Control 8(1):35

    Article  PubMed  PubMed Central  Google Scholar 

  • Neoh H, Tan X, Sapri HF, Tan TL (2019) Pulsed-field gel electrophoresis (PFGE): a review of the “gold standard” for bacteria typing and current alternatives. Infect Genet Evol 74:103935

    Article  CAS  PubMed  Google Scholar 

  • NHSRC (2017) National AMR Action Plan.: Ministry of National Health Services, Regulations, and Coordination. https://www.nih.org.pk/wp-content/uploads/2018/08/AMR-National-Action-Plan-Pakistan.pdf. Accessed 15th February 2019.

  • Nordmann P, Naas T, Fortineau N, Poirel L (2007) Superbugs in the coming new decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp and Pseudomonas aeruginosa in 2010. Curr Opin Microbiol 10(5):436–40

    Article  CAS  PubMed  Google Scholar 

  • Nordmann P, Naas T, Poirel L (2011) Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 17(10):1791–1798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaller MA, Bassetti M, Duncan LR, Castanheira M (2017) Ceftolozane/tazobactam activity against drug-resistant Enterobacteriaceae and Pseudomonas aeruginosa causing urinary tract and intraabdominal infections in Europe: report from an antimicrobial surveillance programme (2012–15). J Antimicrob Chemother 72(5):1386–1395

    Article  CAS  PubMed  Google Scholar 

  • Poonsuk K, Chuanchuen R (2012) Contribution of the MexXY multidrug efflux pump and other chromosomal mechanisms on aminoglycoside resistance in Pseudomonas aeruginosa isolates from canine and feline infections. J Vet Med Sci 74(12):1575–1582

    Article  CAS  PubMed  Google Scholar 

  • Rodulfo H, Arcia A, Hernández A, Michelli E, Martinez DdV, Guzman M, Sharma A, Donato MD (2019) Virulence factors and integrons are associated with MDR and XDR phenotypes in nosocomial strains of Pseudomonas aeruginosa in a Venezuelan university hospital. Rev Inst Med Trop Sao Paulo 61.

  • Romo-Ibáñez Á, Calatrava-Hernández E, Gutiérrez-Soto B, Pérez-Ruiz M, Navarro-Marí JM, Gutiérrez-Fernández J (2020) High clinical impact of rapid susceptibility testing on CHROMID ESBL(®) medium directly from swabs. Ann Transl Med 8(9):604

    Article  PubMed  PubMed Central  Google Scholar 

  • Sabharwal N, Dhall S, Chhibber S, Harjai K (2014) Molecular detection of virulence genes as markers in Pseudomonas aeruginosa isolated from urinary tract infections. Int J Mol Epidemiol Genet 5(3):125

    PubMed  PubMed Central  Google Scholar 

  • Sacha P, Wieczorek P, Hauschild T, Zórawski M, Olszańska D, Tryniszewska E (2008) Metallo-beta-lactamases of Pseudomonas aeruginosa–a novel mechanism resistance to beta-lactam antibiotics. Folia Histochem Cytobiol 46(2):137–142

    Article  CAS  PubMed  Google Scholar 

  • Sader HS, Huband MD, Castanheira M, Flamm RK (2017) Pseudomonas aeruginosa antimicrobial susceptibility results from four years (2012 to 2015) of the International Network for Optimal Resistance Monitoring Program in the United States. Antimicrob Agents Chemother 61(3):e02252-e2316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saleem S, Bokhari H (2019) Resistance profile of genetically distinct clinical Pseudomonas aeruginosa isolates from public hospitals in central Pakistan. J Infect Public Health.

  • Saleem Z, Hassali MA, Hashmi FK (2018) Pakistan’s national action plan for antimicrobial resistance: translating ideas into reality. Lancet Infect Dis 18(10):1066–1067

    Article  PubMed  Google Scholar 

  • Samad A, Ahmed T, Rahim A, Khalil A, Ali I (2017) Antimicrobial susceptibility patterns of clinical isolates of Pseudomonas aeruginosa isolated from patients of respiratory tract infections in a Tertiary Care Hospital. Peshawar Pak J Med Sci 33(3):670–674

    PubMed  Google Scholar 

  • Sepehri S, Poliquin G, Alfattoh N, Boyd D, Mulvey M, Denisuik A, Fanella S, Karlowsky J, Walkty A (2014) Osteomyelitis due to multiple carbapenemase-producing Gram-negative bacteria: the first case report of a GES-13-producing Pseudomonas aeruginosa isolate in Canada. Can J Infect Dis Med Microbiol 25(4):229–231

    Article  PubMed  PubMed Central  Google Scholar 

  • Serra R, Grande R, Butrico L, Rossi A, Settimio UF, Caroleo B, Amato B, Gallelli L, de Franciscis S (2015) Chronic wound infections: the role of Pseudomonas aeruginosa and Staphylococcus aureus. Expert Rev Anti Infect Ther 13(5):605–613

    Article  CAS  PubMed  Google Scholar 

  • Shah DA, Wasim S, Essa Abdullah F (2015) Antibiotic resistance pattern of Pseudomonas aeruginosa isolated from urine samples of Urinary Tract Infections patients in Karachi. Pakistan Pak J Med Sci 31(2):341–345

    PubMed  Google Scholar 

  • Shortridge D, Gales AC, Streit JM, Huband MD, Tsakris A, Jones RN (2019) Geographic and temporal patterns of antimicrobial resistance in Pseudomonas aeruginosa over 20 years from the SENTRY Antimicrobial Surveillance Program, 1997–2016. Open Forum Infectious Diseases 6(Supplement_1):S63–S8

    Article  PubMed  PubMed Central  Google Scholar 

  • Shrivastava SR, Shrivastava PS, Ramasamy J (2018) World health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. J Med Soc 32(1):76

    Article  Google Scholar 

  • Spilker T, Coenye T, Vandamme P, LiPuma JJ (2004) PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 42(5):2074–2079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40(2):175–179

    Article  PubMed  Google Scholar 

  • Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18(3):318–327

    Article  PubMed  Google Scholar 

  • Tomás M, Doumith M, Warner M, Turton JF, Beceiro A, Bou G, Livermore DM, Woodford N (2010) Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 54(5):2219–2224

    Article  PubMed  PubMed Central  Google Scholar 

  • Ullah W, Qasim M, Rahman H, Bari F, Khan S, Rehman ZU, Khan Z, Dworeck T, Muhammad N (2016) Multi drug resistant Pseudomonas aeruginosa: pathogen burden and associated antibiogram in a tertiary care hospital of Pakistan. Microb Pathog 97:209–212

    Article  PubMed  Google Scholar 

  • Vernet G, Mary C, Altmann DM, Doumbo O, Morpeth S, Bhutta ZA, Klugman KP (2014) Surveillance for antimicrobial drug resistance in under-resourced countries. Emerg Infect Dis 20(3):434–441

    Article  PubMed  PubMed Central  Google Scholar 

  • WHO (2015) Global antimicrobial resistance surveillance system: manual for early implementation. World Health Organization. https://apps.who.int/iris/handle/10665/188783. Accessed 10 June 2021.

  • Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y (2002) Imipenem-EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 40(10):3798–801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zolfaghar I, Evans DJ, Fleiszig SMJ (2003) Twitching motility contributes to the role of pili in corneal infection caused by Pseudomonas aeruginosa. Infect Immun 71(9):5389–5393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank Mr. K. F. Kong, School of Biological Sciences, the University of Hong Kong, for facilitating the transportation of samples.

Funding

This research was supported by PhD Indigenous Scholarship Program (PIN:213–66660-2BM2-192) and International Research Support Initiative Program offered by the Higher Education Commission of Pakistan.

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Contributions

A. B. A. designed, performed, and analyzed experiments and wrote the paper; A. Y. designed and supervised the expressional studies; P. S. supervised the biofilm and PFGE studies and provided guidance in statistical analysis and improving the manuscript; Y. S. supervised the phenotypic characterization methods; A. A. designed and supervised the whole project and finalized the manuscript.

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Correspondence to Aamir Ali.

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The study was approved by the Institutional Medical Ethics Committee of National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS) Faisalabad, Pakistan (ID no. 14092015).

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Awan, A.B., Yan, A., Sarwar, Y. et al. Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections. Appl Microbiol Biotechnol 105, 9321–9332 (2021). https://doi.org/10.1007/s00253-021-11680-6

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