Effluents from a university clinical analysis teaching laboratory: an inductive study on adhesion and cellular response of Pseudomonas aeruginosa


  • Débora Conceição da Silva Amaral
  • Hueliton Borchardt
  • Victor Targino Gomes
  • Renan Leite
  • Elisângela Afonso de Moura Kretzschmar
  • Rafael de Almeida Travassos
  • Ian Porto Gurgel do Amaral
  • Ulrich Vasconcelos




Biofilms, Emerging Contaminants, Environmental Stress, Generation of Liquid Effluents, Urban Pollution


Microbial resistance to antibiotics is a global threat and the World Health Organization (WHO) predicts that future pandemics will involve resistant microorganisms. Recently, the WHO published a list of pathogenic microbes whose priority level varies among medium, high and critical. All bacteria in the list can form biofilms. Biofilms are structures made of polymeric adhesive matrix composed by multispecies in close association. Additionally, biofilm provides an important physical barrier. Different phenotypes are found in biofilms, ensuring resistance to oxidative stress, as well as environmental stress factors. It results cell persistence because there is generation of a gradient of chemical inhibitors at subinhibitory levels, which promote the development of resistant subpopulations able to grow and disseminate. Clinical analysis laboratories are sources of selective pressures for potentially pathogenic bacteria because different substances are handled and discarded daily by the effluents generated. This study assessed the activity of a highly toxic leachate on isolates of Pseudomonas aeruginosa recovered from the sinks of a university clinical analysis teaching laboratory. In vitro biofilm formation tests, pyocyanin quantification, cell wall hydrophobicity test, motility test and susceptibility to seven antibiotics were carried out. The isolate with the most antibiotic resistance was assessed. There was a reduction in adhesion of 14 to 430%. Biofilm was formed, however, on all tested materials; the cells preferentially adhered to hydrophilic surfaces, dolomite, followed by galvanized iron and glass. Maximum pyocyanin production was 12 mM. Swimming motility was stimulated, recording a 12-fold increase in speed, while swarming and twitching motility were inhibited by around 20 and 60%, respectively. All isolates were resistant to cephalosporins and one isolate was detected resistant also to ciprofloxacin. The results indicated that P. aeruginosa survived the leachate, triggering different adaptive physiological responses to persist in the stressful environment.


ABOU, P.; EL FEGHALI, R.; NAWAS, T. Extraction and purification of pyocyanin: a simpler and more reliable method. MOJ Toxicol., v. 4, n. 6, p. 412-422, 2018, doi: 10.15406/mojt.2018.04.00139

AKINDUTI, P. A.; GEORGE, O. W.; OHORE, H. U. et al. Evaluation of efflux-mediated resistance and biofilm formation in virulent Pseudomonas aeruginosa associated with healthcare infections. Antibiotics, v. 12, n. 3, p. 626, 2023 doi: 10.3390/antibiotics12030626

ANDRADE, L.; CHIQUE, C.; HYNDS, P. et al. The antimicrobial resistance profiles of Escherichia coli and Pseudomonas aeruginosa isolated from private groundwater wells in the Republic of Ireland. Environ Pollut, v. 317, p. 120817, 2023, doi: 10.1016/j.envpol.2022.120817

APHA, AWWA, WEF. Standard methods for the examination of water and wastewater. 22nd ed. Baltimore: APHA, AWWA, WEF, 2012.

BALASUBRAMANIAN, D.; SCHNEPER, L.; MERIGHI, M. et al. The regulatory repertoire of Pseudomonas aeruginosa AmpC β-lactamase regulator AmpR includes virulence genes. PLoS ONE, v. 7, n. 3, 2012, doi: 10.1371/journal.pone.0034067

BALMURI, S. R.; PHANDANOUVONG-LOZANO, V.; HOUSE, S. D. et al. Mucoid coating provides a growth advantage to Pseudomonas aeruginosa at oil–water interfaces. Appl Bio Mater, v. 5, n. 5, p. 1868-1878, 2022, doi: 10.1021/acsabm.1c01198

BEHZADI, P.; GAJDÁCS, M.; PALLÓS, P. et al. Relationship between biofilm-formation, phenotypic virulence factors and antibiotic resistance in environmental Pseudomonas aeruginosa. Pathogens, v. 11, n. 9, p. 1015, 2022, doi: 10.3390/pathogens11091015

BONINI, P.; PLEBANI, M.; CERIOTTI, F. et al. Errors in laboratory medicine. Clin Chem. v. 48, n. 5, p. 691-698, 2002, doi: 10.1093/clinchem/48.5.691

BUTIUC-KEUL, A.; CARPA, R.; PODAR, D. et al. Antibiotic resistance in Pseudomonas spp. through the urban water cycle. Curr Microbiol., v. 78, n. 4, p. 1227-1237, 2021, doi: 10.1007/s00284-021-02389-w

CLSI. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100, 30th ed. 2020.

DALLANORA, F.; DALLANORA, L. M. F.; DALLANORA, A. F. et al. Multirresistant Pseudomonas aeruginosa from hospital and household effluent of public collecting network. RIES, v. 1, n. 1, p. 46-55, 2023, doi: 10.33362/ries.v1i1.3117

DESTOUMIEUX-GARZÓN, D.; MAVINGUI, P.; BOESTSCH, G. et al. The One Health Concept: 10 years old and a long road ahead. Front Vet Sci., v. 5, p. 14, 2018, doi: 10.3389/fvets.2018.00014

EDWARD, E. A.; EL SHEHAWY M. R.; ABOUELFETOUH, A. et al. Prevalence of different virulence factors and their association with antimicrobial resistance among Pseudomonas aeruginosa clinical isolates from Egypt. BMC Microbiol, v. 23, n. 1, p. 161, 2023, doi: 10.1186/s12866-023-02897-8

FALL, C.; CUENCA-MENDOZA, F.; JIMÉNEZ-MOLEÓN, M. C. Patrón de contaminación de las aguas residuales procedentes de laboratorios universitarios. Inf Tecnol., v. 13, n. 1, p. 55-60, 2002.

FIGUEIREDO, A. R. T; WAGNER, A.; KUMMERLI, R. Ecology drives the evolution of diverse social strategies in Pseudomonas aeruginosa. Molec Ecol., v. 30, n. 20, p. 5214-5228, 2021, doi: 10.1111/mec.16119

GEYTER, D.; VANSTOKSTRAETEN, R.; CROMBÉ, F. et al. Sink drains as reservoirs of VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa in a Belgian intensive care unit: relation to patients investigated by whole-genome sequencing. J Hosp Infect., v. 115, p. 75-82, 2021, doi: 10.1016/j.jhin.2021.05.010

GHSSEIN, G.; EZZEDDINE, Z. A review of Pseudomonas aeruginosa metallophores: pyoverdine, pyochelin and pseudopaline. Biology, v. 11, n. 12, p. 1711, 2022, doi: 10.3390/biology/11121711

GLOAG, E. S.; GERMAN, G. K.; STOODLEY, P. et al. Viscoelastic properties of Pseudomonas aeruginosa variant biofilms. Sci Rep. v. 8, p. 9691, 2018, doi: 10.1038/s41598-018-28009-5

GOUVEIA, C. Q.; GOMES, V. T.; TRAVASSOS, R. A. et al. Coffee breaks the Journey of pseudomonads: a pause for a reflection. Multidiscip Sci Rep. v. 3, n. 2, p. 40, 2023, doi: 10.54038/ms.v3i2.40

HAFIANE, F. Z.; TAHRI, L.; AMEUR, N. et al. Antibiotic resistance of Pseudomonas aeruginosa in well Waters in irrigated zone (Middle Atlas-Morocco). Nat Environ Pollut Technol., v. 18, n. 4, p. 1193-1200, 2019.

HIGHMORE, C. J.; MELAUGH, G.; MORRIS, J. R. et al. Translational challenges and opportunities in biofilm science: a BRIEF for the future. NJJ Biofilms Microbiomes, v. 8, p. 68, 2022, doi: 10.1038/s41522-022-00327-7

HOBAN, S.; ARCHER, F. I.; BERTOLA, L. D. et al. Global genetic diversity status and trends: towards a suite of Essential Biodiversity Variables (EBVs) for genetic composition. Biol Rev., v. 97, n. 4, p. 1511-1538, 2022, doi: 10.1111/brv.12852

IDOWU, A. B.; ADEOYE, O. O.; BELLO, S. A. M. et al. Growth pattern of Pseudomonas aeruginosa in different wastewater media. Int J Environ Agric Biotechnol., v. 2, n. 5, p. 2622-2628, 2017, doi: 10.22161/ijeab/2.5.44

KÜHN, M. J.; TALA, L.; INCLAN, Y. F. et al. Mechanotaxis directs Pseudomonas aeruginosa twitching motility. Biol Sci., v. 118, n. 30, p. e2101759118, 2021, doi: 10.1073/pnas.2101759118

LUCZKIEWICZ, A.; KOTLARSKA, E.; ARTICHOWICZ, W. et al. Antimicrobial resistance of Pseudomonas spp. isolated from wastewater and wastewater-impacted marine coastal zone. Environ Sci Pollut Res., v. 22, p. 19823-19834, 2015, doi: 10.1007/s11356-015-5098-y

MAGALHÃES, M. J.; PONTES, G.; SERRA, P. T. et al. Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants. Microbiology, v. 16, n. 1, p. 193, 2016, doi: 10.1186/s12866-016-0798-0

MAKHARITA, R. R.; EL-KHOLY, I.; HETTA, H. F. et al. Antibiogram and genetic characterization of carbapenem-resistant gram-negative pathogens incriminated in healthcare-associated infections. Infect Drug Resist., v. 13, p. 3991-4002, 2020, doi: 10.2147/IDR.S276975

MAPIPA, Q.; DIGBAN, T. O.; NNOLIM, N. E. et al. Antibiogram profile and virulence signatures of Pseudomonas aeruginosa isolates recovered from selected agrestic hospital effluents. Sci Rep., v. 11, p. 11800, 2021, doi: 10.1038/s41598-021-91280-6

MARUTESCU, L. G.; POPA, M.; GHEORGHE-BARBU, I. et al. Wastewater treatment plants, an “escape gate” for ESCAPE pathogens. Front Microbiol., v. 14, p. 1193907, 2023, doi: 10.3389/fmicb.2023.1193907

MEYER, B. N.; FERRIGNI, N. R.; PUTNAM, J. E. et al. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med., v. 45, n. 5, p. 31-34, 1982, doi: 10.1055/s-2007-971236

MORADALI, M. F.; GHODS, S.; REHM, B. H. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol., v. 7, n. 39, 2017. 10.3389/fcimb.2017.00039

MUES, N.; CHU, H. W. Out-smarting the host: bacteria maneuvering the immune response to favor their survival. Front Imunnol., v. 11, p. 819, 2020, doi: 10.3389/fimmu.2020.0081

NASSRI, I.; TAHRI, L.; SAIDI, A. et al. Prevalence, diversity and antimicrobial resistance of Salmonella enterica and Pseudomonas aeruginosa isolates from spring water in a rural area of northwestern Morocco. Biodiversitas., v. 22, n. 3, p. 1363-1370, 2021, doi: 10.13057/biodiv/d220337

NGUYEN, J.; FERNANDEZ, V.; PONTRELLI, S. et al. A distinct growth physiology enhances bacterial growth under rapid nutrient fluctuations. Nat Comm., v. 12, p. 3362, 2021, doi: 10.1038/s41467-021-23439-8

OLIVEIRA, A. D. L.; VASCONCELOS, U.; CALAZANS, G. M. T. Detection of potential pathogenic Pseudomonas aeruginosa in a hospital water system. Res J Pharm Biol Chem., v. 12, n. 4, p. 132-139, 2021.

OŁDAK, E.; TRAFNY, E.A. Secretion of proteases by Pseudomonas aeruginosa biofilms exposed to ciprofloxacin. Antimicrob Agents Chemother, v. 49, n. 5, p. 3281-3288, 2005, doi: 10.1128/AAC.49.8.3281-3288.2005

PENA, R. T.; BLASCO, L.; AMBROA, A. et al. Relationship between quorum sensing and secretion systems. Front Microbiol, v. 10, p. 1100, 2019, doi: 10.3389/fmicb.2019.01100

PIRZADIAN, J.; VOOR IN ‘T HOLT, A. F.; HOSSAIN, M. et al. Limiting spread of VIM-positive Pseudomonas aeruginosa from colonized sink drains in a tertiary care hospital: A before-and-after study. PLoS One, v. 18, n. 3, p. e0282090, 2023, doi: 10.1371/journal.pone.0282090

QIANG, W.; CHAONENG, Z.; JINZHOU, Z. et al. Numerical simulation of planar hydraulic fracture propagation with consideration to transition from turbulence to laminar flow regime. Eng Fract Mech., v. 262, p. 108258, 2022, doi: 10.1016/j.engfracmech.2022.108258

REYES-LARA, S.; REYES-MAZZOCO, R. Efecto de las cargas hidráulica y orgánica sobre la remoción masica de un empaque estructurado en un filtro percolador. Rev Mex Ing Quím., v. 8, n. 1, p. 101-109, 2009.

RODRIGUES, L. B.; SANTOS, L. R.; TAGLIARI, V. Z. et al. Quantification of biofilm production on polystyrene by Listeria, Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse. Braz J Microbiol., v. 41, n. 4, p. 1082-1085, 2010, doi: 10.1590/S1517-8382201000004000029

ROLLET, C.; GAL, L.; GUZZO, J. Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa. FEMS Microbiol Lett., v. 290, n. 2, p. 135-142, 2009, doi: 10.1111/j.1574-6968.2008.01415.x

ROSSI, C.; SERIO, A.; LÓPEZ, C. C. et al. Biofilm formation, pigment production and motility in Pseudomonas spp. isolated from the dairy industry. Food Control., v. 86, p. 241-248, 2018, doi: 10.1016/j.foodcont.2017.11.018

SADYRBAEVA-DOLGOVA, S.; SÁNCHEZ-SUÁREZ, M. D. M.; REGUERA- MÁRQUEZ, J. A. et al. The challenge of bacteremia treatment due to non-fermenting gram-negative bacteria. Microorganisms, v. 11, n. 4, p. 899, 2023, doi: 10.3390/microorganisms11040899.

SAMPEDRO, I.; PARALES, R. E.; KRELL, T. et al. Pseudomonas chemotaxis. FEMS Microbiol Rev. v. 39, n. 1, p. 17-46, 2014, doi: 10.1111/1574-6976.12081

SOUSA, T.; HEBRAUD, M.; DAPKEVICIUS, M. L. N. E. et al. Genomic and metabolic characteristics of the pathogenicity in Pseudomonas aeruginosa. Int J Mol Sci., v. 22, p. 12892, 2021, doi: 10.3390/ijms222312892

SOUZA, I. A.; GARCIA, D. O.; ANVERSA, L. et al. Comparative analysis of the virulence factors in Pseudomonas aeruginosa strains isolated from clinical and environmental. Colloquium Vitae, v. 11, n. 3, p. 41-50, 2019, doi: 10.5447/cv.v11i3.3267

SPERANZA, G.; GOTTARDI, G.; PEDERZOLLI, C. et al. Role of chemical interactions in bacterial adhesion to polymer surfaces. Biomaterials, v. 25, n. 11, p. 2029-2037, 2004, doi: 10.1016/j.biomaterials.2003.08.061

THI, M. T. T.; WIBOWO, D.; REHM, B. H. A. Pseudomonas aeruginosa biofilms. Int J Molec Sci., v. 21, n. 22, p. 8671, 2020, doi: 10.3390/ijms21228671

TYFA, A.; KUNICKA-STYCZYŃSKA, A.; ZABIELSKA, J. Evaluation of hydrophobicity and quantitative analysis of biofilm formation by Alicyclobacillus sp. Acta Biochim Pol., v. 62, n. 4, p. 785-790, 2015, doi: 10.18338/abp.2015_1133

VIANA, A. A. G.; MARTINS, R. X.; FERREIRA, G. F. et al. Pseudomonas aeruginosa and pyocyanin negatively act on the establishment of Enterobacteriaceae biofilm on a ceramic surface. Int J Eng Res Appl., v. 7, n. 8, p. 23-30, 2017, doi: 10.9790/9622-0708022330

WANG, X.; WANG, J.; LIU, S.-Y. et al. Mechanisms of survival mediated by the stringent response in Pseudomonas aeruginosa under environmental stress in drinking water systems: Nitrogen deficiency and bacterial competition. J Hazard Mater, v. 448, p. 130941, 2023, doi: 10.1016/j.jhazmat.2023.130941

ZHAO, Y.; COCERVA, T.; COX, S. et al. Evidence for co-selection of antibiotic resistance genes and mobile genetic elements in metal polluted urban soils. Sci Total Environ. v. 656, p. 512-520, 2019, doi: 10.1016/j.scitotenv.2018.11.372

ZHOU, Z. Y.; HU, B.; GAO, X. et al. Sources of sporadic Pseudomonas aeruginosa colonizations/infections in surgical ICUs: Association with contaminated sink trap. J Infect Chemother, v. 22, n. 7, p. 450-455, 2016, doi: 10.1016/j.jiac.2016.03.016




How to Cite

Amaral, D. C. da S., Borchardt, H., Gomes, V. T., Leite, R., Kretzschmar, E. A. de M., Travassos, R. de A., Amaral, I. P. G. do, & Vasconcelos, U. (2024). Effluents from a university clinical analysis teaching laboratory: an inductive study on adhesion and cellular response of Pseudomonas aeruginosa. Caderno Pedagógico, 21(6), e4750. https://doi.org/10.54033/cadpedv21n6-044