Bacteria living in biofilms in fluids: can we improve our cultural examination ofsynovial and other organic liquids ?

Introduction

Microorganisms are often considered as freely suspended cells, defined as planktonic, and classified on their growth characteristics in a culture media. However, Antonie Philips van Leeuwenhoek, already in the seventeenth century described the existence of surface-associated microorganisms, growing and living in communities. Biofilms-embedded microorganisms show typical mechanisms for initial attachment to a surface, development of a community structure, and detachment [1] Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis. 2002 Sep;8(9):881-90. doi: 10.3201/eid0809.020063..

A biofilm is an assemblage of microbial cells, associated together on a surface and enclosed in a matrix of primarily polysaccharide material, living like in a proper ecosystem. Depending on the environment in which the biofilm is formed, non-cellular materials such as mineral crystals, clay and silt particles, corrosion particles, and blood components may also be present in the biofilm matrix. Organisms associated with biofilms also differ from planktonic with respect to transcribed genes. Within the ecosystem of the biofilm, bacteria have developed a communication system called “Quorum sensing”, which allows them to coordinate their behavior using chemical molecules as signals. Quorum sensing communication system allows the microorganisms to orchestrate the group behavior, resulting in maximum benefit for the whole bacteria population living in the biofilm. [2] Xie H, Cook GS, Costerton JW, Bruce G, Rose TM, Lamont RJ. Intergeneric communication in dental plaque biofilms. J Bacteriol. 2000 Dec;182(24):7067-9. doi: 10.1128/JB.182.24.7067-7069.2000.

As the knowledge of biofilms improved, it become more and more evident in the last decades that this is the predominant lifestyle of bacteria and fungi and an example of an extremely successful physiological adaptation, as they thrive in most natural environments as well as in harsh conditions.

Unfortunately, biofilms are also often associated with the majority of human infectious diseases and can negatively impact health. Indeed, biofilms offer to microorganisms an enormous capacity to resist host immune system defenses and antimicrobial therapy. In healthcare environments, the persistence of the microorganisms is extended by the formation of biofilms, being responsible for the onset and spread of hospital-care-associated infections (HCAIs) (also referred to as “nosocomial” or “hospital acquired” infections). HCAIs can result in prolonged hospital stays, long-term disabilities, enhanced resistance of the microorganisms to antimicrobials, enormous additional costs for the health care systems, the patients and their families, and increased mortality rates. The prevalence of HCAI is estimated to be between 5.7% and 19.1%.

According to some estimates, 65–80% of total human infections are associated with biofilm formation and include: periodontitis/dental caries, cystic fibrosis lung infection, chronic otitis media, infective endocarditis, chronic osteomyelitis, chronic rhinosinusitis, chronic tonsillitis, chronic peritonitis, chronic prostatitis, chronic wounds, recurrent urinary tract infections (UTIs), bloodstream infections (BSIs), ventilated-associated pneumonia and infections associated with indwelling medical devices (e.g., contact lenses, heart valves, joint prostheses, and other orthopedic implants, intrauterine devices, intravascular catheters, urinary tract catheters, peritoneal catheters, etc.) [3] Pinto H, Simões M, Borges A. Prevalence and Impact of Biofilms on Bloodstream and Urinary Tract Infections: A Systematic Review and Meta-Analysis. Antibiotics (Basel). 2021 Jul 8;10(7):825. doi: 10.3390/antibiotics10070825.

In humans, bacterial and fungal biofilms may form on a wide variety of surfaces, including living tissues, foreign bodies and biomaterials; the solid-liquid interface between a surface and an aqueous medium (e.g., water, blood) provides an ideal environment for the attachment and growth of microorganisms. The spreading of the biofilm on a surface is made possible by dispersion, a process through which bacterial cells leave the biofilms, return to an independent planktonic lifestyle and eventually colonize new surfaces to establish new biofilm-based communities. [4] Singh PK, Bartalomej S, Hartmann R, Jeckel H, Vidakovic L, Nadell CD, Drescher K. Vibrio cholerae Combines Individual and Collective Sensing to Trigger Biofilm Dispersal. Curr Biol. 2017 Nov 6;27(21):3359-3366.e7. doi: 10.1016/j.cub.2017.09.041. Epub 2017 Oct 19.

Recently, the ability of bacteria to form biofilms in fluids, like synovial fluid, blood, urine, cerebrospinal fluids, has been reported. Biofilm formation in liquids proceeds through an initial adhesion process of bacterial cells one to the other, by forming an extracellular polymeric substance, that provides a three-dimensional structure for the bacterial cell’s aggregates, protecting them from the external environment. [5] Drago L, Romanò D, Fidanza A, Giannetti A, Erasmo R, Mavrogenis AF, Romanò CL. Dithiotreitol pre-treatment of synovial fluid samples improves microbiological counts in peri-prosthetic joint infection. Int Orthop. 2023 May;47(5):1147-1152. doi: 10.1007/s00264-023-05714-z. Epub 2023 Feb 22.

Aim of this article is to provide an overview of the bacteria living in human fluids, with a focus on the associated pathologic conditions and the impact that this phenomenon may have on the cultural examination of fluid samples.

Synovial fluid

Periprosthetic joint infections (PJIs) represent a great challenge for the patients and the healthcare systems, due to the increased length of hospital stay, need for complex and expensive surgeries and prolonged antibiotic treatments. [6], Parisi TJ, Konopka JF, Bedair HS. What is the Long-term Economic Societal Effect of Periprosthetic Infections After THA? A Markov Analysis. Clin Orthop Relat Res. 2017 Jul;475(7):1891-1900. doi: 10.1007/s11999-017-5333-6. Epub 2017 Apr 7.[7] Ghirardelli S, Touloupakis G, Antonini G, Violante B, Fidanza A, Indelli PF. Debridement, antibiotic, pearls, irrigation and retention of the implant and other local strategies on hip periprosthetic joint infections. Minerva Orthop 2022;73:409-15. doi: 10.23736/S2784-8469.21.04173-0 Recently, the presence of bacteria and biofilm aggregates floating in the synovial fluids of PJIs has been reported and associated to the resistance of bacterial joint infections to common treatments [8], Dastgheyb SS, Hammoud S, Ketonis C, Liu AY, Fitzgerald K, Parvizi J, Purtill J, Ciccotti M, Shapiro IM, Otto M, Hickok NJ. Staphylococcal persistence due to biofilm formation in synovial fluid containing prophylactic cefazolin. Antimicrob Agents Chemother. 2015 Apr;59(4):2122-8. doi: 10.1128/AAC.04579-14. Epub 2015 Jan 26. [9] Perez K, Patel R. Biofilm-like aggregation of Staphylococcus epidermidis in synovial fluid. J Infect Dis. 2015 Jul 15;212(2):335-6. doi: 10.1093/infdis/jiv096. Epub 2015 Feb 23. . The ability of bacteria to live in biofilm aggregates may explain the limited efficacy of current microbiological investigations of synovial fluids, with reported sensitivities as low as 41.6 to 90% [10], Tande AJ, Patel R. Prosthetic joint infection. Clin Microbiol Rev. 2014 Apr;27(2):302-45. doi: 10.1128/CMR.00111-13. [11] Leggett A, Li DW, Bruschweiler-Li L, Sullivan A, Stoodley P, Brüschweiler R. Differential metabolism between biofilm and suspended Pseudomonas aeruginosa cultures in bovine synovial fluid by 2D NMR-based metabolomics. Sci Rep. 2022 Oct 15;12(1):17317. doi: 10.1038/s41598-022-22127-x. . Moreover, the formation of fluctuating biofilm aggregates may compromise the ability of antibiotics to reach and kill the microbial cells, as it happens in bacteria living in biofilms adhering on a surface. Furthermore, the bacterial-protein interactions in these aggregates changes the production of virulence factors and the phenotype, inducing a marked tolerance to antibiotics [12] Pestrak MJ, Gupta TT, Dusane DH, Guzior DV, Staats A, Harro J, Horswill AR, Stoodley P. Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation. PLoS One. 2020 Apr 17;15(4):e0231791. doi: 10.1371/journal.pone.0231791. Erratum in: PLoS One. 2020 May 14;15(5):e0233534. . Bacteria living in synovial fluids of PJI may substantially contribute to the development of a chronic condition difficult to diagnose and to reat and thus requiring suitable antibiofilm strategies.

Urinary tract

Urinary tract infections (UTIs), one of the most common infections sustained by bacteria, represents a severe public health issue. The operating costs of these infections are estimated around US$3.5 billion per year in the US. UTIs may manifest in different forms such as cystitis, pyelonephritis, prostatitis, urethritis. Ideal environment for attachment and colonization by uropathogens are urinary catheters. The most common agents responsible for complicated UTIs are Escherichia coli, Enterococcus spp., Klebsiella pneumoniae, Candida spp., Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa. All these microorganisms have already been linked to biofilm formation [13] Pelling H, Nzakizwanayo J, Milo S, Denham EL, MacFarlane WM, Bock LJ, Sutton JM, Jones BV. Bacterial biofilm formation on indwelling urethral catheters. Lett Appl Microbiol. 2019 Apr;68(4):277-293. doi: 10.1111/lam.13144. . Large fragments of the biofilms and high concentrations of microbial cells can detach from the catheter and flow into the bladder spreading the infection and leading to bacteriuria. In addition, uropathogens can form biofilm in the bladder and kidney, reducing antibiotic susceptibility and causing recurrent infections. Biofilms play a central role in catheter associated UTIs especially in patients with prolonged catheterization, leading to increased morbidity and mortality [14] Wasfi R, Hamed SM, Amer MA, Fahmy LI. Proteus mirabilis Biofilm: Development and Therapeutic Strategies. Front Cell Infect Microbiol. 2020 Aug 14;10:414. doi: 10.3389/fcimb.2020.00414.. Bacterial populations living in biofilms show a more efficient and adapted behavior, compared to planktonic bacteria, with improved chance of survival while the biofilm community sheds planktonic cells able to further colonize adjacent tissues [15] Pinto H, Simões M, Borges A. Prevalence and Impact of Biofilms on Bloodstream and Urinary Tract Infections: A Systematic Review and Meta-Analysis. Antibiotics (Basel). 2021 Jul 8;10(7):825. doi: 10.3390/antibiotics10070825. .

Blood system

Not less relevant and worrying than UTIs are the blood system infections (BSIs), ranked as the 12th cause of death in the USA, with the estimated mortality rate of 15–30%. Through the bloodstream microorganism can spread from a local infection (endocarditis, meningitis, osteomyelitis…) to distant sites. In addition, intravenous catheters are an important risk factor for BSIs. In fact, bacterial biofilm can easily develop on the surface of this devices, and biofilm fragments or planktonic microorganism may spread into the bloodstream. The most often isolated pathogens in bacteremia are S. aureus, E. coli, K. pneuomoniae, P. aeruginosa, Enterococcus faecalis, Staphylococcus epidermidis, Enterobacter cloacae, Streptococcus pneumoniae, Enterococus faecium and Acinetobacter baumannii [15], Pinto H, Simões M, Borges A. Prevalence and Impact of Biofilms on Bloodstream and Urinary Tract Infections: A Systematic Review and Meta-Analysis. Antibiotics (Basel). 2021 Jul 8;10(7):825. doi: 10.3390/antibiotics10070825. [16], Diekema DJ, Hsueh PR, Mendes RE, Pfaller MA, Rolston KV, Sader HS, Jones RN. The Microbiology of Bloodstream Infection: 20-Year Trends from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother. 2019 Jun 24;63(7):e00355-19. doi: 10.1128/AAC.00355-19.[17] Hattori H, Maeda M, Nagatomo Y, Takuma T, Niki Y, Naito Y, Sasaki T, Ishino K. Epidemiology and risk factors for mortality in bloodstream infections: A single-center retrospective study in Japan. Am J Infect Control. 2018 Dec;46(12):e75-e79. doi: 10.1016/j.ajic.2018.06.019. : all well-known biofilm-producers. As it happens with cultural examination in other fluids, blood cultures may often result in false negative findings.

Cerebrospinl fluid (csf)

Thousands of CSF shunts are implanted every year as a treatment of hydrocephalus. To relieve cranial pressure in fact, CSF is generally shunted, from the cerebral ventricle into the peritoneal cavity. Common complications of this procedure are intraventricular hemorrhage, obstruction, over drainage of CSF and infection. Clinical signs range from local manifestations as ventriculitis, peritonitis, to nephritis or septicemia, leading to high risk of seizures, decreased intellectual performance, and mortality [18] Mounier R, Kapandji N, Birnbaum R, Cook F, Rodriguez C, Nebbad B, Lobo D, Dhonneur G. Biofilm-associated infection: the hidden face of cerebrospinal fluid shunt malfunction. Acta Neurochir (Wien). 2016 Dec;158(12):2321-2324. doi: 10.1007/s00701-016-2977-z. . Correct diagnosis and treatment of device-related infections are notoriously difficult, because of bacteria forming biofilms. Diagnostic cultures of fluid aspirates and swabs are often falsely negative, presumably because of the very low chance to find cells in the planktonic state. [19], Fux CA, Quigley M, Worel AM, Post C, Zimmerli S, Ehrlich G, Veeh RH. Biofilm-related infections of cerebrospinal fluid shunts. Clin Microbiol Infect. 2006 Apr;12(4):331-7. doi: 10.1111/j.1469-0691.2006.01361.x. [20] Benachinmardi KK, Ravikumar R, Indiradevi B. Role of Biofilm in Cerebrospinal Fluid Shunt Infections: A Study at Tertiary Neurocare Center from South India. J Neurosci Rural Pract. 2017 Jul-Sep;8(3):335-341. doi: 10.4103/jnrp.jnrp_22_17. .

Saliva

Van Leeuwenhoek, simply using his microscope, observed for the first-time microorganisms on tooth surfaces, the dental plaque [1] Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis. 2002 Sep;8(9):881-90. doi: 10.3201/eid0809.020063.. Saliva under normal circumstances is sterile until it leaves the salivary duct and enters the oral cavity, where it is quickly contaminated by biofilm-producer microorganisms. Bacterial growth in the form of biofilm has been associated with most ear, nose, and throat infections [21] Perez-Tanoira R, Aarnisalo A, Haapaniemi A, Saarinen R, Kuusela P, Kinnari TJ. Bacterial biofilm in salivary stones. Eur Arch Otorhinolaryngol. 2019 Jun;276(6):1815-1822. doi: 10.1007/s00405-019-05445-1. Epub 2019 Apr 26. . Implanted biomaterials and other inert surfaces with poor host defense, such as salivary calculi, are subject to bacterial attachment and biofilm formation and also in this environment, pharmaceutical treatment and immune system have limited effect on bacteria living in biofilm. Biofilm growth has been associated with chronic otitis media and mastoiditis as well as chronic infections of the adenoid tissue. Planktonic bacteria may shed from mature biofilm and being the cause of an acute phase of infection such as a chronic and recurrent otitis media. Furthermore, the microbial diagnostics in oral cavity are always complicated as the saliva in mouth is contaminated with the oral microbiome [21], Perez-Tanoira R, Aarnisalo A, Haapaniemi A, Saarinen R, Kuusela P, Kinnari TJ. Bacterial biofilm in salivary stones. Eur Arch Otorhinolaryngol. 2019 Jun;276(6):1815-1822. doi: 10.1007/s00405-019-05445-1. Epub 2019 Apr 26. [22] Simon-Soro A, Ren Z, Krom BP, Hoogenkamp MA, Cabello-Yeves PJ, Daniel SG, Bittinger K, Tomas I, Koo H, Mira A. Polymicrobial Aggregates in Human Saliva Build the Oral Biofilm. mBio. 2022 Feb 22;13(1):e0013122. doi: 10.1128/mbio.00131-22. Epub 2022 Feb 22. , which currently form large amount of biofilms.

Tracheal aspirate

Nosocomial pneumonia represents about one quarter of all nosocomial infections and represents the first cause of nosocomial infection in Intensive care unit, contributing to extend the length of hospitalization, mortality and costs of treatment. Tracheal intubation in patient under mechanic ventilation in fact, increases the risk for infection ranging from six to twenty times higher. Bacteriological diagnosis through specific specimen brush, bronchoalveolar lavage, and endotracheal aspirates have been nowadays standardized but lack of specificity, as it is based on the identification of bacteria growing in tracheal secretion. It is necessary to consider that in ventilator-associated pneumonia biofilm plays an important role in the diagnosis as in the treatment. The endotracheal tube allows direct entry of microbial colonization of dental plaque and oropharynx, that has natural ability to form biofilm, into the lower respiratory tract. Then, bacteria within the biofilm can infect the lungs by several ways: through detachment of biofilm portions that then reaches the lungs and by aspiration into deeper airways of aerosolized planktonic pathogens detached from the biofilm [23], Ferreira Tde O, Koto RY, Leite GF, Klautau GB, Nigro S, Silva CB, Souza AP, Mimica MJ, Cesar RG, Salles MJ. Microbial investigation of biofilms recovered from endotracheal tubes using sonication in intensive care unit pediatric patients. Braz J Infect Dis. 2016 Sep-Oct;20(5):468-75. doi: 10.1016/j.bjid.2016.07.003. Epub 2016 Aug 8. [24] Souza LCD, Mota VBRD, Carvalho AVDSZ, Corrêa RDGCF, Libério SA, Lopes FF. Association between pathogens from tracheal aspirate and oral biofilm of patients on mechanical ventilation. Braz Oral Res. 2017 Jun 5;31:e38. doi: 10.1590/1807-3107BOR-2017.vol31.0038. .

Drains

In a wide variety of surgical specialties, closed suction drains are used for prevention of haematoma and fluid accumulation. Nevertheless, more and more evidence, in different surgical field are showing that not always drains are effective, instead they are unnecessary or counterproductive, encouraging local wound complications and infections [25] Dower R, Turner ML. Pilot study of timing of biofilm formation on closed suction wound drains. Plast Reconstr Surg. 2012 Nov;130(5):1141-1146. doi: 10.1097/PRS.0b013e318267d54e. . A recent in vivo drain study demonstrated a significant biofilm formation, as soon as two hours after drain insertion, cocci within clumps of fibrin adherent to the surface of the drain were detected. This finding suggests that drains are contaminated very early, and considering the ideal culture medium that clotted blood represents, combined with a foreign body in the form of the drain, biofilm formation is obviously able to evolve very rapidly [26] De Waele JJ, Boelens J, Van De Putte D, Huis In 't Veld D, Coenye T. The Role of Abdominal Drain Cultures in Managing Abdominal Infections. Antibiotics (Basel). 2022 May 20;11(5):697. doi: 10.3390/antibiotics11050697. . In matter of prevention, even for this reason the drains should be used for the shortest time and if possible no later than 24 hours under strict surveillance.

Is it possible to detach bacteria in fluids from their biofilms? dithiothreitol (dtt) as a diagnostic tool

The presence of biofilm-bacterial aggregates may have a strong impact on pathogen identification, on the bacterial count and antibiogram evaluation performed with traditional cultural techniques, that were designed for planktonic, isolated microorganisms (Figure 1). While physical (sonication) and chemical (Dithiothreitol, DTT) systems have been proposed to dislodge bacteria adhering on a surface from their biofilms, less is known about the possibility to improve microbiological sensitivity by antibiofilm pre-treatment of fluid samples. In fact, to the best of our knowledge, only DTT has been tested to dislodge bacteria from their biofilms in synovial fluid samples with promising results.

As DTT has been demonstrated to be able to release microorganisms from the biofilm produced on prosthetic implants and on human tissues, it has also recently been demonstrated to be effective in disrupting biofilm-bacteria aggregates in fluids, and more specifically it has been shown to be effective in the synovial fluid [5] Drago L, Romanò D, Fidanza A, Giannetti A, Erasmo R, Mavrogenis AF, Romanò CL. Dithiotreitol pre-treatment of synovial fluid samples improves microbiological counts in peri-prosthetic joint infection. Int Orthop. 2023 May;47(5):1147-1152. doi: 10.1007/s00264-023-05714-z. Epub 2023 Feb 22. (Figure 2). DTT is a chemical agent that reduces disulfide bonds in peptides and can indeed alter the matrix of biofilm releasing bacteria without affecting their viability. Through this procedure, bacterial culture is possible and so identification and antibiotic susceptibility tests are easier to perform [27], Giannetti A, Romano J, Fidanza A, Di Mauro M, Brunetti M, Fascione F, Calvisi V (2022) The diagnostic potential of Micro- DTTect compared to conventional culture of tissue samples in orthopedic infections. Lo Scalpello J 36:111–115. https://doi.org/ 10.36149/0390-5276-262.[28], De Vecchi E, Bortolin M, Signori V, Romanò CL, Drago L. Treatment With Dithiothreitol Improves Bacterial Recovery From Tissue Samples in Osteoarticular and Joint Infections. J Arthroplasty. 2016 Dec;31(12):2867-2870. doi: 10.1016/j.arth.2016.05.008. Epub 2016 May 11. [29], Calori GM, Colombo M, Navone P, Nobile M, Auxilia F, Toscano M, Drago L. Comparative evaluation of MicroDTTect device and flocked swabs in the diagnosis of prosthetic and orthopaedic infections. Injury. 2016 Oct;47 Suppl 4:S17-S21. doi: 10.1016/j.injury.2016.07.040. [30] Sambri A, Cadossi M, Giannini S, Pignatti G, Marcacci M, Neri MP, Maso A, Storni E, Gamberini S, Naldi S, Torri A, Zannoli S, Tassinari M, Fantini M, Bianchi G, Donati D, Sambri V. Is Treatment With Dithiothreitol More Effective Than Sonication for the Diagnosis of Prosthetic Joint Infection? Clin Orthop Relat Res. 2018 Jan;476(1):137-145. doi:10.1007/s11999.0000000000000060. .

Conclusion

Diagnosis for biofilm-associated infection (BAI) can be challenging, and even with the correct diagnosis, therapy can be particularly difficult, long and expensive. The fact that bacteria may live in biofilms even in fluid should be taken into account both as to concern our diagnostic and treatment approach. In particular, as to concern microbiological diagnosis, the spontaneous tendency of many bacterial species to aggregate must be considered. In fact, cell disaggregation is often omitted when analyzing bacterial samples and the number of Colony Forming Units (CFUs) is usually taken as the golden standard. However, the CFU count is not an absolute measure of bacteria cells; instead, it represents the number of colonies that can form on an agar plate from a given sample. If bacteria aggregates are cultured as a single cell, the CFU will be falsely low and even false negative results may be reported.

Molecular approaches may at least partially overcome this difficulty; nevertheless, their use is limited due to high costs of the procedure, the level of expertise required and the inability to differentiate living from dead microroganisms. Antibiofilm chemical pre-treatment of fluid samples using DTT can be a low-cost and simple-to-use alternative in BAIs, and probably it should be included as a routine not only for any solid or tissue biological sample but also for the liquid ones. In fact, as antibiofilm pre-treatment of synovial fluid with DTT has been shown to increase the sensitivity of cultural examination by freeing the microorganisms from the biofilm aggregates, it may be expected that a similar result could be obtained by applying the same system to other fluids such as saliva, urine, blood, and cerebrospinal liquid, paving the way to a complete change in the results of the microbiological examinations. Given the high social and economic costs of chronic biofilm-related infections in nearly all the fields of Medicine, we believe that more research on this subject would be greatly beneficial.