|Year : 2017 | Volume
| Issue : 2 | Page : 74-78
Biofilm formation as a virulence factor of Acinetobacter baumannii: An emerging pathogen in critical care units
A Arockia Amala Reena, Anandhalakshmi Subramaniyan, Reba Kanungo
Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry, India
|Date of Submission||10-Nov-2017|
|Date of Acceptance||22-Nov-2017|
|Date of Web Publication||8-Jan-2018|
Dr. Anandhalakshmi Subramaniyan
Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry
Source of Support: None, Conflict of Interest: None
Acinetobacter baumannii, an emerging nosocomial pathogen, is increasingly associated with serious infections among hospitalized patients, especially those on life-support systems. A. baumannii has become resistant to almost all currently available antibacterial agents, including carbapenems, which were once considered the drug of choice for the treatment of infections with multidrug-resistant (MDR) organisms. A. baumannii is notorious in its ability to spread among hospitalized patients and causes outbreaks which have been reported worldwide. The capability of these strains to circulate widely seems to depend on the expression of virulence factors that allow bacterial colonization as well as on the expression of antibiotic resistance. Biofilm production by A. baumannii appears to be one of the major contributing factors in colonization, notably of medical devices. This review explores published literature on the association of biofilms and MDR A. baumannii in hospitalized patients. An online search was made for articles of original work and reviews on biofilms production among Acinetobacter and their association with virulence. The articles were reviewed and results were analyzed based on biofilm production and the factors associated with it, namely biofilm cycle, biofilm-associated protein, chaperone-usher secretion system, and quorum sensing.
Keywords: Acinetobacter, antibiotic resistance and healthcare-associated infections, biofilm, virulence
|How to cite this article:|
Amala Reena A A, Subramaniyan A, Kanungo R. Biofilm formation as a virulence factor of Acinetobacter baumannii: An emerging pathogen in critical care units. J Curr Res Sci Med 2017;3:74-8
|How to cite this URL:|
Amala Reena A A, Subramaniyan A, Kanungo R. Biofilm formation as a virulence factor of Acinetobacter baumannii: An emerging pathogen in critical care units. J Curr Res Sci Med [serial online] 2017 [cited 2020 Feb 17];3:74-8. Available from: http://www.jcrsmed.org/text.asp?2017/3/2/74/222427
| Introduction|| |
Acinetobacter baumannii is emerging as an important nosocomial pathogen causing a variety of infections mostly acquired in the hospital. It is associated with meningitis, endocarditis, wound infections, and bacteremia, especially in patients in the Intensive Care Units, causing ventilator-associated pneumonia, catheter-associated bloodstream, and urinary tract infections. In the past, these organisms were not considered clinically significant. However, developments in resuscitation techniques and use of various devices and prosthetics have changed the natures of infection with Acinetobacter. It is increasingly associated with serious infections among patients on these life-support systems., Till date, some strains of A. baumannii have become resistant to almost all currently available antibacterial agents, including carbapenems, which were once considered the drug of choice for the treatment of multidrug-resistant (MDR) A. baumannii infection., MDR strains of A. baumannii are notorious in their ability to spread among hospitalized patients and cause outbreaks which have been reported worldwide., These resistant strains also pose a therapeutic challenge to the clinician as there are no antimicrobials left in the pipeline. Risk factors for MDR A. baumannii include the severity of a patient's condition, the use of multiple invasive devices and associated comorbid conditions such as diabetes mellitus in addition to indiscriminate use of broad-spectrum antibiotics., However, the ability of MDR A. baumannii strains to circulate widely seems to depend on the expression of virulence factors that allow bacterial colonization as well as on the expression of antibiotic resistance. Despite the problems posed by A. baumannii infection, and possibly due to its nature as an opportunistic pathogen, minimal research has been conducted on the virulence characteristics of this organism.
| Methodology of the Review|| |
An online search was made for articles of original work and reviews on biofilms production among Acinetobacter and their association with virulence.
Keywords used to screen the articles were biofilm, Acinetobacter, virulence, antibiotic resistance, and healthcare-associated infections. Based on the keywords, the number of articles that were screened were 59 out of which 43 articles were then further filtered for biofilm synthesis detection in hospital setups. The articles were reviewed and results were analyzed based on biofilm production and the factors associated with it, namely biofilm cycle, biofilm-associated protein (Bap), chaperone-usher secretion system, and quorum sensing (QS).
Biofilm is a complex aggregation of microorganisms, wherein the cells are embedded in a self-produced matrix of extracellular polymeric substance (EPS). The new definition of a biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other and are embedded in a matrix of extracellular polymeric substances that they have produced and exhibit an altered phenotype with respect to growth rate and gene transcription.
Biofilms are important for survival of bacteria in the environment and A. baumannii cells in biofilms have been shown to have a higher resistance to acid exposure and dehydration. Some clinical strains of A. baumannii have been shown to have a high propensity to form biofilms and this has been linked to their ability to adhere to cells, including human bronchial cells. Proteomic analysis of A. baumannii biofilm cells identified several proteins that are upregulated during biofilm formation, as well as several that are expressed only in a biofilm, including a number of proteins that are involved in antibiotic resistance.,
A. baumannii can form biofilms on several abiotic surfaces, including polystyrene, polypropylene, polytetrafluoroethylene, and glass. Biofilm formation appeared to be positively correlated with multidrug resistance, as well as with the expression of several virulence factors, including the outer membrane protein A (OmpA), the extracellular polysaccharide poly-β-(1,6)-N-acetyl glucosamine (PNAG), type I pili, a homologue of the staphylococcal Bap, the Omp CarO, a QS system, and proteins involved in histidine metabolism, such as urocanase.,,,
In addition, the ability of A. baumannii to adhere and invade epithelial cells has been investigated. Important virulence factors of A. baumannii are the K1 capsular polysaccharide genes, phospholipase D production and OmpA, an Omp., Among these virulence factors, Bap has been demonstrated to play a role in adherence to epithelial cells, and OmpA and phospholipase D have been shown to contribute to invasion of epithelial cells. Biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its perseverance in the hospital environment, increasing the probability of causing nosocomial infections and outbreaks. Recently, a protein called Rec A has been identified to be involved in general stress response and resistance to heat shock and desiccation in A. baumannii.
The aggregations of bacterial cells are natural assemblages of bacteria within the biofilm matrix and it functions as a cooperative consortium, in a relatively complex and coordinated manner. Biofilm phenotype of a pathogen promotes increased colonization and persistence and therefore is the leading cause for device-related infections.
The ability of these pathogens to adhere to human tissues and medical devices to produce biofilms is a major virulence factor that causes increase in antibiotic resistance, reduced phagocytosis, and overall persistence of the bacterial population. Moreover, these biofilms are notoriously difficult to eradicate and are a source of many recalcitrant infections. The medical importance of these biofilms and their architecture is highlighted by the diversity and refractoriness of device-related infections.
| Biofilm Cycle|| |
Biofilm development mechanisms are a multistep process. Attachment to abiotic surface is mainly dependent on cell surface hydrophobicity, whereas surface proteins mediate adhesion to host matrix-covered implants. After adhesion to the surface, exopolysaccharide, specific proteins, and accessory macromolecules aid in intercellular aggregation. Further, at a critical cell density, QS occurs, which is a communication pathway by which cells coordinate resulting in biofilm formation. Further, at a later stage, due to physical forces or intercellular signaling, the cells detach and disperse to colonize new areas.
The three significant factors contributing to the persistence of A. baumannii in the hospital environment are resistance to major antimicrobial drugs, resistance to desiccation, and resistance to disinfectants. This survival property is most likely to play a significant role in the outbreaks caused by this pathogen. Resistance of A. baumannii against a wide range of antibiotics might be explained by its capability to colonize and form biofilm on both abiotic and medical devices.,,, Adherence of A. baumannii to erythrocytes and human bronchial epithelial cells is by means of pilus-like structures., This is considered to be a first step in the colonization process of A. baumannii. Outgrowth on mucosal surfaces and medical devices, such as intravascular catheters and endotracheal tubes can result in A. baumannii biofilm formation, which enhances the risk of infection of the bloodstream and airways.
Interestingly, it has also been demonstrated that biofilm formation in Acinetobacter is phenotypically associated with exopolysaccharide production and pilus formation. A chaperone-usher has been identified as a key factor in pilus and biofilm formation in a pioneer study by Tomaras et al. showing that biofilm production by A. baumannii could promote increased colonization and persistence leading to higher rates of device-related infections.
Identification of genes involved in biofilm formation is required for better understanding of molecular basis of strain variation and various pathogenic mechanisms implicated in chronic Acinetobacter infections.
| Factors Associated With Acinetobacter Baumannii Biofilm Formation|| |
PNAG, a major component of biofilm is involved in host-microbe interaction, virulence, immune evasion, and protection against antibiotics. Pga locus encodes for the proteins involved in the synthesis and translocation of PNAG on to the bacterial surface.,
A. baumannii pga A encodes for a predicted 812 amino acid Omp and it contains a porin domain suggesting that it facilitates PNAG translocation across the outer membrane and a superhelical periplasmic domain that is thought to play a role in protein-protein interaction. Pga B is made up of 510 amino acids with a putative polysaccharide deacetylase domain. It is an outer membrane lipoprotein that along with pga A, is necessary for PNAG export. Pga C encodes for a 392 amino acid N glycosyltransferase that belongs to the glycosyltransferase 2 family. Gene pga D encodes for a 150 amino acid protein which localizes in the cytoplasm and assists pga C in the synthesis of PNAG. One recent investigation speculated that in a more dynamic environment with higher shear forces, PNAG is more essential for maintaining the integrity of A. baumannii biofilms.
| Biofilm-Associated Protein|| |
Bap were first characterized in S. aureus and recent research findings indicated that Acinetobacter has a homologue of Bap protein of Staphylococcus. Bap family members are high-molecular-weight proteins present on the bacterial surface, contain a core domain of tandem repeats, and play a critical role in cell-cell interactions and biofilm maturation. Bap is made up of 8620 amino acids, arranged in tandemly repeated modules A–E. It has a higher proportion of negatively charged amino acids in the tandem repeats compared to nontandem repeat parts. As it has no transmembrane anchoring domain, its interaction with the cell wall is unclear and yet to be investigated.
The mechanism by which the Bap contributes to biofilm development is unknown, though their large size and the presence of a high number of repeats suggest that these proteins could mediate homophilic or heterophilic intercellular interactions. Structural studies suggest that the main target for Bap is carbohydrates, for maintenance of biofilm complex. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrate that Bap mutants are unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. Based on the articles reviewed, it appears that biofilm-associated protein of one class of bacteria is responsible for intracellular adhesion with neighboring organisms. Alternately, cells are linked with one another by extracellular matrix component. The possibility of using Bap as a candidate vaccine can be explored to prevent development of biofilm by organisms in device associated infections.
| Chaperone-Usher Secretion System|| |
To produce biofilm on inanimate surfaces, A. baumannii requires chaperone-usher pili. This secretion system is necessary for the initiation of biofilm formation as it encodes a putative pili-like structure/adhesion protein. There are six ORFs: csu ab-A-B-C-D-E which are clustered in the csu operon. This operon is associated with chaperone-usher pili assembly and is polycistronic in nature. The translational products of csu D and csu E are directly related to chaperone and usher bacterial proteins, respectively, and the four remaining ORFs encode hypothetical proteins potentially involved in pili assembly. The csu operon is regulated by a two-component system, bfm RS. bfmS is a sensor kinase, which senses environmental conditions and activates a response regulator encoded by bfm R. Overexpression of the csu AB operon is caused by higher bfmR intracellular concentration. Due to intensified transcription of the bfmS and bfmR genes, there is enhanced expression of the chaperone-usher secretion system, and this expression is found to cause twitching motility in A. baumannii. Further, the concurrent expression of pili and strain twitching is found to be the key factors enabling A. baumannii to easily adhere on abiotic surfaces and form biofilms.
All the above data suggest that there may be an overlap in factors required for the initiation and maturation of biofilms on abiotic and biotic surfaces, bacterial attachment, and pathogenesis in vivo. It appears that QS mechanism is responsible for the induction of several virulence factors of the organism including development of slime layer, adhesions, and optimum iron uptake for expression of toxicity.
| Quorum Sensing|| |
Many bacteria use cell-to-cell communication to monitor their population density, synchronize their behavior, and socially interact. Such communication used by the bacteria is chemical in nature and generally designated as QS, that is coordinated gene regulation. Small diffusible molecules produced by bacteria are “signals” which can reach other cells and elicit “answers.” A. baumannii predominantly produces small molecules such as acylated homoserine lactones. Sometimes, other signaling molecules such as 2-heptyl-3-hydroxy-4-quinolone and diketopiperazines are also produced by Gram-negative bacteria. When the number of cells in a population increases, the concentration of QS molecules also increases, and once the minimal threshold level crosses, the molecules are recognized by the receptors that trigger signal transduction cascades that result in a population wide change in gene expression. Such molecular cascades enable the population to survive and proliferate.
Depending on the bacterial species, the physiological processes regulated by QS are extremely diverse, ranging from maintaining the biofilms to regulating the antibiotic resistance. A flurry of research, over the past decade, has led to significant understanding of many aspects of QS molecules including their synthesis, their signal transduction process and the interaction of their receptors with the transcriptional machinery.
| Conclusion|| |
Despite studies on the characterization of the A. baumannii, problem still exists in understanding the role of Acinetobacter in causing healthcare-associated infections. Possible role of the diversity of its reservoirs, its resistance to drying, its capacity to accumulate genes and various mechanisms of antimicrobial resistance, and its capacity to cause outbreaks in the hospital are challenges associated with this organism. There is paucity of information on the virulence of the microorganism, and its reservoirs remains to be identified. Strategies to control the spread of multidrug-resistant strains have to be designed and implemented. A daunting task, however, is the challenge in treatment of infections caused by multidrug-resistant strains. An understanding of the diverse nature of A. baumannii will help in meeting some of these challenges.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii
: Emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538-82.
Alsan M, Klompas M. Acinetobacter baumannii
: An emerging and important pathogen. J Clin Outcomes Manag 2010;17:363-9.
Munoz-Price LS, Weinstein RA. Acinetobacter
infection. N Engl J Med 2008;358:1271-81.
Subramaniyan A. Profile of multidrug resistant Acinetobacter baumannii
infections among hospitalized patients. J Med Sci Clin Res 2017;5:23111-5.
Subramaniyan A, Nair S, Devi S, Joseph N, Kenchappa P, Kanungo R. Detection of various resistance mechanisms associated with Acinetobacter
infections in hospitalised patients. Indian J Basic Appl Med Res 2016;5:58-65.
Uma Karthika R, Srinivasa Rao R, Sahoo S, Shashikala P, Kanungo R, Jayachandran S, et al.
Phenotypic and genotypic assays for detecting the prevalence of metallo-beta-lactamases in clinical isolates of Acinetobacter baumannii
from a south Indian tertiary care hospital. J Med Microbiol 2009;58:430-5.
Viehman JA, Nguyen MH, Doi Y. Treatment options for carbapenem-resistant and extensively drug-resistant Acinetobacter baumannii
infections. Drugs 2014;74:1315-33.
Karageorgopoulos DE, Falagas ME. Current control and treatment of multidrug-resistant Acinetobacter baumannii
infections. Lancet Infect Dis 2008;8:751-62.
Manchanda V, Sanchaita S, Singh N. Multidrug resistant Acinetobacter
. J Glob Infect Dis 2010;2:291-304.
Chan JD, Graves JA, Dellit TH. Antimicrobial treatment and clinical outcomes of carbapenem-resistant Acinetobacter baumannii
ventilator-associated pneumonia. J Intensive Care Med 2010;25:343-8.
Beceiro A, Tomás M, Bou G. Antimicrobial resistance and virulence: A successful or deleterious association in the bacterial world? Clin Microbiol Rev 2013;26:185-230.
Donlan RM, Costerton JW. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-93.
Shin JH, Lee HW, Kim SM, Kim J. Proteomic analysis of Acinetobacter baumannii
in biofilm and planktonic growth mode. J Microbiol 2009;47:728-35.
Brossard KA, Campagnari AA. The Acinetobacter baumannii
biofilm-associated protein plays a role in adherence to human epithelial cells. Infect Immun 2012;80:228-33.
Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii
: Involvement of a novel chaperone-usher pili assembly system. Microbiology 2003;149:3473-84.
Choi AHK, Slamti L, Avci FY, Pier GB, Maira-Litrán T. The pgaABCD Locus of Acinetobacter baumannii
encodes the production of Poly-β-1-6-N-acetylglucosamine, which is critical for biofilm formation. J Bacteriol 2009;191:5953-63.
Loehfelm TW, Luke NR, Campagnari AA. Identification and characterization of an Acinetobacter baumannii
biofilm-associated protein. J Bacteriol 2008;190:1036-44.
Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, et al.
Outer membrane protein 38 of Acinetobacter baumannii
localizes to the mitochondria and induces apoptosis of epithelial cells. Cell Microbiol 2005;7:1127-38.
Eijkelkamp BA, Stroeher UH, Hassan KA, Paulsen IT, Brown MH. Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii
. BMC Genomics 2014;15:1020.
Russo TA, Luke NR, Beanan JM, Olson R, Sauberan SL, MacDonald U, et al.
The K1 capsular polysaccharide of Acinetobacter baumannii
strain 307-0294 is a major virulence factor. Infect Immun 2010;78:3993-4000.
Jawad A, Seifert H, Snelling AM, Heritage J, Hawkey PM. Survival of Acinetobacter baumannii
on dry surfaces: Comparison of outbreak and sporadic isolates. J Clin Microbiol 1998;36:1938-41.
Aranda J, Bardina C, Beceiro A, Rumbo S, Cabral MP, Barbé J, et al. Acinetobacter baumannii
recA protein in repair of DNA damage, antimicrobial resistance, general stress response, and virulence. J Bacteriol 2011;193:3740-7.
Prashanth K, Vasanth T, Saranathan R, Makki AR, Pagal S. Antibiotic resistance, biofilms and quorum sensing in Acinetobacter
species. In: Antibiotic Resistant Bacteria – A Continuous Challenge in the New Millennium. InTech; 2012. ISBN 978-953-51-0472-8.
Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: Bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev 2014;78:510-43.
Otto M. Staphylococcus epidermidis – The “accidental” pathogen. Nat Rev Microbiol 2009;7:555-67.
Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999;284:1318-22.
Dijkshoorn L, Aucken H, Gerner-Smidt P, Janssen P, Kaufmann ME, Garaizar J, et al.
Comparison of outbreak and nonoutbreak Acinetobacter baumannii
strains by genotypic and phenotypic methods. J Clin Microbiol 1996;34:1519-25.
Qi L, Li H, Zhang C, Liang B, Li J, Wang L, et al.
Relationship between antibiotic resistance, biofilm formation, and biofilm-specific resistance in Acinetobacter baumannii
. Front Microbiol 2016;7:483.
Gospodarek E, Grzanka A, Dudziak Z, Domaniewski J. Electron-microscopic observation of adherence of Acinetobacter baumannii
to red blood cells. Acta Microbiol Pol 1998;47:213-7.
Lee JC, Koerten H, van den Broek P, Beekhuizen H, Wolterbeek R, van den Barselaar M, et al.
Adherence of Acinetobacter baumannii
strains to human bronchial epithelial cells. Res Microbiol 2006;157:360-6.
Kropec A, Maira-Litran T, Jefferson KK, Grout M, Cramton SE, Götz F, et al.
Poly-N-acetylglucosamine production in Staphylococcus aureus
is essential for virulence in murine models of systemic infection. Infect Immun 2005;73:6868-76.
Vuong C, Kocianova S, Yao Y, Carmody AB, Otto M. Increased colonization of indwelling medical devices by quorum-sensing mutants of staphylococcus epidermidis in vivo
. J Infect Dis 2004;190:1498-505.
Itoh Y, Rice JD, Goller C, Pannuri A, Taylor J, Meisner J, et al.
Roles of pgaABCD genes in synthesis, modification, and export of the Escherichia coli
biofilm adhesin poly-beta-1,6-N-acetyl-D-glucosamine. J Bacteriol 2008;190:3670-80.
Cucarella C, Solano C, Valle J, Amorena B, Lasa I, Penadés JR, et al.
Bap, a Staphylococcus aureus
surface protein involved in biofilm formation. J Bacteriol 2001;183:2888-96.
Rahbar MR, Rasooli I, Mousavi Gargari SL, Amani J, Fattahian Y. In silico
analysis of antibody triggering biofilm associated protein in Acinetobacter baumannii
. J Theor Biol 2010;266:275-90.
Lasa I, Penadés JR. Bap: A family of surface proteins involved in biofilm formation. Res Microbiol 2006;157:99-107.
Gaddy JA, Actis LA. Regulation of Acinetobacter baumannii
biofilm formation. Future Microbiol 2009;4:273-8.
Luo LM, Wu LJ, Xiao YL, Zhao D, Chen ZX, Kang M, et al.
Enhancing pili assembly and biofilm formation in Acinetobacter baumannii
ATCC19606 using non-native acyl-homoserine lactones. BMC Microbiol 2015;15:62.
Smith MG, Gianoulis TA, Pukatzki S, Mekalanos JJ, Ornston LN, Gerstein M, et al.
New insights into Acinetobacter baumannii
pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev 2007;21:601-14.
Ng WL, Bassler BL. Bacterial quorum-sensing network architectures. Annu Rev Genet 2009;43:197-222.
Parsek MR, Greenberg EP. Sociomicrobiology: The connections between quorum sensing and biofilms. Trends Microbiol 2005;13:27-33.
Dou Y, Song F, Guo F, Zhou Z, Zhu C, Xiang J, et al. Acinetobacter baumannii
quorum-sensing signalling molecule induces the expression of drug-resistance genes. Mol Med Rep 2017;15:4061-8.
Holden MT, Ram Chhabra S, de Nys R, Stead P, Bainton NJ, Hill PJ, et al.
Quorum-sensing cross talk: Isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa
and other gram-negative bacteria. Mol Microbiol 1999;33:1254-66.