87

Biofilm Recalcitrance: Theories and Mechanisms

Andrew J. McBain1, Najib Sufya2, and Alexander H. Rickard3

1 School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Manchester, UK2 Microbiology and Immunology Department, Faculty of Pharmacy, University of Tripoli, Tripoli, Libya3 Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA

4

Introduction

It is now generally accepted that in the majority of non-sterile moist environments, bacterial populations develop in associa-tion with surfaces and form biofilms (for an overview see Costerton et al. [1]). Biofilms are functional consortia of micro-bial cells, enveloped within extracellular polymeric matrices, which bind the cells to the colonized surface and which further entrap microbial products such as enzymes and virulence factors [2, 3]. The biofilm matrix is visible in Figure 4.1 which shows a nascent Pseudomonas aeruginosa biofilm imaged using environmental scanning electron microscopy. Biofilms provide for a close proximity of individual cells in association with a surface, which might provide a nutrient source or may simply immobilize the community. Biofilms have been associated with many industrial, medical and environmental problems such as infections of indwelling medical devices, the fouling of pipe work and heat-exchange units, and in the bio-corrosion of materials. From a clinical perspective, they often represent the nidus of infection either to human and animal hosts or to man-ufactured products such as foods and pharmaceuticals. Biofilms are therefore most likely to be encountered in situations where their presence is undesirable, and where disinfection, preserva-tion and chemical treatments are deployed to control or

eradicate their presence. In this respect, it is particularly notable that the susceptibility of biofilms to chemical treatment agents bears little relationship to the susceptibility of the constituent organisms when in suspension (planktonic) or when simply dried onto a surface. Biofilms are often considerably less sus-ceptible than planktonic cells and this phenomenon extends to a wide range of chemically unrelated microbicides, antimicrobi-als and antibiotics (for a review of biofilm resistance, see Gilbert et al. [4]).

Early reports connecting microbial recalcitrance to the forma-tion of biofilms were published in the early 1980s [5–8]. This period was associated with increased use of implanted medical devices and presumably with correspondingly increased surgical site infection rates. In many instances, following implantation of the device, infections were reported that were apparently respon-sive to antibiotic treatment but which recurred after the treatment was stopped. Such infections could often only be resolved when the implanted devices were removed. This remains the case today. In a number of microbiological investigations, the removed devices were found to harbor the implicated pathogens growing as a biofilm [9, 10]. In the 1980s, examples of bacterial “resistance” towards microbicide treatments of inanimate surfaces were also being attributed to the biofilm mode of growth [11]. The range of antimicrobial molecules to which biofilm populations were (and still are) recalcitrant was considerable, and included all of

Russell, Hugo & Ayliffe’s: Principles and Practice of Disinfection, Preservation and Sterilization, Fifth Edition. Edited by Adam P. Fraise, Jean-Yves Maillard,

and Syed A. Sattar.

© 2013 Blackwell Publishing Ltd. Published 2013 by Blackwell Publishing Ltd.

Introduction, 87The matrix, 88Cellular phenotype in biofilm communities

as a moderator of recalcitrance, 89

Biofilms as highly selective environments, 91Conclusions, 92Acknowledgments, 92References, 92

Section 1 Principles

88

their extracellular products. Indeed, early studies of antibiotic action against biofilms attributed their ineffectiveness to the pres-ence of a diffusion barrier [2, 14]. Such explanations have since fallen out of favor because reductions in the diffusion coefficients of antibiotics such as tobramycin and cefsulodin, within biofilms or microcolonies, are insufficient to account for the observed changes in their activity [15]. Even if such changes in diffusivity were large, then they would only delay the achievement of an equilibrium. Ultimately, the concentration of antimicrobial in the bulk fluid phase would equal that at the biofilm – substratum interface. In order for the matrix to protect the enveloped cells, it would be necessary for it to interact with the treatment agent.

Biofilm matrix as an interactive barrier to antibacterial penetrationRetardation of the diffusion of antimicrobial agents therefore alone is insufficient to account for the reduced susceptibility of biofilms. For potent microbicides such as QACs and biguanides, however, or for chemically reactive compounds such as halogens and peroxygens, chemical quenching within the matrix during diffusion may occur. This can either be by adsorption to the charged matrix [16] or by chemical reactions that quench the agent [17, 18]. Whether or not the exopolymeric matrix consti-tutes a physical barrier to the antimicrobial depends upon the nature of the applied agent, the binding capacity of the polymeric matrix, the levels of agent deployed [19], the distribution of biomass and local hydrodynamics [20], together with the rate of turnover relative to antimicrobial diffusion rate [21]. This effect may be more pronounced for charged antibiotics such as the aminoglycosides [22] and cationic microbicides [23]. With chem-ically reactive agents such as halogens, aldehydes and peroxygens, the reaction capacity of the matrix is considerable [24]. If the underlying cells were to be protected through such adsorptive losses, the number of adsorption sites must however be sufficient to deplete the available microbicide within the treatment phase, which will often not be the case.

Enzyme-mediated reaction – diffusion resistanceIn certain cases, the biofilm matrix can contain extracellular enzymes that are capable of degrading a diffusing antimicrobial. If this occurs, reaction – diffusion limitation will be enhanced. In this respect, enzymatic reaction may exacerbate antibiotic pene-tration failure [25], provided that the turnover of substrate by the enzyme is sufficiently rapid. In such respects it is significant that the expression of hydrolytic enzymes, such as β-lactamase, is reportedly induced in adherent populations and in those exposed to sublethal concentrations of β-lactam antibiotics [26]. These enzymes may become trapped and concentrated within the biofilm matrix and are able to further impede the penetration and action of susceptible antibiotics. In taxonomically diverse biofilms, the production of neutralizing enzymes by one species may confer protection upon the remainder [27, 28]. With respect to antibacterial agents used as disinfectants and preservatives, it is notable that the production of aldehyde lyase and aldehyde

the clinically deployed antibiotics and microbicides such as the isothiazolones, halogens, quaternary ammonium compounds (QACs), biguanides and phenolic compounds. The breadth of treatment agents to which biofilms were resistant encouraged intuitively correct, but simplistic, explanations of the responsible mechanisms. Biofilms are enveloped within extensive extracellular polymeric matrices that cement the cells together and trap mol-ecules and ions of environmental and microbial origin [12, 13]. It was thus not surprising that the original hypotheses implicated the matrix and suggested that biofilm communities were impervi-ous because deep-lying cells that survived had evaded exposure.

The matrix

The matrix is heterogeneous, varying in hydration and chemical composition. Organisms within the matrix may be held in close proximity to their congeners. Adsorption sites within the matrices serve to anchor extracellular enzymes from the producer organ-isms, and will also actively concentrate metabolites from the com-munity and ionic materials from the bulk fluid phase. Bound extracellular enzymes may mobilize complex nutrients captured from the fluid phase and may degrade antibacterial substances. The matrix is therefore able to moderate the microenvironments of each of the individual community members.

Biofilm matrix as a barrier to antibacterial agentsMany of the resistance characteristics of biofilm communities are lost when component cells are resuspended and separated from

Figure 4.1 Early stages of biofilm formation of Pseudomonas aeruginosa. The biofilm was imaged using environmental scanning electron microscopy. Ruthenium red stain was used to improve the visualization of the biofilm matrix. The outline of individual cells can be seen (a), as can the enveloping matrix (b). A 20 µm scale bar is shown at the foot of the figure.

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Chapter 4 Biofilm Recalcitrance: Theories and Mechanisms

growth-rate controlled biofilms, and planktonic cells, grown at the same growth rate, possessed similar susceptibilities. In such instances, however, when intact biofilms were tested, the suscep-tibility was decreased from that of planktonic and resuspended biofilm cells. This strongly suggests that some resistance proper-ties are associated with the organization of the cells within an exopolymeric matrix.

Neither the generation of chemical gradients nor physiological gradients provides a complete explanation of the observed resist-ance of biofilm communities. Stewart [42] has developed math-ematical models that incorporate the concepts of metabolism- driven oxygen gradients, growth rate-dependent killing and reaction – diffusion properties of the matrix to explain the insus-ceptibility of S. epidermidis biofilms towards various antibiotics. His model predicted the reductions in susceptibility within thick biofilms through depletion of oxygen. Since nutrient and gaseous gradients increase in extent as biofilms thicken and mature, then growth rate effects become more evident in matured bio-films [43, 44]. This probably accounts for reports that aged biofilms are more recalcitrant to microbicide treatments than younger ones [43].

The contribution of reduced growth rate towards resistance is substantial within biofilm communities. As with reaction – dif-fusion limitation, however, this does not satisfactorily explain the totality of the observed resistance [45]. Physiological gradients depend upon growth and metabolism by cells on the periphery to deplete the nutrients and oxygen as they diffuse towards the more deeply located cells. Peripheral cells will therefore have growth rates and nutrient profiles that are similar to those in the planktonic phase. Consequently, these cells may be relatively sen-sitive to disinfectants and will quickly succumb. Lysis products from such cells will feed survivors within the depths of the biofilm which would, as a consequence, step up their metabolism and growth rate, adopt a more susceptible phenotype, and die [46]. This phenomenon could occur throughout the biofilm, proceed-ing inwards from the outside, until the biofilm was completely killed. Should the antimicrobial agent become depleted from the bulk phase, the biofilm could then re-establish almost as quickly as it was destroyed because of the local abundance of trapped nutrients. Growth rate-related processes might therefore delay the onset of killing in the recesses of a biofilm, but would not nor-mally confer resistance against sustained exposure to antimicro-bial agents.

Reaction – diffusion limitation of the access of agent and the existence of physiological gradients within biofilms therefore provide partial explanations for their reduced susceptibility, but neither explanation, separately or in combination, can fully explain the observation of sustained resistance towards a diverse array of treatment agents. In order for such resistance to be displayed, the biofilm population must either contain cells with uniquely insusceptible phenotypes, or the short-term survivors must adapt to a resistant phenotype during the window of opportunity provided by the buffering effects of diffusion and growth rate (i.e. a rapid response to sublethal treatment).

dehydrogenase enzymes can provide such protection against for-maldehyde and glutaraldehyde [24, 29].

With the exception of the specific examples given above, which involve the presence of drug-inactivating enzymes, the invocation of matrix properties has generally proven to be insufficient to explain the whole panoply of resistance displayed by the biofilm communities [30, 31]. Accordingly, physiological changes in the biofilm cells – mediated through the induction of slow growth rates and starvation responses, together with the induction of separate attachment-specific, drug-resistant physiologies have been considered as further potential mediators of biofilm resist-ance [4].

Cellular phenotype in biofilm communities as a moderator of recalcitrance

Independent of the formation of a biofilm, the susceptibility of bacterial cells towards antibiotics and microbicides may be sig-nificantly affected by the physiological status of the individual cells, which together comprise the biofilm [30, 31]. Numerous phenotypes may be represented within a biofilm, reflecting the chemical heterogeneity of the matrix and the imposition, through cellular metabolism, of chemical, electrochemical, nutrient and gaseous gradients. Changes in antimicrobial susceptibility through alterations in phenotype may relate to growth rate-dependent changes in a variety of cellular components (including membrane fatty acids, phospholipids and envelope proteins) [32] and the production of extracellular enzymes [33] and polysaccharides [34]. Gradients of growth rate and the manifestation of pheno-typic mosaics have been observed [35] and these contribute towards recalcitrance [31, 36, 37]. Within monoculture biofilms, the established physiological gradients are often non-uniform, with pockets of very slow-growing cells juxtaposed with relatively fast-growing areas [38].

A number of studies have associated the interdependence of growth rate and nutrient limitation in biofilms with their anti-microbial susceptibility [39]. These include calculated ratios of isoeffective concentration (growth inhibition and bactericidal activity) for biofilms and planktonic bacteria grown in broth or on catheter disks. In one such study, the calculated ratios corre-lated with those generated between non-growing and actively growing cultures. With the exception of ciprofloxacin, the antibi-otic agents that were most effective against non-growing cultures (i.e. imipenem, meropenem) were also the most active against these biofilms. Other investigators have used perfused biofilm fermenters [40] to directly control and study the effects of growth rate within biofilms. Control populations of planktonic cells were generated in chemostats enabling the separate contributions of growth rate, and association within a biofilm, to be evaluated. Using this approach, decreased susceptibility of Staphylococcus epidermidis to ciprofloxacin [41] and Escherichia coli to cetrimide [36] and tobramycin [37] could be explained to a large extent in terms of specific growth rates, in that cells resuspended from

Section 1 Principles

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ciprofloxacin and observed little or no difference between wild-type and mar-deleted strains [59, 60]. Similar experiments using biofilms constructed from strains in which the efflux pump gene acrAB was either deleted or constitutively expressed showed the acrAB deletion to not significantly affect susceptibility over that of the wild-type strain [61]. Constitutive expression of acrAB protected the biofilm against low concentrations of ciprofloxacin. Studies conducted in continuous culture with a lacZ reporter gene fused to marOII, showed mar expression to be inversely related to specific growth rate [61]. Thus, following exposure of biofilms to sublethal levels of β-lactams, tetracyclines and salicylates, mar expression will be greatest within the depths of the biofilm, where growth rates are suppressed, and might account for the long-term survival of the community when exposed to inducer molecules.

Another study of efflux in biofilms showed that expression of the major multidrug efflux pumps of P. aeruginosa actually decreased as the biofilm developed. Although expression was greatest in the depths of the biofilm, experiments with deletion mutants showed that none of the multidrug efflux pumps were contributing to the general increased resistance to antibiotics exhibited by the biofilm [61].

Quiescence and persistenceThere has been much speculation concerning the ability of non-sporulating bacteria to adopt spore-like qualities while in a qui-escent state. Specific growth rates of such cells approach zero [62] as they undergo reductive divisions in order to complete the segregation of initiated rounds of chromosome replication [62, 63]. Quiescence, as such, is commonly associated with popula-tions that have undergone an extended period of starvation, and has most commonly been associated with bacteria found in oli-gotrophic marine environments [64, 65]. They are, however, also likely to be the dominant forms of bacteria within environments of naturally low nutrient availability. While mainly associated with aquatic Gram-negative bacteria, quiescence has been reported in Gram-positive species [66] and would appear to be a generically widespread response to extreme nutrient stress [67]. This has been termed the general stress response (GSR) and leads to populations of cells that synthesize the highly phosphorylated nucleotides ppApp and pppApp [68] and that, as a consequence, become resistant to a wide range of physical and chemical agents [69]. The GSR is now thought to account for much of the resistance observed in stationary phase cultures. Various terms have been adopted to describe such phenotypes, including “resting” [70], “quiescent” [71], “ultra-microbacteria” [63], “dormant” and “somnicells” [72]. It is also possible that the same or similar phe-nomenon describes the state of viable-but-non-culturable (VBNC) cells [73] since such bacterial cells often fail to produce colonies when transferred directly onto nutrient-rich agar. Indeed, when bacteria are collected and plated from oligotrophic environ-ments then there is often a great disparity between the viable and total cell counts obtained [72, 74]. Even within biofilms that are in eutrophic environments, microenvironments exist where nutri-ents are scarce or even absent. Under such circumstances a small

Drug-resistant phenotypesThe diversity of agents to which biofilms are recalcitrant, together with the long-term survival of biofilms in the face of vast excesses of treatment agent, make it likely that other processes are occur-ring in parallel to the establishment of chemical and physiological gradients. The long-term survival of biofilm populations is often associated with short-term losses in viability of several orders of magnitude. Survival might therefore be related to the presence of a small fraction of the population that expresses highly recalci-trant physiologies. The following sections will consider these, including the hyperexpression of efflux pumps and those induced by exposure to alarmone-signal molecules and dormant, quies-cent or persister cells.

Efflux pumpsAn increasingly studied resistance mechanism is the expression of multidrug efflux pumps [47]. In Gram-negative and Gram-positive bacteria the expression of such pumps may be induced through sublethal exposure to a broad range of agents [48]. These agents include antibiotics and other xenobiotics such as pine oil, salicylate and QACs [49]. Efflux pumps operate in many Gram-negative organisms and may be plasmid or chromosomally encoded [47]. In addition, multidrug efflux pumps such as QacA-G also contribute to microbicide tolerance in Gram-positive bacteria such as Staphylococcus aureus [50]. Sublethal exposure to many antimicrobials of P. aeruginosa MexAB efflux-deleted mutants can select for cells that hyperexpress the alternate efflux pump MexCD [51]. This highlights the multiple redundan-cies of efflux genes and their highly conserved nature.

Several attempts have been made to group efflux pumps into families [52] and to predict the structure and function of the proteins themselves [53]. Four superfamilies of efflux pumps have been recognized [54]. Although the families share no significant sequence identity, substrate specificity is often shared between them [55]. All the efflux superfamilies, however, contain pumps of varying specificity. An efflux pump may therefore primarily mediate the expulsion of endogenous metabolites or, alterna-tively, may primarily mediate the efflux of chemotherapeutic agents. Indeed, it is probable that these exporters were originally developed to expel endogenous metabolites.

Notable among the multidrug resistance operons is mar [48, 56]. The mar locus of E. coli regulates the AcrAB efflux pump and was the first mechanism found to be involved in the chromosoma-lly encoded intrinsic resistance of Gram-negative bacteria to multiple drugs. Homologs have since been described in many Gram-negative bacteria. Moken et al. [57] and McMurry et al. [58] have shown that mutations causing overexpression of MarA or AcrAB are associated with exposure and reduced susceptibility towards a wide range of chemicals and antibiotics. If mar or other efflux systems were induced by growth as a biofilm per se, then this generalized efflux of toxic agents could provide an explana-tion of the ubiquitous observation of biofilm recalcitrance. Maira-Litran et al. [59], however, used perfused biofilm ferment-ers to grow E. coli strains for 48 h with various concentrations of

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[80] presented susceptibility data for four different antimicrobial agents and P. aeruginosa (a very common test bacterium in biofilm studies) and suggested that biofilm recalcitrance is a function of slow growth rate and persister cells. This turned out to be a significant hypothesis and persisters now form an impor-tant part of most explanations of biofilm recalcitrance. The hypothesis suggests that biofilm recalcitrance often represents a Pyrrhic victory. This is because the majority of cells are inacti-vated, but the persister fraction remains to renew the biofilm once the inimical stress has been removed. It is proposed that the per-sisters represent a particularly effective survival mechanism when expressed within biofilms, since biofilms protect the persisters from phagocytes, predation, and physical/chemical stresses in other environments (Figure 4.2).

Biofilms as highly selective environments

Bacterial populations are genetically diverse and a sustained exposure to environmental stress will lead to an expansion of the most well-adapted genotype/phenotype. This is particularly the case for taxonomically diverse biofilms. The exposure of a popu-lation to sublethal concentrations of microbicides or antibiotics may therefore enrich the least susceptible clones. Equally, death and lysis of a subset of cells might lead to the transfer of resistance properties to the survivors.

It is known that pure cultures of bacteria can be “trained” to become less susceptible to antibiotics [81] and microbicides [82]. In such experiments, cultures are either grown in liquid media that contain concentrations of agent that are below the minimum inhibitory concentration (MIC) or they are streaked on to gradient plates that incorporate the agent. At each step in the process, the MIC is determined and the process repeated. Thus, it is relatively easy to select for populations of bacteria that have significantly reduced MIC values towards the selected agents. In some instances the changes in MIC are enough to render the cells resistant to normal treatment regimes. Where groups of agents have common biochemical targets, it is possible for selection by one agent to confer cross-resistance to a third party agent [83]. Such “resistance training” has for many years been regarded an artifact, since it is difficult to imagine a set of circumstances in the environment where bacteria will be exposed to gradually increas-ing concentrations of an inhibitory agent over a prolonged period (see Chapter 6.1). The repeated, sublethal treatment of biofilms in the environment and in infection, however, provides one situ-ation where this might happen [46] as sublethal levels of agent will be sustained within the deeper recesses of the community.

As with any process involving changes in susceptibility to inimical agents, the nature of the genotype/phenotype selected reflects changes in the microbicidal/inhibitory targets, the adop-tion of alternate physiologies that circumvent the target, or to changes in drug access. The latter might be through modifica-tions in the cell envelope [30, 31] or it might reflect active efflux mechanisms [84]. There may be a fitness cost associated with

proportion of the cells present within a mature biofilm may be expressing the GSR regulator and will therefore be relatively recal-citrant to inimical treatments. Similar mechanisms have been proposed for the hostile take-over of batch cultures by killer phe-notypes during the stationary phase [75] induced as part of the GSR in E. coli. This can lead to a phenotype that is not only more competitive in its growth than non-stressed cells, but which can also directly bring about the death of non-stressed ones [75].

The RpoS-encoded sigma factor sigmas is a central regulator in a complex of network of genes moderating the stationary phase in E. coli [69]. In Pseudomonas aeruginosa it appears that two sigma factors, RpoS and AlgU, and the density-dependent cell–cell signaling systems orchestrate such responses [76]. Indeed, there is a hierarchical link between n-acyl homoserine lactones and RpoS expression [77] that might specifically induce the qui-escent state at locations within a biofilm where signals accumulate and where nutrients are most scarce. In such respects it was dem-onstrated by Foley et al. [76] that the GSR response regulator RpoS was highly expressed in all of 19 P. aeruginosa-infected sputum samples taken from cystic fibrosis patients.

The persister hypothesis helps to fill some of the previously unexplained aspects of biofilm recalcitrance and is based on observations that were published over 60 years ago by Joseph Bigger in the Lancet [78]. Bigger demonstrated that although penicillin was bactericidal for Staphylococcus pyogenes, it did not eliminate S. pyogenes in the laboratory or in the patient’s serum. This was described from the presence of “cocci which are called persisters at a frequency of approximately one per million of the staphylococci originally present and for which penicillin is bacte-riostatic and only very slowly, if at all, bactericidal”. It has subse-quently been realized that persisters account for the tailing seen in many bacterial kill curves, which is often erroneously attrib-uted to the antibiotic being expended. Bigger was ahead of his time (or alternatively, biofilm research has been slow off the mark) when he suggested that persisters were dormant forms (and thus indifferent to penicillin) and that they account for the fact that bacteria can be isolated from the pus of penicillin-treated patients, after treatment with penicillinase. Importantly, he sug-gested that failure to cure staphylococcal infections in man with penicillin could be due “to the presence in the body of persisters” [78]. Another important research article was published in the Journal of General Microbiology in 1964 by Gunnison et al. [79]. This made similar observations for S. aureus, again with penicil-lin, and discounted the possibility that persister cells were the result of stable genetic alteration. The recalcitrance of persisters was clearly demonstrated in this report where the proportion of survivors was not changed even by a 1000-fold increase in the dose of penicillin, or the addition of streptomycin. The Gunnison paper reported that persister cells were in a state unfavorable to the initiation of division or cell wall synthesis (i.e. they were drug indifferent).

Continued reports have appeared in the literature relating to bacterial persistence until a firm link between persistence and biofilm resistance was proposed. A paper by Spoering and Lewis

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lying members of the biofilm community are exposed. These cells are typically starved or slow growing, and express stress-associated phenotypes that may include the expression or upregulation of efflux pumps. The phenotype of the deeply seated biofilm com-munity reduces their susceptibility to the treatment agents and exacerbates the likelihood of being exposed to sublethal concen-trations of antimicrobial agent. Deeper-lying cells will often out-survive those at the surface and can proliferate once the bulk of the treatment agent is depleted or if the exposure is only transient. Thus, at the fringes of antimicrobial action, selection pressures may enrich the populations with the least susceptible genotype. It is possible under such conditions for repeated chronic exposure to sublethal treatments to select for a more resistant population. Alternative explanations for the recalcitrance of biofilm commu-nities include the expression of biofilm-specific phenotypes that may be significantly less susceptible than planktonic cells, and the physical protection that the matrix confers on persister cells, which are recalcitrant through metabolic inactivity.

Acknowledgments

The authors would like to acknowledge the substantial contribu-tions of their friend and colleague, Peter Gilbert (1951–2008).

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Figure 4.2 Major mechanisms of biofilm recalcitrance as currently understood, showing: (i) poor antimicrobial penetration; and (ii) gradients of nutrients and oxygen leading to variations in growth rate and other phenotypic variability (including stress-specific phenotypes and dormancy and persisters).

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