Biofilm structural heterogeneity visualized by three microscopic methods

162

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25°C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25°C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 m below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 m. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments. Interventions designed to deliver disinfectants to these locations or to remove viable cells of E. coli O157:H7 and other pathogens from apples by other means need to be developed and validated.

Section: Discussionmentioning

confidence: 50%

Attachment of Escherichia coli O157:H7 to the Surfaces and Internal Structures of Apples as Detected by Confocal Scanning Laser Microscopy

Burnett

Chen

Beuchat

2000

162

“…Recently, scanning confocal laser microscopy has been developed into a powerful tool for elucidating the three-dimensional architecture and species distribution of biofilm systems (5,22) and may be useful for correlating biofilm structure with interspecies relationships. In this study, a triple-labeling technique that was compatible with confocal microscopy was developed to characterize the response of a dual-species biofilm to two compounds that are commonly found in mixed organic chemical waste.…”

mentioning

confidence: 99%

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

Cowan¹,

Gilbert

Liepmann³

et al. 2000

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresoldegrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.Biofilms of environmental and medical significance frequently consist of diverse populations of microorganisms (4, 12). A range of metabolic interactions have been observed among microorganisms in biofilms, including mutualistic and commensal relationships (16,26). Moreover, metabolic interactions within biofilms may be facilitated by the spatial arrangement of interacting cells (11,19,20,27). In biofilms that detoxify mixed organic wastes, the metabolic interactions among bacteria could potentially influence biofilm structure and development, since the metabolism of complex organic pollutants often involves multispecies bacterial consortia (10, 23). For example, fluctuating toxicant concentrations could provide selective pressure that alters the species distribution in a biofilm, ultimately influencing biofilm activity.Another feature of biofilms that may respond to changing toxicant concentrations is the exopolysaccharide (EPS) matrix. EPS is an integral structural and functional component of biofilm systems (6, 24), can account for up to 90% of the organic matter in a biofilm (25), and helps protect organisms in the biofilm community from environmental stresses (1; T. R. Neu, G. Packroff, and J. R. Lawrence, Abstr. 97th Gen. Meet. Am. Soc. Microbiol. 1997, p. 396, 1997. Recent research has characterized the composition and quantity of EPS produced (17, 18), as well as its presence in natural biofilms (8). Few studies have invest...

“…Because the ribosomal content of bacteria can be correlated with the growth rate, metabolic activity in environmental bacteria can be assessed by measuring fluorescence after hybridization (32). Whole-cell in situ hybridizations have been used for the direct identification of ␤ and ␥ proteobacteria in biofilms formed on glass slides in drinking water (26) and for the examination of biofilm structure using embedded biofilm sections (36,37,39).…”

mentioning

confidence: 99%

Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine

Williams

Braun-Howland

2003

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH 2 Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH 2 Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and ␤ and ␦ proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.Assessment of the microbiological safety of drinking water is based largely on the routine monitoring of water supplies for the presence of total coliforms and Escherichia coli. Detection of E. coli is considered indicative of recent fecal contamination and of the potential presence of enteric pathogens, while the presence of total coliforms is indicative of poor water quality.Whereas routine water quality measurements assess the presence of planktonic bacteria, the vast majority of bacteria indigenous to aquatic environments exist attached to solid particles or surfaces. Within water distribution systems, significant bacterial populations exist as complex, structurally heterogeneous biofilms attached to pipe surfaces. Residence within these complex matrices provides organisms with higher localized nutrient concentrations than are commonly found in drinking waters (10,14,18,22), and recent studies have shown that attached bacteria are more metabolically active than are their free-living counterparts (26, 29). Furthermore, biofilms afford bacteria significant protection from disinfecting agents (3, 31, 35), including hypochlorous acid and monochloramine. Biofilms are dynamic ...