Biofilm formation and adherence characteristics of an Elizabethkingia meningoseptica isolate from Oreochromis mossambicus

Abstract

Background: Elizabethkingia spp. are opportunistic pathogens often found associated with intravascular device-related bacteraemias and ventilator-associated pneumonia. Their ability to exist as biofilm structures has been alluded to but not extensively investigated.

Methods: The ability of Elizabethkingia meningoseptica isolate CH2B from freshwater tilapia (Oreochromis mossambicus) and E. meningoseptica strain NCTC 10016(T) to adhere to abiotic surfaces was investigated using microtiter plate adherence assays following exposure to varying physico-chemical challenges. The role of cell-surface properties was investigated using hydrophobicity (bacterial adherence to hydrocarbons), autoaggregation and coaggregation assays. The role of extracellular components in adherence was determined using reversal or inhibition of coaggregation assays in conjunction with Listeria spp. isolates, while the role of cell-free supernatants, from diverse bacteria, in inducing enhanced adherence was investigated using microtitre plate assays. Biofilm architecture of isolate CH2B alone as well as in co-culture with Listeria monocytogenes was investigated using flow cells and microscopy.

Results: E. meningoseptica isolates CH2B and NCTC 10016(T) demonstrated stronger biofilm formation in nutrient-rich medium compared to nutrient-poor medium at both 21 and 37°C, respectively. Both isolates displayed a hydrophilic cell surface following the bacterial adherence to xylene assay. Varying autoaggregation and coaggregation indices were observed for the E. meningoseptica isolates. Coaggregation by isolate CH2B appeared to be strongest with foodborne pathogens like Enterococcus, Staphylococcus and Listeria spp. Partial inhibition of coaggregation was observed when isolate CH2B was treated with heat or protease exposure, suggesting the presence of heat-sensitive adhesins, although sugar treatment resulted in increased coaggregation and may be associated with a lactose-associated lectin or capsule-mediated attachment.

Conclusions: E. meningoseptica isolate CH2B and strain NCTC 10016(T) displayed a strong biofilm-forming phenotype which may play a role in its potential pathogenicity in both clinical and aquaculture environments. The ability of E. meningoseptica isolates to adhere to abiotic surfaces and form biofilm structures may result from the hydrophilic cell surface and multiple adhesins located around the cell.

Figure 1

Microtiter plate adherence of E. meningoseptica isolate CH2B, following exposure to cell-free spent medium supernatants from selected Gram-negative and Gram-positive bacteria, at room temperature (21°C ± 2°C) under static conditions in nutrient-rich (TSB) medium. Bars represent means ± standard deviations for three independent replicate experiments.

Figure 2

Light microscope image depicting polar attachment (arrow) of Elizabethkingia meningoseptica isolate CH2B biofilm cells to glass slide surface following 48 h of incubation in nutrient-poor (EAOB) medium (× 1000 magnification).

Figure 3

Light microscope images depicting Elizabethkingia meningoseptica isolate CH2B cells associated with the glass slide surface as a) microcolonies after 24 h and b) complex biofilm formation following 48 h of incubation in nutrient-rich (TSB) medium, respectively (× 1000 magnification).

Figure 4

Light microscope image depicting the distinctly separate adherence of Elizabethkingia meningoseptica isolate CH2B microcolonies (A) and Listeria monocytogenes NCTC 4885 cells (B) following 48 h in flow cells containing nutrient-poor (EAOB) medium (× 1000 magnification).