Transmission of Yesinia pestis relies primarily on the bite of flea vectors, which is closely related to the biofilm formation of this pathogen[1]. Y. pestis synthesizes heavily the attached biofilms, a population of bacterial colonies embedded in self-produced exopolysaccharide matrix, in the flea proventriculus[1]. The biofilm formation leads to the blockage of fleas, making the fleas feel hungry and repeatedly attempt to feed, and thus the plague bacilli pump into the host body during these futile feeding attempts[1]. Detection of biofilm formation is an important phenotypic measurement for the potential of flea-borne transmission of Y. methods are currently used for detecting Yersinia biofilms[2-5]. However, considerable variations exist for the same method used in the different existing reports, and the experimental protocols are often very brief. The purpose of this study is to optimize the commonly used biofilm detection methods in Y. pestis. The wild-type (WT)Y.pestis used is the biovar Microtus strain 201[6]. The base pairs 146 to 468 of biofilm-required gene hmsS[7] were replaced with the kanamycin resistance cassette[8], generating the hmsS mutant ΔhmsS. The recombinant plasmid pACYC184, which contained the cloned fragment composed of the hmsS coding region together with its 372 bp upstream region and its 233 bp downstream region,was introduced into ΔhmsS, yielding the complemented mutant strain C-hmsS[8]. The incubation temperature of 26 °C was employed for the Y. pestis cultivation. A two-round passage design was employed to prepare the Y. pestis liquid seeds:firstly, the Y. pestis glyceric stock was inoculated into 18 mL of BactoTM Brain Heart Infusion (BHI)or Luria-Bertani (LB)broth for growing with shaking at 230 r/min for 48 h to enter the stationary growth phase; secondly, the resulting cell culture was 20-fold diluted into 18 mL of corresponding fresh BHI or LB, and grew at 230 r/min to reach the mid-exponential phase (an OD620 value of 1.0 to 1.2).

Crystal violet (CV)staining of biofilms The BHI seeds were incubated at 4 °C for 8 to 12 h for cold shock, 20-fold diluted into fresh BHI,transferred into the 24-well tissue culture plates with 1 mL of cultures in each well, and grew at 100 or 230 r/min for 24 or 48 h. The media containing the planktonic cells were removed for determining the OD620 values for the normalization to avoid the effect of growth rate or cell density. The tube with the adherent biofilms was gently washed three times with 2 mL of H2O, and then incubated at 80 °C for 15 min for fixing the attached cells. The attached cells were stained with 2 mL of 0.1% crystal violet for 15 min, and then the tube was washed three times with 2 mL of H2O. Bound dye in the tube was dissolved with 2 mL of dimethylsulfoxide (DMSO).The OD570 values were recorded, and the relative biofilm formation was calculated with the formula:250×OD570/OD620.

We compared the effects of cold-shocked and 26 °C-grown Y. pestis (WT)seeds for inoculation; the cold shock led to much steadier attachment of biofilms, and in contrast the attached biofilms made by the 26 °C-grown seeds fell off from the walls easily during the washing process (data not shown).Therefore, the cold-shocked seeds were routinely employed for the inoculation. After the inoculation,the bacterial strains WT, ΔhmsS, and C-hmsS were incubated in the plates at various rotational speeds(100 and 230 rpm)for different times (24 and 48 h).As shown in Figure 1a, the bacterial growth at 230 rpm for 24 h showed the most stable results for all three strains: compared to WT and C-hmsS with the similar levels of biofilm production, ΔhmsS stained almost no crystal violet (the hmsS deletion abolished the biofilm production[7]). Incubation with a longer time of 48 h or at a lower rotational speed of 100 r/min resulted in the unsteady attachment of biofilms, as characterized by unstable CV staining(Figure 1b to 1d). Therefore, the inoculation of cold-shocked bacterial seeds followed by further incubation at 230 rpm for 24 h was recommended for the CV staining assay.

Caenorhabditis elegans biofilm assays To prepare the nematode eggs, overnight culture of biofilm-negative Escherichia coli OP50 was spread onto an the NGM (Nematode Growth Medium)agar plates, and grew for 24 h to prepare the OP50 lawns as the standard foods for C. elegans. Adult nematode hermaphrodites were placed on the OP50 lawn, and were incubated at 20 °C for about 3 d. The gravid adults and eggs on the lawn were collected in 2 mL of M9 buffer, followed by twice washes with 1 mL of M9 with centrifugation at 1 300×g for 1 min. The washed gravid adults and eggs were re-suspended in 500 μL of various concentrations of sodium hypochlorite (Table 1),and then incubated at room temperature for 10 min with the intermittent up-down reversals to sterilize the residual E. coli and meanwhile to lyze the nematode adults from which the eggs would minimum concentration of 7% was needed to fully kill the residual E. coli; the yield of nematode eggs enhanced with the increase of concentrations from 1% to 7% (the detecting quantity of nematode eggs were 70±34, 127±30, 213±42, and 228±40 for 1%,3%, 5%, and 7%, respectively; assays were performed in triplicate), but a concentration of 9%greatly reduced the yield (98±8). Accordingly, 7%sodium hypochlorite was routinely used.