Introduction {#s1}
============

Phenotype MicroArrays (PM) technology provides a simple tool for testing microbial cells [@pone.0056545-Bochner1], [@pone.0056545-Bochner2], [@pone.0056545-Bochner3] as well as mammalian cells [@pone.0056545-Bochner4] under hundreds or thousands of culture conditions. In 2009, Gardiner and colleagues [@pone.0056545-Gardiner1] reported on the use of PM to examine the effect of many culture condition variables on toxin production by the pathogenic fungus, *Fusarium graminearum*. This fungus is a major pathogen on wheat, with severe agricultural and commercial impact. Over many decades of study, no one was able to find *in vitro* culture conditions that would turn on synthesis of the *F. graminearum* trichothecene mycotoxin. However, the use of PM technology provided the breakthrough, indicating that strong toxin induction could be obtained *in vitro* by simply culturing the fungus with arginine, putrescine, agmatine, or guanine as the nitrogen source. Secondarily they found and confirmed that pH 4.5 produced additional induction [@pone.0056545-Gardiner2].

A different novel approach was taken by Singh [@pone.0056545-Singh1] from the Natural Products Discovery Group at Wyeth Pharmaceuticals, who studied substrate utilization effects on secondary metabolite production in fungal strains with promising commercial potential. He used the 95 substrates of the FF MicroPlate combined with scaled-down LC-MS to quantitatively profile the secondary metabolites directly from the microwell culture supernatants. Singh showed this to be a promising approach for both characterization and optimization of secondary metabolite production by fungi.

To expand upon and generalize these works, we have undertaken a study of bacterial toxin induction and repression using an important human pathogenic bacterium and incorporating a new and generally applicable toxin detection method.

In 1978, *Clostridium difficile*, a Gram-positive, spore-forming anaerobic bacillus, was identified as a gastrointestinal pathogen that frequently causes diarrhea and more seriously pseudomembranous colitis in patients undergoing antibiotic treatment [@pone.0056545-George1], [@pone.0056545-Aronsson1], [@pone.0056545-Bartlett1]. Besides diarrhea, the symptoms of *C. difficile* infection (CDI) include abdominal pain, fever, loss of appetite, nausea, toxic megacolon, and even perforations of the colon and sepsis. Death occurred occasionally. With the emergence of hypervirulent strains, the mortality rate of CDI has risen dramatically. Among serious cases, 15,000--20,000 patients die annually from CDI in the United States [@pone.0056545-Rupnik1]. This bacterium is also an important animal pathogen [@pone.0056545-Songer1].

*C. difficile* is a genetically diverse species with a highly dynamic genome that seems to be evolving rapidly [@pone.0056545-He1], [@pone.0056545-Stabler1], [@pone.0056545-Stabler2], [@pone.0056545-Stabler3], [@pone.0056545-Sebaihia1]. This genetic diversity may be the result of horizontal gene transfer, point mutations, inversions, and large-scale recombination of core chromosomal regions over considerable phylogenetic distance [@pone.0056545-He1], [@pone.0056545-Stabler1], [@pone.0056545-Stabler2], [@pone.0056545-Stabler3]. Disease-causing isolates have arisen not from a single lineage but multiple lineages, suggesting that virulence evolved independently in multiple highly epidemic lineages [@pone.0056545-He1]. These recent findings have provided invaluable insights and significantly advanced our understanding of *C. difficile* pathogenesis and epidemiology.

During the past decade, the prevalence and severity of CDI has increased dramatically worldwide [@pone.0056545-Rupnik1], [@pone.0056545-Warny1], [@pone.0056545-McDonald1], [@pone.0056545-Tae1], [@pone.0056545-Lim1], [@pone.0056545-Loo1]. The emerging epidemic of "hypervirulent" isolates represented by ribotype 027 (also called BI/NAP1/027), which are variant strains of toxinotype III, have been identified as a major culprit in hospital or hospital associated CDI outbreaks [@pone.0056545-Rupnik1]. Comparative genomic analyses showed that the epidemic 027 strains have gained 234 additional genes during the past two decades, which may account for their epidemic proficiency and their higher case-fatality ratio [@pone.0056545-Stabler2], [@pone.0056545-Stabler3].

Nevertheless, the central issue in the pathogenesis of *C. difficle* is its major virulence factors, which have long been linked to the two large toxins, A and B. The cause-effect relationship between the toxins and the pathological changes they engender in animal cells, the cytopathic effects (CPE), have been shown to be due to inactivation of Rho-GTPase through glucosylation by the toxins [@pone.0056545-Just1], [@pone.0056545-Gerhard1], [@pone.0056545-Just2], [@pone.0056545-Just3]. The essential roles the toxins play in *C. difficile* pathogenesis have also been demonstrated in multiple animal models [@pone.0056545-Kuehne1], [@pone.0056545-Czuprynski1], [@pone.0056545-Chang1], [@pone.0056545-Knoop1], [@pone.0056545-Lyerly1], [@pone.0056545-Bongaerts1] and in clinical settings [@pone.0056545-Kyne1], [@pone.0056545-Leav1]. Antibodies against toxins A and B as a supplemental treatment to antibiotic regimens have been shown to reduce recurrence of CDI in patients [@pone.0056545-Lowy1], [@pone.0056545-Hussack1] and to protect intoxicated animals [@pone.0056545-Hussack1]. Identification of *C. difficile* toxin A or B in patients\' diarrheal stool is critical and required for diagnosis of CDI [@pone.0056545-Ananthakrishnan1]. The quality and quantity of the toxins are directly or indirectly determined or regulated by multiple factors such as genetic, environmental, nutritional, and metabolic status. Therefore, monitoring functional toxin production is fundamental in studies of pathogenesis and epidemiology as well as in clinical diagnosis and treatment of CDI.

Cell-based cytotoxicity assay (CCTA) is traditionally regarded as the gold standard assay for *C. difficile* cytotoxin and serves as the reference for other toxin assay methods [@pone.0056545-Planche1]. This assay looks for toxin induced CPE by microscopic detection of a shift from normal to "rounded" morphology using a toxin-sensitive adherent mammalian cell line (an indicator cell, e.g., CHO, Vero, HT-29, foreskin or others) and then verifies that the CPE is prevented by a specific toxin-neutralizing antibody. This gold standard assay is a true test for functional cytotoxin regardless of whether the DNA coding sequence of the toxin or the sequences of regulatory proteins are mutated. Given that *C. difficile* has an extremely dynamic genome [@pone.0056545-He1], [@pone.0056545-Stabler1], [@pone.0056545-Stabler2], [@pone.0056545-Stabler3], [@pone.0056545-Sebaihia1], it is critical to have a reference assay that directly tests the toxin\'s true biological activity. Evidence has shown that, in addition to other factors, virulence is dependent on the autoactivation of a toxin cysteine protease [@pone.0056545-Egerer1], [@pone.0056545-Egerer2], [@pone.0056545-Satchell1], [@pone.0056545-Puri1]. It is also dependent on the ability of the infected host cell to S-nitrosylate *C. difficile* toxins, which attenuates virulence by inhibiting toxin self-cleavage and cell entry [@pone.0056545-Savidge1]. Toxin B from hypervirulent strains (TcdB~HV~) undergoes acid-induced conformational changes at a pH much higher than that of toxin B from historical strains (TcdB~HIST~), which makes TcdB~HV~ enter the cell more rapidly [@pone.0056545-Lanis1]. Further, TcdB~Hv~ is autoprocessed more efficiently than TcdB~HIST~ [@pone.0056545-Lanis2], which may explain why TcdB~HV~ causes increased cytotoxicity. Understandably, such functional differences of the toxin may not be detected by PCR-based toxin assays, or by ELISA- or Western Blot-based assays which do not necessarily measure toxin function. However, CCTA can detect differences that other methods can not because it is a direct functional and phenotypic assay of toxins.

Although authoritative, traditional CCTA also has some pitfalls, including laborious and time consuming steps (usually 2--3 days for direct toxin assay of a faecal sample, not including the culturing step), difficulties in quantitation due to subjective interpretation (grading cell rounding), and requirements for tissue culture facilities and well trained technical staff [@pone.0056545-Wilkins1]. As traditional CCTA is not highly standardized, interpretation of the cytotoxic or cell-rounding activity will be variable [@pone.0056545-Wilkins1]. For example, some consider 50% cell rounding a positive reaction [@pone.0056545-Bouvet1], [@pone.0056545-Florin1] while others consider 100% cell rounding a positive reaction [@pone.0056545-Kushnaryov1], making it difficult to compare the results among various laboratories and studies.

We have used a different approach to overcome the inconvenience and limitations of the traditional CCTA assay cited above. To do so, we have taken advantage of Phenotype MicroArray (PM) technology, which can now be employed with both microbial and mammalian cells. In addition to *C. difficile*, we employ a sensitive mammalian indicator cell line (CHO-k1 or Vero cells) as required by CCTA. Combining microbial and mammalian assay methodologies has allowed us to develop an efficient, reliable, highly informative, and quantitative method to measure *C. difficile* toxin production under hundreds to thousands of different culture conditions. Our new hybrid approach enables high throughput evaluation of the role of environmental factors in stimulation or repression of toxin production by *C. difficile*. Given its high sensitivity and reliability, it may potentially be used for direct clinical faecal sample measurement of the toxins. The same approach can be further generalized to study toxins or other secondary metabolites produced by microbial cells.

Here, we report on toxin production of *C. difficile* type strain ATCC 9689 under hundreds of culture conditions, including variations of basic cellular nutritional components for carbon, nitrogen, phosphorus, and sulfur (768 culture conditions, PM1--8). Also, we demonstrate that this same approach is successful with other toxin-producing clostridia as well as toxin-producing aerobic bacteria.

Results {#s2}
=======

Development of *C. difficile* toxin assays in 96-well format {#s2a}
------------------------------------------------------------

For this study, we purchased purified *C. difficile* toxins A and B which are commercially available in lyophilized form. Once the toxins are dissolved into buffer solutions, their potencies decrease rapidly with noticeable loss by the next day, even if the solutions are stored at 4°C. So, to make standard titration curves for quantification purpose, we used exclusively freshly dissolved purified toxins. We observed that the cytotoxic potency of the supernatants collected from the PM panels also decreased over time. Therefore, the assays of the toxins produced by *C. difficle* strains in PM panels (96-well format) were always carried out with fresh preparations of the supernatants, usually on the same day of collection.

Purified standard *C. difficile* toxin B (Listlab) was used to optimize and calibrate two assays. In a morphological assay the commercial toxin caused cytopathic effects (CPE), seen microscopically as cell rounding changes, in both CHO-k1 and Vero cells. The observed CPE was active in a concentration-dependent manner. The lower panel of [Figure 1A](#pone-0056545-g001){ref-type="fig"} shows an example of a toxin B titration with CHO-k1 cells. Purified toxin A (Listlab) was much less potent than toxin B against both cell lines (data not shown).

![Cytotoxicity and neutralization assay of purified *C. difficile* toxin B with CHO-k1 cell line.\
Serial 3-fold titrations of standard toxin B (ng/ml) in the presence or absence of 2.5 µg/ml neutralizing antibodies IgY specific for toxin B. (A) CHO-k1 cell morphology changed to rounded shapes by toxin B in a dose dependent manner (the lower panel). This was prevented by the neutralizing antibodies (upper panel). (B) CHO-k1 cell dye reduction was reduced by toxin B also in a dose dependent manner (the lower panel), which corresponds to the cell morphological changes. This effect was also prevented by the neutralizing antibodies (the upper panel). (C) Quantification of the dye reduction changes by toxin B, with (Red) or without (Dark blue) 2.5 µg/ml neutralizing antibodies IgY. Light blue: toxin B with control antibodies IgY-010; Purple: no toxin B with no antibodies; Green: no toxin B with control antibodies IgY-010.](pone.0056545.g001){#pone-0056545-g001}

The CHO-k1 and Vero cells could also be employed in a colorimetric assay using Biolog redox dye MB and an OmniLog instrument to quantitatively measure the degree of intoxication of the cells by the toxin. In this assay also, toxin B-treated cells showed concentration-dependent intoxication of cells that resulted in decreasing rates of color formation as the killed cells were incapable of dye reduction and the dying or injured cells were compromised in the reduction (lower panel of [Figure 1B](#pone-0056545-g001){ref-type="fig"}, [Figure 1C](#pone-0056545-g001){ref-type="fig"}, [Materials and Methods](#s4){ref-type="sec"}). Therefore, increasing cell rounding in the morphological assay was correlated with decreasing dye reduction rate in the colorimetric assay ([Figures 1A, 1B, 1C](#pone-0056545-g001){ref-type="fig"}). Neutralization of toxin B with anti-toxin B polyclonal antibodies IgY (Gallus Immunotech) provided complete protection of the indicator cells in both assays ([Figures 1A, 1B, 1C](#pone-0056545-g001){ref-type="fig"}).

The colorimetric dye reduction provided a quantitave assay of toxin over a \>3 log concentration range. From serial titrations of standard toxin B, the dye reduction rates by mammalian cells were calculated using PM Analysis Software. Regression analysis on known (prepared) concentrations of toxin B and corresponding dye reduction rates by CHO-k1 cells could be accurately fit to several regression equations over a range of serial titrations of toxin B, from 800 ng/ml (∼2963 pM) down to approximately 0.122 ng/ml (∼0.45 pM). The predicted toxin concentrations calculated from the regression equations were very close to the prepared concentrations ([Table 1](#pone-0056545-t001){ref-type="table"}).

10.1371/journal.pone.0056545.t001

###### Predicted concentrations of *C. difficile* toxin compared to the true concentrations in cell culture assay medium.

![](pone.0056545.t001){#pone-0056545-t001-1}

  Prepared toxin Conc. (ng/ml)[a](#nt101){ref-type="table-fn"}    Dye Reduction Rate (x)[b](#nt102){ref-type="table-fn"}       Regression Equation         R^2^     Predicted Toxin Conc. (ng/ml) (y)[c](#nt103){ref-type="table-fn"}
  -------------------------------------------------------------- -------------------------------------------------------- ------------------------------ --------- -------------------------------------------------------------------
  800.000                                                                                 28.715                           y = 10.488x^2^−784.98x+14692    0.998                                 799.193
  266.667                                                                                 32.365                                     *ibid.*              *ibid.*                                272.231
  88.889                                                                                  34.955                                     *ibid.*              *ibid.*                                67.808
  29.630                                                                                  35.400                               y = 7E+08x^−4.7608^         0.980                                 29.553
  9.877                                                                                   44.945                                     *ibid.*              *ibid.*                                 9.484
  3.292                                                                                   60.030                                     *ibid.*              *ibid.*                                 2.391
  1.097                                                                                   68.310                               y = 5E+62x^−34.157^         0.999                                  1.093
  0.366                                                                                   70.650                                     *ibid.*              *ibid.*                                 0.346
  0.122                                                                                   72.845                                     *ibid.*              *ibid.*                                 0.122

Standard purified *C. difficile* B toxin.

Dye reduction rate by CHO-k1 cells incubated with the standard purified *C. difficile* toxin B for 20 hr. The dye reduction rate is calculated by using PM Analysis Software from 0 h to 3 h of incubation with Dye MB.

Predicted *C. difficile* toxin B concentration calculated using the equation indicated in the table.

Finally, we showed that toxin preparations taken directly from *C. difficile* cultures could intoxicate indicator cells and be effectively neutralized by anti-toxin B antibody. Both CHO-k1 and Vero cells were intoxicated by crude toxin preparations of *C. difficile* strain ATCC 9689 supernatants from different culture conditions of the PM panels ([Figure 2B](#pone-0056545-g002){ref-type="fig"}). The morphological changes of the CHO-k1 cells were identical to those caused by purified standard toxin B ([Figures 1A](#pone-0056545-g001){ref-type="fig"}, [2A, 2B](#pone-0056545-g002){ref-type="fig"}, [3B](#pone-0056545-g003){ref-type="fig"}). However, the levels of toxin production in the supernatants were distinctively different under different PM culture conditions. This is clearly shown both by the cell culture toxicity assay ([Figures 2A, 2B](#pone-0056545-g002){ref-type="fig"}, [3B](#pone-0056545-g003){ref-type="fig"}) and by the dye reduction assay with the indicator cells ([Figures 2](#pone-0056545-g002){ref-type="fig"}, [3A, 3C, 3D](#pone-0056545-g003){ref-type="fig"}). As seen in the case of purified standard toxin B ([Figure 1](#pone-0056545-g001){ref-type="fig"}), the inverse correlation between the CPE and dye reduction rate of the intoxicated cells was also observed with unpurified toxin preparations (filtered supernatants) from various PM culture conditions ([Figure 2](#pone-0056545-g002){ref-type="fig"}). These adverse effects of unpurified toxin preparations on cell morphology and dye reduction were also specifically prevented by anti-toxin B IgY polyclonal antibodies ([Figures 3A, 3B, 3C, 3D](#pone-0056545-g003){ref-type="fig"}).

![Cellular dye reduction is correlated to cell morphology changes induced by *C. difficile* toxin produced from PM culture conditions.\
A. CHO-k1 cells used in the assay. (a) Upper part: CHO-k1 cell morphological changes 20 h after exposure to *C. difficile* supernatants collected from different wells of PM1. A1 = No PM substrate control; A3 = N-Acetyl-D-Glucosamine; A9 = D-Alanine; B2 = D-Sorbitol. Lower part: corresponding cellular dye reduction by CHO-k1 cells. Pictures taken at the time points indicated after dye addition. (b) Upper panel: cellular dye reduction kinetics over the course of 24 h, automatically recorded by the OmniLog instrument. The numbers are averages of OmniLog Value (OmniLog Unit or Height); Lower panel: dye reduction rate over 0--3 h, an OmniLog parameter calculated and analyzed by PM Analysis Software. Larger rate numbers indicate faster dye reduction, and therefore healthier cells. (c) Upper panel: plots for OmniLog Values; Lower panel: plots for dye reduction rate. B. Similarities of changes seen with both CHO-k1 (left) and Vero (right) cells in cytotoxicity and dye reduction rate by *C. difficile* supernatants from individual wells of PM7.](pone.0056545.g002){#pone-0056545-g002}

![Cytotoxicity and neutralization assays of toxin prepared from *C. difficile* ATCC 9689 grown in PMs.\
Anti-toxin B polyclonal antibodies IgY were used throughout at 2.5 µg/ml for neutralization assays. A. Representative cell-based cytotoxicity and neutralization assays with redox dye MB in PMs 1 and 3 with CHO-k1 cells. B. Typical CHO-k1 cell morphological changes with or without anti-toxin B IgY. PM3: A8 = L-Arginine, B9 = L-Proline, G1 = Xanthine, F2 = Adenine. C. Dye reduction signals by CHO-k1 cells automatically and kinetically collected by OmniLog instrument. The numbers shown are means of dye reduction rates of replicas of each well in PM3, calculated by PM Analysis Software. D. Histograms of the same data of the dye reduction rates as in C. Tan: no *C. difficile*; Red: *C. difficile*; Green: *C. difficile*+anti-toxin B IgY.](pone.0056545.g003){#pone-0056545-g003}

The cell mass under most PM culture condtions peaked at 24 h and thereafter tended to decrease at 48 h and further decrease at 72 h ([Materials and Methods](#s4){ref-type="sec"}, [Figure S1](#pone.0056545.s001){ref-type="supplementary-material"}). In contrast, initial experiments indicated that toxin production was low at 24 h and tended to peak around 72 h.

Toxin production under different PM culture conditions {#s2b}
------------------------------------------------------

To examine the effect of culture conditions on toxin production, *C. difficile* ATCC 9689 was cultured in PM1--8 which constitutes 768 culture conditions with variations in the basic metabolic nutrients, C, N, P, and S ([Table S1](#pone.0056545.s005){ref-type="supplementary-material"}) for 72 h before toxin-containing supernatants were collected. CHO-k1 cells treated with the 768 *C. difficile* supernatants showed different degrees of CPE ([Figures 2A, 2B](#pone-0056545-g002){ref-type="fig"}, [3B](#pone-0056545-g003){ref-type="fig"}). Control experiments (no *C. difficile* control) were also performed to show that the 768 chemicals themselves did not induce toxicity to the CHO-k1 cells (data not shown). Based on statistical analysis (t-test) we consider the differences of dye reduction rate between the two groups (*C. difficile* supernatant group and no *C. difficile* control group) to be significant only if P\<0.05. There were 544 out of 768 culture conditions that significantly induced *C. difficile* toxin production ([Table 2](#pone-0056545-t002){ref-type="table"}). Of these 544 culture conditions, 89 (16.4% of 544) induced toxin production at high levels of  = \>420 ng/ml ([Tables 2](#pone-0056545-t002){ref-type="table"}, [3](#pone-0056545-t003){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}), 192 (35.3% of 544) gave a middle range level of  = \>42 but \<420 ng/ml ([Tables 2](#pone-0056545-t002){ref-type="table"}, [4](#pone-0056545-t004){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}), and 262 (48.2% of 544) gave a low range level of toxin production, \>2.562 but \<42 ng/ml in the supernatants ([Table 2](#pone-0056545-t002){ref-type="table"}). There were 224 out of 768 culture conditions that were not statistically significant in inducing toxin production (P\>0.05 except one PM7 A4 Leu-Trp, P = 0.048). Of these 224, 30 (13.8% of 224) produced lowest toxin levels (\<0.122 ng/ml in CHO-k1 cell assay medium, or possibly 0 ng/ml) ([Tables 2](#pone-0056545-t002){ref-type="table"}, [5](#pone-0056545-t005){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}).

10.1371/journal.pone.0056545.t002

###### *C. difficile* toxin production ranges and statistics under different culture conditions from PM1--8.

![](pone.0056545.t002){#pone-0056545-t002-2}

  Toxin concentration range (ng/ml)[a](#nt104){ref-type="table-fn"}    Number of PM conditions (P\<0.05[b](#nt105){ref-type="table-fn"})   Number of PM conditions (P\>0.05[b](#nt105){ref-type="table-fn"})   Total
  ------------------------------------------------------------------- ------------------------------------------------------------------- ------------------------------------------------------------------- -------
  \> = 420                                                                                            89                                                                   0                                    89
  \> = 42, \<420                                                                                      192                                                                 10                                    202
  \>2.562, \<42                                                                                       262                                                                 184                                   446
  \<0.122[c](#nt106){ref-type="table-fn"}                                                              1                                                                  30                                    31
  Total                                                                                               544                                                                 224                                   768

For measurable toxin levels, the numbers indicate toxin concentrations in supernatants of *C. difficile* grown under different PM conditions.

The P values were obtained from t-test on the dye reduction rates by CHO-k1 cells in the presence vs absence of *C. difficile* supernatants collected from different PM conditions.

Below the measurable limit in CHO-k1 cell assay medium of 0.122 ng/ml. Note that the concentration of the toxin in the cell assay medium is 21-fold diluted from the bacterial supernatant (see [Materials and Methods](#s4){ref-type="sec"}).

10.1371/journal.pone.0056545.t003

###### PM substrates giving highest levels of toxin production by *C. difficile* ATCC 9689.

![](pone.0056545.t003){#pone-0056545-t003-3}

  Plate panel      Well              Chemical                     Category            *C. difficile* Mass[a](#nt107){ref-type="table-fn"}   Toxin (ng/ml)[b](#nt108){ref-type="table-fn"}   P value[c](#nt109){ref-type="table-fn"}
  ------------- ----------- --------------------------- ---------------------------- ----------------------------------------------------- ----------------------------------------------- -----------------------------------------
  PM03              F02               Adenine                    nucleobase                                  0.031                                             \>16800                                     2.10E-04
  PM03              F07              Guanosine                   nucleoside                                 0.0092                                              10402                                      2.45E-05
  PM06              B07               Arg-Asp                    dipeptide                                  0.0444                                              10371                                      2.32E-07
  PM08              G02             γ-D-Glu-Gly                dipeptide, γ-                                0.0519                                              10122                                      3.34E-03
  PM06              B05               Arg-Ala                    dipeptide                                  0.0456                                              10080                                      5.70E-08
  PM03              G02             Xanthosine                   nucleoside                                 0.0516                                              9499                                       6.36E-09
  PM06              B08               Arg-Gln                    dipeptide                                  0.0476                                              8912                                       4.54E-08
  PM06              C05               Arg-Tyr                    dipeptide                                  0.0455                                              8819                                       7.07E-05
  PM06              F10               His-Pro                    dipeptide                                  0.0481                                              8568                                       6.08E-07
  PM06              C04               Arg-Trp                    dipeptide                                  0.0499                                              8423                                       7.81E-07
  PM08              F04              β-Ala-His                 dipeptide, β-                                0.0473                                              8265                                       3.15E-06
  PM06              B06               Arg-Arg                    dipeptide                                  0.0435                                              8229                                       4.65E-07
  PM03              F08               Thymine                    nucleobase                                 0.0338                                              7749                                       2.83E-09
  PM06              C03               Arg-Ser                    dipeptide                                  0.0448                                              7608                                       1.85E-08
  PM07              D08               Pro-Hyp                    dipeptide                                   0.045                                              7523                                       1.04E-06
  PM03              F09              Thymidine                   nucleoside                                 0.0384                                              7336                                       3.22E-08
  PM07              E07               Ser-Pro                    dipeptide                                  0.0472                                              7136                                       2.23E-07
  PM06              F08               His-Lys                    dipeptide                                  0.0453                                              6993                                       1.02E-07
  PM06              C02               Arg-Phe                    dipeptide                                  0.0467                                              6541                                       2.70E-07
  PM03              D05             Methylamine                    amine                                    0.0398                                              6537                                       1.61E-04
  PM07              F07               Trp-Arg                    dipeptide                                  0.0628                                              6468                                       9.53E-07
  PM03              G08         γ-Aminobutyric acid      amino fatty acid, γ-, GABA                         0.0375                                              6329                                       4.45E-11
  PM03              D07            N-Butylamine                  amine, N-                                  0.0407                                              5679                                       3.74E-04
  PM07              A07               Lys-Arg                    dipeptide                                  0.0502                                              5536                                       5.20E-06
  PM03              B09              L-Proline                   amino acid                                 0.0394                                              5343                                       1.07E-10
  PM07              B04               Lys-Trp                    dipeptide                                   0.006                                              5277                                       5.29E-06
  PM07              B02               Lys-Ser                    dipeptide                                  0.0426                                              5105                                       1.25E-07
  PM03              F05              Cytosine                    nucleobase                                 0.0361                                              4809                                       1.11E-09
  PM07              F05               Thr-Pro                    dipeptide                                  0.0355                                              4772                                       3.32E-06
  PM03              G03              Uric acid             nucleobase derivative                            0.0556                                              4289                                       6.27E-07
  PM03              D10           Ethylenediamine                  amine                                    0.0369                                              4133                                       2.78E-05
  PM07              G03               Trp-Trp                    dipeptide                                   0.055                                              4104                                       1.74E-03
  PM06              B09               Arg-Glu                    dipeptide                                  0.0404                                              4059                                       1.70E-09
  PM03              F04              Cytidine                    nucleoside                                 0.0456                                              3952                                       9.78E-12
  PM03              D08             Ethylamine                     amine                                    0.0401                                              3799                                       8.84E-05
  PM06              E11               Gly-Pro                    dipeptide                                  0.0414                                              3798                                       9.96E-07
  PM03              B03             L-Histidine                  amino acid                                 0.0361                                              3640                                       8.27E-05
  PM03              E05              Formamide                     amide                                    0.0418                                              3639                                       1.96E-11
  PM06              F02               Gly-Trp                    dipeptide                                  0.0431                                              3632                                       1.68E-05
  PM03              G09       ε-Amino-N-Caproic acid        amino fatty acid, ε-                            0.0366                                              3389                                       4.02E-05
  PM03              D06             N-Amylamine                  amine, N-                                  0.0433                                              3384                                       9.99E-09
  PM03              C05           D-Aspartic acid              amino acid, D-                               0.0109                                              3334                                       3.33E-04
  PM03              F10               Uracil                     nucleobase                                 0.0375                                              3204                                       2.32E-04
  PM03              C10            L-Citrulline                  amino acid                                 0.0377                                              3086                                       1.56E-03
  PM08              C11               Pro-Arg                    dipeptide                                  0.0442                                              2967                                       2.61E-03
  PM03              E04              Acetamide                     amide                                    0.0458                                              2926                                       7.25E-09
  PM03              E07            D,L-Lactamide                 amide, DL-                                  0.04                                               2859                                       1.50E-07
  PM03              E03              Tyramine              amine, Tyr derivative                            0.0442                                              2772                                       9.91E-07
  PM01              F04             D-Threonine                amino acid, D-                               0.0923                                              2699                                       1.04E-03
  PM01              A03       N-Acetyl-D-Glucosamine       acetyl amino sugar, N-                           0.0955                                              2657                                       5.17E-05
  PM01              G05              L-Alanine                   amino acid                                  0.113                                              2469                                       1.02E-04
  PM01              F05            Fumaric Acid               carboxylic acid                                0.018                                              2284                                       8.73E-04
  PM03              E02         β-Phenylethylamine       amine, Phe derivative, β-                          0.0448                                              2147                                       1.80E-06
  PM01              G06               Ala-Gly                    dipeptide                                  0.0934                                              2130                                       4.82E-07
  PM03              G01              Xanthine                    nucleobase                                 0.2978                                              2079                                       2.83E-05
  PM08              C01               Lys-Gly                    dipeptide                                   0.047                                              1950                                       1.30E-04
  PM08              H03             Gly-Gly-Gly                  tripeptide                                 0.0456                                              1811                                       2.93E-05
  PM03              B06              L-Lysine                    amino acid                                 0.0436                                              1767                                       3.75E-04
  PM03              E09           D-Galactosamine             amino sugar, D-                               0.0374                                              1723                                       3.43E-09
  PM08              F03              β-Ala-Gly                 dipeptide, β-                                0.0389                                              1547                                       2.32E-07
  PM01              G03              L-Serine                    amino acid                                 0.0635                                              1537                                       4.48E-03
  PM08              G04              Gly-D-Asp                   dipeptide                                  0.0467                                              1537                                       3.45E-07
  PM01              F01               Gly-Asp                    dipeptide                                  0.1004                                              1507                                       3.62E-03
  PM03              C07              D-Lysine                  amino acid, D-                               0.0489                                              1472                                       1.00E-04
  PM03              G10      D,L-α-Amino-Caprylic acid     amino fatty acid, DL-α                           0.0484                                               647                                       4.73E-07
  PM01              F06         Bromosuccinic Acid            carboxylic acid                               0.0335                                               615                                       8.93E-05
  PM01              B01              D-Serine                  amino acid, D-                               0.1114                                               588                                       1.19E-03
  PM02              G07            L-Homoserine            amino acid, Thr isomer                           0.0071                                               587                                       7.04E-03
  PM03              E10            D-Mannosamine              amino sugar, D-                               0.0364                                               583                                       1.06E-05
  PM08              G12            D-Ala-Gly-Gly                 tripeptide                                 0.0538                                               515                                       1.09E-05
  PM02              G10              L-Leucine                   amino acid                                  0.089                                               503                                       2.97E-05
  PM03              E01              Histamine             amine, His derivative                            0.0424                                               492                                       3.79E-08
  PM03              F11               Uridine                    nucleoside                                 0.0373                                               484                                       2.62E-04
  PM03              E08            D-Glucosamine              amino sugar, D-                               0.0447                                               476                                       3.86E-06
  PM03              A08             L-Arginine                   amino acid                                  0.046                                               458                                       2.65E-05
  PM03              G11       δ-Amino-N-Valeric acid        amino fatty acid, δ-                            0.0424                                               435                                       5.08E-10
  PM1--8         All wells        All substrates                  control                                   0.0517                                               50                                        3.62E-02
  PM1--8            A1             No substrate                   control                                   0.0517                                               44                                        9.80E-02

OD (750 nm) difference between *C. difficile* under certain PM substrate and the same substrate without *C. difficile*.

Toxin concentrations in *C. difficile* supernatant collected from different PM conditions, which were calculated from the average dye reduction rate by the CHO-k1 cells according to the equations in [Table 1](#pone-0056545-t001){ref-type="table"}.

The P values were obtained from t-test on the dye reduction rates of CHO-k1 cells in the presence or absence of *C. difficile* supernatants collected from different PM conditions.

10.1371/journal.pone.0056545.t004

###### PM substrates giving middle levels of toxin productions by *C. difficile* ATCC 9689.

![](pone.0056545.t004){#pone-0056545-t004-4}

  Plate panel      Well                   Chemical                                 Category                   *C. difficile* Mass[a](#nt110){ref-type="table-fn"}   Toxin (ng/ml)[b](#nt111){ref-type="table-fn"}   P value[c](#nt112){ref-type="table-fn"}
  ------------- ----------- ------------------------------------- ------------------------------------------ ----------------------------------------------------- ----------------------------------------------- -----------------------------------------
  PM07              F06                    Trp-Ala                                dipeptide                                         0.0489                                               415                                       1.08E-04
  PM03              A03                    Nitrite                            inorganic N-source                                    0.0333                                               383                                       3.24E-07
  PM08              B08                    Leu-Asn                                dipeptide                                         0.0482                                               381                                       2.24E-03
  PM06              F07                    His-Leu                                dipeptide                                         0.0495                                               376                                       6.77E-05
  PM03              F03                   Adenosine                               nucleoside                                        0.0472                                               373                                       7.02E-03
  PM01              G01                    Gly-Glu                                dipeptide                                         0.0411                                               366                                       5.53E-04
  PM06              E03                    Gly-Arg                                dipeptide                                         0.0403                                               352                                       6.87E-05
  PM08              B12                    Lys-Asp                                dipeptide                                         0.0525                                               335                                       4.93E-05
  PM03              D11                  Putrescine                                 amine                                           0.0339                                               335                                       7.69E-08
  PM03              G05                   Allantoin                         nucleobase derivative                                   0.0425                                               334                                       1.48E-08
  PM03              D04                 Hydroxylamine                   inorganic base, reducing agent                              0.0228                                               334                                       1.58E-07
  PM01              D02                D-Aspartic Acid                        amino acid, D-Asp                                     0.0269                                               331                                       5.56E-05
  PM02              B02           N-Acetyl-Neuraminic acid             acetyl amino sugar, sialic acid                              0.1163                                               328                                       1.77E-03
  PM04              C12      Cytidine 3′,5′-Cyclic Monophosphate           nucleotide, 3′,5′-cyclic                                 0.0106                                               321                                       8.83E-05
  PM03              A06                    Biuret                        amide, carbamide derivative                                0.0394                                               307                                       3.59E-06
  PM01              G10                Methylpyruvate              carboxylic acid derivative, methyl ester                         0.0795                                               299                                       5.31E-05
  PM08              H08                  Gly-Phe-Phe                              tripeptide                                        0.2751                                               298                                       4.18E-07
  PM08              G05                   Gly-D-Ser                               dipeptide                                         0.0543                                               297                                       8.49E-06
  PM08              H07                  Val-Tyr-Val                              tripeptide                                        0.0382                                               296                                       2.62E-06
  PM03              E06                 Glucuronamide                       sugar acid, amide (6c)                                  0.0369                                               280                                       1.10E-06
  PM07              A08                    Lys-Glu                                dipeptide                                         0.0425                                               271                                       1.01E-04
  PM01              G04                  L-Threonine                           amino acid, Thr                                      0.0643                                               243                                       3.70E-03
  PM07              H05                    Val-Asp                                dipeptide                                         0.0469                                               225                                       1.12E-04
  PM03              C02                   L-Valine                             amino acid, Val                                      0.0431                                               223                                       2.01E-04
  PM08              H05                  Gly-Gly-Leu                              tripeptide                                        0.0305                                               215                                       1.03E-04
  PM02              B08                    Arbutin                   sugar hydroquinone (12c), glycoside                             0.128                                               213                                       5.36E-05
  PM03              C04                 D-Asparagine                          amino acid, D-Apn                                     0.0321                                               196                                       2.31E-04
  PM07              E04                    Ser-Leu                                dipeptide                                         0.0404                                               187                                       3.43E-04
  PM07              G05                    Tyr-Ala                                dipeptide                                         0.0331                                               186                                       5.79E-05
  PM03              H10                    Gly-Glu                                dipeptide                                         0.0472                                               184                                       5.43E-03
  PM08              E03                    Trp-Val                                dipeptide                                         0.0276                                               184                                       1.26E-04
  PM08              E12                    Val-Pro                                dipeptide                                         0.0425                                               183                                       2.01E-04
  PM06              F12                    His-Trp                                dipeptide                                         0.0442                                               178                                       1.49E-04
  PM08              E08                    Val-Glu                                dipeptide                                         0.0481                                               178                                       1.11E-05
  PM07              H02                    Tyr-Tyr                                dipeptide                                         0.0415                                               174                                       2.16E-04
  PM01              H12                2-Aminoethanol                         amine, alcohol, 2-                                    0.0853                                               173                                       1.15E-03
  PM03              C01                  L-Tyrosine                            amino acid, Tyr                                      0.1528                                               172                                       1.52E-05
  PM03              C03                   D-Alanine                           amino acid, D-Ala                                      0.038                                               171                                       1.70E-04
  PM03              B12                 L-Tryptophan                           amino acid, Trp                                      0.0299                                               165                                       2.91E-04
  PM02              E01                  Capric acid                              fatty acid                                         0.014                                               162                                       3.24E-04
  PM02              A06                    Dextrin                          sugar, polysaccharide                                   0.0478                                               162                                       3.99E-03
  PM08              G06                   Gly-D-Thr                               dipeptide                                         0.0549                                               157                                       3.21E-05
  PM03              E12           N-Acetyl-D-Galactosamine                  acetyl amino sugar, N-                                  0.0355                                               153                                       3.06E-04
  PM07              A11                    Lys-Lys                                dipeptide                                         0.0427                                               150                                       5.07E-05
  PM03              C12                  L-Ornithine                           amino acid, Orn                                      0.0369                                               141                                       3.49E-04
  PM02              D02                    Salicin                     sugar phenol, β-glucoside (13c)                              0.1291                                               140                                       3.61E-03
  PM02              C04                   D-Lyxose                              sugar (5c), D-                                      0.1508                                               139                                       3.82E-03
  PM03              D02          N-Phthaloyl-L-Glutamic acid                amino acid derivative                                   0.0344                                               137                                       4.69E-05
  PM02              H04                   L-Valine                             amino acid, Val                                      0.0917                                               135                                       1.28E-02
  PM07              G04                    Trp-Tyr                                dipeptide                                         0.0451                                               134                                       5.89E-05
  PM02              E02                 Caproic acid                              fatty acid                                        0.0568                                               133                                       1.90E-03
  PM02              G12                 L-Methionine                           amino acid, Met                                      0.0818                                               127                                       6.01E-04
  PM06              G04                    Ile-Arg                                dipeptide                                         0.0442                                               122                                       3.82E-03
  PM03              A01               Negative Control                         negative control                                     0.0403                                               122                                       1.90E-04
  PM03              B04                 L-Isoleucine                           amino acid, Ile                                      0.0563                                               122                                       9.75E-04
  PM02              D06                  D-Tagatose                             sugar (6c), D-                                       0.138                                               121                                       8.44E-04
  PM03              C11                 L-Homoserine                        amino acid, Thr isomer                                  0.0293                                               119                                       2.52E-05
  PM01              A09                   D-Alanine                           amino acid, D-Ala                                     0.1233                                               118                                       9.72E-06
  PM07              A03                    Leu-Ser                                dipeptide                                          0.037                                               116                                       1.51E-04
  PM03              C08                   D-Serine                            amino acid, D-Ser                                     0.0465                                               110                                       7.29E-05
  PM03              A11                  L-Cysteine                            amino acid, Cys                                      0.0394                                               108                                       6.64E-05
  PM03              D12                   Agmatine                          amine, Arg derivative                                    0.043                                               108                                       3.06E-04
  PM01              C07                  D-Fructose                             sugar (6c), D-                                      0.1435                                               106                                       1.34E-06
  PM01              H08                 Pyruvic Acid                     carboxylic acid, α-keto acid                                0.093                                               103                                       5.67E-04
  PM02              H02                L-Phenylalanine                         amino acid, Phe                                      0.0714                                               103                                       2.46E-02
  PM1--8         All wells             All substrates                              control                                          0.0517                                               50                                        3.62E-02
  PM1--8            A1                  No substrate                               control                                          0.0517                                               44                                        9.80E-02

OD (750 nm) difference between *C. difficile* under certain PM substrate and the same substrate without *C. difficile*.

Toxin concentrations in C. difficile supernatant collected from different PM conditions, which were calculated from the average dye reduction rate by the CHO-k1 cells according to the equations in [Table 1](#pone-0056545-t001){ref-type="table"}.

The P values were obtained from t-test on the dye reduction rates of CHO-k1 cells in the presence or absence of *C. difficile* supernatants collected from different PM conditions.

10.1371/journal.pone.0056545.t005

###### PM substrates giving lowest levels of toxin production by *C. difficile* ATCC 9689.

![](pone.0056545.t005){#pone-0056545-t005-5}

  PM panel      Well                     Chemical                                    Category                    *C. difficile* mass[a](#nt113){ref-type="table-fn"}   Toxin (ng/ml)[b](#nt114){ref-type="table-fn"}   P value[c](#nt115){ref-type="table-fn"}
  ---------- ----------- ----------------------------------------- -------------------------------------------- ----------------------------------------------------- ----------------------------------------------- -----------------------------------------
  PM01           H11                β-Phenylethylamine                amine, Phe derivative, β-, (C-source)                            0.0477                                             \<0.122                                     9.40E-01
  PM02           H11                   2,3-Butanone                   ketone, methyl ethyl, 2,3-, C-source)                            0.0572                                             \<0.122                                     4.87E-01
  PM04           A01                 Negative Control                           ctrl, no substrate                                     0.0473                                             \<0.122                                     2.59E-01
  PM04           A10            Adenosine 5′-Monophosphate                    nucleotide, (P-source)                                   0.0495                                             \<0.122                                     8.02E-02
  PM04           A03                   Pyrophosphate                         diphosphate, (P-source)                                   0.0075                                             \<0.122                                     1.98E-01
  PM04           A04                 Trimetaphosphate                       metaphosphate, (P-source)                                  0.0493                                             \<0.122                                     1.86E-01
  PM04           B01                   Thiophosphate                         thiophosphate (P-source)                                  0.0373                                             \<0.122                                     1.92E-01
  PM04           H04                   D,L-Lipoamide                amide, 6,8-dithiooctanoic, DL-, (S-source)                         0.0265                                             \<0.122                                     1.16E-01
  PM04           G12               L-Methionine Sulfone                  amino acid derivative (S-source)                              0.0446                                             \<0.122                                     3.84E-01
  PM04           H06                      Taurine                    aminoethanesulfonic acid, 2-, (S-source)                          0.0505                                             \<0.122                                     1.76E-01
  PM04           H05                 Taurocholic acid                        cholyltaurine (S-source)                                  0.0536                                             \<0.122                                     1.64E-01
  PM04           H08           p-Aminobenzene Sulfonic acid              sulfanilic acid, p-, (S-source)                               0.0485                                             \<0.122                                     1.64E-01
  PM04           H07                    Hypotaurine                         sulfinic acid, (S-source)                                  0.0438                                             \<0.122                                     3.83E-01
  PM04           H12              Tetramethylene Sulfone                       Sulfone, (S-source)                                     0.0442                                             \<0.122                                     6.03E-01
  PM04           H09               Butane Sulfonic acid                sulfonic acid derivative, (S-source)                            0.0398                                             \<0.122                                     3.37E-01
  PM04           H10           2-Hydroxyethane Sulfonic acid         sulfonic acid derivative, 2- (S-source)                           0.0428                                             \<0.122                                     2.03E-01
  PM05           A11                     Adenosine                      nucleoside (nutritional stimulant)                             0.0414                                             \<0.122                                     5.88E-01
  PM06           A12                      Ala-Pro                              dipeptide (N-source)                                    0.0507                                             \<0.122                                     2.44E-01
  PM08           H04                    Gly-Gly-Ile                           tripeptide (N-source)                                    0.0421                                             \<0.122                                     1.38E-01
  PM06           E12                      Gly-Ser                              dipeptide (N-source)                                    0.0471                                             \<0.122                                     3.13E-01
  PM06           H09                      Leu-Ile                              dipeptide (N-source)                                    0.0523                                             \<0.122                                     8.95E-02
  PM06           H10                      Leu-Leu                              dipeptide (N-source)                                    0.0530                                             \<0.122                                     1.06E-01
  PM08           H10                    Leu-Leu-Leu                           tripeptide (N-source)                                    0.0463                                             \<0.122                                     1.24E-01
  PM06           H11                      Leu-Met                              dipeptide (N-source)                                    0.0501                                             \<0.122                                     1.12E-01
  PM06           H12                      Leu-Phe                              dipeptide (N-source)                                    0.0538                                             \<0.122                                     7.52E-02
  PM07           A04                      Leu-Trp                              dipeptide (N-source)                                    0.0380                                             \<0.122                                     4.79E-02
  PM07           A02                    L-Glutamine                           amino acid (N-source)                                    0.0339                                             \<0.122                                     7.04E-02
  PM07           A12                      Lys-Phe                              dipeptide (N-source)                                    0.0533                                             \<0.122                                     6.40E-02
  PM08           D12                      Thr-Gln                              dipeptide (N-source)                                    0.0403                                             \<0.122                                     7.74E-02
  PM07           G12                      Tyr-Phe                              dipeptide (N-source)                                    0.0559                                             \<0.122                                     7.37E-02
  PM08           F01                      Val-Ser                              dipeptide (N-source)                                    0.0455                                             \<0.122                                     6.23E-01
  PM6--8         A1                   No PM substrate                                Control                                           0.0489                                              2.690                                      9.80E-02
  PM6--8      All Wells              All PM substrates                               Control                                           0.0467                                              2.423                                      3.62E-02
  PM6--8      All Wells   All PM substrates but no *C. difficile*                    Control                           0.0423[d](#nt116){ref-type="table-fn"}             \<0.122[e](#nt117){ref-type="table-fn"}                        NA

OD (750 nm) difference between *C. difficile* under certain PM substrate (condition) and the same substrate without *C. difficile*.

Toxin concentrations in the CHO-k1 assay medium with *C. difficile* supernatant collected from different PM conditions, which were calculated from the average dye reduction rate by the CHO-k1 cells according to the equations in [Table 1](#pone-0056545-t001){ref-type="table"}.

The P values were obtained from t-test on the dye reduction rates of CHO-k1 cells in the presence or absence of *C. difficile* supernatants collected from different PM conditions.

Average OD (750 nm) value of all uninoculated PM substrates from PM1--8.

This value is a result of calculation based on dye reduction rate of CHO-k1 cells in the presence of PM substrates of PM1--8 without *C. difficile* inoculation. It is a measure of zero toxin.

With glucose as the carbon source, a variety of nitrogen sources from PM3, 6, 7, and 8 gave highest toxin productions ( = \>420 ng/ml) ([Figures 4A, 4C](#pone-0056545-g004){ref-type="fig"}, [Tables 3](#pone-0056545-t003){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}). The nitrogen sources were the predominant class of high toxin level inducers compared to other categories (e.g., carbon, phosphorus, sulfur sources, etc.) of PM culture conditions. The most powerful *C. difficile* toxin inducers (\>2,100 ng/ml) were found in nucleobases, nucleosides, dipeptides, and amine compounds, which included adenine, guanosine, arginine dipeptides, γ-D-Glu-Gly, methylamine and others ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Tables 3](#pone-0056545-t003){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}). Among amino acids as nitrogen sources, L-proline, L-histidine, D-aspartic acid, L-citrulline, L-lysine, and D-lysine were also seen in the group of high toxin inducers ([Figure 4C](#pone-0056545-g004){ref-type="fig"}, [Tables 3](#pone-0056545-t003){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}). Overall, the highest toxin inducer was adenine as a nitrogen source in PM3, which exceeded the upper tested concentration of the standard purified toxin B in this study (800 ng/ml). This corresponds to 16,800 ng/ml or greater (with a 21-fold dilution factor applied) in the bacterial supernatant ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). Guanosine as a nitrogen source induced the second highest toxin production of *C. difficile* ATCC 9689 ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). These were followed by xanthosine, thymine, and thymidine, which induced toxin levels \>7,000 ng/ml, and by others including cytosine, cytidine, uracil, uridine, and xanthine. The purine metabolite uric acid also stimulated high toxin production (4288 ng/ml) ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). Interestingly, whereas multiple arginine dipeptides were among the very top toxin inducers ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}), arginine by itself was not, although it was among the category of high toxin producers ([Figure 4C](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). It induced ∼14--22 times lower than the top toxin-inducing arginine dipeptides ([Figures 4A, 4C](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). Presumably it is not taken up as well as the arginine-containing peptides or arginine dipeptides as intact molecules work differently than arginine itself in stimulating the toxin production.

![Examples of high toxin-inducing PM substrates for *C. difficile* ATCC 9689.\
A. Top toxin inducers. Note that adenine as a nitrogen source is the strongest toxin inducer of all tested PM substrates in this study. g-D-Glu-Gly = γ-D-Glu-Gly; b-Ala-His = β-Ala-His; g-Aminobutyric = γ-Aminobutyric acid. B. High toxin production induced by some carbon sources in PM1 and 2. C. High toxin production induced by some amino acids as nitrogen sources. Note that L-proline is the highest toxin inducer in the amino acid category.](pone.0056545.g004){#pone-0056545-g004}

Carbon sources from PM1 and 2 gave lower levels of toxin production and were less frequent toxin inducers. Though weaker inducers than the nitrogen sources in PM3,6,7,8, D-threonine, N-acetyl-D-glucosamine, L-alanine, Ala-Gly, fumaric acid, L-serine, and Gly-Asp were the highest toxin inducers of the carbon sources in panels PM1--2 ([Figure 4B](#pone-0056545-g004){ref-type="fig"}), and induced toxin levels of greater than 1500 ng/ml. Phosphorus and sulfur sources from PM4 were not seen as significant toxin inducers with *C. difficile* ATCC 9689, although cytidine 3′,5′-cyclic monophosphate as a P-source in PM4 induced a middle range (320 ng/ml) of toxin production. None of the culture conditions in PM5, with low levels of potentially stimulatory nutrients, showed significant toxin induction or repression.

The other interesting class is the PM substrates that gave lowest levels of *C. difficile* toxin production or no toxin at all (\<0.122 ng/ml in CHO-k1 assay medium, or correspondingly \<2.562 ng/ml in bacterial supernatant), which were indistinguishable from no *C. difficile* control ([Table 5](#pone-0056545-t005){ref-type="table"}). The concentrations were so low that they were out of the reliable range of dye reduction measurement. Notable among this group were several leucine dipeptides and the leu-leu-leu tripeptide ([Figure 5](#pone-0056545-g005){ref-type="fig"}, [Table 5](#pone-0056545-t005){ref-type="table"}). Given the average toxin production of 57 ng/ml in the bacterial supernatant (or 2.690 ng/ml in the cell assay medium) from the no PM substrate control and 51 ng/ml (or 2.423 ng/ml in the cell assay medium) from all PM substrate controls of PM 6 through 8 ([Figure 5](#pone-0056545-g005){ref-type="fig"}, [Table 5](#pone-0056545-t005){ref-type="table"}), and also given that the bacterial mass under those conditions were all comparable ([Table 5](#pone-0056545-t005){ref-type="table"}), the substrates that gave very low or no toxin production may in fact repress toxin production by *C. difficile*.

![Repression of *C. difficile* toxin production by leucine dipeptides and the triple leucine tripeptide.\
\* Since the dye reduction rate by CHO-k1 cells under the condition was greater than the upper limit indicated in [Table 1](#pone-0056545-t001){ref-type="table"}, the estimated minimum toxin concentration value (0.12 ng/ml) measurable in this assay was taken for plotting purpose, which was actually \<0.12 ng/ml or could even be 0 ng/ml. \*\* The corresponsing dye reduction rates were within the range covered by the equation in [Table 1](#pone-0056545-t001){ref-type="table"}. Therefore, these toxin concentrations were obtained from the rates by calculation. Because all *C. difficile* supernatants were diluted when tested in CHO-k1 cell assay medium, the actual toxin concentrations in the supernatants were all 21 fold higher than the displayed values.](pone.0056545.g005){#pone-0056545-g005}

Experiments were also performed to compare the results of the type strain *of C. difficile* to the toxinotype strain VPI 10463 (ATCC 43255). These experiments showed that the toxin levels were greatly elevated in the toxinotype strain which we estimate at 100--1000 times higher than ATCC 9689 (data not shown).

Additional experiments were performed to demonstrate the general applicability of this assay technology to diverse toxin producing bacteria. Using CHO-k1 or Vero cells we could demonstrate variable toxicity from the PM panel supernatants of *C. perfringens*, *C. tetani*, *C. sordellii*, *Bacillus cereus*, *Escherichia coli O157* (Vero), *Shigella dysenteriae* (Vero), and *Listeria monocytogenes* ([Figure 6](#pone-0056545-g006){ref-type="fig"}).

![Cell-based cytotoxicity assay of other bacteria.\
A. Cytotoxin assay comparison of Clostridial species. Five µl of bacterial supernatant from each well of the PM of each Clostridial species, *C. difficile* (ATCC 9689), *C. perfringens* (ATCC 25763), and *C. sordellii* (ATCC 9714), were used in this assay. CHO-k1 is the indicator cell in this dye reduction assay. Upper panel: supernatants from PM1; Lower panel: supernatants from PM6. In the lower panel, the wells above the lines contain arginine dipeptides as the nitrogen source. B. Aerobic bacterial toxin assays and comparison of supernatants from PM1 between toxigenic and non toxigenic strains or species. Vero is the indicator cell for *E. coli* strains and *Shigella* species. CHO-k1 is the indicator cell for *Listeria* species. Upper panel: toxigenic strains or species; Lower panel: non-toxigenic strains or species. For all: darker color indicates higher dye reduction rate and less toxin produced in that well; lighter color indicates lower dye reduction rate and more toxin produced in that well.](pone.0056545.g006){#pone-0056545-g006}

Discussion {#s3}
==========

Reliable, quantitative, and robust assays for functional toxins are essential in scientific research and clinical practice. The roots of our new assay extend back by several decades. In 1983, Mosmann published a highly cited, quantitative and colorimetric assay using the tetrazolium salt MTT to measure mammalian cell proliferation and cytotoxicity [@pone.0056545-Mosmann1]. Since then, it has been applied in numerous research studies. In addition to its applications in anti-cancer drug research and other toxicity studies, it has also been applied to bacterial cytotoxin studies [@pone.0056545-Chung1] including measurement of cytotoxcity of *C. difficile* toxin [@pone.0056545-Savidge1], [@pone.0056545-Yang1], [@pone.0056545-Mahida1], [@pone.0056545-Mullan1]. Rothman similarly reported quantitation of cell responses to *C. difficile* toxin by crystal violet staining of mammalian cells [@pone.0056545-Rothman1]. The application of colorimetric dyes significantly reduces the labor time because quantitation of color change avoids the laborious and subjective counting of rounded cells. However, the prior colorimetric assays are also inconvenient in that they require solubilization of dye and reading of the plates manually with a microplate reader. This compromises the efficiency of the assays and does not allow one to collect high throughput kinetic data for analysis.

To develop a new, reliable, quantitative, and robust cytotoxicity assay, we started with the gold standard approach by observing the reliable morphological changes of intoxicated cells by *C. difficile* toxin B, the cell rounding, or cytopathic effect (CPE). We confirmed that the cell rounding caused by *C. difficile* toxin B is concentration-dependent and specifically prevented by IgY anti-toxin B polyclonal antibodies. Then, we employed a new proprietary Dye Mix MB [@pone.0056545-Bochner4], that forms a water soluble formazan and does not require solubilization. We also employed the OmniLog instrument which enables the automatic collection of kinetic dye reduction data directly from all wells of 96-well plates in a high throughput format and calculated reduction rates using PM Analysis Software. This enabled measurement of the CPE as decreased cell viability resulting in decreased redox dye reduction rates. Through quantitative analysis, we showed that the CPE is inversely correlated with dye reduction rate of the cells: the stronger the CPE, the lower the dye reduction rate. Furthermore, both toxin-induced CPE and decreased dye reduction rate were specifically and simultaneously prevented by the anti-toxin antibodies. The close correlation between the predicted toxin concentrations calculated from the regression equations and the prepared concentrations ([Table 1](#pone-0056545-t001){ref-type="table"}) indicates that the equations obtained using the standard toxin are reliable and accurate over a wide range (\>3 logs) of toxin concentration. Thus, the basis of determining the levels of *C. difficile* toxin production under various culture conditions in this study has been established.

It is estimated that toxin B of *C. difficile* is 1000 times more potent than toxin A [@pone.0056545-Rothman2]. We also observed that the indicator cell lines (CHO-k1 and Vero) were much more sensitive to toxin B than to toxin A (data not shown). Because of this large difference, trace or equivalent amounts of toxin A contamination in toxin B preparations would be of no consequence in the cell rounding assay [@pone.0056545-Rothman2]. Therefore, in this study, we employed exclusively IgY anti-toxin B polyclonal antibodies which were capable of completely protecting the cells from purified standard toxin B ([Figure 1](#pone-0056545-g001){ref-type="fig"}). As expected, the anti-toxin B IgY almost completely protected the cells from the crude toxin preparations collected from 96-well PM panels as well ([Figures 3A, 3B, 3C, 3D](#pone-0056545-g003){ref-type="fig"}).

As a cell-based cytotoxicity assay, this new method has gold standard reliability and makes the traditional cytotoxicity assay objectively quantifiable, more efficient, shorter in turn-around time (1 day rather than 2 or 3 days), and amenable to high throughput testing. By eliminating subjective scoring of cell rounding, it allows results from different laboratories to be compared.

Combining this toxin assay method with various PM culture conditions (in 96-well plates) provides another unique advantage over the traditional methods used in toxin research. It allows scientists to simultaneously study hundreds to thousands of culture conditions that may positively or negatively affect toxin production by *C. difficile* or other toxigenic microorganisms. Using this approach, we measured toxin production by *C. difficile* ATCC 9689 under very diverse nutritional conditions, including 768 carbon, nitrogen, phosphorus, sulfur, and other nutrient sources ([Table S1](#pone.0056545.s005){ref-type="supplementary-material"}). Measuring hundreds of culture conditions in a quantitative and high throughput manner provides a broad perspective on toxin regulation and thus increases the probability of meaningful discoveries in toxin research. It provides another dimension beyond the effects of genetic changes.

Research has shown that toxin A and B production by *C. difficile* VPI 10463 is regulated by temperature through TcdR activity, the alternative sigma factor positively regulating *tcdA* and *tcdB* expression [@pone.0056545-Karlsson1]. At 37°C, toxin expression was highest compared to 22 or 42°C. This temperature regulated toxin production is positively correlated to the regulation of butyric acid production, but not to other short chain fatty acids [@pone.0056545-Karlsson1]. With a robust assay capability on hundreds of different PM culture conditions, this assay technology can enable simultaneously studies of toxin regulation in cells with different or same genetic backgrounds and at different temperatures. This kind of multi-dimensional analysis would greatly enrich our understanding of the bacterium, its metabolism, and environmental factors affecting regulation of toxin production.

Influences of genetic factors on toxin production, in some cases, seem very clear. For example, for some non-toxic strains examined by Hammond and Johnson, the pathogenicity locus (PaLoc) is absent and a very short fragment (127 bp) occupies the same chromosomal location [@pone.0056545-Hammond1]. Emergent in North America, Europe, and Asia since 2003 [@pone.0056545-Rupnik1], [@pone.0056545-Warny1], [@pone.0056545-McDonald1], [@pone.0056545-Tae1], [@pone.0056545-Lim1], [@pone.0056545-Loo1], hypervirulent strains, represented and dominated by ribotype 027 or BI/NAP1/027, produce high levels of toxin A and toxin B, which is presumably due to their harboring a *tcdC* repressor gene mutated at several distinct sites (an 18-bp, a 36-bp, a 39-bp deletion, or a single base pair deletion at position 117 that causes a frameshift introducing a stop codon at position 196 [@pone.0056545-Warny1], [@pone.0056545-McDonald1], [@pone.0056545-Bouvet1], [@pone.0056545-MacCannell1], [@pone.0056545-Dupuy1], [@pone.0056545-Curry1], [@pone.0056545-Wolff1], [@pone.0056545-Matamouros1]). However, some conflicting evidence has shown that truncation of the repressor gene *tcdC* does not cause higher toxin production in ribotype 027-related strains [@pone.0056545-Murray1]. In addition, other groups have reported a lack of association of *tcdC* mutation type with disease severity in toxigenic *C. difficile* [@pone.0056545-Verdoorn1], [@pone.0056545-Goldenberg1], [@pone.0056545-Cloud1], [@pone.0056545-Wilson1], [@pone.0056545-Sirard1]. Therefore, regulation by genetic factors may be more complicated than expected. A comparable and direct functional or phenotypic toxin assay, such as the assay that we have demonstrated, should be helpful in sorting this out.

Spo0A, the master regulator for sporulation initiation, positively regulates toxin production [@pone.0056545-Underwood1]; and SigH, the key phase transcriptional factor, negatively regulates the toxin expression [@pone.0056545-Saujet1]. CodY, a global regulator of gene expression, directly binds to the promoter of *tcdR* with high affinity, down-regulating toxin genes *tcdA* and *tcdB* [@pone.0056545-Dineen1]. This binding is enhanced by GTP and branched-chain amino acids (leucine, isoleucine, and valine). Therefore, CodY may integrate toxin production with the nutrient status of *C. difficile*. [@pone.0056545-Dineen1], [@pone.0056545-Dineen2]. In our studies with the type strain of *C. difficile*, ATCC 9689, we found that toxin B production was most strongly repressed by some dipeptides containing leucine and the triple-leucine tripeptide as nitrogen sources in the presence of glucose. This appears to agree with the observation of leucine\'s enhancement of down-regulation via CodY. Leucine peptides may be taken up more efficienty than leucine. It would be very interesting to quantitatively measure functional toxin production with isogenic mutants of relevant regulatory genes under hundreds of nutritional culture conditions. This could provide new insights into understanding the coordination of nutrient metabolism and toxin production by these regulatory genes. Furthermore, if the leucine peptides are broadly and rapidly active in suppressing toxin production, they could perhaps be utilized to quell *C. difficile* induced toxicity in patients.

Over the past decades, there have been some prior studies of the effects of nutritional factors on *C. difficile* toxin production. It is well known that biotin deficiency increases toxin A and toxin B production significantly in some strains [@pone.0056545-Yamakawa1], [@pone.0056545-Yamakawa2] whereas glucose has been shown to reduce toxin production [@pone.0056545-Karlsson2], [@pone.0056545-Karlsson3], [@pone.0056545-Antunes1], [@pone.0056545-Dupuy2]. In our studies, we used vitamin sufficient defined media by adding 0.5× of a vitamin mix (RPMI1640 vitamins, Sigma, used for mammalian cell culture). For biotin, the concentration used for *C. difficile* culture was 410 nM. Therefore, in these studies, biotin deficiency was not a factor. Looking at diverse carbon sources, we confirmed that glucose gives relatively low toxin production. Compared to N-Acetyl-D-Glucosamine (2657.19 ng/ml, PM1 A3), glucose gave a much lower level of toxin production (78.86 ng/ml, PM1 C9), only slightly higher than that of no substrate control of the panel (57.83 ng/ml, PM1 A1). The inhibition of *C. difficile* toxin production by glucose may be due to carbon catabolite repression through catabolite control protein A, CcpA [@pone.0056545-Antunes1]. On the other hand, glucose limitation reduced toxin production dramatically [@pone.0056545-Yamakawa1], [@pone.0056545-Yamakawa2], [@pone.0056545-Karlsson3], which was also true in our observations when *C. difficile* was inoculated in PM3 with reduced glucose as carbon source (data not shown).

An influence of arginine on *C. difficile* toxin production has been noted previously, but the effect has not been consistent in reports: (1) toxin production increases with arginine addition in complex medium [@pone.0056545-Osgood1]; (2) toxin production increases with the absence or insufficiency of arginine [@pone.0056545-Karasawa1]. In our assay, some arginine dipeptides increased toxin production dramatically ([Figures 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}), with less increase by arginine (458 ng/ml, [Figure 4C](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). This increase in toxin production by arginine or arginine dipeptides agrees with results of Osgood et al [@pone.0056545-Osgood1] and results from a plant pathogenic fungus using PM panels [@pone.0056545-Gardiner1], where arginine and amines were strong toxin inducers in *Fusarium graminearum*. Culture conditions with nitrogen sources other than arginine or arginine dipeptides, can also produce high toxin levels, some of which are even higher ([Figures 4A, 4C](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}, [S2](#pone.0056545.s006){ref-type="supplementary-material"}). This agrees with the results of Karasawa et al [@pone.0056545-Karasawa1] who found that arginine is not absolutely required for increased toxin production. Not surprisingly, the regulation appears to be multifactorial. For example, arginine dipeptides containing branched-chain amino acids (Leu, Ile, and Val), gave much lower levels of toxin production than other arginine dipeptides, even lower than that of arginine itself. For example, *C. difficile* ATCC 9689 produced 282 ng/ml on Arg-Ile; 122 on Ile-Arg, 67 on Leu-Arg, 54 on Arg-Leu, 36 on Val-Arg, and 32 on Arg-Val ([Table S2](#pone.0056545.s006){ref-type="supplementary-material"}). These low toxin production levels could be explained, at least in part, by the branched-chain amino acids enhancing CodY\'s ability to down-regulate *tcdA* and *tcdB* [@pone.0056545-Dineen1]. Other interesting observations may have different underlying explanations. For example, while Arg-Ala and Lys-Arg gave very high toxin production, 10,080 and 5536 ng/ml, respectively ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}), Ala-Arg gave a strikingly low level toxin production of 34 ng/ml ([Table S2](#pone.0056545.s006){ref-type="supplementary-material"}) and Arg-Lys gave a middle level of 308 ng/ml ([Table S2](#pone.0056545.s006){ref-type="supplementary-material"}). Presumably the specificity of toxin induction reflects differences in the transport and hydrolylsis of the specific peptides by *C. difficile*.

To our knowledge, this is the first report that nucleobase and nucleoside biochemicals are among the strongest *C. difficile* toxin inducers. In particular, adenine and guanosine top all substrates measured in this study, giving very high levels of toxin production (\>168,000 ng/ml and 10402 ng/ml, respecitively) when they serve as nitrogen source in the presence of 5 mM glucose. In this nucleobase and nucleoside substrate group, many derivatives of purines and pyrimidines can stimulate high *C. difficile* toxin production as well ([Figure 4A](#pone-0056545-g004){ref-type="fig"}, [Table 3](#pone-0056545-t003){ref-type="table"}). The reason is not known, but it is clearly not a random phenomenon. Maegawa et al [@pone.0056545-Maegawa1] report that there is a linkage between toxin production and purine biosynthesis in *C. difficile*. Further investigation of these nucleobases and nucleosides inducing high levels of toxin may provide a new insight into the biochemical and molecular basis of *C. difficile* toxin regulation and may also help in explaining the clinical severity of CDI. Given induction of high toxin production by uric acid, a metabolite of purine, a question could be rationally asked: what is the correlation between circulating level of uric acid and severity or even mortality of CDI in patients?

The nutrients in the basal medium used in this study were present at low concentrations and systematically varied. Each well of panels PM3, 6, 7, and 8 contains these same components except one nitrogen source which differs from well to well (Materials & Methods, [Table S1](#pone.0056545.s005){ref-type="supplementary-material"}). So, the differences in toxin production are truly related to the specific difference of the nitrogen source among the wells. This is in contrast with previous studies where much richer and more complex media were typically used. For example, amino acids were added up to 35 mM plus 27.78 mM glucose and vitamins and minerals [@pone.0056545-Haslam1], or 39 mM amino acids plus 11.11 mM glucose and other nutrients [@pone.0056545-Karasawa2], or 82 mM amino acids plus 11.11 mM glucose and other nutrients [@pone.0056545-Yamakawa1]. BHI plus 20.83 mM glucose (0.375%) or TY (3% bacto tryptose and 2% yeast extract) plus 55.5 mM glucose were used [@pone.0056545-Dineen1]; PT (peptone-yeast extract) plus 50 mM glucose (PT~0~G) or PT plus 10 mM cysteine (PY~10~) were reported [@pone.0056545-Karlsson2]. In our study, the relatively low nutritional PM culture conditions may partially contribute to the high toxin production, which agrees well with the notion that the toxin production increases when the bacteria are under stresses including environmental and nutritional factors. In fact, nutrients in the colon are at low levels based on human feces analysis [@pone.0056545-Goldblith1] and *C. difficile* must compete for nutrients against other flora [@pone.0056545-Wilson2], [@pone.0056545-Wilson3], [@pone.0056545-Wilson4]. According to Goldblith\'s analysis [@pone.0056545-Goldblith1], vitamins account on the average for about 0.01% of the feces in human and biotin is about 0.133 mg in feces collected over a period of 24 h (∼544 nM, given 0.133 mg biotin collected and assuming 1000 g wet weight of feces over 24 h). Therefore, the levels of nutrients of our PM culture conditions seem more close to the natural nutrient levels in the colon.

It is reported that before antibiotic treatment the average toxin B concentration in CDI patients\' stools was 58.29 pM (∼15.68 ng/ml). After antibiotic treatment the toxin level was 3.3 pM (∼0.89 ng/ml) [@pone.0056545-Ryder1]. Human colonic mucosae *ex vivo* testing showed damaging effects after 5 h of exposure to toxin B at a concentration about 0.2 nM (53.8 ng/ml) [@pone.0056545-Riegler1]. The TcdB~HV~ toxin can cause broad tissue damage to zebrafish embryos at concentrations about 1 nM (∼270 ng/ml) and is cytotoxic to CHO-k1 cells with TCD~50~ of 23.7 pM (∼6.4 ng/ml) [@pone.0056545-Lanis1]. In this study, the lowest concentration of *C. difficile* toxin B that can be reliably detected and quantified is as low as 0.45 pM (∼0.122 ng/ml). Therefore, our method could find applications in direct measurement of clinical samples from patients or animals to improve diagnosis, treatment, and epidemiologic studies of CDI.

*Clostridium difficile* strain VPI 10463 (ATCC 43255) is a high toxin producer [@pone.0056545-Merrigan1] and is the toxinotype reference strain, classified as toxinotype 0 [@pone.0056545-Rupnik2], [@pone.0056545-Rupnik3]. Strain ATCC 9689 is the *C. difficile* type strain, sharing the same toxinotype with VPI 10463. Presumably the toxins produced by the two strains are similar. Indeed, the neutralizing antibody raised against toxin B protein from VPI 16403 (Gallus Immunotech) is highly effective in neutralizing toxins from both strains. In a comparison study, we examined toxin production in the bacterial supernatants of the two strains under identical PM culture conditions. From this we estimate that VPI 10463 produced ∼100 to ∼1000 times more toxin in quantity (data not shown). However, some of the highest toxin-producing substrates (e.g., adenine and guanosine) already stimulate very high toxin production by ATCC 9689 and a further induction of 100--1000 fold in VPI 10463 seems less likely. A more likely explanation is that the toxin B molecules produced by VPI 10463 are more potent than those of ATCC 9689.

The toxins A and B we purchased (Listlab) have been subjected to purification and lyophilization processes whereas the toxin produced in this study are crude toxin protein preparations that are just filtered to remove cells and then assayed quickly. Given the large size of toxins A and B (300 kDa and 270 kDa, respectively), it is possible that the purified proteins may suffer some damage or conformational changes by physical or chemical forces during the processes of purification and lyophilization. Crude toxin proteins tested immediately or within a very short period of time without freezing may be more intact and therefore may retain more functionality and potency. The intactness of the toxin molecules may be particularly important for toxin autoprocessing for the toxin to become cytotoxic. If the toxin molecules in a sample are less damaged than the standard purified toxins against which the sample toxin is quantified, it may lead to assigning an artificially high toxin concentration to the sample. The opposite may also be true. Our PM culture conditions usually produce high level (or high potency) toxin at very low *C. difficile* mass, suggesting that we are more likely to be overestimating the toxin level as our preparations may contain higher levels of intact toxin molecules.

To generalize this assay technology, we also tested some other species of clostridia and other bacterial genera that are known or potential producers of cytotoxins. Species tested in this study that demonstrated cytotoxicity include *C. perfringens*, *C. tetani*, *C. sordellii*, *Bacillus cereus*, *Escherichia coli* O157:H7, *Shigella dysenteriae*, and *Listeria monocytogenes*. Species tested that did not demonstrate cytotoxicity include *E. coli* BW30270, *S. sonnei*, *L. innocua*. Thus, our new cytotoxicity assay has been further validated with diverse bacterial species.

In addition to studying the regulation of toxin production in species and strains that are known producers, the sensitive and general nature of this approach will also enable the discovery of novel toxin-producing strains and novel toxins, especially toxins that are produced only under a very restricted set of culture conditions, as was the case with the *F. graminearum* toxin [@pone.0056545-Gardiner2] which inspired these studies.

In summary, this is the first demonstration of simultaneous study of a large number of culture conditions influencing bacterial toxin production in diverse bacteria. Toxin production is a central issue in pathogenesis of *C. difficile* and other pathogenic microorganisms. Reliable, quantitative, sensitive, and robust assays for functional toxins are essential and critical in scientific research and clinical practice. The functional toxin assay method presented here is such an assay technology. It is based on the gold standard cell-based cytotoxicity assay and also takes advantage of PM technology. With hundreds to thousands different culture conditions, this toxin assay technology can provide a wealth of information and insights into the regulation of the toxin production and pathogenesis of toxingenic microorganisms. It may also find beneficial applications in clinical and epidemiological research.

Materials and Methods {#s4}
=====================

Phenotype MicroArray (PM) panels, chemicals, toxins, anti-toxins, bacterial strains, and mammalian cells lines {#s4a}
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PM panels, inoculating fluid IF-0a GN/GP Base (or IF-0a for short), Redox Dye Mix MB and inoculating fluid IF-M1 are from Biolog, Inc. (Hayward, CA, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. Yeast extract (YE) was from Oxoid (UK). *Clostridium difficile* toxin A and toxin B were from List Biological Laboratory (Listlab, Campbell, CA, USA). It is important to use antibody of high quality and antibody from some vendors was not satisfactory. We ultimately sourced anti-toxin A and anti-toxin B polyclonal chicken IgYs from Gallus Immunotech (Fergus, Ontario, Canada). *Clostridium difficile* strains: type strain ATCC 9689 and Toxinotype 0 strain VPI 10463 (ATCC 43255) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Some other bacteria obtained through commercial sources inlude: *C. perfringens* (ATCC 25763), *C. tetani* (ATCC 19406), *C. sordellii* (ATCC 9714), *Bacillus cereus* (ATCC 14579), *Escherichia coli* O157:H7 (ATCC 43894), *Shigella dysenteriae* (ATCC 11835), *S. sonnei* (ATCC 25931), *L. innocua* (ATCC 33090). Others were obtained as gifts, including *E. coli* BW30270 (from Barry Wanner, USA), *Listeria monocytogenes* strains P14 and P14-A (from Jose Vasquez-Boland, UK). Mammalian cell lines CHO-k1, Vero, HT-29, and A549 were also purchased from ATCC. Cell growth medium RPMI 1640, fetal bovine serum (FBS), Penicillin-Streptomycin, glutamine, and trypsin were purchased from Invitrogen. Glucose solution and RPMI 1640 vitamins stock solution were from Sigma-Aldrich. For preparation of bacterial culture supernatants we used 96-well filter plates (Pall, 0.2 uM pore size, PN 8015). For cell-based cytotoxicity assay and subsequent cellular dye reduction assay we used tissue culture treated 96-well plates (BD Falcon 353072).

Work flow {#s4b}
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[Figure 7](#pone-0056545-g007){ref-type="fig"} depicts and summarizes the testing and assay for measuring *C. difficile* toxin production under different culture conditions in a 96-well panel format. This process is applied to all anaerobic bacteria. The incubation time for different bacteria may vary before toxins are collected. To collect toxins from aerobic bacteria, the process is the same as that for anaerobic bacteria except bacteria are incubated in an aerobic incubator instead of an anaerobic chamber.

![Flow chart of the process used in this study to quantitate toxin production by *C. difficile* under hundreds of culture conditions using PM technology.](pone.0056545.g007){#pone-0056545-g007}

Bacterial preculture and inoculum preparation {#s4c}
---------------------------------------------

*C. difficile* and other anaerobic bacterial strains were routinely pre-cultured on BUA+B agar (Biolog) inside an anaerobic chamber (Bactron IV,) at 36°C with a gas atmosphere of 5% H~2~, 5% CO~2~, and 90% N~2~. *C. difficile* fresh cultures (20--24 h) on BUA+B were used for PM panel inoculation. The inoculum of *C. difficile* was from fresh cultures in late log phase ([Figure S2](#pone.0056545.s002){ref-type="supplementary-material"}) which were examined and found to be free of endospores ([Figure S3](#pone.0056545.s003){ref-type="supplementary-material"}). Inocula were conveniently prepared by removing colonies from a BUA+B agar plate using a swab, and resuspending in IF-0a inoculating fluid. The bacterial suspension was adjusted in IF-0a to achieve a 40% transmittance (T40) using a Biolog Turbidimeter, which was measured spectrophotometrically as 0.139±0.002 O.D. (at 750 nm in a Multiskan Ascent). The suspension was further diluted 1∶16 in IF-0a. This cell density was directly used for PM panel inoculation. This inoculum (T40 1∶16) when plated on BUA+B gave a count of 9.07E+07 CFUs/ml.

PM panel special pretreatment for anaerobic bacteria {#s4d}
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Prior to their use with anaerobic bacteria, all PM panels were converted to an anaerobic state by thorough deoxygenation. To do this, the PM panel packaging bags are cut to open one end. Two oxygen absorbers (Ageless sachets, Mitsubishi) are inserted into the bag along with the original desiccate sachet and then the bag is resealed with a heat sealer. If the bag has a good seal, the sachets will absorb the air in the bag so that the packaging appears as if it is shrink-wrapped. This operation can be done on the lab bench. The resealed PM panels can be kept at room temperature for 1 day and then stored in the refrigerator (2--8°C) for additional days or months before use. The thoroughly deoxygenated panels can then be warmed up to room temperature before being used in an experiment. It is critical that the panels are resealed well, which allows the Ageless sachets to completely remove oxygen from the panels. It is not recommended to deoxygenate the PM panels by putting them in the anaerobic chamber because the moisture inside the chamber may destabilize some substrates. To deoxygenate Biolog IF-0a GN/GP Base, the bottle caps were loosened and the bottles were placed inside the anaerobic chamber for 3 days before use.

PM media and bacterial toxin collection {#s4e}
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PM panels are 96-well microplates containing a different substrate in each well. PM1 and PM2 are carbon source panels. PM3, 6, 7, and 8 are nitrogen source panels. PM4 contains various phosphate and sulfur sources; PM5 contains various biosynthesis pathway endproducts and nutrient supplements. The substrates in the PM wells are shown in [Table S1](#pone.0056545.s005){ref-type="supplementary-material"}. In addition to a unique substrate, each well of these metabolic panels also contains the needed minimal medium components [@pone.0056545-Bochner2] but without ferric chloride, tetrazolium violet, and sodium pyruvate added.

To produce and collect bacterial culture supernatants containing toxins, *C. difficile* or other bacterial species were inoculated in duplicate or triplicate into panels PM1--8 at T40 1∶16 and incubated for an optimum length of time, e.g., 3 days for *C. difficile*. The inoculating fluid for *C. difficile* consists of Biolog IF-0a GN/GP Base, 0.5× RPMI 1640 vitamins, and 0.2% yeast extract (sterilized by filtration) for PM1, 2 or 0.05% yeast extract for PM3, 4, 5, 6, 7, and 8. To provide the carbon source in PM3--8, glucose at a final concentration of 5 mM was added to the inoculating fluid. To obtain a crude toxin preparation free of cells, the bacterial liquid cultures were transferred to a 96-well filter plate (Pall) and filtered by centrifugation at 2000 rpm for 5 minutes (Hermle Z 360 K, rotor model C-0360-50). The filtrates were used immediately (the same day) or kept in a sterile 96-well plate (Biolog) sealed with tape for brief storage at 4°C before use. No bacteria controls (i.e. no toxin controls, or PM substrate controls) were PM substrate solutions rehydrated from the corresponding PM panels, obtained by following the same procedure for bacterial supernatants described above except no bacteria inoculated into the PM panels. All experiments described above were done multiple times (2--4) as independent replicates (2--3) and each was followed by cytotoxicity assays on the bacterial filtrates collected.

All aerobic bacteria tested in this study were handled on the bench and grown on Biolog agar medium (BUG+B) as a preculture and then inoculated and incubated aerobically in PM panels without shaking. After 24 h of incubation in the panels at 37°C, the bacterial supernatants were collected using the 96-well filter plates and centrifugation, and then handled and tested as described above.

The toxin-containing supernatants should be used as soon as possible after harvest because refrigeration temperature does not completely maintain the toxins during long term storage and the potency of the toxins was observed to decrease over time. Better long term storage of the toxins requires purification and lyophilization. These same procedures were followed for preparing PM substrate suspension controls without *C. difficile*.

Determination of bacterial mass in wells of PM panels {#s4f}
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To deteremine bacterial mass kinetics, PM panels inoculated with *C. difficile* in triplicate were removed from the anaerobic chamber at 24, 48, or 72 h of incubation. Bacterial mass was determined by measuring the optical density of wells at 750 nm using a microplate reader (Multiskan Ascent). Compared to 48 h or 72 h, the values of OD-750 at 24 h of incubation were the highest under most PM well conditions. With a few exceptions, all the OD-750 values decreased more or less at 48 h and further decreased at 72 h ([Figure S1](#pone.0056545.s001){ref-type="supplementary-material"}). Given the nutritional limitations of the media, the growth was limited, but the kinetic trends were clear and similar to each other among the various culture conditions ([Figure S1](#pone.0056545.s001){ref-type="supplementary-material"}).

To determine the bacterial mass at the end point and before harvesting toxin-containing supernatants, the PM panels with *C. difficile* in replicates were removed from the anaerobic chamber at 72 h and the mass was deteremined as decribed above. To obtain net bacterial mass, the OD-750 value from the corresponding well of an uninoculated plate is subtracted from the OD-750 value of the inoculated well. These bacterial net mass values can be used to normalize toxin production when a specific cell productivity measurement is needed.

Mammalian cell lines and their cytotoxicity and neutralization assays {#s4g}
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Cell lines CHO-k1, Vero, HT-29, and A549 were grown at 37°C in an incubator atmosphere of 5% CO~2~ in standard T75 cell culture flasks with RPMI 1640 medium plus 10% FBS and 1× Pen/Strep, without phenol red. The cells were allowed to grow for 20--24 h before harvesting for experiments. This young cell culture was then prepared in an assay medium (Biolog IF-M1 inoculating fluid plus 2 mM glucose, 2 mM glutamine, 2% FBS, and 1× Pen/Strep) at a density of 200,000 cells per ml. A 100 µl aliquot of this cell suspension was plated into each well of tissue culture treated 96-well microplates, the assay plate. Five microliters of toxin preparation either from serial titrations of purified standard toxin (Listlab) or from supernatants of microbial culture filtrates collected from each PM culture condition were transferred to each well of the assay plate immediately following the cell plating. This makes the toxin preparation a 21-fold dilution \[(5+100)/5\]. Five µl of corresponding PM substrate solution were used as no toxin control. The assay plates of the treated indicator cells were then incubated at 37°C with 5% CO~2~ for 18--20 h.

For neutralization experiments, a neutralizing antibody was mixed with the indicator cell suspension immediately prior to cell plating. The cell plating was followed immediately by addition of the toxin preparation to the plated cells. After 18--20 h of incubation, cell morphologies were observed under the microscope (10×10 magnification) and images were recorded with a digital camera. These same cells were further tested for their ability to reduce the Biolog Dye Mix MB. Twenty µl of the dye solution was transferred to each well. The plates were then placed into the OmniLog PM instrument (Biolog, Inc.) for incubation and kinetic data collection for 3 h or longer. As the dye is reduced, a purple color is irreversibly developed. The healthier the cells are, the more NADH they produce and the higher the rate of dye reduction [@pone.0056545-Bochner4].

Determination of dye reduction rate by the mammalian cells {#s4h}
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The dye reduction by indicator cells was kinetically measured during the incubation period by the OmniLog instrument. The resulting dye reduction values were imported into a PM analysis program, which can calculate a rate of dye reduction based on a linear regression algorithm. The rate of dye reduction within the first few hours of incubation (e.g., 0 through 3 h) was calculated. The effects of standard *C. difficile* toxin B or *C. difficile* supernatants collected from different PM conditions on the rate were studied. The average rates of the standard toxin or of the supernatants were used in all subsequent calculations.

To correct for the influence (positive or negative) of a given PM substrate on the rate, the dye reduction rate of the PM substrate control is measured. A ratio of a PM substrate is derived from the control set and defined as a quotient of the average rate of all wells for a given PM panel and its replicas (e.g., an average of 3 PM1 panels is obtained from 3×96 wells) divided by the average of a given well in that panel. So, if a PM substrate has a positive influence on the dye reduction rate of the indicator cell line, the ratio will be smaller than 1, otherwise greater than 1. For example, D-glucose ([Table S1](#pone.0056545.s005){ref-type="supplementary-material"}, PM1 C9) could positively influence dye reduction because the substrate control experiments showed it was associated with higher dye reduction rate than the panel average (78.48 vs 74.93). So, the ratio for D-glucose is smaller than 1 (0.9547). The average dye reduction rate of CHO-k1 in the presence of *C. difficile* supernatant from D-glucose is 57.19, but the corrected dye reduction rate is 57.19×0.9547 = 54.6.

In general, the rate of dye reduction by cells can be affected by many factors, e.g., cell lines, cell number and fitness, adverse environmental conditions (e.g., toxic chemicals), nutrient or energy source used to support cell respiration, the type of redox dyes, and so on. In these experiments, however, all factors and conditions given are controlled except the levels of toxins produced by the microorganism under the different culture conditions in the PM panels.

As mentioned above, all experiments were done multiple times and as independent replicates. From these, the averages and standard deviations of dye reduction rates were calculated, which are expressed as a mean +/− standard deviation and ploted as histograms exemplified by [Figrure 3D](#pone-0056545-g003){ref-type="fig"}. Statistical analyses were perfomed using Microsoft Office Excel TTEST via Excel automation on the rates between the experimental set and the control set on a well-by-well basis. The data were treated as two-tailed distributions with unequal variance. The difference is considered significant only if the probability of no difference between the means of the two sets is smaller than 5%, P\<0.05.

Having measured the effect of the toxin as a dye reduction rate, the next step is to convert the rate into a concentration value for the toxin.

Determination of toxin production by *C. difficile* and other Clostridium species {#s4i}
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Anaerobic bacteria used in this study including *C. difficile*, *C. perfringens*, *C. tetani*, and *C. sordellii*, were incubated for three days before removal from the anaerobic chamber for cell mass measurement and toxin collection. (1) Establishment of standard curves of toxin and dye reduction rate. *C. difficile* toxins A and B from Listlab were used as standards to determine the relationship between toxin concentration and the corresponding level of inhibition of dye reduction rate by the indicator mammalian cells. Cytotoxicity and dye reduction assays with serial 2-fold or 3-fold standard toxin titrations were performed multiple times. For a given indicator cell line, the known concentrations of the toxin (ng/ml) used in the assay were plotted on the Y-axis with a log~10~ scale against corresponding dye reduction rates plotted on the X-axis ([Figure S4](#pone.0056545.s004){ref-type="supplementary-material"}). Using Microsoft Office Excel 2002 software, the plots were fitted to curves by non-linear regression analyses and the equations for predicting concentrations from the rates were generated ([Figure S4](#pone.0056545.s004){ref-type="supplementary-material"}, [Table 1](#pone-0056545-t001){ref-type="table"}). (2) Determination of toxin production by *C. difficile* under different PM culture conditions. The corrected average dye reduction rates from the *C. difficile* supernatants were used to calculate the toxin concentration according to the equations ([Figure S4](#pone.0056545.s004){ref-type="supplementary-material"}, [Table 1](#pone-0056545-t001){ref-type="table"}). The calculations were performed robustly by employing Excel worksheet functions.

Supporting Information {#s5}
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***C. difficile*** **mass kinetics in wells of PM panels.** The bacterial mass was determined at 24, 48, and 72 h of incubation as described in [Materials and Methods](#s4){ref-type="sec"}. A, PM1. B, PM3.

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**Growth curve of** ***C. difficile*** **colonies on BUA+B.** Single colonies of *C. difficile* (ATCC 9689) grown on BUA+B were picked up and cell counts were made at the time points, 18, 24, 48, and 72 h. As the colony size was very small at 18 h, 4 colonies were used instead of one, but the count of colony forming units (CFUs) was normalized to a single colony by dividing by 4. To count the viable cells, the colonies were suspended in IF-0a followed by serial dilution and plating on BUA+B. The number of viable cells was determined by counting CFUs.

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**Endospore formation of** ***C. difficile*** **(ATCC 9689) at different ages on BUA+B.** At 24, 48, or 72 h of incubation on BUA+B, a cell suspension was made from a single colony in 1 ml IF-0a inoculating fluid. The cell suspension was then incubated in an eppendorf tube without or with 200 proof ethanol (cell suspension ∶ ethanol = 1∶1) for 1 h inside the anaerobic chamber. Then, 30 µl of each suspension was plated on a BUA+B plate. All plates were incubated in the chamber at 36°C for 24 h and then photographed. As shown, without ethanol, a bacterial lawn was formed from cultures of different ages on BUA+B (24, 48, and 72 h). Endospores appear as colonies that survive the ethanol treatment. Note that no endospores were formed from the 24 h culture but then the number of endospores increased as the culture age increased (72 h\>48 h).

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**Plots of dye reduction rate by CHO-k1 cells against corresponding standard** ***C. difficile*** **toxin B.** A serial 3-fold titrations of standard toxin B (Listlab) was performed on CHO-k1 cells. From top panel to bottom: The toxin concentrations decreased. Dye reduction kinetics were recorded by the OmniLog instrument and reduction rates (0--3 h) at different toxin concentrations were calculated using PM Analysis Software. The known standard toxin concentrations (Y-axis) were plotted against the corresponding Mean ± Stdev of the rates (X-axis). Regression analyses on the data were performed using Microsoft Excel 2002 software. Each equation given was suitable only for a certain range of toxin level as illustrated, and may be suitable only for the given conditions (cell line, assay conditions, type of reducible dye, etc.) as described in the text.

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Plate maps of Phenotype MicroArrays microplate panels PM1 through PM8.

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Toxin production of *C. difficile* ATCC 9689 under different PM conditions.

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The authors would like to thank Eric Olender, Ph.D., for his help with data analysis.

[^1]: **Competing Interests:**This study was partly funded by Biolog Inc. Xiang-He Lei is employed by Biolog Inc. Barry Bochner is the CEO and CSO of Biolog Inc., has ownership of stock and made the decision to publish the manuscript. Patents 6,271,022, 6,387,651, 6,436,631, 6,472,201, 6,686,173, 6,696,239 and 6,727,076 have been issued to Biolog, Inc., and the Phenotype MicroArrays described in this manuscript are produced and marketed by Biolog, Inc. There are no further patents, products in development or marketed products to declare. This does not alter the authors\' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. The authors include the names of the patents (Journal requirement 3) as follows: 6,271,022: Device for Incubating and Monitoring Multiwell Assays; 6,387,651: Comparative Phenotype Analysis of Two or More Microorganisms Using a Plurality of Substrates within a Microwell Device; 6,436,631: Comparative Phenotype Analysis for Assessment of Biological Active Compounds such as Antimicrobials; 6,472,201: Comparative Phenotype Analysis; 6,686,173: Comparative Phenotype Analysis of Two or More Microorganisms Using a Plurality of Substrates within a Multiwell Testing Device; 6,696,239: Comparative Phenotype Analysis for Assessment of Biological Active Compounds such as Antimicrobials; 6,727,076: Comparative Phenotype Analysis.

[^2]: Conceived and designed the experiments: XHL BB. Performed the experiments: XHL. Analyzed the data: XHL BB. Contributed reagents/materials/analysis tools: XHL. Wrote the paper: XHL BB.
