Strain-to-strain variation of Rhodococcus equi growth and biofilm formation in vitro

Objective Rhodococcus equi is an opportunistic pathogen that causes disease worldwide in young foals and immunocompromised humans. The interactions of R. equi with the host immune system have been described; however, most studies have been conducted using a few well-characterized strains. Because biological differences between R. equi strains are not well characterized, it is unknown if experimental results will replicate when different strains are used. Therefore, our objective was to compare the growth and biofilm formation of low-passage-rate clinical isolates of R. equi to higher-passage-rate, commonly studied isolates to determine whether strain-to-strain variation exists. Results Twelve strains were used: 103+, ATCC 33701, UKVDL206 103S harboring a GFP-expressing plasmid, a plasmid-cured 33701 (higher-passage-rate) and seven low-passage clinical isolates. Generation time in liquid revealed fast, moderate-fast, moderate-slow, and slow-growing isolates. The higher-passage-rate isolates were among the moderate-slow growing strains. A strain’s rate of growth did not correspond to its ability to form biofilm nor to its colony size on solid media. Based on our results, care should be taken not to extrapolate in vitro work that may be conducted using different R. equi strains. Further work is needed to evaluate the effect that the observed differences may have on experimental results. Electronic supplementary material The online version of this article (10.1186/s13104-019-4560-1) contains supplementary material, which is available to authorized users.


Introduction
Rhodococcal pneumonia is an important cause of morbidity and mortality in young foals worldwide and an emerging opportunistic pathogen of immunocompromised humans [1]. The etiologic agent, Rhodococcus equi, is a Gram-positive, aerobic, soil-dwelling opportunistic pathogen [2][3][4]. Inside the host, it is a facultative intracellular pathogen that replicates within macrophages, where it prevents acidification of the phagolysosome [5,6]. Despite characterization of the immune system's interactions with this organism and the identification of some virulence factors of R. equi [7], understanding of its pathogenesis is limited. The virulence-associated plasmid (VAP), and VapA in particular, is critical for virulence in horses and is routinely used to differentiate pathogenic from non-pathogenic strains [8,9]. Virulence determinants appear to be conserved in the core genome of R. equi [10,11], but differences in their expression have yet to be characterized.
Several genotypically distinct isolates of R. equi have been obtained from soil, air, water, and feces at horse breeding farms [12,13], but the majority of studies to date have been conducted using a few relatively well characterized strains [2,7,14,15]. The advantage to this approach is reproducibility among studies and the ability to build on previous work. However, it is hard to predict if the observed results will be replicable using different strains. Therefore, our objective was to compare the growth and biofilm formation of low-passage-rate clinical isolates of R. equi to commonly studied and higher-passage-rate isolates to determine whether strain-to-strain variation exists.

Growth
All isolates were grown three ways: in liquid media with shaking to measure the rate of planktonic growth; on solid media to measure colony appearance and size over time; and in static culture in 96-well microtiter plates incubated for up to 72 h, to evaluate biofilm formation.
Growth in liquid media For each strain, a single colony isolate from a BHI agar plate (cultured as described above) was standardized and inoculated to ~ 10 5 colony forming units (CFU) per mL in 5 mL of pre-warmed BHI broth in a 17 × 100 mm plastic culture tube (Falcon, Corning, NY). Cultures were incubated at 37 °C with 100 rpm shaking for 40 h (until growth plateaued), OD 600nm was measured, and cells were enumerated by serial dilution and plate counts to determine CFU/mL immediately after inoculation (0 h post-inoculation; hpi), at 4 h intervals from 0 to 24 hpi, and again at 40 hpi. All growth curves were repeated three times for strains that grew consistently and up to six times for strains that exhibited some variation in their growth curves.
Growth on solid media To measure colony size development over time on solid media, BHI broth cultures for each strain were serially diluted in PBS, 100 µL was spread onto BHI agar plates and incubated at 37 °C for 48 h. Colony diameters were measured every 4 h from 24 to 48 hpi using a ruler and calipers, as previously described [21]. Experiments were repeated on three separate occasions, and each colony was treated as a biological replicate.

Biofilm assay
A biofilm formation assay was conducted as previously described [22], with some modifications. Briefly, cultures were inoculated as described for liquid growth curves to standardize the inoculum to an input CFU of ~ 10 5 CFU/mL, which was confirmed by serial dilution and plate counts. For each strain, 100 µL of bacterial culture at 0 hpi were added to four replicate wells of a 96-well plate (CoStar, tissue culture-treated, Corning, NY). Two , BHI wells were used to blank experimental wells and absorbance results were standardized to input CFU for each strain as previously described [22]. The assay was repeated four separate times per strain. Four empty wells per plate were also stained with crystal violet alongside experimental wells in order to confirm that the plasticware did not retain any stain.

Statistics
Data were analyzed using a commercial software (Sig-maPlot, SPSS, Chicago, Illinois, USA). The Shapiro-Wilk test was used to test for normality. CFU/mL, colony size, and absorbance were compared using one-way analysis of variance (ANOVA) or Kruskal-Wallis one-way ANOVA on ranks. Multiple comparisons were performed using Fisher LSD or Dunn's methods, respectively. Significance was set at p < 0.05. All raw data collected and used for statistical analyses can be accessed in Additional file 1.

Results and discussion
The purpose of this study was to determine whether low-passage-rate R. equi clinical isolates were phenotypically different than the higher-passage-rate isolates. R. equi is a prevalent opportunistic pathogen that causes a high financial burden on the horse industry worldwide, but there is limited information on potential differences between R. equi strains and the implications this may have on disease development in foals. Most studies to date have been performed using a few strains of R. equi (namely 103+, 33701, or UKVDL206), but here we demonstrate that there are differences between strains in the three phenotypic characteristics we evaluated. The implications these differences may have in in vitro studies remain undefined.
The low-passage-rate strains (WSU001-007) were isolated from foals except for WSU001, which was isolated from a human. All isolates were confirmed to be R. equi as they tested positive for choE by PCR. All isolates except for ATCC 33701 pc also tested positive for the vapA virulence-associated gene (data not shown) and are therefore considered pathogenic to horses.
To determine whether measurable differences among strains existed, isolates were grown in liquid and on solid media, and their ability to form biofilms in vitro was measured (summarized in Table 2 and Additional file 2: Table S1). A study of Pseudomonas aeruginosa using the same three growth methods employed here found that biofilms more closely resemble exponentially growing planktonic cells and that solid and liquid media growth yield similar protein expression profiles [23], suggesting that these three modes of growth may provide a better in vitro model of bacterial growth than employing one method alone. Using data from liquid growth curves (Additional file 2: Fig. S1; Additional file 2: Table S2), generation times were calculated and sorted from fastest-to slowest-growing isolates (Fig. 1). Natural breakpoints in generation times divided isolates into four groups: fast (0.9-0.93 h generation times), moderatefast (1.01-1.05 h), moderate-slow (1.12-1.16 h), or slow (1.22-1.31 h) growers. The fast and moderate-fast groups were comprised entirely of low-passage-rate isolates. The moderate-slow group contained the higher-passagerate isolates 103+, UKVDL206, ATCC 33701, and 33701 pc. The slow group had one low-passage isolate and the sub-cultured 103S-GFP laboratory strain. The three fast strains grew significantly (p < 0.001) faster than the moderate-slow strains. The fast and moderate-fast isolates all grew significantly (p < 0.001) faster than slow isolates.
Differences were observed in the rate of colony appearance on solid media for the isolates studied; therefore, colony sizes were measured from 20 to 48 h to see whether this corresponded with liquid culture generation times (Table 2; Additional file 2: Fig. S2). The slow-grower 103S-GFP had the smallest colonies on solid media, but no other obvious commonalities were observed between solid and liquid growth. There have been no reports in the literature on the different rates of growth of R. equi strains on solid media, but colony size can be affected by the amount of capsule or extracellular polymeric substance (EPS) produced by a given strain [24]. Further examination of capsule expression would be required to validate this hypothesis.
The ability of R. equi to form biofilms has been previously reported [25,26], therefore biofilm formation was examined here. Biofilm production varied greatly between strains (Fig. 1). UKVDL206 formed the most biofilm, while slow-grower WSU006 formed the least. Biofilm formation at 24 and 48 h showed no significant differences among isolates (data not shown). Using the cut-off imposed by Gressler et al. [25], whereby R. equi strains were deemed to be biofilm-producers if the crystal violet staining exceeded the negative control, all 12 isolates in our study were considered biofilm-producers. While this is consistent with results reported by others [27], some have reported R. equi strains that lack the ability to form biofilms in vitro [26]. In the present study, 103 + and 103S-GFP were relatively high biofilm formers, suggesting that the presence of the GFP plasmid did not affect this phenotype. The loss of the VAP did not affect the rate of biofilm formation of 33701, suggesting that most biofilm-associated genes are chromosomally encoded rather than plasmid-encoded. The lack of difference in growth rates between the plasmid-cured and VAP-carrying 33701 strains may be due to the fact that isolates were only grown at one temperature (37 °C). While plasmid-bearing cells have been shown to replicate slower than plasmid-cured derivatives, such differences were dependant on incubation temperature [28]. Overall, there were no obvious shared trends between growth and biofilm experiments under the conditions examined.
In summary, the 103 isolates grew more slowly in liquid, formed smaller colonies on solid media, but formed more biofilm than most strains tested. UKVDL206 grew more slowly in liquid, formed smaller-than-average colonies on solid media, but produced the most biofilm. WSU006 grew slowly and plateaued earlier than most isolates in liquid culture, produced the least biofilm, but formed slightly largerthan-average colonies on solid media. Strains 33701 and 33701 pc grew moderately-slowly but had larger colonies on solid media; however, both were only average biofilm formers. Finally, WSU007 was among the moderately-fast-growing strains but plateaued and declined early in liquid culture, had only moderatelylarge colonies on solid media, and was a moderate biofilm former.