Repeated laboratory passage of pathogens in the presence of rifampicin has been used to generate live-attenuated vaccines [1, 7–12], but the mechanism of rifampicin-induced attenuation remains unknown. There are a variety of potential confounding factors with this type of passage experiment that make cause-and-effect interpretations challenging. Passage can produce changes in global protein expression profiles [12, 13] or altered lipopolysaccharide (LPS) biosynthesis/colony roughness [7]. These changes could be attributed to altered activity of RpoB in the Rif resistant DNA-dependent RNA polymerase (RNAP) present in rifampicin resistant bacteria [14]. That is, the presence and activity of the mutated rpoB is one hypothesis for the mechanism of attenuation. Alternatively, rifampicin-associated attenuation may result from accumulation of spontaneous mutations acquired in the course of in vitro passaging. Serial passage of a pathogenic bacterium without any antibiotics can also induce changes in LPS profile and colony roughness, which have been correlated with attenuation [18]. This finding is consistent with the role of random mutations in the attenuation process, although the rate of mutation may differ depending on the stress imposed on the passaged microorganism [20, 21]. For example, for the current study the two strains that were passaged with rifampicin, CR and TR, accumulated more SNPs compared to their matched strains that were passaged without the antibiotic (CN and TN, 33.3 and 29.4 % respectively).

In the present study, passage did not affect in vitro growth characteristics except for the CR strain, which was compromised to some extent (Fig. 1). Others have reported that acquisition of rifampicin resistance can negatively impact growth rates although this probably depends on the exact mutation that is acquired in rpoB [22–24]. When grown as a colony on agar plates, the CR colonies had smooth edges and the CR strain lacked gliding motility, both changes that could be explained by either altered function of a mutated rpoB or other SNPs in relevant genes. For example, colonies of the CR grown on TYES and GMA media have reduced yellow pigmentation compared to the CW and CN strains. This phenotype may be attributed to the Thr365Lys mutation in the phytoene desaturase (crtI) gene, which encodes an enzyme involved in carotenoid biosynthetic pathway in Flavobacterium sp. [25–27]. Microscopic analysis of gliding motility and colony morphologies of TW, TN and TR strains did not reveal any phenotypic changes, despite the large number of SNPs that were accumulated by the TN and TR strains.

Polyacrylamide gel electrophoresis of LPS fractions revealed no obvious differences in LPS profiles, including no apparent changes from an Ile376Leu substitution in O-antigen acetylase (FPSM_01556) in the TR strain. 2D-PAGE analyses of proteins revealed probable differences in protein synthesis among rifampicin resistant F. psychrophilum strains (B17, CR and TR), which is consistent with our previous studies [14]. It is important to emphasize that while rpoB mutations might alter transcriptional regulation, other mutations in these strains could have contributed to this effect. Specifically, SNPs in proteins directly involved in gene/protein expression such as DNA primase, ribosome recycling factor, elongation factor P (EF-P), ATP-dependent RNA helicase (FPSM_01657) in the B17 or transcriptional regulator FPSM_00453 and ATP-dependent RNA helicase DbpA (FPSM_01345) in the TR strain.

Remarkably, considerable reduction in virulence of the CN (28.1 % mortality) and CR (4 % mortality) strains occurred despite relatively few SNPs leading to nonsynonymous amino acid changes (5 and 8, respectively). There were overlapping synonymous mutations among these strains and synonymous SNPs have been reported to potentially affect protein function [28]. Aside from SNPs occurring in the rpoB sequence, there was no overlap in SNPs for the attenuated B17 strain and the CR and CN strains developed in the current project. Noteworthy, 3 genes (nrdA – encoding a large chain of ribonucleotide-diphosphate reductase, yihA – encoding a GTP-binding protein and a putative membrane spanning protein FPSM_02361) out of 4 that harbor SNPs in the CN strain are identical with the CR strain. We surmise that the probability of these identical SNPs arising independently is unlikely. Instead, they most likely arose from the original culture used to initialize the passage experiments (i.e., a founder effect), or a cross-over contamination event occurred sometime early in the course of the experiment; if this was a contamination event, it must have occurred early in the experiment because these strains have distinct rpoB mutations making it easy to differentiate the final cultures. One of these shared SNPs is in nrdA, which has been implicated in pathogenesis of Pseudomonas aeruginosa [29]. Additionally, in Escherichia coli yihA is a GTPase that is essential for normal cell morphology and coordination of division; thus SNPs in this gene may contribute to attenuation [30, 31]. Because adhesion is an essential process in host-pathogen interaction and pathogenesis, mutation in the putative membrane spanning protein FPSM_02361 may have a direct effect on reduced virulence of CN and CR strains. Collectively, these findings are consistent with involvement of mutational events in the process of attenuation.

The CR strain was mostly attenuated (4 % mortality) and was characterized by slower growth in culture, lost gliding motility, three observable changes in protein synthesis, and altered carotenoid metabolism that is probably due to mutated phytoene desaturase (ctrI). As part of a gene cluster involved in carotenoid biosynthesis, mutations in ctrI were shown to alter colony pigmentation of other pathogens [32]. Importantly, mutations of ctr genes are able to decrease resistance to oxygen radicals and reduce growth in macrophages of the fish pathogen, Mycobacterium marinum [33]. Additionally, golden pigment synthesized through carotenoid pathway in Staphylococcus aureus enhances virulence by promoting resistance to respiratory burst of neutrophils, and the ∆crtM strain of S. aureus was also attenuated in mice model [34].

Additional SNPs unique to the attenuated CR strain included those found in a hypothetical protein (FPSM_01200), a transcriptional regulator (TetR family protein, FPSM_02533) and the Ser492Phe substitution in rpoB. The FPSM_01200 hypothetical protein has a predicted function of an N-acetylglucosamine (NAG) kinase, an enzyme important for bacterial cell wall and LPS metabolism. Moreover, importance of NAG metabolism was implied in initiation of murine intestine colonization by E. coli [35] and in bacterial signaling and growth in mucus of P. aeruginosa [36]. Together, these missense mutations may affect the virulence of the CR strain and its ability to colonize host and survive in challenged fish. In our analysis, however, we cannot discount the possible effects of silent SNPs on phenotype changes as described by Kimchi-Safraty and co-workers [28].

Our alternative hypothesis that rpoB can be directly involved in attenuation is potentially supported by the fact that RpoB is crucial in bacterial transcription and allosteric changes of the mutant protein may produce global alterations such as phenotype changes. The potential involvement of rpoB Ser492Phe mutation in attenuation of the CR strain is supported by existence of phenotypic (e.g. loss of gliding motility) and proteomic changes, and by a Gln474Arg rpoB mutation in the completely attenuated B17 strain [13, 14]. Furthermore, in other bacteria different rpoB mutations lead to different phenotypic changes [22–24, 37–39]. In Brucella spp. virulence and colony roughness of Rif^R phenotypes also varied depending on the position and character of single amino acid substitutions of RpoB [17]. Mutations providing resistance to rifampicin are clearly not universally responsible for attenuation because presumptively virulent Rif^R strains of Mycobacterium tuberculosis have been described [40–42].

Unlike attenuated strains derived from CSF 259–93, the TN and TR strains from wild-type THC 02–90 accumulated a large number of SNPs from serial passage regardless of the presence of rifampicin, and yet these changes had no apparent effect on virulence. The large numbers of SNPs present in the TN and TR strains as compared to the CN and CR strains may be attributed to the fact that the CSF 259–93 and THC 02–90 belong to different genetic lineages of F. psychrophilum. These two lineages are generally associated with different fish hosts, and the lineage to which THC 02–90 belongs is amenable to some genetic manipulations, which have not been successful with the lineage of strains to which CSF 259–93 belongs [19, 43–46]. It is notable that 54 and 46 % of the nonsynonymous mutations found in the TN and TR strains, respectively, occurred in subtilisin-like proteases that are putative cell surface proteins with predicted leucine-rich repeats. Repeat regions in DNA sequences are notoriously difficult to assemble correctly; particularly when using short read technologies such as employed herein [47, 48]. Notably, however, these same regions are also found in the CSF 259–93 genome and yet there were no mutations found in these gene sequences for the CN and CR strains. Given that all of these passaged strains introduced in this study were developed and sequenced at the same time using the same chemistries, we submit that this is not a sequencing artifact but that there is probably a distinctly different mutation process underway between the CSF 259–93 strains and the THC 02–90 strains. More strains from the two lineages need to be tested to determine if this is a lineage-level difference or if THC 02–90 is unique in this regard.

Additional changes at the SNP level, such as a SNP in nrdA in CR, and mutation in the FPSM_00026 gene encoding multimodular transpeptidase-transglycosylase PBP 1A and mreC in the B17 also support a mutation-dependent attenuation hypothesis because these genes have been associated with virulence (e.g. nrdA in P. aeruginosa [29], PBP 1A in group B streptococci [49], and MreC in Salmonella [50]). Alternatively, if rpoB mutation is more important, then the exact position of the mutations may be important. For example, TR was not attenuated, but it also had distinctly different mutations in the rpoB (Asp477Tyr and Pro496Ser) as compared to single amino acid substitutions of the mostly attenuated CR (Ser492Phe) and the fully attenuated B17 (Gln474Arg) strains. The large differential in SNP accumulation between passaged strains of CSF 259–93 and THC 02–90 are consistent with differential mutation process and coincidently may reflect differences at the lineage level [19].

Unfortunately, there are no molecular tools allowing for universal allelic exchange of genes in F. psychrophilum [44, 51]. Therefore, there is no direct way of assessing rpoB involvement in the process of rifampicin-induced attenuation. Introduction of the mutant rpoB allele from the B17, CR and TR into the wild-type F. psychrophilum would be the most direct method to investigate the role of rifampicin-resistant RNA polymerase in attenuation of these bacterial strains. Other options to characterize of attenuation in the CN, CR and B17 strains would be comparison of transcriptomes or more in-depth analysis of DNA methylation patterns between these and the virulent CSF 259–93 strain to examine possible global transcriptional changes from rpoB mutations or altered DNA methylation that might contribute to differential transcriptional regulation.

Differential DNA methylation may be associated with loss of virulence as altered DNA methylation affects these traits in other bacterial species [52, 53]. Others have found evidence for different methylation patterns among F. psychrophilum strains using restriction enzyme analysis [44, 54, 55]. Consequently, it was not entirely unexpected to find differences in methylation motifs based on the PacBio analysis (Additional file 1: Tables S3 and S4). It is remarkable, however, that there were no shared motifs between these two strains. Unfortunately, this also means that the comparison of the CR and TR strains provides no insight into the potential contribution of methylation to attenuation. Nevertheless, given that methylation and restriction enzymes can function to defend bacteria from foreign DNA, it is tempting to speculate that the two lineages of F. psychrophilum have diverged due to the influence of different phage communities. CSF 259–93 is most closely associated with freshwater fisheries whereas the THC 02–90 lineage is most closely associated with anadromous fish. It is likely that phage communities differ substantially between these ecosystems.