L-cysteine transporter-PCR to detect hydrogen sulfide-producing Campylobacter fetus

Introduction

Campylobacter fetus is best known as a major veterinary pathogen that has a detrimental effect on reproductive efficiency of herds. However, in humans, this bacterium can also cause intestinal illness and, occasionally, severe systemic infections and thus the products from cattle and sheep are suspected as sources of transmission (Wagenaar et al., 2014). The classification of C. fetus subspecies relies on clinical features, host specificity, and phenotypic traits. Despite technical limitations and variable success, hydrogen sulfide (H₂S) production as well as tolerance to glycine and NaCl, selenite reduction and resistance to antibiotics are the available biochemical tests currently employed as differential diagnosis of C. fetus (OIE, 2018; Schulze et al., 2006).

Members of C. fetus have different tropism, as evidenced in veterinary practice and in the diagnosis. The subspecies C. fetus venerealis (Cfv) is restricted to the bovine reproductive tract, and is associated to the venereal disease bovine genital campylobacteriosis (BGC), whereas C. fetus fetus (Cff) is mainly intestinal and is usually related to sporadic abortion. To date, the bovine products are subjected to strict regulations by the World Organization for Animal Health (OIE) and must be tested for the presence of C. fetus subsp. venerealis before international trading (OIE, 2018). Therefore, its differentiation at the subspecies level is critical. The isolation of the bacteria can confirm BGC and subsequently biochemical tests can determine the particular different isolates. Among the biochemical tests, glycine resistance and hydrogen sulfide(H₂S) production are two of the best biochemical performing tests. For example, Cff strains show 1% glycine resistance and produce H₂S in L-cysteine (L-Cys) enriched media. By contrast, Cfv strains fail to grow in 1% glycine-containing media and to produce H₂S (Véron & Chatelain, 1973). Hence, these traits allow their discrimination. A glycine-tolerant variant of Cfv (C. fetus venerealis biovar intermedius, Cfvi) are frequently isolated in some countries such as USA, UK, South Africa and Argentina, which complicates their accurate identification (Schmidt, Venter & Picard, 2010; Van Bergen et al., 2005; Iraola et al., 2013). A third-host associated subspecies, C. fetus subsp. testudinum, completes the list of subspecies of C. fetus. This subspecies has been isolated from reptiles and humans (Fitzgerald et al., 2014) and therefore would not be relevant for animal production.

In a previous wide genome association study, Van der Graaf-van Bloois et al. (2016a) described a recent diversification of mammalian C. fetus and implicated a genetic factor associated to H₂S production. They described a deletion in an ATP-binding cassette-type L-Cys transporter in Cfv strains. The operon structure of this L-Cys transporter has five coding sequences and three of them code for different molecular components of the transporter: the ATP-binding protein, the permease, and the substrate-binding protein (locus tags CFF8240_RS03845, CFF8240_RS03850 and CFF8240_RS03855 in C. fetus 82-40 genome, respectively). This L-Cys importer could be part of the Class 3 ABC-transporters (Licht & Schneider, 2011) and in Cfv the permease and the extracellular binding domain coding genes are deleted. This deletion may impair the transporter assembly, affecting the up-take of L-Cys. This therefore could explain the impaired production of H₂S from this amino acid in Cfv strains. On these bases, we aimed to develop a simple molecular technique for detecting the L-Cys transporter-deletion polymorphism with the main purpose of identifying H₂S-producing C. fetus strains.

Materials and Methods

L-Cys transporter-PCR

One forward and two reverse primers (Fwd 5′-gtccatttacttatcacgataacagtgg-3′, Rev1 5′-gatattaggctaagaggaatggtgtattg-3′ and Rev2 5′-ctcccgtatctacatgaaagctaatatc-3′) were designed for a multiplex-PCR format using open source Unipro UGENE 1.31 (Okonechnikov et al., 2012) (Fig. 1A). The amplification mix consisted of 1 × GoTaq green Reaction buffer (1.5 mM MgCl₂), 0.25 mM of each dNTP, 0.1 μM of each primer, and 1.25 U Taq polymerase (Promega Corp., Madison, WI, USA), nuclease-free water to reach a final volume of 25 µl and Campylobacter DNA template.

The touch-down amplification program consisted of an initial step at 94 °C for 3 min, 10 cycles at 94 °C for 1 min, followed by annealing temperatures starting at 55 °C for 1 min and decreasing 1 °C per cycle from 55 to 45 °C. Then, an extension step was performed at 72 °C for 1 min, followed by 30 cycles with an annealing at 51 °C, and a final termination step at 72 °C for 8 min.

Under these conditions, the absence of the expected product of 1,390 bp makes the interpretation of the PCR results easy. A product of 714-bp is indicative of Cff and Cfvi strains (which have a complete version of the operon and are H₂S-producing strains), whereas a 310 bp product refers to Cfv strains (which contain a partly deleted operon and are non- H₂S-producing strains). All the products were resolved in 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR-products were submitted to the UGB unit-INTA to confirm their identity through Sanger sequencing.

Results

Discussion

To date, phenotypic tests are among the most valuable methods to identify and differentiate microorganisms. However, these tests are usually time-consuming, because they are growth-rate dependent, and the whole process depends on the objectivity and skills of the operator. Furthermore, a proper standardization, which is essential to obtain reliable and reproducible results, is often absent. Despite all this, the replacement of these phenotypic tests by molecular techniques is not always an alternative to date. The antimicrobial resistance constitutes a good example of complementary testing, and this particular phenotypic trait can be tested by bacteriological methods and at molecular level in several pathogens (Fluit, Visser & Schmitz, 2001).

Over the last years, researchers have proposed many genotypic tests to facilitate C. fetus differentiation. For example, different studies have employed molecular techniques such as PCR based on different target genes to differentiate Cfv from Cff (Hum et al., 1997; Van Bergen et al., 2005; Abril et al., 2007). However, to date there is no clear consensus on the best method to assess C. fetus subspecies. The main problems rise from the limited number of tested strains, the failure to identify Cfvi strains and the low concordance with other techniques such as AFLP and, mainly, biochemical tests (Willoughby et al., 2005; Schulze et al., 2006; Schmidt, Venter & Picard, 2010).

A genome-wide association study has proposed the association between candidate gene loci coding for the L-Cys transporter and the H₂S production, which together to glycine resistance is one of the phenotypic traits available for assessing C. fetus subspecies to date. According to this, H₂S-producing C. fetus strains, commonly classified as Cff and Cfvi, have a complete L-Cys transporter operon, whereas the non-producing H₂S C. fetus strains, classically classified as Cfv, have a deleted L-Cys transporter. It is important to mention that C. fetus subsp. testudinum (Cft), the last subspecies proposed of C. fetus, has a complete version of the operon. This is the case of the strain Cft 03/427 (whose genome is the representative of the species) which has been concordantly described as an H₂S-producing strain elsewhere (Van der Graaf-van Bloois et al., 2016a). To date, this subspecies has not been described in cattle, and for this reason it was excluded of this study.

In this work, we have designed a multiplex-PCR protocol to provide a molecular tool to contribute to C. fetus characterization and differentiation. This L-Cys transporter-PCR showed an excellent correlation with the H₂S production test according to both wet lab and in silico approaches. As other molecular techniques, this PCR failed to differentiate Cff from Cfvi strains. This will limit its use in countries where this biovar is prevalent. However, until more discriminative techniques are developed, its usefulness could be further enhanced by combining this technique with other complementary test, such as the glycine resistance assay.

In addition to practical implications of this tool in the laboratory, this study also contributes to the existing debate around C. fetus subspecies classification.

In this study, we tested C. fetus strains isolated and typed at the Bacteriology Unit of INTA-Balcarce (Argentina), which has a long history in culturing this bacterium and in performing its biochemical based classification. In this way, the wet-lab approach showed a perfect correlation not only with the H₂S production test, but also with the C. fetus subspecies. Indeed, a CFF-CFVI pattern, which is indicative of L-Cys complete transporter, was associated with H₂S-producing strains typically classified as Cff or Cfvi. By contrast, a CFV pattern, which is indicative of a deleted transporter, was exclusively associated with H₂S-non-producing strains, which are typically classified as Cfv.

On the other hand, when we performed the in silico study, we analyzed genomic data from strains classified elsewhere by both molecular based approaches and/or biochemical tests and, as mentioned above, both techniques frequently displayed discordant results. In this way, we have obtained a perfect correlation with the H₂S production test, but not with the reported subspecies of the strains.

This discordancy is well reflected by the strain 98-25. Researchers from the Bacteriology Unit of INTA-Balcarce isolated this strain in 1998 from aborted foetus, and originally typed it as Cfv because of its glycine sensitivity and its inability to produce H₂S. This strain was included in this study and the PCR-L-Cys result was concordant with the phenotype of this strain. Later studies have also tested this strain and successfully sequenced its genome. Indeed, Van Bergen et al. (2005) typed it as glycine sensitive and H₂S positive (typical traits of Cfvi strains). Later, Van der Graaf-van Bloois et al. (2014) reported it as non-H₂S-producing strain. However, in this latter work, it has been called Cfvi 98-25 regardless the biochemical traits reported. In our study, the in silico sequence data analysis revealed the polymorphism of the L-Cys transporter (CFV pattern) typical of the non- H₂S- producing strains.

Therefore, the same strain could display different biochemical traits when assayed in different labs -or time- and this is the classical bottle-neck of phenotypic tests.

We initially had other discrepancies with some isolates. Remarkably, 14 out of 43 Cfv genomic sequences tested in silico showed a complete version of the L-Cys transporter (CFF/CFVI pattern). Hence, at first glance, the hypothesis that all Cfv isolates do have a deleted L-Cys transporter appeared as not valid, according to the in silico analysis. However, when we searched the biochemical tests reported for some of these strains, we concluded that the in silico results presented here were concordant with the H₂S production test. This discrepancy with the subspecies assigned could be due to the classification method of the strains that is frequently based on molecular techniques regardless the biochemical test results and moreover; the chosen method is not always specified (Iraola et al., 2017). Altogether, the in silico analysis also supports the hypothesis that states the occurrence of a deletion in the transporter operon in non-H₂S producing strains, which are classified as Cfv according to biochemical methods.

As was mentioned earlier, it is important to highlight that the strains from databases are not typed by the same methodology and this fact is not always taken into account. Consequently, this could be problematic as our study showed. The most widespread molecular-based method is the multiplex-PCR described by Hum et al. (1997). This PCR targets the parA gene to identify Cfv strains. This transfer-associated gene is harbored in a pathogenicity island which encodes a Type 4 Secretion System (T4SS). Although the presence of a T4SS has been previously associated to Cfv strains (Gorkiewicz et al., 2010), it has also been demonstrated later that some Cff strains can harbor the T4SS and their related genes (Van der Graaf-van Bloois et al., 2016b). Furthermore, distinct phylogenetic analyses of C. fetus suggest that the current classification in subspecies must be redefined. A phylogenomic study based on the core genome have shown that the strains are divided in two clusters. While all the Cfv and Cfvi strains were grouped in one genome cluster, the Cff strains were equally distributed in both clusters (Van der Graaf-van Bloois et al., 2014). Additionally, a phylogenetic reconstruction based on the divergence acquired by recombination have also shown that Cfv and Cff strains share the same clade, which differs clearly from the clade of Cft strains of reptile origin (Gilbert et al., 2018). This emphasizes a real need to go further toward current C. fetus classification and identification, which will have a significant impact on the diagnostic practice. As mentioned above, although this issue has been addressed in the literature and genomic studies have broadened and strengthened our knowledge of this bacterium (Van der Graaf-van Bloois et al., 2014, 2016a; Iraola et al., 2017), a concerted action toward C. fetus subspecies classification and differentiation has been neglected. There are no molecular markers associated to tropism or virulence of Cfv that could help with a differential diagnosis of the BGC (Gilbert et al., 2018).

Consequently, in light of these evidences, more research is essential to determine, as a first step, whether the differential diagnosis should be promoted and, if so, to improve or replace definitively the tests currently available. Another point to consider is that, veterinary diagnostic laboratories from developing countries are often refractory to replace those methods that have proven be useful, even if they are not the most suitable ones. Because of that, the adoption of genetic or even genomic-based methods has been delayed. One possible reason is the cost related to each method. The second main reason is the time lapse it takes for scientific knowledge to reach end users. Interdisciplinary research combining genomics, biochemistry, epidemiology and the provision of updated information and training to end users could shed light on this matter in the near future.

Conclusions

Biochemical tests including tolerance to glycine and H₂S production are currently recommended by the OIE (2018) for C. fetus subspecies differentiation and are still employed in laboratories around the world. Thus, a molecular tool linked to a phenotypic trait is a valuable tool that could be more accurate and less time consuming than the available phenotypic tests. Mutagenesis and functional studies are essential to associate definitely this putative L-Cys transporter with the H₂S production. Meanwhile, this study shows that this transporter constitutes a good marker that is useful for detecting H₂S-producing C. fetus. Future actions will be addressed to test the L-Cys-PCR in clinical samples to propose it not only as a typing method, but as a detection technique and, as a second phase of validation, to transfer this technology to other labs to test the reproducibility of the results.

Finally, this work provides a molecular tool linked to H₂S production in C. fetus and supports the findings of the pioneering study of Van der Graaf-van Bloois et al. (2016a).

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