Changes in physiology and virulence during the selection of resistant Yersinia ruckeri mutants under subinhibitory cefotaxime concentrations.

Bacterial antibiotic resistance is one of the main healthcare problems currently. Apart from reducing antibiotic efficacy, it has awakened the interest of scientists due to its association with bacterial fitness and virulence. Interestingly, antibiotic resistance can be a source of both increased fitness and decreased fitness, even though the molecular basis of these relationships remains unknown. The aim of this work is to define the effects of sub-MIC concentrations of cefotaxime, an antibiotic extensively used in clinical practice, on the physiology and virulence of Yersinia ruckeri and to determine the importance of these sub-MIC concentrations for the selection of antibiotic-resistant mutants in the aquatic environment. Results indicated that exposure to sub-MIC concentrations of cefotaxime selected Y. ruckeri populations with irreversible alterations in the physiology, such as slow growth, aggregation in liquid cultures and modification of the colony morphology. These bacteria also displayed changes in the OMPs and LPS profiles and a full attenuation of virulence. An overexpression of the envelope stress regulator RpoE was also detected after exposure to the antibiotic. In conclusion, exposure to cefotaxime selected, at high frequency, Y. ruckeri strains that survive the antibiotic stress at the expense of a fitness cost and the loss of virulence.

The Infection Process of Yersinia ruckeri: Reviewing the Pieces of the Jigsaw Puzzle.

Finding the keys to understanding the infectious process of Yersinia ruckeri was not a priority for many years due to the prompt development of an effective biotype 1 vaccine which was used mainly in Europe and USA. However, the gradual emergence of outbreaks in vaccinated fish, which have been reported since 2003, has awakened interest in the mechanism of virulence in this pathogen. Thus, during the last two decades, a large number of studies have considerably enriched our knowledge of many aspects of the pathogen and its interaction with the host. By means of both conventional and a variety of novel strategies, such as cell GFP tagging, bioluminescence imaging and optical projection tomography, it has been possible to determine three putative Y. ruckeri infection routes, the main point of entry for the bacterium being the gill lamellae. Moreover, a wide range of potential virulence factors have been highlighted by specific gene mutagenesis strategies or genome-wide transposon/plasmid insertion-based screening approaches, such us in vivo expression technology (IVET) and signature tagged mutagenesis (STM). Finally, recent proteomic and whole genomic analyses have allowed many of the genes and systems that are potentially implicated in the organism's pathogenicity and its adaptation to the host environmental conditions to be elucidated. Altogether, these studies contribute to a better understanding of the infectious process of Y. ruckeri in fish, which is crucial for the development of more effective strategies for preventing or treating enteric redmouth disease (ERM).

Temperature-Dependent Gene Expression in Yersinia ruckeri: Tracking Specific Genes by Bioluminescence During in Vivo Colonization.

Yersinia ruckeri is a bacterium causing fish infection processes at temperatures below the optimum for growth. A derivative Tn5 transposon was used to construct a library of Y. ruckeri mutants with transcriptional fusions between the interrupted genes and the promoterless luxCDABE and lacZY operons. In vitro analysis of ß-galactosidase activity allowed the identification of 168 clones having higher expression at 18°C than at 28°C. Among the interrupted genes a SAM-dependent methyltransferase, a diguanylated cyclase, three genes involved in legionaminic acid synthesis and three transcriptional regulators were defined. In order to determine, via bioluminescence emission, the in vivo expression of some of these genes, two of the selected mutants were studied. In one of them, the acrR gene coding a repressor involved in regulation of the AcrAB-TolC expulsion pump was interrupted. This mutant was found to be highly resistant to compounds such as chloramphenicol, tetracycline, and ciprofloxacin. Although acrR mutation was not related to virulence in Y. ruckeri, this mutant was useful to analyze acrR expression in fish tissues in vivo. The other gene studied was osmY which is activated under osmotic stress and is involved in virulence. In this case, complemented mutant was used for experiments with fish. In vivo analysis of bioluminescence emission by these two strains showed higher values for acrR in gut, liver and adipose tissue, whereas osmY showed higher luminescence in gut and, at the end of the infection process, in muscle tissue. Similar results were obtained in ex vivo assays using rainbow trout tissues. The results indicated that this kind of approach was useful for the identification of genes related to virulence in Y. ruckeri and also for the in vivo and in vitro studies of each of the selected genes.

Comparative genome analysis reveals important genetic differences among serotype O1 and serotype O2 strains of Y. ruckeri and provides insights into host adaptation and virulence.

Despite the existence of a commercial vaccine routinely used to protect salmonids against Yersinia ruckeri, outbreaks still occur, mainly caused by nonmotile and lipase-negative strains (serotype O1 biotype 2). Moreover, epizootics caused by other uncommon serotypes have also been reported. At the moment, one of the main concerns for the aquaculture industry is the expanding range of hosts of this pathogen and the emergence of new biotypes and serotypes causing mortality in fish farms and against which the vaccine cannot protect. The comparative analysis of the genome sequences of five Y. ruckeri strains (150, CSF007-82, ATCC29473, Big Creek 74, and SC09) isolated from different hosts and classified into different serotypes revealed important genetic differences between the genomes analyzed. Thus, a clear genetic differentiation was found between serotype O1 and O2 strains. The presence of 99 unique genes in Big Creek 74 and 261 in SC09 could explain the adaptation of these strains to salmon and catfish, respectively. Finally, the absence of 21 genes in ATCC29473 which are present in the other four virulent strains could underpin the attenuation described for this strain. The study reveals important genetic differences among the genomes analyzed. Further investigation of the genes highlighted in this study could provide insights into the understanding of the virulence and niche adaptive mechanisms of Y. ruckeri.

Genome Sequence of the Fish Pathogen Yersinia ruckeri Strain 150, Isolated from Diseased Rainbow Trout.

We present here the draft genome of a pathogenic Yersinia ruckeri strain, isolated from rainbow trout (Oncorhynchus mykiss) affected by enteric redmouth disease. The chromosome has 3,826,775 bp, a GC content of 46.88%, and is predicted to contain 3,538 coding sequences. The data will be useful for comparative pathogenicity studies.

Temperature-dependent expression of virulence genes in fish-pathogenic bacteria.

Virulence gene expression in pathogenic bacteria is modulated by environmental parameters. A key factor in this expression is temperature. Its effect on virulence gene expression in bacteria infecting warm-blooded hosts is well documented. Transcription of virulence genes in these bacteria is induced upon a shift from low environmental to a higher host temperature (37°C). Interestingly, host temperatures usually correspond to the optimum for growth of these pathogenic bacteria. On the contrary, in ectothermic hosts such as fish, molluscs, and amphibians, infection processes generally occur at a temperature lower than that for the optimal growth of the bacteria. Therefore, regulation of virulence gene expression in response to temperature shift has to be modulated in a different way to that which is found in bacteria infecting warm-blooded hosts. The current understanding of virulence gene expression and its regulation in response to temperature in fish-pathogenic bacteria is limited, but constant extension of our knowledge base is essential to enable a rational approach to the problem of the bacterial fish diseases affecting the aquaculture industry. This is an interesting issue and progress needs to be made in order to diminish the economic losses caused by these diseases. The intention of this review is, for the first time, to compile the scattered results existing in the field in order to lay the groundwork for future research. This article is an overview of those relevant virulence genes that are expressed at temperatures lower than that for optimal bacterial growth in different fish-pathogenic bacteria as well as the principal mechanisms that could be involved in their regulation.

The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence.

BACKGROUND: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. RESULTS: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. CONCLUSIONS: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.

Flavobacterium psychrophilum vaccine development: a difficult task.

Bacterial cold water disease (BCWD) is a globally distributed freshwater fish disease caused by the Gram-negative bacterium Flavobacterium psychrophilum. It is a particularly devastating infection in fry salmonids and may lead to high levels of mortality. In spite of its economic impact on fish farms, neither the biology of the bacterium nor the bacterium-host interactions are well understood. This review provides a synopsis of the major problems related to critical remaining questions about research into the use of vaccines against F. psychrophilum and the development of a commercial vaccine against this disease. Studies using sera from convalescent rainbow trout have shown the antigenic properties of different proteins such as OmpH, OmpA and FspA, as well as low and high molecular mass lipopolysaccharide of F. psychrophilum, which are potential candidates for subunit vaccines. Inactivated F. psychrophilum bacterins have been successfully tested as vaccines under laboratory conditions by both immersion and intraperitoneal routes. However, the efficacy and the practical usefulness of these preparations still have to be proved. The use of attenuated and wild-type strains to immunize fish showed that these systems offer high levels of protection. Nevertheless, their application clashes with the regulations for environmental protection in many countries. In conclusion, protective vaccines against BCWD are theoretically possible, but substantial efforts still have to be made in order to permit the development of a commercial vaccine.

The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.

In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.