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Shewanella khirikhana sp. nov. – a shrimp pathogen isolated from a cultivation pond exhibiting early mortality syndrome

Summary Early mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pirvp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 105 colony‐forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA–DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012T genome revealed no homologues of the Pirvp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp.

sequences in S. litorisediminis SMK1-12 T and S. litorisediminis TBRC 5001 were obtained by sequencing PCR amplicons we produced using specific PCR primers that we designed from the sequences of TH2012 T (Supporting Information Table S1) and the PCR condition is described in Supporting Experimental Procedures. The sequences have been deposited at GenBank The genome assembly of TH2012 T contained eight copies of the 16S rRNA gene, each comprising 1533 nucleotides (nt). Seven shared an identical sequence, while the remaining one showed three (3) nt differences from it. A neighbor-joining phylogenetic tree based on a comparison of nearly-complete and complete 16S rRNA sequences revealed that TH2012 T resided in a clade (bootstrap re-sampling value 100%) together with other three Shewanella strains (Supporting Information Figure S1). TH2012 T was most closely linked to TBRC 5001, next with the type strain of S. amazonensis SB2B T and then with the type strain of S. litorisediminis SMK1-12 T (bootstrap 100%). The node where SMK1-12 T was clustered with a subclade of the other three strains was also supported by trees of the other three algorithms with 99-100% bootstrap values. The cluster of TH2012 T with TBRC 5001 also arose in a tree constructed using the minimum evolution algorithm, but the trees constructed by maximumlikelihood and maximum parsimonious algorithms placed TBRC 5001 in a cluster with SB2B T and then with TH2012 T . The available partial 16S rRNA sequences of S. sp. AK55 (1487 nt; HG529988) and S. litorisediminis LV 5 (1178 nt; KF801478) clustered with TH2012 T (Supporting Information Figure S2). The former showed 100% sequence identity with the seven identical copies of TH2012 T 16S rRNA, whereas the latter had three different nucleotide positions to each of the two sets of TH2012 T 16S rRNAs. This suggests that TH2012 T , S. sp. AK55 and S. litorisediminis LV 5 might be representatives of a single species. On the other hand, the TH2012 T 16S rRNA sequences exhibited 98-99% identity to the nearly-full 16S rRNA sequence of TBRC 5001 (99.18-99.25%) and of the type strains of S. litorisediminis  and to the available full sequence of S. amazonensis SB2B T (98.5-98.7%).
The atpA, mreB and rpoA genes of TH2012 T obtained from genome assembly contained 1545, 1050, 990 nt, respectively, and homologous sequences amplified in TBRC 5001 and SMK1-12 T contained 75-89% of the corresponding TH2012 gene length (see Supporting   Information Table S2). All neighbor-joining trees of atpA, mreB and rpoA genes placed TH2012 T as a sister taxon of a subclade containing TBRC 5001, SMK1-12 T and S. amazonensis SB2B T (Supporting Information Figures S3, S4 and S5). Similarly, a placement of TH2012 T as a sister taxon with a subclade containing TBRC 5001, SMK1-12 T and S. amazonensis SB2B T was also recovered by all of the other three algorithms (the minimum evolution, maximum-likelihood and maximum-likelihood algorithms). The atpA, mreB and rpoA nucleotide sequences of TH2012 T exhibited 91-97% identities to the partial sequences of TBRC 5001 and SMK1-12 T and 91-96% identities to the complete sequences of S. amazonensis SB2B T . TH2012 T tended to show higher percentage identities to TBRC 5001 (93-97%) than to SMK1-12 T and SB2B T (91-96%) based on the three genes studied (Supporting Information Table S2).

Morphological and physiological characteristics of TH2012 T
Morphology of the isolated bacterium by Gram staining and of colonies on TSA are shown in Supporting Information Figures S6 and S7, respectively. Physiological characteristics of TH2012 T were investigated using various temperatures, salinities, pHs and growth profiles. To investigate the effects of temperature on growth, TH2012 T were cultured in TSB overnight, inoculated in new fresh TSB media with an initial optical density at 600 nm (OD600) of 0.05 and cultured to reach the OD600 of 0.6 (~ 10 8 colony-forming units per mL [CFU/mL]). Then, the cultures were used for the colony plate counts on TSA using 100 µL aliquots of 10 5 -fold dilution that were incubated at different temperatures (4,25,30,37 and 42 ºC;see Supporting Experimental Procedures). The cell counts were performed after an overnight incubation. TH2012 T could grow at 25, 30 and 37 ºC and did not grow at 4 and 42 ºC (Supporting Information Table S3). To investigate the effects of salinity on TH2012 T growth, overnight TH2012 T TSB culture was inoculated into new fresh TSB supplemented by various NaCl concentrations (0, 1, 1.5, 2, 3, 4, 5, 6, 7 and 8 %; see Supporting Experimental Procedures). TH2012 T grew best at supplemented with 1 and 1.5% NaCl (highest OD at hour 18), grew relatively well with 0, 2, and 3% supplementation (highest OD at hours 5 and 7), grew poorly at 4 and 5% supplementation, but could not grow with NaCl supplementation between 6 to 8% (Supporting Information Figure S8). Since TSB already contained 0.5% NaCl, TH2012 T could grow at salinities 0.5-5.5% NaCl and grew optimally at 1.5-2% NaCl. pH also affected TH2012 T growth (Supporting Information Figure S9; see Supporting Experimental Procedures) for investigation of pH effect between 6 to 9.5. TH2012 T grew best at pH 7, 7.5 and 8, grew poorer at pH 6.5, and 8.5, less at pH 9 and 9.5, and not at all at pH 6 (Supplementary Figure S9). TH2012 T growth curves were investigated by measuring OD600 and viable cell counts in TSB medium supplemented with 1.5% NaCl at pH 7.5 and 30 °C (Supporting Experimental Procedures). For OD600, TH2012 T entered the exponential growth phase at the first hour and entered the stationary phase after the forth hour (Supporting Information Figure S10). For viable cell counts, during exponential phase (hours 2, 3, 4, and 5) see Supporting Information Figure   S11.

Histopathology of moribund shrimp from TH2012 T bacterial challenge
Altogether, 40 shrimp were examined from three challenge experiments carried out independently using three different sources of shrimp. Shrimp examined in the first two challenge experiments (total 15 each) included three control shrimp and 12 moribund shrimp (four each from immersion challenges at 10 4 to 10 6 CFU/mL) and in the third experiment (total 10 shrimp), three control shrimp and seven moribund shrimp (three each from immersion challenges 10 5 and 10 6 CFU/mL and one from immersion challenge 10 6 since all others from that group had died before they could be retrieved for fixation while still alive).
In the main manuscript, we found that the most consistent and prominent histopathological abnormality in the moribund shrimp challenged by immersion exposure to TH2012 T as bacteria-free lesions in the ventral nerve cord and the lymphoid organ (LO) . The next most consistent and prominent histopathological abnormalities in the moribund shrimp consisted of unusual vacuolization of the epithelial cells of the digestive system and the hematopoietic tissue. In the digestive system these included 1) the anterior midgut cecum (AMC) (22/26 = 85% of specimens with AMC tissue in the section) (Supporting Information Figure   S13), 2) nearby sub-cuticular epithelial cells of the stomach (24/26 = 92% of specimens with appropriate tissue in the section) (Supporting Information Figure S14) and 3) E-cells of the tubule epithelium of the hepatopancreas (HP) (22/31 = 71% of specimens) (Supporting Information Figure S15). In the control shrimp, vacuolated cells were not seen in E-cells of the HP for 9/9 specimens, but they did occur in the AMC for 2/3 specimens and in the stomach epithelial cells for 3/3 specimens from the third challenge experiment, indicating little diagnostic value for these features, either because of low prevalence (HP) or possible presence in normal shrimp (AMC and stomach). In addition, the occurrence of prominent eosinophilic inclusions within the vacuoles of these tissues from the moribund shrimp was 16/25 = 65% for the AMC, 2/22 = 9% for the HP and 1/22 = 4.5% for the stomach epithelium. As far as we know, the occurrence of prominent vacuoles in the cytoplasm of epithelial cells of the AMC, E-cells of the shrimp HP and epithelial cells of the stomach (normally characterized by dense cytoplasm devoid of prominent vacuoles) has not been previously reported as a pathological sign of a specific shrimp disease. However, even if they have some association to challenge with S. khirikhana, they appear to be less specific and less reliable for use in presumptive diagnosis for disease caused by it when compared to the lesions described herein for nerve tissue and LO tissue.
Similarly, to our knowledge, lesions showing prominent eosinophilic, cytoplasmic inclusions (sometimes within vacuoles) in cells of the hematopoietic tissue (HT) (20/31= 65% of moribund shrimp specimens examined) (Supporting Information Figure S16) have never been previously reported as a pathological sign for any shrimp disease as far as we know. Thus, they may be unique to disease caused by S. khirikhana. However, based on prevalence, they are less reliable for presumptive diagnosis when compared to the nerve cell and LO lesions. Their reliable detection is also more difficult because it requires use of an 100x oil immersion lens in contrast to the 40x lens sufficient for examination of nervous and LO tissues. On the other hand, they could be used together with the nerve cell and LO lesions to support a presumptive diagnosis of S. khirikhana infection.
We also found focal gill lesions that were characterized by the presence of pyknotic and karyorrhetic nuclei, eosinophilic cytoplasmic inclusions and loss of gill structure (25/31 = 81% of moribund shrimp; data not shown). As with the lesions in other tissues, no bacterial cells were present. However, the scattered nature of the lesions and their highly variable extent from specimen to specimen means that excessive time is needed for analysis when compared to examination of specific organs such as the ventral nerve cord and LO in cephalothorax sections.
In addition, it is normal practice to prepare midline, lateral sections of the shrimp cephalothorax for routine histological examination and these do not usually include gill tissue unless specifically requested.

Bacteria in the hepatopancreatic lumens of challenged and control shrimp
During histological analysis of the hepatopancreas of control and challenged shrimp, two full, adjacent but non-overlapping sweeps were made from the anterior end to the posterior end and back across the widest possible section of the hepatopancreas using an oil emersion lens to search for any evidence of bacterial presence. None were found in any of the control shrimp specimens examined. However, 11 of the 31 moribund shrimp (35.5%) from the bacterial challenge tests did. Of these 11 specimens, two showed focal lesions of relatively large numbers of bacterial cells of mixed morphology, sometimes in clumps in the HP tubule lumens, especially in or near the central lumen of the HP (Supporting Information Figure S17A). These were associated with damage to the HP tubule epithelium. For the remaining nine specimens, the numbers of bacteria were very low with consistent, rod-shaped (uncurved) morphology and not associated with lesions in the nearby HP tubule epithelial cells (Supporting Information Figure   S17B). They measured 0.5±0.05 X 1.95±0.46 µm (n = 100). This corresponded to 0.58±0.05 X 2.02±0.38 (n = 50) for S. khirikhana in Gram-stained smears of cells from a broth culture (Supporting Information Figure S6B). None of the control shrimp or moribund shrimp specimens with or without bacteria showed abnormal hemocyte aggregation or nodule or granuloma formation that are often associated with bacterial infections in the shrimp HP. These observations for absence of bacterial cells at the sites of lesions in the nerve cord, LO, etc. and the low frequency and low numbers of bacterial cells seen in the HP support the suggestion that the pathological effects from S. khirikhana arise from toxins produced by the cells located in the culture water and/or in the shrimp stomach.

Free sample testing for TH2012 T
We would welcome samples of approximately 100-500 ng of the ethanol purified DNA extract in buffer spotted on sterile filter paper and mailed to us by ordinary airmail. We would test such samples for S. khirikhana and inform the sender of the confidential result for free. To do this, any potential sender should make advance arrangements with us by e-mail so that we are ready to receive samples.

Genome Sequencing and Genome Properties
The complete genome sequence of TH2012 T was previously described (Wechprasit et al., 2019), and a summary of the project information is given in Supporting Information Table S4.
Supporting Information Table S5 shows the distribution of predicted protein-coding genes into COG functional categories. The graphical representation of open reading frames (ORFs) were annotated by colors in Fig. 5 in the main manuscript and Supporting Information Figure S18 with Circos (Krzywinski et al., 2009).

Supporting Experimental Procedures
Note that all tryptic soy broth (TSB) and tryptic soy agar (TSA) used in this study were supplemented by 1.5% NaCl; otherwise indicated. TH2012 T was revived from glycerol stocks by culturing aerobically at 30°C in TSB under shaking conditions before subsequent uses.

Genomic DNA extraction
For use as PCR templates, total genomic DNA of each bacterial isolate from an overnight culture in TSB was extracted from cells using the standard phenol-chloroform method (Sambrook and Russel, 2001).

Tests for enzymatic activities
API®biomerieux API 20E stripe tests were used according to the manufacturer's instruction for TH2012 T and KCTC 23961 T (= SMK1-12 T ) cells grown in TSA at 37°C for 16-24 h prior to strip inoculation. After inoculation and 24 h incubation at 37°C, the tests were evaluated simultaneously for both TH2012 T and SMK1-12 T . The following additional tests were carried out for TH2012 T . Casein hydrolysis tests were carried out on 4% skim milk powder supplemented TSA plates and measured by clear zone surrounding bacterial colonies on the second day after spotting at the middle of the plates with 10 µl TH2012 T TSB culture (OD600 = 0.5). Lipase activity was verified similarly by 1% Tween 20 supplemented TSA plates spotted with serially-diluted overnight cultures. Similar to the casein hydrolysis test, the gelatin hydrolysis test was carried out using 0.5% gelatin supplemented TSA plates and measured by clear zones at the seventh day after spotting. The clear zones were measured at the second minute after pouring saturated ammonium sulfate (80%) in distilled water on top of the agar plates.

Effects of temperatures on TH2012 T growth
TH2012 T TSB culture was added into 100 mL of fresh TSB medium to obtain the optical density at 600 nm (OD600) of ~0.05, and then incubated at 30 °C with 250 rpm shaking until OD600 reaches 0.6. Then 100 µL of 10 5 -fold diluted culture medium was spread on TSA plates in two replicates. The plates were cultured overnight (~18 h) at 4, 25,30, 37 and 42 ºC. Bacterial colonies were counted and recorded to calculate colony-forming units per mL (CFU/mL).

Effects of NaCl on TH2012 T growth
An aliquot of 100 µL of overnight culture broth of TH2012 T was added to 100 mL TSB supplemented with NaCl ranging from 0-8 % (w/v) (2 replicates) followed by culture at 30 °C with 250 rpm shaking. OD600 of each culture was measured at 0, 1, 3, 5, 7 and 18 h.

Effects of pH on TH2012 T growth
TSB were prepared and adjusted to pH of 6, 6.5, 7, 7.5, 8, 8.5, 9 and 9.5 by addition of acetic acid and Na2CO3 buffers. TH2012 T from overnight culture in TSB was added into 100 mL of each pH-adjusted medium to obtain OD600 of ~0.05 (2 replicates for each pH) followed by culture at 30 °C with 250 rpm shaking. OD600 of each culture was measured at 0, 1, 3, 5, 7 and 18 h.

Growth profiles
TH2012 T was added into 100 mL of fresh TSB medium (1.5% NaCl and pH 7.5) to obtain the OD600 of ~0.05 followed by culture at 30 °C with 250 rpm shaking for 20 h. OD600 of the inoculum culture was monitored every thirty minutes during the first five hours and then again at 10 and 20 h. Viable bacteria cell counts on TSA were carried out by spreading 100 µL of 10-fold serial dilutions (2 replicates) at each time point of the above OD600 measures, except at 0, 1 and 1:30 h. Counts were made after incubation at 30 ºC for 16-18 h.

Histopathology examination
Whole moribund shrimp and normal control shrimp were fixed in Davidson's fixative for histopathological analysis according to Bell and Lightner (Bell and Lightner, 1988). After 24 h in Davidson's fixative, shrimp organs were transferred to 70% ethanol for tissue preservation. The rostrum, walking legs and the abdominal region were removed and the whole cephalothorax was cut off at center of cephalothorax before tissue processing. Tissues were dehydrated, paraffin embedded, sectioned at 5 µm and stained with hematoxylin and eosin (H&E) as described by Bell and Lightner (Bell and Lightner, 1988). Stained tissue sections were examined by light microscopy and recorded as digital images.

PCR conditions and PCR product sequencing
The primer sets for atpA, mreB and rpoA genes were designed based on the sequences of strain TH2012 T using Primer3 (ref. (Untergasser et al., 2012)). Sequences of all the specific primer sets used in this study are listed in Supporting Information Table S1. The PCR reactions for atpA, mreB and rpoA genes were performed in a 25 µl mixture containing 1X PCR buffer The ligated products were transformed into E. coli DH5α that was spread onto LB agar containing 100 µg/mL of ampicillin and then incubated at 37ºC overnight. Plasmids containing the inserted fragment were identified by EcoRI digestion. Three clones of each bacterial isolate were sent for sequencing at Macrogen, South Korea. Chromatograms were processed by Phred and phd2fasta (Ewing and Green, 1998) for base-calling and cross_match (Gordon) for detecting primer or vector sequences. Fig. S1: Phylogeny of 16S rDNA genes of Shewanella khirikhana TH2012 T and other Shewanella species. The neighbour-joining phylogenetic tree with Jukes-Cantor distance was based on 1,452 aligned positions of 16S rDNA gene sequences. Moritella marina ATCC 15381 was used as an outgroup. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Most of the nodes were also recovered in the trees generated with the minimum evolution (Jukes-Cantor distance), maximum-likelihood (Jukes-Cantor distance) and maximum parsimonious algorithms based on the same set of sequences, except those with Filled circles, or Filled squares were not recovered in the trees of maximum parsimonious, or both maximum parsimonious and maximum-likelihood algorithms, respectively. Scale bar indicates estimated sequence divergence (substitutions per nucleotide position). Sequence accession numbers are shown in the parentheses after the species names. Fig. S2: Phylogeny 16S rDNA genes of Shewanella khirikhana TH2012 T and other Shewanella species including strains LV 5 and AK55. The neighbour-joining phylogenetic tree with Jukes-Cantor distance was based on 1,176 aligned positions of 16S rDNA gene sequences. Moritella marina ATCC 15381 was used as an outgroup. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Most of the nodes were also recovered in the trees generated with the minimum evolution (Jukes-Cantor distance), maximum-likelihood (Jukes-Cantor distance) and maximum parsimonious algorithms based on the same set of sequences, except those with Filled circles, or Opened squares were not recovered in the trees of maximum parsimonious, or maximum-likelihood algorithms, respectively. Scale bar indicates estimated sequence divergence (substitutions per nucleotide position). Sequence accession numbers are shown in the parentheses after the species names. Fig. S3: Phylogeny of atpA genes of Shewanella khirikhana TH2012 T and other Shewanella species. The neighbour-joining phylogenetic tree with Jukes-Cantor distance was based on 1,262 aligned positions of atpA gene sequences. Moritella marina ATCC 15381 was used as an outgroup. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Most of the nodes were also recovered in the trees generated with the minimum evolution (Jukes-Cantor distance), maximum-likelihood (Jukes-Cantor distance) and maximum parsimonious algorithms based on the same set of sequences, except those with Opened squares, or Filled squares were not recovered in the trees of maximum-likelihood, or both maximum parsimonious and maximum-likelihood algorithms, respectively. Scale bar indicates estimated sequence divergence (substitutions per nucleotide position). Sequence accession numbers are shown in the parentheses after the species names. Fig. S4: Phylogeneny of mreB genes of Shewanella khirikhana TH2012 T and other Shewanella species. The neighbour-joining phylogenetic tree with Jukes-Cantor distance was based on 785 aligned positions of mreB gene sequences. Moritella marina ATCC 15381 was used as an outgroup. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Most of the nodes were also recovered in the trees generated with the minimum evolution (Jukes-Cantor distance), maximum-likelihood (Jukes-Cantor distance) and maximum parsimonious algorithms based on the same set of sequences, except those with Filled circles, or Filled squares were not recovered in the trees of maximum parsimonious, or both maximum parsimonious and maximum-likelihood algorithms, respectively. Scale bar indicates estimated sequence divergence (substitutions per nucleotide position). Sequence accession numbers are shown in the parentheses after the species names. Fig. S5: Phylogeny of rpoA genes of Shewanella khirikhana TH2012 T and other Shewanella species. The neighbour-joining phylogenetic tree with Jukes-Cantor distance was based on 789 aligned positions of rpoA gene sequences. Moritella marina ATCC 15381 was used as an outgroup. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Most of the nodes were also recovered in the trees generated with the minimum evolution (Jukes-Cantor distance), maximum-likelihood (Jukes-Cantor distance) and maximum parsimonious algorithms based on the same set of sequences, except those with Filled circles, Opened squares, or Filled squares were not recovered in the trees of maximum parsimonious, maximum-likelihood, or both maximum parsimonious and maximum-likelihood algorithms, respectively. Scale bar indicates estimated sequence divergence (substitutions per nucleotide position). Sequence accession numbers are shown in the parentheses after the species names. Fig. S11: Graph showing Shewanella khirikhana TH2012 T viable bacterial cell counts (CFU/mL) vs. OD600 during hours 2 to 4 of the growth curve experiment (hours are indicated by colors) in Fig. S10.The grey area denotes the 95% confidence level interval for predictions from a linear model of log10 CFU/mL versus log10 OD600. R-squared = 0.7814, P = 4.902 x 10 -8 . Fig. S12: Graph showing the regression line for shrimp mortality versus bacterial concentration (log10 CFU/mL) for Shewanella khirikhana TH2012 T immersion bioassay up to 60 hours post immersion. The grey area denotes the 95% confidence level interval for predictions from a linear model. R-squared = 0.6945, P = 0.03933.  Figure S18: Graphical map of the plasmid pSTH1. From outside to the center: Genes on forward strand (colored by COG categories), Genes on reverse strand (colored by COG categories). COG color and functional designations are described in Supporting Information Table S5.  42 1 UD 2 UD Supporting Information Table S4. Shewanella khirikhana TH2012 T genome project information. The total is based on the total number of protein coding genes in the genome.