Detection and genetic analysis of group II capsules in Aeromonas hydrophila

Y. L. Zhang1, Y. L. Lau1, E. Arakawa2 and K. Y. Leung1,3

1 Department of Biological Sciences, Faculty of Science, The National University of Singapore, Singapore 117543
2 Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan 162-8640
3 Tropical Marine Science Institute, The National University of Singapore, Singapore 117543

Correspondence
K. Y. Leung
dbslky{at}nus.edu.sg


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The genetic organization and sequences of the group II capsule gene cluster of Aeromonas hydrophila PPD134/91 have been determined previously. The purified capsular polysaccharides can increase the ability of avirulent strain PPD35/85 to survive in naive tilapia serum but have no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells. In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was examined by electron microscopy and genetic analysis. Ten strains were found to produce group II capsules. A PCR detection system was developed to identify two types of group II capsules (IIA and IIB) based on their genetic organization in the region II gene clusters. Group IIA capsules in the authors' collection of A. hydrophila strains are mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules are found in the O : 21 and O : 27 serogroups. The presence of group II capsules in A. hydrophila strongly correlates with the serum and phagocyte survival abilities (seven out of ten strains). The results indicate that the authors' PCR detection system can constitute a reliable assay for the classification of group II capsules in A. hydrophila.


The GenBank accession numbers for the DNA sequences of A. hydrophila PPD11/90, PPD64/90, PPD88/90 and JCM3983 region II capsule gene clusters are AY144595, AY177618, AY177619 and AY177683, respectively.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The expression of polysaccharide capsules is a common feature of many bacteria (Roberts, 1996). These extracellular polysaccharides play a key role in interactions between pathogen and host, such as protecting bacterial cells from complement-mediated serum killing and desiccation, acting as adhesion factors, and aiding survival in phagocytes (Moxon & Kroll, 1990; Roberts, 1996).

All of the known capsule assembly systems seen in Gram-negative bacteria are well represented in Escherichia coli. Capsules in E. coli have been categorized into four groups based on genetic and biosynthetic criteria (Whitfield & Roberts, 1999). The genes for group II type capsules can be divided into three distinct regions (Roberts et al., 1988; Roberts, 1996). Genes in regions I and III are for capsule transport, and genes in region II are for capsule biosynthesis. Regions I and III are conserved among various group II capsule clusters of a broad range of bacterial species, while region II is serotype specific (Roberts, 1996). These unique characteristics can be used to develop a DNA-based detection method for various bacterial species. The presence of capsules in motile aeromonads was first reported by Martinez et al. (1995). The first capsule gene cluster was cloned, sequenced and identified as the group II type from Aeromonas hydrophila PPD134/91 in our laboratory (Zhang et al., 2002). The purified capsular polysaccharides of PPD134/91 were found to be capable of conferring resistance to serum-mediated killing of avirulent strain PPD35/85, but had no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells.

In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was identified by electron microscopy and genetic analyses. Two types of group II capsules (IIA and IIB) were identified based on their genetic organization in region II. Group IIA capsules in our randomly chosen A. hydrophila strains were mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules were found in the O : 21 and O : 27 serogroups. The presence of these group II capsules strongly correlated with the serum and phagocyte survival abilities in A. hydrophila.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, growth conditions and serotyping.
The A. hydrophila strains used in this study are listed in Table 1. The characteristics and virulence of the A. hydrophila strains studied have been described previously (Zhang et al., 2000, 2002). Strains with the prefix ‘JCM’ were from the Japan Collection of Microorganisms (http://www.jcm.riken.go.jp/JCM/catalogue.html). Cultures were routinely grown in tryptic soy broth (TSB, Difco) or on tryptic soy agar (TSA, Difco) at 25 °C. Stock cultures were maintained at -80 °C in TSB containing 25 % (v/v) glycerol. Preparation of antibodies and agglutination tests for serotyping of A. hydrophila were performed according to the protocols described by Sakazaki & Shimada (1984).


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Table 1. Detection of group II capsules in A. hydrophila

 
Examination of the bacterial capsules by electron microscopy.
The glutaraldehyde/ruthenium red/uranyl acetate method was used to stain the A. hydrophila cells (Mustaftschiev et al., 1982). A drop of bacterial culture was placed on a carbon-coated grid and surplus fluid was removed with filter paper. The grid was then floated on the top of an equal-volume mixture of 1 % glutaraldehyde in 0·2 M cacodylate buffer (pH 7) with the specimen side down. A drop of 1 % ruthenium red in water was added and this was followed by staining with 0·05 % uranyl acetate. The sample was dried and observed by transmission electron microscopy.

Survey of the distribution of group II-like capsules.
Oligonucleotide primers used in this study (Fig. 1, Table 2) were designed based on regions I and III of the group II capsule gene cluster of PPD134/91 (Zhang et al., 2002). Primers 79DP5S1 and 79DP5 were used to detect ORFC, and primers 79A5 and CPS13 for ORFL. PCR was performed by using Advantage Genomic Polymerase Mix (Clontech), following a three-step cycle protocol: 25 s at 94 °C; 32 cycles of 15 s at 94 °C, 30 s at 62 °C, and 4 min at 72 °C. PPD134/91 was used as the positive control. Only those samples which had one band at the same position as the positive control were taken as positive.



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Fig. 1. Comparison of the A. hydrophila PPD134/91 capsule cluster with those of A. hydrophila PPD64/90, PPD88/90, PPD11/90 and JCM3983. The organization of the locus into three regions (I, II and III) is indicated by the boxes. ORFs within the operon, with the direction of transcription, are indicated by open arrows. The corresponding gene designation is shown under each ORF. Flanking genes and their transcriptional directions are shown by filled arrows. The small arrows above and below the PPD134/91 and PPD11/90 capsule clusters indicate the forward and reverse primers, respectively, for the PCR analysis.

 

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Table 2. Primers used in the detection of group II capsule genes

 
Determining the organization of regions I and III genes of group II capsules.
PCR was performed on 10 strains that had group II-like capsules to determine the genetic organizations of regions I and III of the capsule gene clusters. For region I, one forward primer, CPS1, was designed based on the 5' end of ORFA and three reverse primers, 79DP5S2, 79DP5 and 79DP4, were designed based on the 3' end sequences of ORFB, ORFC and ORFD, respectively (Fig. 1, Table 2). For region III, 79A5 was designed based on the 5' end of ORFL and CPS15 was designed based on the 3' end sequence of ORFM (Fig. 1, Table 2). The sizes of the PCR fragments were used to decide the genetic organization. PPD134/91 was used as the positive control. Only those samples which had one band at the same position as the positive control were taken as positive. Southern blots were performed using PCR products of regions I and III DNA sequences as probes, following the method of Zhang et al. (2000).

Determining the organization of region II genes of group II capsules.
Seven pairs of primers were designed based on the sequences of the seven genes in region II (ORFE–K) of the PPD134/91 capsule gene cluster (Fig. 1, Table 2). PCR was performed to detect their presence in nine capsule gene clusters of A. hydrophila. Only those samples which had one band at the same position as that of PPD134/91 were taken as positive. The 3' and 5' end sequences of each PCR product were sequenced to confirm the identities of these genes. DNA sequencing and analysis were carried out as described previously (Srinivasa Rao et al., 2001).

Cloning the capsular biosynthesis regions from motile A. hydrophila strains.
Region II of group II capsule gene clusters from A. hydrophila was cloned using long-range PCR with primers 79B1 (ORFD) and 79B2 (ORFL) (Fig. 1, Table 2). PCR was performed by using Advantage Genomic Polymerase Mix (Clontech) and carried out under the following conditions: one hold at 94 °C for 1 min followed by 32 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 10–20 min. The amplified fragments were purified using QIAquick PCR Purification Kit (Qiagen) and cloned into the pGEM-T Easy Vector (Promega). The recombinant DNA molecules were transformed into E. coli DH5{alpha} competent cells and sequenced.

Survival assay in tilapia serum and phagocyte intracellular replication assay.
Naive tilapia [Oreochromis aureus (Steindachner)] serum was used to perform the serum resistance assay. Bacteria were prepared and treated with 50 % tilapia serum as described previously (Leung et al., 1995). The survival of A. hydrophila was calculated by dividing the number of viable bacteria after a 1 h serum treatment by the number of bacteria before treatment. Bacteria with survival values greater than 1 were considered serum resistant, while those with values below 1 were considered serum sensitive. The intracellular replication assay was performed as described by Srinivasa Rao et al. (2001). Phagocytes were isolated from the head kidney of naive blue gourami [Trichogaster trichopterus (Pallas)]. Thirty minutes after infection, the phagocytes were washed once with Hanks' balanced salt solution (Sigma) and then incubated for 1·5 h in fetal calf serum-supplemented fresh L-15 medium with 100 µg gentamicin ml-1. The gentamicin treatment killed extracellular bacteria but did not affect the viability of intracellular organisms. The intracellular population of bacteria was assayed at 2 and 6·5 h. The phagocytic index was calculated by dividing the intracellular population of A. hydrophila at 6·5 h by the population at 2 h. The data were obtained from three independent experiments.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Electron microscopy observation of capsules
Thirty-three strains from 26 serogroups of A. hydrophila were randomly chosen and negatively stained with ruthenium red. Capsules were seen as electron-dense reticulated networks surrounding the bacteria. In total, 17 (52 %) strains were found to produce capsules (Fig. 2, Table 1), indicating that capsule production is quite common among A. hydrophila.



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Fig. 2. Transmission electron micrographs of negatively stained cells of A. hydrophila: PPD35/85 (a), PPD88/90 (b), JCM3996 (c) and JCM3983 (d). Note the presence of a fuzzy coat around the three strains (b, c and d) that produce capsules. PPD35/85 is a capsular-polysaccharide-negative control.

 
Distribution of group II capsules
Group II capsules have been found to have common genetic organizations in various bacteria (Roberts, 1996). Regions I and II contain the genes involved in the maturation and export of the capsular polysaccharides, and are highly conserved. Therefore, two pairs of primers were designed based on ORFC (region I) and ORFL (region III) of PPD134/91 (Fig. 1) to survey the presence of group II capsule gene clusters among 33 strains of A. hydrophila. In strain PPD134/91, the PCR products of ORFC and ORFL are 946 bp and 1863 bp, respectively (Table 2). Ten strains produced the same PCR products as PPD134/91, indicating that these strains may produce group II-like capsules (Table 1).

Genetic organization of regions I and III of the group II capsule
To further verify the presence of group II capsule gene clusters in these ten A. hydrophila strains, the genetic organization of regions I and III of these strains was further examined. DNA fragments for ORFA–B, ORFA–C and ORFA–D in region I, and ORFL–M in region III, were amplified using various sets of primers and the sizes were compared with those in PPD134/91 (Fig. 1, Table 2). All of the ten strains except JCM3990 had the same PCR profiles as PPD134/91. The results indicate that all the capsule gene clusters in nine of these strains share a common genetic organization in their regions I and III and consequently confirm them as having PPD134/91-like group II capsule gene clusters. JCM3990 and six other strains that produced non-group II capsules did not give any PCR products of expected sizes. A Southern blot analysis using probes prepared from regions I and III of PPD134/91 further confirmed the presence of group II capsule gene clusters in these nine strains, but not in JCM3990 and the six other non-group II capsule-producing strains (Table 1).

Two types of capsule biosynthesis gene clusters in nine group II capsule-producing strains
Region II DNA from the ten group II capsular positive strains (including PPD134/91) was cloned by long-range PCR using primers 79B1 and 79B2 (Table 1, Fig. 1). These strains were divided into two types based on the sizes of their PCR products. The first type included PPD134/91, PPD64/90, JCM3980, JCM3996, 307-01 and 309-01. They had similar PCR bands as PPD134/91, which was about 10 kb in size (data not shown). The other type consisted of four strains, namely PPD88/90, PPD11/90, JCM3983 and SL118-79, which had band sizes of about 5 kb in their PCR products (data not shown).

Seven pairs of primers were designed based on the seven capsule biosynthesis genes in region II of PPD134/91 (Fig. 1, Table 2). PCR was performed on all the nine strains of A. hydrophila that produced group II capsules to survey the distribution of the biosynthesis genes among these strains. The results (Table 3) showed that, except for ORFG' in PPD64/90, PCR products of similar size were present in six group II capsule-producing strains (first type). The 3' and 5' end sequences of these capsule biosynthesis genes were also sequenced to confirm their identities (data not shown). Our results confirmed that all of the PPD134/91 group II capsule biosynthesis genes were present in JCM3980, JCM3996, 307-01, 309-01 and PPD64/90. Due to the similarity in their capsule gene organization, these six strains were named group IIA capsule-producing strains. None of the capsule biosynthesis (region II) genes in PPD134/91 was present in the other three strains, namely PPD11/90, JCM3983 and SL118-79 (Table 3), and their region II gene clusters were much shorter than those of group IIA capsule-producing strains. These were named group IIB capsule-producing strains. The genetic organization of the region II gene cluster in PPD88/90 was unusual. Four out of seven genes in PPD134/91 (ORFE, F, G, K) were found in PPD88/90, with three genes (ORFH–J) deleted and an addition of an ORF with unknown function (ORFN, position 1618–2361, in accession no. AY177619) (Fig. 1, Table 3). This could reflect the heterogeneity of capsule biogenesis in A. hydrophila.


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Table 3. Distribution of capsule region II ORFs of PPD134/91 among various strains of A. hydrophila

Internal primers were designed for the seven capsule region II ORFs. PCR was performed using genomic DNAs for different A. hydrophila strains as templates in order to survey the distribution among these strains. Those samples which had only one band of the same size as the coding gene in PPD134/91 were taken as positive. Symbols: +, positive; -, negative; (+), strain that was positive but which had a larger size of PCR product; (-), strain that was negative but found to have a similar coding gene as PPD134/91 based on DNA sequencing.

 
The ORFG' of PPD64/90 was a larger fragment (2932 bp) than the ORFG of PPD134/91 (1257 bp; Zhang et al., 2002). The sequence analysis of ORFG' (positions 1–150 and 2143–2898 in accession no. AY177618) showed that it was similar to the ORFG of PPD134/91 (positions 8754–10010 in accession no. AF375657), which encoded UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase (Zhang et al., 2002) with 62 % amino acid identity (Fig. 1). Two additional ORFs, ORFG'a and G'b (positions 235–870 and positions 1252–1908 in accession no. AY177618), were found inside ORFG', which showed high similarity with the transposase (AF450275, 56 % amino acid identity and E value of 2e-60) and transpositional helper protein (AF450275, 68 % amino acid identity and E value of 2e-5) of Ralstonia solanacearum. The transposase and transpositional helper protein jumped from one strain to another and this could affect the putative function of ORFG', which is to catalyse the synthesis of UDP-ManNAcA from UDP-GlcNAc (Lo et al., 2001).

Cloning and sequence analysis of region II genes of PPD11/90 and JCM3983
Genes in region II of PPD11/90 and JCM3983 were sequenced for detailed analysis of the group IIB capsule clusters. Region II of PPD11/90 was 5424 bp long, including five ORFs, namely ORFO, P, Q, R and S (Fig. 1, Table 4; accession no. AY144595). The first ORF of region II, ORFO, was separated from region I by 79 bp. The second ORF (ORFP) was separated from the first one by 53 bp. ORFQ was separated from ORFP by 91 bp, and from ORFR by 361 bp. The last ORF (ORFS) of region II was separated from ORFR by 69 bp, and from the first ORF of region III (ORFL) by 205 bp. The mean G+C content of PPD11/90 region II cluster was 35 mol%, which is low compared to the mean G+C content for A. hydrophila. The G+C contents of ORFO, P, Q, R and S were 41 %, 35 %, 36 %, 33 % and 34 %, respectively.


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Table 4. Properties of ORFs in the region II capsule clusters of A. hydrophila PPD11/90 and JCM3983

 
The region II capsule cluster of JCM3983 was 5520 bp long and included four ORFs (Fig. 1, Table 4; accession no. AY177683): ORFO, P, Q'R' and T. The first ORF, ORFO, was separated from region I by 67 bp, and from ORFP by 57 bp. ORFP was 6 bp from ORFQ'R'. ORFQ'R' was separated from ORFT by 57 bp. There were 104 bp between ORFT and the first gene of region III, ORFL (kpsC). The mean G+C content of JCM3983 region II cluster was 36 mol%. The G+C contents of ORFO, P, Q'R' and T were 44 %, 38 %, 33 % and 32 %, respectively. The nucleotide and amino acid sequences of the putative ORFs from PPD11/90 and JCM3983 were used to search available databases for an indication of possible functions and the results are summarized in Table 4.

Five pairs of primers were designed based on the five ORFs of region II genes of PPD11/90. PCR was performed on PPD134/91, PPD88/90, JCM3983, SL118-79 and PPD11/90. Four of the region II genes in PPD11/90 (ORFO, P, Q, R) were present in JCM3983 and SL118-79. None of them was present in PPD134/91 and PPD88/90, suggesting that PPD88/90 is closer to the group IIA rather than the group IIB capsule. Only four ORFs were found in the region II gene cluster of strain JCM3983. However, the corresponding ORFQ and R of PPD11/90, which encoded putative glycosyltransferase and putative galactosyl phosphotransferase, were merged to form a putative bifunctional enzyme (ORFQ'R') in JCM3983. This ORF might have evolved from ORFQ and ORFR by mutation or recombination.

Predicted gene function of region II genes in the group IIB capsule
The first ORFs (ORFO) of region II in PPD11/90 and JCM3983 share about 70 % identity in their amino acid sequences with GalU of Yersinia pestis and other bacteria such as E. coli and Salmonella enterica subsp. enterica serovar Typhi (Table 4). These two ORFs share 77 % identity in their DNA sequences and 91 % identity in their amino acid sequences. galU encodes the enzyme UTP-glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, UDPG : PP), which catalyses the formation of UDP-glucose (UDP-Glc) from glucose 1-phosphate and UTP. UDP-Glc is required for the interconversion of glucose and galactose (Gal) by the Leloir pathway (Frey, 1996) and is also the substrate for the synthesis of UDP-galacturonic acids (UDP-GalA). Glc, Gal and UDP-GalA are the common components for the biosynthesis of capsular polysaccharides (Mollerach et al., 1998). A defect in UDPG : PP can impair the biosynthesis of capsules (Mollerach et al., 1998) and decrease the virulence of bacteria (Genevaux et al., 1999). It is reasonable to predict that UDPG : PP is a key component in the biosynthesis pathways of PPD11/90 and JCM3983 capsules.

ORFP in PPD11/90 and JCM3983 have 76 % and 81 % identities in their nucleotide and amino acid sequences, respectively. Both of them have sequence similarities to LcbA of Neisseria meningitidis, the capsular polysaccharide synthesis gene of Actinobacillus pleuropneumoniae, SacB of N. meningitidis serogroup A, and hexose transferase of Streptococcus thermophilus NCFB2393. Specific functions have not been assigned to these proteins. However, the functions of SacB of N. meningitidis serogroup A and hexose transferase of S. thermophilus have been postulated (Swartley et al., 1998; Almiron-Roig et al., 2000). Mutational studies have shown that SacB is essential for the expression of group A capsules in N. meningitidis. It is hypothesized that SacB is a polymerase linking individual UDP-ManNAc monomers together. The sequences of orfP from PPD11/90 and JCM3983 are much shorter than SacB (by 186 and 182 aa). Detailed biochemical analysis will be required to confirm the functions of ORFP.

The amino acid sequence of the product of ORFQ of PPD11/90 showed similarity to glycosyltransferase of S. thermophilus (EpsH) (Germond et al., 2001) and Lactobacillus delbrueckii subsp. bulgaricus (EpsJ) (Lamothe et al., 2002). A conserved domain database search (http://www.ncbi.nlm.nih.gov/BLAST/) showed that ORFQ (of 144 aa) has sequence similarity to putative glycosyltransferase of Rhizobium meliloti ExpA2 (Becker et al., 1997) and other bacterial glycosyltransferases. Thus, ORFQ may be a putative glycosyltransferase. ORFR of PPD11/90 had sequence similarity to EpsJ of Lactococcus lactis subsp. cremoris (van Kranenburg et al., 1999), which appeared to be involved in its exopolysaccharide biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier. ORFS of PPD11/90 had sequence similarity to O-acetyltransferase of Vibrio cholerae type 2 strain V208 (Nesper et al., 2002). This protein may play a role in the modification of the capsular polysaccharides of this bacterium.

A conserved domain database search using the amino acid sequence of ORFQ'R' of JCM3983 showed that there is a glycosyltranferase domain in its N-terminus (173 aa, positions 2071–2589 in accession no. AY177683). The N-terminal sequence (213 aa, positions 2062–2700 in accession no. AY177683) had similarity to glycosyltransferase of Strep. thermophilus (EpsH) and Lb. delbrueckii subsp. bulgaricus (EpsJ) (Table 4). The 330 aa sequence (positions 2059–3048 in accession no. AY177683) from the N-terminus of ORFQ'R'shared 70 % amino acid identity when compared with ORFQ of PPD11/90. The 266 aa sequence (positions 3493–4290 in accession no. AY177683) from the C-terminus of ORFQ'R' shared 55 % amino acid identity with ORFR of PPD11/90. The C-terminus sequence (343 aa, positions 3211–4239 in accession no. AY177683) had sequence similarity to galactosyl phosphotransferase of Lc. lactis subsp. cremoris (EpsJ) (Table 4). Thus, ORFQ'R' may be a bifunctional glycosyltransferase and galactosyl phosphotransferase. The function of ORFT of JCM3983 could not be predicted since no good sequence similarity was obtained from searches in available databases using both its DNA and amino acid sequences.

In summary, the G+C content and database search results indicated that large portions of genes in these region II gene clusters of PPD11/90 and JCM3983 are homologous and may have evolved from the same source.

Serum resistance and phagocyte killing
Capsulation is a crucial virulence determinant for a number of bacterial species, providing protection from serum and phagocyte killing. The capsule increased the degree of virulence of Pasteurella piscicida for fish and conferred resistance to serum killing (Magarinos et al., 1996). Acapsular mutants of P. multocida were removed efficiently from the blood and other host organs, and were readily taken up by murine peritoneal macrophages, but the capsulated wild-type showed significant resistance to phagocytosis (Boyce & Adler, 2000). Differences in capsule types were also found to have significant effects on pneumococcal (Kadioglu et al., 2002) and pathogenic E. coli infections (Kim et al., 1992; Russo et al., 1994). Furthermore, certain group II capsular serotypes in pathogenic E. coli have been documented to contribute to pathogenesis in systemic models of infection (Kim et al., 1992; Russo et al., 1994).

Similarly, the group II capsules of A. hydrophila were found to act as important anti-serum killing and anti-phagocytic factors in our infection model. Table 5 shows that seven out of ten group II capsule-producing A. hydrophila strains (those other than PPD64/90, JCM3983 and SL118-79) were resistant to serum and phagocyte killing. On the other hand, four out of six randomly chosen acapsular strains were sensitive to serum and phagocyte killing. Two acapsular strains, namely L31 and PPD122/91, and a non-group II capsule-producing strain (PPD70/91), also survived in serum and phagocyte killing, suggesting that the group II capsule may not be the only factor contributing to the resistance. It is interesting to note that most of the group IIA capsule-producing strains belong to the O : 18 and O : 34 serogroups in our 33 randomly chosen A. hydrophila isolates, while the group IIB strains belong to the O : 21 and O : 27 serogroups. Among the group II capsule-producing strains, group IIA capsules have a stronger correlation with conferring resistance to serum and phagocyte killing than do group IIB capsules. It is unclear as to the correlation of O-serogroups and types of capsules, and the exact virulence properties of groups IIA and IIB capsules. More strains and further experiments are needed to draw any conclusion.


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Table 5. Serum survival ability and intracellular multiplication in phagocytes of various A. hydrophila strains

 
Three of the group II capsule-producing strains are sensitive to serum and phagocyte killing. For strain PPD64/90, this may be the result of the insertion of putative transposase and putative transpositional helper protein in ORFG' (UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase). This mutation may have inhibited the synthesis of UDP-ManNAcA and the genes that are downstream due to the polar effect, thus affecting the biogenesis of normal capsular polysaccharides. On the other hand, all the genes of O-acetyltransferase in group IIB capsule-producing strains (such as ORFS of PPD11/90) were deleted in the region II gene clusters of JCM3983 and SL118-79. It has been suggested that O-acetylation in bacterial polysaccharides plays a critical pathological and biological role due to its precise sugar(s) modification property. For example, studies of bacteraemia caused by E. coli K1 strains have revealed that O-acetylated K1 strains are more virulent than non-O-acetylated K1 isolates (Frasa et al., 1993). The loss of O-acetylation in Staphylococcus aureus capsular polysaccharide significantly reduced the resistance of the bacterium to opsonophagocytic killing (Bhasin et al., 1998). In the absence of UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase and O-acetyltransferase, capsular polysaccharides may be altered structurally and chemically and hence produce significant changes in the composition and architecture of the group II capsules. As a result, capsular polysaccharide may interrupt the virulence ability of A. hydrophila PPD64/90, JCM3983 and SL118-79. Further experiments are needed to confirm the exact nature of these genes as regards serum and phagocyte sensitivity.

Conclusion
We have reported a DNA-based method for the detection of group II capsules in A. hydrophila. These group II capsules can be divided into two types, IIA and IIB, with group IIA-producing strains having a strong correlation with conferring resistance to serum and phagocyte killing. Knowledge of the composition of the various capsular polysaccharides and of the function of the encoded proteins of each type is important. This will provide insight into the dynamics of the capsule biosynthesis pathway, the evolution of serotypes, and their roles in bacterial pathogenesis. However, the functional relevance of these capsule genes and the mechanisms by which the capsular polysaccharides are synthesized remain to be determined.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 22 November 2002; revised 15 January 2003; accepted 17 January 2003.



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