Routine isolation and identification of Yersinia enterocolitica from naked carp of Qinghai Lake
Colony morphology and culture characteristics
Red bull-eye colonies appeared on CIN-1. Colorless and transparent, non-sticky colonies appeared on modified Y. Gram staining microscopic examination showed short rod-shaped Gram-negative bacilli or cocci, mostly scattered individually, sometimes arranged in short chains or piles, without spores and capsules.
Screening of bacteria
After preliminary screening tests, 20 strains of KIA with yellow and non-gas-producing strains were obtained on the slope and bottom of KIA. Fifteen urea-positive strains were obtained by rescreening. Two rescreening yielded 5 strains of 26 ° C cultured with motility, and 37 ° C cultured non-motility strains, which were classified as Y1301, Y1302, Y1303, Y1304, Y1305.
Biochemical identification
See Table 1 for details.
Table 1. The result of biochemical characterisitic of Yersinia enterocolitica
Items
|
Y1301
|
Y1302
|
Y1303
|
Y1304
|
Y1305
|
GLU
|
+
|
+
|
+
|
+
|
+
|
LAC
|
-
|
-
|
-
|
-
|
-
|
MAL
|
-
|
-
|
-
|
-
|
-
|
SUC
|
+
|
+
|
+
|
+
|
+
|
Trehalose
|
+
|
-
|
+
|
+
|
-
|
RHA
|
-
|
-
|
-
|
-
|
-
|
Raffinose
|
-
|
-
|
-
|
-
|
-
|
ARA
|
+
|
-
|
+
|
+
|
-
|
XYL
|
-
|
-
|
-
|
-
|
-
|
MON
|
+
|
+
|
+
|
+
|
+
|
SOR
|
+
|
+
|
+
|
+
|
+
|
DUL
|
-
|
-
|
-
|
-
|
-
|
SAL
|
-
|
-
|
-
|
-
|
-
|
INO
|
+
|
+
|
+
|
+
|
+
|
ODC
|
+
|
+
|
+
|
+
|
+
|
TRP
|
-
|
-
|
-
|
-
|
-
|
CIT
|
-
|
-
|
-
|
-
|
-
|
NTT
|
+
|
-
|
+
|
+
|
-
|
URE
|
+
|
+
|
+
|
+
|
+
|
Semi-solid
|
26℃
|
+
|
+
|
+
|
+
|
+
|
37℃
|
-
|
-
|
-
|
-
|
-
|
GEL
|
+
|
+
|
+
|
+
|
+
|
MR
|
-
|
-
|
-
|
-
|
-
|
V-P
|
26℃
|
+
|
+
|
+
|
+
|
+
|
37℃
|
-
|
-
|
-
|
-
|
-
|
H2S
|
-
|
-
|
-
|
-
|
-
|
IND
|
-
|
-
|
-
|
-
|
-
|
Note: "+" means positive, "-" means negative
Drug sensitive test
The results are as follows (Tab.2)
Table 2. Sensitive Test of 14 kinds of Medicine for 5 strains Yersinia enterocolitic
Antibiotics
|
Y1301
|
Y1302
|
Y1303
|
Y1304
|
Y1305
|
Resistance
|
intermediary
|
Sensitivity
|
AZI
|
S
|
R
|
R
|
S
|
R
|
60%
|
0.00%
|
40%
|
GMIO
|
S
|
S
|
S
|
S
|
I
|
0.00%
|
20%
|
80%
|
CH
|
R
|
R
|
R
|
R
|
R
|
100%
|
0.00%
|
0.00%
|
E
|
R
|
R
|
S
|
I
|
R
|
60%
|
20%
|
20%
|
AN
|
S
|
R
|
S
|
R
|
I
|
40%
|
20%
|
40%
|
CAZ
|
R
|
R
|
I
|
R
|
S
|
60%
|
20%
|
20%
|
PIP
|
R
|
S
|
R
|
S
|
I
|
40%
|
20%
|
40%
|
TE
|
I
|
I
|
S
|
R
|
I
|
20%
|
60%
|
20%
|
TM
|
S
|
S
|
S
|
R
|
S
|
20%
|
0.0%
|
80%
|
CMZ
|
R
|
R
|
R
|
R
|
S
|
80%
|
0.00%
|
20%
|
K
|
R
|
R
|
R
|
S
|
I
|
60%
|
20%
|
20%
|
S300
|
R
|
R
|
R
|
I
|
R
|
80%
|
20%
|
0.00%
|
SXT
|
R
|
R
|
R
|
R
|
I
|
80%
|
20%
|
0.00%
|
C
|
R
|
S
|
R
|
R
|
S
|
60%
|
0.00%
|
40%
|
Pathogenicity test
All mice in the test group died after 48 hours, and the control mice remained alive after one week of feeding. It was proved that the isolated strain had a lethal effect on mice. Examination of dead mice showed hepatomegaly, dark spleen, a small amount of fluid in the pericardium, and intestinal necrosis in some sections. Aseptic examination of the liver and spleen with contact pad staining microscopy showed short rod-shaped or oval Gram-negative Spores and capsular bacteria, and the pathogenic bacteria were isolated from dead rat livers, and then biochemically identified. The results showed that the biochemical results of pathogenic bacteria isolated from the liver of dead mice and isolated from naked carp of Qinghai Lake were completely consistent.
16S rRNA gene colon
PCR amplification of 16S rRNA gene of Yersinia enterocolitica from naked carp of Qinghai Lake (Fig 1).
PCR specificity test of wild isolates and control bacteria
The PCR specificity test of 16s rRNA gene was performed on Y1301 and Y1302 strains with P1 and P2 primers, respectively. The target fragment of 1419 bp in size consistent with the expected results was obtained (Fig.2).
Sensitivity test of isolated strains
A single clone of Y1301 was aseptically inoculated into LB Amp + liquid medium, cultured at 26 ° C for 12 hours, and genomic DNA was extracted. The extracted DNA was diluted 10-fold to 10-10 in turn, and P1 and P2 primer pairs were applied respectively. The PCR amplification of 16S rRNA gene was performed, and the obtained PCR product was subjected to electrophoresis, and the results showed that a dilution of 10-6 was detectable (Fig.3).
Homologous analysis of sequence of Y.e 16S rRNA gene from naked carp of Qinghai Lake
The nucleotide sequence comparison showed that the nucleotide homology of Y1301, Y1303 and Y1304 was 100%; the homology of Y1302 and Y1305 was 100%, and there were no mutation sites. AJ639645, HE803738, HE803739, HE803740, HE803741, HE803742, HE803743, HE803744, HE803745, HE803748, HE803750, HE803756, HE803758, HE803762, HE803792 are in sites of 28th, 72th, 75th, 81th, 84th, 101th, 112nd, 154 th, 299 th, 325 th, 346 th, 347 th, 351st , 352nd , 359 th, 361 st, 362 nd, 366 th, 367 th, 481 st, 485 th, 491 st, 507 th, 511 st, 515 th, 529 th, 540 th, 702 nd, 729 th, 739 th, 806 th, 816 th, 867 th, 892 nd, 893 th, 897 th, 912 nd, 913 th, 928 th, 929 th, 992-997 th, 998 th -1006 th, 118 th, 1010 th -1012 nd, 1016 th -1017 th 1020 th -1025 th, 1041st -1042 nd, 1046 th, 1050 th - 1055 th, 1057 th -1067 th, 1070 th -1073 th, 1076 th -1077 th, 1078 th -1079 th, and 1081st -1082nd have changed. Y1301 had base deletions at 915 and 976, and Y1302 had base deletions at 816 and 867, respectively(Fig.4, Fig.5).