Distribution and expression of virulence genes in potentially pathogenic bacteria isolated from seafood in Thailand

Figures & data

Table 1. Targeted virulence genes and related information.

Tabla 1. Genes de virulencia seleccionados e información relacionada

Virulence gene	Mechanism	Primer sequence	Size of the amplified fragment (bp)	Bacteria harboring virulence gene	Reference
aerA	synthesis of aerolysin	aerAF (5ʹ AAC CGA ACT CTC CAT) aerAR (5ʹ CGC CTT GTC CTT GTA 3ʹ)	301	A. hydrophila	Li et al. (2011)
ahp	synthesis of cytotoxic enterotoxin	ahpF (5ʹ ATT GGA TCC CTG CCT A 3ʹ) ahpR (5ʹ GCT AAG CTT GCA TCC G 3ʹ)	911
alt	synthesis of serine protease	altF (5ʹ TGA CCC AGT CCT GG 3ʹ) altR (5ʹ GGT GAT CGA TCA CC 3ʹ)	442
csgD	synthesis of curli	csgDF (5ʹ TGA AAR YTG GCC GCA TAT CAA TG 3ʹ) csgDR (5ʹ ACG CCT GAG GTT ATC GTT TGC C 3ʹ)	355	E. cloacae	Akbari et al. (2015)
cesT/eae	interaction with host cell	cesT/eaeF (5ʹ GTT TGC AGA GAA TGG TGG CCC 3ʹ) cesT/eaeR (5ʹ TAG CTT ATG CTT GTG CCG GGT 3ʹ)	333	E. coli	Sandner et al. (2001)
espB		espBF (5ʹ GCC GCT CTG ATT GGT GGT GCT 3ʹ) espBR (5ʹ TGG CGT TGA ACC GGA AAT CCT 3ʹ)	387
cnf1	synthesis of cytotoxic necrotizing factor	cnf1F (5ʹ AAG ATG GAG TTT CCT ATG CAA GGA G 3ʹ) cnf1R (5ʹ CAT TCA GAT CCT GCC CTC ATT ATT 3ʹ)	498		Yamamoto et al. (1995)
cnf2		cnf2F (5ʹ GTG AGG CTC AAC GAG ATT ATG CAC TG 3ʹ) cnf2R (5ʹ CCA CGC TTC TTC TTC AGT TGT TCC TC 3ʹ)	839		Pass et al. (2000)
eaeA	interaction with host cell	eaeAF (5ʹ TGA GCG GCT GGC ATG AGT CAT AC 3ʹ) eaeAR (5ʹ TCG ATC CCC ATC GTC ACC AGA GG 3ʹ)	241
LTI	synthesis of thermolabile toxin	LTIF (5ʹ TGG ATT CAT CAT GCA CCA CAA GG 3ʹ) LTIR (5ʹ CCA TTT CTC TTT TGC CTG CCA TC 3ʹ)	360
STII		STIIF (5ʹ CCC CCT CTC TTT TGC ACT TCT TTC C 3ʹ) STIIR (5ʹ TGC TCC AGC AGT ACC ATC TCT AAC CC 3ʹ)	423
vt1	synthesis of verotoxin	vt1F (5ʹ ACG TTA CAG CGT GTT GCR GGG ATC 3ʹ) vt1R (5ʹ TTG CCA CAG ACT GCG TCA GTR AGG 3ʹ)	121
vt2e		vt2eF (5ʹ CCA GAA TGT CAG ATA ACT GGC GAC 3ʹ) vt2eR (5ʹ GCT GAG CAC TTT GTA ACA ATG GCT G 3ʹ)	322
kfu	iron uptake system	kfuF (5ʹ GAA GTG ACG CTG TTT CTG GC 3ʹ) kfuR (5ʹ TTT CGT GTG GCC AGT GAC GAC TC 3ʹ)	797	Klebsiella	Aher et al. (2012)
magA	synthesis of capsular polysaccharide	magAF (5ʹ GGT GCT CTT TAC ATC ATT GC 3ʹ) magAR (5ʹ GCA ATG GCC ATT TGC GTT AG 3ʹ)	1,280
uge	synthesis of lipopolysaccharide	ugeF (5ʹ TCT TCA CGC CTT CCT TCA CT 3ʹ) ugeR (5ʹ GAT CAT CCG GTC TCC CTG TA 3ʹ)	534
ssaO	type III secretion system apparatus	ssaOF (5ʹ ATG GAA ACT TTG CTG GAG A 3ʹ) ssaOR (5ʹ TCA ACT TTG GTA ATA CGC AT 3ʹ)	378	Salmonella	Bhowmick et al. (2011b)
sscB	synthesis of chaperone	sscBF (5ʹ ATG ATG ATG AAA GAA GAT CA 3ʹ) sscBR (5ʹ TTA AGC AAT AAG AGT ATC AA 3ʹ)	435
sseG	secretion system effector	sseGF (5ʹ ATG AAA CCT GTT AGC CCA AA 3ʹ) sseGR (5ʹ TTA CTC CGG CGC ACG TTG TT 3ʹ)	690
tdh	synthesis of thermolabile haemolysin	tdhF (5ʹ GTA AAG GTC TCT GAC TTT TGG AC 3ʹ) tdhR (5ʹ TGG AAT AGA ACC TTC ATC TTC ACC 3ʹ)	269	Vibrio	Panicker et al. (2004)
tlh		tlhF (5ʹ AAA GCG GAT TAT GCA GAA GCA CTG 3ʹ) tlhR (5ʹ GCT ACT TTC TAG CAT TTT CTC TGC 3ʹ)	450
trh		trhF (3ʹ TTG GCT TCG ATA TTT TCA GTA TCT 3ʹ) trhR (5ʹ CAT AAC AAA CAT ATG CCC ATT TCC G 3ʹ)	500

Table 2. Average numbers of presumptive target bacteria per sample of the same seafood species sold in Thailand.

Tabla 2. Número promedio de bacterias presuntamente seleccionadas por muestra de la misma especie de marisco o pescado vendida en Tailandia

Seafood species		Average number of presumptive target bacteria per sample of the same seafood species (log CFU/g)* (number of positive sample)
Common name (number of sample)	Scientific name
		Presumptive E. coli on EMB agar	Presumptive Salmonella and Shigella on SS agar	Presumptive Vibrio on TCBS agar
Blue swimming crab (6)	Portunus pelagicus	0.7 ± 1.1 (1)	under the detection limit (< 1.2)	0.9 ± 1.1 (2)
Banana shrimp (5)	Fenneropenaeus merguiensis	0.9 ± 1.2 (1)	2.0 ± 2.1 (2)	0.8 ± 1.1 (1)
Splendid squid (4)	Loligo formosana	1.1 ± 1.4 (1)	1.7 ± 2.0 (1)	1.3 ± 1.6 (1)
Spotted babylon (3)	Babylonia areolata	under the detection limit	1.9 ± 2.2 (1)	0.8 ± 1.0 (1)
Scallop (3)	Argopecten purpuratus	under the detection limit	1.9 ± 2.1 (1)	under the detection limit
Barramundi (2)	Lates calcarifer	1.5 ± 1.6 (1)	2.3 ± 0.8 (2)	1.5 ± 1.6 (1)
Bigfin reef squid (2)	Sepioteuthis lessoniana	1.8 ± 0.7 (2)	2.3 ± 1.0 (2)	2.1 ± 1.5 (2)
Short-bodied mackerel (2)	Rastrelliger brachysoma	under the detection limit	2.1 ± 2.3 (1)	under the detection limit
Silver pomfret (2)	Pampus argenteus	under the detection limit	under the detection limit	1.5 ± 1.6 (1)
Kuruma prawn (1)	Marsupenaeus japonicus	under the detection limit	under the detection limit	under the detection limit
Blood cockle (1)	Tegillarca granosa	under the detection limit	under the detection limit	under the detection limit
Mangrove crab (1)	Scylla serrata	under the detection limit	under the detection limit	under the detection limit
Cuttle fish (1)	Sepia officinalis	under the detection limit	2.3 ± 1.4 (1)	under the detection limit
John’s snapper (1)	Lutjanus johnii	1.8 ± 0.9 (1)	2.4 ± 1.3 (1)	1.7 ± 1.4 (1)
Green tiger prawn (1)	Penaeus semisulcatus	under the detection limit	2.4 ± 1.5 (1)	under the detection limit

*In cases that there were more than one sample of the same seafood species, the values shown are the means of all samples assayed ± standard deviations. In cases that there was only one sample of the same seafood species, the values shown are the means of three replicates ± standard deviations.

*En los casos en que hubo más de una muestra de la misma especie de mariscos o pescados, los valores mostrados son las medias de todas las muestras ensayadas ± desviaciones estándar. En los casos en que sólo hubo una muestra de la misma especie de mariscos o pescado, los valores mostrados son las medias de tres réplicas ± desviaciones estándar.

Figure 1. The UPGMA dendrogram constructed from ERIC-PCR patterns of 40 individual strains.

Figura 1. Dendrograma UPGMA construido a partir de patrones ERIC-PCR de 40 cepas individuales

Table 3. The number of strain in each genus or species that possessed virulence gene.

Tabla 3. Número de cepas de cada género o especie que poseen el gen de virulencia

Genus/Species	Number of strain	Number of strain possessing virulence gene
		cnf2	csgD	kfu	LTI	ssaO	tlh	uge	vt2e
Aeromonas spp.	9	0	0	0	0	0	0	0	0
Citrobacter spp.	5	0	5	0	1	0	0	0	0
Enterobacter spp.	7	0	7	0	0	0	0		0
E. coli	2	2	0	0	2	0	0	0	2
H. alvei	2	0	0	0	2	0	0	0	0
K. pneumoniae	3	0	2	1	0	0	0	1	0
M. morganii	4	0	0	0	0	0	0	0	0
P. hauseri	2	0	0	0	0	0	0	0	0
P. rustgianii	1	0	0	0	0	0	0	0	0
S. enterica	2	0	0	0	0	2	0	0	0
V. parahaemolyticus	3	0	0	0	0	0	3	0	0
Total	40	2 (5.0%)	14(35.0%)	1 (2.5%)	5 (12.5%)	2(5.0%)	3(7.5%)	5 (12.5%)	2(5.0%)

Table 4. Detected virulence genes and % identities to the reference sequences.

Tabla 4. Genes de virulencia detectados y porcentaje de identidades respecto a las secuencias de referencia

Detected virulence gene	A representative strain of each species that harbored virulence gene	% identity to the reference sequence	GenBank accession number of the reference sequence
cnf2	E. coli SFEMB6	99.15%	U01097
csgD	C. braakii SFEMB5	99.68%	CP010376
	C. freundii SFEMB1	97.41%	CP010376
	C. koseri SFEMB2	96.66%	CP024680
	E. cloacae SFSS14	100%	CP010376
	E. ludwigii SFSS13	100%	CP001918
	K. pneumoniae SFSS17	99.02%	CP024675
kfu	K. pneumoniae SFSS17	99.20%	AP018753
LTI	C. braakii SFEMB5 E. coli SFEMB6 H. alvei SFSS20	99.71%	EU113255
ssaO	S. enterica SFSS26	99.17%	CP043662
tlh	V. parahaemolyticus SFTCBS4	100%	CP006007
uge	E. cloacae SFSS14	82.82 to 98.38%	CP001891
	E. ludwigii SFSS13
	K. pneumoniae SFSS19
vt2e	E. coli SFEMB6	98.03%	FM998849

Strain names refer to kinds of media used for isolation of the strains. SFEMB: EMB agar; SFSS: SS agar; SFTCBS: TCBS agar.

Los nombres de las cepas hacen referencia a los tipos de medios utilizados para su aislamiento. SFEMB: agar EMB; SFSS: agar SS; SFTCBS: agar TCBS.

Figure 2. The PCR products of the detected virulence genes.

Figura 2. Productos de la PCR de los genes de virulencia detectados

Lane M: 100 bp DNA ladder.; 1: 839-bp PCR product of the cnf2 gene; 2: 360-bp PCR product of the LTI gene; 3: 355-bp PCR product of the csgD gene; 4: 797-bp PCR product of the kfu gene; 5: 534-bp PCR product of the uge gene; 6: 378-bp PCR product of the ssaO gene; 7: 450-bp PCR product of the tlh gene; 8: 322-bp PCR product of the vt2e gene. Arrows indicate PCR products of virulence genes.Carril M: escalera de ADN de 100 pb [par de bases]. 1: 839 pb producto de PCR del gen cnf2; 2: 360 pb producto de PCR del gen LTI; 3: 355 pb producto de PCR del gen csgD; 4: 797 pb producto de PCR del gen kfu; 5: 534 pb producto de PCR del gen uge; 6: 378 pb producto de PCR del gen ssaO; 7: 450 pb producto de PCR del gen tlh; 8: 322 pb producto de PCR del gen vt2eLas flechas indican los productos de la PCR de los genes de virulencia.

Table 5. Expression of virulence genes under the desired conditions.

Tabla 5. Expresión de los genes de virulencia en las condiciones deseadas

Condition of cultivation	Gene expression
	cnf2	csgD	kfu	LTI	ssaO	tlh	uge	vt2e
1. LB without NaCl, 37°C, microaerophilic, 12 h	-	-	+	-	-	+	-	-
2. LB with 0.17 M NaCl, 37°C, microaerophilic, 12 h	-	-	+	-	-	+	+	-
3. LB with 0.3 M NaCl, 37°C, microaerophilic, 12 h	-	-	+	-	-	+	+	-
4. M63 without NaCl, 37°C, microaerophilic, 12 h	-	+	-	-	-	+	-	-
5. M63 with 0.17 M NaCl, 37°C, microaerophilic, 12 h	-	-	-	-	-	+	-	-
6. M63 with 0.3 M NaCl, 37°C, microaerophilic, 12 h	-	-	-	-	-	+	-	-
7. LB with 0.17 M NaCl, 37°C, aerobic, 12 h	-	-	+	-	-	+	-	-
8. LB with 0.17 M NaCl, 25°C, microaerophilic, 12 h	-	-	+	-	-	+	-	-
9. LB with 0.17 M NaCl, 37°C, microaerophilic, 6 h	-	-	-	-	-	+	-	-
10. LB with 0.17 M NaCl, 37°C, microaerophilic, 16 h	-	-	+	-	-	+	-	-

+: gene was expressed; -: gene was not espressed.

+: el gen se expresó; -: el gen no se expresó.

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