Characterization of biofilms in biliary stents and potential factors involved in occlusion

Table 1 Primer sequences used for polymerase chain reaction

Sr. No	Organism	Primer sequences
1	Pseudomonas	F: 5'-GACGGGTGAGTAATGCCTA- 3'
		R: 5'-CACTGGTGTTCCTTCCTATA -3'
2	Staphylococcus	F: 5'- AAC TCT GTT ATT AGG GAA GAA CA- 3'
		R: 5'- CCA CCT TCC TCC GGT TTG TCA CC-3'
3	E. coli	F: 5'-GAAGCTTGCTTCTTTGCT- 3'
		R: 5'-GAGCCCGGGGATTTCACAT- 3'
4	Enterococcus	F: 5'- GTTTATGCCGCATGGCATAAGAG -3'
		R: 5'-CCGTCAGGGGACGTTCAG -3'
5	Citrobacter	F: 5′-TCAGATTTGAACGCTGGCGGCA -3′
		R: 5′-CGTATTACCGCGGCTGCTGCCAC -3′
6	Proteus	F: 5'-AGA GTT TGA TCC TGG CTC AG-3'
		R: 5'-AAG GAG GTG ATC CAG CC-3'
7	Klebsiella	F: 5'-AGA GTT TGA TCC TGG CTC AG-3'
		R: 5'-AAG GAG GTG ATC CAG CC-3'
8	Clostridium	F: 5'-TGG CTC AGA TTG AAC GCT GGC GGC -3'
		R: 5'-TAC CTT GTT ACG ACT TCA CCA CA-3'
9	Bacillus	F:5'- AGA GTT TGA TCC TGG CTC AG -3'
		R: 5'- AAG GAG GTG ATC CAG CCG CA-3'
10	Vibrio	F: 5'-AGA GTT TGA TCA TGG CTC AG -3'
		R:5'-GAA ATT CTA CCC CCC TCT ACA G-3'
11	Aeromonas	F: 5'-GCT GGT CTG AGA GGA TGA TC-3'
		R: 5'-CTT TAC GCC CAG TAA TTC CG-3'
12	Bacteroides	F: 5'- ATT CTA GAG TTT GAT CAT GGC TCA-3'
		R: 5'-ATG GTA CCG TGT GAC GGG CGG TGT GTA-3'
13	Enterobacter	F: 5'-AGTTTGATCCTGGCTCAG -3'
		R: 5'-TAC CTT GTT ACG ACT TCG TCC CA-3'
14	Streptococcus	F: 5'-TAA CCA GAA AGG GAC GGC TA-3'
		R: 5'-CAC TCT CCG CTT CTG CAC TC-3'
15	Serratia	F : 5'-GCGGTTTGTTAAGTCAGATG -3'
		R: 5'-CGAATTAAACCACATGCTCC-3'
16	Yersinia	F : 5′-AAT ACC GCA TAA CGT CTT CG-3′
		R: 5′-CTT CTT CTG CGA GTA ACG TC-3′

Table 2 Organisms identified by polymerase chain reaction and sequencing (accession Nos. KP198519-43; KP205043-80; KP2)

Sr. No.	Organisms identified by touchdown PCR and sequencing	Organisms identified by PCR alone
BF 1	Stenotrophomonas maltophilia	Klebsiella
BF 2	Pseudomonas stutzeri	Pseudomonas, Proteus, Aeromonas
BF 3	Bacillus tequilensis	Staphylococcus, Bacillus
BF 4	Uncultured bacterium clone DolRC 17069	Streptococcus, Aeromonas, Serratia
BF 5	Bacillus cereus	Staphylococcus, Bacillus
BF 6	Micrococcus yunnanensis	Streptococcus, Proteus, Serratia
BF 7	Staphylococcus epidermidis	Staphylococcus, Bacillus
BF 8	Citrobacter sp.	Citrobacter, Escherichia coli
BF 9	Stenotrophomonas maltophilia	Proteus
BF 10	Anaerosalibacter sp.	Pseudomonas, Citrobacter
BF 11	Enterobacteriales bacterium	Staphylococcus, Streptococcus, Klebsiella
BF 12	Uncultured bacterium clone PS B346	Staphylococcus, Aeromonas
BF 13	Stenotrophomonas maltophilia	——
BF 14	Stenotrophomonas maltophilia	Streptococcus
BF 15	Stenotrophomonas maltophilia	Serratia
BF 16	Uncultured organism clone ELU0026	Citrobacter
BF 17	Uncultured organism clone ELU0020	——
BF 18	Stenotrophomonas maltophilia	Citrobacter, Streptococcus
BF 19	Uncultured bacterium clone ELU0020	Klebsiella, Aeromonas, Enterococcus
BF 20	Bacillus mojavensis	Pseudomonas, Bacillus, Enterobacter
BF 21	Paenibacillus sp. A1006	Proteus, Klebsiella, Serratia
BF 22	Bacillus cereus	Streptococcus, Serratia
BF 23	Stenotrophomonas maltophilia	——
BF 24	Bacillus sp.	Proteus, Yersinia, Aeromonas
BF 25	Pseudomonas stutzeri	Pseudomonas, Escherichia coli
BF 26	Enterobacteriales bacterium	Pseudomonas, Proteus, Klebsiella
BF 27	Uncultured bacterium clone DolRc DL35rect19C08	Proteus, Klebsiella
BF 28	Enterococcus faecalis	Citrobacter, Enterococcus
BF 29	Micrococcus luteus	——
BF 30	Staphylococcus epidermidis	Staphylococcus, Serratia
BF 31	Staphylococcus epidermidis	Citrobacter, Escherichia coli, Staphylococcus
BF 32	Enterococcus durans	Escherichia coli, Klebsiella, Enterococcus
BF 33	Enterococcus durans	Staphylococcus, Streptococcus, Serratia
BF 34	Stenotrophomonas maltophilia	Pseudomonas
BF 35	Uncultured bacterium clone B64	Citrobacter, Proteus, Klebsiella
BF 36	Pseudomonas otitidis	Pseudomonas, Aeromonas
BF 37	Enterobacteriales bacterium	Streptococcus, Klebsiella, Enterobacter
BF 38	Pseudomonas alcaligenes	Citrobacter, Escherichia coli, Yersinia
BF 39	Enterococcus faecalis	Citrobacter, Streptococcus, Klebsiella
BF 40	Enterococcus faecalis	Streptococcus, Klebsiella , Aeromonas
BF 41	Enterococcus sp.	Citrobacter, Enterobacter
BF 42	Bacillus subtilis	Pseudomonas, Streptococcus, Aeromonas
BF 43	Pseudomonas aeruginosa	Pseudomonas, Escherichia coli, Yersinia
BF 44	Uncultured bacterium	Pseudomonas, Citrobacter, Yersinia
BF 45	Uncultured bacterium	Pseudomonas, Citrobacter, Escherichia coli
BF 46	Pseudomonas sp.	Escherichia coli, Enterobacter
BF47	Bacillus cereus	Proteus, Klebsiella
BF 48	Stenotrophomonas maltophilia	Pseudomonas, Citrobacter, Streptococcus
BF 49	Stenotrophomonas maltophilia	Pseudomonas, Staphylococcus, Proteus
BF 50	Bacillus cereus	Pseudomonas , Staphylococcus
BF 51	Pseudomonas aeruginosa	Pseudomonas, Citrobacter, Escherichia coli
BF 52	Pseudoxanthomonas icgebensis	Citrobacter, Serratia
BF 53	Enterobacteriales bacterium	Klebsiella, Enterobacter, Vibrio
BF 54	Citrobacter freundii	Citrobacter, Staphylococcus
BF 55	Uncultured bacterium clone PS	Staphylococcus, Serratia
BF 56	Enterobacteriales bacterium	Pseudomonas, Citrobacter, Streptococcus
BF 57	Morganella morganii	——
BF 58	Pseudomonas aeruginosa	Pseudomonas, Escherichia coli, Staphylococcus
BF 59	Pseudomonas sp.	Pseudomonas, Staphylococcus, Serratia
BF 60	Pseudomonas putida	Pseudomonas, Klebsiella , Aeromonas
BF 61	Morganella morganii	——
BF 62	Enterobacteriales bacterium	Pseudomonas, Aeromonas
BF 63	Enterococcus faecalis	Enterococcus
BF 64	Pseudomonas sp.	Pseudomonas, Staphylococcus, Serratia
BF 65	Bacillus sp.	Citrobacter, Enterobacter
BF 66	Uncultured organism clone	——
BF 67	Pseudomonas sp.	Pseudomonas
BF 68	Bacillus licheniformis	Pseudomonas, Enterobacter
BF 69	Enterococcus sp.	Klebsiella , Serratia
BF 70	Enterococcus faecalis	Citrobacter, Enterobacter, Serratia
BF 71	Pseudomonas stutzeri	Pseudomonas, Staphylococcus, Aeromonas
BF 72	Escherichia coli	Escherichia coli
BF 73	Klebsiella sp.	Pseudomonas, Klebsiella
BF 74	Uncultured Klebsiella sp.	Escherichia coli
BF 75	Enterobacteriales bacterium	Escherichia coli, Enterobacter, Yersinia
BF 76	Stenotrophomonas maltophilia	——
BF 77	Enterobacteriales bacterium	Klebsiella
BF 78	Citrobacter sp. enrichment clone	Citrobacter
BF 79	Serratia marcescens	Staphylococcus, Serratia
BF 80	Klebsiella sp. BAB-3527	Klebsiella, Aeromonas
BF 81	Exiguobacterium aurantiacum	Klebsiella

PCR: Polymerase chain reaction.

Table 3 Microbial species detected from the biliary stents (n = 81)

	Number of stents positive for the isolates
Gram-positive microorganism
Bacillus sp.	11
Enterococcus sp.	9
Micrococcus sp.	2
Streptococcus sp.	11
Staphylococcus sp.	17
Gram-negative microorganism
Citrobacter sp.	20
Escherichia coli	12
Enterobacter sp.	9
Klebsiella sp.	19
Morganella morganii	2
Proteus sp.	10
Pseudomonas sp.	27
Serratia sp.	15
Stenotrophomonas maltophila	11
Vibrio sp.	1
Yersinia sp.	5
Aeromonas sp.	12

Table 4 Various factors in relation to protein and polysaccharide concentrations

Parameters	Protein (mg/ml)	P value	Polysaccharide concentration (mg/mL)	P value
Gender
Male (n = 41)	0.547 ± 0.242	0.115	0.052 ± 0.021	< 0.0001
Female (n = 40)	0.458 ± 0.259		0.049 ± 0.016
Age
Below 60 (n = 60)	0.386 ± 0.238	0.205	0.038 ± 0.016	0.011
Above 60 (n = 21)	0.468 ± 0.295		0.051 ± 0.026
Etiology of stenting
Cholangitis (n = 50)	0.555 ± 0.225	0.018	0.0512 ± 0.021	0.790
No cholangitis (n = 31)	0.419 ± 0.276		0.050 ± 0.014
Indication of stent insertion
CBD stone (n = 46)	0.518 ± 0.256	0.530	0.051 ± 0.022	0.785
Benign stricture (n = 29)	0.453 ± 0.256		0.050 ± 0.012
Indwelling stent size
7 Fr (n = 62)	0.541 ± 0.238	0.005	0.049 ± 0.015	0.674
10 Fr (n = 19)	0.356 ± 0.252		0.052 ± 0.020
Duration of indwelling stents
< 3 mo (n = 39)	0.481 ± 0.242	0.472	0.0489 ± 0.015	0.385
≥ 3 mo (n = 42)	0.523 ± 0.264		0.0525 ± 0.022
< 6 mo (n = 65)	0.476 ± 0.251	0.060	0.0501 ± 0.017	0.560
≥ 6 mo (n = 16)	0.609 ± 0.240		0.0533 ± 0.026
No of microorganisms detected monomicrobial (n = 13)	0.502 ± 0.263	0.996	0.051 ± 0.018	0.968
Polymicrobial (n = 68)	0.501 ± 0.050		0.049 ± 0.015