Relationship Between Drug Resistance Characteristics and Biofilm Formation in Klebsiella Pneumoniae Strains

Figures & data

Table 1 Primer Sequences Used in This Study

Target Genes	Primer Sequence (5’–3’)	Amplicon Size (bp)	Tm (°C)	Reference
TEM	F:CATTTCCGTGTCGCCCTTATTC	800	56.2	[14]
	R:CGTTCATCCATAGTTGCCTGAC
SHV	F:CACTCAAGGATGTATTGTG	885	52	[15]
	R:TTAGCGTTGCCAGTGCTCG
CTX-M	F:ACAGCGATAACGTGGCGATG	216	58.3	[16]
	R:TCGCCCAATGCTTTACCCAG
K1	F:GGTGCTCTTTACATCATTGC	1283	53	[17]
	R:GCAATGGCCATTTGCGTTAG
K2	F:GACCCGATATTCATACTTGACAGAG	641	54	[17]
	R:CCTGAAGTAAAATCGTAAATAGATGGC
K5	F:TGGTAGTGATGCTCGCGA	280	56	[17]
	R:CCTGAACCCACCCCAATC
K20	F:CGGTGCTACAGTGCATCATT	741	58	[17]
	R:GTTATACGATGCTCAGTCGC
K57	F:CTCAGGGCTAGAAGTGTCAT	1037	56	[17]
	R:CACTAACCCAGAAAGTCGAG
rmpA	F:ACTGGGCTACCTCTGCTTCA	535	54.1	[16]
	R:CTTGCATGAGCCATCTTTCA
fimH	F:TGCTGCTGGGCTGGTCGATG	550	61	[16]
	R:GGGAGGGTGACGGTGACATC
mrkD	F:CCACCAACTATTCCCTCGAA	226	54	[16]
	R:ATGGAACCCACATCGACATT
ybtS	F:GACGGAAACAGCACGGTAAA	242	52	[18]
	R:GAGCATAATAAGGCGAAAGA
allS	F:CCGAAACATTACGCACCTTT	508	55	[12]
	R:ATCACGAAGAGCCAGGTCAC
kfu	F:GGCCTTTGTCCAGAGCTACG	638	59	[18]
	R:GGGTCTGGCGCAGAGTATGC
aerobactin	F:GCATAGGCGGATACGAACAT	556	54	[12]
	R:CACAGGGCAATTGCTTACCT
entB	F:GTCAACTGGGCCTTTGAGCCGTC	400	60	[18]
	R:TATGGGCGTAAACGCCGGTGAT

Figure 1 The value of biofilm formation of strains from different sources.

Table 2 Distribution of the Lowest Inhibitory Concentrations of 96 Strains of Klebsiella Pneumoniae to 16 Antibiotics

Drug	Number of Isolates with MIC (μg/mL)
	1	2	4	8	16	32	64	128	256	512	1024
Piperacillin				1	3	10	32		9	15	26
Ampicillin/ Sulbactam		13	17	4	1		2	10	21	25	3
Cefazolin			9	28	7			10	32	9	1
Ceftazidime	1	27	30		1	16	16	4	1
Ceftriaxone	32	8	6	20	15	13	2
Cefotaxime					3	17	21	13	8	12	22
Cefepime	1	2	36	20		3	7	17	10
Cefoxitin		9	20	31	1	2	1	7	23	2
Aminotransol	1	5	38	12	2			1	20	17
Imipenem	78	1		5	10	2
Meropenem	80			4	11	1
Gentamicin	12	37	8	5	7	26	1
Tobramycin	11	32	10	9	4	5	24	1
Ciprofloxacin	10	33	11	9	4	21	4	3
Cotrimoxazole						1	20	32	9	29	5
Furantoin			1	6	23	3	3	7	33	18	2

Table 3 Resistance Rates of hvKP Vs cKP, n=96

Antibacterial Drugs	hvKP (n=32)	cKP (n=64)	P-value
	No. (%)	No. (%)
Piperacillin	8 (16.7)	42 (65.6)	<0.001
Ampicillin / Sulbactam	13 (40.6)	48 (75.0)	0.001
Cefazolin	10 (31.3)	42 (65.6)	0.001
Ceftazidime	9 (28.1)	29 (45.3)	0.105
Ceftriaxone	9 (28.1)	41 (64.1)	0.001
Cefotaxime	9 (28.1)	38 (59.4)	0.004
Cefepime	7 (21.9)	30 (46.9)	0.018
Cefoxitin	9 (28.1)	24 (37.5)	0.362
Aminotransol	9 (28.1)	29 (45.3)	0.105
Imipenem	4 (12.5)	13 (20.3)	0.345
Meropenem	3 (9.4)	13 (20.3)	0.175
Gentamicin	6 (18.8)	33 (51.6)	0.002
Tobramycin	3 (9.4)	31 (48.4)	<0.001
Ciprofloxacin	6 (18.8)	27 (42.2)	0.023
Cotrimoxazole	3 (9.4)	31 (48.4)	<0.001
Furantoin	16 (50.0)	47 (73.4)	0.023
MDR	9 (28.1)	44 (68.8)	<0.001
ESBLs	5 (15.6)	28 (43.8)	0.006
Carbapenemase	3 (9.4)	12 (18.8)	0.233

Table 4 Distribution of Virulence Genes in cKP Strains and hvKP Strains, n=96

Toxicity Factor	hvKP (n=32)	cKP (n=64)	P-value
	No. (%)	No. (%)
fimH	31 (96.9)	57 (89.1)	0.361
mrkD	32 (100.0)	63 (98.4)	1.000
entB	22 (68.8)	23 (35.9)	0.002
rmpA	28 (87.5)	12 (18.8)	<0.001
aerobactin	23 (71.9)	8 (20.7)	<0.001
ybtS	17 (53.1)	34 (53.1)	1.000
allS	6 (18.8)	9 (14.1)	0.551
kfu	9 (28.1)	6 (9.4)	0.017
K1	9 (28.1)	5 (7.8)	0.019
K2	8 (25.0)	3 (4.7)	0.009
K5	1 (3.1)	3 (4.7)	1.000
K20	0 (0.0)	2 (3.1)	0.551
K57	5 (15.6)	1 (1.6)	0.025

Figure 2 Agarose gel electrophoresis of ESBLs genes and virulence factors.

Notes: M, DNA marker; N, negative control; P, positive control; S, strain to be tested; (A) blaTEM (800bp); (B) blaSHV (885bp); (C) blaCTX-M (216bp); (D) K1 (1283bp); (E) K2 (641bp); (F) K5 (280bp); (G) K20 (741bp); (H) K57 (1037bp); (I) rmpA (535bp); (J) fimH (550bp); (K) mrkD (226bp); (l) ybtS(242bp); (M) allS (508bp); (N) kfu (638bp); (O) aerobactin (556bp); (P) entB (400bp).

Table 5 Distribution of ESBLs Genes in ESBL-Positive cKP Strains and hvKP Strains, n=54

ESBL Gene	cKP (n=45)	hvKP (n=9)
	No. (%)	No. (%)
TEM	1 (2.2)	–
SHV	2 (4.4)	–
CTX-M	1 (2.2)	–
TEM+SHV	2 (4.4)	–
TEM+CTX-M	1 (2.2)	–
SHV+CTX-M	11 (24.4)	5 (55.6)
TEM+SHV+CTX-M	27 (60.0)	4 (44.4)

Table 6 Correlation of hvKP Strains with ESBL-Producing Strains

Virulence Profiles	TEM (n=35)	SHV (n=51)	CTX-M (n=49)
hypermucoviscosity	4 (P = 0.001)	9 (P = 0.001)	9 (P = 0.001)
fimH	30	46	45
mrkD	35	51	49
entB	3 (P < 0.001)	5 (P < 0.001)	5 (P < 0.001)
rmpA	6 (P < 0.001)	10 (P < 0.001)	9 (P < 0.001)
aerobactin	2 (P < 0.001)	2 (P < 0.001)	2 (P < 0.001)
ybtS	15	27	26
allS	3	4 (P = 0.025)	4 (P = 0.040)
kfu	0 (P = 0.001)	0 (P < 0.001)	0
K1	2	2 (P = 0.002)	2 (P = 0.003)
K2	1	2 (P = 0.014)	2 (P = 0.021)
K5	0	1	0
K20	0	0	0
K57	0	0 (P = 0.023)	0 (P = 0.031)

Table 7 Statistics of the Relationship Between the Genes Carrying Different ESBLs and Biofilm Formation

ESBL Gene and Enzyme	Biofilm Formation (n=72)	Non Biofilm Formation (n=24)	P-value
	No. (%)	No. (%)
TEM	29 (40.3)	6 (25.0)	0.178
SHV	44 (61.1)	7 (29.2)	0.007
CTX-M	43 (59.7)	6 (25.0)	0.003
ESBLs	29 (40.3)	4 (16.7)	0.035
Carbapenemase	13 (18.1)	2 (8.3)	0.417

Figure 3 The results of double-disc synergy test (DDST) and modified carbapenem inactivation method (mCIM).

Notes: N, negative control; P, positive control; S, strain to be tested; (A) positive result of DDST; (B) negative result of DDST; (C) the result of mCIM.

Table 8 Statistics on the Relationship Between Biofilm Formation Level and Drug Resistance

Biofilm Formation	Cephalosporin (n=72)		P-value	rs
	Sensitive	Resistant
Weak	10	16	0.034	−0.251
Moderate	10	17
Strong	14	5

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