Virulence-Related Gene Distribution Among Shigella Isolates in Anhui, China: The Association with Antimicrobial Resistance

Figures & data

Table 1 Sequences of the Primers Used for PCR Amplification

Target Gene	Primer	Sequence (5′–3′)	Annealing Temperature	Size (bp)
ipaH	ipaH-F	TGGAAAAACTCAGTGCCTCT	57 °C	423
	ipaH-R	CCAGTCCGTAAATTCATTCT
ipaA	ipaA-F	CCTGTGTCCCCGAGAAAGAGA	60 °C	628
	ipaA-R	TGACGCACAGGCAAAACTTG
ipaB	ipaB-F	CAAGCCCTGAATCCGATCAT	60 °C	204
	ipaB-R	TGCTGCTGCCTGTTTACCAA
ipaC	ipaC-F	CCTCACCACAAACTAACTCTAGCA	60 °C	93
	ipaC-R	GAGAAGTTTTATGTTCAGTTGACAGGGATA
ipaD	ipaD-F	AAGAAGCCGAGCTTGATGGAG	60 °C	450
	ipaD-R	CCTCGCCATTTCCACCTAGA
ial	ial-F	CTGGATGGTATGGTGAGG	57 °C	320
	ial-R	GGAGGCCAACAATTATTTCC
virB	virB-F	CGCGCGAGACAGATTCTCTT	60 °C	488
	virB-R	TGGTGGATTTGTGCAACGAC
virF	virF-F	AGCTCAGGCAATGAAACTTTGAC	57 °C	618
	virF-R	TGGGCTTGATATTCCGATAAGTC
set	set-F	TCCCTTCATACTGGCTCCTG	57 °C	553
	set-R	AACACTCTGGGGGGAACAG
sen	sen-F	ATCTCCTTGAGGCCAGCAAA	58 °C	296
	sen-R	GGAAGGAATGGGAGGACGAA
icsA	icsA-F	CCAACCCCTCTCATGCAT	60 °C	83
	icsA-R	ATCACCAGCACCACCATGAC
icsB	icsB-F	GGCCTGCATCAAGTCTTTCG	60 °C	280
	icsB-R	GGCATCGGTACAGCCAAAAA
sigA	sigA-F	ACCGACTTCTCACTTTCTCCCG	58 °C	430
	sigA-R	CCATCCAGCTGCATAGTGTTTG
sat	sat-F	TCAGAAGCTCAGCGAATCATTG	59 °C	930
	sat-R	CCATTATCACCAGTAAAACGCACC
pic	pic-F	ACTGGATCTTAAGGCTCAGGAT	58 °C	570
	pic-R	GACTTAATGTCACTGTTCAGCG
sepA	sepA-F	GCAGTGGAAATATGATGCGGC	58 °C	789
	sepA-R	TTGTTCAGATCGGAGAAGAACG

Figure 1 The yearly distribution of the proportions of the Shigella isolates.

Figure 2 Age and gender distribution of Shigella isolates in China from 2011 to 2015. (A) The distribution of the proportions of Shigella isolates by age group. (B) Comparison of the proportion of S. flexneri and S. sonnei by gender group.

Table 2 MIC₅₀ and MIC₉₀ of 17 Antimicrobial Agents Against S. Flexneri and S. Sonnei

Antimicrobial Agent	S. flexneri (n=449)			R	S. sonnei (n=68)			R	x^2	P-value
	MIC Range	MIC50	MIC90		MIC Range	MIC50	MIC90
	(μg/mL)	(μg/mL)	(μg/mL)	(%)	(μg/mL)	(μg/mL)	(μg/mL)	(%)
AMP	0.5 – >256	>256	>256	97.3	32 – >256	>256	>256	100
NAL	1 – >256	>256	>256	96.9	1 – >256	128	>256	98.5
TCY	0.25 – >128	128	>128	93.8	1 – >128	64	>128	94.1
CHL	0.5 – >256	64	128	86.6	1 – >256	4	16	8.8	198.334	<0.0001*
SXT	0.125 – >32	16	32	76.2	0.125–32	16	32	92.6	9.449	0.002*
CTX	0.0625 – >32	16	64	53	0.0625 – >32	16	>32	52.9
CIP	0.0625 – >32	4	16	49.7	0.125 – >32	0.25	16	19.1	22.213	<0.0001*
CRO	0.0625 – >32	0.125	32	45.7	0.0625 – >32	0.0625	16	33.8
LVX	0.125–64	4	16	30.5	0.125 – >64	0.5	8	10.3	12.013	0.001*
GEN	0.25 – >128	1	>128	28.7	0.5 – >128	64	128	92.6	103.345	<0.0001*
FOS	4 – >2048	64	>2048	24.5	16 – >2048	128	256	26.5
FEP	0.5–256	1	32	21.6	0.5–128	0.5	32	19.1
CAZ	0.25–128	0.5	16	21.2	0.25–128	0.25	16	17.6
FOX	0.5 – >256	4	8	3.3	0.5 – >256	4	16	1.5
AMK	1 – >512	4	8	2	1–16	4	8	0
IPM	0.0625–8	1	2	1.8	0.25–16	0.5	1	1.5
TZP	1–128	8	16	0.7	2–32	8	16	0

Abbreviations: AMP, ampicillin; NAL, nalidixic acid; TCY, tetracycline; CHL, chloramphenicol; SXT, trimethoprim/sulfamethoxazole; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; CIP, ciprofloxacin; LEV, levofloxacin; GEN, gentamicin; AMK, amikacin; TZP, piperacillin–tazobactam; FOS, fosfomycin; IMP, imipenem; MIC, minimal inhibitory concentration; R, resistance.

Note: *Chi-square test.

Table 3 Distribution of Virulence-Related Genes Among Shigella Isolates in Anhui, China

Gene	Total
	S. flexneri	S. sonnei	P-value
	n=449	n=68
ipaH	449 (100%)	68 (100%)
ial	431 (96.0%)	51 (75.0%)	<0.0001^#
ipaA	442 (98.4%)	65 (95.6%)
ipaB	448 (99.8%)	67 (98.5%)
ipaC	443 (98.7%)	66 (97.1%)
ipaD	443 (98.7%)	65 (95.6%)
virB	439 (97.8%)	66 (97.1%)
virF	431 (96.0%)	51 (75.0%)	<0.0001^#
set	420 (93.5%)	0
sen	413 (92.0%)	60 (88.2%)
icsA	448 (99.8%)	67 (98.5%)
icsB	439 (97.8%)	66 (97.1%)
sigA	415 (92.4%)	64 (94.1%)
sat	421 (93.8%)	0
pic	396 (88.2%)	0
sepA	394 (87.8%)	0

Note: ^#Fisher’s exact test.

Figure 3 Comparison of the antimicrobial resistance rates between virulence-related gene (VRG)-positive (ial, virF, sepA, pic, sat, and set) and VRG-negative S. flexneri. (A) Comparison of the antimicrobial resistance rates between ial-positive and ial-negative S. flexneri. (B) Comparison of the antimicrobial resistance rates between virF-positive and virF-negative S. flexneri. (C) Comparison of the antimicrobial resistance rates between sepA-positive and sepA-negative S. flexneri. (D) Comparison of the antimicrobial resistance rates between pic-positive and pic-negative S. flexneri. (E) Comparison of the antimicrobial resistance rates between sat-positive and sat-negative S. flexneri. (F) Comparison of the antimicrobial resistance rates between set-positive and set-negative S. flexneri. *, P<0.05; **, P<0.001.

Figure 4 Comparison of the antimicrobial resistance rates between virulence-related gene (VRG)-positive (ipaA, ipaC, ipaD, virB, icsB, sigA, and sen) and VRG-negative S. flexneri. (A) Comparison of the antimicrobial resistance rates between ipaA-positive and ipaA-negative S. flexneri. (B) Comparison of the antimicrobial resistance rates between ipaC/ipaD-positive and ipaC/ipaD-negative S. flexneri. (C) Comparison of the antimicrobial resistance rates between virB-positive and virB-negative S. flexneri. (D) Comparison of the antimicrobial resistance rates between icsB-positive and icsB-negative S. flexneri. (E) Comparison of the antimicrobial resistance rates between sigA-positive and sigA-negative S. flexneri. (F) Comparison of the antimicrobial resistance rates between for sen-positive and sen-negative S. flexneri. *, P<0.05; **, P<0.001.