Microbiological features and clinical impact of the type VI secretion system (T6SS) in Acinetobacter baumannii isolates causing bacteremia

Figures & data

Table 1. Antibiotic resistance rates of A. baumannii isolates according to the presence of the hcp gene.

	Number of resistant isolates (%)
Antimicrobial agent	Total (n = 162)	hcp+ (n = 51)	hcp– (n = 111)
Imipenem	137 (84.6)	46 (90.2)	91 (82.0)
Ceftazidime	141 (87.0)	47 (92.2)	94 (84.7)
Ampicillin/sulbactam	138 (85.2)	46 (90.2)	92 (82.9)
Piperacillin/tazobactam	140 (86.4)	46 (90.2)	94 (84.7)
Gentamicin	129 (79.6)	45 (88.2)	84 (75.7)
Ciprofloxacin	138 (85.2)	46 (90.2)	92 (82.9)
Trimethoprim/sulfamethoxazole	143 (88.3)	48 (94.1)	95 (85.6)
Colistin	6 (3.7)	3 (5.9)	3 (2.7)
Tigecycline	15 (9.3)	3 (5.9)	12 (10.8)

Table 2. Genotypes and distribution of A. baumannii isolates.

ST	Allelic profile^a	Total (n = 162) (%)	hcp+ (n = 51) (%)	hcp–(n = 111) (%)	Imipenem resistance (%)
GC2		132 (81.5)	41 (31.1)	91 (68.9)	132 (100)
ST191	1–3–3–2–2–94–3	66 (40.7)		66 (100)	66 (100)
ST208	1–3–3–2–2–97–3	14 (8.6)	13 (92.9)	1 (7.1)	14 (100)
ST357	1–12–3–2–2–145–3	11 (6.8)	11 (100)		11 (100)
ST368	1–3–3–2–2–140–3	6 (3.7)		6 (100)	6 (100)
ST369	1–3–3–2–2–106–3	11 (6.8)	3 (27.3)	8 (72.7)	11 (100)
ST451	1–3–3–2–2–142–3	13 (8.0)	13 (100)		13 (100)
ST784	1–3–3–2–2–107–3	6 (3.7)		6 (100)	6 (100)
ST1114	1–12–3–2–2–79–3	1 (0.6)	1 (100)		1 (100)
ST1141	1–3–3–104–2–140–3	1 (0.6)		1 (100)	1 (100)
ST1144	1–3–3–102–2–94–3	2 (1.2)		2 (100)	2 (100)
ST1316	1–35–3–2–2–107–3	1 (0.6)		1 (100)	1 (100)
Others
ST681	1–102–59–28–1–83–45	1 (0.6)		1 (100)
ST734	1–1–5–59–28–4–157–45	1 (0.6)		1 (100)
ST1319	1–15–59–28–4–269–45	1 (0.6)		1 (100)
ST503	1–62–80–28–35–164–45	1 (0.6)		1 (100)
ST1077	1–62–80–28–35–178–45	1 (0.6)		1 (100)
ST435	1–54–80–28–48–127–45	1 (0.6)		1 (100)
ST552	21–35–2–28–1–145–4	1 (0.6)		1 (100)
ST1318	21–35–2–28–1–268–4	1 (0.6)		1 (100)
ST711	21–12–2–28–1–109–4	1 (0.6)		1 (100)
ST1262	21–35–2–28–22–23–4	1 (0.6)		1 (100)
ST447	1–15–13–12–4–106–2	1 (0.6)		1 (100)
ST1313	1–15–13–12–4–94–2	1 (0.6)		1 (100)
ST1317	1–15–13–12–4–163–112	1 (0.6)		1 (100)
ST1315	1–1–13–12–92–16–2	1 (0.6)		1 (100)
ST751	33–31–2–28–1–83–5	1 (0.6)		1 (100)
ST1181	33–31–2–28–1–144–5	1 (0.6)		1 (100)
ST229	1–15–2–28–1–107–32	4 (2.5)	4 (100)		4 (100)
ST491	10–53–4–11–4–98–5	1 (0.6)	1 (100)		1 (100)
ST373	1–12–12–11–4–103–3	5 (3.1)		5 (100)
ST613	15–48–58–42–36–54–41	2 (1.2)	2 (100)
ST1131	60–17–140–1–4–103–107	1 (0.6)	1 (100)
ST1312	1–53–135–12–1–110–110	1 (0.6)	1
ST1314	21–15–2–28–35–193–111	1 (0.6)		1 (100)
ST1320	1–102–80–85–28–214–113	1 (0.6)	1

Note.

^a gltA-gyrB-gdhB-recA-cpn60-gpi-rpoD.

Figure 1. Diagram of the distribution of the hcp gene according to sequence type (ST). The numbers in the circles are the STs. The size of each circle represents the proportion of each ST, which are the marks within the circles. Solid line linkages indicate a single-locus variant, and dotted-line linkages indicate differences in 2 or 3 alleles.

Figure 2. Relative hcp transcript levels in A. baumannii isolates as measured by qRT PCR. hcp transcription levels are shown relative to that in A. baumannii ATCC 19606. The dashed line indicates 0.01-fold the expression levels of hcp in the reference strain, ATCC19606. The asterisk (*) and pound (#) symbols indicate the isolates with high and low hcp expression, respectively, that were used in the western blotting, competition, biofilm formation, and serum resistance assays.

Figure 3. Detection of Hcp, with RNA polymerase (RNAP) as a lysis control. (A) Three isolates showing high hcp transcript levels; (B) 2 isolates showing low hcp transcript levels, (i.e., less than 0.01-fold that of ATCC 19606). Immunoblots show Hcp in whole cells and concentrated culture supernatants prepared from cultures of clinical isolates.

Table 3. Profiles of A. baumannii isolates used in western blotting and analyses of microbiologic characteristics.

				MIC (μg/mL) of antimicrobial agents ^b
Strain	Genotype	Date of isolation	Site of Infection^a	IMI	CAZ	A/S	P/T	GEN	CIP	SXT	COL	TGC
T6SS+ (active hcp+)
B849	ST357	2013 SEP 3	SSTI	>64	>64	64/32	<256/4	>64	>64	>32/608	2	1
B1144	ST357	2014 JUL 2	CRBSI	>64	>64	64/32	<256/4	>64	>64	>32/608	1	1
14AC-06	ST208	2014 JAN 9	VAP	>64	>64	>64/32	<256/4	>64	64	>32/608	1	1
T6SS– (inactive hcp+)
B876	ST357	2013 SEP 14	CRBSI	64	>64	>64/32	<256/4	2	>64	>32/608	2	4
13AC-06	ST369	2013 MAR 3	CRBSI	>64	>64	64/32	<256/4	>64	64	>32/608	1	1
T6SS– (hcp–)
AC12	ST191	2013 MAY 14	IAI	>64	>64	>64/32	<256/4	8	>64	>32/608	1	2
B764	ST191	2013 JUL 25	IAI	>64	>64	>64/32	<256/4	>64	>64	>32/608	1	4
13AC-45	ST369	2013 SEP 22	Primary bacteremia	>64	>64	>64/32	<256/4	>64	>64	>32/608	1	1

Notes.

^a SSTI, skin and soft tissue infection; CRBSI, catheter-related bloodstream infection; VAP, ventilator-associated pneumonia; IAI, intra-abdominal infection

^b MIC, minimum inhibitory concentration; IMI, imipenem; CAZ, ceftazidime; A/S, ampicillin-sulbactam; P/T, piperacillin-tazobactam; GEN, gentamicin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; COL, colistin; TGC, tigecycline

Figure 4. Results of competition, biofilm formation, and serum resistance assays. These results came from 3 T6SS+ and 2 T6SS- isolates. (A) Relative in vitro competition indexes (CIs) for A. baumannii isolates by the presence of T6SS (T6SS+ vs. T6SS–). The CI values shown are the number of A. baumannii divided by the number of E. coli MG1655. Each circle represents the CI of each in vitro replicate. (B) Relative quantity of biofilm in A. baumannii isolates compared with that of ATCC 19606. (C) Serum resistance in A. baumannii isolates of each T6SS+ and T6SS– replicate. Serum resistance as a viability ratio (colony forming units [CFUs] of serum-bacterial suspension/ CFUs of bacterial suspension without normal human serum). The error bars indicate the standard deviations. *, Statically significant difference (P < 0.05).

Table 4. Clinical characteristics of patients with T6SS+ A. baumannii bacteremia.^a

	Total(n = 162)	T6SS+(n = 45, 28.4%)	T6SS–(n = 117, 71.3%)	P-value
Age, years	62.2 ± 14.7	61.6 ± 12.5	62.5 ± 15.5	0.710
Male gender	94 (58.0)	27 (60.0)	67 (57.3)	0.859
Underlying disease
Immunocompromised status	109 (67.3)	35 (77.8)	74 (63.2)	0.093
Solid tumor	47 (29.0)	11 (24.1)	36 (30.8)	0.449
Hematologic malignancy	32 (19.8)	10 (22.2)	22 (18.8)	0.662
HSCT	10 (6.2)	6 (13.3)	4 (3.4)	0.029^c
Immunosuppressive agents	75 (46.3)	28 (62.2)	47 (40.2)	0.014
Neutropenia	32 (19.8)	8 (17.8)	24 (20.5)	0.827
Chronic renal disease	52 (32.1)	14 (31.1)	38 (32.5)	1.000
Hemodialysis	17 (10.5)	8 (17.8)	9 (7.7)	0.084^c
Congestive heart failure	23 (14.2)	4 (8.9)	19 (16.2)	0.317
Liver cirrhosis	18 (11.1)	5 (11.1)	13 (11.1)	1.000
Diabetes mellitus	52 (32.1)	14 (31.1)	38 (32.5)	1.000
Comorbid conditions
Septic shock	80 (49.4)	21 (46.7)	59 (50.4)	0.727
Acute kidney injury	68 (42.0)	16 (35.6)	52 (44.4)	0.375
Mechanical ventilation care ^b	81 (50.0)	15 (33.3)	53 (45.3)	0.214
Surgery within 30 d	45 (27.8)	12 (26.7)	33 (28.2)	1.000
Pitt bacteremia score	3.3 ± 2.9	3.1 ± 2.6	3.4 ± 3.0	0.515
Charlson WIC	3.2 ± 2.6	3.4 ± 2.9	3.1 ± 2.4	0.525
Site of infection
Pneumonia	59 (36.4)	12 (26.7)	47 (40.2)	0.145
CRBSI	41 (25.3)	20 (44.4)	21 (17.9)	0.001
Intra-abdominal infection	34 (21.0)	4 (8.9)	30 (25.6)	0.303
Primary bacteremia	17 (10.5)	4 (8.9)	13 (11.1)	0.782^c
Skin and soft tissue infection	8 (4.9)	4 (8.9)	4 (3.4)	0.219^c
Urinary tract infection	3 (1.9)	1 (2.2)	2 (1.7)	1.000
Polymicrobial bacteremia	32 (19.8)	9 (20.0)	23 (19.7)	1.000
Bacteremic episodes in ICU	99 (61.1)	29 (64.4)	70 (59.8)	0.719
Hospital-acquired infection	149 (92.0)	44 (97.8)	105 (89.7)	0.114^c
Appropriate antibiotics ≤ 48 h	57 (35.2)	16 (35.6)	41 (35.0)	1.000
Antibiotic resistance
MDR	144 (88.9)	41 (91.1)	103 (88.0)	0.781
CR	137 (84.6)	40 (88.9)	97 (82.9)	0.468
XDR	132 (81.5)	40 (88.9)	92 (78.6)	0.176
30-day mortality	94 (58.0)	25 (55.6)	69 (59.0)	0.725

Notes.

^a Data are expressed as no. (%) of patients, unless otherwise indicated. Continuous variables expressed as mean and standard deviation. Bold indicates P < 0.05. HSCT, haematopoietic stem cell transplantation; WIC, weighted index of comorbidity; CRBSI, catheter-related bloodstream infection; ICU, intensive care unit; MDR, multidrug-resistant; CR, carbapenem-resistant; XDR, extensively drug-resistant;

^b Mechanical ventilation care before bacteremia onset;

^c Fisher's exact test