Bacterial membrane vesicles from Acinetobacter baumannii induced by ceftazidime are more virulent than those induced by imipenem

Figures & data

Figure 1. Cytokine expression in macrophages after treatment with bacterial membrane vesicles (BMVs) from antimicrobial-treated Acinetobacter baumannii. BMVs induced by ceftazidime (CAZ) treatment led to higher expression of IL-1β (a) and IL-6 (c) in murine RAW264.7 macrophages than BMVs induced by imipenem (IMP) treatment or BMVs from untreated A. baumannii (LB). BMVs from ceftazidime and imipenem-treated cultures induced similar levels of TNF-α (b), IL-12 (d), and IFN-ϒ (e) expression, which were similar to that induced by untreated supernatant (LB). RAW264.7 macrophages treated with LPS plus ATP were used as a positive control, and phosphate-buffered saline (PBS) were used as negative controls. * P < 0.05 between CAZ-BMV and IMP-BMV

Figure 2. IL-1β and TNF-α expression in macrophages after treatment with bacterial membrane vesicles (BMVs) and culture supernatants from antimicrobial-treated Acinetobacter baumannii. BMVs pretreated with polymyxin B (PMB) did not induce IL-1β (a) or TNF-α (b) expression. Supernatants from ceftazidime and imipenem-treated cultures induced similar levels of IL-1β (c) and TNF-α (d) expression, which were similar to that induced by untreated supernatant (LB). RAW264.7 macrophages treated with LPS plus ATP were used as a positive control, and IL-4 and phosphate-buffered saline (PBS) were used as negative controls. LB-BMV-PMB, CAZ-BMV-PMB, and IMP-BMV-PMB are BMVs pretreated with polymyxin B (25 μg/ml). Shown are the mean and SD of results from triplicate studies. * P < 0.05 between two compared groups

Figure 3. Bacterial membrane vesicles (BMVs) induced by ceftazidime (CAZ) caused higher mortality in mice than BMVs induced by imipenem (IMP) treatment. In the study, mice were inoculated with 30 μl (a), 48.3 μl (b), or 77.5 μl (c) of BMVs derived from treatment of A. baumannii with CAZ, IMP, or no antimicrobial (LB). In the second study, the mice were inoculated with BMVs that carried the same amount of LPS (270 EU) (d). * P < 0.05 vs. BMVs derived from A. baumannii grown in Luria-Bertani broth without antibiotics (LB-BMV)

Figure 4. (a) Cytokine expression in mice serum after inoculated with bacterial membrane vesicles (BMVs) from antimicrobial-treated Acinetobacter baumannii. BMVs induced by ceftazidime (CAZ) treatment led to higher expression of many cytokines in mice than BMVs induced by imipenem (IMP) treatment or BMVs from untreated A. baumannii (LB). Phosphate-buffered saline (PBS) were used as negative controls. * P < 0.05 between CAZ-BMV and IMP-BMV. (b) Cytokine expression in mice serum after inoculated with bacterial membrane vesicles (BMVs) from antimicrobial-treated Acinetobacter baumannii. BMVs induced by ceftazidime (CAZ) treatment led to higher expression of many cytokines in mice than BMVs induced by imipenem (IMP) treatment or BMVs from untreated A. baumannii (LB). Phosphate-buffered saline (PBS) were used as negative controls. * P < 0.05 between CAZ-BMV and IMP-BMV

Figure 4. Continued

Figure 5. Lung pathology study showed that more neutrophil infiltration was found in lung interstitium of mice treated with ceftazidime-induced bacterial membrane vesicles (CAZ-BMV) than those treated with imipenem-induced bacterial membrane vesicles (IMP-BMV). Increased neutrophil infiltration occurred in the CAZ-BMV group (a), whereas no significant neutrophil recruitment in the IMP-BMV (b), LB-BMV (c), and control (d) group. Mice intraperitoneally administered 30 μl of phosphate-buffered saline (PBS) were used as control group

Figure 6. Treatment with 1/2 the minimal inhibitory concentration of ceftazidime (CAZ) induced the release of a higher number of bacterial membrane vesicles (BMVs) than treatment with imipenem (IMP). The number of BMVs (a), surviving colonies (b), and secreted BMVs per surviving colony (c) of A. baumannii after CAZ or IMP treatment, or without antimicrobial treatment (LB). Shown are the mean and SD of results from triplicate studies. *P < 0.05 vs. the sample derived from A. baumannii grown in Luria-Bertani broth without antimicrobials (LB)

Figure 7. Treatment with 1/2 the minimal inhibitory concentration of ceftazidime (CAZ) increased the amount of lipopolysaccharide (LPS) carried by bacterial membrane vesicles (BMVs) but not the amount in supernatants, compared to treatment with imipenem (IMP). LPS was detected with an anti-LPS antibody by western blot analysis. Cell lysate of ATCC 17978 was used as a positive control

Table 1. The top 15 proteins with the greatest increase in the bacterial membrane vesicles (BMVs) from Acinetobacter baumannii induced by antimicrobial treatment when compared to BMVs collected from an untreated LB culture

CAZ-BMV			IMP-BMV
Annotation and locus	Category	Fold change (CAZ-BMV/LB-BMV)	Annotation and locus	Category	Fold change (IMP-BMV/LB-BMV)
TonB-dependent siderophore receptor family protein [A0A009HBC2_ACIBA]	Iron chelate transport	104.7541022	Uncharacterized protein [A0A013RMZ9_ACIBA]	Other	6.212754
Autotransporter beta-domain protein [A0A009PWL1_ACIBA]	Channels or transporters other than iron transport	57.06149634	Autotransporter beta-domain protein [A0A009PWL1_ACIBA]	Channels or transporters other than iron transport	6.125172
Putative selenocysteine synthase [A0A009HT31_ACIBA]	Other	42.43002751	D-Alanyl-D-alanine carboxypeptidase family protein [A0A009ISR9_ACIBA]	Other	5.037994
Ligand-gated channel protein [A0A0H4UM69_ACIBA]	Channels or transporters other than iron transport	37.41420083	Putative selenocysteine synthase [A0A009HT31_ACIBA]	Other	4.35946
OmpW family protein [L9NZT1_ACIBA]	Iron chelate transport	36.97469299	TonB-dependent siderophore receptor family protein [A0A009HBC2_ACIBA]	Iron chelate transport	4.312864
Biopolymer transport protein ExbB [A0A1G5M1H5_ACIBA]	Iron chelate transport	33.53059039	Uncharacterized protein [A0A1E3MBA5_ACIBA]	Other	4.076264
TonB-dependent siderophore receptor family protein [A0A009KCF1_ACIBA]	Iron chelate transport	30.60025788	VacJ like lipofamily protein [A0A009HTJ0_ACIBA]	External encapsulating structure	3.60731
Putative outer membrane protein [Q4A209_ACIBA]	External encapsulating structure	29.02662837	LPS-assembly protein LptD [A0A0J0ZUK2_ACIBA]	External encapsulating structure	3.551672
VacJ like lipofamily protein [A0A009HTJ0_ACIBA]	External encapsulating structure	28.80445093	OprF membrane domain protein [A0A009K329_ACIBA]	Channels or transporters other than iron transport	3.339952
Outer membrane porin, OprD family protein [A0A062FDT6_ACIBA]	Channels or transporters other than iron transport	28.42551339	Outer membrane efflux family protein [A0A009IJT3_ACIBA]	Channels or transporters other than iron transport	3.339501
Uncharacterized protein [A0A1E3MA30_ACIBA]	Other	26.98190015	Glutaminase [A0A0B2XJ85_ACIBA]	Other	3.327919
TonB dependent receptor family protein [A0A062FR71_ACIBA]	Iron chelate transport	26.44018082	Quinoprotein glucose dehydrogenase [A0A0E1FTP2_ACIBA]	Other	3.248067
TonB-dependent receptor [A0A1E3M8J1_ACIBA]	Iron chelate transport	25.55863382	Uncharacterized protein [A0A062ID41_ACIBA]	Other	3.14646
Porin [S3TYL6_ACIBA]	Channels or transporters other than iron transport	25.18439556	Outer membrane protein assembly factor BamA [A0A062IKF0_ACIBA]	External encapsulating structure	3.08583
Uncharacterized protein [A0A1E3M6B5_ACIBA]	Other	24.61266347	Uncharacterized protein [A0A062F965_ACIBA]	Other	3.081454

Table 2. The proteins with the greatest decrease in the bacterial membrane vesicle (BMVs) from Acinetobacter baumannii induced by antimicrobial treatment when compared to BMVs collected from an untreated LB culture

CAZ-BMV			IMP-BMV
Annotation and locus	Category	Fold change (CAZ-BMV/LB-BMV)	Annotation and locus	Category	Fold change (IMP-BMV/LB-BMV)
Putative major capsid protein [A0A062EZ25_ACIBA]	Phage protein	0.57897	Putative major capsid protein [A0A062EZ25_ACIBA]	Phage protein	0.380593
Uncharacterized protein [A0A062ID26_ACIBA]	Other	0.628035	Uncharacterized protein [A0A009GER5_ACIBA]	Other	0.424309
Uncharacterized protein [B0VT25_ACIBS]	Other	0.956489	Succinate dehydrogenase iron-sulfur subunit [A0A009TAP4_ACIBA]	Other	0.545266
50S ribosomal protein L5 [A0A0B2XT25_ACIBA]	Peptide biosynthetic process	0.988407	50S Ribosomal protein L15 [A0A062DKQ2_ACIBA]	Peptide biosynthetic process	0.545366
			Uncharacterized protein [A0A009SPB4_ACIBA]	Other	0.545674
			30S Ribosomal protein S3 [A0A022IKC9_ACIBA]	Peptide biosynthetic process	0.555256
			Small GTP-binding domain protein [A0A062L8H2_ACIBA]	Other	0.561308
			ATP synthase subunit b [A0A009SUU5_ACIBA]	Other	0.572067
			Bacterioferritin [A0A1S2FNX1_ACIBA]	Iron chelate	0.593761
			50S ribosomal protein L6 [A0A022II07_ACIBA]	Peptide biosynthetic process	0.604319
			Elongation factor Tu [A0A062IYJ9_ACIBA]	Other	0.604711
			50S ribosomal protein L5 [A0A0B2XT25_ACIBA]	Peptide biosynthetic process	0.625656
			50S ribosomal protein L20 [A0A062IGI6_ACIBA]	Peptide biosynthetic process	0.625876
			NADH-quinone oxidoreductase subunit C [A0A062M7K5_ACIBA]	Other	0.62749
			CTP synthase [A0A062FS34_ACIBA]	Other	0.643215

LB-BMV indicates BMVs derived from A. baumannii grown in Luria-Bertani broth without antibiotic treatment. CAZ-BMV indicates BMVs derived from A. baumannii grown in Luria-Bertani broth with ceftazidime. IMP-BMV indicates BMVs derived from A. baumannii grown in Luria-Bertani broth with imipenem.