Biosafety assessment of Acinetobacter strains isolated from the Three Gorges Reservoir region in nematode Caenorhabditis elegans

Acinetobacter isolates from the TGR region

In the TGR region, we isolated 21 Acinetobacter strains (one A. johnsonii, one A. haemolyticus and 19 Acinetobacter sp. strains) (Table S1). Based on phylogenetic analysis after 16S rRNA gene sequencing, these 21 isolates belong to the genus Acinetobacter, exhibiting a similarity of 95.38–99.93% with known Acinetobacter strains in GenBank (Table S1). In phylogenetic tree (N-J) constructed with both isolated and known Acinetobacter strains, these 21 isolates branched deeply with three Acinetobacter clusters consisting of important clinical Acinetobacter species, such as A. johnsonii H10 (FJ009371), A. junii NH88-14 (FJ447529), A. baumannii ATCC19606^T (HE651907), A. lwoffii DSM2403^T (X81665) and A. haemolyticus TTH04-1 (KF704077) (Fig. 1). Five reference Acinetobacter strains were selected and used²¹. Currently, the genus Acinetobacter comprises 68 species with validly-published names (https://apps.szu.cz/anemec/Classification.pdf, May 25, 2021). Among the named species, A. baumannii is the most studied species associated with clinical infections followed by the non-A. baumannii species A. haemolyticus, A. junii, A. johnsonii, and A. lwofii²¹.

Effect of different Acinetobacter strains isolated from the TGR region and reference strains on lifespan of nematodes

L4-larvae were exposed to different Acinetobacter strains for 24-h. Totally 21 Acinetobacter strains isolated from the TGR region and 5 reference strains of Acinetobacter species were used for the lifespan analysis. Based on the comparison of lifespan curves, exposure to Acinetobacter strains of AC2, AC3, AC4, AC5, AC6, AC7, AC8, AC9, AC10, AC11, AC12, AC13, AC14, AC16, AC17, AC19, AC20, A. johnsonii H10, and A. haemolyticus TTH0-4 could not alter lifespan curve (Fig. 2). Similarly, Acinetobacter strains of AC2, AC3, AC4, AC5, AC6, AC7, AC8, AC9, AC10, AC11, AC12, AC13, AC14, AC16, AC17, AC19, AC20, A. johnsonii, and A. haemolyticus also could not influence mean lifespan (Fig. 2). Different from these, the lifespan curves of nematodes exposed to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii ATCC 19606^T, A. junii NH88-14, and A. lwoffii DSM 2403^T were significantly (P < 0.01) different from that in control nematodes (Fig. 2). Additionally, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii, and A. lwoffii significantly decreased the mean lifespan (Fig. 2). Thus, Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii, and A. lwoffii potentially resulted in adverse effects on lifespan of nematodes.

Effect of exposure to different Acinetobacter strains isolated from the TGR region and reference strains on locomotion behavior of nematodes

Locomotion behavior is more sensitive than lifespan for assessing toxicity of environmental toxicants or stresses²⁵. After exposure for 24-h, Acinetobacter strains of AC2, AC3, AC4, AC5, AC6, AC7, AC8, AC9, AC10, AC11, AC12, AC13, AC14, AC16, AC17, AC19, AC20, A. johnsonii, and A. haemolyticus did not obviously affect locomotion behavior (Fig. 3). In contrast, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii, and A. lwoffii significantly decreased locomotion behavior (Fig. 3).

Effect of exposure to different Acinetobacter strains isolated from the TGR region and reference strains in inducing activation of oxidative stress of nematodes

Oxidative stress is one cellular contributor to toxicity of exposure to toxicants or stresses^25,26,27. We further employed the ROS production to examine effect of Acinetobacter strains in inducing oxidative stress. Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii, and A. lwoffii for 24-h resulted in obvious induction of ROS production (Fig. 4A).

SOD-3/Mn-SOD provides a molecular basis for antioxidation defense response²⁵. Moreover, we observed that exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii, and A. lwoffii for 24-h further led to significant increase in expression of SOD-3::GFP (Fig. 4B).

Effect of exposure to different Acinetobacter strains isolated from the TGR region on expressions of antimicrobial genes in nematodes

In nematodes, intestine is the important organ to activate innate immune response to pathogen infection⁹. F55G11.4, dod-22, lys-8, lys-1, spp-12, lys-7, dod-6, and spp-1 are most studied intestinal anti-microbial genes^{28,29,30,31,32,33,34}. We next selected these 8 intestinal antimicrobial genes to determine effect of different Acinetobacter strains isolated from the TGR region on innate immune response. The increase in these 8 intestinal antimicrobial genes function to be against pathogen infection and environmental stress^{28,29,30,31,32,33,34}. After exposure to Acinetobacter strains of AC1, AC15, AC18, or AC21 for 24-h, expressions of some of these antimicrobial genes could be noticeably increased. Among these 8 antimicrobial genes, exposure to strain AC1 significantly increased the expressions of spp-1, lys-8, lys-7, lys-1, spp-12, dod-6, dod-22, and F55G11.4, exposure to strain AC15 significantly increased the expressions of F55G11.4, lys-8, dod-6, and lys-7, exposure to strain AC18 significantly increased the expressions of lys-8, lys-7, and spp-12, and exposure to strain AC21 significantly increased the expressions of dod-6, lys-7, spp-12, lys-1, dod-22, spp-1, and F55G11.4 (Fig. 5). In nematodes, LYS-8, LYS-7, and LYS-1 are lysozymes, SPP-12 is a saposin-like protein, DOD-6 and DOD-22 are proteins downstream of DAF-16, SPP-1 is a caenopore, and F55G11.4 is a protein containing CUB-like domain.

Morphological and biochemical properties of Acinetobacter strains of AC1, AC15, AC18, and AC21

For the Acinetobacter strains of AC1, AC15, AC18, and AC21, they did not show obvious difference in morphological properties of cell shape, arrangement of cell, Gram staining, and colony morphology (Table 1). The Acinetobacter strains of AC1, AC15, AC18, and AC21 also did not exhibit the obvious difference in biochemical properties of hydrothion, phenylalanine, gluconate, oxidase, nitrate reduction, catalase, peptone water, semi-solid agar, glucose, ornithine, raffinose, sorbitol, side calendula, and xylose (Table 1). Different from this, the Acinetobacter strains of AC1 and AC21 showed the negative reactions for the biochemical properties of l-arginine, l-lactic acid, d-fucose, l-histidine, l-malic acid, and d-serine (Table 1). The Acinetobacter strains of AC15 and AC18 exhibited the positive reactions for the biochemical properties of l-arginine, l-lactic acid, d-fucose, l-histidine, l-malic acid, and d-serine (Table 1). Additionally, the Acinetobacter strains of AC1 and AC21 showed the negative reactions for the biochemical properties of glucopeptone water, citrate, and gelation, whereas the Acinetobacter strain of AC15 exhibited the positive reactions for the biochemical properties of glucopeptone water, citrate, and gelation (Table 1).

Differences of main virulence genes among Acinetobacter strains

For understanding of differences of virulence genes from these pathogenic Acinetobacter strains, we checked for the presence of 14 main virulence genes (Table S4) in pathogenic strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii and A. lwoffii and nonpathogenic strains of AC2, AC12, AC14, AC17, A. haemolyticus, and A. johnsonii by PCR. Distribution of virulence genes in tested Acinetobacter strains was different and pathogenic Acinetobacter strains generally had more virulence genes than nonpathogenic strains (Table 2). 10 or more virulence genes were detected from pathogenic strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii and A. lwoffii (Table 2).

Source: Ecology - nature.com