Keywords

1 Introduction

China is a country in the production and consumption of a large amount of aquatic products, accounting for about two-thirds of the total amount in the world’s total. In recent years, aquaculture constantly tended to operate in the ways with high density, industrialization and intensification, for increasing the output of aquatic products. However, the water rich in nutrients led to pathogenic bacteria blooming in aquaculture. Annual losses from aquaculture diseases accounts for about 30% of production, while pathogenic bacterial infection diseases accounts for about half.

The major aquatic bacterial pathogens mainly comprise multiple genera of both Gram-negative and Gram-positive bacteria, such as Vibrio, Aeromonas, Escherichia coli, Streptococcus iniae, Salmonella, Edwardsiella tarda, Pleisionomas, Flavobacterium, Acinetobacter, Pasteurella, Pseudomonas, Staphylococcus aureus, Mycobacterium, Clostridium and others [1]. Aeromonas, including 25 different species, is the important pathogen of fish, shrimp and other animals [2, 3], causing serious economic losses [4].

In this work, fish of Tianjin and Xiamen, Fujian, China, Penaeus vannamei shrimp seeds of Hangu, Tianjin, China, and seawater samples of Jiyun River and Bohai Bay were collected in Tianjin. The epidemiology of aquatic pathogens was isolated with the selective media. The isolates were identified with the 16S rRNA sequencing analysis [5]. Since 16S rRNA gene sequence is highly homologous among most Aeromonas species, the Aeromonas isolates in this study were also analyzed with the multilocus sequence typing (MLST) method for further discrimination of the Aeromonas isolates [6].

2 Materials and Methods

2.1 Samples, Media and Bacteria Isolation

Freshwater fishes were obtained from five local markets and two fish farms in Tianjin, China from March through May 2012. The fishes belonged to 11 different species, including Hypophthalmichthys molitrix (4), Ctenopharyngodon idella (4), Carassius auratus (4), Hypophthalmichthys nobilis (4), Megalobrama amblycephala (2) and Ephippus orbis (2). Penaeus vannamei shrimp seeds (2) and Saltwater fish Epinephelus awoara (12) were respectively obtained from Hangu, Tianjin, and Xiamen, Fujian, China, during March 2012. Seawater samples (4) were obtained from in Tianjin, China from March through May 2012.

The gastrointestinal tract was removed from fish and the content was collected and stored at 4 °C. The content sample was serially diluted in phosphate buffered saline and spread on three types of agar plates, including SS (Salmonella Shigella agar), TCBS (thiosulfate citrate bile salts sucrose agar) [7, 8], MAC (MacConkey agar) [9], CIN-1 (Cepulodin Irgasan Novobiocin Agar). L-agar plates containing ampicillin (20 μg/ml), erythromycin (20 μg/ml), and kanamycin (20 μg/ml) were also used for screening the possible bacterial pathogens in the gut samples.

2.2 The Identification of Isolated Strains

The genomic DNA was extracted from the isolated strains. The DNA was used as template for PCR amplification of the 16S rRNA gene with primers described previously [10]. The PCR product was subjected to sequencing directly. The obtained sequences were analyzed by the BLAST program on the NCBI website. MEGA 5 was used to construct the phylogenetic trees [11].

2.3 MLST Analysis

Multilocus sequence typing (MLST) was proposed as a universal method to characterize bacteria based on sequence polymorphisms within internal fragments of housekeeping genes. The primers for amplifying a set of six housekeeping genes, namely, gyrB, groL, gltA, metG, ppsA, and recA, was synthesized for MLST of the Aeromonas isolates [12]. The sequences of distinct alleles were deposited in the Aeromonas MLST database (http://pubmlst.org/aeromonas). Allele number and sequence type (ST) number were determined by described previously [12].

3 Results

3.1 Strain Separation

120 bacteria strains were isolated from 37 different samples with various media. Morphology of bacterial colonies grown on the TCBS plate were mostly yellow and green, smooth, and moist; on the SS plate were mostly yellow, pink, colorless with black center; on the MAC plate mostly were pink and red, flat; on the CIN-1 plate mostly were pink, big, and smooth.

3.2 Identification of Isolated Strains

120 isolated strains were identified with the comparative sequence analysis of the 16S rRNA gene. The 16S rRNA sequences were deposited in GenBank with accession no KC210757-KC210826, KC210827- KC210872 and KC252599-KC252602. All the isolates were tentatively classified into 15 genera of seven families, including Aeromonas spp. (53), Vibrio spp. (19), Proteus spp. (5), Citrobacter spp. (9), Hafnia spp. (8), Providencia spp. (6), Pseudomonas spp. (3), Kluyvera spp. (2), Enterobacter spp. (2), Bacillus spp. (2), Pantoea sp. (1), Leclercia sp. (1), and Acinetobacter sp. (1). Aeromonas strains were not isolated from all the samples except from Epinephelus awoara from samples of Xiamen, Fujian, China. The Vibrio isolates include V. parahaemolyticus (2), V. alginolyticus (5), V. azureus (2), V. anguillarum (9), and V. fluvialis (1), found in the samples from Tianjin and Xiamen, Fujian, China (Table 1).

Table 1 Comparative analysis of 16S rRNA gene sequences of the isolated
Full size table

3.3 Phylogenetic Analysis

Based on the 16S rRNA gene sequences, the phylogenetic tree of all isolated strains was constructed. As shown in Fig. 1, Aeromonas isolates, Enterobacteriaceae isolates, Vibrio isolates, and Shewanella isolates were in grouped in one cluster, respectively; 9 C. freundii isolates were also grouped into two clades, one comprising of 4 members and the other comprising of 5 members. The phylogenetic analysis result indicated that the isolated strains within the same genus or species might belong to different lineages (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree based on 16S rRNA gene sequences of the isolates

Full size image

Since 16S rRNA gene sequence is highly homologous among most Aeromonas species, the Aeromonas isolates in this study were identified only at genus level [6].

3.4 MLST Analysis

The fragments of the six housekeeping genes were successfully amplified and sequenced in all 53 Aeromonas strains. The sequence alignment results showed only 21 out of the 318 alleles was known alleles in the database, including gyrB22 (LSB2), gyrB22 (MY-1), gyrB97 (MJ-4), gyrB22 (MY), groL42 (MJ-1), groL96 (CH13), groL96 (SH11), gltA56 (MY-1), gltA56 (MJ-4), gltA51 (ML-1), gltA56 (MY), gltA56 (SJ1), gltA51 (SL-1), gltA99 (TB2), metG64 (TY-4), ppsA67 (LSJ1), ppsA40 (MJ-1), ppsA40 (SL1), ppsA40 (TL1), ppsA40 (TY-2) and recA42 (MJ-1). The remaining 297 sequences represented the novel alleles. Correspondingly, only 8 STs were known sequence types and 45 STs were novel. The results suggested the highly genetic diversity of the Aeromonas isolates in our work.

3.5 Distribution of Aeromonas in Worldwide

By analyzing the database of all 398 Aeromonas STs, Aeromonas strains geographic isolation was found. As shown in Fig. 2a, the Asia, Europe, North America and South America were respectively discovered 195, 181, 8 and 1 unique STs, only one ST is the North American and Asian shared, the ST is famous ST 251 [13].

Fig. 2
figure 2

Distributions of Aermonas STs across China (a) and the world (b)

Full size image

As shown in Fig. 2b, a total of 171 unique ST were found in the Chinese region, Beijing, Tianjin, Jilin, Guangdong and Zhejiang as the main source of the data, and was found to also have geographic isolation; no duplicate ST occurs among strains isolated in different regions.

3.6 Aeromonas Relationship Between the STs

Relationships among the STs deposited in the Aeromonas MLST database were analyzed by using the eBURST algorithm [9]. The eBURST analysis revealed that 42 irrelevant clonal complexes (CCs) and 81 singletons were identified. Figure 3 is a schematic diagram of eBURST analysis, the identified 45 STs distributed in the 8 of 42 clonal complexes, and the low correlation between strains, indicating a high degree of biological diversity of Aeromonas strains. Additionally, in this study only one allele number difference was identified between ST127 (MY-1) and ST 140 (MY).

Fig. 3
figure 3

eBURST algorithm analysis of 398 Aermonas STs. CC clonal complexes; Transparent circle is 398 Aermonas STs, grey circle is grouped by eBURST algorithm analysis 398 Aermonas STs; Fraction = (Number of Aermonas CCs in this study and CCs)/(Number of Aermonas STs in this CCs)

Full size image

4 Discussion

Aquatic pathogenic bacteria can cause fish diseases, leading to the death of aquatic animals, bringing economic losses to farmers. Deng et al. [14] collected samples from a variety of aquatic animals from 64 aquaculture farms in Guangdong, China, and isolated 112 Aeromonas strains with multidrug-resistant phenotype and Class I integrons. Adesoji et al. [15] isolated 108 tetracycline resistant strains from water distribution systems in southwestern Nigeria, including Aermonas spp., Alcaligenes spp., Bacillus spp., Klebsiella spp., Leucobacter spp., Morganella spp., and Proteus spp..

MLST was performed to further discriminate the 53 Aeromonas isolates. Totally 297 new alleles and 45 novel STs were identified in our study. The data indicated the strains of the Aeromonas genus from different sources were highly genetically diversified [16, 17]. By eBURST algorithm analysis 398 Aeromonas STs were found belonging to 42 CCs, further suggesting the diversity of all Aeromonas isolates across the world. That may be due to the small number of Aeromonas isolates in the MLST database, and more deposited strains will better unveil the distribution patterns of Aeromonas strains.

This study has identified a number of novel alleles and STs of the Aeromonas spp. The relevant information has been deposited in the Aermonas MLST database. The data obtained in our study will provide more support for the study of the Aeromonas epidemiology.