Taxonomy, virulence genes and antimicrobial resistance of Aeromonas isolated from extra-intestinal and intestinal infections

Overall, 1286 stool samples were collected from adults over 14 years old presenting with acute diarrhea at a general hospital in Beijng, China, between June and July 2015, 2017. Epidemiology related medical records were completed to assess clinical history and physical fitness of patients (Additional file 1). Samples of Stool were enriched in alkaline peptone water broth (Beijing landbrige, China) for 8 h at 37 °C, and a loop of the resulting mixture was subcultured on a blood agar plate (Oxoid, UK) supplemented with 20% ampicillin (Sigma, USA) for 16–24 h at 37 °C [28]. An oxidase test (BioMerieuX, France) was performed to select the colonies which were different from Enterobacteriaceae. Microorganisms were identified by use of an automatic bacteriologic analyzer (VITEK2 Compact, BioMerieuX, France). Salmonella spp, Shigella spp and Vibrio spp were also detected on a routine basis. simultaneously.

Extra-intestinal infections due to Aeromonas were monitored and the strains were isolated between 2015 and 2017. Clinical samples of blood or bile were cultured in a BACTEC FX400 (BD Diagnostic Instrument Systems, USA). Samples positive for Aeromonas were simultaneously subcultured on a blood agar plate and a Maconkey agar plate (BioMerieuX, France). Identification of the isolated microorganisms was completed by use of an automatic bacteriologic analyzer (VITEK2 Compact, BioMerieuX, France). Concurrently, medical records of the patients with extra-intestinal infections due to species of Aeromonas were reviewed and age, gender, underlying conditions, microbiological findings and outcome were assembled.

Strains were stored in a Luria broth: glycerol mixture (80:20) at − 80 °C until identification was performed.

Molecular identification and subtyping of Aeromonas isolates was completed by use of 16S rRNA sequencing and MLPA. Total chromosomal DNA from Aeromonas was prepared by use of the DNA purification kit (Tiangen Biotech, China) as specified by the manufacturer. PCR amplification was performed by use of 2 × Taq PCR MasterMix (Tiangen Biotech, China). Primers synthesis and sequencing of PCR products were conducted (Shanghai Sangon Biotech, China). Due to the limitations of molecular identification by 16S rRNA sequencing, phylogenetic analysis of the seven selected housekeeping genes gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD was completed to identify strains of Aeromonas. Primers [5] used for PCR amplification are provided in Additional file 2. Concatenated 7-gene phylogenetic trees were constructed and compared with representative species by use of MLPA as previously described [5]. Unrooted neighbour-joining phylogenetic trees were prepared by use of MEGA 5.0 software with Bootstrap values calculated by use of 1000 replicates.

The presence of 10 genes encoding virulence factors was determined by use of PCR. Primers are listed in Additional file 2, including alt [29], ast [30], hlyA, aerA, act, ascF-G of TTSS, laf [14], lip, fla, and ela [31]. PCR amplification reactions were performed at a final volume of 40 μl, containing 20 μl of Taq PCR MasterMix (2×), 1 μl 10 μM primer, 1 μl DNA template (~ 30-40 ng), and 17 μl ddH₂O. Cycling conditions consisted of an initial single cycle at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing was completed at 55 °C–60 °C for 30 s, elongation was completed at 72 °C for 1 min and followed by a final cycle at 72 °C for 7 min. The PCR products were sequenced for further confirmation.

Antibiotic susceptibility tests were performed by use of the microbroth dilution method according to guidelines of the current Clinical and Laboratory Standards Institute (CLSI). Minimum inhibitory concentrations (MIC) of strains of Aeromonas strains to 15 antibiotics were determined and included; gentamycin (GEN), imipenem (IPM), ampicillin (AMP), cefoxitin (FOX), ceftriaxone (CRO), amoxicillin-clavulanate (AMC), nalidixic acid (NAL), ciprofloxacin (CIP), chloramphenicol (CHL), tetracycline (TCY), doxycycline (DOX), azithromycin (AZM), cefepime (FEP), sulfonamides (Sas) and trimethoprim-sulfamethoxazole (SXT). E. coli ATCC 25922 was used as the quality-control strain for susceptibility testing.

Multiple drug resistance (MDR) was was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, according the criteria for defining MDR, XDR and PDR in Enterobacteriaceae [32].

Intestinal infections related to a strain of Aeromonas were diagnosed as patients presenting with acute diarrhea and a sample culture positive for a strain of Aeromonas. Extra-intestinal infections related to a strain of Aeromonas were diagnosed as patients presenting with inflammation in a region not identified as intestinal and a sample culture positive for a strain of Aeromonas.

Data were analyzed by use of the x² test and Fisher’s exact test (SPSS 15.0), When P < 0.05 results were considered statistically significant.

Aeromonas spp. were identified as the causative agent of diarrhea in 98 (7.6%) of 1286 patients. Clinical and epidemiological characteristics were shown in Additional file 3. Gender ratio (male: female) was 0.94 (46/49) among 98 patiens presenting with diarrhea caused by strains of Aeromonas. Sources of infections were largely unknown and likely originate from contaminated food. However, 3% of patients identified seafood, cooked food or frozen drinks as likely sources. Twenty percent of patients presented with vomiting, 35.8% abdominal pain, 11.6% fever (body temperature ≥ 37.7 °C), and 9.1% had mild dehydration. Approximately 70% of patients presenting with diarrhea caused by a strain of Aeromonas had loose stools for ≥3 times per day, 29.4% had watery stools, and 1.0% had mucus-like stool. Erythrocytes and leukocytes were present in 28.4 and 11.6% of samples of stool collected from patients infected by strains of Aeromonas when observed by use of high magnification (HP, × 40). In addition, 6.3% of stool samples presented with erythrocytes and leukocytes. Infection of patients by other enteropathogens was observed in three patients (3/98, 3.1%). Combinations of infectious species included; Salmonella typhimurium with A.caviae, Vibrio fluvialis with A. veronii and Vibrio parahaemolyticus with A. veronii.

Between 2015 and 2017, 17 strains of Aeromonas causing extra-intestinal infections were identified (Table 1 and Additional file 4). With the excepted for 3 children accepting a liver transplant (age < 4 years), the average age of the 14 patients was 58.5 years old. The gender ratio (male: female) was 1.83 (11/6). None of the 17 patients were ICU admissions nor was there any acute respiratory failure or mortality. Six (35.3%) patients suffered from Aeromonas-related cholecystitis following a liver transplant and 6 (35.3%) pantients presented with malignant tumors. Overall, the most common underlying conditions of patients presenting with Aeromonas infections were liver transplantation and malignancy (12/17), In addition, patients presenting with Aeromonas related infections were associated with increased prevalence of lung cancer in our study.

Eleven patients presented with monomicrobial related Aeromonas infections, and 6 patients presented with polymicrobial Aeromonas infections (Table 1). Of polymicrobial infections, two consisted of A.caviae and Klebsiella pneumoniae; and two were A.aquariorum with Klebsiella pneumoniae. One patient presented with A.aquariorum and Stenotrophomonas maltophilia and one patient with A.veronii and Proteus mirabilis. Klebsiella pneumoniae was the most common combined pathogen (66.7%, 4/6).

Results of MLPA performed with the concatenated 7-gene phylogenetic tree analysis classified 113 of 115 (98.3%) Aeromonas isolates to 8 different species (Fig. 1). The four most prevalent species of Aeromonas were A. caviae (41.7%), A. veronii (31.3%), A. dhakensis (13.9%), and A. hydrophila (5.2%). As presented in Table 2, comparative analysis of genotyping demonstrated differences between intestinal and extra-intestinal isolates was completed. Overall, there was a significant difference in the assemblage of isolates as intestinal isolates generally contained species of A. caviae (43.9%), A. veronii (35.7%), and A. dhakensis (12.2%). In contrast, extra-intestinal isolates generally contained A. hydrophila (29.4%), A. caviae (29.4%), and A. dhakensis (23.5%). There was significant difference between intestinal and extra-intestinal isolates for the species A. veronii and A. hydrophila (P < 0.05, x² test). Five of 6 strains of A. hydrophila were isolated from patients with solid tumors, while only 1 strain of A. hydrophila was associated with an intestinal infection.

Presence of multiple virulence genes was common among isolates of Aeromonas and 40 virulence combinations of 10 putative virulence genes were identified. The predominant combination (i.e. pattern) of virulence genes was alt/ela/lip/fla (pattern1), which presented in 27.0% of patients presenting with an infection related to a isolate of Aeromonas. In addition, the patterns of act/fla (pattern 2), alt/ela/lip (pattern 3) and act/ascF-G/fla (pattern 4) were prevalent among patients.. As presented in Table 3, the pattern of virulence genes varied among genus’s. Of the four most prevalent species, the haemolytic genes act was prevalent in A. veronii and A. dhakensis. The haemolytic genes hlyA was prevalent in A. hydrophila and A. dhakensis, and the haemolytic genes aerA was more prevalent in A. dhakensis. The enterotoxin gene ast was identified primarily in A. hydrophila. The enterotoxin gene alt, extracellular protease genes ela and lip were less prevalent in A. veronii. The TTSS genes (ascF-G) was prevalent in A. hydrophila. A. veronii carried pattern 2 and 4 and A. caviae carried pattern1 and 3. The species A. dhakensis and A. hydrophila had diverse virulence patterns, 93.3% A. dhakensis and 100% A. hydrophila had 5 or more virulence genes.

Resistance profiles of the 115 Aeromonas isolates to 15 antimicrobial agents were shown in Table 4. High resistance to ampicillin (93.9%) and Nalidixic acid (54.8%) was observed in Aeromonas isolates. The majority of isolates (≥80%) were susceptible to chloramphenicol, gentamicin and the new generation antibiotics ciprofloxacin, ceftriaxone, cefepime, imipenem, sulfonamides, trimethoprim- sulfamethoxazole, doxycycline and azithromycin. Resistance was most prevalent for ciprofloxacin, amoxicillin-clavulanate, cefoxitin, ceftriaxone, sulfonamides, gentamicin and azithromycin in A. hydrophila, as a resistance of 66.7, 100.0, 66.7, 66.7, 50.0, 50.0, and 66.7% was observed, respectively. Higher rates of resistance to cefoxitin was also observed in A. dhakensis (87.5%). Only 3 strains exhibited resistance to imipenem, all of which were identified as belonging to A. dhakensis. Significantly higher rates of resistance to 10 antibiotics (ciprofloxacin, nalidixic acid, amoxicillin-clavulanate, cefoxitin, ceftriaxone, cefepime, sulfonamides, trimethoprim-sulfamethoxazole, gentamicin and azithromycin) were found among extra-intestinal isolates when compared with intestinal isolates (P < 0.05, x² test).

Of the 115 strains, 33 strains (28.7%) exhibited 35 multiple-drug resistance (MDR) patterns to 15 antimicrobial agents. Eighty-three percent (5/6) of strains of A. hydrophila and 81.2% (13/16) of strains of A. dhakensis presented with MDR, while less MDR isolates were found in A.caviae (39.6%, 19/48) and A.veronii (16.7%, 6/36). Intestinal strains (30.6%, 30/98) presented with significantly less rates of MDR when compare to isolates from extra-intestinal strains (82.3%, 14/17), indicating acquisition of MDR was likely from in the hospital.