Virulence characteristics and antimicrobial resistance of Aeromonas veronii biovar sobria 312M, a clinical isolate

Abstract

Aeromonas are bacteria widely distributed in the environment, and some species are able to cause infections in humans, of which diarrhea is the most common. The objective of this study was to evaluate the presence of virulence and antimicrobial resistance associated characteristics in A. veronii biovar sobria strain 312M isolated from diarrheal stools. For this, the genome sequencing and phenotypical tests were performed. The draft genome annotation revealed several complete pathways associated with carbon metabolism and a mucin-desulfating sulfatase which may contribute to intestine colonization, and a large number of virulence-associated genes encoding structures associated with adhesion, toxins, and secretion systems. The strain exhibited swimming and swarming motility, biofilm formation, and hemolytic activity. It was resistant to ampicillin, ampicillin/sulbactam, and amoxicillin-clavulanic acid. Although a cphA gene encoding a narrow-spectrum carbapenase was identified in the strain genome, no carbapenemase activity was detected in the antimicrobial susceptibility test. When compared with other A. veronii with complete genomes, the main differences in virulence characteristics are related to lateral flagella and type III and VI secretion systems; the antimicrobial resistance spectrum also varied among strains. The results indicated that A. veronii biovar sobria 312M presents high virulence potential and resistance to limited classes of antimicrobials.

Introduction

Aeromonas are gram-negative rods, facultative anaerobic, glucose fermenters, and oxidase-positive. These bacteria are ubiquitous in aquatic habitats and have been found in foods, such as seafood, raw milk, chicken, meat, and vegetables [1]. There are about 30 Aeromonas species (http://www.bacterio.net/aeromonas.html), some of which are pathogens of fish, and at least 12 can cause infections in humans.

Of these, A. hydrophila, A. caviae, and A. veronii biovar sobria are responsible for the majority of human infections caused by bacteria of this genus [1]. The virulence of Aeromonas is multifactorial, and several virulence-associated characteristics have been identified in these bacteria including pili, flagella, extracellular enzymes, several toxins, and secretion systems [2, 3]. In the last 13 years, the genomes of several Aeromonas species have been sequenced and published, increasing the knowledge concerning these bacteria and allowing for the identification of other virulence factors, such as the Flp pilus and a type VI secretion system, by sequence homology with other pathogens [4, 5]. The comparison of Aeromonas genomes revealed that the distribution of virulence characteristics is heterogeneous among the strains and pathotypes with distinct virulence signatures were identified [2]. While there are published data on the molecular determinants of virulence in clinical isolates of A. hydrophila and A. caviae, they are lacking for clinical A. veronii biovar sobria [2]. This biovar of A. veronii is associated with several types of infections in humans, including wound infections, septicemia, meningitis, peritonitis, hepatobiliary disease, and diarrhea [1, 6, 7]. Here we present the molecular determinants of virulence and antimicrobial resistance, as well as some phenotypic features of the clinical isolate A. veronii biovar sobria strain 312M (AVS 312M).

Material and methods

Bacteria source and identification

The bacterium was recovered as the single pathogen from stools of 1-year-old Brazilian child with diarrhea. The clinical isolate was identified through conventional biochemical tests as A. veronii biovar sobria [8]. Here, the identity of the isolate was again analyzed utilizing conventional microbiology assays [9] in addition to a molecular assay based on the restriction fragment length polymorphism of PCR-amplified 16S rRNA genes [10].

Genomic DNA extraction

Genomic DNA was extracted from an overnight culture of the bacteria on BHI broth (Himedia, Mumbai, India), using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions.

Genome sequencing and analysis

Genome shotgun sequencing was performed using the 454 GS Junior Titanium series (Roche, Branford, USA) and the Illumina MiSeq (Illumina, San Diego, USA) sequencers according to the manufacturer’s protocol. Sequences from the 454 GS Junior and Illumina were assembled using Newbler 2.7 (GS Data Analysis assembly software, Roche, Branford, USA), CLC Genomics Workbench 6.5.1 (CLC bio, Aarhus, Denmark), SPAde 3.10 [11], and Velvet 1.2.10 [12]. The genome assemblies were improved with GFinisher [13] using multiple complete genomes of Aeromonas species deposited in NCBI as reference.

Assessment of genome similarity using average nucleotide identity

The assessment of the AVS 312M genome similarity was determined using average nucleotide identity (ANI) with the online ANI calculator [14] in pairwise comparisons with complete genomes of several other Aeromonas species.

Genome annotation

Annotation was performed with Rapid Annotations using Subsystems Technology (RAST) [15] and the KEGG database [16] to determine the metabolic pathways present in the genome. The ARAGORN software [17] was used to determine the presence of tRNAs.

Virulence factors

Identification of virulence-associated genes was done by RAST annotation as well as a manual search of Aeromonas virulence genes using NCBI BLASTp [18]. The search for Aeromonas virulence genes was performed using as reference the loci associated with motility and adhesion [4, 5, 19]; toxins and some extracellular proteins considered putative virulence factors [4, 19, 20]; and secretion systems and quorum sensing [4, 19, 21, 22]. Sequence alignments containing at least 75% identity and 75% coverage were used to determine the presence of virulence-associated genes. Additionally, ResFinder-2.1 [23] was used for the identification of antimicrobial resistance genes, where an identity of 90% and an overlapping length of 60% were used as the minimum limit.

Motility assays

The swimming and swarming motility were performed as described [2], except that LB medium and BBL agar granulated (Becton Dickinson, Cockeysville, MD, USA) were used for both assays. Additionally, 0.005% (vol/vol) Tween 80 (J.G. Shaw, personal communication) was added to LB medium for swarming motility testing. Swimming motility was determined by the development of motility zones from the stabs inoculated into the center of the swimming plates (contains 0.3% agar), after the incubation. The growth from the edge of the swimming plates was the inoculum for swarming motility testing that was inoculated on the surface at the center of the swarming plates (contains 0.5% agar); swarming zones were evaluated after the incubation. Aeromonas hydrophila ATCC 7966 was used as control.

Biofilm formation

The ability of biofilm formation was evaluated using the method described by [6] with modifications. Briefly, polypropylene tubes were inoculated with 200 μL of a fresh bacterial suspension (1.5 × 10⁸ CFU) in LB medium and incubated at 30 °C for 24 h. After, the content was discarded and the tubes were rinsed vigorously twice with water to remove nonadherent bacteria. The biofilm was stained with 200 μL of 1% crystal violet for 10 min, then rinsed, dried, and solubilized with 400 μL of 95% ethanol for 15 min. The supernatant was transferred to a 96-well microplate and the optical density determined at 570 nm. The assay was performed three times in triplicate. An uninoculated broth control was included in each experiment, and Pseudomonas aeruginosa ATCC 27853 was used as a positive control.

Hemolytic activity

The hemolytic activity was assayed in 5% sheep blood agar [9].

Antimicrobial susceptibility tests

Antimicrobial susceptibility testing was performed by a disk diffusion method [24, 25] and Vitek 2 Compact card AST-GN09 with the Vitek 2 Compact automated system for microbial identification (bioMérieux, Marcy l’Etoile, France). The following antimicrobials were tested: amikacin, ampicillin, ampicillin/sulbactam, amoxicillin-clavulanic acid, aztreonam, cefepime, cefoxitin, cefotetan, ceftazidime, ceftriaxone, cefuroxime, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, imipenem, levofloxacin, meropenem, nalidixic acid, nitrofurantoin, piperacillin, piperacillin-tazobactam, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole.

Results

To confirm the identity of AVS 312M, a 16S rRNA restriction fragment length polymorphism assay was used [10]. This test was developed for the identification of Aeromonas at the species level [10] and confirmed that the clinical isolate belongs to the species A. veronii. However, A. veronii contains two biovars, named biovar sobria and biovar veronii, which are not differentiated by this assay [10], but they can be distinguished through the activity of ornithine decarboxylase and arginine dihydrolase [9]. The biochemical test results, ornithine decarboxylase negative and arginine dihydrolase positive, indicated that the clinical isolate belongs to the biovar sobria.

Genome of AVS 312M

The AVS 312M genome was sequenced using 454 GS Junior and Illumina sequencers which provided 43,500,289 bp in 96,886 reads and 1,464,278,530 bp in 9,199,874 reads, respectively. The contigs obtained by assemblies were 526 with Newbler, 90 with CLC, and 186 with SPAdes 3.10. The Velvet generated between 32,770 (21-mer) and 70,333 (103-mer) contigs. The contigs were submitted to GFinisher, which resulted in 14 contigs. The AVS 312M genome contains 4.57 Mbp and GC content of 58.6% and showed a high coverage with more than 320 depth. The AVS 312M genome sequence was deposited in GenBank under accession number RHDQ00000000.

The ANI results (Table 1), determined for AVS 312M in pairwise comparisons with Aeromonas species with complete genomes, show that values above 95% are observed only with A. veronii strains and also confirm that AVS 312M belongs to this species.

Table 1.

Average nucleotide identity (ANI) values determined for A. veronii biovar sobria 312M relative to other Aeromonas species

Aeromonas species	BioProject	ANI value (%)
A. aquatica MX16A	PRJNA260478	87.40
A. caviae 8LM	PRJNA277314	86.04
A. caviae FDAARGOS_72	PRJNA231221	86.09
A. caviae NCTC12244	PRJEB6403	85.99
A. caviae R25-2	PRJNA428427	85.98
A. caviae R25-6	PRJNA428427	86.03
A. caviae T25-39	PRJNA428427	86.01
A. dhakensis KN-Mc-6U21	PRJNA400818	86.96
A. hydrophila 4AK4	PRJNA210524	85.92
A. hydrophila ATCC7966	PRJNA16697	87.30
A. hydrophila AH10	PRJNA278509	87.38
A. hydrophila AHIN1	PRJNA273636	87.24
A. hydrophila AL06-06	PRJNA227242	87.37
A. hydrophila AL09-71	PRJNA227037	87.47
A. hydrophila D4	PRJNA308632	87.49
A. hydrophila GYK1	PRJNA323754	87.49
A. hydrophila J1	PRJNA227242	87.47
A. hydrophila JBN2301	PRJNA302121	87.45
A. hydrophila KN-Mc-1R2	PRJNA438415	87.33
A. hydrophila ML09-119	PRJNA188141	87.45
A. hydrophila MX16A	PRJNA353572	87.38
A. hydrophila NJ-35	PRJNA226230	87.41
A. hydrophila Pc104A	PRJNA227038	87.43
A. hydrophila WCHAH045096	PRJNA415336	87.48
A. hydrophila WCX23	PRJNA530076	87.46
A. hydrophila YL17	PRJNA234473	87.10
A. hydrophila ZYAH72	PRJNA339368	87.32
A. hydrophila ZYAH75	PRJNA339336	87.34
A. media WS	PRJNA170164	86.01
A. salmonicida 01-B526	PRJNA264317	85.63
A. salmonicida 34mel	PRJNA299262	85,83
A. salmonicida A449	PRJNA16723	85.68
A. salmonicida A527	PRJNA264317	85.79
A. salmonicida O23A	PRJNA383853	85.76
A. salmonicida RFAS1	PRJNA342065	85.78
A. salmonicida S121	PRJNA391818	85.68
A. salmonicida S44	PRJNA391845	85.69
A. salmonicida S68	PRJNA391844	85.67
A. schubertii WL1483	PRJNA297116	82.03
A. veronii 17ISAe	PRJNA438884	95.55
A. veronii AVNIH1	PRJNA279607	95.62
A. veronii B565	PRJNA63671	95.60
A. veronii CB51	PRJNA319612	95.59
A. veronii FC951	PRJNA428153	95.32
A. veronii MS-18-37	PRJNA504296	95.65
A. veronii TH0426	PRJNA293940	95.63
A. veronii X11	PRJNA419084	95.63
A. veronii X12	PRJNA419086	95.52

Only Aeromonas complete genomes were used in the analysis; ANI values between genomes of the same species are above 95%

RAST annotation identified a total of 4262 coding sequences of which 36% (1502) were classified in 360 subsystems. The major gene categories were associated with metabolism of amino acids and derivatives (378), carbohydrates (272), protein (268), cofactors, vitamins, prosthetic groups, pigments (184), and membrane transport (174). Genome annotation indicated the presence of complete metabolic pathways involving glycolysis and gluconeogenesis, tricarboxylic acid cycle, utilization of fructose, galactose, mannitol, sucrose, maltose, and N-acetyl-D-glucosamine. Also, the presence of 130 RNAs, including 101 tRNAs, was identified.

Virulence and antimicrobial resistance genes

The virulence-associated genes found in the genome of AVS 312M are indicated in Table 2. Additionally, RAST annotated a gene cluster coding for colicin V and ResFinder analysis indicated the presence of genes encoding the β-lactamases AmpS and CphA.

Table 2.

Virulence associated characteristics of AVS 312M

Function	Identity (%)	Coverage (%)	Locus	Reference
Motility and adhesion
Lateral flagellaa	76–95	86–98	ASA_0346–0386	[5]
Polar flagella	80–100	87–100	B565_1098–1124; B565_1452–1458; B565_2588–2590; B565_2596–2611; B565_3200–3201	[19]
Tap type IV pilus	94–100	96–100	B565_0359–0357; B565_0964–0968; B565_3513–3512; B565_3469–3464; B565_1398; B565_3454; B565_1560; B565_2370
Flp type IV pilus	96–100	97–100	B565_2723–2735
Msh type IV pilusa	79–99	84–100	B565_3654–3670
Type I fimbriae	97–100	98–100	B565_0475–0480
Toxin/ other putative virulence features
Cytotonic enterotoxin thermo labile (Alt)	81	87	L77573	[20]
Cytotoxic enterotoxin (Act)	99	99	B565_3626	[19]
Thermostable hemolysin	95	97	B565_0938
Hemolysin HlyA	100	100	B565_2574
Hemolysin III	98	98	B565_0799
Hemolysin	78	88	AHA_0229	[4]
Hyaluronidase	85	92	AHA_1474
Serine protease	80	99	AHA_2687
Enolase	100	100	AHA_0821
DNA adenine methylase (Dam)	95	98	AHA_3186
Glucose-inhibited division protein (GidA)	96	98	AHA _4273
Mucin-desulfating sulfatase	75	84	AHA _0617
Collagenase	99	99	B565_0474	[19]
Secretion system
T2SS	93–100	95–100	B565_2887–2900; B565_0357	[19]
T3SSa	89-100	83-100	ABP51916.1- ABP51951.1	[21]
T6SSa	82-98	88-100	AHA_1826–1848	[22]
Quorum sensing				[4]
Autoinducer synthase (AhyI)	75	86	AHA_0556
Transcriptional activator protein (AhyR)	90	94	AHA_0557
QseBC QS system	81–89	88–94	AHA3223–3222
Ribosylhomocysteine lyase (LuxS)	97	98	AHA_0700

The Aeromonas virulence features searched for are described with the identity and coverage results from Blastp. Type I fimbriae, Msh type IV pilus, type II secretion system, and type VI secretion system were also annotated by RAST

^aSome genes had identity and/or coverage lower than 75%; in these cases, the gene presence was confirmed with other Aeromonas genome references

Phenotypical characteristics

AVS 312M displayed β-hemolytic activity and exhibited swimming and swarming motility (Fig. 1). The ability of AVS 312M to form biofilms on a polypropylene surface corresponded to 40% of that observed for P. aeruginosa, indicating a moderate activity. However, the biofilm formation may be influenced by type of surface used in the study. For example, Escherichia coli 0157:H7 exhibited little or no biofilm formation on a polypropylene surface, but displayed strong biofilm formation on borosilicate glass [26]. Thus, it is possible that AVS 312M may show a more intense biofilm activity on another type of surface. Results of the antimicrobial susceptibility tests showed that AVS 312M is resistant to ampicillin and sensitive to cephalothin. These results are consistent with what has been described for this species reported as fully resistant to ampicillin and fully sensitive to cephalothin [9]. Additionally, AVS 312M presents resistance to ampicillin/sulbactam and amoxicillin-clavulanic acid and was sensitive to all of the other antimicrobials tested.

Fig. 1.

Motility and hemolytic activity of A. veronii 312M. a, b Swimming motility (growth spreads from the stab occupying all the plate) of A. hydrophila ATCC 7966 (control) and A. veronii 312M, respectively; c no swarming, only growth at the inoculum point, of A. hydrophila ATCC 7966; d swarming motility (growth from the point of inoculation and spreading on the plate) of A. veronii 312M; e growth of A. veronii 312M on sheep blood agar exhibiting β-hemolysis

Discussion

A. veronii biovar sobria can cause several types of infections in humans [1]; here we present genomic and phenotypical characteristics of AVS 312M which was isolated from a patient with diarrhea. The AVS 312M genome size and GC content are similar to that of other strains of the same species recovered from different sources [19, 21, 27, 28], but its genome size, 4.57 Mbp, is smaller than of the strain TH0426 which contains 4.92 Mbp [29].

In order to cause gastroenteritis, bacteria need to compete against the commensal microbiota and establish themselves in the intestine. This process involves the production of bacteriocin as well as several interrelated steps, including motility, attachment, biofilm formation, colonization, and elaboration of virulence factors [1]. Furthermore, the ability to utilize nutrients available in the infection site is critical for host colonization and metabolic flexibility provides a competitive advantage in the intestine [30, 31].

The finding of several complete metabolic pathways related to the carbon metabolism in AVS 312M genome is in agreement with the bacterium ability to utilize several carbohydrates as the sole carbon source for growth [32]. In addition, galactose, mannose, N-acetyl-glucosamine, glucose, glutamic acid, and serine, some of the carbohydrates and amino acids that AVS 312M can use for growth [32], are found in the mucin of the intestinal tract. Colonization studies [31] performed in mice using the commensal Escherichia coli MG1655 and the enteropathogen E. coli EDL933 and their mutants for intestinal mucus-derived sugars indicated mutations that affected the intestine colonization showing that arabinose, fucose, and N-acetylglucosamine are used for both bacteria. Besides, MG1655 used gluconate and N-acetylneuraminic acid, while EDL933 utilized galactose, mannose, and ribose, not used by MG1655. The utilization of several different sugars suggests a strategy for enteric pathogens to invade and colonize the intestine in the presence of commensal strains [31]. Thus, the AVS 312M ability to utilize components which are found in the intestinal mucin [32], including some used by E. coli EDL933, may contribute to host colonization and its metabolic flexibility may facilitate the survival in the intestine. Moreover, a mucin-desulfating sulfatase is encoded in AVS 312M genome (Table 2) and may facilitate mucin degradation [4], which could be useful for colonization of the intestine. Additionally, a gene cluster encoding for the bacteriocin colicin V is present in AVS 312M genome. The production of colicin V may be a tactic used by AVS 312M to compete with the resident microbiota during the colonization process.

Several virulence-associated genes were found in the genome of AVS 312M (Table 2), and some of these may potentially be associated with the development of diarrhea. Among them are the genetic loci encoding polar flagella (Table 2) which were found in all Aeromonas genomes [2]. Additionally, genes encoding lateral flagella, found in species or strains considered highly pathogenic [2], are also present in AVS 312M (Table 2). The expression of both flagellar types in AVS 312M was inferred from positive results in both the swimming and swarming assays associated, respectively, with the presence of polar and lateral flagella. In addition to motility, Aeromonas also utilizes flagella as adhesins, which not only facilitate adherence to human intestinal cells but also participate in biofilm formation, a characteristic also exhibited by AVS 312M, both of which are important for intestinal colonization [6]. Other structures that may be associated with adhesion and biofilm formation, fimbriae and pili are also encoded in AVS 312M (Table 2). Type I pili are widely distributed among Aeromonas but were reported to not being involved in binding to human intestinal cells. However, in A. salmonicida, type I pili are the primary adhesin used to attach to intestinal epithelial cell in salmon and improved the efficiency of infection [33]. Tap and Msh (Bfp) pili are common in Aeromonas species [2]. While Msh (Bfp) pili have been shown to be required for bacterial adhesion to human intestinal cells and biofilm formation, Tap pili were shown to not playing a role in the adherence of Aeromonas to human intestinal cells [7, 34]. However, in contrast, Tap pili contribute to the virulence of A. salmonicida in Atlantic salmon infection [35]. The Flp type pilus, though, is not very common among Aeromonas [2]. It was reported to contribute minimally to the virulence of A. salmonicida in Atlantic salmon [35], and its involvement in human infections has not yet been demonstrated. Although Tap pili apparently do not contribute to diarrhea, and the function of Flp pili is unknown, it is possible that these pili may contribute to the adhesion to other cell types or hosts and may represent an advantage for AVS 312M survival. Genes coding for several other virulence-factor were identified in the genome of AVS 312M (Table 2). Among them are two enterotoxins, Act, which has hemolytic and cytotoxic activities, and Alt. Both Act and Alt cause intestinal fluid secretion and have been shown to cause diarrhea in a murine model [3]. Moreover, hemolysins, serine protease, collagenase, and hyaluronidase (Table 2) are also encoded in the genome of AVS 312M. They have the potential to cause tissue damage, enabling the dissemination of bacteria within the host, and simultaneously providing nutrients for growth [1]. AVS 312M displayed β-hemolytic activity indicating the expression of at least one of the hemolysins. Three secretion systems are encoded in the genome of AVS 312M (Table 2) and play a role in Aeromonas virulence. The type II secretion system secretes the Act toxin as well as other virulence factors such as proteases [3]. The type III and type VI secretion systems deliver effector proteins into the cytosol of host cells. Genetic mutants of these secretion systems exhibited virulence attenuation and reduced toxicity to murine macrophages and human cells, and caused reduced mortality in mice [3, 22]. Furthermore, studies have shown that the Act toxin and type III or type VI secretion system (or a combination of them), all encoded in AVS 312M genome, are present in the most virulent Aeromonas in murine septicemic or intramuscular infection models [2]. Other virulence-associated factors identified in the genome of AVS 312M include genes encoding Dam and GidA which control Act expression, and Quorum Sensing systems which regulate the type VI secretion system, the cytotoxic enterotoxin, swimming and swarming motility, protease, and biofilm formation [3].

Finally, AVS 312M showed resistance to ampicillin, ampicillin/sulbactam, and amoxicillin-clavulanic acid; these results are in agreement with the activity of β-lactamase. Genes encoding β-lactamases AmpS and CphA were found in the genome of AVS 312M. AmpS is a class D oxacillinase with penicillinase profile. However, CphA is a class B narrow-spectrum carbapenemase [36] and no carbapenemase activity was detected in the antimicrobial susceptibility test. This result is in agreement with other study [37] which showed that CphA is not detected in majority of Aeromonas strains using the disc diffusion assay with the standard inoculum, as observed for AVS 312M.

When AVS 312M was compared with other A. veronii with complete genomes including the fish pathogen strains TH0426 [29] and MS-18-37 [28], and strain B565 recovered from aquaculture pond sediment [19] regarding the virulence characteristics, the main differences were in the distribution of lateral flagella and type III and VI secretion systems present only in AVS 312M and TH0426. Differences were also observed in the antimicrobial resistance profile in which strain MS-18-37 presented resistance to a higher number of antimicrobial classes.

In conclusion, results indicate that the clinical isolate AVS 312M genome encodes several characteristics potentially associated with host colonization and is also well-equipped with potential virulence features including several adhesin and toxin types, polar and lateral flagella, and type III and VI secretion systems, suggesting that the strain is a versatile bacterium with high virulence potential while resistant to limited classes of antimicrobials.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.