Characterization and Whole Genome Sequencing of Chromobacterium violaceum OUAT_2017: A Zoonotic Pathogen Found Fatal to a Wild Asiatic Elephant

Abstract

This study reports a rare fatal case of Chromobacterium violeceum OUAT_2017 strain infection in an Asiatic elephant calf in India. Necropsy revealed pus-filled nodules in liver, spleen, and lungs. Nutrient broth cultures of nodule content showed sediment of violet pigment whereas smooth, non-diffusible, violet-pigmented, homogeneous colonies appeared on nutrient agar. The organism was found to be non-haemolytic and resistant to 8 of the 24 antibiotics tested in vitro. Partial 16S rRNA gene sequence measuring 1410 bp revealed 97% homology with C. violeceum. The bacterial genome composed of 64.87% of G + C content with total size of 4,681,202 bp. The genome annotation has 42 genes responsible for multidrug antibiotic resistance with the presence of Aminoglycoside-modifying enzymes (AAC (6’)) that targets streptomycin and spectinomycin. Our findings corroborated the lethal effect of C. violeceum in a new host (elephant) that enriched scientific information on epidemiological picture and whole genome sequencing as well.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01047-4.

Introduction

Chromobacterium violaceum commonly present in soil and stagnant water bodies in tropical and subtropical regions. This bacterium is characterized as Gram negative rod-shaped facultative anaerobe. It is an opportunistic pathogen that causes rare but fatal infections in humans and animals. The ailment was first described in 1872 as a tropical pathogen potentially harmful to man and animals [1]. So far, fewer than 300 cases with high mortality have been reported in the literature worldwide [2] Chromobacteriosis has been reported in pigs, non-human primates, sheep, and dogs [3]. C. violaceum is transmitted through the exposure of skin wounds and traumatic lesions to soil and water contaminated with C. violaceum [4–6]. The bacteria can cause septicemia with necrotizing metastatic lesions. There are several reports suggesting that the bacteria were responsible for fatal multiorgan failure due to formation of abscesses in internal organ such as the liver, lung, spleen, skin, lymph nodes, and brain [7–9]. Early identification of the organism and targeted therapy is the key for its successful cure. In this study, we report a rare and first case of C. violeceum infection in a wild elephant calf that was diagnosed on the basis of postmortem examination, microbiological and molecular tests. We performed whole genome sequencing of C. violeceum isolate.

Materials and Methods

History

A 4/5-year-old free-ranging male Asian elephant (Elephas maximus) calf died on the morning of June 29, 2017 at the Baniabandha Reserve Forest, Orda, Khuntuni, Odisha, India (20.5658° N, 85.7244° E) after a brief period of illness for two days with signs of slow movement and poor food intake.

Necropsy Examination

A systematic postmortem examination of both external and internal organs was carried out at the site of death on the date of death and findings were noted. Tissue samples were collected aseptically for laboratory investigation. The carcass was disposed of by deep burial method.

Microbiological Examination

Contents of freshly cut pus filled nodules from liver, spleen, and lungs were inoculated independently into nutrient both and incubated at 37° C for 24 h of incubation period. After 24 h of incubation the broths were examined for bacterial growth. The broths, those were showing bacterial growth were streaked on nutrient agar plates and incubated for 24 h at 37° C to isolate single bacterial colony. Colonies from nutrient agar plates were streaked on the Sheep blood agar plates to determine the hemolytic activity of the bacteria.

Another set of such samples were processed to know the presence of Mycobacterium spp. by following the protocol described by Hota et al. [10]. Samples were incubated at room temperature in presence of equal volume of 4% NaOH. After that, the sample was centrifuged at 3000 g for 15 min, and the supernatant was discarded keeping the pallet. The pellet was resuspended in 15 ml of sterile distilled water. The above mixer was again centrifuged at 3000 g for 15 min and the supernatant was discarded. Two Lowenstein-Jensen agar slants were prepared separately with glycerol and sodium pyruvate in McCartney tubes. The sample was streaked on the Lowenstein-Jensen slants and incubated at 37° C in an inclined position overnight and vertically for 8–12 weeks. The slants were examined weekly for bacterial growth starting from three days post inoculation.

Antibiotic Sensitivity

Kirby Bauer’s disk diffusion susceptibility test was performed to obtain antibiogram profiling of the C. violeceum isolate. The interpretation of the results for other non-Enterobacteriaceae family were done by following the guidelines of Clinical and Laboratory Standards Institute (CLSI) [11].

Molecular Analysis

DNA was extracted from pure bacterial colony using Purelink™ Microbiome DNA purification kit (Invitrogen, ThermoFisher Scientific, USA) according to the manufacture’s instruction. The whole 16S rRNA gene of C. violeceum was amplified in thermal cycler using universal 16S primers [12] from extracted DNA. The amplified product was sequenced by Sanger sequencing.

Whole Genome Sequencing

Library Preparation and Sequencing

The above extracted DNA sample was submitted to Pathcare life sciences, Hyderabad, India for whole genome sequencing. By following manufacturer instructions, an Illumina sequencing library was prepared using Illumina TruSeq DNA HT Library Preparation Kit (Illumina, San Diego, CA), with pair end indexing. The final library was spiked into 5% PhiX and subjected to proper quality control procedures recommended by Illumina, San Diego, CA. The sequencing run was performed on the Illumina NextSeq500 sequencing platform (Illumina, San Diego, CA). The genomic sequence reads (FASTQ) files were obtained for further analysis after automated cluster generation.

Bioinformatic Analysis

We used a program called Trimmomatic to filter out poor quality reads and bases from genomic sequence reads generated through Illumina sequencing. Filtered reads were subjected to de novo assembly for contig generation using unicycler [13]. Rapid Annotation using Subsystem Technology (RAST) [14] was used for further annotation of genome assembly from the SEED database. The annotation analysis assigns the predicted proteins to functional families such as genus-specific protein families (PLFams), and cross-genus protein families (PGFams) [15]. The contigs were annotated using resources available in Pathosystems Resource Integration Center (PATRIC, https://www.patricbrc.org) [16]. The k-mer based detection method was used to classify the genes associated in antimicrobial resistance (AMR) mechanism with reference to the data available in PATRIC resource center. The assembled sequences were also screened using antiSMASH version 6.1.1 to identify secondary metabolite regions [17].

The closest reference and representative genomes were identified by MinHash [18] from National Center for Biotechnology Information (NCBI) database to generate comprehensive phylogenetic analysis. PATRIC global protein families (PGFams) [16] were selected from representative genomes to determine the phylogenetic placement of C. violaceum strain OUAT_2017 genome. The protein sequences from these families were aligned with MUSCLE, and the nucleotides for each of those sequences were mapped to the protein alignment. The joint set of amino acid and nucleotide alignments were concatenated into a data matrix. This matrix was analyzed by RaxML [19] to generate the genome based phylogenetic tree.

Results

Postmortem examination of external body parts did not reveal any appreciable skin lesions. liver, spleen, and lungs showed multiple pus-filled nodules (Supplement Fig. 1). The broth cultures of such individual tissue/pus samples showed sediment of violet pigment. A smooth, non-diffusible, violet-pigmented, homogeneous colonies appeared were appeared on the nutrient agar plates after incubation for 24 h at 37° C. The bacteria was found to be non-haemolytic in nature on Sheep blood agar plate (Fig. 1). Gram staining revealed the presence of Gram negative coccobacilli. The bacterial isolate was resistant to eight antibiotics tested, e.g., Penicillin-G, Amoxicillin, Ampicillin, Cloxacillin, Bacitracin, Cefalexin, Cefuroxime and Ceftriaxone/Sulbactam. Ceftriaxone and Enrofloxacin showed intermediate patterns of sensitivity (Supplement Table 1). Lowenstein-Jensen agar slant with either glycerol or sodium pyruvate after 6–10 weeks of incubation at 37° C showed no growth of bacteria signifies the absence of Mycobacterium tuberculosis and Mycobacterium bovis in pus/nodule samples.

Fig. 1.

Multiple pus-filled nodules in (A, B) spleen and its cut surface; C, D liver and its cut surface; and E abdominal cavity

Partial 16S rRNA gene sequence, obtained through Sanger sequencing, measured 1410 bp and revealed 97% homology with C. violaceum. The sequence was submitted in the NCBI database with accession number ‘LC314498’. Subsequently, complete genome sequencing of the C. violaceum strain OUAT_2017 was performed. The curated data was deposited to GenBank with the accession JAIUJK000000000, BioProject PRJNA764546 and BioSample SAMN21508251. Based on whole genome phylogenetic analysis it confims that the C. violeceum is similar to strain C. violeceum ATCC 12,472 (Accesion number 243365.4) (Supplement Fig. 2). The genome analysis revealed that our isolate genome has an average G + C content of 64.87% and a genome length of 4,681,202 bp. There was no extra-chromosomal DNA (plasmid) detected in the strain OUAT_2017. Annotation by RAST indicated that the genome contains 4628 protein coding domain sequences (CDS), 4 ribosomal RNA (rRNA) coding genes, and 82 transfer RNA (tRNA) coding genes (Table 1). The annotation also classifies 1298 and 3330 proteins with hypothetical and functional assignments simultaneously. The proteins with functional assignments represent 830 with Gene Ontology (GO) assignments, 995 proteins assigned with Enzyme Commission numbers (EC) based on the chemical reactions they catalyze, and 729 proteins mapped to KEGG pathways that were actively involved in the metabolic pathway. Again, the genome annotation of C. violeceum OUAT_2017 divided into two protein families groups, i.e. genus specific protein families (PLFams-4334) and cross genus protein families (PGFams-4,393). Genome-based phylogenetic analysis using TYGS showed that the genome of this isolate was very similar to that of C. violeceum; thus, it was classified into the same clade (Fig. 2). Based on subsystem annotation analysis, the CDS distribution on the genome was presented in a circular graphical figure (Supplement Fig. 3). The k-mer-based genome annotation of C. violeceum OUAT_2017 revealed the genes responsible for antimicrobial resistance (AMR) (Table 2). In addition to that the proteins involved in either biological process or structural complex were analyzed from annotated genome based on subsystem analysis. The subsystems include 283 categories that were representing a total of 1818 genes (Supplement Fig. 4). The subsystem category distributed 748 genes for metabolism, 264 genes for energy generation, 226 genes for protein processing, 188 genes for cellular processing, 154 genes responsible for defence, stress response and virulence, and 150 genes for active and passive membrane transport. The antiSMASH analysis using strict protocol detected several islands of secondary metabolite regions (Table 3).

Table 1.

Genome characteristics

Assembly details
Contigs	58
GC Content	64.87
Plasmids	0
Contig L50	5
Genome length	4,681,202 bp
Contig N50	379,345
Chromosomes	0
Annotated genome features
CDS	4,628
Repeat regions	141
tRNA	82
rRNA	4
Partial CDS	0
Miscellaneous RNA	0

Fig. 2.

Delineation of species and subspecies of Chromobacterium violaceum based on the GBDP phylogenetic analyses retrieved from the TYGS website. a GBDP phylogenetic analyses based on whole genome sequence (WGS) and b 16S rDNA. The tree was inferred with FastME from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications

Table 2.

Antimicrobial resistance genes

AMR Mechanism	Genes
Antibiotic inactivation enzyme	AAC(6′)-Ic,f,g,h,j,k,l,r-z, ChpA family
Antibiotic target in susceptible species	Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, inhA, fabI, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, S12p
Antibiotic target protection protein	BcrC
Efflux pump conferring antibiotic resistance	EmrAB-OMF, EmrAB-TolC, MacA, MacB, MdfA/Cmr, MdtABC-OMF, MdtABC-TolC
Gene conferring resistance via absence	gidB
Protein altering cell wall charge conferring antibiotic resistance	GdpD, PgsA
Regulator modulating expression of antibiotic resistance genes	EmrAB-TolC, OxyR

Table 3.

Identified secondary metabolite regions using antiSMASH version 6.1.1

Region	Type	From (bp)	To (bp)	Most similar known cluster	Similarity
2.1	Betalactone	230,913	253,880
4.1	RiPP-Like	3,667	14,497
4.2	Indole	244,626	267,612	Violacein	100%
9.1	NRPS	1	39,107	Salinichelins	30%
10.1	Terpene	26,264	47,992	Surfactin	8%
12.1	NRPS	39,994	83,902	Turnerbactin	30%
13.1	NRPS	91,963	114,564
14.1	Hserlactone	1	17,114
31.1, 35.1, 38.1	NRPS	1	2099	Rhizomide A/B/C	100%

Discussion

Chromobacterium violaceum, isolated from the present study, is a facultative, anaerobic, catalse-positive, Gram negative motile rod-shaped bacterium that can grow rapidly on sheep blood agar and MacConkey II agar and produce brilliant violet pigment, violacein, after anaerobic incubation for 24 h [20]. Chromobacterium violaceum is widely distributed in natural aquatic environments and is sensitive to temperature; therefore, it has a predilection to the tropical and subtropical areas [21]. Though most of the C. violaceum infection in humans are confined between latitudes of 35° north and 35° south of the tropics [22], cases have been reported from colder regions, such as Japan and Europe [23]. Apart from humans, there are few reports available on C. violaceum septicemic infection in non-human primates such as baboon and macaques [24].

Microbiological and molecular analysis of the contents of multiple nodules present in liver, lungs and spleen of dead elephant revealed presence of C. violaceum. In the current study we found that there was absence of other secondary pathogens along with M. tuberculosis and M. bovis. In contrast to our findings, Liu et al. [24] reported the presence of multiple bacteria accompanied with C. violaceum infection in the lungs and liver of non-primate organism. Multiple abscesses in various internal organs like lungs, liver and spleen as recorded in the present study corroborated with earlier reports in medical fields [2]. There are reports of osteomyelitis, chronic granulomatosis, cellulitis, and periorbital and ocular infections [25]. Septicemia caused by Chromobacterium leads to death in untreated cases is an unavoidable consequence [22]. C. violaceum enters body through skin injury or intake of water contaminated with C. violaceum [24]. The soil is a natural habitat where C. violaceum can be isolated in abundant numbers [21, 26]. It is therefore reasonable to correlate the elephant calf’s infection through the soil exposure and thereafter its entry to the body in either of the above routes.

In order to understand the biology and pathogenicity of isolated C. violaceum strain, a whole genome sequencing (WGS) was conducted. Phylogenetic tree based on WGS and 16S rDNA showed clustering with its closest strain C. violaceum (ATCC 12,472). Pair-wise genome comparison of our isolate with ATCC 12,472 showed in-silico dDDH score of 87.9%; scores over 70% and 79% indicate the same species and sub-species, respectively.

Because of the rapidly progressive nature of this infection, the elephant calf succumbed to death in a short period of illness. Similarly, in Florida, C. violeceum infection was responsible for killing one of two infected dogs though the bacterium was susceptible to fluoroquinolones, carbapenems, aminoglycosides, chloramphenicol, tetracycline, and cotrimoxazole and resistant against penicillin and cephalosporins [3]. The phenotypic character of antibiotic resistance of the C. violeceum OUAT_2017 corresponds to the AMR genes found through genome annotation. This finding confers that the C. violeceum OUAT_2017 susceptible to broad-spectrum chemotherapeutic bactericidal drugs that share core structure related to the substance 4-quinolone.

Of the two species of elephants on planet earth, the Asian elephant (E. maximus) is found in 13 countries across tropical south and Southeast Asia [27]. Currently, India has over 50% of global elephant population. Odisha, an Eastern state of India, is home to nearly 10% of the elephant population in India. Human development has led to fragmentation of sizable patches of natural habitats, thereby pushing elephant population towards inbreeding depression. Continuous erratic changes in climate with concomitant high environmental temperature make the habitat nonconductive for normal physiological activities of these animals. Exposure to insecticides and pesticides through modern agricultural practices, a stress linked phenomenon, is also a matter of concern. Decreased availability of fodder and water in the forest, coupled with the loss of tranquility by anthropogenic activities, has forced these animals to move to nearby human habitat thereby making them more prone to man-elephant conflict and livestock-elephant interface leading to stress in these animals. Because of global climate change and precipitation of associated risk factors including habit of regular bath in stagnated water bodies, the epidemiological pattern of this pathogen is likely to change in near future with high incidence of this fatal disease in multiple hosts.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.