Phenotypic plasticity and characterization of Chromobacterium isolates from aquatic environment

The genus Chromobacterium spp. are gram-negative bacilli, which may or may not have characteristic purple pigmentation, and are mainly isolated from soil, water and infected patients. The main representative species of this genus is C. violaceum , responsible for a high mortality rate among those infected. The aim of this study was to genetically characterize and broadly compare phenotypic characteristics between the genus Chromobacterium species described in the literatureand bacterial isolates. This study is an experimental research, in which sequencing and phenotypic tests of bacterial isolates were carried out for comparison with species of the genus Chromobacterium . Two strains were identified, CRJL01 and CRJL02 that have characteristics of Chromobacterium spp species. These isolates showed high resistance to antibiotics, tolerance and resistance to metals, biochemical and physiological versatility of the CRJL01 and CRJL02 strains. In the sequencing of the 16S rRNA gene, the CRJL01 strain showed similarity with the C. piscinae strain. The CRJL02 strain showed similarity with the C. subtsugae strain. This work is the first report in 40 years of Chromobacterium spp. in the Brazilian Midwest – Goiás, in water samples. This isolated genus has a wide applicability for the pharmaceutical, food and cosmetic industries, due to the production of its purple/violet pigment known as violacein, and its bioprospecting is of great importance. Thus, this study is a kick-off for exploring your produced pigment.


Introduction
The first species of the genus Chromobacterium isolated, was identified in 1880, diagnosed as Chromobacterium violaceum Bergonzini (1881) (Euzeby, 1998). According to the List of prokaryotic names (LPSN) the genus Chromobacterium belongs to the Neisseriaceae family, class Betaproteobacteria and has 14 species, being them C.
Bacteria characterized with violet pigments of the Chromobacterium genus include opportunistic pathogens that can cause septicemia, highly resistant to antibiotics and with high rates of fatal infections in humans and animals, being considered an emerging pathogen (Chandler, 2019;Durán & Menck, 2001;Kothari et al., 2017;Yang & Li, 2011). The species described in the literature causing infections is C. violaceum, in which acquisition and/or maintenance of the pathogenicity island containing the type III secretion system (T3SS) provides the characteristic of virulence for this bacterium, thus changing from an environmental microorganism for an opportunist (Batista & Neto, 2017).
Infection by C. violaceum is associated with healthy and/or young people, and may have some predisposing factors such as: trauma, exposure to water or soil, or both (Yang & Li, 2011). Infections are difficult to treat, with cases of necrotizing metastatic lesions, abscesses and rapid progression to sepsis, the most common symptoms being fever and pain in the infected area (Kothari et al., 2017;Yang & Li, 2011). Reports associate C. violaceum with chronic granulomatous disease, indicating that this bacterium may be an indicator for this pathology (Justo & Durán, 2017;Meher-Homji et al., 2017).
In view of the biotechnological applications with industrial and medicinal potential of the pigment produced by the species of Chromobacterium spp. and its great importance due to its pathogenicity (Santos et al., 2018) and also the growing interest in bioprospecting new pigment-producing strains. This study aimed to characterize and identify two isolated strains suggestive of the genus Chromobacteium as they are purple pigment-producing strains in water samples from Ribeirão João Leite, Goiás, Brazil.

Isolation
During a characterization study of fertilizing bacteria from the João Leite stream, Goiás-Brazil, bacteria were bioprospected in MacConkey and R2A media present in the water. Among the syndicates, two colonies with violet/violet morphology were observed and examined for this study.
The presence of colonies with the production of purple/violet color pigments was isolated, which belonged to points A (16°34'30.54"S; 49°13'55.02"O) e B (16°28'25.05"S; 49°6'43.87"O) described in the map of Figure 1, from which the CRJL01 and CRJL02 strains were respectively isolated. A. Map of Brazil, highlighting the state of Goiás. B. Map of the state of Goiás, highlighting the basin in which Ribeirão João Leite is located, in the center of the state. C. Map of the cities and basin in which the Ribeirão João Leite and its tributaries pass, highlighting the course of the river and points A and B where colonies were isolated with the production of rock-colored pigments. Source: own author.

Chromosomal DNA extraction, sequencing and phylogenetic analysis
For the molecular identification of bacterial isolates, amplification of the 16S rRNA gene, sequencing and analysis of this sequence were used. For this, genomic DNA extraction was performed, following the methodology proposed by Soolingen et al. (1994), with the adaptations suggested by Oliveira et al. (2012).
The sequences obtained were analyzed according to their quality and later joined in contig, using the CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA). The homology of the contig was compared with the GenBank National Center for Biotechnology Information (NCBI) database using the nucleotide tool Basic Local Alignment Search Tool (BLASTn). The hits that presented the highest identities were listed as possible identifications of bacterial isolates.
The sequences obtained were deposited at Genbank NCBI.
The analysis of species diversity was performed through the construction of the phylogenetic tree using the Neighborjoining method in the MEGA X software.

Morphological, phenotypic and biochemical properties of cultures
To carry out the morphological and physiological characterization of the isolated bacterial cultures, the manual of the National Health Surveillance Agency was followed (Brasil, 2013). They were compared with other species found of the same bacterial genus identified. The comparison was made by searching the species of this genus described in the LPSN (List of Prokaryotic names with Standing in Nomenclaturehttps://www.bacterio.net/) together with the search for the valid publication of this species, in which all the morphological, phenotypic and biochemical information of these species, with this search being carried out in September 2021.
To test the susceptibility of these strains to antimicrobials, it was performed and interpreted using the agar fusion disk µg). Resistance status was assessed using the classification system that resistance to ≥3 classes of antibiotics defining multiresistance status (MDR) (Magiorakos et al., 2012).

Results and Discussion
The 16S rRNA sequences have been deposited with the NCBI under accession number MN134084.1 for CRJL01 and accession number MN134085 for CRJL02. When compared to the database, the CRJL01 strain had the highest identity Research, Society and Development, v. 11, n. 15, e28111536821, 2022 (CC BY 4. The multiple alignment was performed based on the sequences obtained from the 16S rRNA and the phylogenetic tree was constructed (Figure 2). The bootstrap values (rooting) 100 homology of strain CRJL01 with C. Piscinae strain LMG 3947 is 49% homology between strain CRJL02 and C. subtsugae, lineage PRAA4-1. The sequences used are available in the NCBI database and the CRJL01 and CRJL02 strains were sequenced by Sanger and then contigs were formed using the clustal W. The statistical method used was the Neighbor-joining, the phylogenetic test based on Booktstrap method with 1000 replications and the Jukes-Cantor replacement model were executed using the software package MEGA_X_10.0.5_win64_setup. Source: Own author. , indicates that in its isolates of Chromobacterium spp. despite having high similarity above 97% between 16S rRNA sequences with those of C. Piscinae, the strains showed high genetic and phenotypic diversity, which they attributed to adaptations to the habitat (ecological barriers), suggesting the origin of new species or representing bacterial ecotypes. Thus, the data presented here indicate that the CRJL01 and CRJL02 strains are of the Chromobacterium genus. It can suggest that CRJL01 is a C. piscinae and CRJL02 is a C. subtsugae. However, when deposited at the NCBI, CRJL02 was described as Chromobacterium sp., as the phylogenetic analysis did not give a high value (49) to confirm the species of this strain.
A comparison was made between CRJL01 and CRJL02 strains and other species of Chromobacterium spp. previously described (14 species described in the LPSN). The location and characteristics of the bacterial colony were described, the data are shown in Table 1. It can be seen that most strains were isolated from environmental samples, water and soil. A comparison of the biochemical and physiological characteristics of the CRJL01 and CRJL02 strains, described in Table 2, was also performed. The data were also associated with the described characterizations of the other species of Chromobacterium spp.
In the Brazilian Midwest (Goiás), the first report of isolation of Chromobacterium spp. was described by Reis et al. (1972), isolated from the waters of the Ribeirao Dois Irmãos in Goiânia, Goiás, Brazil. In subsequent years, Da Freitas et al. (1974), isolated in water samples from three municipalities in the state of Goiás (Hidrolândia, Ipameri and Cavalcante).
Subsequently, there was a last report of Rodrigues (1979), in water from slaughterhouses in the municipalities of Catalão and Luziânia, assigning a new species, called C. goianiensis. This described species obtained in its biochemical tests growth in macConkey médium agar, hemolysis, did not produce hydrogen sulfide (H2S), fermented only glucose, catalase, urease and oxidase positive and conversion of tryptophan to indole, production of phenylalanine deaminase and negative citrate assimilation, differing from CRJL01 and CRJL02 isolates ( In the study of Freitas et al. (2019), which isolated a strain of the genus Chromobacterium spp. in an Amazon lake, in Brazil, indicated that this strain has resistance to carbapenems. Another study of Gudeta et al. (2016), indicated that the genus Chromobacterium spp. have high identity and were phylogenetically related to KPC (class A carbapenemase) suggesting that this bacterial genus may have played a role in the evolution of KPC.
Reports of Chromobacterium spp. resistant must be careful. Although the infection is rare in humans, its evolution is rapid, causing abscess in vital organs, with high mortality due to high resistance to antibiotics. The genera related to infections mentioned in the literature are C. violaceum (Alves De Brito et al., 2004;Anuradha et al., 2018;Madi et al., 2015;Martinez et al., 2000;Zala et al., 2018) and C. haemolyticum (Han et al., 2008;Miki & Okada, 2014;Okada et al., 2013). However, there is no study of other species of Chromobacterium described, they have pathogenicity. Plus its high resistance in the environment can serve as a reservoir and dissemination of antibiotic resistance genes (Gudeta et al., 2016). When performing the MIC test for metals, it was observed that barium, cobalt, iron, mercury and zinc, in high concentrations, partially or totally inhibited the production of purple pigment of the CRJL01 and CRJL02 strains. New tests must be carried out to verify if there is any relationship between metal concentrations and pigment production. It is known that the production of violacein is controlled by the quorum sensing (de Oca-Mejía et al., 2014) and a study suggests that cadmium disrupts quorum-sensing-related proteins (Newaj-Fyzul et al., 2008;Thornhill et al., 2017). Suggesting that stress caused by metals may inhibit violacein production. However, for proof, additional studies must be carried out.
The strain C. pseudoviolaceum GCC-SO4, showed tolerance to metals cadmium, lead, iron and copper and resistance to antibiotics methicillin and penicillin (Nath et al., 2019). Under alkaline conditions, cyanogenic bacteria, including C.
violaceum, had the ability to leach the metals copper, iron, silver, gold and zinc; exhibiting maximum bioliability compared to the other bacteria tested in the study (Pradhan & Kumar, 2012). C. violaceum is one of the most cited and studied bacterial species with potential for metal bioremediation (de Alencar et al., 2017). In this way, the resistance and tolerance to the metals tested here, the biotechnological potential for metal degradation of the CRJL01 and CRJL02 strains can be explored.

Conclusion
Considering the data presented here, this study is an indication that Chromobacterium spp. are circulating in the aquatic environment of central western Brazil -Goiás. Being the first report of Chromobacterium spp. in the last 40 years, in water samples, showing MDR to the tested antimicrobials, also a high resistance and tolerance to metals and a great phenotypic and biochemical diversity. The report of this study is of paramount importance, given the great potential for infection and mortality that this bacterial genus can induce.
The sample isolation site is a river, which serves to supply the population of the state of Goiás and is also used in general for recreation and leisure activities, for water collection by industries and agriculture. Thus, the population has had direct contact with this isolation habitat of the CRJL01 and CRJL02 strains.
Another important point is the biotechnological potential that the strains have and studies are being carried out on the production of pigment and the use of the strain for bioremediation of metals. In-depth studies should be taken into consideration for future work on the circulation of bacterial genus in the state of Goiás and its potential in bioremediation and application of the pigment violacein produced, which has great application in the pharmaceutical, cosmetic and food industries.
Our study group is investigating the biotechnological potential of these strains, and future studies should be published soon.