Antibioresistance and Virulence Factors Spectrum in Several Bacterial Strains Isolated from Polluted Environments

Most studies concerning antibiotic resistance are performed on strains isolated from the clinical environment. This phenomenon should not be neglected in bacterial strains isolated from natural or polluted environments, because the phenomenon of virulence and antibiotic resistance occurred in these environments and can contribute to maintaining a reservoir of virulence and antibiotic resistance. This study aimed to assess the resistance factors as well as the antibiotic resistance pattern in bacterial strains isolated from environments polluted with oil products or heavy metals.The strains under study exhibited virulence factors, 85% of the strains had the ability to synthesize amylases, 65% to hydrolyze esculin and gelatin, 50% to produce lecithinases, and hemolysins, and 40% to metabolize casein. To some bacterial straina, the blaNDM and blaIMP resistance genes were identied. These microorganisms showed intermediate resistance to Carbapenem, Monobactam and Aminoglycoside antibiotic classes. Pollutants exert selective pressure on native microorganisms contributing to the development of defense mechanisms against pollution stress, these mechanisms can be used in virulence and antibiotic resistance. The study was carried out on 20 bacterial strains isolated from environments polluted with oil products or heavy metals, belonging to the Department of Microbiology of the Bucharest Institute of Biology (IBB) collection; 17 strains are classied in the Pseudomonasgenus, and the other three in Shewanella, Alcaligenis and Serratiagenera, respectively. The taxonomic classication of the strains was made in advance of the present study. The antibiotic susceptibility/resistance spectrum of the strains under study was assessed by the Kirby-Bauer disc diffusion method, following EUCAST provisions.


Introduction
The phenomenon of antibiotic resistance is a topic the real interest, being studied extensively especially in the last decades, in connection with the increasing use of antibiotics [1].
Uncontrolled use of antibiotics in human and veterinary medicine has a major impact on human health, as well as implications on the ecosystems, leading to the selection of antibiotic-resistant bacterial strains, which represent today a real problem for the effective treatment of bacterial infections [2]. Martinez, 2009, mentioned the adverse effects of antibiotics upon aquatic ecosystems, causing imbalances in the speci c microbiota and increasing the incidence of antibiotic-resistant bacteria.
Internationally, most studies concerning antibiotic resistance are performed on strains isolated from the clinical environment. The rst references to the phenomenon of antibiotic resistance in strains isolated from the external environment were made by EUCAST (European Committee for Antimicrobial Susceptibility Testing), the environmental microorganisms representing true reservoirs of resistance genes. This is why the expending of researches on antibiotic resistance and virulence of microorganisms from polluted environments becomes a must.

Biological material
The study was carried out on 20 bacterial strains isolated from environments polluted with oil products or heavy metals, belonging to the Department of Microbiology of the Bucharest Institute of Biology (IBB) collection; 17 strains are classi ed in the Pseudomonasgenus, and the other three in Shewanella, Alcaligenis and Serratiagenera, respectively. The taxonomic classi cation of the strains was made in advance of the present study.
The antibiotic susceptibility/resistance spectrum of the strains under study was assessed by the Kirby-Bauer disc diffusion method, following EUCAST provisions.
Inoculation was performed by the pour plate technique on the Müller-Hinton medium with a bacterial suspension corresponding to 0.5 McFarland turbidity standard. Antibiotic-impregnated discs (bioMérieux), standardized (according to CLSI 2018 Clinical Laboratory Standards Institute) were applied to the inoculated plates. The plates were incubated for 18-24 hours at 37°C, and the results read by measuring the diameters of the growth inhibition zones generated by different antibiotics, according to CLSI 2018 tables, with standardized critical points for the diffusimetric method.
Antibiotic resistance was also being considered when bacterial colonies visible to the naked eye appeared in the area of inhibition.
Highlighting the expression of soluble virulence factors was performed by biochemical tests, using speci c culture media, which were incubated at 37°C for 24-72h according to the methodology described by [4].
Hemolysin production was assessed by inoculating the strains in agar with 5% sheep blood. The bacterial strains that can produce hemolysins show a transparent halo.
The presence of lecithinases was estimated by the interest bacterium onto an solid medium containing egg yolk. Lecithinase synthesis results in a white opaque zone of precipitation that spreads beyond the edge of the colony.
Lipase detection was carried out by the spot method on agar supplemented with 1% Tween 80 (sorbitol monooleate). The presence of an opaque precipitation zone around the growth area, given by the formation of insoluble calcium oleate crystals (crystals formed between the released fatty acids and Ca 2+ ) was considered a positive reaction.
Assessment of amylases was performed on 1% starch supplemented agar. After the spot-seeding of the bacterial strains and incubation, the Petri dishes were ooded with Lugolol solution. The formation of a yellow ring around the inoculation sites is considered a positive reaction.
To highlight the proteases (caseinase and gelatinase), the strains were seeded on agar added with 15% casein and 3% gelatin, respectively. The strains that secrete caseinase have a precipitation zone around the culture spots due to the formation of calcium para-caseinate. Strains that can synthesize gelatinase liquefy entirely or partially the medium.
DNase presence was assessed by growing the microorganisms on agar medium with DNA and toluidine blue. After incubation, a pink halo is observed around the culture spot of positive strains, while the rest of the medium remains blue [4,5].
Hydrolysis of esculin was estimated by cultivation on esculin and iron citrate supplemented medium. The strains that have the ability to hydrolyze esculin cause blackening of the medium, due to the esculetol resulted from the hydrolysis of esculin and its combination with the iron salts present in the medium [4,5].

Highlighting of antibiotic resistance genes by PCR method
In order to perform PCR reactions, the following mixture was used: 12.5 µl Maxter Mix Promega, 20 µM forward primer − 1 µl, 20 µM reverse primer − 1 µl, DNA − 1 µl, and sterile Mili-Q water up to 25 µl / tube. The PCR reaction was performed with a Mastercycler pro S equipment (Eppendorf, Hamburg, Germany). The ampli cation conditions for the three pairs of primers were: initial denaturation at 93°C − 5 min, followed by 35 cycles of 92°C for 1min, 50°C for 50s, 72°C for 1.30 min. and a nal extension of72°C for 10 min.

Results And Discussion
The strains under study were Gram-negative, oxidase-positive, and catalase-negative. Most microbial strains were sensitive to the tested antibiotics (Table 1), and only six were resistant to one or more antibiotics, representing 30% of the tested strains. Pseudomonas spp. (M27) and Pseudomonas spp. (Ps 4) were resistant to one antibiotic and the strains Alcaligenisspp. and Pseudomonasspp (CMM2)were resistant to two antibiotics: the rst to IMP and ATM and the latter to ATM and MEM. T Pseudomonas spp. (Ps3c) and Pseudomonas aeruginosa (C16) had multiple antibiotic resistance, being resistant to seven and eight antibiotics, respectively, with a similar phenotypic pattern of resistance. The resistance level of the strains isolated from polluted environments (Fig. 1)  Analyzing the resistance level of the studied strains, an insigni cant resistance was found to EFF (4th generation In the present study, we analyzed eight virulence factors, these being more or less present with different levels of expression.

Hydrolysis of esculin
In both natural and interstitial environments, the amounts of Fe 2+ required for bacterial metabolism were low, and the microorganisms have developed speci c mechanisms for the acquisition of iron ions. Thus, the biochemical property of esculin hydrolysis (a complex heteroside) to esculetin may be a nonspeci c mechanism of iron acquisition, representing a virulence factor as well.
65% of the studied strains, could hydrolyze esculin ( Table 2,). Because they are strains isolated from polluted environments, this property can favor the development of bacteria in the external environment poor in iron ions, but it can also be a virulence factor when invading the human body.
Amylase was produced by 85% of the studied strains isolated from various polluted environments. The bacteria that have the ability to synthesize this enzyme have the advantage that they can survive in environments with limited resources ( Table 2).  Instead, caseinase was synthesized by 8 bacterial strains, representing 40% of the tested strains. This extracellular protease hydrolyzes proteins to peptides and amino acids, giving a competitive advantage to the producing strains for colonization of a particular niche and survival in different environments.
Gelatinase gives the strains the ability to invade and disseminate in the host organism. Also, this enzyme is involved in the survival and maintenance of the reservoir of microorganisms in the external environment.
Out of the 20 strains under test, 13 produced gelatinase, which means 65% of the strains can degrade gelatin.
Four bacterial strains among the tested ones synthesized a low amount of enzyme because they did not completely liquefy the whole amount of culture medium on which they grew.
Hemolysins(extracellular enzymes) were present in 50% of the strains isolated from polluted environments. These enzymes cause tissue damage, as an expression of the pathogenicity of various bacterial species.
On egg yolk containing medium, 10 strains exhibited the capacity to synthesize lecithinases, representing 50% of the tested strains.
Neither strain under test possessed the ability to synthesize DNase, this being a competitive disadvantage in the ecological niche of origin.
All tested strains showed one or more virulence factors, the strain Pseudomonas spp. (D7.2) exhibiting the most, six out of the eight tested.

Investigation of molecular markers of antibiotic resistance
In the present study, we aimed to investigate the blaVIM, blaIMP, blaNDM genes; these genes are encoders of metallo-βlactamases (carbapenemases), which contain Zn 2+ at the active site [10]. Bacterial strains that synthesize these enzymes are resistant to penicillins, cephalosporins, carbapenems, and β-lactamase inhibitors. They do not hydrolyze aztreonam and are sensitive to bivalent metal chelating agents, such as EDTA (ethylenediaminetetraacetic acid).
For the blaNDM gene, in addition to the amplicon of interest indicated in the literature with a size of 475 bp [11], 2014) secondary amplicons were obtained in most of the tested strains (Fig. 2).  [12,13], in three of the four positive strains, the amplicon was much larger, over 1000 bp, while the amplicon obtained in the case of Pseudomonas spp (Ps 4) was about 250 bp (Fig. 3).
In the case of the blaVIM gene ampli cation, the results were negative in all 20 bacterial strains isolated from polluted environments. The blaVIM gene was rarely reported in bacterial strains isolated from the external environment, being frequently found in the clinical environment [14,15] and wastewaters from hospitals [16].

Conclusions
The tested strains showed intermediate resistance to antibiotics from the Carbapenel, Monobactam, and Aminoglycoside classes; The tested bacterial strains exhibited at least one virulence factor; Similar to strains isolated from the clinical environment, the strains isolated from contaminated environments possess the blaNDM and blaIMP resistance genes; Bacterial strains from polluted environments can be a reservoir of virulence and antibiotic resistance and can contribute to the vertical and horizontal transmission of these phenomena; Declarations