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The Toxic Effect of Heavy Metal on B.intermedia and B. rhizospherae and Its Relation with the Seasonal Environmental Factors

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28 December 2023

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29 December 2023

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Abstract
This investigation aimed to knowledge of the presence of Brucella spp .in the samples from Babylon province and the relationship with environmental factors. The seasonal collection of animal soil samples for various villages in the middle, south, and north of Babylon province, Hilla City, Iraq, with physicochemical parameters (air and soil temperature, pH, EC, TDS, salinity, TOC), as well as heavy metals (Fe, Cu, Cd, and Pb). The results showed the presence of Brucella species in some of soil samples and its percentage varies according to the four seasons, where the highest percentage of Brucella spp. was in summer season and the lowest in winter season and these percentages were related to seasonal environmental indicators. After sending the positive bacterial samples isolated from the soil for sequencing analysis, it was found that the bacterial samples isolated from the soil have new strains recorded in Genbank as following: five strains for Brucella intermedia and one strain for Brucella rhizosphaerae, Also, phylogenetic analysis was for the drawing of phylogenetic tree and the determination of phylogenetic relationship for Knowledge the degree of convergence between species and their source. Through our research, we concluded that proportion of Brucella spp. presence in summer was high and also environmental factors which included (Air and Soil temperature, pH, EC, TDS and Salinity) were high, except TOC was lower in summer season whereas heavy metals for Cu, Fe, Cd were lower in summer except Pb was high. This indicated that differences of seasons affect to Brucella ratio in soil. The findings of sequencing for the bacterial samples from soil demonstrated that B. intermedia was found in both the spring and fall seasons while B.rhizosphaerae found in the spring season , These types of Brucella had genetic variations. Also concluded that during phylogenetic analysis of the 16S rRNA sequences indicated that B.intermedia was nearest to Pakistan and other strain of B.intermedia was nearest to Russia while B.rhizosphaerae was nearest to Ukraine and Pakistan.
Keywords: 
Subject: Biology and Life Sciences  -   Animal Science, Veterinary Science and Zoology

Introduction

Brucella belongs to the Brucellaceae family, the orders Rhizobiales, Daeguia, Crabtreella, Mycoplana, Pseudochrobactrum, and Paenochrobactrum are all members of the Alpha proteobacteria class1. It is heat sensitive and can survive for several weeks in water. The survival of Brucella abortus in the environment is influenced by sunlight and temperature2. Bacterial development and diversity are linked to organic matter; thus, microbial numbers are greatest at the soil's surface (10 cm) and decrease with depth3. Identify different ecotypes and the potential for new strains, giving them insight into the environmental maintenance and transmission of emerging and re-emerging disease risks in Iraq. The presence of Brucella species in soil is one of the risk factors for the disease's spread to animals and humans4. Brucellosis is a zoonotic infection that can affect both animals and humans and it is still prevalent in the United States5. Brucella species are pathogenic bacteria that adapt to new hosts and are naturally transmitted to their primary hosts by direct or indirect contact, as well as to other vulnerable hosts unwittingly6. The use of cows, buffaloes, sheep, and goats in mixed farming has increased the risk of brucellosis with small ruminants acting as primary hosts and cattle acting as an overflow host for B. melitensis7. The current study investigates the association between soil risk variables (physical and chemical) and the occurrence of Brucella species. the epidemiological relevance of brucellosis in terms of risk of transmission to humans and cattle8. Seiler & Berendonk, (2012)9 showed that the risk of metal resistance in the environment was assessed based on heavy metal concentrations, Analyses of the data indicate that agricultural and aquacultural practices represent major sources of soil and water contamination with moderately to highly toxic metals such as mercury (Hg), cadmium (Cd), copper (Cu), and zinc (Zn). If those metals reach the environment and accumulate to critical concentrations, they can trigger co-selection of antibiotic resistance. Li et al.,(2013)10 found that incidence of brucellosis were strongly associated with lower temperatures and less sunshine in the winter and spring, climatic factors likely influence the ecology of brucellosis both directly and indirectly by affecting several parameters, including the growth and reproduction dynamics of domestic animals, interactions between sheep, goats and humans, pathogen replication, and population immunity11. Ahmed et al., (2017)12 discovered DNA of Brucella species in soil samples (n=1280) from nine districts in Punjab.

Material and methods

The Soil Samples Collection

The animals soil samples collected seasonally from February to November, 2021 were taken the samples from the upper layer for bacteriological study and from the deepest layer (25-30 cm in depth) for environmental study, dried by air and sieved (to ~ 2 mm particle size) during the collecting the samples measured air temperature and soil temperature and determination the sites by GPS.
Total Organic Carbon is the wet oxidation technique was utilized exothermic heating and oxidation of organic carbon of the sample with potassium dichromate by the titration13, Heavy Metals include soils samples for heavy metals determination were digested according to the procedure described Sharidah (1999)14. One gram of dried soil samples were digested and determined with by Flame Atomic absorption - spectrophotometer (Type Aa 7000),Shimdzu /Japan was used to determine the concentrations (mg/L) of the elements15.
Preparation of Culture Media:
Culture media used in this study were prepared according to the manufacturer’s instructions:
Brucella Agar Base
It was prepared according to Himedia manufacturing company (India) to Isolation and detection of Brucella spp: suspend(21.55gm) in (500ml) D.W,heating to boiling to dissolve the medium completely. sterilize by autoclaving at (150C) Ibs pressure (1210C) for 15 minutes, Cool to (45-500C) and aseptically add sterile (5%) v\v inactivated horse serum (RM 1239, inactivated by heating at (560C) for (30 minutes) and then add the antibiotics (Polymxin B sulphate, Vancomycin, Bacitracin, Nystatin, Nalidixic acid, Cycloheximide) as supplement.
Brain Heart Infusion Broth (BHI)
This media were prepared according to the manufacturer’s instruction. It was used for preservation of bacterial isolates as stock for long time16.
Brucella isolation from soil samples
After samples collection in sterile bags from sites taken (1gm) from soil's sample and put in plain tube completed the size to (5ml) by normal saline then mixed by vortex and leave to settle after that incubation for (15) minute in Incubator. Two hundred (200 μl) were taken from mixing by micropipette and published in petridishs where all samples subcultured in selective Brucella agar base media with Polymyxin B (2,500IU), Vancomycin(10.0mg), Bacitracin (12,500IU), Cycloheximide(50.0mg), Nystatin(50,000 IU) and Nalidixic acid (2.5mg) and then5% of inactivated horse serum. Bacterial cultures were incubated for 14 days at (37°C) and 10% carbon dioxide until appearance of growth17,18,19.

Molecular diagnostic methods

Methods are also currently being used for the detection of Brucella spp. in various samples20.
Bacterial DNA Extraction:
Genomic DNA extracted from bacterial isolates cultured from the soil according to the manufacturer's protocol FavorPrepTM Blood/ Cultured Cells Genomic DNA Extraction Mini Kit.
Conventional PCR for Bacteria Extracted from Soil
Conventional PCR were used to amplify the target bacterial DNA using specific primer pairs. It includes three consecutive steps that repeated for specific number of cycles to get PCR product which can be finally visualized after agarose gel electrophoresis. The primer sequence, PCR product size and thermal cycling conditions mentioned in (table 1-1) and (table 1-2).
Table 1-1. The Sequence of Primers.
Table 1-1. The Sequence of Primers.
Primer Sequence Primer sequence Tm
(ᵒC) 		GC% Size of Product (bp)
16s rRNA
bacterial primers 		27F
 5'- AGAGTTTGATCCTGGCTCAG- 3' 54.3 50.0 1250
Srinivasan et al., (2015)21
1392R 5'- GGTTACCTTGTTACGACTT- 3’ 49.4 42.1
Table 1-2. The Optimum Conditions for Detection the Bacterial Isolates from Soil (Stages and Temperature of PCR for 16s rRNA gene).
Table 1-2. The Optimum Conditions for Detection the Bacterial Isolates from Soil (Stages and Temperature of PCR for 16s rRNA gene).
Temperature 0C Time cycle
Stage 1 Initial Denaturation 95ºC 5 min 1
Stage 2 Denaturation 95ºC 45 sec
annealing 56ºC 45 sec 35
Extension 72ºC 1 min
Stage 3 Final Extension 72ºC 5 min 1

16S rRNA Sequence Analysis and Phylogenetic Tree

Sequencing method was performed for study of genetic changes and phylogenetic tree draw of 16SrRNA gene in some local Brucella isolates by comparing with NCBI-GenBank Brucella isolates. The sequencing of the 16SrRNA gene were done after assurance in presence amplification of PCR products for required volume; These products were sent to company (Macrogen) in Korea for performing Sanger sequence, after getting on nitrogenous bases sequence for 16S rRNA gene amplified products of Brucella isolates. This sequence analyzed by NCBI-blast programme for purpose compared homology or diversity degree to local Brucella isolates with the world isolates recorded in NCBI-GenBank. Also phylogenetic analysis for draw of phylogenetic tree and determination phylogenetic relationship used (MEGA 6) programme to compare local one strain with strains all of the world states.
Recording of Iraqi Brucella isolates in gene bank –NCBI
Sequences of Brucella isolates were isolated from animals soils in Al-Hilla city\Iraq and each sequences have variations.

Results

Soil Samples
The current study results showed that Brucella spp. was found in all seasons,
Genetic Detection
Conventional PCR for Bacterial Isolates and 16SrRNA Sequencing
After the culture for both samples from the soil on Brucella agar base media and DNA extraction from bacterial isolates and electrophoresis of DNA as in Figure (1-1) after that, identified the positive samples by conventional PCR to knowledge Brucella sp., as in Figure (1-2).
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Figure (1-1). Gel electrophoresis for Extracted DNA from soil bacterial isolates, (Agarose 1%, at 70 volts, 60 min). Visualized after staining with ethidium bromide stain.
Figure (1-1). Gel electrophoresis for Extracted DNA from soil bacterial isolates, (Agarose 1%, at 70 volts, 60 min). Visualized after staining with ethidium bromide stain.
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Figure (1-2). Agarose gel electrophoresis (1%) for 16s-rRNA bacterial primer-bacteria isolated from the soil samples (1250 bp), Primer Ta at (560C ), at (65Amp ,70 volts, 60min). It was visualized under U.V light after staining with Eco Safe dye , Lane M 100 bp DNA Ladder.
Figure (1-2). Agarose gel electrophoresis (1%) for 16s-rRNA bacterial primer-bacteria isolated from the soil samples (1250 bp), Primer Ta at (560C ), at (65Amp ,70 volts, 60min). It was visualized under U.V light after staining with Eco Safe dye , Lane M 100 bp DNA Ladder.
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After Sequencing analysis for both bacteria isolated from soil and blood samples found that Brucella isolates from soil samples for the four seasons and sented for sequencing was 6 new strains recorded in GenBank mentioned in table (1-3). These samples divided to (5) new strains for Brucella intermedia in Summer and in Autumn, one species of Brucella rhizosphaerae in Spring.
Table 1-3. Brucella isolates recorded in GenBank for Brucella spp. isolated from soil.
Table 1-3. Brucella isolates recorded in GenBank for Brucella spp. isolated from soil.
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The results of 16SrRNA sequencing revealed the presence variation for Brucella isolates from soil established (6 new strains) for two species, indicating the presence of genetic diversity among the isolates.
Table (1-4). The relationship between B.intermedia and environmental factors, Mean ±S.E. p≤0.05.
Table (1-4). The relationship between B.intermedia and environmental factors, Mean ±S.E. p≤0.05.
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This table (1-4) showed that all isolates have significant differences (p<0.05) between genotype and environmental factors that included physical and chemical parameters (air and soil temperature, pH, EC, TDS, salinity, TOC) and heavy metals (Cu, Fe, Cd, Pb) according to statistical analysis.
Table (1-5). The relationship between B.rhizosphaerae and environmental factors, Mean ±S.E. p≤0.05.
Table (1-5). The relationship between B.rhizosphaerae and environmental factors, Mean ±S.E. p≤0.05.
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While Brucella rhizosphaerae mentioned in tables (1-5) do not exhibit significant differences (p>0.05) between genotype and environmental parameters, this suggests that genetic diversity is present but has no impact on the makeup of the bacterium. B.rhizosphaerae founded one in the spring season.

Phylogenetic Tree Draw

Sanger sequencing was successful for soil samples with an expected PCR product size (1250 bp) by 16S rRNA bacterial primers. The sequences were identified as belonging to Brucella spp. following similarity searched by blast (sequence identity of 99%). 16S rRNA sequencing method was performed for study of genetic changes and phylogenetic tree draw of 16SrRNA gene in some local Brucella species isolates by compared with NCBI-GenBank Brucella species. The sequences were deposited in GenBank with accession numbers as mentioned in table (1-6).
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Figure 1-3. Evolutionary analysis (phylogenetic tree of Brucella isolates isolated from soil)) of 16S rRNA gene sequences at compared of B.intermedia with different states. This figure(1-3) found that B.intermedia was nearest to Pakistan.
Figure 1-3. Evolutionary analysis (phylogenetic tree of Brucella isolates isolated from soil)) of 16S rRNA gene sequences at compared of B.intermedia with different states. This figure(1-3) found that B.intermedia was nearest to Pakistan.
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Figure (1-4). Evolutionary analysis (phylogenetic tree of Brucella isolates isolated from soil) of 16S rRNA gene sequences of B.intermedi and B.rhizosphaerae at compared with different state. This figure(1-4) found that B.intermedia was nearest to Russia while B.rhizosphaerae was nearest to Ukraine and Pakistan.
Figure (1-4). Evolutionary analysis (phylogenetic tree of Brucella isolates isolated from soil) of 16S rRNA gene sequences of B.intermedi and B.rhizosphaerae at compared with different state. This figure(1-4) found that B.intermedia was nearest to Russia while B.rhizosphaerae was nearest to Ukraine and Pakistan.
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The Relationship between the Environmental Factors and Brucella species

Our results proved existence relation between the presence of Brucella and the physical, chemical properties.
Table 1-6. The Relationship between the Four Seasons and the Physical, Chemical Parameters and the Percentage of Brucella species (Mean ± S.D). p≤0.05.
Table 1-6. The Relationship between the Four Seasons and the Physical, Chemical Parameters and the Percentage of Brucella species (Mean ± S.D). p≤0.05.
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This table(1-6) noted that proportion of Brucella presence in summer is higher that it is in relation with environmental parameters which include (Air and Soil temperature, pH, EC, TDS and Salinity) that are high, except TOC is low while proportion of Brucella presence in winter is lower, also relate with environmental parameters include (Air and Soil temperature, pH, EC, TDS and Salinity), these the parameters (the physical and chemical) is low except TOC which is high. This indicated that variation of seasons affect to Brucella ratio in soil.

Relationship of the Heavy Metals with Brucella Species.

Our results confirmed existence relation between the presence of Brucella spp. and the heavy metals, as mentioned in table (1-7).
Table 1. The Relationship between the Four Seasons with Brucella species (%) and the Concentrations of Heavy Metals (Mean±S.D). p≤0.05.
Table 1. The Relationship between the Four Seasons with Brucella species (%) and the Concentrations of Heavy Metals (Mean±S.D). p≤0.05.
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In the current reseach, it was observed that heavy metals affect the percentage of Brucella presence in soil, The high amounts of Cu, Fe, Cd are in winter, where the percentage of Brucella is low, except for lead (Pb), which is low in the presence of little Brucella. while low amounts of copper, iron, cadmium be in the proportion of the presence of high Brucella except for lead (Pb), which is high in the presence high Brucella.

Discussion

A microbiological study is necessary for detecting cases of Brucella spp. because the symptoms are varied and non-specific, as shown by Bonaventura et al.,(202122. Typical zoonotic illnesses Brucella spp. was the source of the disease brucellosis, which continues to pose a serious health risk in many underdeveloped nations worldwide23. The 16S rRNA gene was the target of the PCR technique which was used to identify the bacteria in the samples24. Srinivasan et al., (2015)25 who utilized 10 ng of genomic DNA extracted from each strain to amplify this gene. Sanger sequences have been created. Isolated 16S rRNA sequences with a genus and species name (Isolated named- strains 16S aligned. fasta) were obtained from the Greengenes database26. PCR was used to diagnose the risk of brucellosis in people who were exposed to animals at work. It is a more accurate for finding Brucella spp. To tackle this potentially hazardous zoonotic disease in Pakistan, especially where brucellosis is common in animals, there is an urgent need for more accurate and focused diagnostic tools like PCR27, as mentioned in figures (1-1),(1-2). The 16S rRNA gene was the target of the PCR technique which was used to identify the bacteria in the samples28. A final diagnosis of the condition is made using either serological or cultural methods, or both29. As a result, PCR-based approaches have improved in accuracy, sensitivity, speed, and ability to work with DNA rather than highly contagious live cultures as a result of their increased using for detecting and identifying Brucella species30. In particular, for slow-growing bacteria like Brucella, Rahman et al., (2014)31 found that the PCR assays were efficient for speedy and precise diagnoses. It was shown by Manivannan et al., (2021)32 to be a sensitive and specific approach for identifying Brucella spp.
In table (1-4) showed that all isolates of B.intermedia has significant differences (p<0.05) between genotype and environmental factors, This indicates presence of genetic variation in them. This implies the presence of genetic variation in them and suggests that these bacteria changed as a result of polymorphism caused by environmental indicators and heavy metals. This caused differences between them, allowing them to adapt to and live in harsh environments. In addition, to survive in this soil, its resistance to the poisonous heavy metals may be changed into new strains. The psychological differences amongst microorganisms are caused by genetic changes. while table (1-5) mentioned that B.rhizosphaerae, does not exhibit significant differences (p>0.05) between genotype and environmental parameters, This may be because B. rhizosphaerae is related to roots, B. rhizosphaerae is a gram-negative, oxidase-positive bacterium discovered in the rhizosphere of a potato in Austria33.
There was many ways to become infected by the dangerous bacteria known as Brucella. Long-lasting resistance was possible in both inside and outside of mammalian hosts, even in harsh circumstances. It can linger in food for up to 15 months under adverse conditions such as acidity and temperatures ranging from 11 to 14 °C or for a few days if kept below 37 °C for up to two months in the winter and for only a few hours if exposed to direct sunshine, Brucella can also survive in contaminated manure and aborted infected feti34. B. intermedia is a bacterium in the genus Brucella35. Velasco et al., (1998)36 described it initially. Only one case of cholangitis following liver transplantation has been reported in humans37.
Exposure to Heavy metal persists and is rising in many regions of the world the fact that heavy metals were known to have several negative health impacts and that these effects could endure for a very long time. For ecological, evolutionary, nutritional and environmental reasons, heavy metals were significant environmental contaminants and their toxicity is a problem that is becoming more and more important38,39. The oxidative degradation of biological macromolecules were mostly caused by heavy metal binding to DNA where it displaced original metals from their native binding sites and caused malfunction to cells and finally became toxic40.
The phylogenetic tree of Ochrobactrum spp. already shows the presence of the A44T-related strains in a separate branch41. Ramette et al., (2011)42 based on the concatenated sequences of 16S rRNA and genes to acquire higher phylogenetic resolution within this group, which reveals that O. rhizosphaerae PR17T is the closest relative of A44T. The strain Ochrobactrum sp. was identified in the rhizosphere of a field-grown potato in Gelderland, the Netherlands. According to phylogenetic analysis based solely on the 16S rRNA gene as in figures (1-3),(1-4).

Conclusions

  • In this research, it was noted that proportion of Brucella spp. presence in summer was high and also environmental parameters.
  • Brucella spp. survived in local soils due to environmental conditions that being variations in Brucella species, which led to a change in their properties and make it resistance. Recording new nucleotides sequencing in soil samples and presence of new isolates of Brucella in local soils samples. Registered new strains in soil were two new species of Brucella (B. rhizosphaerae, and B. intermedia).
  • Phylogenetic analysis of the 16S rRNA sequences noted that B.intermedia was nearest to Pakistan, and the other B.intermedia was nearest to Russia, while B.rhizosphaerae was nearest to Ukraine and Pakistan in animals soils samples, these stated consider one of the sources for the infection.

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