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Characterization of Aeromonas hydrophila type strains isolates from industrial wastewater Hilla -

Iraq

Suad Ghali Kadhim ALahmed1, Anwar Kadhim AL-saffar2

Abstract-Twenty one strains of motile Aeromonas species were isolated from Industrial wastewater Al- Furat Company Hilla- Iraq. The Study aimed to evaluate the possible ecological effect of the industrial waste water released from Company . Samples were monthly taken started from may 2013 to October 2013 . The results showed that the natural properties of waste water were variable due to the nature of the materials dumping in the derange stream . It was found the pH ranged from (1.5 - 5.2 ) , conductivity reached to 3100 Ms / cm while the higher concentration of COD, TDS , Cl , SO4 , Ca and Mg were 140 , 1953 , 335 , 738, 200 and 104 mg/l respectively

.A. hydrophila suspected isolale was screening by traditionally tests and then confirmed by Vitek 2 system and PCR technique (16S r RNA gene).

In this study A. hydrophil strains are highly resistance to several antibiotics that was considered as pathogen for human being causing several

disease.

Keywords- Aeromonas hydrophila, W astewater, Al-Furat Company.

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1 INTRODUCTION

ndustrial wastewater is one of the important pollution sources in the pollution of the water environment .During the last century a huge amount of industrial wastewater was discharged into rivers, lakes and coastal areas . this resulted in serious pollution problems in the water environment and caused negative effects to eco-system and humans life.[1,2 ]
The chemical industry is critical for the economic development of any country chemicals are a signify cant contributor to national economies across the life cycle - from extraction to disposal - is essential al to avoid signify cant risks to human health and the environment . [3,4]
Industrial Wastewater leads to septic conditions in the environment and consequently leads to the deterioration of surface and groundwater quality and pollutes the soil. wastewater is rich with nitrogen and phosphorus (N, P) and leads to the phenomena of Eutrophication. Industrial wastewater is rich with organic matter which consumes oxygen in aquatic environment . wastewater may contains toxic gases and volatile organic matter.[ 5,6].
————————————————
• Suad Ghali Kadhim ALahmed, lecturer, MSc
.Microbiology, College of Sciences, Babylon University,
Babylon, Iraq
Suad_chaly2000@yahoo.com.
• Anwar Kadhim AL-saffar2, Assist.Prof.,, MSc
.Microbiology, College of Sciences, Babylon University,
Babylon, Iraq
Anwaralsafar78@gmail.com

the chemicals industry has made good progress reducing its overall environmental footprint, chemicals can also create a negative impact on human health and the environment when their production and use are not managed responsibly. Although the impacts are complex and often unknown or sometimes open to debate, some negative effects are well documented, such as chemicals found in the environment that are persistent, bioaccumulative and/or toxic (e.g. PCBs, dioxins). Most recently, concern has been expressed about chemicals which interfere with the normal function of hormonal systems of humans and animals (i.e. endocrine disrupters), and substances which impact on children’s health . [7,8]

Suad_chaly2000@yahoo.comhe chemicals industry is very diverse, comprising basic or commodity chemicals;
speciality chemicals derived from basic chemicals (adhesives and sealants, catalysts, coatings, electronic chemicals, plastic additives, etc.); products derived from life sciences (pharmaceuticals, pesticides and products of modern biotechnology); and consumer care products (soap, detergents, bleaches, hair and skin care products, fragrances, etc.). The global chemicals industry today produces tens of thousands of substances (some in volumes of millions of metric tonnes, but most of them in quantities of less than 1000 tonnes per year. [ 9, 10] .
The term pollutant is a broad term that refers to a wide range of compounds, from a superabundance of nutrients giving rise to enrichment of ecosystems to toxic compounds that may be carcinogenic, mutagenic, or teratogenic. Chemicals - with units on all the major organic and inorganic building

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blocks such as ethane, sulfuric ,hydrolytic acid . Effects on ecosystem resources can include contaminate on of air, water, and soil, as well as adverse effects on wildlife. Human health effects can include both acute and chronic diseases and disorders. [ 11].
Recently, research for new and innovative technologies has
centered on the biological treatment methods [12].
Bioremediation is the use of microorganisms to break
down toxic and hazardous compounds in the environment
[13]. It generally utilizes microbes (bacteria, fungi, yeast,
and algae), although higher plants are used in some applications.
Microorganisms have the ability to accommodate a variety of pollutants, both organic and inorganic .. Bioremediation has already proven itself to be a cost-effective and
beneficial addition to chemical and physical methods of managing wastes and environmental pollutants. New bioremediation approaches are emerging based on advances in molecular biology and process engineering. Recently developed rapid-screening assays can identify organisms capable of degrading specific wastes and new gene-probe methods can ascertain their abundance at specific sites. [14]. Bacterial survival in stressful environments is an intriguing biological problem with applications toward understanding pathogenic and environmentally important micro-organisms. [15 , 16] . Aeromonas hydrophila, a gastrointestinal pathogen of humans, was shown to exhibit a significant adaptive acid tolerance response (ATR) capable of protecting cells from severe acid at a pH of 3.5.
Adaptation required protein Synthesis since treatment
with chloramphenicol during adaptation to pH 5-0 prevented the development of acid tolerance. The adaptation to acid environment was found to be a non- transient phenomenon. Also, iron was not required for acid adaptation in A. hydrophila to adapt and survive in acid environments by producing ‘protective proteins. The adaptation and survival of this pathogen in low pH may provide valuable information about its ability to withstand acid environments in nature and in the human gastrointestinal tract [17] .
the genus Aeromonas are good examples of such bacteria. Regardless of the ubiquity of aeromonads in aquatic environments and the possibility to develop antimicrobial resistance, the patterns of resistance of Aeromonas genus present in wastewater are not fully documented in scientific literature. In this study, the ability of chitosan-immobilized Acinetobacter haemolyticus as biosorbent for chromium (VI) biosorption in batch system was investigate. Optimized parameter namely pH, contact time, biosorbent dosage and initial metal concentration obtained from the experiment was then applied for electroplating wastewater treatment. [18]
Chemicals are an integral part of daily life in today’s
world. There is hardly any industry where chemicals are
not used and there is no single economic sector where
chemicals do not play an important role.The natural and the zymogene microorganisms(which come in waters by pollution) constitute a diverse microbiota adapted to the different physical - chemical wastewater conditions, being very important for biodegradation [19 ].

2. MATERIALS AND METHODS

- study of physical - chemical wastewater:
The present study carried out from May 2013 to October
2013 pH , electric conductivity (by EC meter type
HANNA) , salinity (calculated from EC value ) chemical oxygen demand were measured at the field according to standard method [20] Total hardness, T.D.S , calcium, magnesium, and alkalinity were determined according[
21 ].
- Bacterial isolates:
Aeromonas hydrophila, A. caviae and A. sobria were
isolate from industrial wastewater of the Al-Furat
Company from Hilla. Bacterial isolates were identified to
level of species and subspecies by using the
morphological and traditional biochemical tests
according to standard methods described by [22,23 ]. Vitek System Identification
The Vitek 2 system assay has been used to confirm identification of all bacterial isolates . This system performed according to the manufacturer's instructions
(Biomerieux Company, France).
- Antimicrobial susceptibility testing
Antimicrobial susceptibility testing of A. hydrophila was
carried out against different antibiotics (Ciprofloxacin,
Gentamycin, ,Rifampin, Colstin, Rifampin, Tetracycline,
Cefoyoxime and Ampicillin ) using disc diffusion method on Muller-Hinton agar medium. The cultured was incubated at 37 C o for 18 hr under aerobic condition and bacterial growth inhibition zones around the discs were measured. Results were interpreted as recommended by [24].
- Bacterial DNA isolates.
This method was performed according to the genomic
DNA purification Kit supplemented by the
manufacturing company.
-Gel electrophoresis
The extracted DNA was analyzed by DNA gel electrophoresis as described by [25] .
Molecular Identification:
A polymerase chain reaction (PCR) technique was used
to identify A.hydrophila by amplify genes of 16Sr RNA
gene from genomic DNA. DNA extraction from Gram negative bacteria was performed according to the genomic DNA purification kit supplemented by the manufacturing company (Geneaid/Taiwan). Gel electrophoresis has been used for detection of DNA by UV transilluminator[26]. The primers selection according to [22],[27] recommendations and used for diagnosis A.hydrophila. These primers synthesized by AccuOligo- Bioner Company, Korea, as shown in table (1)
Table 1: The Sequence of Forward and Reverse Primers

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Primer

Type
Primer Sequence Produc t Size
current. As for the salinity of the wastewater study area is very high water salinity irrigation water according to the
Forward
16Sr RNA
– F
Reverse
16Sr RNA

– R
5- CCAGCAGCCGCG GTAATACG-3
5- TACCAGGGTATCT AATCC-3
300 bp
classification based on the U.S. Salinity Laboratory
classification . The extent to which a broad range by the
values of pH in the terminals is due to the clear influence of the industrial wastewater company .
As for the solids in the wastewater of the main contaminants are found in different shapes and different
PCR Mixture solution was according to information of manufacturing company (Master mix, Geneaid/Taiwan) and PCR Program conditions was listed in table (2). Ten ml standard molecular weight of DNA ladder was loaded in first well on 1% agarose gel and each well has been loaded with 10ml of PCR product.
Electrophoresis runs at 80 volt/cm for 1hr. loaded in first
well on 1% agarose gel and each well has been loaded
with 10ml of PCR product [28].
Table 2:Amplication Conditions
Steps Temperature Time No.of
amounts can be dissolved in water consisting of negative
ions for vehicles combined with the positive ions of the
elements may be solids suspended in the water column itself. Was measured by chemical oxygen requirement (COD) through oxidation of organic matter oxidizing chemicals has been observed through the current study COD rates at all stations exceeded the determinants of international
The wastewater is under study in station were very

hardness .Thus, for the ionic calcium and magnesium as
concentration hardness closely associated with the

Cycles concentration of TDS and salinity as the TDS is a

Initial
denaturation
94 oC 3 min
carbonate and bicarbonate and chloride and sulfate and
30 nitrate and sodium and potassium, calcium, and
Denaturation 94 oC 30sec
Annealing 52 oC 30 sec
Elongation 72 oC 30 sec
cycle
magnesium, and an increase of the concentration of TDS
increasingly saline and hardness .
Sulphates with limited solubility in wastewater, so there
Final
elongation
72 oC 10
min

are usually low concentrations in surface water, with the exception of areas rich in the findings of the current study
. The high sulphate in industrial wastewater. The reason

3.RESULT & DISCCUSION

Result optioned from this work showed that increased in pollutants due to the presence of acids and bases and inorganic salts raised .Fig (1:A,B,C,D,E,F,G,H,I,J,K,L and M) summaries the physical and chemical characteristics of studied area .pH value was noticed , the sampling monthes varying from (1.5 - 5.2 ) ,these result also recorded electrical conductivity ranged between (1005 -
3100) while wastewater salinity ranged between (643 –
2058 ) . The present study shows closely values of chemical dissolved oxygen sites .The lowest COD( 45 mg/l) was recorded while the highest value was (140 mg/l) .
Total hardness ranged between (320 -663 mg/l) while calicium and magnesium hardness (65 – 200 mg/l) , (57 -
104 mg/l) respectively and T.D.S (629 – 1953 mg/l) .
Total alkalinity ranged between (57 - 128 mg/l) while
nitrate ranged between (3.1- 8.2 mg/l)

The result recorded sulphate range between (235 – 738
for this may be due to the large number of chemicals containing sulfates raised by factories and laboratories such as sulfuric acid , as well as the launch quantities of ferrous sulfate and iron sulfide increased to ask sulfate sodium
The chlorides have recorded high values. The
concentrations of chlorides in Iraq are generally higher
than in the countries of the world . The nitrates within the parameters for wastewater And that the change in the concentration of nitrates during the study period due to several reasons, including changes in the amount and the presence of water and the growth of plants , chapters heat
, add pollutants and denitrification . The phosphate has recorded high values him at all stations during different months of the study period and so maybe attributed to the occurrence of pollution in the wastewater detergent phosphorus-rich . These results agree with other studies [5, 29, 30, 31].
mg/l) while phosphate ranged between (0.11 -0.53 6
mg/l) . finally recorded range between (126 - 335 mg/l) 5
. 4
Connectivity is defined as a numerical value 3
refers to the ability of water to carry electric current and
this value depends on the concentration of ions and equal 2
dissolved in water is acid and inorganic bases and salts 1 A
dissolved in water are good conductors of electrical 0

4.837

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3500

3000

2500

2000

1500

1000

500

5.038

0 1 2 3 4 5 6 7

Months

300

200

100

5.829

0 1 2 3 4 5 6 7

Months

2500

2000

1500

1000

500

4.866

0 1 2 3 4 5 6 7

Months

150

100

10.440

0 1 2 3 4 5 6 7

Months

2500

2000

1500

1000

500

5.067

800

600

400

200

5.459

0 1 2 3 4 5 6 7

D Months

i 0

0 1 2 3 4 5 6 7

800

600

400

200

7.769

0 1 2 3 4 5 6 7

150

100

t= 6.882

0 1 2 3 4 5 6 7

Months

150

100

8.957

0 1 2 3 4 5 6 7

400

300

200

100

t= 7.188

Months

K 0 1 2

M3 onth4s

5 6 7

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0.6

t= 5.403

Table (3): Antibiotic susceptibility testing of A. hydro

0.5

0.4

0.3

0.2

0.1

0 1 2 3 4 5 6 7

Antibiotic

Ciprofloxacin Gentamycin Ampicillin Rifampin Tetracycline Cefotoxime Clostin
Susceptibility testing
S R R R R R R

150

100

Months

t= 6.138

0 1 2 3 4 5 6 7

Months

Resistance :R Sensitive : S
The results related to the antimicrobial susceptibility to the seventh tested antibiotics are given in the Table 3.The antibiotic resistance levels are expressed in percentages of the whole population and as antibiotic
resistance index numbers. Due to the fact that the
isolation medium already contained Gentamycin, Ampicillin, Rifampin, Colstin, Tetracycline and Cefotoxime, absolute resistance to all of these drugs was expected to be confirmed and therefore it was not practical to consider these resistance levels [32]. Where as it sensitive to Ciprofloxacin [33] .
With regard to extract DNA of this bacteria according to
the genomic DNA purification Kit and after the DNA
extracted agarose gel electrophoresis , showed the results
of this study contain only Chromosomal DNA (Fig 2).
Fig(1:A,B,C,D,E,F,G,H,I,J,K,Land M):Monthly variation of pollutionts industrial wastewater of site study.
In This study involved 21 bacterial strains, including 9
Aeromonas hydrophila, 7 A. caviae and 5 A. sobria, this research has show that A. hydrophila strain dominated in industrial wastewater and further more highly resistance to several antibiotic and A. hydrophila considered as pathogen for human being causing several disease.
.Aeromonas organisms are nonsporulating Gram negative rods that are ubiquitous inhabitants of fresh water sources. These hardy organisms multiply and grow under a variety of conditions and temperatures. A.hydrophila showed a yellow shine colour on TCBS agar, pale (non -lactose fermenters) on the MacConkey agar, smooth, convex, rounded, β-hemolytic colonies and pale white to grey colour on blood agar[22]. In terms of, initial biochemical tests A. hydrophila showed a positive result to each of catalase, oxidase, Indole, methyl red, simmone citrateand gelatin liquefaction. The current results of the biochemical tests in this study are almost finding in the other researchers reports [17] While, A. hydrophila was gave variable results to vogues- proskauer. Vitek 2 system is an efficient biochemical test to confirm identification of A. hydrophila [19].

Chromosomal DNA

Fig (2) : Electrophoresis of whole DNA for Aeromonas hydrophila (0.7%) agarose gel for 1.5 hr. 60 v In this study a polymerase chain reaction (PCR) technique was used to identify A.hydrophila by amplify genes of 16Sr RNA gene from genomic DNA of A. hydrophila isolate. This isolate has given a positive results for 16Sr RNA (300)bp (Fig 3). In addition to that,

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they reported the ribosomal mainly 16Sr RNA gene has confirmed to be a stable and specific molecular marker for the identification of A. hydrophila bacteria. Amplification of specific DNA by PCR provides a highly sensitive and specific tool for the detection of A. hydrophila from different sources.

1000

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[5]- Fahid, R. (2003 ). Unit 1. Physical, chemical and biological Characteristics of Wastewater
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[6]- Daniel, J. ; Hans, W.; Robert, W. and Donald, F.
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Figure 3: Agarose Gel Electrophoresis of PCR Amplified of Gene product 300 bp of Aeromonas hydrophila Isolates for 1 hr at 80 Volt.. Lane 1 Amplify of gene product.
Industrial wastewater aeromonads were confirmed as relevant agents for antimicrobial resistance spreading in the environment. The Patterns of Aeromonas species and antimicrobial resistance varied over different parts of the industrial wastewater.

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