A First Record of Sphingobacterium thalpophilum Isolated from Coptodon zillii and Oreeochromis aureus in the Al-Diwaniya River/Iraq

This investigation was done to determine whether C. zillii and O. aureus in the Al-Diwaniya Stream included every possibly harmful species of Gram-negative microbes. 100 C. zillii and 100 O. aureus fish samples were taken from the Al-Diwaniya River between Apr 2021 and Mar 2022. bacteria he was found us the VITEK 2 system. Here, we reported the discovery of S. thalpophilum, which was isolated from the intestine, fins, and gills of C. zillii and O. aureus, for the first time in Iraq. S. thalpophilum were found in C. zillii with a different proportion as follows: skin 1%, gills 70%, fins 24%, and intestine 5%; and in O. aureus with a different percentage as follows: skin 1%, gills 49%, fins 30%, and intestine 20%. Additionally, the VITEK 2 system was utilized to antibiotic sensitivity. The findings showed that the bacteria were susceptible to minimum inhibitory concentration (MIC) of imipenem, ciprofloxacin, and levofloxacin (0.25 g/ml), as well as to S. thalpophilum, but resistant to cefazolin (>= 61 g/ml).


Introduction
Fish diseases are one of the main problems that have a significant impact on fish farms [1][2][3][4][5].S. thalpophilum is a facultative gram-negative bacteria.It is hardly documented as a human pathogen [6].The majority of the clinical samples from which this new strain of the Flavobacterium genus was primarily isolated came from wounds and blood [7].Accordingly of the discovery that this cell wall contained animportantnumber of sphingolipids, Sphingobacterium was adopted as the new name for the group.Sphingobacteria typically have modest pathogenicity, but in patients with impaired immune systems, they can produce severe symptoms [8].are among the 15 species that make up this [9,10].Farming fish diseases effect by wide ecological and natural features, multiplication and controlling practices [11].According to Douglas [12], the microbes are transmitted by fish that have come into connection with other diseased fish.Fish bacterial diseases and infections are most common and are among the greatestproblematicto the health deals with.entering of bacteria to fish's body done through the gills or skin, or they can stay on the body surface.bacterial diseases causes aquaculture major economic losses.Seasonal changes in water quality and difficult stock conditions cause tension, which permits the most commonly cultivated carp to repeatedly become infected with opportunistic microorganisms [3,13].

Study Location
The Diwaniya Stream, which is a branch of the Euphrates River in central Iraq, is joined by.It measures 123 km in length, 25-30 m in width, and 3-5 m in depth.The main source of water for the city of Al-Diwaniya is this stream.Taken of samples were from two locations in the Stream between Al-Daghrah barricade, passing through Al-Saniya zone and go up to Al-Diwaniya city (Fig. 1).

Figure 1.
A map of the Al-Diwaniya River's field sample locations.

Sample Collection
A total of 100 C. zillii fish and 100 C. aureus fish total, Fish were sampled using cast nets (9m), gill nets (25m), and seine nets (3m).The sampling procedure was completed between April 2021 and March 2022.The fish was between 16 and 30 cm long and weighed between 70 and 470 g.Before being delivered to the lab at the College of Veterinary Medicine at Al-Qassim Green University and Ethical standards were considered in all steps of performing procedures and animal handling and the study was approved by the Ethical Committee [Approval letter No. UoM.Dent/A.L.66/21].No. Um.VET.2021.5.

Bactria Isolation and Characterization
The bacteria were removed aseptically from the dissected fish using a sterile ring fashioned of the fish's skin, gills, fins, and intestines.Using MacConkey agar medium, bacteria were isolated.For 24 hours, the infected plate was incubated at 37 °C.

Test Antibiotic Susceptibility
The results demonstrated that the bacteria S. thalpophilum were susceptible to imipenem, ciprofloxacin, and levofloxacin at their minimum inhibitory concentrations (MICs) of 0.25 g/ ml, as well as to S. thalpophilum, but hardy resistance to cefazolin at their maximum inhibitory concentration (MIC) of >61 g/ ml (Tables 2).
As a result, it has been proposed that these two medications may be useful for S. thalpophilum prophylaxis.However, using antibiotics frequently carries some hazards.

The Quality of Water
In Al-Diwaniya Stream, the quality of the water parameters varies (Fig. 4).The basic water Tempe ranges the lowest value of 11.2°C in February to the highest value of 33.8°C in August.The values of liquefied oxygen ranged from 4.5.0 mg/L in the month of August to 9.0 mg/L cm in the month of February.In April, the saltiness readings were 0.50°C, while in October, they were 0.69°C.In AL-Diwaniya Stream, there was only a small range of pH values over study period, mean value being 8 in Mar and the lowest being 6.1 in Aug.

Discussion
Identification S. thalpophilum as Gram-negative a human microbe, despite being extremely rare [6].B. Holmes et al. first reported it as such in 1983.In this study, S. thalpophilum was isolated for the first time from C. zillii and O. aureus of Iraq, and it was diagnosed in this study by Vitek2 device.This may be due to pollution in the riverbed due to agricultural and human activities along the river and the lack of sewage systems in the river.The cities that the river passes through, as well as the dumping of medical waste in the river.In a study by Marcel, Sabri [14] on the occurrence of pathogens in red tilapia fish elevated in cages in two water bodies in Malaysia with the isolation of spp, Micrococcus revealed no clinical indications, but the somatic lesions included engorgement or congestion of kidney with paleness of the liver.M. luteus was isolated from the skin and gills in the current study.In Beheira city that lies in the Egypt [15] isolated several types of Enterobacteriaceae in O. niloticus that suffer from cutaneous bleedings and sores with marked focal bleedings and parts of necrotic in the liver and congestion in the gills and spleen.These species are K. aerogenes and E. cloacae, C. freundii, E. coli, Klebsiella sp and P. vulgaris.Microbial load differs in the four parts of the skins, gills, fins and intestine.The microbial load was highest (gills and fins) and may be attributed to the highest ambient river temperature.Microbial load in fish may rise with increasing temperature of water [16,17].In study [18] acquired eighty isolates of P. aeruginosa from samples of fish market specimens' gills, skin, gut, and muscles.The gills had the largest proportion of isolation at 42.5%, followed by the skin at 37.5%, the intestine at 15%, and the muscles at 5%.P. aeruginosa was widespread, with a prevalence incidence of 26.6%.Antibiotics are even more important in aquaculture chemicals for more than half a century all over the world.Antibiotics are organic or chemical compounds They are also used that kill [19].DiMasi, Grabowski [20] described that prevention or treatment the spread of disease in fish farming by commonly using of antibiotics.However, development of antibiotic-resistant pathogens that can infect farmed animals as well as humans was carried by the extensive use of antibiotics in fish farming.The maximum sensitivity of drug screening in different organisms is a reasonable technique that can significantly reduce the dose of antibiotics.After starting therapy, isolates that were initially susceptible or intermediately resistant to one antibiotic may develop resistance [18].Ipenem and gentamicin were both ineffective against 96% of the isolates, whereas doxycycline, ciprofloxacin, trimethoprim-sulfamethoxazole, norfloxacin, and ceftriaxone were next in line.None of them were levofloxacin-resistant on Disease Aeromonas hydrophila [21].Infrequently exceed biological barriers to destroy bacterial organism in tissues, particularly the bloodbrain barrier (BBB) [22].The long-term antibiotics using causes bacterial resistance, drug remains in fishes and environmental health problems [23].According to Skwor, Shinko [24].increased antibiotic resistance in aquatic bacteria due to continuous antibiotic using.Thus, antimicrobial susceptibility tests are important for effective treatment.Drug screening with high sensitivity in many organisms is a applied technique that can significantly reduce antibiotic doses.Water quality is a very important factor in fish farming.The poor environmental conditions surrounding the farming sites affect the water quality, and this frequently reduces the immune status of the farmed fish, causing bacterial infections that lead to disease outbreaks [25].Kiriratnikom, et al [26] definite that microorganisms multiply in water is better when there is a high levels of organic substance, salinity, water Tempe 25°C to 32°C and pH of 5-9

Conclusions
The outcomes of the current study illustrated that S. thalpophilum microbe were present in the AL-Diwaniya River.Therefore, in order to confirm the quality of the fish.On other hand, bacterial resistance associated with long-term use of antibiotics, drug residues in fish and problems to the environmental health.
Isolatation of Sphingobacterium thalpophilum for the first time from C. zillii and O. aureus Iraq fish, and it was diagnosed in this study by Vitek2 device.Isolatation of S. thalpophilum from C. zillii and O. aureus skin, gills, fin and intestine of in AL-Diwaniya River exhibited clinical indications of diseases fish such as redden patches along the operculum and lower abdomen.These microorganisms were well-known with a different percentage in C. zillii proportion as follows: skin 1%, gills 70%, fins 24%, and intestine 5%; and in O. aureus with a different percentage as follows: skin 1%, gills 49%, fins 30%, and intestine 20% (Figures 2 & 3).

Figure 4 .
Figure 4. Changes in water Tempe, oxygen, salinity, and pH in Al-Diwaniya River.

Table 1 .
Biochemical details isolated of the S. thalpophilum.