**2.4** *Salmonella* **spp. analyses for seawater samples**

*Salmonella* spp. analyses depend on identification with biochemical and serological tests of suspicious colonies from selective solid medium after selective enrichment and unselective prior enrichment at 37 0C in liquid medium in the seawater samples (APHA, 2000).

Then the colonies were restreaked several times to obtain pure cultures and the pure isolates of *Salmonella* spp. were identified using GN cards in the automated biochemical identification system VITEK 2 Compact 30 (Biomereux, France). The identification cards are based on established biochemical methods and newly developed substrates. There are

The Occurrence of Salmonella in Various Marine Environments in Turkey 81

Diluted homogenous solutions of samples taken from those parts that are edible were prepared with 0.1% buffered peptone water: 10:100 for the *E. coli* total coliform and fecal coliform analyses. Sample dilutions of 10–1, 10–2, and 10–3 with buffered peptone water were transferred to three series of test tubes, each containing 10 mL of Modified Lauryl

Analyses were done according to the three tube most probable number method (MPN) using Brilliant green bile broth (BGLB), EC broth, Eosin methylene blue agar medium, Plate

The percentage of bacteria in the samples which exhibited antibiotic resistance was measured on Nutrient agar plates supplemented with Imipenem, Ampicillin, Cefotaxim,

**3. Occurrence of** *Salmonella* **spp. in the samples of seawater, shellfish and** 

The frequency of *Salmonella* spp. according to their exposure to environmental factors in the areas from which they were isolated were shown in Table 1 in the form of summary data of

No *Salmonella* spp. was detected in the samples which were taken from the western Black

The presence of *Salmonella* spp. in seawater from the four stations was significantly different (p < 0.05) in the Golden Horn Estuary, Istanbul from 2002 to 2003. Eleven of 44 seawater samples were found positive for *Salmonella* spp. The number of *Salmonella* spp. positive

The percentage distribution of the values for the ratio of fecal coliform to fecal streptococci in the surface water of the Aegean Sea and their relation with *Salmonella* spp. was also investigated. The contribution of fecal coliform bacteria to fecal streptococci (FC/FS > 0.7) showed that the sources of fecal contamination were anthropological in this area in 2006- 2008. Seven of the 22 unit seawater samples were found positive for *Salmonella* spp. in the sea water samples which were taken from the coastal areas of the Aegean Sea, *Salmonella*  spp. positive samples were positive correlated with the indicator bacteria count. In the five stations which have higher number of indicator bacteria than the other stations *Salmonella* spp. were found positive. The percentages of *Salmonella* spp. among the total enteric bacteria

*Salmonella* spp. was not isolated in the seawater samples which were taken from the

Four units of 14 seawater samples tested which were taken from coastal areas of eastern

Eight units of 83 seawater samples tested which were taken from 0-30 cm to 500 meters were found positive for *Salmonella* spp. in the samples of 0-30 cm, 50 meters and 100 meters in the

June 2006. *Salmonella* spp. was only isolated in the summer period during the study.

Mediterranean were found positive for *Salmonella* spp. in August 2007-2008.

the level of coliform and fecal coliform bacteria and the occurrence of *Salmonella* spp.

**2.6.2 The most probable number method** 

For characterization of coliform, Endo agar was used.

Ceftriaxon, Ceftazidim media (NCCLS 1999).

samples was highest in the inner part of the estuary.

were between 25% and 37% in these stations.

Sulphate Triptose Broth.

count agar medium (FDA, 1998).

**2.7 Antibiotic resistance test** 

**fish** 

**3.1 Seawater** 

Sea in 1998-1999.

offshore areas.

biochemical tests (47 tests for GN) measuring carbon source utilization, enzymatic activities, inhibition, and resistance (Pincus, 2005).

#### **2.5** *Salmonella* **analyses for shellfish samples**

In the analyses, 94 groups were used; 6 individuals were accepted as a group, and a total of 10 g (25 g for *Salmonella* spp.) was taken from each of these groups to form a sample group. In accordance with the purpose of the test, diluted homogenous solutions of samples taken from those parts that are edible, were prepared with 0.1% buffered peptone water: 25:225 for the *Salmonella* spp.

Analyses depend on identification with current biochemical and serologic tests of suspicious colonies from selective solid medium after selective enrichment for 24 h in Selenith cystine broth at a temperature of 350C, and unselective prior enrichment for 18–24 h at 370C in buffered peptone water 25:225 (w/v) (FDA, 1998). To further identify and characterize the strains that were detected and isolated, commercially available API test system (BioMerieux, France) was used. The biochemical reactions tested with API test are: production of indole; utilization of citrate; production of nitrite; fermentations of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdaline, and arabinose; production of H2S; activities of beta-galactosidase, tryptophane desaminase, gelatinase, arginine dihydrolase, lysine decarboxylase, and ornithine decorboxylase; formation of acetoin from pyruvate and oxidase (MacDonell et al.1982, Oberhofer 1983). When there was a need to further identification, the pure isolates of suspicious colonies were identified using GN cards in the automated biochemical identification system VITEK 2 Compact 30 (Biomereux, France).

The identification cards are based on established biochemical methods and newly developed substrates. There are biochemical tests (47 tests for GN) measuring carbon source utilization, enzymatic activities, inhibition, and resistance (Pincus, 2005).

#### **2.6 Indicator bacteria analyses**

Two different methods were used for indicator bacteria analyses in various sampling periods in 1998-2010.

#### **2.6.1 Membrane filtration method**

The water samples were taken from 0-30 cm surface and from various depths ranging from 1 to 50 meters. Water samples were filtered through a 0.45 μm membrane filter with a metal vacuum filtering set (Millipore, Germany) and then the membrane filters were placed on m-Endo, m-FC and Azide-NKS for total coliform, fecal coliform and fecal streptococci. The plates were incubated for 48 h (at 37±0.1°C and 44.5±0.1°C) and the colonies on the plates were evaluated (APHA 1998; EPA 2006). Following the correction tests on suspicious colonies which grew after incubation, the average of three parallel tests was used for the numerical identification (cfu/100 mL: colony formed unit/100 mL). Brown-red colonies which grew on Azide medium were evaluated as fecal streptococci suspicious; blue colonies which grew on m-FC medium were evaluated as fecal coliform suspicious; pink-red colonies with yellow-green metallic shinyness which grew on m-Endo medium were evaluated as coliform suspicious. cytochrome oxidase test (API Strep, BioMereux ) was applied to coliform suspicious colonies and oxidase negative colonies were counted. cytochrome oxidase (API Strep, BioMereux ) and indole (HIMEDIA) tests were applied to fecal coliform suspicious colonies, and oxidase negative and indole positive colonies were counted. (MacFaddin 1980, APHA 2000).
