**3.1 Seawater**

80 Salmonella – A Dangerous Foodborne Pathogen

biochemical tests (47 tests for GN) measuring carbon source utilization, enzymatic activities,

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

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

Two different methods were used for indicator bacteria analyses in various sampling

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.

utilization, enzymatic activities, inhibition, and resistance (Pincus, 2005).

inhibition, and resistance (Pincus, 2005).

the *Salmonella* spp.

**2.6 Indicator bacteria analyses** 

**2.6.1 Membrane filtration method** 

(MacFaddin 1980, APHA 2000).

periods in 1998-2010.

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

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

No *Salmonella* spp. was detected in the samples which were taken from the western Black Sea in 1998-1999.

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 samples was highest in the inner part of the estuary.

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 were between 25% and 37% in these stations.

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

Four units of 14 seawater samples tested which were taken from coastal areas of eastern Mediterranean were found positive for *Salmonella* spp. in August 2007-2008.

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.

The Occurrence of Salmonella in Various Marine Environments in Turkey 83

While the highest Multiple Antibiotic Resistance (MAR) was found in the bacteria isolated in seawater which was taken from the Golden Horn Estuary, Istanbul, the bacteria isolated from northern part of the Sea of Marmara and coastal areas of Istanbul

samples

10-<102 0 0 102 - <103 14 21.8 103-<104 17 26.5 >104 33 51.5

10-<102 0 0 102 - <103 1 12.5 103-<104 2 25 >104 5 83.3

10-<102 0 0 102 - <103 0 0 103-<104 3 100 >104 0 0

samples: 94 3 (2.13% of the 94 samples)

specimens:832 75 (9.01% of the 832 samples)

Table 2. The frequency of *Salmonella* spp. (cfu/25 ml; cfu/25 g) and fecal coliform bacteria

64 (13% of the 495 samples)

8 (3.3% of the 243 samples)

R*elation (*%) *between the fecal coliform level and the number of Salmonella* (+) samples

Sample Type F. coliform Number of *Salmonella* (+)

respectively followed it.

Sea Water

Number of seawater samples: 495

Shellfish

Number of shellfish samples: 243

Fish

Number of fish

Total number of

(cfu/100 ml) in the samples

Fourteen of 80 seawater samples which were taken from 30 cm to 50 meter were positive for *Salmonella* spp. in July 2006 in southern part of the Sea of Marmara. Also, three seawater samples were found *Salmonella* spp. positive in June 2007. During this study *Salmonella* spp. was isolated only in July 2006 and June 2007.

Sixty four of the 495 unit seawater samples tested was found positive for *Salmonella* spp. (13%) in the stations. Thirty three of the 64 unit *Salmonella* spp. positive samples of seawater (51.5 %) which have been recorded in the stations indicator bacteria were > 104 fecal coliform /100 ml.

Twenty two of 136 unit seawater samples which were taken from 0-30 cm in the Sea of Marmara were found positive for *Salmonella* spp. in the July 2009 and June 2010 period. *S. enterica* ssp. *arizonae, S. enteritidis and S. typhimurium* were the most identified isolates in the samples. *S. typhimurium* represented 64.3% of all *Salmonella* spp. strains and was identified in the seawater samples.

The frequency of *Salmonella* spp. related to fecal coliform bacteria in the seawater samples was summarized in the Table 2. Biochemical details of two of isolated *Salmonella* spp. was summarized in Table 3.
