Bacteriological Studies on Wastewater in Some Cities in Egypt

INTRODUCTION            

          Wastewater is defined as any stormwater runoff, as well as industrial, domestic or commercial sewage or any combination thereof carried by water.  The importance of adequate wastewater management is one of the key steps in protecting and ensuring the supply of safe drinking water and the maintenance of good public health       (Shalinee and Ademola, 2014). Groundwater is an important source of drinking water and its quality is currently threatened by the combination of chemical pollution and microbiological contamination, especially microbes of sewage origin (Reid et al., 2003). Generally, the European Union regulation requires that the heterotrophic plate count (HPC) be assessed at two recovery temperatures: 22º C for 72 hrs and 37º C for 24 hrs. The 37º C plate count was believed to fast-growing bacteria more likely to be related to pathogenic types and 22º C. Plate count was used for enumeration of characteristic water bacteria that tend to develop slowly (Ramalho et al., 2001). The heterotrophic plate count (HPC), gives a valuable indication of general microbiological quality of water (WHO, 2001). Drinking water may be contaminated by bad bacteria resulting in health problems. The presence of different bacterial genera in the water is due to direct contamination caused by human activities and an indirect effects by ecological disturbances.  The World Health Organization (WHO) reported that nearly half of the population in developing countries suffers from health problems associated with a lack of drinking water or with microbiologically contaminated water (WHO, 1992). The enteric bacteria are perhaps the most common pathogens present in wastewater. These microorganisms maintain their viability in wastewater, and it has been reported (Akin et al., 1978) that these entric bacteria may be found in raw sewage. Enteric bacteria are defined as rod-shaped Gram-negative organisms which ferment lactose. Afify et al., (2021) showed that there is a difference between bacterial isolates sensitivity and different groups of antibiotics.  

           In a previous study for the survey bacterial pollution of drinking water there were contaminated by enteric and pathogenic bacterial genera. This work aims to indicate that the source of these enteric and pathogenic bacteria is wastewater

MATERIALS AND METHODS

Source of wastewater samples      

        Wastewater samples were collected for microbiological examinations. The samples were collected from five different cities: Tanta, Elmehala Elkobra, Mehalet Abo Ali, Samanod and Mehalet Roh during four seasons (summer, autumn, winter and spring) at El Gharbia Governorate, Egypt. Samples were collected in liter sterile glass bottles and then transferred from the sites to the lab in an ice box and examined within 8 hours. All analyses were carried out in the microbiological laboratory of the Microbiology Department, Faculty of Agriculture, Mansoura University, Mansoura city, Egypt (Abdallah, 2016). 

Total bacterial count

Collected wastewater samples were analyzed for total viable bacterial count using the poured plates on nutrient agar medium (OXOID, CM0003, UK). Three plates for replicates and incubated at 37º C for 24 hrs and other plates were incubated at 20º C for 48hrs (AHPA, 2005).

Identification bacterial isolates 

                Bacterial isolates from colonies total count plates were obtained for purification and identification according to morphological, biochemical and physiological characteristics of purified bacterial isolates as follows:

Morphological characterizations of bacterial isolates

               All the isolates were examined for their cell size, cell shape, gram stain, capsulation, endospore formation and motility according to Bergey's Manual of Systematic Bacteriology 2005.

Biochemical, physiological characterizations and antibiotic susceptibility of bacterial isolates

              All the isolates were examined for different biochemical, physiological characters and antibiotic susceptibility by using the biomerieux VITEK^® 2 System David H. Pincus bioMerieux, Inc Hazelwood, MO, USA (Barnett et al., 2000). In the VITEK System there are currently four reagent cards available for the identification of different organism classes as follows:

1-GN: Gram-negative fermenting and non-fermenting bacilli.

2-GP: Gram-positive cocci and non spore-forming bacilli.

3-YST: Yeasts and yeast-like organisms.

4-BCL: Gram-positive spore-forming bacilli.

Total count of bacterial indicators 

Aeromonas sp. count

           An aliquot from each sample (0.1 ml) was streaked onto a hydrophila agar plate and incubated at 37º C for 24 hrs, after which the grown yellow colonies were counted as Aeromonas count/ 100 ml of original sample (Rippey and Cabelli, 1979).

Staphylococcus sp. count

           Staphylococci count was determined as described in medium staph 110 (Murray et al., 2007). 

Salmonella sp. count

         The enumeration of Salmonella was performed using the bismuth sulfite agar medium. Colonies producing diffusible black pigment across the membrane filters with or without metallic sheen were counted and considered as salmonella count (Engelbrecht et al., 1977). 

Proteus sp. count

        Proteus was determined as described in selective medium heart infusion agar Difeco plates and incubated at 37º C for 24 hrs (Baird-Parker 1962).

Serratia sp. count

        Serratia was determined as described in selective medium SD medium (Grimont et al., 1988).

Pantoea sp. count

          Pantoea was determined as described in nutrient agar (NA) medium (Walcott et al., 2002).

RESULTS AND DISCUSSION

             The bacteriological examinations of the collected wastewater samples were evaluated by monitoring the colonies counts as (colony forming unit, cfu) (total bacteria, enteric and/or pathogenic bacterial indicators) and identification of these bacteria to appear the source of bacterial pollution for drinking water (Afify et al., 2016) at the five cities of El Gharbia Governorate, Egypt.

Total bacterial counts

              The results in Figs. (1&2) show that, the counts determination at 20ºC and 37ºC. Generally, the maximum value of total bacterial counts at 20ºC (Fig.1) during summer at El mehala El kobra (6.1 cfu x 10⁹ /100ml) and the minimum value was in the spring at Samanod (0.4 cfu x 10⁹ /100ml). On the other hand, total bacterial counts at 37ºC (Fig.2) varied from 0.02 to 5.7 cfu x 10¹⁰ /100ml, the maximum value was in summer at Mehalet Roh but the minimum value was in spring at Samanod city. Among of all wastewater samples of different sites the detected total bacterial counts at 20ºC and 37ºC were greater in summer which might be attributed to high temperature and the discharged wastewater during this season.

            This phenomenon may be due to the nutrients availability for growth of the bacteria. Niemi and Niemi (1991) & Putheti and Lebure (2009) reported that domestic and industrial wastewater, agriculture waste environment are sources of bacteria. In Uruguay (Laguna de Rocha) Piccini et al., (2006) found that bacterial counts were higher average three times in the brackish southern part of the lagoon than the freshwater, in addition they suggested that this may be a consequence of better growing conditions.

Fig. 1:  Total bacterial counts (cfu/100ml) in wastewater at 20ºC of some cities at ElGharbia Governorate.

Fig.2:  Total bacterial counts (cfu/100ml) in wastewater at 37ºC of some cities at El Gharbia Governorate.

Identification of bacterial isolates

Morphological characterizations

Results in Table (1) showed six representative pure bacterial isolates to examine for their morphological characters: cell size, cell shape, gram stain, capsulation, endospore formation and motility according to Bergey’s Manual of Systematic Bacteriology 2005.

Table 1:  Morphological characterizations of bacterial isolates from wastewater  

 (+): Positive                 ;   (-): Negative   

Biochemical, physiological characterization and antibiotic susceptibility 

              Through the different biochemical, physiological characters and antibiotic susceptibility by using the biomerieux VITEK^® 2 System David H. Pincus bioMerieux, Inc Hazelwood, MO, USA (Barnett et al., 2000), tests were performed as follows:

                  Isolate No. 1 is positive with catalase, indole, citrate, acetate, V.P., gelatin, and lipase. While, negative with urea, malate and does not produce acid from cellobiose, inositol, melibiose, raffinose and sorbitol. Table (2) presented the results of antibiotic susceptibility. These results indicated that the isolate is identified as Aeromonas hydrophila. Similar results were reported by (Schubert, 1991) confirmed the presence of Aeromonas hydrophila in water with other enteric pathogens reflecting contamination of the environment. 

Table 2:  Antibiotic susceptibility of isolate No.1

 (MIC):  Minimum Inhibitory Concentrations;  (R): Resistant;  (S): Sensitivity 

Isolate No.2 colonies representing white yellow or orange colored, cells arranged in clusters and positive with catalase, phosphatase, fermented sucrose, lactose, mannitol, mannose, maltose and trehalose. But negative with ribose, raffinose, proline, xylose, lactose, urease, galactose and growth at 6.5% NaCl. Table (3) shows the results of antibiotic susceptibility. The isolate was identified as Staphylococcus aureus. Staphylococci are every now detected in the gastrointestinal tract and can be detected in sewage. Staphylococcus aureus can release by way of human contact into water environments. It has additionally been detected in drinking water supplies (Anti, 1987).

Table 3:  Antibiotic susceptibility of isolate No.  2  

 (MIC):  Minimum Inhibitory Concentrations ;  (R): Resistant;  (S): Sensitivity    

         Isolate No.3 can grow in the presence of NaCl 0.4 to 4.0%, grow at temperature range from 5 to 47 ºC but is sensitive to heat killed at 70ºC or above and facultative anaerobe, optimum pH 6.7-7.5, negative with gelatinase and urease while, positive with oxidase and catalase.  No fermented lactose, sucrose, use citrate as sole of carbon source and reduced nitrate to nitrite. The results in Table (4) shows that antibiotic susceptibility. Therefore, the isolate identified as Salmonella sp. Salmonella spp.  are solely human pathogens. Infection occurs due to ingestion of food or water contaminated with human waste (Scherer and Miller, 2001).  

Table 4:  Antibiotic susceptibility of isolate No. 3

 (MIC):  Minimum Inhibitory Concentrations ;  (R): Resistant;  (S): Sensitivity 

           Isolate No. 4 grows on several media, reduced nitrate and production H₂S, positive with phosphatase, lipase, urease, catalase but not oxidase and fermented glucose, while negative with proline, sucrose, lactose, glycine, maltose, mannitol, trehalose, mannose, citrate, galactose, cellobiose and sorbitol. The supplementation of the medium with bile salts lithium chloride sodium thiosulfate and sodium citrate at the concentrations stated above produced the selective growth of Proteus mirabilis. In addition, the antibiotic susceptibility in Table (5) results indicated that the isolate was identified as Proteus mirabilis (Janak, 2012). Ali et al., (2008) recorded, the characteristic spectrum of bacterial strains in water such as enteric and fecal bacteria and showed that these bacteria were the most contaminated source of drinking water.

Table 5:  Antibiotic susceptibility of isolate No.4

(MIC):  Minimum Inhibitory Concentrations ;  (R): Resistant;  (S): Sensitivity

Isolate No. 5 comparison of biochemical tests the following: H₂S production, positive with urease, phosphatase, lipase positive for glucose, sucrose, inositol and arabinose fermentation, citrate utilization and no fermented proline, lactose, maltose, trehalose, mannose, cellobiose, lactose, sorbitol and galactose, variable for raffinose. The strain was identified in parallel using the MicroScan Walk Away system and by standard reference procedures (Nieto et al.,1984 & Amos, 1985). In the comparison between MicroScan WalkAway system and conventional laboratory tests to evaluate important biochemical characteristics for the identification of S. plymuthica strains, all the tests presented a correlation of the 100% except the raffinose test (91%) (Jose et al., 2000). Table (6) shows that the antibiotic susceptibility and the strain were routinely cultured on trypticase soy agar at 37º C for 24 hr and stored on TSA slants at 4º C under mineral oil and frozen at 70º C with 15% glycerol., Serratia plymuthica.

Table 6:   Antibiotic susceptibility of isolate No.  5

(MIC):  Minimum Inhibitory Concentrations ;  (R): Resistant;  (S): Sensitivity 

             Isolate No. 6 isolate was positive with catalase, glucosidase, xylosidase, galactosidase, urease fermented sucrose, maltose, mannitol, trehalose, mannose, sorbitol and cellobiose. But negative with H₂S production, lipase, oxiase, proline, lactose, decarboxylase, citrate, urease, lactate and phosphatase. Pantoea agglomerans is a member of Enterobacteriaceae that inhabits plants, soil, water and such species include bacteria reported as both commensal and pathogen of animals and humans (Gavini et al., 1989). This isolate in Table (7) shows the antibiotic susceptibility and identified according to USFDA (2002) as Pantoea agglomerans.   

Table 7:   Antibiotic susceptibility of isolate No.  6

(MIC):  Minimum Inhibitory Concentrations ;  (R): Resistant;  (S): Sensitivity 

Previous studies have shown that entric and some pathogenic bacteria can survive in wastewater as sources of contamination (Cabral, 2010). Those bacteria that include several species of the Enterobacteriaceae family and live in the human and animal intestine (Ashbolt, 2004).  The enumeration of either Staphylococci or specific Staph. aureus appears to provide a useful index for the water quality level (Kamel, 2005).    

Total count of bacterial indicators

Aeromonas sobria and Staph. aureus  counts in wastewater

           In addition, the determination of total bacterial count which is considered new indicator of pollution of water, the water is also tested to determinate occurrence of Aeromonas sobria and Staph. aureus bacterial counts.

          Figs. (3&4) shows the comparison between the results of Aeromonas sobria and Staph. aureus  bacterial counts  at El Gharbia Governorate. Among of all wastewater samples Fig. (3) counts of Aeromonas sobria were almost in the summer (2.1 x10⁵ cfu/100ml). However, the winter samples contained the minimum value (1.6 x10⁵ cfu/100ml). In the city, Tanta recorded the highest values of Aeromonas sobria (2.9 x10⁵ cfu/100ml). These results are in agreement with El-Taweel (2003) reported that high counts of Aeromonas spp. in aquatic environments might be referred to the water polluted by surface seepage of sewage from septic tanks and sewers lines or from land applications. At the Fig. (4) Staph. aureus counts are recorded minimum value in autumn (1.5 x10⁷ cfu/100ml) and maximum value in summer    (6.4 x10⁷ cfu/100ml) at El mehala Elkobra. The means values ranged between 4.3 and 3.5 x10⁷ cfu/100ml in summer and spring respectively. The obtained results were in harmony with Vaerewijck et al (2005)

Fig.3:  Total counts of Aeromonas sobria in wastewater of some cities at El Gharbia Governorate.  

Fig.4:  Total counts of Staph. aureus in wastewater of some cities at El Gharbia Governorate.  

Pathogenic bacteria indicators counts in wastewater

        Salmonella sp. and Proteus mirabillis counts were determined in this investigation as pathogenic bacteria.

         Fig. (5) presents values of Salmonella sp. counts in wastewater. The minimum value was 1.7cfu x 10⁴/100ml, and it was in the winter at Samanod, but the maximum value was 4.5 cfu x 10⁴/100ml during summer at Tanta. Means value fluctuated between (3.6 and 2.7 cfu x 10⁴/100ml) in summer and spring respectively. The enteric bacteria are perhaps the most common pathogens present in wastewater, and of these, Salmonella species occur most frequently. These microorganisms maintain their viability in wastewater, and it has been reported (Akin et al., 1978) that Salmonella densities of 5,000 per liter may be found in raw sewage. 

Fig. (6) shows the maximum count of Proteus mirabillis was 4.2 cfu x107 107/100ml, in summer at Mehalet Roh while, the minimum count was    0.8 cfu x 107/100ml, in winter at Samanod, respectively. Genus Proteus is a source of enteric bacteria. 

        Fig. (6) shows the maximum count of Proteus mirabillis was 4.2 cfu x10⁷ 10⁷/100ml, in summer at Mehalet Roh while, the minimum count was    0

Fig.5:  Total counts of Salmonella sp. in wastewater of some cities at El Gharbia Governorate.

Fig.6:  Total counts of Proteus mirabillis  in wastewater of some cities at El Gharbia Governorate.

Also, Serratia plymuthica and Pantoea agglomerans  counts were determined in this investigation as pathogenic bacteria. Fig. (7) indicated that Serratia plymuthica values of counts in wastewater. The minimum value (0.7 cfu x 10⁵/100ml), it was in the spring at Samanod. While, the maximum value was 3.1 cfu x 10⁵/100ml during summer at Tanta. Means values were fluctuated between 2.4 and 1.7 cfu x 10⁵/100ml during summer and winter respectively.

        In Fig. (8) shows the maximum count of  Pantoea agglomerans  was 1.2 cfu x 10⁶/100ml in the summer at Mehalet Abo Ali and the minimum count was  0.01 cfu x 10⁶/100ml during autumn at Tanta. Means values fluctuated between 0.4 and 0.1 cfu x 10⁶/100 ml  during summer and autumn respectively.

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Fig.7:  Total counts of Serratia plymuthica in wastewater of some cities at El Gharbia

           Governorate.   

Fig.8:  Total counts of Pantoea agglomerans  in wastewater of some cities at El  Gharbia Governorate.   

Our results are in agreement with recently published data and the greatest microbial risks are associated with ingestion of water that is contaminated with human or animal feces. Wastewater discharges in fresh waters and coastal seawaters are the major source of fecal microorganisms, including pathogens (WHO, 2008). In general terms, show that all water sources were grossly polluted, and water was not fit for drinking (Madhab et al.,2010). Neweigy et al., (2010) reported that most of the ground water was not treated or disinfected before pumping to the drinking water pipeline. Groundwater contamination is nearly always the result of human activities. When ground water becomes contaminated, it is difficult and expensive to make it pure. Liquid waste discharged on to soil initiates solute and microbial movement may contaminate groundwater. This could be due to mixing of sewage water with drinking water as a result of leakage in the pipeline. A regular monitoring of the water quality for improvement not only prevents disease and hazards but also checks the water resources from going further polluted (Manjula  et al., 2011). Also, Abd-elhameed et al., (2021) reported that some studies such as domestic and industrial wastewater, agriculture waste environment are sources of fecal bacteria in rivers.

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الملخص العربى

دراسات بكتيريولوجية على مياه الصرف الصحى فى بعض مدن مصر

عايده حافظ عفيفى¹ - أحمد محمد عبد الله²- محمد خضر² - أحمد محمود السواح¹

• قسم الميكروبيولوجى – كلية الزراعه –  جامعة المنصوره –  المنصوره- مصر
• قسم النبات و الميكروبيولوجى – كلية العلوم – جامعة الأزهر – القاهره - مصر

  فى دراسة لقياس جودة مياه الشرب بكتيريواوجيا ثم إجراء مقارنه بمياه الصرف الصحى لمعرفة مصدر التلوث البكتيرى  وذلك فى مدن مختلفه بمحافظة الغربية ( طنطا- المحله الكبرى- محلة أبو على- سمنود- محلة روح) بمصر . ففى هذه الدراسة تم إجراء عد كلى للبكتيريا فى عينات مياه الصرف الصحى وكذلك عد لبعض الأنواع البكتيريه المعوية والمرضية على بيئاتها المتخصصه وذلك فى فصول السنه الأربعة ( الصيف والخريف والشتاء و الربيع) وثم تعريف تلك العزلات البكتيريه مورفولوجيا وفسيولوجيا وكيميائيا مع إجراء إختبارات تأثير المضادات الحيويه بجهاز الفيتك  . أظهرت النتائج أن أكبر عدد للبكتيريا كان فى فصل الصيف فى مدينة المحلة الكبرى وبتعريف العزلات البكتيريه حصلنا على الأنواع التالية :

Aeromonas sobria, Staphylococcus aureus, Salmonella sp., Proteus mirabilis, Serratia plymuthica and Pantoea agglomerans .

وعند إجراء العد لهذه البكتيريا فى فصول السنه الأربعه كانت أعلى متوسط الأعداد البكتيريه فى فصلى الصيف والربيع ربما يرجع ذلك لمناسبة درجات حرارة الفصلين لنمو مثل هذه البكتيريا . من هذه الدراسه يمكن القول أن مصدر التلوث الميكروبى لمياه الشرب يكون مصدره الرئيسى مياه الصرف الصحى وبالتالى لابد من المحافظة على المصادر الرئيسية لمياه الشرب بعدم إلقاء مخلفات المنازل وكذلك مخلفات المصانع والمخلفات الزراعية فى المصادر الرئيسيه لمياه الشرب  للمحافظه على صحه الإنسان بالإضافة إلى المحافظة على البيئه من التلوث .