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**3**

**Influence of a Salt Water Regulator on**

**the Survival Response of** *Salmonella*

*Paratyphi* **in Vembanadu Lake: India**

Chandran Abhirosh1,2, Asit Mazumder1, Sherin Varghese2,

*2School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala*

Contamination of environmental water by pathogenic microorganisms and subsequent infections originated from such sources during different contact and non- contact recreational activities are a major public health problem worldwide particularly in developing countries. The main pathogen frequently associated with enteric infection in developing countries are *Salmonella* enterica serovar typhi and paratyphi. Although the natural habitat of *Salmonella* is the gastrointestinal tract of animals, it find its way into natural water through faecal contamination and are frequently identified from various aquatic environments (Baudart *et al*., 2000; Dionisio *et al*., 2000; Martinez -Urtaza *et al*., 2004., Abhirosh *et al*., 2008). Typhoid fever caused by *S. enterica* serotype typhi and paratyphi are a common infectious disease occurring in all the parts of the world with its highest endemicity in certain parts of Asia, Africa, Latin America and in the Indian subcontinent with an estimated incidence of 33 million cases each year with significant morbidity and mortality (Threlfall, 2002). In most cases the disease is transmitted by polluted water (Girard *et al*., 2006) because of the poor hygienic conditions, inadequate clean water supplies and sewage treatment facilities. However in developed countries the disease is mainly associated

*Salmonella*, since being allochthonous to aquatic environments, the potential health hazard is dependent on their period of survival outside the host and retention of critical density levels in the receiving water in a given time frame during transmission via the water route. In general, the major environmental factors influencing the enteric bacterial survival following their exposure to aquatic environments are water temperature (Anderson *et al*., 1983), adsorption and sedimentation processes (Auer & Niehaus, 1993), sunlight action (Sinton *et al*., 1999), lack of nutrients (Sinclair & Alexander, 1984),

with food (Bell *et al*., 2002) especially shellfish (Heinitz *et al.*, 2000).

**1. Introduction**

 A.P Thomas2 and A.A.M Hatha3 *1Water and Aquatic Sciences Research Lab*

 *and Technology, Cochin, Kerala*

*1Canada 2,3India*

*Department of Biology, University of Victoria, Victoria*

*3School of Marine Sciences, Cochin University of Science*

### **Influence of a Salt Water Regulator on the Survival Response of** *Salmonella Paratyphi* **in Vembanadu Lake: India**

Chandran Abhirosh1,2, Asit Mazumder1, Sherin Varghese2, A.P Thomas2 and A.A.M Hatha3 *1Water and Aquatic Sciences Research Lab Department of Biology, University of Victoria, Victoria 2School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala 3School of Marine Sciences, Cochin University of Science and Technology, Cochin, Kerala 1Canada 2,3India*

#### **1. Introduction**

46 Salmonella – A Diversified Superbug

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(2011). Systems analysis of multiple regulator perturbations allows discovery of virulence factors in Salmonella. *BMC systems biology*. Vol. 5, No. 1, (June 2011) pp. Contamination of environmental water by pathogenic microorganisms and subsequent infections originated from such sources during different contact and non- contact recreational activities are a major public health problem worldwide particularly in developing countries. The main pathogen frequently associated with enteric infection in developing countries are *Salmonella* enterica serovar typhi and paratyphi. Although the natural habitat of *Salmonella* is the gastrointestinal tract of animals, it find its way into natural water through faecal contamination and are frequently identified from various aquatic environments (Baudart *et al*., 2000; Dionisio *et al*., 2000; Martinez -Urtaza *et al*., 2004., Abhirosh *et al*., 2008). Typhoid fever caused by *S. enterica* serotype typhi and paratyphi are a common infectious disease occurring in all the parts of the world with its highest endemicity in certain parts of Asia, Africa, Latin America and in the Indian subcontinent with an estimated incidence of 33 million cases each year with significant morbidity and mortality (Threlfall, 2002). In most cases the disease is transmitted by polluted water (Girard *et al*., 2006) because of the poor hygienic conditions, inadequate clean water supplies and sewage treatment facilities. However in developed countries the disease is mainly associated with food (Bell *et al*., 2002) especially shellfish (Heinitz *et al.*, 2000).

*Salmonella*, since being allochthonous to aquatic environments, the potential health hazard is dependent on their period of survival outside the host and retention of critical density levels in the receiving water in a given time frame during transmission via the water route. In general, the major environmental factors influencing the enteric bacterial survival following their exposure to aquatic environments are water temperature (Anderson *et al*., 1983), adsorption and sedimentation processes (Auer & Niehaus, 1993), sunlight action (Sinton *et al*., 1999), lack of nutrients (Sinclair & Alexander, 1984),

Influence of a Salt Water Regulator on the Survival

Fig. 1. Map showing Vembanadu Lake

**2.1 Test organism and water sample**

**2. Materials and methods**

Response of *Salmonella Paratyphi* in Vembanadu Lake: India 49

A pure culture of *S. paratyphi* isolated from the Vembanadu lake was used for the survival experiments. All experiments were conducted in filter sterilized lake water in order to avoid the effect of predation. When saltwater regulator is closed the saline intrusion from northern part is prevented and the water on the southern part becomes freshwater. Therefore, to

predation by bacteria or protozoa (Hahn & Hofle, 2001), bacteriophage lysis (Ricca & Cooney, 1999), competition with autochthonous microbiota (McCambridge & McKeekin, 1981) and antibiosis (Colwell, 1978).

Although *Salmonella* spp. has been isolated from fresh, estuarine and marine waters, they showed differential survival response to those aquatic environments and the results were sometimes contradictory in relation to salinity. For instance, it has been reported that *Salmonella* showed very low survival in sea water (Lee *et al*., 2010) on the contrary Sugumar & Mariappan (2003) found that they exhibited very long survival up to 16 to 48 week in sea water. But it is also documented that it survived for 54 days (Moore *et al*., 2003) and 58 days in freshwater Sugumar & Mariappan (2003). However, when *Salmonella* suspended in stabilization ponds effluent and rapidly mixed with brackish water, survival time was particularly short, whereas it was prolonged when the bacteria was submitted to a gradual increase in salinity (Mezrioui *et al*., 1995). Therefore the survival of pathogenic bacteria in estuarine environments in response to varying saline concentration due to the mixing of salt water with freshwater has of particular health significance especially in locations where contact and non recreation takes place.

Hence the present study has been carried out in Vembanadu Lake that lies 0.6-2.2 m below mean sea level (MSL) along the west coast of India (9o35'N 76o25'E) and has a permanent connection with the Arabian Sea (Fig.1). As the north-east monsoon recedes, the area is exposed to tidal incursion of saline water from the Arabian Sea. In order to prevent the saline incursion during certain periods of the year, a salt water regulator is constructed in the lake. It divides the lake into a freshwater region on the southern part and a saline lagoon on the northern part. As a result, during the closure and opening of the regulator the water quality on both regions of the regulator may change in terms of its salinity and a progressive saline gradient may occur throughout the lake when the regulator is open. On the other hand over 1.6 million people directly or indirectly depend on it for various purposes such as agriculture, fishing, transportation and recreation. As a result water related diseases are very common in this region particularly in young children but none of them were reported officially. Enteric fever caused by *Salmonella* enterica serovars paratyphi *A*, B and C and Newport have been reported in India (Misra *et al*., 2005; Gupta *et al*., 2009).

Since die-off of enteric bacteria in aquatic environment could be attributed to a variety of interacting physical, chemical and biological factors and processes (Rhoder & Kator, 1988), in our previous studies in the Vembanadu lake we have evaluated the effect of sunlight, chemical composition of the estuarine water (Abhirosh & Hatha, 2005) effect of biological factors such as protozoan predation, predation by bacteriophages, autochthonous bacterial competition (Abhirosh *et al*., 2009) on the survival of *Salmonella* and other organisms. However, the effect of salinity, since being important on the survival of enteric bacteria has not been evaluated in Vembanadu lake. As we already reported the presence of different *Salmonella* serotypes such as *Salmonella paratyphi* A, B, C and *Salmonella Newport* in Vembanadu lake (Abhirosh *et al*., 2008), in this study our aim was to evaluate the health risk associated with *S. paratyphi* when released into the water by studying the survival responses to the salinity changes (saline gradient) caused by the saltwater regulator in Vembanadu lake using microcosm experiments at 20oC and 30oC.

predation by bacteria or protozoa (Hahn & Hofle, 2001), bacteriophage lysis (Ricca & Cooney, 1999), competition with autochthonous microbiota (McCambridge & McKeekin,

Although *Salmonella* spp. has been isolated from fresh, estuarine and marine waters, they showed differential survival response to those aquatic environments and the results were sometimes contradictory in relation to salinity. For instance, it has been reported that *Salmonella* showed very low survival in sea water (Lee *et al*., 2010) on the contrary Sugumar & Mariappan (2003) found that they exhibited very long survival up to 16 to 48 week in sea water. But it is also documented that it survived for 54 days (Moore *et al*., 2003) and 58 days in freshwater Sugumar & Mariappan (2003). However, when *Salmonella* suspended in stabilization ponds effluent and rapidly mixed with brackish water, survival time was particularly short, whereas it was prolonged when the bacteria was submitted to a gradual increase in salinity (Mezrioui *et al*., 1995). Therefore the survival of pathogenic bacteria in estuarine environments in response to varying saline concentration due to the mixing of salt water with freshwater has of particular health significance especially in locations where

Hence the present study has been carried out in Vembanadu Lake that lies 0.6-2.2 m below mean sea level (MSL) along the west coast of India (9o35'N 76o25'E) and has a permanent connection with the Arabian Sea (Fig.1). As the north-east monsoon recedes, the area is exposed to tidal incursion of saline water from the Arabian Sea. In order to prevent the saline incursion during certain periods of the year, a salt water regulator is constructed in the lake. It divides the lake into a freshwater region on the southern part and a saline lagoon on the northern part. As a result, during the closure and opening of the regulator the water quality on both regions of the regulator may change in terms of its salinity and a progressive saline gradient may occur throughout the lake when the regulator is open. On the other hand over 1.6 million people directly or indirectly depend on it for various purposes such as agriculture, fishing, transportation and recreation. As a result water related diseases are very common in this region particularly in young children but none of them were reported officially. Enteric fever caused by *Salmonella* enterica serovars paratyphi *A*, B and C and Newport have been reported in India (Misra *et*

Since die-off of enteric bacteria in aquatic environment could be attributed to a variety of interacting physical, chemical and biological factors and processes (Rhoder & Kator, 1988), in our previous studies in the Vembanadu lake we have evaluated the effect of sunlight, chemical composition of the estuarine water (Abhirosh & Hatha, 2005) effect of biological factors such as protozoan predation, predation by bacteriophages, autochthonous bacterial competition (Abhirosh *et al*., 2009) on the survival of *Salmonella* and other organisms. However, the effect of salinity, since being important on the survival of enteric bacteria has not been evaluated in Vembanadu lake. As we already reported the presence of different *Salmonella* serotypes such as *Salmonella paratyphi* A, B, C and *Salmonella Newport* in Vembanadu lake (Abhirosh *et al*., 2008), in this study our aim was to evaluate the health risk associated with *S. paratyphi* when released into the water by studying the survival responses to the salinity changes (saline gradient) caused by the saltwater regulator in Vembanadu

1981) and antibiosis (Colwell, 1978).

contact and non recreation takes place.

*al*., 2005; Gupta *et al*., 2009).

lake using microcosm experiments at 20oC and 30oC.

Fig. 1. Map showing Vembanadu Lake

#### **2. Materials and methods**

#### **2.1 Test organism and water sample**

A pure culture of *S. paratyphi* isolated from the Vembanadu lake was used for the survival experiments. All experiments were conducted in filter sterilized lake water in order to avoid the effect of predation. When saltwater regulator is closed the saline intrusion from northern part is prevented and the water on the southern part becomes freshwater. Therefore, to

Influence of a Salt Water Regulator on the Survival

Freshwater 30oC■ and 20oC □; mixing water 30oC ▲and 20oC ∆

**Days Freshwater**

Response of *Salmonella Paratyphi* in Vembanadu Lake: India 51

Fig. 2. The survival curves of *S. paratyphi* in freshwater and mixing water at 20oC and 30oC

0 0.00 0.00 0.00 0.00 2 -1.64 -0.74 -2.48 -2.05 4 -1.59 -1.23 -3.75 -2.59 6 -1.75 -1.25 -2.96 -2.54 8 -1.86 -1.46 -2.96 -2.51 10 -1.78 -1.66 -2.95 -2.45 13 -1.80 -1.53 -3.04 -2.79 16 -1.84 -1.50 -2.76 -2.65 22 -1.86 -1.65 -2.75 -2.64 28 -2.57 -1.75 -2.67 -2.63 34 -3.78 -1.90 -2.76 -2.65

K value -0.11 -0.07 -0.13 -0.12

T99 17.68 25.99 15.20 16.37 Table 1. Inactivation rates of *S. paratyphi* in freshwater and mixing water at 30oC and 20oC

**30oC Freshwater 20oC Mixing water 30oC Mixing water**

**20oC**

imitate the actual condition on the southern part of the lake, experiments were conducted in water collected from the lake when the salinity was 0 ppt (freshwater microcosm). To study the survival of the test organisms during mixing of water from northern and southern part of the Vembanadu lake, experiments were conducted in mixing water samples collected when the regulator was open (mixing water microcosm). Besides, in order to study the survival in all possible saline gradient throughout the year, survival experiments were conducted in lake water with salinity concentration ranged from 0-25 ppt. The test solutions of desired saline concentrations were prepared using fresh lake water with NaCl.

#### **2.2 Preparation of inocula**

The inocula were prepared as previously described by Abhirosh & Hatha (2005). *S. paratyphi* was grown in Tryptone Soy Broth (TSB) and incubated at 37oC for 24 h. After incubation, the cells were concentrated by centrifugation at 1400 × g for 15 min and washed twice with sterile isotonic saline. After the final wash, the cells were re-suspended in sterile isotonic saline for inoculation into the microcosms. Then 1 ml washed cell suspension of *S. paratyphi* was inoculated into each microcosm containing different test solution (250 ml Erlenmeyer flask with 100 ml) at a concentration of 106-7 CFU/ mL. All inoculated microcosm were incubated at 20oC and 30oC. The microcosms were incubated at 20oC in order to find out the survival at low temperature as the temperature goes down to 20oC in winter as well as at a certain depth. The enumeration of culturable bacteria were done after 2, 4, 6, 8, 10, 13, 16, 22, 28 and 34 days using spread plate technique on TSA agar plates and the colony forming units were counted.

#### **2.3 Decay rate and statistical calculation**

The decay rates of culturable *S. paratyphi* cells were calculated as per first order decay model using the following equation Log Nt/N0= -kt, where *Nt* is the number of bacteria at time *t, N*0 is number of bacteria at time 0, and *t* is expressed in days; *k* is the first-order constant calculated by linear regression technique. T99 (time required for 2 log reduction) values were calculated using the decay constant (*k*) in the following equation, T99=- 2/*k*.The difference in the survival at different salinities and temperature was analysed using two way analysis of variance (ANOVA).

#### **3. Results and discussion**

The survival curves of *S. paratyphi* in freshwater and mixing water at 20oC and 30oC are given in Fig. 2 and the inactivation rates and T99 values are given in Table 1. The results revealed that *S. paratyphi* showed significantly (p<0.01) higher survival at 20oC (T99= 25.99) compared to 30oC (T99= 17.68) in freshwater water indicating their better survival capacity at low temperature. However *S. paratyphi* did not show much difference in the survival response in mixing water at both temperature and the T99 respectively was 16.37 days at 20oC and 15.12 days at 30oC. The results revealed that *S. paratyphi* cells remained viable until 34 days at a high density of 105 CFU/mL. The salinity of the mixing water when it was collected was 12.77 ppt and the average saline concentration of the lake water was 12.5ppt when it was monitored over 2hr interval in a day.

imitate the actual condition on the southern part of the lake, experiments were conducted in water collected from the lake when the salinity was 0 ppt (freshwater microcosm). To study the survival of the test organisms during mixing of water from northern and southern part of the Vembanadu lake, experiments were conducted in mixing water samples collected when the regulator was open (mixing water microcosm). Besides, in order to study the survival in all possible saline gradient throughout the year, survival experiments were conducted in lake water with salinity concentration ranged from 0-25 ppt. The test solutions

The inocula were prepared as previously described by Abhirosh & Hatha (2005). *S. paratyphi* was grown in Tryptone Soy Broth (TSB) and incubated at 37oC for 24 h. After incubation, the cells were concentrated by centrifugation at 1400 × g for 15 min and washed twice with sterile isotonic saline. After the final wash, the cells were re-suspended in sterile isotonic saline for inoculation into the microcosms. Then 1 ml washed cell suspension of *S. paratyphi* was inoculated into each microcosm containing different test solution (250 ml Erlenmeyer flask with 100 ml) at a concentration of 106-7 CFU/ mL. All inoculated microcosm were incubated at 20oC and 30oC. The microcosms were incubated at 20oC in order to find out the survival at low temperature as the temperature goes down to 20oC in winter as well as at a certain depth. The enumeration of culturable bacteria were done after 2, 4, 6, 8, 10, 13, 16, 22, 28 and 34 days using spread plate technique on

The decay rates of culturable *S. paratyphi* cells were calculated as per first order decay model using the following equation Log Nt/N0= -kt, where *Nt* is the number of bacteria at time *t, N*0 is number of bacteria at time 0, and *t* is expressed in days; *k* is the first-order constant calculated by linear regression technique. T99 (time required for 2 log reduction) values were

the survival at different salinities and temperature was analysed using two way analysis of

The survival curves of *S. paratyphi* in freshwater and mixing water at 20oC and 30oC are given in Fig. 2 and the inactivation rates and T99 values are given in Table 1. The results revealed that *S. paratyphi* showed significantly (p<0.01) higher survival at 20oC (T99= 25.99) compared to 30oC (T99= 17.68) in freshwater water indicating their better survival capacity at low temperature. However *S. paratyphi* did not show much difference in the survival response in mixing water at both temperature and the T99 respectively was 16.37 days at 20oC and 15.12 days at 30oC. The results revealed that *S. paratyphi* cells remained viable until 34 days at a high density of 105 CFU/mL. The salinity of the mixing water when it was collected was 12.77 ppt and the average saline concentration of the lake water was 12.5ppt

2/*k*.The difference in

of desired saline concentrations were prepared using fresh lake water with NaCl.

TSA agar plates and the colony forming units were counted.

calculated using the decay constant (*k*) in the following equation, T99=-

**2.3 Decay rate and statistical calculation**

when it was monitored over 2hr interval in a day.

variance (ANOVA).

**3. Results and discussion**

**2.2 Preparation of inocula**

Freshwater 30oC■ and 20oC □; mixing water 30oC ▲and 20oC ∆

Fig. 2. The survival curves of *S. paratyphi* in freshwater and mixing water at 20oC and 30oC

Table 1. Inactivation rates of *S. paratyphi* in freshwater and mixing water at 30oC and 20oC

Influence of a Salt Water Regulator on the Survival

submitted to a gradual increase in salinity.

(Mean ±SD, n = 4).

Response of *Salmonella Paratyphi* in Vembanadu Lake: India 53

25ppt at 20oC and 30oC and the results are represented in Fig 3-8 and the inactivation rates are given in Table 2 and 3. When the saltwater is closed the saline concentration on Northern part was reported to a maximum of 20ppt. Even though no significant variation in the survival response of *S. paratyphi* was noticed at 0, 5, 10, 15 and 20 ppt (p>0.05), they exhibited an extended survival for 34 days at 20oC and 30oC. They showed enhanced survival in water at 0 ppt at both temperatures as evident from T 99 values and it was 25.99 days at 20oC and 17.68 days at 30oC (Table 2 and 3). However as time goes depending on the increasing saline concentration from 5to 25 ppt it showed gradual decrease in the T99 values at both temperatures. The lowest T99 was observed at 25 ppt (8.61 and 7.25) and showed a significant (p<0.0001) decline of cultural cells at both temperature indicating the deleterious effect of high saline concentration. However the most suitable condition for their growth was found to be at 0 and 5 ppt and suggests that they can survive well at low salinity levels in Vembanadu lake. The results indicate that *Salmonella* can survive well in water weakly diluted or with gradually increasing saline concentrations. In agreement with our results Mezrioui *et al*. (1995) reported that when *Salmonella* suspended in stabilization ponds effluent and rapidly mixed with brackish water survival time was particularly short as we found at 25 ppt where it showed a sudden decline at both temperature, whereas it was prolonged when the bacteria was

Fig. 3. Survival curves of *S. paratyphi* in fresh sterile water at 0 ppt at 20oC (□)and 30oC (▲)

Even though the survival time was longer, in agreement with our results Sugumar & Mariyappan (2003) reported that *Salmonella* survived up to 24 weeks in sterile freshwater microcosm at 30oC but at low temperature it survived for 58 weeks. It is also documented that it survived for 54 days (Moore *et al*., 2003) in freshwater. Since *S. paratyphi* did not show much difference in survival response in mixing water at both temperatures, similar to our results Rhodes and Kator (1988) reported that *Salmonella* populations exhibited significantly less die-off in filtered estuarine water at temperatures of <10°C. In sterile estuarine water virtually unaltered bacterial densities over a 10-day period have also been reported by McCambridge & McMeekin (1980a,b). It has been documented in other studies that low temperature is favorable for the survival of *Salmonella* in (Vasconcelos & Swartz, 1976; Hernroth *et al*., 2010) and other enteric bacteria in aquatic environments (Craig *et al*., 2004; Sampson *et al*., 2006; Silhan *et al*., 2006).

The aim of conducting this survival experiments in freshwater and mixing water was to evaluate the public health risk associated with *S. paratyphi* in Vembanadul lake during the closure and subsequent opening of the regulator. While addressing this issue it has been noticed that similar to other studies *S. paratyphi* could survive very long time in freshwater and mixing water until the end of the experimental period. Therefore the log term survival potential *S. paratyphi* in freshwater may pose health risk since people use this region for their freshwater needs and we have already recorded high abundance of indicator bacteria and enteric pathogens (*Salmonella* serotypes such as *S. paratyphi* A, B, C and S. Newport) on the southern part during the closure of the saltwater regulator (Abhirosh *et al*., 2008). During the closure of the saltwater regulator the water on southern part of the lake become fresh and the natural flow is prevented which results in the accumulation of organic load in the southern part of the lake, giving proper environmental conditions for the multiplication of bacteria. Besides, the high survival capacity noticed at low temperature further increases the health risk during monsoon season because of the drop down of the water temperature to nearly 20oC and we already reported high prevalence of indicator and pathogenic bacteria in southern part of the lake during monsoon season (Abhirosh *et al*., 2008) and every year waterborne disease outbreaks occur during monsoon season. Prolonged survival of *S. paratyphi* in mixing water suggests that it can remain viable in water at high concentration (105CFU/ml) when the saltwater is open. It was almost similar to the results we obtained for *S. typhimurium* in Cochin estuary where we found it remained viable at even higher density (106 CFU/mL) until the end of experiment (Abhirosh & Hatha . 2005) at 20oC and 30oC. Our results are also in agreement with other studies that better survival of enteric bacteria in estuarine and other aquatic environments (Rhodes and Kator, 1988; Placha *et al*., 2001).

It has been reported that *Salmonella* may be of prolonged public health significance once it is introduced into tropical surface waters than *E. coli* (Jimenez *et al.,* 1989). Sporadic outbreaks of enteric fever due to *S. enterica* serovars paratyphi A, have been reported in India with an annual incidence of 3 million cases (Threlfall,2002; Misra *et al*., 2005). *S. enterica* serovar paratyphi A has emerged as an important cause of enteric fever in India Gupta *et al*. (2009). These reports suggest that the high survival of *S. paratyphi* in Vembanadu lake could be a public health concern.

In order to assess the survival in all possible saline concentrations on both sides of the salt water regulator, survival experiment were conducted in lake water at 5, 10, 15, 20, and

Even though the survival time was longer, in agreement with our results Sugumar & Mariyappan (2003) reported that *Salmonella* survived up to 24 weeks in sterile freshwater microcosm at 30oC but at low temperature it survived for 58 weeks. It is also documented that it survived for 54 days (Moore *et al*., 2003) in freshwater. Since *S. paratyphi* did not show much difference in survival response in mixing water at both temperatures, similar to our results Rhodes and Kator (1988) reported that *Salmonella* populations exhibited significantly less die-off in filtered estuarine water at temperatures of <10°C. In sterile estuarine water virtually unaltered bacterial densities over a 10-day period have also been reported by McCambridge & McMeekin (1980a,b). It has been documented in other studies that low temperature is favorable for the survival of *Salmonella* in (Vasconcelos & Swartz, 1976; Hernroth *et al*., 2010) and other enteric bacteria in aquatic environments (Craig *et al*., 2004;

The aim of conducting this survival experiments in freshwater and mixing water was to evaluate the public health risk associated with *S. paratyphi* in Vembanadul lake during the closure and subsequent opening of the regulator. While addressing this issue it has been noticed that similar to other studies *S. paratyphi* could survive very long time in freshwater and mixing water until the end of the experimental period. Therefore the log term survival potential *S. paratyphi* in freshwater may pose health risk since people use this region for their freshwater needs and we have already recorded high abundance of indicator bacteria and enteric pathogens (*Salmonella* serotypes such as *S. paratyphi* A, B, C and S. Newport) on the southern part during the closure of the saltwater regulator (Abhirosh *et al*., 2008). During the closure of the saltwater regulator the water on southern part of the lake become fresh and the natural flow is prevented which results in the accumulation of organic load in the southern part of the lake, giving proper environmental conditions for the multiplication of bacteria. Besides, the high survival capacity noticed at low temperature further increases the health risk during monsoon season because of the drop down of the water temperature to nearly 20oC and we already reported high prevalence of indicator and pathogenic bacteria in southern part of the lake during monsoon season (Abhirosh *et al*., 2008) and every year waterborne disease outbreaks occur during monsoon season. Prolonged survival of *S. paratyphi* in mixing water suggests that it can remain viable in water at high concentration (105CFU/ml) when the saltwater is open. It was almost similar to the results we obtained for *S. typhimurium* in Cochin estuary where we found it remained viable at even higher density (106 CFU/mL) until the end of experiment (Abhirosh & Hatha . 2005) at 20oC and 30oC. Our results are also in agreement with other studies that better survival of enteric bacteria in estuarine and other aquatic environments (Rhodes and Kator, 1988; Placha *et al*., 2001).

It has been reported that *Salmonella* may be of prolonged public health significance once it is introduced into tropical surface waters than *E. coli* (Jimenez *et al.,* 1989). Sporadic outbreaks of enteric fever due to *S. enterica* serovars paratyphi A, have been reported in India with an annual incidence of 3 million cases (Threlfall,2002; Misra *et al*., 2005). *S. enterica* serovar paratyphi A has emerged as an important cause of enteric fever in India Gupta *et al*. (2009). These reports suggest that the high survival of *S. paratyphi* in Vembanadu lake could be a

In order to assess the survival in all possible saline concentrations on both sides of the salt water regulator, survival experiment were conducted in lake water at 5, 10, 15, 20, and

Sampson *et al*., 2006; Silhan *et al*., 2006).

public health concern.

25ppt at 20oC and 30oC and the results are represented in Fig 3-8 and the inactivation rates are given in Table 2 and 3. When the saltwater is closed the saline concentration on Northern part was reported to a maximum of 20ppt. Even though no significant variation in the survival response of *S. paratyphi* was noticed at 0, 5, 10, 15 and 20 ppt (p>0.05), they exhibited an extended survival for 34 days at 20oC and 30oC. They showed enhanced survival in water at 0 ppt at both temperatures as evident from T 99 values and it was 25.99 days at 20oC and 17.68 days at 30oC (Table 2 and 3). However as time goes depending on the increasing saline concentration from 5to 25 ppt it showed gradual decrease in the T99 values at both temperatures. The lowest T99 was observed at 25 ppt (8.61 and 7.25) and showed a significant (p<0.0001) decline of cultural cells at both temperature indicating the deleterious effect of high saline concentration. However the most suitable condition for their growth was found to be at 0 and 5 ppt and suggests that they can survive well at low salinity levels in Vembanadu lake. The results indicate that *Salmonella* can survive well in water weakly diluted or with gradually increasing saline concentrations. In agreement with our results Mezrioui *et al*. (1995) reported that when *Salmonella* suspended in stabilization ponds effluent and rapidly mixed with brackish water survival time was particularly short as we found at 25 ppt where it showed a sudden decline at both temperature, whereas it was prolonged when the bacteria was submitted to a gradual increase in salinity.

Fig. 3. Survival curves of *S. paratyphi* in fresh sterile water at 0 ppt at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Influence of a Salt Water Regulator on the Survival

(Mean ±SD, n = 4).

(Mean ±SD, n = 4).

Response of *Salmonella Paratyphi* in Vembanadu Lake: India 55

Fig. 6. Survival curves of *S. paratyphi* in sterile water at 15 ppt at 20oC (□)and 30oC (▲)

Fig. 7. Survival curves of *S. paratyphi* in sterile water at 20 ppt at 20oC (□)and 30oC (▲)

Fig. 4. Survival curves of *S. paratyphi* in sterile water at 5 ppt at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Fig. 5. Survival curves of *S. paratyphi* in sterile water at 10 ppt at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Fig. 4. Survival curves of *S. paratyphi* in sterile water at 5 ppt at 20oC (□)and 30oC (▲) (Mean

Fig. 5. Survival curves of *S. paratyphi* in sterile water at 10 ppt at 20oC (□)and 30oC (▲)

±SD, n = 4).

(Mean ±SD, n = 4).

Fig. 6. Survival curves of *S. paratyphi* in sterile water at 15 ppt at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Fig. 7. Survival curves of *S. paratyphi* in sterile water at 20 ppt at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Influence of a Salt Water Regulator on the Survival

high saline concentration.

Response of *Salmonella Paratyphi* in Vembanadu Lake: India 57

Days 0 ppt 5 ppt 10ppt 15ppt 20ppt 25 ppt 0 0.00 0.00 0.00 0.00 0.00 0.00 2 -1.64 -0.97 -0.53 -1.52 -0.94 -1.98 4 -1.59 -0.98 -1.40 -2.14 -1.07 -2.68 6 -1.75 -0.98 -1.39 -1.95 -1.32 -2.74 8 -1.86 -1.35 -1.56 -1.70 -1.37 -2.94 10 -1.78 -1.47 -1.68 -1.67 -1.85 -2.85 13 -1.80 -1.56 -1.51 -1.85 -1.97 -3.20 16 -1.84 -1.53 -1.61 -1.69 -1.74 -7.22 22 -1.86 -1.52 -1.63 -1.72 -2.28 - 28 -2.57 -1.83 -2.02 -2.01 -3.45 - 34 -3.78 -2.00 -2.15 -2.52 -4.90 -

*K* -0.11 -0.07 -0.08 -0.09 -0.13 -0.27

*T99* 17.68 26.14 24.03 21.56 14.95 7.25 Table 3. Inactivation rates of *S. paratyphi* in water at different saline concentration at 30oC

We clearly observed that the decline of cell density with increasing saline concentration. Similar results were reported previously when a freshwater bacteria was exposed to brakish water, Painchaud *et al*. (1987, 1995; Painchaud and Therriault 1989). Similar gradients were reported in other estuaries (Albright, 1983) Rivers (Prieur, 1987). Painchaud *et al*.( 1995) reported that no mortality resulted from exposure to water with a salinity of >10ppt and high bacterial count at saline concentration between 0-5. He also reported drastic decline of bacteria at higher salinity (20ppt). This is in agreement with our results that we observed high survival rate at 0 and 5 ppt which was found to be the most suitable condition for the growth whereas at 25 ppt a drastic decline was noticed indicates the deleterious effect of

At higher saline concentration, for example in sea water, enteric bacteria are subjected to an immediate osmotic upshock, and their ability to overcome this by means of several osmoregulatory systems could largely influence their subsequent survival in the marine environment (Gauthier *et al*., 1987; Davies *et al*., 1995). This osmotic shock might be the reason for the sudden decline of cells at 25ppt. However there are contradicting reportes related to enteric bacterial survival in sea water. Lee *et al* (2010) Gerba and McLeod, (1976) reported that noh halophilic bacterial like *Salmonella* and *E. coli* do not survive well in seas water whereas Sugumar & Mariappan (2003) reported very long survival up to 16 to 48 week in sea water. Upon an osmotic upshift, bacterial cells accumulate or synthesize specific osmoprotectant molecules, in order to equalize osmotic pressure and avoid drastic loss of water from the cytoplasm (Csonka & Epstein, 1996). Although the accumulation or synthesis

Saline concentration

Fig. 8. Survival curves of *S. paratyphi* in sterile water at 25 ppt at at 20oC (□)and 30oC (▲) (Mean ±SD, n = 4).

Table 2. Inactivation rates of *S. paratyphi* in water at different saline concentration at 20oC

Fig. 8. Survival curves of *S. paratyphi* in sterile water at 25 ppt at at 20oC (□)and 30oC (▲)

0 0.00 0.00 0.00 0.00 0.00 0.00 2 -0.74 -0.31 -0.58 -0.85 -1.10 -1.26 4 -1.23 -0.43 -0.59 -0.82 -1.19 -1.49 6 -1.25 -0.78 -0.79 -0.96 -1.40 -1.67 8 -1.46 -0.92 -1.14 -1.44 -1.87 -2.54 10 -1.66 -1.13 -1.10 -1.44 -1.95 -2.46 13 -1.53 -1.12 -1.28 -1.47 -2.07 -2.80 16 -1.50 -1.34 -1.39 -1.65 -1.96 -3.35 22 -1.65 -1.38 -1.53 -1.67 -2.20 -5.07 28 -1.75 -1.59 -1.72 -2.03 -2.44 -7.22 34 -1.90 -1.66 -1.87 -2.19 -2.76 -

*k* -0.07 -0.06 -0.06 -0.08 -0.10 -0.23

*T99* 25.99 31.74 28.85 24.54 19.23 8.61

Table 2. Inactivation rates of *S. paratyphi* in water at different saline concentration at 20oC

Saline concentration 0 ppt 5 ppt 10ppt 15ppt 20ppt 25 ppt

(Mean ±SD, n = 4).

Days

Table 3. Inactivation rates of *S. paratyphi* in water at different saline concentration at 30oC

We clearly observed that the decline of cell density with increasing saline concentration. Similar results were reported previously when a freshwater bacteria was exposed to brakish water, Painchaud *et al*. (1987, 1995; Painchaud and Therriault 1989). Similar gradients were reported in other estuaries (Albright, 1983) Rivers (Prieur, 1987). Painchaud *et al*.( 1995) reported that no mortality resulted from exposure to water with a salinity of >10ppt and high bacterial count at saline concentration between 0-5. He also reported drastic decline of bacteria at higher salinity (20ppt). This is in agreement with our results that we observed high survival rate at 0 and 5 ppt which was found to be the most suitable condition for the growth whereas at 25 ppt a drastic decline was noticed indicates the deleterious effect of high saline concentration.

At higher saline concentration, for example in sea water, enteric bacteria are subjected to an immediate osmotic upshock, and their ability to overcome this by means of several osmoregulatory systems could largely influence their subsequent survival in the marine environment (Gauthier *et al*., 1987; Davies *et al*., 1995). This osmotic shock might be the reason for the sudden decline of cells at 25ppt. However there are contradicting reportes related to enteric bacterial survival in sea water. Lee *et al* (2010) Gerba and McLeod, (1976) reported that noh halophilic bacterial like *Salmonella* and *E. coli* do not survive well in seas water whereas Sugumar & Mariappan (2003) reported very long survival up to 16 to 48 week in sea water. Upon an osmotic upshift, bacterial cells accumulate or synthesize specific osmoprotectant molecules, in order to equalize osmotic pressure and avoid drastic loss of water from the cytoplasm (Csonka & Epstein, 1996). Although the accumulation or synthesis

Influence of a Salt Water Regulator on the Survival

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of such molecules (trehalose, glycine betaine, glutamic acid) has been reported in *Salmonella* spp. in estuarine waters, in the present study *S. paratyphi* might not overcome the stress caused by the high saline concentration at 25ppt whereas all other saline concentration tested were not found to be lethal.

The maximum saline concentration during the closure of the regulator on the Northern part of the Vembanadu lake is 20 ppt and minimum is 0ppt. Therefore in a year the possible seasonal salinity changes in Vembanadu lake could be between 0-20ppt. It has been generally assumed that when the regulator is closed the bacterial density on the Northern part would be very low because of the increasing saline concentration compared to Southern part. But it has been clearly observed that *S. paratyphi* exhibited high survival capacity in all possible saline gradients from 0 to 20ppt. The result indicates that *S. paratyphi* could survive very long time throughout Vembanadu lake irrespective of the saline concentration. Since the opening and closing of the regulator related to water quality and recreational activities has always been a topic of endless debate, the results indicates that the opening and closing of the salt water regulator does not have any significant impact on the survival (reduction in survival) of the enteric pathogens in relation to saline concentration in Vembanadu lake. However, if the saline concentration reaches 25 ppt it will negatively affect their survival (p<0.0001) but the maximum salinity so far reported is 20ppt. Since the lake is being used for various recreational activities the long term survival of *S. paratyphi* in all season regardless of saline concentration in Vembanadu lake could be a public health concern.

#### **4. Conclusions**

The results of the microcosm experiment revealed that *S. paratyphi* has a better survival capacity over a wide range of saline concentration from 0 to 20 ppt in Vembanadu lake. It exhibited significantly higher survival at 20oC compared to 30oC. It also showed prolonged survival in all other saline concentration at a higher density at both the temperature and the most suitable saline concentration was found to be 5 ppt. The result indicates that *S. paratyphi* could survive very long time throughout Vembanadu lake irrespective of the saline concentration. The opening and closing of the salt water regulator does not have any significant impact on the survival (reduction in survival) of the enteric pathogens in relation to saline concentration in Vembanadu lake. However, if the saline concentration reaches 25 ppt it will negatively affect their survival but the maximum salinity so far reported is 20ppt. Since the lake is being used for various recreational activities the long term survival of *S. paratyphi* in all season regardless of saline concentration in Vembanadu lake could be a public health concern.

#### **5. References**

of such molecules (trehalose, glycine betaine, glutamic acid) has been reported in *Salmonella* spp. in estuarine waters, in the present study *S. paratyphi* might not overcome the stress caused by the high saline concentration at 25ppt whereas all other saline concentration

The maximum saline concentration during the closure of the regulator on the Northern part of the Vembanadu lake is 20 ppt and minimum is 0ppt. Therefore in a year the possible seasonal salinity changes in Vembanadu lake could be between 0-20ppt. It has been generally assumed that when the regulator is closed the bacterial density on the Northern part would be very low because of the increasing saline concentration compared to Southern part. But it has been clearly observed that *S. paratyphi* exhibited high survival capacity in all possible saline gradients from 0 to 20ppt. The result indicates that *S. paratyphi* could survive very long time throughout Vembanadu lake irrespective of the saline concentration. Since the opening and closing of the regulator related to water quality and recreational activities has always been a topic of endless debate, the results indicates that the opening and closing of the salt water regulator does not have any significant impact on the survival (reduction in survival) of the enteric pathogens in relation to saline concentration in Vembanadu lake. However, if the saline concentration reaches 25 ppt it will negatively affect their survival (p<0.0001) but the maximum salinity so far reported is 20ppt. Since the lake is being used for various recreational activities the long term survival of *S. paratyphi* in all season regardless of saline concentration in

The results of the microcosm experiment revealed that *S. paratyphi* has a better survival capacity over a wide range of saline concentration from 0 to 20 ppt in Vembanadu lake. It exhibited significantly higher survival at 20oC compared to 30oC. It also showed prolonged survival in all other saline concentration at a higher density at both the temperature and the most suitable saline concentration was found to be 5 ppt. The result indicates that *S. paratyphi* could survive very long time throughout Vembanadu lake irrespective of the saline concentration. The opening and closing of the salt water regulator does not have any significant impact on the survival (reduction in survival) of the enteric pathogens in relation to saline concentration in Vembanadu lake. However, if the saline concentration reaches 25 ppt it will negatively affect their survival but the maximum salinity so far reported is 20ppt. Since the lake is being used for various recreational activities the long term survival of *S. paratyphi* in all season regardless of

Abhirosh, C.; & Hatha, A.A.M. (2005). Relative survival of *Escherichia coli* and *Salmonella*

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pathogenic bacteria in Vembanadu Lake: a function of salt water regulator, along

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tested were not found to be lethal.

Vembanadu lake could be a public health concern.

**4. Conclusions**

**5. References**

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**Part 2**

**Antimicrobial Chemotherapy**

## **Part 2**

**Antimicrobial Chemotherapy**

62 Salmonella – A Diversified Superbug

Sugumar, G.; & Mariappan, S. (2003) Survival of *Salmonella* sp. in Freshwater and Seawater

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in food- and water-borne infections. FEMS Microbiology Reviews, Vol. 26, No.

chamber app.aratus in situ. *Applied and Environmental Microbiology* , Vo. 31,No. 6,

**4**

*Nigeria*

**Antibiotic Resistance and the**

**Prospects of Medicinal Plants in**

*1Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto 2Department of Microbiology, Usmanu Danfodiyo University, Sokoto*

*Salmonella enterica* serotype typhi is the aetiological agent of typhoid fever, a multisystemic disease with protean manifestations and initial lesions in the bowel. Typhoid fever still remains a major public health problem in developing countries even in the twenty first century (Lin et al., 2000; Otegbayo et al., 2003). This was also the case in America and Europe three centuries ago, until measures for sanitary disposal and supply of potable water were put in place. Unacceptable morbidity and mortality are still recorded in developing countries in spite of availability of several drugs over the years for the treatment of typhoid fever. There is enough evidence to show that the prevalence of typhoid fever in any community is an index of communal hygiene and effectiveness of sanitary disposal. In Nigeria, as in other developing countries of the world, studies have estimated over 33 million cases and 500,000 deaths due to typhoid fever per year (Institute of Medicine, 1986). Otegbayo (2005) enumerated several factors responsible for the failure of public health measures to tame the tide of the continuing rise in the incidence, prevalence, morbidity and

Salmonellosis is an infection with Salmonella bacteria, often restricted to the gastrointestinal tract and is often a self limiting disease. Most individuals infected with *Salmonella typhimurium* experience mild gastrointestinal illness involving diarrhoea, chills, abdominal cramps, fever, head and body aches, nausea, and vomiting (Honish, 1999). Infections are usually self-limiting, and antimicrobial treatment is not recommended for uncomplicated illnesses (Aserkoff and Bennet, 1969; Gill and Hammer, 2001). However, extraintestinal infection can occur, particularly in very young, elderly, and immunocompromised patients (Angulo and Swerdlow, 1995; Thuluvath and McKendrick, 1998). In these cases, effective antimicrobial treatment is essential (Cruchaga et al., 2001). Every year, approximately 40,000 cases of salmonellosis are reported in the United States. The actual number of infections may be thirty or more times greater (CDC, 2006). In many parts of the world, such cases are either not documented or because many milder cases are not diagnosed or reported. Cases, however, of systemic disease due to *Salmonella typhimurium* and other salmonellae have been reported (Panhot and Agarwal, 1982; Varma et al., 2005). Salmonelloses have been

**1. Introduction**

mortality of typhoid fever.

**the Treatment of Salmonellosis**

A.A. Aliero1 and A.D. Ibrahim2

### **Antibiotic Resistance and the Prospects of Medicinal Plants in the Treatment of Salmonellosis**

A.A. Aliero1 and A.D. Ibrahim2 *1Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto 2Department of Microbiology, Usmanu Danfodiyo University, Sokoto Nigeria*

#### **1. Introduction**

*Salmonella enterica* serotype typhi is the aetiological agent of typhoid fever, a multisystemic disease with protean manifestations and initial lesions in the bowel. Typhoid fever still remains a major public health problem in developing countries even in the twenty first century (Lin et al., 2000; Otegbayo et al., 2003). This was also the case in America and Europe three centuries ago, until measures for sanitary disposal and supply of potable water were put in place. Unacceptable morbidity and mortality are still recorded in developing countries in spite of availability of several drugs over the years for the treatment of typhoid fever. There is enough evidence to show that the prevalence of typhoid fever in any community is an index of communal hygiene and effectiveness of sanitary disposal. In Nigeria, as in other developing countries of the world, studies have estimated over 33 million cases and 500,000 deaths due to typhoid fever per year (Institute of Medicine, 1986). Otegbayo (2005) enumerated several factors responsible for the failure of public health measures to tame the tide of the continuing rise in the incidence, prevalence, morbidity and mortality of typhoid fever.

Salmonellosis is an infection with Salmonella bacteria, often restricted to the gastrointestinal tract and is often a self limiting disease. Most individuals infected with *Salmonella typhimurium* experience mild gastrointestinal illness involving diarrhoea, chills, abdominal cramps, fever, head and body aches, nausea, and vomiting (Honish, 1999). Infections are usually self-limiting, and antimicrobial treatment is not recommended for uncomplicated illnesses (Aserkoff and Bennet, 1969; Gill and Hammer, 2001). However, extraintestinal infection can occur, particularly in very young, elderly, and immunocompromised patients (Angulo and Swerdlow, 1995; Thuluvath and McKendrick, 1998). In these cases, effective antimicrobial treatment is essential (Cruchaga et al., 2001). Every year, approximately 40,000 cases of salmonellosis are reported in the United States. The actual number of infections may be thirty or more times greater (CDC, 2006). In many parts of the world, such cases are either not documented or because many milder cases are not diagnosed or reported. Cases, however, of systemic disease due to *Salmonella typhimurium* and other salmonellae have been reported (Panhot and Agarwal, 1982; Varma et al., 2005). Salmonelloses have been

Antibiotic Resistance and the Prospects of

(Oluwatuyi et al., 2004).

**3. Problems of antibiotic resistance**

Medicinal Plants in the Treatment of *Salmonellosis* 67

negative organisms (Norrby et al., 2005). As resistance to old antibiotics spreads, the development of new antimicrobial agents has to be expedited if the problem is to be contained. However, the past record of rapid, widespread and emergence of resistance to newly introduced antimicrobial agents indicates that even new families of antimicrobial agents will have a short life expectancy (Coates et al., 2002). Confronted with a possible shortage of new antimicrobials, there is need to ensure a careful use of our available drugs. This has led to calls for controlled use of antibiotics through the reduction of dosage used per regime of treatment or by regulating prescriptions in areas such as animal husbandry and aquaculture (Hernandez, 2005). While, reduced use could lead to delayed resistance development, the emergence of resistant strains is from an evolutionary viewpoint inevitable. It becomes imperative therefore that alternative approaches are explored. Targeting and blocking resistance processes could be an attractive approach. The presence of efflux pumps and multidrug resistance (MDR) proteins in antibiotic resistant organisms contribute significantly to the intrinsic and acquired resistance in these pathogens. The discovery and development of new compounds that either block or circumvent resistance mechanisms could improve the containment, treatment, and eradication of these strains

The origin of antibiotic resistance extends much further back in evolutionary terms and reflects the attack and counter-attack of complex microbial flora in order to establish ecological niches and survive. Early treatment failures with antibiotics did represent a significant clinical problem because other classes of agents, with different cellular targets, were available. The emergence of multiple resistances is causing major problems in the treatment options today. Several factors drove this situation in the 1970s and 1980s, including the introduction of extended-spectrum agents and advances in medical techniques such as organ transplantation and cancer chemotherapy. The net result has been a huge selective pressure in favour of multiply resistant species. Coupled with this, there has been a sharp decline in the introduction of agents acting on new cellular targets over the last 30 years compared with the 20-year period following World War II. Smith (2004) reported that the resistant organisms causing concern among Gram-positive organisms at present are methicillin resistant *Staphylococcus aureus* (MRSA) and coagulasenegative staphylococci, glycopeptides intermediate sensitivity *S. aureus* (GISA), vancomycin-resistant *Enterococcus* (VRE) species and penicillin-resistant *Streptococcus pneumoniae*. Concerns among the Gram-negative organisms include multidrug-resistant *Pseudomonas aeruginosa, Stenotrophomonas maltophilia* and *Acinetobacter baumannii* and

members of the Enterobacteriaceae with extended-spectrum beta- lactamases.

might hold a promise for combating the problem of antibiotic resistance.

When infections become resistant to first choice or first line antimicrobials, treatment has to be switched to second-or third-line drugs, which are nearly always expensive (Sibanda and Okoh, 2007). In many poor countries in developing nations, the high cost of such replacement drugs is not easy to come-by, with the result that some diseases can no longer be treated in areas where resistance to first-line drugs is widespread (WHO, 2002). The alarming challenges facing physicians and pharmacist now, is the need to develop alternative approaches in addition to the search for new antimicrobial compounds (Sibanda and Okoh, 2007). Plants

reported to be season dependent and occur more in the winter than summer and often referred to as gastroenteritis or diarrheoa. Likewise more cases of diarrhoea caused by enterobacteriacea especially *E. coli*, occurring more during wet season than dry season (Olowe et al., 2003). Children are the most likely to get salmonellosis, however the elderly, and the immunocompromised are the most likely to have severe infections. It is estimated that approximately 600 persons die each year with acute salmonellosis as reported by Centre for disease control (CDC, 2006).

#### **2. Multidrug-resistant (MDR) strains of** *Salmonella*

Multidrug-resistant (MDR) strains of *Salmonella* are now encountered frequently and the rates of multidrug-resistance have increased considerably in recent years (CDC, 2006). Even worse, some variants of *Salmonella* have developed multidrug-resistance as an integral part of the genetic material of the organism, and are therefore likely to retain their drug-resistant genes even when antimicrobial drugs are no longer used, a situation where other resistant strains would typically lose their resistance (CDC, 2006). Most of the strains of *Salmonella typhimurium* isolated in a study in western part on Nigeria were resistant to drugs like streptomycin, amoxicilin, tetracycline, ampicillin, kanamycin and chloramphenicol (Olowe et al., 2007). This data is alarming since the isolates were already showing high resistance to drugs that are meant as alternate therapy to salmonellosis treatment; especially isolates from blood were resistant to the commonly used antibiotics. Drug-resistant *Salmonella* emerged in response to antimicrobial usage in food animals, which has also contributed or resulted in major outbreaks of salmonellosis (Olowe et al., 2007). Selective pressure from the use of antimicrobials is a major driving force behind the emergence of resistance, but other factors also need to be taken into consideration. Four types of species namely *S. typhi* (55.5%) *S. paratyphi* A (48.1%), *S. paratyphi* B (25.9%) and *S. typhimurium* (22.2%) were isolated from food samples in Namakkal and the isolates showed multiple drug resistance, and 100 % resistant to Vancomycin, Novobiocin, Nitrofurantoin, Ciproflaxacin, and Methicillin (Jegadeeshkumar et al., 2010). Jegadeeshkumar et al. (2010) also observed increasing resistance for Amoxiclave (92.55 %) and Bacitracin (78.57 %). Akinyemi et al. (2000) reported that out of the total blood samples cultured, 101 (15.9%) isolates of *Salmonella* species were isolated of which 68 (67.3%) were *S. typhi*, 17 (16.8%) and 16 (15.8%) were *S. paratyphi A*  and *S. arizonae* respectively. All the *S. typhi* and *S. paratyphi* isolates showed resistance to two or more of the 10 of 12 antibiotics tested particularly the 3-first-line antibiotics commonly used (chloramphenicol, ampicillin and cotrimoxazole) in the treatment of typhoid fever in Nigeria.

Since the discovery of antibiotics and their uses as chemotherapeutic agents, there was a belief in the medical fraternity that this would lead to the eradication of infectious diseases. However, diseases and disease agents that were once thought to have been controlled by antibiotics are returning in new forms resistant to antibiotic therapies (Levy and Marshall, 2004). Incidents of epidemics due to such drug resistant microorganisms are now a common global problem posing enormous public health concerns (Iwu et al., 1999). The global emergence of multi-drug resistant bacterial strains is increasingly limiting the effectiveness of current drugs and significantly causing treatment failure of infections (Hancock, 2005). Examples include methicillin-resistant staphylococci, pneumococci resistant to penicillin and macrolides, vancomycin-resistant enterococci as well as multidrug resistant gram-

reported to be season dependent and occur more in the winter than summer and often referred to as gastroenteritis or diarrheoa. Likewise more cases of diarrhoea caused by enterobacteriacea especially *E. coli*, occurring more during wet season than dry season (Olowe et al., 2003). Children are the most likely to get salmonellosis, however the elderly, and the immunocompromised are the most likely to have severe infections. It is estimated that approximately 600 persons die each year with acute salmonellosis as reported by Centre

Multidrug-resistant (MDR) strains of *Salmonella* are now encountered frequently and the rates of multidrug-resistance have increased considerably in recent years (CDC, 2006). Even worse, some variants of *Salmonella* have developed multidrug-resistance as an integral part of the genetic material of the organism, and are therefore likely to retain their drug-resistant genes even when antimicrobial drugs are no longer used, a situation where other resistant strains would typically lose their resistance (CDC, 2006). Most of the strains of *Salmonella typhimurium* isolated in a study in western part on Nigeria were resistant to drugs like streptomycin, amoxicilin, tetracycline, ampicillin, kanamycin and chloramphenicol (Olowe et al., 2007). This data is alarming since the isolates were already showing high resistance to drugs that are meant as alternate therapy to salmonellosis treatment; especially isolates from blood were resistant to the commonly used antibiotics. Drug-resistant *Salmonella* emerged in response to antimicrobial usage in food animals, which has also contributed or resulted in major outbreaks of salmonellosis (Olowe et al., 2007). Selective pressure from the use of antimicrobials is a major driving force behind the emergence of resistance, but other factors also need to be taken into consideration. Four types of species namely *S. typhi* (55.5%) *S. paratyphi* A (48.1%), *S. paratyphi* B (25.9%) and *S. typhimurium* (22.2%) were isolated from food samples in Namakkal and the isolates showed multiple drug resistance, and 100 % resistant to Vancomycin, Novobiocin, Nitrofurantoin, Ciproflaxacin, and Methicillin (Jegadeeshkumar et al., 2010). Jegadeeshkumar et al. (2010) also observed increasing resistance for Amoxiclave (92.55 %) and Bacitracin (78.57 %). Akinyemi et al. (2000) reported that out of the total blood samples cultured, 101 (15.9%) isolates of *Salmonella* species were isolated of which 68 (67.3%) were *S. typhi*, 17 (16.8%) and 16 (15.8%) were *S. paratyphi A*  and *S. arizonae* respectively. All the *S. typhi* and *S. paratyphi* isolates showed resistance to two or more of the 10 of 12 antibiotics tested particularly the 3-first-line antibiotics commonly used (chloramphenicol, ampicillin and cotrimoxazole) in the treatment of

Since the discovery of antibiotics and their uses as chemotherapeutic agents, there was a belief in the medical fraternity that this would lead to the eradication of infectious diseases. However, diseases and disease agents that were once thought to have been controlled by antibiotics are returning in new forms resistant to antibiotic therapies (Levy and Marshall, 2004). Incidents of epidemics due to such drug resistant microorganisms are now a common global problem posing enormous public health concerns (Iwu et al., 1999). The global emergence of multi-drug resistant bacterial strains is increasingly limiting the effectiveness of current drugs and significantly causing treatment failure of infections (Hancock, 2005). Examples include methicillin-resistant staphylococci, pneumococci resistant to penicillin and macrolides, vancomycin-resistant enterococci as well as multidrug resistant gram-

for disease control (CDC, 2006).

typhoid fever in Nigeria.

**2. Multidrug-resistant (MDR) strains of** *Salmonella*

negative organisms (Norrby et al., 2005). As resistance to old antibiotics spreads, the development of new antimicrobial agents has to be expedited if the problem is to be contained. However, the past record of rapid, widespread and emergence of resistance to newly introduced antimicrobial agents indicates that even new families of antimicrobial agents will have a short life expectancy (Coates et al., 2002). Confronted with a possible shortage of new antimicrobials, there is need to ensure a careful use of our available drugs. This has led to calls for controlled use of antibiotics through the reduction of dosage used per regime of treatment or by regulating prescriptions in areas such as animal husbandry and aquaculture (Hernandez, 2005). While, reduced use could lead to delayed resistance development, the emergence of resistant strains is from an evolutionary viewpoint inevitable. It becomes imperative therefore that alternative approaches are explored. Targeting and blocking resistance processes could be an attractive approach. The presence of efflux pumps and multidrug resistance (MDR) proteins in antibiotic resistant organisms contribute significantly to the intrinsic and acquired resistance in these pathogens. The discovery and development of new compounds that either block or circumvent resistance mechanisms could improve the containment, treatment, and eradication of these strains (Oluwatuyi et al., 2004).

#### **3. Problems of antibiotic resistance**

The origin of antibiotic resistance extends much further back in evolutionary terms and reflects the attack and counter-attack of complex microbial flora in order to establish ecological niches and survive. Early treatment failures with antibiotics did represent a significant clinical problem because other classes of agents, with different cellular targets, were available. The emergence of multiple resistances is causing major problems in the treatment options today. Several factors drove this situation in the 1970s and 1980s, including the introduction of extended-spectrum agents and advances in medical techniques such as organ transplantation and cancer chemotherapy. The net result has been a huge selective pressure in favour of multiply resistant species. Coupled with this, there has been a sharp decline in the introduction of agents acting on new cellular targets over the last 30 years compared with the 20-year period following World War II. Smith (2004) reported that the resistant organisms causing concern among Gram-positive organisms at present are methicillin resistant *Staphylococcus aureus* (MRSA) and coagulasenegative staphylococci, glycopeptides intermediate sensitivity *S. aureus* (GISA), vancomycin-resistant *Enterococcus* (VRE) species and penicillin-resistant *Streptococcus pneumoniae*. Concerns among the Gram-negative organisms include multidrug-resistant *Pseudomonas aeruginosa, Stenotrophomonas maltophilia* and *Acinetobacter baumannii* and members of the Enterobacteriaceae with extended-spectrum beta- lactamases.

When infections become resistant to first choice or first line antimicrobials, treatment has to be switched to second-or third-line drugs, which are nearly always expensive (Sibanda and Okoh, 2007). In many poor countries in developing nations, the high cost of such replacement drugs is not easy to come-by, with the result that some diseases can no longer be treated in areas where resistance to first-line drugs is widespread (WHO, 2002). The alarming challenges facing physicians and pharmacist now, is the need to develop alternative approaches in addition to the search for new antimicrobial compounds (Sibanda and Okoh, 2007). Plants might hold a promise for combating the problem of antibiotic resistance.

Antibiotic Resistance and the Prospects of

this group of organisms (Lomovskaya and Bostian, 2006).

force-dependent antiport mechanism (Brown and Skurray, 2001).

complement killing) genes (Guiney et al., 1994).

Medicinal Plants in the Treatment of *Salmonellosis* 69

The consequence of this drug extrusion is that, it leads to a reduced intracellular concentration of the antimicrobial such that the bacterium can survive under conditions of elevated antimicrobial concentration (Marquez, 2005). The MIC of the drug against such organisms will be higher than predicted. Multi-drug resistance efflux pumps are ubiquitous proteins present in both gram-positive and gram-negative bacteria as either chromosomally encoded or plasmid encoded (Akama et al., 2005). Although, such proteins are present constitutively in bacteria, the continued presence of the substrate induces over-expression (Teran et al., 2003). This increased transcription is responsible for the acquired resistance. In gram negatives bacteria, the effect of the efflux pumps in combination with the reduced drug uptake due to the double membrane barrier is responsible for the high inherent and acquired antibiotic resistance often associated with

The NorA protein which is the best studied chromosomally encoded pump in pathogenic gram positive bacteria (Hooper, 2005) has been reported to be present in *S. epidermidis* but appears to be absent in *Enterococcus faecalis* or in gram-negative organisms, such as *E. coli* and *K. pneumoniae* (Kaatz et al., 1993). Over expression of the NorA gene in *S. aureus* confers resistance to chloramphenicol and hydrophilic fluoroquinolone antimicrobials (Hooper, 2005; Kaatz and Seo, 1995; Hooper, 2005 instead of Hooper, 2005; Kaatz and Seo, 1995). QacA is a member of the major facilitator super-family of transport proteins, which are involved in the uniport, symport, and antiport of a wide range of substances across the cell membrane (Mitchell et al., 1998). The QacA multidrug exporter from *S. aureus* mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds. QacA provides resistance to these various compounds via a proton motive

Baucheron et al. (2004) reported the resistance of *Salmonella enteric* serovar Typhimurium to fluororoquinolones, chloramphenicol-florfenicol and tetracycline is highly dependent on the presence of AcrAB-TolC efflux pump. *S. enterica* includes nontyphoidal Salmonella belonging to different serotypes on the basis of the flagellar and somatic antigens, and represents one of the most important food-borne pathogens causing gastroenteritis in humans (Neidhardt, 1996). Nontyphoidal *S. enterica* strains are easily passed from animals to humans and are thus classified as zoonotic pathogens. They can colonize or infect humans as well as a variety of domesticated and wild animals ranging from mammals to birds and reptiles. Most infections are related to ingestion of contaminated food products rather than person-to-person transmission or direct fecal-oral transmission (Mead et al., 1999). Several *S. enterica* isolates are characterized by the presence of host-adapted virulence plasmids encoding genes contributing to colonization and resistance to complement killing, such as the spvA, spvB and spvC (salmonella plasmid virulence) and the rck (resistance to

In the last few years, Salmonella shows increasing antimicrobial resistance rates in isolates obtained from both food animals and humans. *S. enterica* strains belonging to different serotypes and showing multiple antibiotic resistance (to four or more antimicrobials) are now widespread in both developed and developing countries, most of these strains are zoonotic in origin, acquire their resistance in the food-animal host and cause human infections through the food chain (Threlfall, 2002). Salmonella infections have been associated with the ingestion of poultry, meat, milk and dairy products (Bean et al., 1996).

#### **4. Mechanisms of antibiotic resistance in pathogenic bacteria**

Resistance to antimicrobial agents typically occurs as a result of four main mechanisms namely enzymatic inactivation of the drug (Davies, 1994), alteration of target sites (Spratt, 1994), reduced cellular uptake (Smith, 2004) and extrusion by efflux (Nakaido, 1994). It has been reported that chemical modifications could be significant in antibiotic resistance, though exclusion from the cell of unaltered antibiotic represents the primary means in denying the antibiotic access to its targets and this is believed to enhance resistance even in cases where modification is the main mechanism (Li et al., 1994b).

#### **4.1 Alteration of target site**

Chemical modifications in the antibiotic target may result in reduced affinity of the antibiotic to its binding site (Lambert, 2005). These mechanisms have been reported to be employed by a number of pathogenic bacteria in over-powering the effect of antibiotics and are usually mediated by constitutive and inducible enzymes. Resistance to macrolides, lincosamide and streptogramin B antibiotics (MLSB resistance) in pathogenic *Streptococcus*  species is a result of methylation of the N6 amino group of an adenine residue in 23S rRNA. This is presumed to cause conformational changes in the ribosome leading to reduced binding affinity of these antibiotics to their binding sites in the 50S ribosomal subunit (Seppala et al., 1998; Kataja et al., 1998). Beta-lactams antibiotics function by binding to and inhibiting the biosynthetic activity of Penicillin Binding Proteins (PBPs), thereby blocking cell wall synthesis.

#### **4.2 Enzymatic inactivation**

The production of hydrolytic enzymes and group transferases is a strategy employed by a number of pathogens in evading the effect of antibiotics (Wright, 2005). Genes that code for antibiotic degrading enzymes are often carried on plasmids and other mobile genetic elements. The resistance to-lactam antibiotics by both gram negative and gram positive bacteria has long been attributed to -lactamases (Frere, 1995). These enzymes confer significant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the four membered-lactam ring (Wilke et al., 2005). Resistance to aminoglycosides in gramnegative bacteria is most often mediated by a variety of enzymes that modify the antibiotic molecule by acetylation, adenylation or phosphorylation (Over et al., 2001).

#### **4.3 Antibiotic efflux pump**

It is now widely recognized that constitutive expression of efflux pump proteins encoded by house-keeping genes that are widespread in bacterial genomes are largely responsible for the phenomenon of intrinsic antibiotic resistance (Lomovskaya and Bostian, 2006). Several studies have shown that active efflux can be a mechanism of resistance for almost all antibiotics (Li et al., 1994a; Gill et al., 1999; Lin et al., 2002). The majority of the efflux systems in bacteria are non-drug-specific proteins that can recognize and pump out a broad range of chemically and structurally unrelated compounds from bacteria in an energy-dependent manner, without drug alteration or degradation (Kumar and Schweizer, 2005).

Resistance to antimicrobial agents typically occurs as a result of four main mechanisms namely enzymatic inactivation of the drug (Davies, 1994), alteration of target sites (Spratt, 1994), reduced cellular uptake (Smith, 2004) and extrusion by efflux (Nakaido, 1994). It has been reported that chemical modifications could be significant in antibiotic resistance, though exclusion from the cell of unaltered antibiotic represents the primary means in denying the antibiotic access to its targets and this is believed to enhance resistance even in

Chemical modifications in the antibiotic target may result in reduced affinity of the antibiotic to its binding site (Lambert, 2005). These mechanisms have been reported to be employed by a number of pathogenic bacteria in over-powering the effect of antibiotics and are usually mediated by constitutive and inducible enzymes. Resistance to macrolides, lincosamide and streptogramin B antibiotics (MLSB resistance) in pathogenic *Streptococcus*  species is a result of methylation of the N6 amino group of an adenine residue in 23S rRNA. This is presumed to cause conformational changes in the ribosome leading to reduced binding affinity of these antibiotics to their binding sites in the 50S ribosomal subunit (Seppala et al., 1998; Kataja et al., 1998). Beta-lactams antibiotics function by binding to and inhibiting the biosynthetic activity of Penicillin Binding Proteins (PBPs), thereby blocking

The production of hydrolytic enzymes and group transferases is a strategy employed by a number of pathogens in evading the effect of antibiotics (Wright, 2005). Genes that code for antibiotic degrading enzymes are often carried on plasmids and other mobile genetic elements. The resistance to-lactam antibiotics by both gram negative and gram positive bacteria has long been attributed to -lactamases (Frere, 1995). These enzymes confer significant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the four membered-lactam ring (Wilke et al., 2005). Resistance to aminoglycosides in gramnegative bacteria is most often mediated by a variety of enzymes that modify the antibiotic

It is now widely recognized that constitutive expression of efflux pump proteins encoded by house-keeping genes that are widespread in bacterial genomes are largely responsible for the phenomenon of intrinsic antibiotic resistance (Lomovskaya and Bostian, 2006). Several studies have shown that active efflux can be a mechanism of resistance for almost all antibiotics (Li et al., 1994a; Gill et al., 1999; Lin et al., 2002). The majority of the efflux systems in bacteria are non-drug-specific proteins that can recognize and pump out a broad range of chemically and structurally unrelated compounds from bacteria in an energy-dependent manner, without drug alteration or degradation (Kumar and

molecule by acetylation, adenylation or phosphorylation (Over et al., 2001).

**4. Mechanisms of antibiotic resistance in pathogenic bacteria**

cases where modification is the main mechanism (Li et al., 1994b).

**4.1 Alteration of target site**

cell wall synthesis.

**4.2 Enzymatic inactivation**

**4.3 Antibiotic efflux pump**

Schweizer, 2005).

The consequence of this drug extrusion is that, it leads to a reduced intracellular concentration of the antimicrobial such that the bacterium can survive under conditions of elevated antimicrobial concentration (Marquez, 2005). The MIC of the drug against such organisms will be higher than predicted. Multi-drug resistance efflux pumps are ubiquitous proteins present in both gram-positive and gram-negative bacteria as either chromosomally encoded or plasmid encoded (Akama et al., 2005). Although, such proteins are present constitutively in bacteria, the continued presence of the substrate induces over-expression (Teran et al., 2003). This increased transcription is responsible for the acquired resistance. In gram negatives bacteria, the effect of the efflux pumps in combination with the reduced drug uptake due to the double membrane barrier is responsible for the high inherent and acquired antibiotic resistance often associated with this group of organisms (Lomovskaya and Bostian, 2006).

The NorA protein which is the best studied chromosomally encoded pump in pathogenic gram positive bacteria (Hooper, 2005) has been reported to be present in *S. epidermidis* but appears to be absent in *Enterococcus faecalis* or in gram-negative organisms, such as *E. coli* and *K. pneumoniae* (Kaatz et al., 1993). Over expression of the NorA gene in *S. aureus* confers resistance to chloramphenicol and hydrophilic fluoroquinolone antimicrobials (Hooper, 2005; Kaatz and Seo, 1995; Hooper, 2005 instead of Hooper, 2005; Kaatz and Seo, 1995). QacA is a member of the major facilitator super-family of transport proteins, which are involved in the uniport, symport, and antiport of a wide range of substances across the cell membrane (Mitchell et al., 1998). The QacA multidrug exporter from *S. aureus* mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds. QacA provides resistance to these various compounds via a proton motive force-dependent antiport mechanism (Brown and Skurray, 2001).

Baucheron et al. (2004) reported the resistance of *Salmonella enteric* serovar Typhimurium to fluororoquinolones, chloramphenicol-florfenicol and tetracycline is highly dependent on the presence of AcrAB-TolC efflux pump. *S. enterica* includes nontyphoidal Salmonella belonging to different serotypes on the basis of the flagellar and somatic antigens, and represents one of the most important food-borne pathogens causing gastroenteritis in humans (Neidhardt, 1996). Nontyphoidal *S. enterica* strains are easily passed from animals to humans and are thus classified as zoonotic pathogens. They can colonize or infect humans as well as a variety of domesticated and wild animals ranging from mammals to birds and reptiles. Most infections are related to ingestion of contaminated food products rather than person-to-person transmission or direct fecal-oral transmission (Mead et al., 1999). Several *S. enterica* isolates are characterized by the presence of host-adapted virulence plasmids encoding genes contributing to colonization and resistance to complement killing, such as the spvA, spvB and spvC (salmonella plasmid virulence) and the rck (resistance to complement killing) genes (Guiney et al., 1994).

In the last few years, Salmonella shows increasing antimicrobial resistance rates in isolates obtained from both food animals and humans. *S. enterica* strains belonging to different serotypes and showing multiple antibiotic resistance (to four or more antimicrobials) are now widespread in both developed and developing countries, most of these strains are zoonotic in origin, acquire their resistance in the food-animal host and cause human infections through the food chain (Threlfall, 2002). Salmonella infections have been associated with the ingestion of poultry, meat, milk and dairy products (Bean et al., 1996).

Antibiotic Resistance and the Prospects of

Villa et al., 2000; Mulvey et al., 2003).

horizontally transmitted to *S. enteriditis* strains.

Medicinal Plants in the Treatment of *Salmonellosis* 71

Extended-spectrum β-lactamase (ESBL), evolved from the blaTEM-1, blaTEM-2, and blaSHV-1 genes, extending resistance to new third-generation cephalosporins. During the last decade, infections caused by *S. enterica* carrying ESBLs have been reported, and most of the ESBL-producing strains were found to carry plasmids encoding the blaTEM- 1, and blaSHV-1 gene derivatives (Hammani et al., 1991; Morosini et al., 1995; Tassios et al., 1997;

In Southern Italy, during the period 1990 to 1998, several epidemiologically unrelated *S. enteriditis* isolates showing resistance to expanded-spectrum cephalosporins were recurrently isolated from ill patients. Most of these strains carried the blaSHV-12 gene located on conjugative plasmids (Villa et al., 2002a). Notably, this was the first case of acquisition of the blaSHV-12 gene by Salmonella in Italy and worldwide. However, SHV-12 encoding plasmids were previously encountered in *K. pneumoniae* isolated from hospitals throughout Italy (Laksai et al., 2000; Pagani et al., 2000). Therefore, it is plausible that SHV-12-encoding plasmids originated from nosocomial bacterial pathogens and were

In 1999, the spread of a *S. typhimurium* clone resistant to third generation cephalosporins has been reported in Russia, Hungary and Greece. In this case non distinguishable institutional and community outbreak of *S. typhimurium* isolates harboured a transferable plasmid containing the blaCTX-M gene (Tassios et al., 1999). The relatedness of resistance plasmids harboured by strains of various origins can be demonstrated by incompatibility grouping, restriction fragmentation pattern analysis and identification of specific resistance determinants located within the plasmids. These analyses may allow a better understanding

The *S. typhimurium* pSEM plasmid has a very similar restriction pattern to that of the *K. oxytoca* plasmid, pACM1. The pSEM plasmid was identified in 1997 in *S. typhimurium* strains isolated from children in Albania (Villa et al., 2000), while pACM1 was isolated from *K. oxytoca* strains responsible of a nosocomial outbreak in the USA (Preston et al., 1997; Preston et al., 1999). Both plasmids belonged to the same IncL/M group and conferred resistance to expanded-spectrum cephalosporins by the blaSHV-5 gene. Both plasmids carried a class 1 integron conferring aminoglycoside resistance by the aacA4, aacA1 and aadA1 resistance gene cassettes (Villa et al., 2000; Preston et al., 1997). Thus, these plasmids could be members of a family of broad-host-range replicons widely spreading among Gram-negative pathogens. Other plasmids of the IncL/M group showing similar restriction profiles, and carrying the blaSHV-5 gene and a class 1 integron, were previously described in several countries in Europe from clinical isolates of *P. aeruginosa* and *K. pneumoniae* (de Champs et al., 1991; Petit et al., 1990; de Champs et al., 1991; Prodinger et al., 1996; Preston et al., 1997 instaed of de Champs et al., 1991; Petit et al., 1990; Preston et al., 1997, Prodinger et al., 1996). The identification of a family of related plasmids has serious public health implications, since it demonstrates that broad-host-range plasmids carrying resistance to clinically relevant antibiotics can spread worldwide among bacteria responsible of both nosocomial and community-acquired infections. The fact that conserved plasmids can be identified in a wide variety of pathogens isolated in different countries, illustrates the important role of plasmids in the dissemination of antimicrobial resistance among Gram-negative bacteria.

Recently, a case of treatment failure due to ceftriaxone resistant *S. anatum* has been reported in Taiwan (Su et al., 2003). In this study, ceftriaxone-susceptible *S. anatum* was

of how resistance plasmids propagate, helping to trace their evolution.

Most infections result in self-limiting diarrhea and do not require antimicrobial treatment. However, severe life-threatening bacteremias and other deep-seated infections do occur, particularly in children and immunocompromised hosts and in these cases an antimicrobial therapy is recommended (Blaser and Feldman, 1981). Good drugs for Salmonella infections include fluoroquinolones, ampicillin, trimethoprimsulfamethoxazole or third-generation cephalosporins. Rising rates of resistance to ampicillin and trimethoprimsulfamethoxazole have significantly reduced their efficacy and fluoroquinolones are not approved for the use in children. Consequently, extended-spectrum cephalosporins have become the current drugs of choice for the treatment for invasive infections in children (Hohmann, 2001). The emergence of Salmonella species that are resistant to extended-spectrum cephalosporins (Herikstad et al., 1997; Threlfall et al., 1997) is cause of worldwide concern.

#### **4.4 Importance of plasmids in the dissemination of resistance in** *S. enterica*

Since the aminoglycoside antibiotic apramycin was licensed for veterinary use in the 1980s, resistance to apramycin and the related antibiotic gentamicin, one the most frequently used aminoglycoside in human therapy (Sibanda and Okoh, 2007). In the United Kindom, during the period 1982-84, the incidence of resistance to apramycin in salmonellas increased from 0.1% in 1982 to 1.4% in 1984 (Wray et al., 1986). Resistance to both apramycin and gentamicin was detected in different Salmonella serotypes, as well in *Escherichia coli*. In particular, the incidence of *S. enterica* (S.) *typhimurium* definitive type 204c (DT204c) from calves showed a dramatically increase (Wray et al., 1986). In *S. typhimurium* DT204c the gentamicin resistance was specified by three types of plasmids of the I1 incompatibility group, which also conferred resistance to apramycin (Threlfall et al., 1986). Most of these plasmids produced the enzyme aminoglycoside 3-N-acetyltransferase IV and the resistance was transferable by conjugation in most of the strains examined. The increasing incidence of the gentamicin-resistant *S. typhimurium* DT204c was also observed in humans, providing the first evidence that the use of apramycin in animal husbandry gave rise to resistance to gentamicin, an antimicrobial used for human therapy (Threlfall et al., 1986).

In the 1990s, the increasing frequency of *E. coli* and Salmonella with plasmids conferring resistance to gentamicin and apramycin was reported in other European countries (Chaslus-Dancla et al., 1991; Pohl et al., 1993). Gentamicin- and apramycin- resistant strains were isolated from both humans and cattle in France and Belgium and six different types of replicons were identified (Pohl et al., 1993). During the 1990-1997 alarming reports pointed out the rapid development in several countries of resistance to β-lactam antibiotics in Salmonella (Threlfall et al., 1997). A survey conducted between 1987 and 1994 in France, demonstrated a dramatic increase (from 0 to 42.5%) in the prevalence of β-lactam resistance among Salmonella isolates. Several types of β-lactamases were found on plasmids belonging to different incompatibility groups Q, P, F and HI (Llanes et al., 1999).

Resistance to β-lactams in Gram-negative bacteria is mediated predominantly by two major types of β- lactamases: the chromosomally-encoded enzymes of the Amber class C (e.g. AmpC β-lactamase in *Citrobacter, Enterobacter, Serratia spp, Morganella morganii* and *Pseudomonas aeruginosa*) or by plasmid-encoded enzymes of the Amber class A, in species that do not produce AmpC β-lactamases, such as *E. coli, Salmonella spp*., and *Shigella spp*. (Bauernfeind et al., 1998a).

Most infections result in self-limiting diarrhea and do not require antimicrobial treatment. However, severe life-threatening bacteremias and other deep-seated infections do occur, particularly in children and immunocompromised hosts and in these cases an antimicrobial therapy is recommended (Blaser and Feldman, 1981). Good drugs for Salmonella infections include fluoroquinolones, ampicillin, trimethoprimsulfamethoxazole or third-generation cephalosporins. Rising rates of resistance to ampicillin and trimethoprimsulfamethoxazole have significantly reduced their efficacy and fluoroquinolones are not approved for the use in children. Consequently, extended-spectrum cephalosporins have become the current drugs of choice for the treatment for invasive infections in children (Hohmann, 2001). The emergence of Salmonella species that are resistant to extended-spectrum cephalosporins

(Herikstad et al., 1997; Threlfall et al., 1997) is cause of worldwide concern.

gentamicin, an antimicrobial used for human therapy (Threlfall et al., 1986).

to different incompatibility groups Q, P, F and HI (Llanes et al., 1999).

(Bauernfeind et al., 1998a).

**4.4 Importance of plasmids in the dissemination of resistance in** *S. enterica*

Since the aminoglycoside antibiotic apramycin was licensed for veterinary use in the 1980s, resistance to apramycin and the related antibiotic gentamicin, one the most frequently used aminoglycoside in human therapy (Sibanda and Okoh, 2007). In the United Kindom, during the period 1982-84, the incidence of resistance to apramycin in salmonellas increased from 0.1% in 1982 to 1.4% in 1984 (Wray et al., 1986). Resistance to both apramycin and gentamicin was detected in different Salmonella serotypes, as well in *Escherichia coli*. In particular, the incidence of *S. enterica* (S.) *typhimurium* definitive type 204c (DT204c) from calves showed a dramatically increase (Wray et al., 1986). In *S. typhimurium* DT204c the gentamicin resistance was specified by three types of plasmids of the I1 incompatibility group, which also conferred resistance to apramycin (Threlfall et al., 1986). Most of these plasmids produced the enzyme aminoglycoside 3-N-acetyltransferase IV and the resistance was transferable by conjugation in most of the strains examined. The increasing incidence of the gentamicin-resistant *S. typhimurium* DT204c was also observed in humans, providing the first evidence that the use of apramycin in animal husbandry gave rise to resistance to

In the 1990s, the increasing frequency of *E. coli* and Salmonella with plasmids conferring resistance to gentamicin and apramycin was reported in other European countries (Chaslus-Dancla et al., 1991; Pohl et al., 1993). Gentamicin- and apramycin- resistant strains were isolated from both humans and cattle in France and Belgium and six different types of replicons were identified (Pohl et al., 1993). During the 1990-1997 alarming reports pointed out the rapid development in several countries of resistance to β-lactam antibiotics in Salmonella (Threlfall et al., 1997). A survey conducted between 1987 and 1994 in France, demonstrated a dramatic increase (from 0 to 42.5%) in the prevalence of β-lactam resistance among Salmonella isolates. Several types of β-lactamases were found on plasmids belonging

Resistance to β-lactams in Gram-negative bacteria is mediated predominantly by two major types of β- lactamases: the chromosomally-encoded enzymes of the Amber class C (e.g. AmpC β-lactamase in *Citrobacter, Enterobacter, Serratia spp, Morganella morganii* and *Pseudomonas aeruginosa*) or by plasmid-encoded enzymes of the Amber class A, in species that do not produce AmpC β-lactamases, such as *E. coli, Salmonella spp*., and *Shigella spp*. Extended-spectrum β-lactamase (ESBL), evolved from the blaTEM-1, blaTEM-2, and blaSHV-1 genes, extending resistance to new third-generation cephalosporins. During the last decade, infections caused by *S. enterica* carrying ESBLs have been reported, and most of the ESBL-producing strains were found to carry plasmids encoding the blaTEM- 1, and blaSHV-1 gene derivatives (Hammani et al., 1991; Morosini et al., 1995; Tassios et al., 1997; Villa et al., 2000; Mulvey et al., 2003).

In Southern Italy, during the period 1990 to 1998, several epidemiologically unrelated *S. enteriditis* isolates showing resistance to expanded-spectrum cephalosporins were recurrently isolated from ill patients. Most of these strains carried the blaSHV-12 gene located on conjugative plasmids (Villa et al., 2002a). Notably, this was the first case of acquisition of the blaSHV-12 gene by Salmonella in Italy and worldwide. However, SHV-12 encoding plasmids were previously encountered in *K. pneumoniae* isolated from hospitals throughout Italy (Laksai et al., 2000; Pagani et al., 2000). Therefore, it is plausible that SHV-12-encoding plasmids originated from nosocomial bacterial pathogens and were horizontally transmitted to *S. enteriditis* strains.

In 1999, the spread of a *S. typhimurium* clone resistant to third generation cephalosporins has been reported in Russia, Hungary and Greece. In this case non distinguishable institutional and community outbreak of *S. typhimurium* isolates harboured a transferable plasmid containing the blaCTX-M gene (Tassios et al., 1999). The relatedness of resistance plasmids harboured by strains of various origins can be demonstrated by incompatibility grouping, restriction fragmentation pattern analysis and identification of specific resistance determinants located within the plasmids. These analyses may allow a better understanding of how resistance plasmids propagate, helping to trace their evolution.

The *S. typhimurium* pSEM plasmid has a very similar restriction pattern to that of the *K. oxytoca* plasmid, pACM1. The pSEM plasmid was identified in 1997 in *S. typhimurium* strains isolated from children in Albania (Villa et al., 2000), while pACM1 was isolated from *K. oxytoca* strains responsible of a nosocomial outbreak in the USA (Preston et al., 1997; Preston et al., 1999). Both plasmids belonged to the same IncL/M group and conferred resistance to expanded-spectrum cephalosporins by the blaSHV-5 gene. Both plasmids carried a class 1 integron conferring aminoglycoside resistance by the aacA4, aacA1 and aadA1 resistance gene cassettes (Villa et al., 2000; Preston et al., 1997). Thus, these plasmids could be members of a family of broad-host-range replicons widely spreading among Gram-negative pathogens. Other plasmids of the IncL/M group showing similar restriction profiles, and carrying the blaSHV-5 gene and a class 1 integron, were previously described in several countries in Europe from clinical isolates of *P. aeruginosa* and *K. pneumoniae* (de Champs et al., 1991; Petit et al., 1990; de Champs et al., 1991; Prodinger et al., 1996; Preston et al., 1997 instaed of de Champs et al., 1991; Petit et al., 1990; Preston et al., 1997, Prodinger et al., 1996). The identification of a family of related plasmids has serious public health implications, since it demonstrates that broad-host-range plasmids carrying resistance to clinically relevant antibiotics can spread worldwide among bacteria responsible of both nosocomial and community-acquired infections. The fact that conserved plasmids can be identified in a wide variety of pathogens isolated in different countries, illustrates the important role of plasmids in the dissemination of antimicrobial resistance among Gram-negative bacteria.

Recently, a case of treatment failure due to ceftriaxone resistant *S. anatum* has been reported in Taiwan (Su et al., 2003). In this study, ceftriaxone-susceptible *S. anatum* was

Antibiotic Resistance and the Prospects of

**overcome resistance**

**overcoming resistance**

Medicinal Plants in the Treatment of *Salmonellosis* 73

The selection pressure exerted by the continued presence of bactericidal or bacteriostatic agents facilitates the emergence and dissemination of antibiotic resistance genes. Over generations, the genotypic makeup of bacterial populations is altered (Taylor et al., 2002). The clinical implications of this are that many infections become untreatable resulting in serious morbidity and mortality. Although the introduction of new compounds into clinical use has helped to curtail the spread of resistant pathogens, resistance to such new drugs, has developed in some cases. It has been observed by several studies that antibiotic combinations can have synergistic benefits and interactions between existing antibiotics (Bayer et al., 1980; Hallander et al., 1982; Hooton et al., 1984; Cottagnoud et al., 2000; instaed of Bayer et al., 1980; Hooton et al., 1984; Cottagnoud et al., 2000; Hallander et al., 1982.). Several current therapeutic regimes are based on synergistic interactions between antibiotics with different target sites. As new antimicrobial compounds are discovered, there is need to assess their potentials in combination therapies with old antibiotics that have been rendered ineffective by the development of resistant strains, even when such compounds are not directly evidently inhibitory. Taylor et al. (2002) suggested that the use of agents that do not kill pathogenic bacteria but modify them to produce a phenotype that is susceptible to the antibiotic could be an alternative approach to the treatment of infectious disease. Such agents could render the pathogen susceptible to a previously ineffective antibiotic, and because the modifying agent applies little or no direct selective pressure, this concept could slow down or prevent the emergence of resistant genotypes. The inhibition of resistance expression approach was successfully used in the production of Augmentin, a combination of amoxycillin and clavulanic acid (Reading and Cole, 1977). In this case, clavulanic acid is an inhibitor of class-Alactamases which is coadministered with amoxicillin. The combination has been used clinically since the late 1970s (Neu et al., 1993). A similar approach can be used for target-modifying enzymes and for efflux systems. A number of *in vitro* studies have reported the use of plant extracts in combination with antibiotics, with significant reduction in the MICs of the antibiotics against some resistant strains (Al-hebshi et al., 2006; Darwish et al., 2002; Al-hebshi et al., 2006; Betoni et al., 2006 instead of Al-hebshi et al., 2006; Darwish et al., 2002; Betoni et al., 2006). The curative effect of plant extracts in this combination study has been variably referred to as resistance modifying/modulating activity (Gibbons, 2004). This ability of plant extracts to potentiate antibiotics has not been well explained. It is speculated that inhibition of drug efflux, and alternative mechanisms of action could be responsible for the synergistic interactions between plant extracts and antibiotics (Lewis and Ausubel, 2006; Zhao et al., 2001;

**4.6 The use of resistance modifying agents in combination with antibiotics to**

Lewis and Ausubel, 2006; instead of Lewis and Ausubel, 2006; Zhao et al., 2001).

**4.7 Efflux pump inhibition in combination with antibiotics as a strategy for**

The discovery and development of clinically useful Efflux Pump Inhibitors (EPIs) that decrease the effectiveness of efflux pumps represents a significant advance in the development of therapeutic regimes for the treatment of MDR-related conditions. This approach termed the EPI strategy (Lomovskaya and Bostian, 2006), is based on blocking the activity of the pumps, resulting in the accumulation of the antibiotic inside the bacterial cell, consequently increasing access to its target sites. In addition, this will lead to increased susceptibility of the bacterium, thus implying that the therapeutic effect of the drug is

initially isolated from the urine of a 70-year-old diabetic patient hospitalized for the treatment of a large pressure sore in the sacral area and urinary tract infection. The unexpected emergence of the resistance during the treatment with ceftriaxone led to systemic bacteraemia by *S. anatum* and to the fatal outcome in the patient. The emergence of the resistance has been linked to the *in vivo* acquisition of a resistance plasmid carrying the CTX-M3 β-lactamase by the susceptible *S. anatum* strain. In the same hospital this βlactamase has been previously identified in clinical isolates of *E. coli, K. pneumoniae* and *Enterobacter cloacae*, suggesting that such bacteria may have acted as reservoirs of the resistance plasmid (Su et al., 2003).

#### **4.5 Plasmid-mediated resistance to expanded-spectrum cephalosporins encoded by the CMY-2 AmpC β- lactamase**

The ampC genes were regarded as exclusively chromosomal until 1989, when an AmpCtype β-lactamase was found for the first time on transmissible plasmids (Bauernfeind et al., 1998a). Plasmid-mediated AmpC β-lactamases belong to the homogeneous group of genes related to the chromosomal ampC gene of *Citrobacter freundii* (cmy-2, bil-1 and lat genes), the cmy-1, fox and mox family, or originate from the *Morganella morganii* AmpC βlactamase (DHA-1) (Bauernfeind et al., 1998a). The latter was identified on a plasmid in *S. enteriditis* (Barnaud et al., 1998; Verdet et al., 2000). The first case of plasmid-mediated CMY-2 in Salmonella was reported on a conjugative plasmid of *S. senftenberg* recovered in 1994 from stool of an Algerian child (Koeck et al., 1997). Review of 1996 data from the National Antimicrobial Resistance Monitoring System (NARMS) in the United States identified only 1 (0.1%) Salmonella isolate among 1272 human Salmonella isolates showing expanded spectrum cephalosporin resistance (Dunne et al., 2000). However, in 1999, NARMS reported the emergence of domestically acquired broad-spectrum cephalosporin resistant Salmonella, most of them producing the CMY-2 AmpC βlactamase (Dunne et al., 2000).

*S. typhimurium* strains carrying indistinguishable CMY-2-encoding plasmids were isolated in Nebraska from a patient and cattle during a local outbreak of salmonellosis, demonstrating that the Salmonella-resistant strain evolved primarily in livestock (Fey et al., 2000). Since 1998, *Salmonella* of human and animal origin were reported to show resistance to expanded-spectrum cephalosporins, in Iowa (Winokur et al., 2000; Winokur et al., 2001). Molecular studies demonstrate the emergence of plasmid encoded CMY-2 β-lactamase in most cephalosporin resistant isolates. During the 1998-1999 periods, nearly 16% of *E. coli* isolates and 5.1% of *Salmonella* isolates from clinically ill animals in Iowa produced CMY-2 (Winokur et al., 2000; Winokur et al., 2001).

Similar plasmids, carrying the cmy-2 gene were also reported in Salmonella isolated from animals in Illinois (Odeh et al., 2002). The spreading of the CMY-2-carrying plasmids in the USA was confirmed by molecular analysis of domestically acquired Salmonella strains of human origin isolated in nine different States, representing the 87% of the total expandedspectrum cephalosporin resistant Salmonella collected by the Center for Disease Control and Prevention (CDC) during the 1996-1998 surveillance periods. The isolates were distinguishable by their chromosomal DNA patterns, thus demonstrating that they did not represent the epidemic spread of a clonal strain (Dunne et al., 2000).

initially isolated from the urine of a 70-year-old diabetic patient hospitalized for the treatment of a large pressure sore in the sacral area and urinary tract infection. The unexpected emergence of the resistance during the treatment with ceftriaxone led to systemic bacteraemia by *S. anatum* and to the fatal outcome in the patient. The emergence of the resistance has been linked to the *in vivo* acquisition of a resistance plasmid carrying the CTX-M3 β-lactamase by the susceptible *S. anatum* strain. In the same hospital this βlactamase has been previously identified in clinical isolates of *E. coli, K. pneumoniae* and *Enterobacter cloacae*, suggesting that such bacteria may have acted as reservoirs of the

**4.5 Plasmid-mediated resistance to expanded-spectrum cephalosporins encoded by**

The ampC genes were regarded as exclusively chromosomal until 1989, when an AmpCtype β-lactamase was found for the first time on transmissible plasmids (Bauernfeind et al., 1998a). Plasmid-mediated AmpC β-lactamases belong to the homogeneous group of genes related to the chromosomal ampC gene of *Citrobacter freundii* (cmy-2, bil-1 and lat genes), the cmy-1, fox and mox family, or originate from the *Morganella morganii* AmpC βlactamase (DHA-1) (Bauernfeind et al., 1998a). The latter was identified on a plasmid in *S. enteriditis* (Barnaud et al., 1998; Verdet et al., 2000). The first case of plasmid-mediated CMY-2 in Salmonella was reported on a conjugative plasmid of *S. senftenberg* recovered in 1994 from stool of an Algerian child (Koeck et al., 1997). Review of 1996 data from the National Antimicrobial Resistance Monitoring System (NARMS) in the United States identified only 1 (0.1%) Salmonella isolate among 1272 human Salmonella isolates showing expanded spectrum cephalosporin resistance (Dunne et al., 2000). However, in 1999, NARMS reported the emergence of domestically acquired broad-spectrum cephalosporin resistant Salmonella, most of them producing the CMY-2 AmpC β-