Occurrence of Klebsiella pneumoniae and salmonella spp. in environmental samples

The present study was undertaken for the isolation and identification of Klebsiella pneumoniae and Salmonella spp. from environmental samples. A total of 120 soil and 120 water samples were collected from ponds and rivers in Thrissur. The samples were subjected to conventional culture techniques and K. pneumoniae and Salmonella spp. isolates were detected in 57.50 and 14.58 per cent samples, respectively. There were no significant differences between the occurrence of K. pneumoniae and Salmonella spp. in different soil and water samples, (p≥0.05). The isolates were subjected to molecular confirmation targeting rpoB gene for K. pneumoniae and invA gene for Salmonella spp. Out of the 138 K. pneumoniae and 35 Salmonella spp. isolates analysed, 86.96 and 97.14 per cent isolates were found positive on PCR. The results of the study revealed that environment acts as an important reservoir of K. pneumoniae and Salmonella spp.


abstract
The present study was undertaken for the isolation and identification of Klebsiella pneumoniae and Salmonella spp.from environmental samples.A total of 120 soil and 120 water samples were collected from ponds and rivers in Thrissur.The samples were subjected to conventional culture techniques and K. pneumoniae and Salmonella spp.isolates were detected in 57.50 and 14.58 per cent samples, respectively.There were no significant differences between the occurrence of K. pneumoniae and Salmonella spp. in different soil and water samples,(p≥0.05).The isolates were subjected to molecular confirmation targeting rpoB gene for K. pneumoniae and invA gene for Salmonella spp.Out of the 138 K. pneumoniae and 35 Salmonella spp.isolates analysed, 86.96 and 97.14 per cent isolates were found positive on PCR.The results of the study revealed that environment acts as an important reservoir of K. pneumoniae and Salmonella spp.

Keywords: Klebsiella pneumoniae, Salmonella spp., environment, water
Gram negative bacterial infections are highly significant public health concern worldwide.Among the Gram negative bacterial agents, K. pneumoniae and Salmonella spp.are important in terms of virulence and pathogenicity.These bacteria are often detected in the environment from which they enter the human body directly and through contamination of food materials.Klebsiella pneumoniae is an opportunistic pathogen and causes several types of infections in humans, including respiratory tract, urinary tract and bloodstream infections.These infections usually occur but are not limited to hospitalised or immunocompromised patients.The pneumonia caused by Klebsiella spp.causes mortality in as high as 50 per cent of the patients (He et al., 2016).In addition, they have an ability to adhere to the surfaces of urinary catheters and ventilators, forming biofilms causing catheter-associated urinary tract infections and ventilator-associated pneumonia, making it the common cause of death in patients admitted to intensive care units.The bacteria are main agents in the transmission and maintenance of antimicrobial resistance (AMR).The species  (Grimont and Weill, 2007).Considering the gravity of the infections caused by these bacteria, the present study was carried out to study the occurrence of K. pneumoniae and Salmonella spp. in environmental samples.

Materials and methods
A total of 120 water samples were collected from Thrissur district, which included pond water samples (60) and river water samples (60).From the respective pond sides and river banks, 60 soil samples each were also collected.The samples were collected during a span of 10 months, from September 2019 to July 2020.Sampling was performed across the three river basins in Thrissur district viz., Chalakkudy, Karuvannur and Kechery.
Approximately 250 mL water sample and 100g soil sample each were collected from each sampling site in sterile containers.The samples were then transported and processed in the Quality Control Laboratory, Department of Veterinary Pubic Health, College of Veterinary and Animal Sciences, Mannuthy.All the samples were subjected to isolation and identification of K. pneumoniae (Kumar et al., 2013) and Salmonella spp.(APHA, 2012) by conventional culture techniques with some modifications.
For isolation of K. pneumoniae, samples were enriched on nutrient broth (NB).Five grams of soil sample was transferred to 45 mL NB, which was incubated at 37°C for 24 h.In case of water samples, five millilitres of each sample was transferred to 45 mL NB.This was followed by selective plating onto MacConkey agar (MCA) and incubation at 37°C for 24 h.Characteristic large, pink mucoid colonies were selected and further confirmed by biochemical tests (Fig. 1).For isolation of Salmonella spp., the samples were enriched on tetrathionate (TT) broth, followed by selective plating on Xylose Lysine Deoxycholate (XLD) agar and incubated at 37°C for 24 h.Characteristic red colonies with black centres were selected and later confirmed by biochemical tests (Fig. 2).The significance of occurrence of K. pneumoniae and Salmonella spp.among different sources was analysed using chi-square test with the help of IBM -SPSS version 24.0.
The isolates were further subjected to molecular confirmation by polymerase chain reaction (PCR) targeting rpoB gene for K. pneumoniae (Bobbadi et al., 2020) and invA gene for Salmonella spp.(Amini et al., 2010).The primer sequences and cyclical conditions of the genes are mentioned in Table 1 and Table  2.

Results and discussion
Out of the 240 samples analysed, 138 (57.50 per cent) were found positive for K. pneumoniae by conventional culture techniques (Fig. 1).Of this, 54.35 per cent were from water samples and the rest 45.65 per cent were isolated from soil samples.On statistical analysis, there was no significant difference (p≥0.05) between the occurrence of K. pneumoniae in different water and soil samples (Table 3).Similar results were obtained in studies conducted by Podschun et al. (2001) in Germany and Tafoukt et al. (2017) in Algeria.
In case of Salmonella spp., 35 out of the 240 samples analysed (14.58 per cent) were found positive (Fig. 2).Of this, water samples and soil samples constituted 57.14 per cent and 42.86 per cent, respectively.There was no significant difference (p≥0.05) between the occurrences of Salmonella spp. in different water and soil samples (Table 4).In a study The isolates were then subjected to PCR for molecular confirmation.To confirm K. pneumoniae isolates, the house keeping gene rpoB was used.He et al. (2016) reported this gene to be a suitable PCR target for species level differentiation of K. pneumoniae.Of the 138 K. pneumoniae isolates identified by culture techniques, 120 isolates (86.96 per cent) were found positive (Fig. 3) by PCR.In a study conducted by Bobbadi et al. (2020) in Andhra Pradesh, 90 per cent of K. pneumoniae isolates were confirmed by PCR targeting rpoB gene.
To confirm Salmonella spp.isolates, conserved virulence gene invA was used.Of the 35 isolates identified as Salmonella spp.by conventional culture techniques, 34 (97.14 per cent) generated amplicons specific for invA (Fig. 4).Chaudhary et al. (2015) from Ahmedabad analysed different environmental samples for the presence of invA and concluded that it was the major gene coding for the virulence of Salmonella spp.
The results of the present study show that environment could be a potent source of K. pneumoniae and Salmonella spp.The pathogens gain entry to the human body directly, causing infections in susceptible patients.In addition, pond and river water are routinely used for the irrigation of agricultural fields, paving way for the contamination of fresh produce by the bacteria.There is also high chance of acquisition of resistant infections as more often than not, these bacteria may contain resistance genes, making the infections untreatable with many of the routine antibiotics.

conclusion
The present study demonstrated that antibiotic resistant K. pneumoniae and Salmonella spp.are present in the environment in abundance.A coordinated One Health approach is required at local and national level taking into account the role of environment in antibiotic resistance transmission, so that appropriate control measures can be put in place to limit the emergence and spread of AMR.This entails an integrated approach of surveillance at human, animal and environmental systems which should be coupled with awareness and education of public and professionals to contain global resistance load.

acknowledgement
The authors are thankful to the Kerala Veterinary and Animal Sciences University for providing the facilities needed for carrying out the research.

table 1 .
Primers used for PCR

table 3 .
Occurrence of K. pneumoniae in water and soil samples (Ko et al., 2002)ant, ns -Non significant table 4. Occurrence of Salmonella spp. in water and soil samples K. pneumoniae is again constituted by three different subspecies; K. pneumoniae subsp.pneumoniae,K.pneumoniae subsp.ozaenaeandK. pneumoniae subsp.Rhinoscleromatis(Ko et al., 2002).