Prevalence of Pseudomonas aeruginosa and other Microorganisms from Mastitis Milk and Their Antimicrobial Resistance Pattern

Sampling
 
101 milk samples (15-20 ml) from mastitis affected cattle and buffaloes from farms in and around Ludhiana and mastitis cases presented in Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India were collected aseptically (after discarding first few streaks of milk) from August 2018- April 2019. 76 samples were collected from cattle of different breeds mostly Holstien Friesian, Holstien Friesian cross and Sahiwal and 25 samples were collected from buffaloes of mostly Murrah and Nili Ravi breeds. The milk samples collected were tested immediately using Sodium Lauryl Sulphate test (SLS test) and positive samples were transported to the laboratory on ice for further possessing.
 
Infectious agent isolation and identification
 
Milk samples brought to the laboratory were inoculated onto Brain Heart Infusion (BHI) Agar, Eosin Methylene Blue (EMB) Agar, MacConkey Lactose (MLA) Agar, Cetrimide Agar, Baird Parker Agar (BPA) supplemented with egg-yolk tellurite emulsion, Edward’s medium and Blood Agar (BA) at 37°C for 18-24 h. The isolated bacterial colonies were examined culturally, microscopically and biochemically (catalase, oxidase, indole, methyl red, voges proskauer’s, citrate and triple sugar iron test) for the confirmation of the organism (Markey et al., 2013).
 
Extraction of bacterial genomic DNA
 
The extraction of bacterial genomic DNA was done as per the method of Sambrook and Russels (2001). In brief, 1.8 ml of overnight incubated individual bacterium in Luria-Bertani (LB) broth was centrifuged and to the pellet, 50 µl each of 10% sodium dodecyl sulphate (SDS) and proteinase K (200 µg/ml) were added and incubated at 60°C for 1h. 500 µl of phenol: chloroform: isoamyl alcohol (PCI; 25:24:1) was added and mixed by vortexing and centrifuged at 10,000x g for 10 minutes to collect the supernatant into a fresh tube. PCI step was repeated and to the supernatant equal volume of isopropanol and one-tenth volume of supernatant, 3M Sodium acetate (pH-5.2) was added, mixed and kept at -20°C for overnight incubation. Next day, it was centrifuged at 10,000x g for 20 minutes to collect the pellet and the pellet was washed twice with 500 µl of 70% ethanol and centrifuged at 10,000x g for 10 minutes and after removal of residual ethanol the pellet was reconstituted into 50 µl of nuclease free water (NFW) and stored at -20°C for further use.
 
Polymerase Chain Reaction (PCR)
 
The PCR assay was carried out for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus agalactiae identification (Table 3). A 25 µl PCR reaction mixture was formulated using 12.5 µl of master mix (2X one Taq Master Mix, New England Bio Labs, USA), 0.5 µl of 20 pmol/ul of each forward and reverse primers (Cusabio, China), 1.0 µl of template DNA and finally the reaction volume was made up to 25 µl using NFW (New England Bio Labs, USA). PCR was performed using thermocycler (Veriti, Applied Biosystems, USA) with the following conditions; an initial denaturation at 94°C for 5 minutes and later 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 1 minute and extension at 72°C for 1 minute. This was followed by a final extension at 72°C for 10 minutes. The PCR products were run on 1.5% agarose gel along with 100 bp DNA molecular weight marker (New England Biolabs, USA) at 5V/cm and visualized using gel documentation system (AlphaImager, Alpha Innotech, USA).
 

 
Antibiotic sensitivity test
 
The test was performed as per the protocol of National Committee for Clinical Laboratory Standards (NCCLS, 2002). In brief, overnight culture of individual bacteria in LB broth was spread on a Muller Hinton Agar plate with the help of a sterilized cotton swab and fifteen antibiotic discs viz., chloramphenicol (30 mcg), erythromycin (15 mcg), tetracycline (30 mcg), amoxycillin (10 mcg), co-trimoxazole (25 mcg), ciprofloxacin (5 mcg), gentamicin (10 mcg), cephalexin (30 mcg), ofloxacin (5 mcg), sparfloxacin (5 mcg), gatifloxacin (5 mcg), teicoplanin (30 mcg), azithromycin (15 mcg), vancomycin (30 mcg) and doxycycline (30 mcg) were placed equidistantly and incubated overnight. After incubation, the zone of inhibition was measured using scale and recorded in millimeters (mm). Later, these reading were compared with the manufacturers chart.

Isolation and identification of the causative agents
 
In the present study out of a total of 101 milk samples collected, 92 samples were SLS positive (91.1%). All the SLS positive samples when subjected to bacterial isolation 86 samples yielded positive bacterial growth (93.48%) whereas 6 samples yielded no growth on any medium (specific or non specific) (6.52%). From 101 milk samples, 76 samples were collected from cattle, of which 68 samples showed positive SLS test results (89.47%) and 63 of them yielded positive bacterial growth (92.65%) whereas 25 samples were collected from buffalo, whereof which 24 gave positive SLS test results (96%) and 23 yielded positive bacterial growth (95.83%). The results of our study are similar to findings of a study done by Hussein, (2012) that reported a positive bacterial isolation of 47 samples out of the total 61 milk samples that showed positive results by the CMT. Singh et al., (2019) reported from a study that out of a total of 69 SLS positive samples 50 samples showed positive bacterial growth (72.46%) and Kulaste, (2019) observed 93.47% correlation between bacterial isolation and SLS test results. Similarly, very high correlation between isolation rate and CMT positive too has been reported by Heleili et al., (2012) and Saidi et al., (2013) of 87.25% and 98% respectively which is absolutely in tandem to the findings of this study.

Out of 86 samples yielding bacterial growth, 76 (88.37%) showed single bacterial growth and 10 showed growth of more than one type bacteria (11.63%). Among 63 samples collected from cattle showing bacterial growth, 57 had single bacterial growth (90.47%) and 6 had mixed bacterial growth (9.52%) whereas in 23 samples yielding bacterial growth collected from buffalo, 19 had single bacterial growth (82.61%) and 4 had mixed bacterial growth (17.39%) (Table 1 and 2). In our present study a combination of two or more than two organisms were observed in 10 (11.63%) samples out of a total of 86 samples that exhibited bacterial growth. The above finding was similar to the findings of Hawari and Al-Dabbas (2008), Rahaman et al., (2010), Sayed et al., (2014) and Kulaste, (2019) indicating that there was presence of mixed infection. In another study Ranjan et al., (2011) reported a mixed bacterial infection of 4.74% among 190 milk samples tested which too is similar to the findings of the present study.
 

 

 
Over all it was observed that 86 samples yielded a total of 97 bacterial isolates in which 33 (34.02%) were Staphylococcus aureus isolates, 24 E. coli isolates (24.74%), 16 Klebsiella pneumoniae (16.49%) isolates, 5 P. aeruginosa (5.15%) isolates and 2 Streptococcus agalactiae (2.06%) isolates. Along with these organisms 12 (12.37%) Bacillus spp. and 5 (5.15%) Serratia marcescens were also isolated in the present study. These organisms were identified on the basis of cultural characters, gram’s staining and biochemical tests. On subjecting these individual bacterium to PCR using species specific primers (Table 3), we could amplify at the desired product size (Fig 1). Microbial culturing is considered as gold standard for diagnosis. Out of all the organisms isolated Staphylococcus aureus was the predominant bacteria followed by E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus agalactiae. The high rate of isolation of Staphylococcus aureus may be attributed to the fact that Staphylococcus species reservoirs are present on the skin of the udder and milk of the infected gland (Ranjan et al., 2010). Pseudomonas aeruginosa  had a prevalence of 5.15% and in a study by Sumathi et al., (2008) reported the prevalence of major pathogens isolated from clinical mastitis cases were Staphylococcus aureus (24%) and Escherichia coli (20%) followed by Staphylococcus epidermidis (16%), Streptococcus spp. (16%), Klebsiella spp. (10.67%), Bacillus spp. (4%), Proteus spp. and Pseudomonas spp. (1.33%). Pseudomonas aeruginosa is a metabolically versatile ubiquitous gram negative bacterium. It is an opportunistic pathogen present in soil and aquatic habitats and colonizes the animate surfaces of plants, animals and humans (Ramos, 2004).
 
Antibiotic sensitivity test
 
All the bacterial isolates (except for Bacillus spp. and Serratia marcescens) were subjected to antimicrobial sensitivity test individually against 15 commonly available antibiotics (Table 4) and it was observed that antibiotic sensitivity test on the isolated Staphylococcus aureus revealed sensitivity to sparfloxacin and doxycycline (75.8%) each, co-trimoxazole (63.6%), teicoplanin, chloramphenicol and ofloxacin  (57.6%) each, azithromycin (54.6%), for E. coli  sensitivity was seen for sparfloxacin (70.83%), ofloxacin (66.67%), followed by azithromycin, gatifloxacin and tetracycline (62.5%) each, chloramphenicol and doxycycline (58.33%) each.  Klebsiella pneumoniae showed sensitivity to chloramphenicol (75%), followed by azithromycin (68.7%), doxycycline and sparfloxacin (56.3%) each. Streptococcus agalactiae exhibited sensitivity to sparfloxacin and teicoplanin (100%) each, followed by gatifloxacin and doxycycline (50%) each. For Pseudomonas aeruginosa out of 15 antibiotics, resistance was observed for almost all the antibiotics and sensitivity was observed for only two antibiotics which were ofloxacin (80%), followed by gentamicin (40%) which is very alarming.
 

        
Overall, it was observed that maximum resistance was observed for amoxicillin, followed by cephalexin, erythromycin, vancomycin, gentamicin, co-trimoxazole ciprofloxacin, tetracycline and teicoplanin. The results were similar to the study of Kour et al., (2017; 2020) where sensitivity to chloramphenicol, cotrimoxazole, ofloxacin, gentamicin, ciprofloxacin, doxycycline and sparfloxacin resistance against penicillin, methicillin, amoxycillin and teicoplanin was observed. Similarly, Sarangi et al., (2009) observed that the use of newer drugs such as levofloxacin, enrofloxacin, chloramphenicol and gentamicin were effective in treatment of Staphylococcus mastitis which is relevant with the findings of current study too.
        
Also, from the study multidrug resistance was observed in Pseudomonas aeruginosa to almost all the antibiotics which is alarming and possesses a great therapeutic challenge. The ability of Pseudomonas aeruginosa to develop resistance against multiple classes of antimicrobials even during course of treatment causes greater complications (Lister et al., 2009). The resistance could either be chromosomally encoded as observed in the periplasmic β-lactamases or could be acquired via plasmids, transposons and bacteriophages. It could also arise from the changes in the outer-membrane permeability/changes or malfunctioning of energy-dependent multidrug efflux pump (Lambert, 2002; Chuanchuen et al., 2008). Though, the mechanism of occurrence of antibiotic resistance could vary but from the study it seems that Pseudomonas aeruginosa was most resistant among all the bacterial isolates which is a cause of concern. Thus, further more intensive studies to isolate, identify Pseudomonas aerugninosa along with its antimicrobial resistance is suggested.

It could be concluded from the study that the main microorganisms isolated from mastitis cases were Staphylococcus aureus, followed by E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Streptococcus agalactiae along with Bacillus spp. and Serratia marcescens occurring either singly or in combination. The prevalence of Pseudomonas aeruginosa was found to be 5.15% and on the basis of antibiotic sensitivity test. Pseudomonas aeruginosa showed multidrug resistance to almost all the antibiotics tested in the present study.

The authors are grateful to the University Grant Commission (UGC), India for the financial support MRP-MAJOR-VETE-201331898 (General). The authors are also thankful to the Director Research, GADVASU for providing the necessary laboratory facilities.