A total of 4378 cattle and buffalo were screened during period of study (July 2018-June 19). Out of which 27 Pseudomonas aeruginosa were isolated. The isolates were confirmed phenotypically based on pigment production on nutrient agar. These were then confirmed by PCR amplification of species specific oligonucleotide sequences. All the 27 isolates amplified 956bp amplicon 16srRNA Pseudomonas aeruginosa species specific nucleotide sequence. The isolates were also checked for exo and agl D virulence associated genes. All of them amplified 540bp and 313bp amplicon of exo gene and agl D gene. All the isolates were subjected to antibiotic sensitivity testing. Most of the isolates showed highest sensitivity for levofloxacin, streptomycin and enrofloxacin followed by gentamicin, moxifloxacin and amikacin. Neomycin, cefoperazone and ceftriaxone were intermediate in action.
Pseudomonas aeruginosa is a gram’s negative opportunistic environmental pathogen and can cause severe clinical diseases in both animals and humans. Prevalence of pseudomonas mastitis is only sporadic (<10% prevalence) as reported previously, but it may be a serious herd problem due to persistence of high antibiotic resistance strains (Livermore, 2002; Sharma and Sindhu, 2007). It is typically found in soil, water, skin flora and most man-made environments as it requires minimal amounts of oxygen for growth. Thereby allowing it to colonize a multitude of both natural and artificial environments (Hameed et al., 2007). Contaminated teat dips, teat wipes, and dry-cow intramammary treatment tubes have also been implicated in outbreaks of acute and subacute mastitis in dairy cows, sheep, and goats. Contaminated dry-cow intramammary infusion tubes have been found to be the cause of P. aeruginosa mastitis following calving in a dairy herd in Australia with the outbreak resulting in the loss of almost one-third of the herd (death or culling) (Kelly and Wilson, 2016). The presence of unsanitary housing and bedding conditions can contribute to occasional outbreaks of P. aeruginosa infections.
When the host defences are decreased by stress, concomitant disease, or by nutritional imbalances, P. aeruginosa efficiently attacks weak or injured tissues of teats or mammary gland causing acute mastitis with systemic signs as well as subclinical chronic mastitis. In severe cases, the affected gland may be necrotic or even gangrenous and the blood stained milk. Most P. aeruginosa strains possess the type III secretion system (TTSS), which may increase somatic cell counts (SCCs) in milk from mastitis-affected cows. Moreover, most of P. aeruginosa bacterial cells can form biofilms, thereby reducing antibiotic efficacy (Park et al., 2014). The pathogen uses a wide range of virulence factors, including proteins capable of inducing toxicity, which effectively damage tissues (Lyczak et al., 2000). Some of these toxic effector proteins are injected into the cytosol of host cells through the type III secretion system (TTSS) which are involved in the disruption of epithelial surface and cell death. These TTSS proteins include cytotoxin (Exo U), ADP-ribosylating enzymes (Exo S and Exo T) and adenylcyclase (Exo Y). These cytotoxinsare involved in adhesion, phagocytosis and systemic spread of the bacterial cells together (Vance et al., 2005; Engel and Balachandran, 2009; Hauser, 2009).
How to cite this article: Yadav, R., Chhabra, R., Shrinet, G. and Singh, M. (2020). Isolation of Pseudomonas aeruginosa from bovine mastitic milk sample along with antibiogram study. J. Anim. Res.,10(2): 269-273.
Outer membrane of P. aeruginosa bacterium creates permeability barrier, resultant became naturally resistant to many antibiotics. Moreover, Pseudomonas maintains antibiotic resistance plasmids, R-factors and resistance transfer fragment (RTFs) and it is able to transfer these genes by horizontal gene transfer (HGT), mainly transduction and conjugation (Livermore, 2002; Kobayashi et al., 2013). This high level of multidrug resistant P. aeruginosa strains are major concern to dairy industries. Although, incidence of P. aeruginosa infection are reported very few (less than 3%) if udder is already infected by Staphylococcus or Streptococcus etc. pathogens, but mastitis remained incurable due to presence of multidrug resistance strains of P. aeruginosa(Ama et al., 2016). On keeping above facts in mind the present study was designed to isolates and identify virulent P. aeruginosa from mastitic milk of cattle and buffalo along with their antibiogram.
Materials and methodsSample collection, isolation and identification
Mastitic milk samples of 4378 cattle and buffalo during year 2018-19 were processed for isolation and identification of Pseudomonas aeruginosa isolates. All the samples were plated onto Nutrient agar and incubated at 370C for 24 h. The presumptive isolates were phenotypically characterized on the basis of colony morphology, catalase, oxidase, pigmentation and gram’s staining.
Primer sequence for PCR amplification
Molecular confirmation and virulence gene detection of P. aeruginosa isolates
Presumptive Pseudomonas aeruginosa isolates were confirmed by genus and species specific 16S rRNA nucleotide sequence (Spilker et al. 2004). The virulence associated genes were also detected molecularly by exo S and agl D nucleotide sequence as shown in table 1. The reaction mixture (total volume 25μl) was prepared by mixing 12.5μl GoTaq® Green Master Mix (Promega), 1μl Primer-1 and 2 each (10 pM/μl), 3μl Template DNA (25ng/ μl) and 7.5μl Nuclease free water (upto final volume 25μl). PCR Amplification was carried out in ‘Thermal cycler gradient (BR Gradient Thermal Cycler)’ as follows: initial cycle of denaturation at 95 °C for 5min, 30 cycle at (denaturation at 95 °C for 45 s, primer annealing at 55 °C for 45 s and primer extension at 72 °C for 60 s), and final extension at 72 °C for 5 min. The PCR products were analyzed by electrophoresis on 1-1.5% agarose gel with ethidium bromide (0.5 μg/ml) in 1X TAE buffer for 60 min at 100 V. The gel was then visualized by Azure C-15 UVP gel documentation system.
Determination of antibiotic resistant strains of P. aeruginosa isolates
The antibiotic sensitivity testing was carried out as per standard protocol describe by Kirby et al. (1966) against 16 different antibiotics. Briefly, the isolates were inoculated in sterile BHI broth (5 ml) and incubated for 18-24 h at 37°C. The opacity of 0.5 McFarland opacity standards (Quinn et al., 2002) was well spread over the Muller Hinton agar with the help of sterilized swab and after that antibiotic discs were carefully placed, then incubated for 24 h at 37°C. The zone of inhibition was measured for each disc and interpretation was done as per Clinical and Laboratory Standards Institute (CLSI) guidelines (Wayne, 2009). All Multidrug resistant (MDR) isolates were evaluated for their multiple antibiotic resistances (MAR) index (Krumperman, 1983).
Results and Discussion
Pseudomonas aeruginosa is becoming a threat to dairy animals causing mastitis by drug resistance strain, since it requires few nutrients to multiply. It is the most commonly isolated microorganisms from clinical specimens, usually responsible for nosocomial infections (Chattergee et al., 2016). In the present study, 27 (17 from buffalo and 10 from cattle origin) Pseudomonas aeruginosa were isolated from mastitic milk samples of 4378 cattle and buffalo during year 2018-19. All the isolates were catalase and oxidase positive and produce green pigmentation on nutrient agar plate. Further, molecular confirmation was done by PCR amplification of genus and species specific oligonucleotide sequences (Fig. 1). All 27 isolates amplified 618bp amplicon and 956 bp amplicon of 16S rRNA Pseudomonas genus specific and 16S rRNA P. aeruginosa species specific nucleotide sequence. The prevalence of P. aeruginosa was detected as 0.61% in the present study. In agreement to our study, Nam et al. (2009) reported 5.6% prevalence of P. aeruginosa from bovine mastitis between 2003 and 2008 in Korea and Vasquez-Garcia et al. (2017) detected 9.6% prevalence of P. aeruginosa from bovine in Brazil. Banerjee et al. (2017) also detected 5.4% cases of bovine subclinical mastitis in South Bengal, India were associated with P. aeruginosa. Contrarily, Scaccabarozzi et al. (2015) detected high prevalence (nearly 27%) of P. aeruginosa as causative agent of mastitis in goats.
All the 27 P. aeruginosa isolates amplified 313bp amplicon of agl D virulence associated genes and 540bp amplicon of exoenzyme, exo S gene which helps in invasion. exo S and exo T are two homologous bifunctional Type III Secretion System (T3SS) virulence factors that induce apoptosis in target host cells (Kaminski et al. 2018).
Growth of P. aeruginosa on nutrient agar plate, PCR amplification of 16SrRNA genus specific (B), 16SrRNA P. aeruginosa specific specific (C) and virulence genes exoS and aglD gene (D)sequences
Similar to our observation Park et al. (2014) also detected TTSS-related genes (exo U and/or exo S genes) in 82.7% of the isolates including the invasive (exo U-/exo S+, 69.4%), cytotoxic (exo U+/exo S-, 8.3%) and cytotoxic/ invasive strains (exo U+/ exo S+, 5.0%). The agl D gene of P. aeruginosa is responsible for synthesis of the capsular polysaccharide alginate, an important virulence factor expressed in cystic fibrosis (CF) cases (Baynham et al. 1996). Similar to present study, Taee et al. (2014) 98% detection of agl D gene by PCR amplification from P. aeruginosa of clinical isolates.
All the isolates were tested against 16 antibiotics for antibiotic sensitivity. All the isolates showed absolute resistant to chloramphenicol, penicillin-G, cloxacillin, tetracycline, amoxycillin, kanamycin and ampicillin. The isolates were most sensitive to levofloxacin, streptomycin, enrofloxacin followed by gentamicin, moxifloxacin and amikacin (Fig. 2). Neomycin, cefoperazone and ceftriaxone were intermediate in action. Standard antibiotic regimes against P. aeruginosa are increasingly becoming ineffective due to the rise in drug resistance (Chattergee et al., 2016). Park et al. (2014) detected efficiency of antibiotic therapy against P. aeruginosa related bovine mastitis and found that majority of isolates were sensitive to gentamicin, amikacin, meropenem and ciprofloxacin. They also suggested that determination of efficacy could also be improved by MIC analysis of the isolates.
In contrast to our observation, Smith et al. (2012) reported that all the 20 P. aeruginosa isolates from cystic fibrosis cases (humans) showed resistance to gentamicin (80%) and ciprofloxacin (70%). However, detection of resistance towards amoxycillin (100%), tetracycline (95%), augmentin (95%), ofloxacin (80%), nalidixic Acid (100%), was similar to observations in the present study. Imipenem was the drug of choice for treatment of cystic fibrosis previously but increasing trends in imipenem resistance leaves fluoroquinolones only in terms of susceptibility. The cephalosporins tested were showing emergence of resistance due to indiscriminate use.
Graphical representation of antibiotic sensitivity and resistance patterns of P. aeruginosa isolates
Conclusion
From this study, majority of isolates were highly pathogenic on the basis of detection of exo S and agl D virulence genes and also resistant to majority of antibiotics (eight antibiotics) signifying constant antibiotic screening must be done before mastitis treatment for Pseudomonas aeruginosa infections.
Conflict of Interest
Authors don’t have any conflict of interests.
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