Polymerase chain reaction for the identification of bacteria.

<p>Genomic DNA was isolated from the obtained isolates as well as reference strains, and subjected to mono- or multi-plex PCR as described in the Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142717#pone.0142717.t001" target="_blank">Table 1</a>. The experiments were repeated at least three times and representative gel pictures are shown. Note that each panel is composed from two separate gels since all the samples could not be accommodated in a single gel. <b>(A) PCR for genus-specific <i>tuf</i> genes of streptococci and staphylococci.</b> Lane designation: M, 100 bp ladder; 1–5, <i>Streptococcus</i> spp. isolates; 6, Reference strain Streptococcus AD1; 7, No template control for streptococcus; 8, Negative control (<i>S</i>. <i>aureus</i>, <i>E</i>. <i>coli</i>); 9, Reagent control; 10, Reference strain <i>S</i>. <i>aureus</i> 96; 11, No template control for staphylococcus; 12–18: <i>Staphylococcus</i> spp. isolates. <b>PCR for <i>S</i>. <i>aureus nuc</i> (lanes 1–11) and <i>E</i>. <i>coli alr</i> (lanes 12–21) genes.</b> Lane designation: M, 100 bp ladder; 1–8, <i>S</i>. <i>aureus</i> test isolates; 9, Reference strain SAU-3; 10, Negative control (<i>E</i>. <i>coli</i>); 11, No template control; 12, Negative control (<i>S</i>. <i>aureus</i>); 13, Reference strain EC11 (<i>E</i>. <i>coli</i>); 14–16, Test isolates of <i>E</i>. <i>coli</i>; 17, No template control; 18–20, Test isolates; 21, Negative control (streptococcus). <b>(B) PCR for the identification of CoNS species.</b> Lane designation: M, 100 bp ladder; 1, <i>S</i>. <i>haemolyticus</i> (MTCC 3383) control; 2, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 3, <i>S</i>. <i>saprophyticus</i> (MTCC 6155) control; 4, <i>S</i>. <i>arlettae</i> (JQ764624) control; 5, <i>S</i>. <i>chromogenes</i> (MTCC 3545) control; 6, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 7, <i>S</i>. <i>xylosus</i> (FJ90627.1) control; 8, <i>S</i>. <i>simulans</i> (AF495498.1) control; 9, <i>S</i>. <i>epidermidis</i> (MTCC 3615) control; 10, <i>S</i>. <i>haemolyticus</i> (MTCC 3383) control; 11, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 12, <i>S</i>. <i>saprophyticus</i> (MTCC 6155) control; 13, <i>S</i>. <i>arlettae</i> (JQ764624) control; 14, <i>S</i>. <i>chromogenes</i> (MTCC 3545) control; 15, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 16, <i>S</i>. <i>simulans</i> (AF495498.1) control; 17, <i>S</i>. <i>xylosus</i> (FJ90627.1) control; 18, <i>S</i>. <i>epidermidis</i> (MTCC 3615) control. This Panel represents two mutually exclusive pictures depicting the results of the standardization of one tube each of the two-tube multiplex PCR. In the left panel, primers for <i>S</i>. <i>arlettae</i>, <i>S</i>. <i>chromogenes</i>, <i>S</i>. <i>sciuri</i>, <i>S</i>. <i>epidermidis</i> and <i>S</i>. <i>saprophyticus</i> were used, and <i>S</i>. <i>haemolyticus</i>, <i>S</i>. <i>xylosus</i> and <i>S</i>. <i>simulans</i> DNA served as negative controls. In the right panel, primers for <i>S</i>. <i>equorum</i>, <i>S</i>. <i>haemolyticus</i>, <i>S</i>. <i>xylosus</i>, <i>S</i>. <i>simulans</i> and <i>S</i>. <i>fluerettii</i> were used, and <i>S</i>. <i>sciuri</i>, <i>S</i>. <i>sapryphyticus</i>, <i>S</i>. <i>arlettae</i>, <i>S</i>. <i>chromogenes</i> and <i>S</i>. <i>epidermidis</i> DNA served as negative controls. Numbers in parentheses indicate the GenBank Accession numbers or the MTCC culture designations. <b>(C) PCR for the identification of <i>Streptococcus</i> species.</b> Lane designation: M, 100 bp ladder; 1–20, Test streptococcal isolates streptococci (no amplification); 21, Negative control (<i>S</i>. <i>aureus</i>); 22, Negative control (<i>E</i>. <i>coli</i>); 23 & 24, No template control; 25, Tube 2 positive control (<i>Streptococcus</i> reference strain AD3); 26, Tube 1 positive controls (<i>Streptococcus</i> reference strains AD1 and AD6).</p

Isolation, biochemical and molecular identification, and in-vitro antimicrobial resistance patterns of bacteria isolated from bubaline subclinical mastitis in South India

Buffaloes are the second largest source of milk. Mastitis is a major impediment for milk production, but not much information is available about bubaline mastitis, especially subclinical mastitis. The aim of this study was to (a) investigate the application of various tests for the diagnosis of bubaline subclinical mastitis, (b) identify the major bacteria associated with it, and (c) evaluate the antibiotic resistance pattern of the bacteria. To this end, 190 quarter milk samples were collected from 57 domesticated dairy buffaloes from organized (64 samples) and unorganized (126 samples) sectors. Of these, 48.4%, 40.0%, 45.8%, 61.1%, and 61.6% were positive for subclinical mastitis by somatic cell count, electrical conductivity, California mastitis test, bromothymol blue test, and N-acetyl glucosaminidase test, respectively. As compared to the gold standard of somatic cell count, California mastitis test performed the best. However, a combination of the two methods was found to be the best option. Microbiological evaluation, both by biochemical methods as well as by monoplex and multiplex polymerase chain reaction, revealed that coagulase-negative staphylococci were the most predominant (64.8%) bacteria, followed by streptococci (18.1%), Escherichia coli (9.8%) and Staphylococcus aureus (7.3%). Most of the pathogens were resistant to multiple antibiotics, especially to β-lactam antibiotics. We propose that California mastitis test be combined with somatic cell count for diagnosis of subclinical mastitis in domestic dairy buffaloes. Further, our results reveal high resistance of the associated bacteria to the β-lactam class of antibiotics, and a possible major role of coagulase-negative staphylococci in causing the disease in India