Manoj Kumar Mohan Nair

Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.

Cloning and Sequencing of the ompA Gene of Enterobacter sakazakii and Development of an ompA-Targeted PCR for Rapid Detection of Enterobacter sakazakii in Infant Formula

ABSTRACT Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 103 CFU/ml of E. sakazakii bacteria in infant formula directly and 10−1 CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.

Role of bacterial OmpA and host cytoskeleton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii.

Enterobacter sakazakii is an emerging pathogen in neonates and infants. Interactions of E. sakazakii with intestinal epithelium could be vital in the pathogenesis of enteric infections and in its systemic dissemination. The present study investigated the interaction of E. sakazakii with human intestinal epithelial (INT407) cells and the role of bacterial outer membrane protein A (OmpA) and host cytoskeleton in these interactions. E. sakazakii invaded INT407 cells with moderate efficiency. An ompA– mutant of E. sakazakii was significantly attenuated in its invasiveness, and complementation restored the invasive phenotype significantly. Drugs acting on host cell microfilaments (MF) and microtubules (MT) significantly inhibited bacterial invasion. Localization of both microfilaments (MF) and microtubules (MT) was observed in INT407 cells following E. sakazakii infection. The results suggest that E. sakazakii invasion of INT407 cells involves participation of both MF and MT and bacterial OmpA plays a critical role in invasion.

Outer Membrane Protein A (OmpA) of Cronobacter sakazakii Binds Fibronectin and Contributes to Invasion of Human Brain Microvascular Endothelial Cells

Cronobacter sakazakii is an emerging foodborne pathogen that causes severe meningitis and meningoencephalitis in neonates. Currently there is a dearth of information available on the virulence factors of C. sakazakii and the pathogenic mechanisms involved in its neonatal infections. The invasion and translocation of the blood-brain barrier formed by brain microvascular endothelial cells (BMEC) is critical in the pathogenesis of neonatal bacterial meningitis. Because bacterial binding of fibronectin is an initial step in the invasion of BMEC, the role of a major surface-expressed fibronectin-binding protein of C. sakazakii in invasion of BMEC was investigated. Outer membrane protein A was identified as a major fibronectin-binding protein of C. sakazakii, and an isogenic ompA mutant of C. sakazakii exhibited significantly (p < 0.05) attenuated invasion in BMEC compared with the wild-type strain. The findings of this study indicate that outer membrane protein A is one of the determinants that contribute to C. sakazakii invasion of human BMEC in vitro, and may potentially play a role in the pathogenesis of neonatal meningitis caused by this organism.

Inactivation of Enterobacter sakazakii in Reconstituted Infant Formula by Monocaprylin

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children, with a mortality rate of 14%. Epidemiological studies have implicated dried infant formula as the principal source of the pathogen. Caprylic acid is a natural eight-carbon fatty acid present in breast milk and bovine milk and is approved as generally recognizable as safe by the U.S. Food and Drug Administration. The objective of this study was to determine the antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula. A five-strain mixture of E. sakazakii was inoculated into 10-ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23 degrees C for 0, 1, 6, and 24 h and at 8 or 4 degrees C for 0, 6, 24, and 48 h, and the surviving populations of E. sakazakii at each sampling time were counted. The treatments containing monocaprylin significantly reduced the population of E. sakazakii (P < 0.05) compared with the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23 degrees C and by 24 h of incubation at 8 or 4 degrees C. Results indicate that monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula; however, sensory studies are warranted before its use can be recommended.

In Vitro Inactivation of Escherichia coli O157:H7 in Bovine Rumen Fluid by Caprylic Acid

The antibacterial effect of caprylic acid (35 and 50 mM) on Escherichia coli O157:H7 and total anaerobic bacteria at 39 degrees C in rumen fluid (pH 5.6 and 6.8) from 12 beef cattle was investigated. The treatments containing caprylic acid at both pHs significantly reduced (P < 0.05) the population of E. coli O157:H7 compared with that in the control samples. At pH 5.6, both levels of caprylic acid killed E. coli O157:H7 rapidly, reducing the pathogen population to undetectable levels at 1 min of incubation (a more than 6.0-log CFU/ml reduction). In buffered rumen fluid at pH 6.8, 50 mM caprylic acid reduced the E. coli O157:H7 population to undetectable levels at 1 min of incubation, whereas 35 mM caprylic acid reduced the pathogen by approximately 3.0 and 5.0 log CFU/ml at 8 and 24 h of incubation, respectively. At both pHs, caprylic acid had a significantly lesser (P < 0.05) and minimal inhibitory effect on the population of total anaerobic bacteria in rumen compared with that on E. coli O157:H7. At 24 h of incubation, caprylic acid (35 and 50 mM) reduced the population of total anaerobic bacteria by approximately 2.0 log CFU/ml at pH 5.6, whereas at pH 6.8, caprylic acid (35 mM) did not have any significant (P > 0.05) inhibitory effect on total bacterial load. Results of this study revealed that caprylic acid was effective in inactivating E. coli O157:H7 in bovine rumen fluid, thereby justifying its potential as a preslaughter dietary supplement for reducing pathogen carriage in cattle.

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey.

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.

Inactivation of Listeria monocytogenes on frankfurters by monocaprylin alone or in combination with acetic acid.

The antilisterial activity of monocaprylin (MC) and its combination with acetic acid (AA) on frankfurters was investigated. Each frankfurter was surface inoculated with a three-strain mixture of Listeria monocytogenes to obtain an inoculation level of 4.0 log CFU per frankfurter, and then dipped for 35 s in sterile deionized water (45 or 50 degrees C) containing 1% ethanol (control), 50 mM MC plus 1% ethanol, 1% AA plus 1% ethanol, or 50 mM MC plus 1% AA plus 1% ethanol. Samples were vacuum packaged, stored at 4 degrees C for 77 days, and analyzed for L. monocytogenes. Sensory odor and color of frankfurters were evaluated using a 9-point hedonic scale. Color was also objectively measured using the Minolta Chroma Meter. From day 0 to day 77, population counts of L. monocytogenes on frankfurters dipped in antimicrobial solutions at 50 degrees C were consistently lower than the control counts. Similar results were observed for samples treated at 45 degrees C. However, L. monocytogenes grew readily on control samples at both temperatures. Dipping of frankfurters in antimicrobial solutions (45 or 50 degrees C) significantly reduced (P < 0.05) the populations of L. monocytogenes. After 70 days of storage, L. monocytogenes was completely killed in samples dipped in MC+AA solution at 50 degrees C. The antimicrobial treatments did not affect the odor or color of the samples (P > 0.05). Overall, results indicated that dipping of frankfurters with MC reduced L. monocytogenes, and inclusion of AA further enhanced MC antilisterial activity, without any negative effect on odor or color.

Antibacterial effect of monocaprylin on Escherichia coli O157:H7 in apple juice.

The antibacterial effect of low concentrations of monocaprylin on Escherichia coli O157:H7 in apple juice was investigated. Apple juice alone (control) or containing 2.5 mM (0.055%) or 5 mM monocaprylin was inoculated with a five-strain mixture of E. coli O157:H7 at approximately 6.0 log CFU/ml. The juice samples were stored at 23 or 4 degrees C for 14 or 21 days, respectively, and the population of E. coli O157:H7 was determined on tryptic soy agar plates supplemented with 0.6% yeast extract. At both storage temperatures, the population of E. coli O157:H7 in monocaprylin-supplemented juice samples was significantly lower (P < 0.05) than that in the control samples. The concentration of monocaprylin and the storage temperature had a significant effect on the inactivation of E. coli O157:H7 in apple juice. Monocaprylin at 5 mM was significantly more effective than 2.5 mM monocaprylin for killing E. coli O157:H7 in apple juice. Inactivation of E. coli O157:H7 by monocaprylin was more pronounced in juice stored at 23 degrees C than in the refrigerated samples. Results of this study indicated that monocaprylin is effective for killing E. coli O157:H7 in apple juice, but detailed sensory studies are needed to determine the organoleptic properties of apple juice containing monocaprylin.