Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia).

AIMS: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. METHODS AND RESULTS: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. CONCLUSIONS: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR.

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef.

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.

Thermal resistance of wild-type and antibiotic-resistant Listeria monocytogenes in meat and potato substrates.

AIMS: This study aimed to elucidate the relationship, if any, between the acquisition/possession of antibiotic resistance in strains of Listeria monocytogenes and the resistance of such strains to heat stress. METHODS AND RESULTS: D-values calculated using a linear survival model were used to compare the heat resistance of two wild-type (WT) and two antibiotic (streptomycin)-resistant (AR) mutant strains of L. monocytogenes measured in minced beef and potato substrates at 55 degrees C, with and without prior heat shock at 48 degrees C. In both minced beef and potato, no significant differences (P < 0.05) between D-values of AR and WT strains were noted. Heat shock did not significantly increase D-values of WT or AR strains in minced beef, while in potato slices, D-values in almost all cases were significantly higher in samples which had received heat-shock treatment. In minced beef, the use of a non-selective/overlay recovery medium did not result in higher D-values for any strains, while in potato, significantly higher (P < 0.05) D-values were obtained in most cases. CONCLUSION: The presence or absence of antibiotic resistance genes did not modulate the heat resistance of the strains examined in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated that heat shock, and the type of media used to determine bacterial numbers during heat processing, can significantly affect the D-values obtained.

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems.

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P-) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 degrees C in supplemented phosphate-buffered saline, buffered peptone water, cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P- strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 degrees C, the lag phase duration of the P+ strain was significantly longer than P-. In all but the most selective medium, P+ strains had slower growth rates that P- strains at 37 degrees C, probably due to the increase metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P-. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phase than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 degrees C. No significant differences were observed between the length of lag phases of P+ and P strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one of more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.