pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni.

The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.

Susceptibility of Pediococcus spp. to antimicrobial agents.

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva.

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.

Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese.

AIMS: A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. METHODS AND RESULTS: In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied. Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). CONCLUSION: While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.

IL-6 and IL-8 levels after cardiopulmonary bypass are not affected by surface coating.

UNLABELLED: BACKGROUND.:Contact of blood with the surfaces of the cardiopulmonary bypass (CPB) circuit has been implicated as a cause of the inflammatory response. We undertook a prospective randomized trial of 200 pediatric patients, all with a calculated total bypass flow of less than 2.3 L/min (< 0.96 L/m2/min). METHODS: Patients were randomly assigned to 1 of 4 CPB groups: (1) Nonheparin-bonded circuit with no albumin preprime; (2) Nonheparin-bonded circuit with albumin preprime; (3) Heparin-bonded circuit with no albumin preprime; (4) Heparin-bonded circuit with albumin preprime. Measurements of cytokines, (interleukin [IL]-6, IL-8) and blood cell counts were made prebypass and 6 and 24 hours after institution of cardiopulmonary bypass. RESULTS: Analysis of variance showed no significant difference in any of the clinical or biochemical characteristics of the 4 groups. The interaction between heparin-bonded oxygenators and albumin preprime was not significant. No important differences in IL-6 or IL-8 concentrations were noted after CPB using either heparin or nonheparin-bonded oxygenators with albumin or albumin free preprime using two-way analysis of variance. CONCLUSIONS: Albumin preprime and heparin-bonding do not attenuate the inflammatory response component attributable to the concentration of these markers.

Centrifugal ventricular assist in children under 6 kg.

OBJECTIVE: The clinical application of centrifugal ventricular assist devices (VAD) has generally been limited to adults and large paediatric patients. In our experience neonates and small paediatric patients requiring ventricular support post-cardiopulmonary bypass are well supported by VAD. In this study we analyse our experience. METHODS: We have examined the records of our VAD patients who weighed less than 6 kg. Thirty-four patients, ranging in age from 2 to 258 days (median 60 days) and weight from 1.9 to 5.98 kg (median 3.7 kg), underwent 35 VAD procedures. One patient was supported on VAD twice. RESULTS: All patients had congenital heart lesions and were placed on VAD either because they could not be weaned from cardiopulmonary bypass after repair or palliation of the lesion (71.5%), or for support in the post-operative period due to refractory low cardiac output (28.5%). Twenty-two of the 35 VAD procedures (0.63, 95% CI: 0.45-0.78) resulted in successful weaning and decannulation, this was similar to the weaning probability for patients greater than 6 kg (P = 0.07). There were 10 late deaths in this group, with a 1-year KM survival of 0.31 (95% CI: 0.17-0.47). Most late deaths were related to irreversible cardiac disease processes as were the elective discontinuance of VAD outcomes. Neither weight, age, VAD duration, CPB duration, X clamp duration, univentricular anatomy or TGA anatomy predicted successful discharge from hospital (P > 0.05)--Weight P = 0.576; Age P = 0.532; VAD duration P = 0.181; CBP duration P = 0.549; X clamp duration P = 0.984; Univentricular anatomy P = 0.481; TGA anatomy P = 0.099. CONCLUSION: We believe centrifugal ventricular assist is a realistic option in very small patients who require post-cardiopulmonary bypass support. It is relatively easy to establish and manage, the results, although showing no factors predictive of successful discharge, are encouraging.