Technological performance of the enterocin A producer Enterococcus faecium MMRA as a protective adjunct culture to enhance hygienic and sensory attributes of traditional fermented milk `Rayeb?

Enterococcus faecium MMRA is an enterocin A producer isolated from ‘Rayeb’, a Tunisian fermented milk drink. In this work, safety aspects and its behaviour in raw milk were investigated to assess its suitability as a protective adjunct culture. E. faecium MMRA showed interesting features such as the absence of several virulence traits, susceptibility to vancomycin and other clinically relevant antibiotics, and lack of haemolytic activity. To evaluate its performance as an adjunct culture for Rayeb, changes in the overall composition of control (non-inoculated) and experimental Rayeb (inoculated with 1%, v/v, E. faecium MMRA) were determined throughout duplicate fermentations of raw milk using microbiological, chemical, HPLC and headspace GC-MS analyses. E. faecium MMRA could multiply in raw milk and produced enterocin A. Interestingly, a higher content of volatile compounds including ethanol, diacetyl and 2-propanol was observed in the presence of this bacteriocin producer. Furthermore, this strain was capable of inhibiting the growth of Listeria monocytogenes CECT 4032 in pasteurised milk, although total killing was not achieved. Further experiments confirmed the development of resistant variants to enterocin A. On similar challenge assays, L. monocytogenes CECT 5672 growth was halted by the presence of the enterocin producer, but viability was only slightly reduced during cold storage. According to our results, E. faecium MMRA meets the criteria for an autochthonous protective adjunct culture to enhance both the hygienic and the sensory attributes of Rayeb.This work has been partially funded by grant BIO2007-65061 from Ministerio de Ciencia e Innovación (Spain)Peer Reviewe

Technological performance of the enterocin A producer Enterococcus faecium MMRA as a protective adjunct culture to enhance hygienic and sensory attributes of traditional fermented milk `Rayeb?

Enterococcus faecium MMRA is an enterocin A producer isolated from ‘Rayeb’, a Tunisian fermented milk drink. In this work, safety aspects and its behaviour in raw milk were investigated to assess its suitability as a protective adjunct culture. E. faecium MMRA showed interesting features such as the absence of several virulence traits, susceptibility to vancomycin and other clinically relevant antibiotics, and lack of haemolytic activity. To evaluate its performance as an adjunct culture for Rayeb, changes in the overall composition of control (non-inoculated) and experimental Rayeb (inoculated with 1%, v/v, E. faecium MMRA) were determined throughout duplicate fermentations of raw milk using microbiological, chemical, HPLC and headspace GC-MS analyses. E. faecium MMRA could multiply in raw milk and produced enterocin A. Interestingly, a higher content of volatile compounds including ethanol, diacetyl and 2-propanol was observed in the presence of this bacteriocin producer. Furthermore, this strain was capable of inhibiting the growth of Listeria monocytogenes CECT 4032 in pasteurised milk, although total killing was not achieved. Further experiments confirmed the development of resistant variants to enterocin A. On similar challenge assays, L. monocytogenes CECT 5672 growth was halted by the presence of the enterocin producer, but viability was only slightly reduced during cold storage. According to our results, E. faecium MMRA meets the criteria for an autochthonous protective adjunct culture to enhance both the hygienic and the sensory attributes of Rayeb.This work has been partially funded by grant BIO2007-65061 from Ministerio de Ciencia e Innovación (Spain)Peer Reviewe

Production of enterocin A by Enterococcus faecium MMRA isolated from 'Rayeb', a traditional Tunisian dairy beverage

[Aims] Characterization and purification of a bacteriocin produced by a wild Enterococcus faecium strain, isolated from a Tunisian traditional fermented milk.[Methods and Results] Enterococcus faecium MMRA was selected on the basis of its strong anti-Listeria activity. The antibacterial activity was sensitive to proteases, confirming its proteinaceous nature. It was extremely heat stable (15 min at 121°C), remained active over a wide pH range (2-12), and also after treatment with lipase, amylase, organic solvents, detergents, lyophilisation and long-term storage at -20°C. Production of the bacteriocin occurred throughout the logarithmic growth phase, it did not adhere to the surface of the producer cells and the mode of action was bactericidal. After partial purification of the active supernatants, a 4-kDa band with antibacterial activity was revealed by SDS-PAGE electrophoresis and bioassay. Tryptic digestion followed by MALDI-TOF mass spectrometry identified the peptide as enterocin A.[Conclusions] The inhibitory activity of Ent. faecium MMRA, a wild strain isolated from the artisan dairy beverage 'Rayeb', is due to the synthesis of an enterocin A.[Significance and Impact of the Study] Traditional fresh Tunisian fermented dairy products are generally manufactured with raw milk that can be used as a source of uncharacterized wild lactic acid bacteria strains. To our knowledge, this is the first report on the isolation of an enterocin A producing Ent. faecium from 'Rayeb'. This bacteriocin or the producing strain might have a promising potential in biopreservation to enhance the hygienic quality of this dairy product. © 2009 The Society for Applied Microbiology.The research was partially supported by the AECID (Ministerio de Asuntos Exteriores y de Cooperación, Spain) as R.A. was a recipient of a MAEC-AECID fellowship and grant BIO2007-65061 from Ministerio de Educación y Ciencia (Spain).Peer Reviewe

High prevalence of gut microbiota colonization with broad-spectrum cephalosporin resistant Enterobacteriaceae in a Tunisian intensive care unit

Healthcare-associated infections due to cefotaxime-resistant Enterobacteriaceae (CRE) have become a major public health threat, especially in intensive care units (ICUs). Often acquired nosocomially, CRE can be introduced initially by patients at admission. This study aimed to determine the prevalence and genetic characteristics of CRE-intestinal carriage in ICU patients, to evaluate the rate of acquisition of these organisms during hospitalization, and to explore some of the associated risk factors for both carriage and acquisition.Between December 2014 and February 2015, the 63 patients admitted in the ICU of Charles Nicolle hospital were screened for rectal CRE colonization at admission and once weekly thereafter to identify acquisition. CRE fecal carriage rate was 20.63% (13/63) at admission and the acquisition rate was 42.85% (15/35). Overall, 35 CRE isolates were collected from 28 patients (25 Klebsiella pneumoniae, 7 Escherichia coli and 3 Enterobacter cloacae strains). Seven patients were simultaneously colonized with 2 CRE isolates. CTX-M-15 was detected in most of the CRE isolates (30/35, 88.23%).Three strains co-produced CMY-4 and 22 strains were carbapenem-resistant and co-produced a carbapenemase OXA-48 (n=13) or NDM-1 (n=6). All isolates were multidrug resistant. Molecular typing of K. pneumoniae strains, revealed 8 Pulsed field gel electrophoresis (PFGE) patterns and 4 sequence types (ST) ST101, ST147, ST429 and ST336. However, E. coli isolates were genetically unrelated and belonged to A (n=2), B1 (n=2) and B2 (n=3) phylogenetic groups and to ST131 (2 strains), ST572 (2 strains), ST615 (one strain) and ST617 (one strain). Five colonized patients were infected by CRE (4 with the same strain identified from their rectal swab and 1 with a different strain). Whether imported or acquired during the stay in the ICU, colonization by CRE is a major risk factor for the occurrence of serious nosocomial infections. Their systematic screening in fecal carriage is mandatory to prevent the spread of these multidrug resistant bacteria

Assessment of potential probiotic properties and multiple bacteriocin encoding-genes of the technological performing strain Enterococcus faecium MMRA

In the present study, Enterococcus faecium MMRA, a safety and technological performing strain, previously isolated from a well-known fermented dairy product of Tunisia, was evaluated for certain properties relevant to probiotic including acid and bile tolerance, gastrointestinal juice resistance and adhesive properties. Acidic pH (2.0-5.0) and bile salt concentrations of 0.3% were used as stress conditions. The adhesive properties were assessed by determination of bacterial hydrophobicity. The strain MMRA showed interesting features such as high tolerance to gastrointestinal tract transit, an extremely high survival rates under stress caused by acidic pHs or bile salts, and a high adhesive potential linked to the elevated percentage of hydrophobicity. Furthermore, the genome of this strain was examined for the occurrence of known enterocin (enterococcal bacteriocins) genes by means of specific PCR assays and this study revealed the genes coding for enterocins A, B, P and X. Although the joint production of these bacteriocins has not been verified, producers of multiple enterocins could have a great technological potential as protective and/or probiotic cultures in the food industry. The results suggested that E.faecium MMRA should be considered as a potential probiotic which meets the criteria to enhance healthy attributes of fermented dairy product besides both of hygienic and sensory aspects. © 2013 Elsevier Ltd.A part of the research presented in this paper was financially supported by the Spanish Agency for International Cooperation (AECI), Spain within the Inter-University Cooperation Programme Cooperation Programme and Scientific Research (PCI-Mediterranean) between Spain and Tunisia (project A/5664/06). Amel Rehaiem is the recipient of a MAEC-AECID fellowship (Spain).Peer Reviewe

Treatment of Helicobacter pylori infection 14‐day concomitant quadruple therapy versus triple therapy: A parallel double‐blind randomized controlled trial

Abstract Background and Aims Successful Helicobacter pylori (Hp) eradication with the traditional 7‐day course of proton pump inhibitor triple therapy is declining. Prolonging therapy to 14 days is associated with better eradication rates. Most learned societies recommend concomitant quadruple therapy (QC) as a first‐line alternative therapy for this bacterial infection. The aim of this study is to compare the efficacy and safety of triple therapy (TT) and QC for the eradication of Hp infection. Methods A parallel double‐blind randomized controlled trial was conducted. The diagnosis of Hp infection was made by pathological examination of gastric biopsies. Patients were randomly assigned to two treatment groups: either QC (esomeprazole 80 mg, amoxicillin 2000 mg, clarithromycin 1000 mg, and metronidazole 1000 mg daily) or triple therapy (esomeprazole 80 mg, amoxicillin 2000 mg, and clarithromycin 1000 mg daily in divided doses) for 14 days. The efficacy of the treatment is defined by Hp eradication attested by a negative breath test performed 6 weeks after the completion of treatment. Treatment outcomes were compared using the chi‐square test, while binary logistic regression identified predictors of treatment failure. Results Ninety‐two patients were included. Forty‐two patients belonged to the QC group and 50 to the TT group. No significant difference was noted between the two groups concerning the rate of Hp eradication either by intention to treat (81% vs. 72% respectively, p = 0.31) or per protocol (81.6% vs. 76.1% respectively, p = 0.54). Likewise, there was no difference between the two groups in terms of tolerance to treatment (59.5% for QC vs. 58% for TT, p = 0.88). No factor has been associated with treatment failure. Conclusion There was no significant difference in the rate of HP eradication between the QC and the 14‐day triple therapy. Neither regimen should be used topically because of their low eradication rates