Evaluation of a cocktail of three bacteriophages for the biocontrol of Salmonella of wastewater

Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30 °C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 10⁰, 10², and 10⁴ PFU/CFU. At 30, and 37 °C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 10⁰ or 10 ² PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species.     

Isolation and characterization of a new dimeric esterase from Lactobacillus sakei 23K

The M_w of a Lactobacillus sakei intracellular esterase, determined by gel filtration, was compared to those obtained from SDS-PAGE or MALDI-TOF, pointing to a dimeric structure. Its N-terminal sequence and peptide mass fingerprint suggest that it is the putative LSA044 protein from L. sakei 23K genome.     

Method for reliable isolation of Lactobacillus sakei strains originating from Tunisian seafood and meat products

In Tunisia, several food products derived from meat or seafood are naturally processed, without any addition of bacterial starters. Such fermented, dried-cured, salted, or marinated products, as well as the raw meat or fish may thus provide a source to isolate the natural microflora colonizing such environments. We isolated lactic acid bacteria from a representative range of flesh-foods sold or manufactured in different parts of Tunisia, and selectively searched for Lactobacillus sakei, a lactic acid bacterium potentially useful as starter or protective culture. Eighty six (86) strains were isolated from various seafood (anchovy, sardine, sole, mullet, and octopus), or meat (pork, veal, beef, sheep, chicken, and turkey) products that were either fresh, or transformed by different traditional processes. Several methods were used in order to develop a rapid and reliable protocol for the direct identification of L. sakei. Amplified ribosomal DNA restriction analysis (ARDRA) classified the various isolates into 9 distinct groups. Search for the presence of the L. sakei specific katA gene indicated that all positive isolates were grouped in the same ARDRA group. Sequencing of 16S rDNA confirmed those isolates as L. sakei. Those 22 different L. sakei strains represent 25.6% of the total isolates, while other isolates found in the different ARDRA groups were tentatively ascribed to Lactobacillus plantarum, Lactococcus lactis/garviae, Enterococcus avium, Streptococcus parauberis, Hafnia alvei, Pediococcus pentosaceus, and Lactobacillus curvatus through 16S rDNA sequencing. A fast and reliable method to isolate and discriminate L. sakei from complex food environments is proposed.     

Biofilm formation,virulence gene and multi-drug resistance in Salmonella Kentucky isolated in Tunisia

Food-borne diseases caused by Salmonella enterica are a significant public health concern around the world. Since 2002, S. enterica serovar Kentucky has shown an increase in several countries with the concurrent emergence of multidrug-resistant isolates. The spread of such strains in the environment poses a major public health problem. A total of 57 Salmonella Kentucky strains isolated from different sources during the period 2005 to 2008 in Tunisia, were characterized by their antimicrobial and mercury resistance profiles; ability to form a biofilm; virulence invA/spvC genes and quorum sensing sdiA gene. A total of 10.6% of the isolates demonstrated multidrug-resistance against 3 to 13 antibiotics with ciprofloxacin resistance occurring in 33% of human isolates. In addition, 37% of the isolates exhibited minimum inhibitory concentrations value to mercuric chloride, ranging from 8 to 32 μg ml^?1 and were considered as resistant strains. The majority of strains tested were able to form a biofilm, especially for environmental and animal derived isolates. Therefore, the biofilm seems to comprise a normal and favorable capability in the life of Salmonella Kentucky in the environment. Interestingly, all the isolates possessed the sdiA gene, 87.7% of isolates possessed the invA gene, and no isolate harbored the spvC gene.The emergence of resistance to ciprofloxacin in human Salmonella Kentucky isolates, added to the presence of invA and sdiA genes, and the production of biofilm could be the decisive factors in the dissemination of S. Kentucky strains on a large scale.     

Epidemiology and antibiotic resistance of Salmonella enterica Serovar Kentucky isolates from Tunisia: The new emergent multi-drug resistant serotype

Since 2002, the emergence of multidrug resistant Salmonella enterica serovar Kentucky (S. Kentucky) and the associated salmonellosis with treatment failure were declared in different parts of the world and were in most of the case contracted during travels to Northeast and Eastern Africa. In the present work, we reported an epidemiological study of S. Kentucky isolated from different environmental and clinical origins in Tunisia, using Pulsed-field gel electrophoresis (PFGE); Enterobacterial Repetitive Intergenic Consensus (ERIC-2) fingerprinting; Plasmid profiling; and antibiotic resistance profiles. ERIC-2 fingerprinting allowed the differentiation of 14 different patterns versus only 4 pulsotypes. Besides, a high proportion of strains were found to be nontypeable by XbaI-PFGE and/or by plasmid profiling (plasmid-free strains). The antibiotic resistance was mainly detected against streptomycin (80.7%), sulfonamides (42.1%) and tetracycline (15.7%). Furthermore, two avian strains were shown to be resistant to trimethoprim–sulfamethoxazole and three clinical strains have demonstrated multidrug-resistant phenotypes (against 5 to 10 antibiotics) and all of them exhibited resistance against nalidixic acid, ciprofloxacin, ofloxacin and tetracycline.ERIC-2 PCR was found to be the most discriminative. However, combination of the three typing methods offer a better mean for differentiating S. Kentucky isolates, monitoring the multiresistant types and determination of their origin.