Validation of a Modified Version of Actero™ Salmonella Enrichment Media for Rapid Detection of Salmonella spp. in Environmental and Food Samples.

BACKGROUND: Actero™ Salmonella Enrichment Media1 (Actero™ Salmonella) is a culture broth developed to recover Salmonella spp. from foods and environmental surfaces. Performance of Actero™ Salmonella broth has already been assessed and validated (AOAC Performance Tested MethodSM 041403) for the detection of Salmonella spp. in various foods, feeds and environmental samples. OBJECTIVE: This study aimed to validate the performance of a modified version of Actero™ Salmonella broth by incorporating one of the two liquid supplements into the powdered formula. METHODS: Inclusivity, exclusivity, stability, and lot-to-lot studies were carried out. Raw ground beef, chicken carcass rinse, dry pet food and stainless steel samples were enriched for 14-20 h at 35-39°C and analyzed using real-time PCR assay as well as by direct plating. RESULTS: The Probability of Detection assay confirmed the equivalent performance of the alternative methods as compared to the reference methods. All Salmonella strains, except Salmonella II : 57: z29:-, were able to grow in Actero™ Salmonella broth. One-half of the non-target strains did not grow in Actero™ Salmonella broth, whereas the atypical for Salmonella growth was observed for other non-target microorganisms subsequently plated onto selective and differential agars. Lot-to-lot consistency was demonstrated for three consecutively manufactured lots of the broth. The liquid broth was proven to be stable at 4°C for up to 9 weeks of storage. CONCLUSIONS AND HIGHLIGHTS: The incorporation of one of the two specific supplements into a powdered formula of Actero™ Salmonella broth made it more convenient to use without compromising the performance and accuracy.

SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA.

Transcription termination is a critical step in the control of gene expression. One of the major termination mechanisms is mediated by Rho factor that dissociates the complex mRNA-DNA-RNA polymerase upon binding with RNA polymerase. Rho promotes termination at the end of operons, but it can also terminate transcription within leader regions, performing regulatory functions and avoiding pervasive transcription. Transcription of rho is autoregulated through a Rho-dependent attenuation in the leader region of the transcript. In this study, we have included an additional player in this pathway. By performing MS2-affinity purification coupled with RNA sequencing (MAPS), rho transcript was shown to directly interact with the small noncoding RNA SraL. Using bioinformatic in vivo and in vitro experimental analyses, SraL was shown to base pair with the 5'-UTR of rho mRNA upregulating its expression in several growth conditions. This base pairing was shown to prevent the action of Rho over its own message. Moreover, the results obtained indicate that both ProQ and Hfq are associated with this regulation. We propose a model that contemplates the action of Salmonella SraL sRNA in the protection of rho mRNA from premature transcription termination by Rho. Note that since the interaction region between both RNAs corresponds to a very-well-conserved sequence, it is plausible to admit that this regulation also occurs in other enterobacteria.

GcvB small RNA uses two distinct seed regions to regulate an extensive targetome.

GcvB small RNA is described as post-transcriptional regulator of 1-2% of all mRNAs in Escherichia coli and Salmonella Typhimurium. At least 24 GcvB:mRNA interactions have been validated in vivo, establishing the largest characterized sRNA targetome. By performing MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we identified seven additional mRNAs negatively regulated by GcvB in E. coli. Contrary to the vast majority of previously known targets, which pair to the well-conserved GcvB R1 region, we validated four mRNAs targeted by GcvB R3 region. This indicates that base-pairing through R3 seed sequence seems relatively common. We also noticed unusual GcvB pairing sites in the coding sequence of two target mRNAs. One of these target mRNAs has a pairing site displaying a unique ACA motif, suggesting that GcvB could hijack a translational enhancer element. The second target mRNA is likely regulated via an active RNase E-mediated mRNA degradation mechanism. Remarkably, we confirmed the importance of the sRNA sponge SroC in the fine-tuning control of GcvB activity in function of growth conditions such as growth phase and nutrient availability.

An in vivo reporter assay for sRNA-directed gene control in Gram-positive bacteria: identifying a novel sRNA target in Staphylococcus aureus.

Bacterial small regulatory RNAs (sRNAs) play a major role in the regulation of various cellular functions. Most sRNAs interact with mRNA targets via an antisense mechanism, modifying their translation and/or degradation. Despite considerable progresses in discovering sRNAs in Gram-positive bacteria, their functions, for the most part, are unknown. This is mainly due to difficulties in identifying their targets. To aid in the identification of sRNA targets in Gram-positive bacteria, we set up an in vivo method for fast analysis of sRNA-mediated post-transcriptional control at the 5΄ regions of target mRNAs. The technology is based on the co-expression of an sRNA and a 5΄ sequence of an mRNA target fused to a green fluorescent protein (GFP) reporter. The system was challenged on Staphylococcus aureus, an opportunistic Gram-positive pathogen. We analyzed several established sRNA-mRNA interactions, and in addition, we identified the ecb mRNA as a novel target for SprX2 sRNA. Using our in vivo system in combination with in vitro experiments, we demonstrated that SprX2 uses an antisense mechanism to prevent ecb mRNA translation initiation. Furthermore, we used our reporter assay to validate sRNA regulations in other Gram-positive organisms, Bacillus subtilis and Listeria monocytogenes. Overall, our method is broadly applicable to challenge the predicted sRNA-mRNA interactions in Gram-positive bacteria.

Identification of unknown RNA partners using MAPS.

Recent advances in high-throughput sequencing have led to an explosion in the rate of small regulatory RNAs (sRNAs) discovery among bacteria. However, only a handful of them are functionally characterized. Most of the time, little to no targets are known. In Lalaouna et al. (2015), we proposed a new technology to uncover sRNAs targetome, which is based on the MS2-affinity purification (MAPS). We were able to prove its efficiency by applying it on well-characterized sRNAs of Escherichia coli. Thereafter, we adapted the procedure to other kind of RNA (mRNAs and tRNA-derived RNA fragments) and bacteria (pathogenic or Gram-positive strains). Here, we clearly report all improvements and adjustments made to MAPS technology since it was originally reported.

A small RNA controls a protein regulator involved in antibiotic resistance in Staphylococcus aureus.

The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen.