Locus of Heat Resistance (LHR) in Meat-Borne \u3ci\u3eEscherichia coli\u3c/i\u3e: Screening and Genetic Characterization

Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR1 E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR+ isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response. IMPORTANCE Currently, a “multiple-hurdle” approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438–1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments

An ∼140-kb Deletion Associated with Feline Spinal Muscular Atrophy Implies an Essential LIX1 Function for Motor Neuron Survival

The leading genetic cause of infant mortality is spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of disorders. Previously we described a domestic cat model of autosomal recessive, juvenile-onset SMA similar to human SMA type III. Here we report results of a whole-genome scan for linkage in the feline SMA pedigree using recently developed species-specific and comparative mapping resources. We identified a novel SMA gene candidate, LIX1, in an ~140-kb deletion on feline chromosome A1q in a region of conserved synteny to human chromosome 5q15. Though LIX1 function is unknown, the predicted secondary structure is compatible with a role in RNA metabolism. LIX1 expression is largely restricted to the central nervous system, primarily in spinal motor neurons, thus offering explanation of the tissue restriction of pathology in feline SMA. An exon sequence screen of 25 human SMA cases, not otherwise explicable by mutations at the SMN1 locus, failed to identify comparable LIX1 mutations. Nonetheless, a LIX1-associated etiology in feline SMA implicates a previously undetected mechanism of motor neuron maintenance and mandates consideration of LIX1 as a candidate gene in human SMA when SMN1 mutations are not found

Genetic and expression studies of SMN2 gene in Russian patients with spinal muscular atrophy type II and III

<p>Abstract</p> <p>Background</p> <p>Spinal muscular atrophy (SMA type I, II and III) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (<it>SMN1</it>). <it>SMN2 </it>is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two <it>SMN2 </it>copies while most SMA type II patients carry three <it>SMN2 </it>copies and SMA III patients have three or four <it>SMN2 </it>copies. The <it>SMN1 </it>gene produces a full-length transcript (FL-SMN) while <it>SMN2 </it>is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMNΔ7 mRNA.</p> <p>Methods</p> <p>In this study we performed quantification of the <it>SMN2 </it>gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively) by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the <it>SMN1 </it>gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMNΔ7 mRNA ratio as SMA biomarker.</p> <p>Results</p> <p>Comparison of the <it>SMN2 </it>copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III) and presence of four copies of the <it>SMN2 </it>gene. In both asymptomatic cases we found an increased number of <it>SMN2 </it>copies in the healthy carriers and a biallelic <it>SMN1 </it>absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls.</p> <p>Conclusions</p> <p>We suggest that the <it>SMN2 </it>gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMNΔ7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy.</p

SMA CARNI-VAL TRIAL PART II: A Prospective, Single-Armed Trial of L-Carnitine and Valproic Acid in Ambulatory Children with Spinal Muscular Atrophy

Multiple lines of evidence have suggested that valproic acid (VPA) might benefit patients with spinal muscular atrophy (SMA). The SMA CARNIVAL TRIAL was a two part prospective trial to evaluate oral VPA and l-carnitine in SMA children. Part 1 targeted non-ambulatory children ages 2–8 in a 12 month cross over design. We report here Part 2, a twelve month prospective, open-label trial of VPA and L-carnitine in ambulatory SMA children.This study involved 33 genetically proven type 3 SMA subjects ages 3–17 years. Subjects underwent two baseline assessments over 4–6 weeks and then were placed on VPA and L-carnitine for 12 months. Assessments were performed at baseline, 3, 6 and 12 months. Primary outcomes included safety, adverse events and the change at 6 and 12 months in motor function assessed using the Modified Hammersmith Functional Motor Scale Extend (MHFMS-Extend), timed motor tests and fine motor modules. Secondary outcomes included changes in ulnar compound muscle action potential amplitudes (CMAP), handheld dynamometry, pulmonary function, and Pediatric Quality of Life Inventory scores.Twenty-eight subjects completed the study. VPA and carnitine were generally well tolerated. Although adverse events occurred in 85% of subjects, they were usually mild and transient. Weight gain of 20% above body weight occurred in 17% of subjects. There was no significant change in any primary outcome at six or 12 months. Some pulmonary function measures showed improvement at one year as expected with normal growth. CMAP significantly improved suggesting a modest biologic effect not clinically meaningful.This study, coupled with the CARNIVAL Part 1 study, indicate that VPA is not effective in improving strength or function in SMA children. The outcomes used in this study are feasible and reliable, and can be employed in future trials in SMA

Determining the Impact of Probicon L28 and BioPlus 2B on Finishing Pig Growth Performance and Carcass Characteristics

These data represent the growth performance of pigs enrolled in a study to determine the impact of two direct fed microbial products on Salmonella and Escherichia coli prevalence pre- and post-harvest. A total of 650 finishing pigs in two groups were randomly assigned to pen via a completely randomized design, and pens were assigned to one of three treatments: 1) a control treatment with pigs fed a standard corn-soybean meal finishing diet (with no added probiotic); 2) the control diets with Probicon L28 (NexGen Innovations, LLC, Lubbock, TX) supplemented through water lines using a water medicator system at a target concentration of 1.0 × 106 CFU/head/day; and 3) the control diet with added BioPlus 2B (5.0 × 108 CFU/lb of feed; ~3.0 × 109 CFU/ head/day; CHR Hansen, Inc, Milwaukee, WI). No evidence of difference (P \u3e 0.10) between treatments was observed for overall ADG, ADFI, or F/G or any of the carcass traits. However, there was a tendency for a treatment effect for loin depth (P = 0.070). Pigs fed the BioPlus 2B treatment had numerically greater loin depth compared to other treatments, but there were no significant pairwise differences between treatments (P \u3e 0.05). The results of this study suggested that probiotics used in this study and supplied through the water or feed had no impact on growth or carcass characteristics of finishing pigs

Exploring the Use of Probicon L28 and BIOPLUS 2B as Direct-Fed Microbials to Reduce Salmonella and Shiga Toxin-Producing Escherichia coli in Market Pigs

Pigs are hosts for Salmonella and Shiga toxin-producing Escherichia coli (STEC) and these pathogens can commonly be isolated from the pig farm environment. Pigs can carry pathogens to the abattoir and contaminate pork products, posing a risk to public health. Identifying an intervention that effectively reduces pathogens in commercial pigs before harvest is imperative. Due to the need for effective pre-harvest interventions in the pig industry, the objective of this study was to investigate BIOPLUS 2B (Bacillus licheniformis and Bacillus subtilis) and Probicon L28 (Lactobacillus salivarius L28) as pre-harvest interventions to reduce Salmonella and STEC in commercial growing-finishing pigs. Two groups of pigs (group 1, N = 294; group 2, N = 356, initial body weight = 106.6 lb) were fed a standard corn-soybean meal (SBM) finishing diet according to the following treatments: Probicon L28 supplementation through water lines at 1.0 × 106 CFU/head/day (Probicon); BIOPLUS 2B supplemented at 3.0 × 109 CFU/head/day (BIOPLUS 2B); and a control with no added probiotic (Control). With each group of pigs, 12 pens were used per treatment (N = 24 total), for a total of 36 pens per group (N = 72 pens total). Each group was sampled upon arrival/baseline, midway through the grow-finish phase/6 weeks post-placement, and prior to loadout/13 weeks post-placement to collect fecal samples (4 pigs/pen), boot covers (2/pen), and ropes (1/pen). Market pigs were followed to the abattoir and superficial inguinal lymph nodes (SILNs) were collected. All samples were analyzed for STEC (stx, eae genes, and O157:H7, and O26, O111, O121 O45, O103, and O145 serogroups) and Salmonella using the BAX System (real-time polymerase chain reaction). Overall, Salmonella and O111 prevalence was very low for all sample types, and Escherichia coli O157:H7 was not detected in any samples throughout the study. When compared to the control, there was no evidence (P \u3e 0.05) that BIOPLUS 2B and Probicon L28 impacted the prevalence of STEC (stx and eae genes) or serogroups O26, O121, O45, O103, and O145 in feces, boot covers, ropes, and SILNs of market pigs