The growing prevalence of antibiotic-resistant Staphylococcus aureus strains mandates selective susceptibility testing and epidemiological investigations. It also draws attention to an efficient typing strategy. Whole genome sequencing helps in genetic comparison, strain differentiation, and typing; however, it is not that cost-effective. In comparison, Multi-Locus Sequence Typing (MLST) is an efficient typing method employed for bacterial strain typing and characterizations. In this paper, a comprehensive pangenome and phylogenetic analysis of 502/1279 S. aureus genomes is carried out to understand the species divergence. Additionally, the current Multi-Locus Sequence Typing (MLST) scheme was evaluated, and genes were excluded or substituted by alternative genes based on reported shortcomings, genomic data, and statistical scores calculated. The data generated were helpful in devising a new Multi-Locus Sequence Typing (MLST) scheme for the efficient typing of S. aureus strains. The revised scheme is now a blend of previously used genes and new candidate genes. The genes yQil, aroE, and gmk are replaced with better gene candidates, opuCC, aspS, and rpiB, based on their genome localization, representation, and statistical scores. Therefore, the proposed Multi-Locus Sequence Typing (MLST) method offers a greater resolution with 58 sequence types (STs) in comparison to the prior scheme’s 42 STs.
Heart rate is an important physiological index of overall health and is primarily controlled by the parasympathetic nervous system, which suppresses heart rate at rest and during cardiac reflexes. The nucleus ambiguus (nAmb), a region found within the brainstem, is a primary source of parasympathetic input to the heart. It also innervates striated muscles of the larynx, pharynx, and esophagus to regulate the motor function of speech and swallowing. To identify candidate cardiovagal neurons and differentiate them from neighboring motor neurons, we profiled genome-wide RNA expression in ~1,250 nAmb neurons by single nuclei RNA-sequencing. Clustering these neurons based on their transcriptomic similarity revealed six candidate molecular subtypes, which we annotated according to their subtype-specific marker genes. Based on their gene expression profiles, we hypothesized that three subtypes may be cardiovagal neurons. Three other subtypes, marked by Vipr2 or Crhr2 expression, likely correspond to the neurons that innervate the upper airways and esophagus, as shown by our previous work. Two of the three potential cardiovagal subtypes were specifically marked by their expression of Vip and Adcyap1, respectively, and the other subtype co-expressed Npy2r and Adcyap1. Fluorescence RNA in situ hybridization validated that Npy2r and Adcyap1 transcripts mark partially overlapping nAmb neurons residing throughout its rostrocaudal axis. To determine whether these neuron subtypes can control heart rate, we targeted expression of a calcium-translocating channelrhodopsin variant (CatCh) to the Npy2r+ and Adcyap1+ nAmb neuron subtypes using intersectional genetics. We then activated CatCh+ neuron subtypes using laser light transmitted via a fiber optic implanted over the nAmb and measured the effect on heart rate (HR) in awake, behaving mice. Our results show an immediate and dramatic decrease in HR upon activation of Npy2r+nAmb neurons (n=6 Npy2r-Cre:Chat-Flp:CatCh mice; laser-on HR= 58 ± 17% of laser-off HR; p<0.0001), or Adcyap1+nAmb neurons (n=5 Adcyap1-Cre:Chat-Flp:CatCh mice; laser-on HR= 34 ± 13% of laser-off HR; p<0.0001). The HR decrease depended on the frequency of stimulation (n=3 mice per genotype) and was not observed in the absence of CatCh expression (n=4 mice; laser-on HR = 100 ± 0% of laser-off HR; p>0.05). Blocking muscarinic receptors with atropine prevented the bradycardic effect of activating Npy2r+nAmb neurons (n=5 mice), suggesting a parasympathetic mechanism. These results indicate that Npy2r+nAmb neurons and Adcyap1+nAmb neurons are capable of suppressing HR, which builds on our previous observation of bradycardia when activating all nAmb neurons but not when activating the upper airway and esophageal projecting subtypes (recently published) Vipr2+nAmb neurons and Crhr2+nAmb neurons, respectively. Together, our results identify cardiovagal neuron subtypes in the nAmb based on their molecular and functional properties. The authors gratefully acknowledge Maisie Crook for her technical assistance. Funding was provided by National Institutes for Health (NIH) F31 HL158187 to T.C.C.; NIH R01 HL148004 to S.B.G.A.; and an American Diabetes Association Pathway to Stop Diabetes Initiator Award 1-18-INI-14 and NIH R01 HL153916 to J.N.C. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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