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Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Human newborns exhibit increased vulnerability and risk of mortality from infection that is consistent with key differences in the innate and adaptive immune responses relative to those in adult cells. We have previously shown an increase in the immune suppressive cytokine, IL-27, in neonatal cells and tissues from mice and humans. In a murine model of neonatal sepsis, mice deficient in IL-27 signaling exhibit reduced mortality, increased weight gain, and better control of bacteria with reduced systemic inflammation. To explore a reprogramming of the host response in the absence of IL-27 signaling, we profiled the transcriptome of the neonatal spleen during Escherichia coli -induced sepsis in wild-type (WT) and IL-27Rα-deficient (KO) mice. We identified 634 genes that were differentially expressed, and those most upregulated in WT mice were associated with inflammation, cytokine signaling, and G protein coupled receptor ligand binding and signaling. These genes failed to increase in the IL-27Rα KO mice. We further isolated an innate myeloid population enriched in macrophages from the spleens of control and infected WT neonates and observed similar changes in gene expression aligned with changes in chromatin accessibility. This supports macrophages as an innate myeloid population contributing to the inflammatory profile in septic WT pups. Collectively, our findings highlight the first report of improved pathogen clearance amidst a less inflammatory environment in IL-27Rα KO. This suggests a direct relationship between IL-27 signaling and bacterial killing. An improved response to infection that is not reliant upon heightened levels of inflammation offers new promise to the potential of antagonizing IL-27 as a host-directed therapy for neonates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Povroznik, Akhter, Vance, Annamanedi, Dziadowicz, Wang, Divens, Hu and Robinson.)

Background: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India., Results: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes., Conclusion: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains., (© 2023. The Author(s).)

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC + , 44 (26.5%) isolates were mecA + , with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA + but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates., (© 2021. The Author(s).)

Context: Infections with methicillin-resistant Staphylococcus aureus (MRSA) greatly influence clinical outcome. Molecular characterisation of MRSA can help to predict their spread and to institute treatment and hospital protocols., Aim: The aim of this study is to understand the diversity of MRSA in a tertiary care hospital in Hyderabad, India., Settings and Design: Samples collected at Gandhi Medical College, Hyderabad, and designed to assess hospital-or community-associated MRSA (HA-MRSA or CA-MRSA)., Subjects and Methods: MRSA were subjected to antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and staphylococcal cassette chromosome-mec (SCCmec) typing., Statistical Analysis Used: Discriminatory index and 95% confidence interval., Results: Of the 30 MRSA, (a) 18 and 12 were HA-MRSA and CA-MRSA, respectively, and (b) 23.3% and 6.6% displayed induced clindamycin and intermediate vancomycin resistance, respectively. Genetic diversity was evident from the presence of (a) 20 pulsotypes, (b) eight spa types, with the predominance of t064 (n = 9) and (c) seven sequence types (ST), with the preponderance of ST22 and ST8 (9 each). ST22 and ST8 were the most prevalent among HA-MRSA and CA-MRSA, respectively. SCCmec type IV was the most frequent (n = 8). 44.4% of HA-MRSA belonged to SCCmec IV and V, whereas 33.3% of CA-MRSA belonged to SCCmec I and III; 33.3% (5/15) of the isolates harbouring the pvl gene belonged to SCCmec IVC/H., Conclusions: ST8 was a dominant type along with other previously reported types ST22, ST239, and ST772 from India. The observations highlight the prevalence of genetically diverse clonal populations of MRSA, suggesting potential multiple origins., Competing Interests: None

In Taiwan, the incidence rate of oral cancer is constantly increasing. Polymorphisms and lifestyle habits are major contributing factors to the development of oral cancer in such cases. Casein kinase 1 epsilon ( CK1ε ) gene expression plays a role in numerous cancers, and the knockdown of CK1ε induces tumor cell-selective cytotoxicity. The present study was designed to determine the effects of CK1ε gene polymorphisms combined with environmental carcinogens on susceptibility to developing oral squamous cell carcinoma and its clinicopathological status. Four single-nucleotide polymorphisms (SNPs) in CK1ε gene (rs135745, rs135764, rs1997644 and rs2075984) from 741 oral cancer patients and 462 healthy controls were analyzed using real-time polymerase chain reaction. Our results shown that variant types (GC) of CK1ε polymorphic rs135745 exhibited a significantly higher risk of 1.41 (95% confidence interval [CI]: 1.036-1.919) for oral cancer than did wild type alleles. Furthermore, these CK1ε gene SNPs along with betel-quid chewing and/or tobacco use further increased susceptibility to oral cancer. Moreover, variant genotypes (GC+CC) of CK1ε rs135745 were significantly associated with lymph node metastasis. These results suggested that the CK1ε gene polymorphism is associated with the clinicopathological development of oral cancer and increases individuals' susceptibility to environmental carcinogens (e.g., smoking and betel-quid chewing) in terms of developing oral cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Treatment of multidrug resistant bacterial infections has been a great challenge globally. Previous studies including our study have highlighted the use of celecoxib, a non-steroidal anti-inflammatory drug in combination with antibiotic has decreased the minimal inhibitory concentration to limit Staphylococcus aureus infection. However, the efficacy of this combinatorial treatment against various pathogenic bacteria is not determined. Therefore, we have evaluated the potential use of celecoxib in combination with low doses of antibiotic in limiting Gram-positive and Gram-negative bacteria in vivo in murine polymicrobial sepsis developed by cecum ligation and puncture (CLP) method and against clinically isolated human ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , and Enterobacter species). The in vivo results clearly demonstrated a significant reduction in the bacterial load in different organs and in the inflammatory markers such as COX-2 and NF-κB via activation of SIRT1 in mice treated with imipenem, a choice of antibiotic for polymicrobial sepsis treatment. Combinatorial treatment of ampicillin and celecoxib was effective on clinical isolates of ESKAPE pathogens, 45% of tested clinical isolates showed more than 50% reduction in the colony forming units when compared to ampicillin alone. In conclusion, this non-traditional treatment strategy might be effective in clinic to reduce the dose of antibiotic to treat drug-resistant bacterial infections.

We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S. aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1.