The purpose of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), a potent methane inhibitor, on total and metabolically active methanogens in the rumen of dairy cows over the course of the day and over a 12-wk period. Rumen contents of 8 ruminally cannulated early-lactation dairy cows were sampled at 2, 6, and 10 h after feeding during wk 4, 8, and 12 of a randomized complete block design experiment in which 3-NOP was fed at 60 mg/kg of feed dry matter. Cows (4 fed the control and 4 fed the 3-NOP diet) were blocked based on their previous lactation milk yield or predicted milk yield. Rumen samples were extracted for microbial DNA (total) and microbial RNA (metabolically active), PCR amplified for the 16S rRNA gene of archaea, sequenced on an Illumina platform, and analyzed for archaea diversity. In addition, the 16S copy number and 3 ruminal methanogenic species were quantified using the real-time quantitative PCR assay. We detected a difference between DNA and RNA (cDNA)-based archaea communities, revealing that ruminal methanogens differ in their metabolic activities. Within DNA and cDNA components, methanogenic communities differed by sampling hour, week, and treatment. Overall, Methanobrevibacter was the dominant genus (94.3%) followed by Methanosphaera, with the latter genus having greater abundance in the cDNA component (14.5%) compared with total populations (5.5%). Methanosphaera was higher at 2 h after feeding, whereas Methanobrevibacter increased at 6 and 10 h in both groups, showing diurnal patterns among individual methanogenic lineages. Methanobrevibacter was reduced at wk 4, whereas Methanosphaera was reduced at wk 8 and 12 in cows supplemented with 3-NOP compared with control cows, suggesting differential re-sponses among methanogens to 3-NOP. A reduction in Methanobrevibacter ruminantium in all 3-NOP samples from wk 8 was confirmed using real-time quantitative PCR. The relative abundance of individual methanogens was driven by a combination of dietary composition, dry matter intake, and hydrogen concentrations in the rumen. This study provides novel information on the effects of 3-NOP on individual methanogenic lineages, but further studies are needed to understand temporal dynamics and to validate the effects of 3-NOP on individual lineages of ruminal methanogens.

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Ruminants are one of the largest sources of global CH 4 emissions. This enteric CH 4 is exclusively produced by methanogenic archaea as a natural product during microbial fermentation in the reticulorumen. As CH 4 formation leads to a gross energy loss for the ruminant host and is also an environmental issue, several CH 4 mitigation approaches have been investigated, but results have been inconsistent, which may be partially attributed to a lack of understanding of the mechanistic basis of methanogenesis and the effect of inhibitors on individual methanogenic lineages and other fermenting microbes in the rumen. Methanogenic archaea are obligatory anaerobes that can reduce CO 2 , methanol, or methylamines or cleave acetate to form CH 4 . Although methanogens work toward a common goal of generating energy through the formation of CH 4 , individual methanogenic lineages differ in their physiological and metabolic capabilities, which can differentially affect H 2 transactions and CH 4 formation. Using advanced omic approaches, recent research has revealed that less abundant methanol-utilizing Methanosphaera and methylamine-and methanol-utilizing Methanomassiliicoccales lineages are positively correlated with CH 4 emissions and may have a greater share in overall CH 4 production compared with more abundant CO 2 -reducing methanogens than previously thought. These data imply that the diversity as well as the abundance of methanogens is important in CH 4 formation, and that this diversity is influenced by H 2 availability and interactions within and between H 2 -producing microbes in the rumen. These complex interactions between microbes and H 2 are further influenced by variations in dietary, host, and environmental conditions. This review discusses critical knowledge gaps underlying methanogen diver-sity and its link to CH 4 formation, formation of specific bacteria-archaeal cohorts, and how H 2 production and utilization are regulated between these cohorts during normal and inhibited methanogenesis. Addressing these knowledge gaps has the potential to lead to the development of novel strategies or to complement existing strategies to effectively reduce CH 4 formation while also improving productivity in dairy cows.

Aims: To elucidate the antibiotic resistance and virulence genes of nisinresistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin-sensitive and -resistant E. faecalis on the innate immunity of rats. Methods and Results: Slanetz-Bartley agar plates containing nisin were used to isolate nisin-resistant E. faecalis. The virulence factors were ascertained using quantitative real-time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin-resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced b-galactosidase. Conclusions: Nisin-resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin-resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity. Significance and Impact of the Study: This study elucidated the contamination of raw buffalo milk by nisin-resistant E. faecalis, which may pose food safety risk.Enterococcus faecalis is prevalent in human and animal faeces and is a major cause of contamination in dairy products (Giraffa et al. 1997). Enterococcus faecalis collected from the milk of healthy animals and humans has also been reported as a reservoir of virulence and antibiotic-resistant genes, which exhibit resistance to clinically used drugs such as tetracycline, quinolones, vancomycin, macrolides, streptogramins and glycopeptides (Jim enez et al. 2008;Klibi et al. 2015). There are complex relationships between multidrug-resistant and virulence genes in bacteria. Schaufler et al. (2016) reported that extended spectrum b-lactamase plasmids for resistance enhance virulence in the carrier strains by affecting gene expression. Enterococcus faecalis harbours many virulence factors including aggregation substances, as-I, as-II and asa-Journal of Applied Microbiology 127, 897--910

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant E. faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome-c assay were used for characterization. Growth kinetics, HPLC, qPCR, and western-blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phospho-transferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin-resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants.