Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Impacts of feeding a Saccharomyces cerevisiae fermentation product on productive performance, and metabolic and immunological responses during a feed-restriction challenge of mid-lactation dairy cows.

Saccharomyces cerevisiae fermentation products are commonly used in dairy cattle ration to improve production efficiency and health. However, whether these benefits will persist during feed-restriction-induced negative energy balance is unknown. The objective of this experiment was to examine the effect of a Saccharomyces cerevisiae fermentation product (NT, NutriTek, Diamond V) on performance, metabolic, inflammatory, and immunological responses to a feed-restriction challenge in mid-lactation dairy cows. Sixty Holstein cows were blocked by parity, days in milk, and milk yield and then randomly assigned to 1 of the 2 supplements: NT or placebo (CTL). The supplements were mixed in total mixed ration before feeding at a rate of 19 g/d per cow. The production phase of the experiment lasted 12 wk. Intake and milk yield were recorded daily, and milk composition was measured weekly. After the production trial, a subset of cows (NT: n = 16; CTL: n = 16) were immediately enrolled in a 5-d feed-restriction challenge with 40% ad libitum intake followed by a 5-d realimentation. Milk yield and composition were measured at each milking from d -2 to 10 relative to feed restriction. Blood samples were collected on d -2, -1, 1, 2, 3, 4, 5, 6, 8, and 10 relative to the initiation of feed restriction to measure circulating metabolites, insulin, cortisol, IL-10, tumor necrosis factor-α, lipopolysaccharide binding protein, and haptoglobin. Immune function assessments, including peripheral mononuclear cell proliferation and functional assays of circulating granulocytes, were performed on d -3 and 4 of the feed restriction. No differences were observed in dry matter intake, milk yield, or concentrations or yield of components except for fat yield. An interaction of parity and treatment was observed for milk fat yield that was lower for CTL than NT in primiparous cows, but no differences were observed among treatments in milk fat yield of multiparous cows. Feed restriction successfully induced negative energy balance and its associated metabolic changes, including reduced concentrations of plasma glucose and increased nonesterified fatty acids and ß-hydroxybutyrate. Cows fed NT had a similar decrease in milk yield but had a more pronounced reduction in plasma glucose concentration and greater ß-hydroxybutyrate concentration during feed restriction than those fed CTL. Feed restriction did not induce evidence of systemic inflammation but did reduce granulocyte functional activity. Compared with CTL, feeding NT improved the reactive oxygen species production by granulocytes after stimulation by extracellular antigens. In conclusion, feeding NT increased milk fat production of first-lactation cows but did not affect overall productive performance. However, supplementation with NT improved induced granulocyte oxidative burst. This may explain the greater glucose utilization by cows fed NT rather than CTL during feed restriction.

Saccharomyces cerevisiae fermentation products reduce bacterial endotoxin concentrations and inflammation during grain-based subacute ruminal acidosis in lactating dairy cows.

Subacute ruminal acidosis (SARA) is a metabolic disorder in dairy cows that is associated with dysbiosis of rumen and hindgut microbiomes, translocation of immunogenic compounds from the gut lumen into blood circulation, and systemic inflammatory response. In this study we hypothesized that Saccharomyces cerevisiae fermentation products (SCFP) attenuate the increases in ruminal and peripheral bacterial endotoxin concentrations and the inflammation resulting from repeated induction of SARA. Lactating Holstein dairy cows (parity 2 and 3+, n = 32) were fed diets with or without SCFP (all from Diamond V) and subjected to 2 episodes of SARA challenges. Cows received a basal total mixed ration (TMR) containing 34% neutral detergent fiber and 18.6% starch, dry matter (DM) basis. Treatments were randomly assigned to control (basal TMR and 140 g/d of ground corn with no SCFP) or 1 of 3 SCFP treatments: basal TMR and 14 g/d Original XPC (SCFPa), 19 g/d NutriTek (SCFPb-1×), or 38 g/d NutriTek (SCFPb-2×) mixed with 126, 121, or 102 g/d of ground corn, respectively. Treatments were implemented from 4 wk before until 12 wk after parturition. During wk 5 (SARA1) and wk 8 of lactation (SARA2), grain-based SARA challenges were conducted by gradually replacing 20% of DM of the basal TMR over 3 d with pellets containing 50% wheat and 50% barley. Ruminal fluid, fecal, and blood samples were collected weekly during Pre-SARA1 (wk 4, as baseline), Post-SARA1 (wk 7), and Post-SARA2 (wk 10 for blood and wk 12 for rumen and fecal parameters) stages, and twice a week during the challenges SARA1 and SARA2. Rumen papillae samples were taken only during Pre-SARA1 and Post-SARA2. We measured the concentrations of free lipopolysaccharides (LPS) in the rumen fluid and feces; free LPS and lipoteichoic acid (LTA) endotoxins in peripheral plasma; interleukin (IL)-1ß and IL-6 in peripheral serum; acute-phase proteins, serum amyloid A (SAA), and LPS-binding protein in peripheral plasma; haptoglobin (Hp) in peripheral serum; and myeloperoxidase (MPO) in rumen papillae. Induction of SARA episodes increased free LPS concentrations in rumen fluid and tended to increase LTA in peripheral plasma. The SARA episodes increased concentration of circulating SAA and tended to increase that of IL-1ß compared with Pre-SARA1. Induction of SARA did not affect the concentrations of circulating IL-6, Hp, and MPO. The SCFP supplementation reduced plasma concentrations of LTA and SAA and serum concentration of IL-1ß compared with control. Additionally, SCFPb-2× tended to reduce ruminal LPS in second-parity cows compared with control. Overall, SCFP supplementation appeared to stabilize the rumen environment and reduce proinflammatory status, hence attenuating adverse digestive and inflammatory responses associated with SARA episodes.

Effect of Saccharomyces cerevisiae fermentation product on oxidative status, inflammation, and immune response in transition dairy cattle.

Dairy cattle are subjected to oxidative stress, inflammation, and altered immune function during the transition to lactation. The objective of this study was to evaluate the effects of a dietary Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V) on oxidative status, inflammation, and innate and adaptive immune responses during the transition period. Holstein cows were blocked by parity, expected calving date, and previous milk yield and then randomly assigned to treatment within block. Treatment was a control total mixed ration (n = 30) or SCFP total mixed ration (n = 34) fed from -29 ± 5 to 42 d relative to calving (RTC). Blood was sampled during wk -4, -2, 1, 2, and 5 and liver tissue at wk -3 and 2 RTC. Oxidative status was evaluated in plasma by retinol, α-tocopherol, and malondialdehyde concentrations, glutathione peroxidase activity, and Trolox equivalent antioxidant capacity, and in liver by mRNA abundance of nuclear factor E2-related factor 2 (NFE2L2), metallothionein 1E (MT1E), and glutathione peroxidase 3 (GPX3). Inflammation was evaluated in plasma by haptoglobin (HP) and serum amyloid A (SAA) concentrations and in liver by mRNA abundance of HP, serum amyloid A3 (SAA3), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB1). Innate immune response was measured by stimulated oxidative burst of polymorphonuclear cells (neutrophils) isolated from blood. Ovalbumin (OVA) was administered with adjuvant on d 7 and 21 RTC, and adaptive immune response was evaluated by serum anti-OVA IgG content on d 28 and 35. Mixed models were used to assess effects of treatment, time, parity, and all interactions. We previously reported that SCFP had limited effects on productivity in this cohort, although milk fat yield was transiently increased and subclinical ketosis incidence was increased. Supplementation with SCFP did not affect overall oxidative, inflammatory, or immune parameters. The only treatment × week interaction detected was for plasma α-tocopherol concentration, which tended to be greater in control cows during wk 2 RTC. A tendency for a treatment × parity interaction was detected for serum anti-OVA IgG titer, which tended to be greater for SCFP than for controls among primiparous cows. Plasma inflammatory biomarkers were not affected by SCFP but, unexpectedly, plasma HP was elevated at both prepartum time points and plasma SAA was elevated during wk -2 RTC compared with the expected increases in both biomarkers postpartum. In this cohort of transition cows with low disease incidence, SCFP generally did not affect oxidative, inflammatory, or immune parameters.

The insect peptide CopA3 blocks programmed cell death by directly binding caspases and inhibiting their proteolytic activation.

Caspases play essential roles in apoptotic processes, which is necessary for cellular homeostasis. However, over-activation of caspases and subsequent excessive apoptosis is considered a main cause of Parkinson's disease and liver diseases. Here, we found that the insect-derived peptide, CopA3, which has shown antiapoptotic effects in many apoptosis models, directly binds to caspases. The resulting complexes do not dissociate during denaturing polyacrylamide gel electrophoresis, as evidenced by a distinct shift in the migration of caspase reflecting an increase in their molecular weight. Surface plasmon resonance and experiment using cysteine-substituted mutants of CopA3 collectively revealed that binding of CopA3 to caspases is dependent on an internal cysteine residue. Notably, CopA3 binding significantly inhibited proteolytic activation of downstream caspases by upstream caspases. In summary, the demonstration that CopA3 directly binds to caspases and inhibits their activating cleavage suggests a possible therapeutic approach for treating human diseases resulting from uncontrolled apoptosis.

A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development.

Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor. Whole-genome sequencing was performed on 3,505 A. baumannii isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. Isolates were cultured from clinical samples at health care facilities around the world between 2001 and 2017. Core-genome multilocus sequence typing and high-resolution single nucleotide polymorphism (SNP)-based phylogenetic analyses were used to select a final panel of 100 strains that captured the genetic diversity of the collection. Comprehensive antibiotic susceptibility testing was also performed on all 100 isolates using 14 clinically relevant antibiotics. The final 100-strain diversity panel contained representative strains from 70 different traditional Pasteur scheme multilocus sequence types, including major epidemic clones. This diversity was also reflected in antibiotic susceptibility and antimicrobial resistance (AMR) gene content, with phenotypes ranging from pansensitive to panresistant, and over 100 distinct AMR gene alleles identified from 32 gene families. This panel provides the most diverse and comprehensive set of A. baumannii strains for use in developing solutions for combating antibiotic resistance. The panel and all available metadata, including genome sequences, will be available to industry and academic institutions and federal and other laboratories free of charge.

Effects of a Saccharomyces cerevisiae fermentation product on heat-stressed dairy cows.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP) on body temperature indices, metabolism, acute phase protein response, and production variables during heat stress (HS). Twenty multiparous lactating Holstein cows (body weight = 675 ± 12 kg; days in milk = 144 ± 5; and parity = 2.3 ± 0.1) were used in an experiment conducted in 2 replicates (10 cows/replicate). Cows were randomly assigned to 1 of 2 dietary treatments: control diet (CON; n = 10) or the CON diet supplemented with 19 g/d of SCFP (n = 10; NutriTek, Diamond V, Cedar Rapids, IA). Cows were fed their respective diets for 21 d before initiation of the study. The experiment consisted of 2 periods: thermoneutral (period 1; P1) and heat stress (period 2; P2). During P1 (4 d), cows were fed ad libitum and housed in thermoneutral conditions for collecting baseline data. During P2 (7 d), HS was artificially induced using an electric heat blanket (EHB; Thermotex Therapy Systems Ltd., Calgary, AB, Canada). Cows were fitted with the EHB for the entirety of P2. Rectal temperature, respiration rate, and skin temperature were obtained twice daily (0600 and 1800 h) during both periods. Overall, HS increased rectal temperature, skin temperature, and respiration rate (1.4°C, 4.8°C, and 54 breaths/min, respectively) relative to P1, but no dietary treatment differences were detected. Compared with P1, HS decreased dry matter intake and milk yield (36 and 26%, respectively), and the reductions were similar between dietary treatments. Relative to P1, HS increased milk fat content and milk urea nitrogen (17 and 30%, respectively) and decreased milk protein and lactose contents (7 and 1.4%, respectively). Overall, HS increased (52%) plasma cortisol concentrations of CON, but circulating cortisol did not change in SCFP-fed cows. Heat stress increased circulating lipopolysaccharide binding protein and serum amyloid A (SAA; 2- and 4-fold, respectively), and SCFP supplementation tended to decrease peak SAA (∼33%) relative to CON cows. Overall, although HS did not influence circulating white blood cells and neutrophils, SCFP increased circulating white blood cells and neutrophils by 9 and 26%, respectively, over CON in P2. In conclusion, HS initiated an acute phase protein response and feeding SCFP blunted the cortisol and SAA concentrations and altered some key leukocyte dynamics during HS.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the transition period on rumen fermentation of dairy cows fed fresh diets differing in starch content.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the transition period (d -28 ± 3 to 23 ± 3 relative to calving) on rumen fermentation and mRNA abundance of genes in the rumen epithelium of fresh cows (d 1 to 23 ± 3 after calving) fed diets differing in starch content. Eighteen ruminally cannulated multiparous Holstein cows were fed diets with SCFP (n = 9) or without (CON; n = 9) throughout the experiment. All cows were fed a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) before calving. Cows within each treatment (CON or SCFP) were fed either a low-starch (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet during the fresh period. Cows were assigned to treatment after balancing for parity, body condition score, and expected calving date. Rumen pH was measured continuously for 72 h starting on d -10, -3, 1, 7, and 21 relative to calving date. Rumen papillae were collected on d -10 and 21 relative to calving. Supplementation of SCFP had no effect on rumen pH during d -10 to -8, but mean rumen pH tended to be higher (6.64 vs. 6.49) for SCFP cows than for CON cows during d -3 to -1. Feeding SCFP decreased the range of rumen pH variation compared with CON within the HS group during both d 7 to 9 (1.08 vs. 1.38) and d 21 to 23 (1.03 vs. 1.30) after calving. In addition, nadir rumen pH tended to be higher (5.64 vs. 5.44) and duration of pH below 5.8 tended to be shorter (116 vs. 323 min/d) for the SCFP group than for the CON group during d 21 to 23 after calving. Supplementation of SCFP increased the mRNA abundance of insulin-like growth factor-6 (1.10 vs. 0.69) before calving and decreased the mRNA abundance of putative anion transporter isoform 1 (1.12 vs. 2.27) after calving. Nadir rumen pH tended to be higher during d 1 to 3 (5.63 vs. 5.41) for LS cows than for HS cows, but rumen pH was not affected by dietary starch content during other time periods. Dietary starch content had no effect on mRNA abundance of genes in the rumen epithelium after calving. These results suggest that supplementation of SCFP may reduce the range of variation in rumen pH in fresh cows fed HS diets and the duration of subacute ruminal acidosis by the end of the fresh period regardless of dietary starch content and that decreasing dietary starch content during the fresh period may reduce the decrease in rumen pH immediately after parturition.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on the immune response of dairy cows fed fresh diets differing in starch content.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d -28 ± 3 to 44 ± 3 relative to calving) on mRNA abundance of genes in the rumen epithelium, inflammation indicators, oxidative status, and adaptive immunity of dairy cows fed diets with different starch content after calving. From d 28 ± 3 (± standard deviation) before the expected calving date to calving, Holstein cows (n = 38) received a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) with (SCFP; n = 19) or without (CON; n = 19) SCFP, and cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period). There were 4 treatment groups: LS + CON (n = 9), LS + SCFP (n = 10), HS + CON (n = 10), and HS + SCFP (n = 9). From d 24 ± 3 to 44 ± 3 after calving, all cows were fed the HS diets (post-fresh period). Animal assignment to treatments was balanced for parity, body condition score, and expected calving date. An interaction was observed between dietary starch content and SCFP on indices of oxidative stress; plasma concentrations of total antioxidant capacity tended to be reduced on d 21 after calving for SCFP compared with CON cows when a LS fresh diet was fed, but did not differ for cows fed HS fresh diets. Regardless of starch content, SCFP supplementation increased plasma concentrations of malondialdehyde at d 21 after calving compared with CON. Supplementing with SCFP reduced serum concentrations of haptoglobin on d 7 after calving, indicating reduced inflammation, and feeding LS fresh diets reduced mRNA abundance of IL receptor associated kinase-1 in rumen tissue at d 21 after calving, suggesting reduced immune activation in rumen tissue. Other than the anti-inflammatory effects indicated by lower serum haptoglobin concentration, no other effects of treatment on adaptive immunity were detectable. These results indicate that supplementing SCFP through the transition period and feeding low-starch diets during the fresh period may reduce inflammation.

Effect of Saccharomyces cerevisiae fermentation product on feed intake parameters, lactation performance, and metabolism of transition dairy cattle.

The transition period in dairy cattle is characterized by many stressors, including an abrupt diet change, but yeast product supplementation can alter the rumen environment to increase dairy cattle productivity. Saccharomyces cerevisiae fermentation product (SCFP) was fed from -29 ± 5 to 42 d relative to calving (RTC) to evaluate the effects on feed intake, milk production, and metabolism. Treatments were control (n = 30) or SCFP (n = 34) incorporated into a total mixed ration. Cows were individually fed 3×/d prepartum and 2×/d postpartum. Blood samples were collected once during each of the following time points RTC: d -28 to -24 (wk -4), d -14 to -10 (wk -2), d 3 to 7 (wk 1), d 12 to 16 (wk 2), and d 31 to 35 (wk 5). Liver biopsies were taken once between d -19 and d -12 (wk -3) and at 14 d in milk. Cows were milked 2×/d, and samples were taken 2 d/wk for composition analysis. Dry matter intake did not differ by treatment, but SCFP increased meals per day and decreased time between meals. Body weight (measured at enrollment, d 0, and d 42 RTC) and body condition score (scored weekly) were not affected by treatment. Milk, energy-corrected milk, and fat-corrected milk yields did not differ by treatment. Milk fat concentration was greater for SCFP, with significant differences in wk 4 and 5. Milk lactose concentration tended to be greater for the control and milk urea nitrogen tended to be lesser for the control, but there were no treatment effects on milk protein concentration or somatic cell count. Assuming equal digestibility, energy balance deficit was greater for SCFP than for the control (-6.15 vs. -4.34 ± 0.74 Mcal/d), with significant differences in wk 4 and 5. Plasma concentrations of free fatty acids, ß-hydroxybutyrate, glucose, and insulin did not differ with treatment, but cholesterol was greater for SCFP. Liver triglyceride increased and liver cholesterol decreased with time. Liver triglyceride did not differ by treatment, but liver cholesterol tended to be lesser in SCFP. Relative mRNA abundance of cholesterol-related genes (SREBF2, HMGCS1, HMGCR, MTTP, SPOB100, APOA1), FGF21, and CPT1A did not differ by treatment, but PCK1 tended to be greater for SCFP. The ketogenic transcript HMGCS2 was greater for SCFP, which aligns with SCFP increasing incidence of subclinical ketosis; however, BDH did not differ between treatments. In conclusion, SCFP supplementation increased meals per day with less time between meals, increased milk fat concentration, altered cholesterol metabolism, and increased incidence of subclinical ketosis, but early-lactation milk yield and metabolism were generally unaffected.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d -28 ± 3 to 44 ± 3 relative to calving) on dry matter intake (DMI), milk production, apparent total-tract nutrient digestibility, and postpartum ovarian activity of dairy cows fed fresh diets varying in starch content. From d 28 ± 3 before the expected calving date until d 44 ± 3 after calving, 117 Holstein cows were fed diets with SCFP (SCFP; n = 59) or without (control, CON; n = 58). A common, basal, controlled-energy close-up diet (net energy for lactation: 1.43 Mcal/kg; 13.8% starch) was fed before calving. Cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period), resulting in 4 treatment groups: LS-CON (n = 30), LS-SCFP (n = 29), HS-CON (n = 28), and HS-SCFP (n = 30). All cows were fed the HS diets from d 24 ± 3 to 44 ± 3 after calving (post-fresh period). Cows were assigned to treatment balanced for parity, body condition score, body weight, and expected calving date. Milk yield was higher for cows fed the LS diets compared with those fed the HS diets during the fresh period (34.1 vs. 32.1 kg/d), whereas DMI and 3.5% fat-corrected milk yield (FCM) were not affected by dietary starch content, and LS cows tended to lose more body condition than HS cows (-0.42 vs. -0.35 per 21 d) during the fresh period. Overall DMI during the close-up and fresh periods did not differ between SCFP and CON cows. However, SCFP supplementation transiently increased DMI on d 1 (13.0 vs. 11.9 kg/d) and 5 (15.5 vs. 14.1 kg/d) after calving compared with CON. During the post-fresh period, SCFP cows tended to eat less than CON cows (19.8 vs. 20.6 kg/d) but had similar 3.5% FCM (44.9 vs. 43.6 kg/d), resulting in greater feed efficiency for SCFP cows (FCM/DMI; 2.27 vs. 2.13). Neither starch content of fresh diets nor SCFP supplementation affected the interval from calving to first ovulation or the incidence of double ovulation. These findings suggest that feeding low-starch diets during the fresh period can increase milk production of dairy cows during the fresh period, and that supplementation of SCFP may increase feed intake around calving and feed efficiency in the post-fresh period.

Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa.

Whole-genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was nonsusceptible to colistin by broth microdilution, and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin-nonsusceptible P. aeruginosa.

Analysis of Serial Isolates of mcr-1-Positive Escherichia coli Reveals a Highly Active ISApl1 Transposon.

The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1 Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.

Insulin-sparing and fungible effects of E4orf1 combined with an adipocyte-targeting sequence in mouse models of type 1 and type 2 diabetes.

Obesity impairs glycemic control and causes insulin resistance and type 2 diabetes. Adenovirus 36 (Ad36) infection can increase the uptake of excess glucose from blood into adipocytes by increasing GLUT4 translocation through the Ras-Akt signaling pathway, which bypasses PI3K-Akt-mediated insulin receptor signaling. E4orf1, a viral gene expressed early during Ad36 infection, is responsible for this insulin-sparing effect and may be an alternative target for improving insulin resistance. To deliver the gene to adipocytes only, we connected the adipocyte-targeting sequence (ATS) to the 5' end of E4orf1 (ATS-E4orf1). In vitro transfection of ATS-E4orf1 into preadipocytes activated factors for GLUT4 translocation and adipogenesis to the same extent as did Hemagglutinin (HA)-E4orf1 transfection as positive reference. Moreover, the Transwell migration assay also showed that ATS-E4orf1 secreted by liver cells activated Akt in preadipocytes. We used a hydrodynamic gene delivery technique to deliver ATS-E4orf1 into high-fat diet-fed and streptozotocin-injected mice (disease models of type 2 and type 1 diabetes, respectively). ATS-E4orf1 improved the ability to eliminate excess glucose from the blood and ameliorated liver function in both disease models. These findings suggest that ATS-E4orf1 has insulin-sparing and fungible effects in type 2 and 1 diabetes independent of the presence of insulin.

The American cockroach peptide periplanetasin-4 inhibits Clostridium difficile toxin A-induced cell toxicities and inflammatory responses in the mouse gut.

Many reports have shown that crude extracts of the American cockroach have therapeutic effects on inflammation. In a previous study, our research group showed that an antimicrobial peptide (Periplanetasin-2) derived from the American cockroach via de novo transcriptome analysis inhibited apoptosis of human colonocytes and inflammatory responses of the mouse gut caused by Clostridium difficile toxin A. Here, we examined whether Periplanetasin-4 (Peri-4), another antimicrobial peptide identified via de novo transcriptome analysis of the American cockroach, could also inhibit the various toxicities induced by C. difficile toxin A. We found that Peri-4 significantly reduced the cell viability loss and cell apoptosis caused by toxin A in vitro. Peri-4 also ameliorated the severe inflammatory responses seen in the toxin A-induced mouse enteritis model, rescuing the villus disruption and interleukin-6 production induced by luminal injection of toxin A into the mouse gut. Mechanistically, we found that Peri-4 could reduce toxin A-induced reactive oxygen species production to inhibit the activations of p38MAPK and p21Cip1/Waf1 , which are critical for the cell damages induced by toxin A. These results collectively suggest that the Peri-4 may be a potential therapeutic agent for treating toxin A-induced pseudomembranous colitis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Influence of Saccharomyces cerevisiae fermentation products, SmartCare in milk replacer and Original XPC in calf starter, on the performance and health of preweaned Holstein calves challenged with Salmonella enterica serotype Typhimurium.

This study was designed to investigate the effects of supplementing SmartCare (SC; Diamond V, Cedar Rapids, IA) in milk replacer and Original XPC (XPC; Diamond V) in calf starter on performance and health of preweaned calves following an oral challenge with Salmonella enterica. The study was performed in two 35-d periods with 30 Holstein bull calves (2 ± 1 d of age) per period. In each period, calves were blocked by location in the barn and randomly assigned to treatments that included control, base milk replacer and calf starter with no added Saccharomyces cerevisiae fermentation products; SC, milk replacer with 1 g of SC/calf per day and base calf starter; and SC+XPC, milk replacer with 1 g of SC/calf per day and calf starter with 0.5% XPC on a dry matter basis. Calves were fed 350 g of milk replacer solids at 14% dry matter twice daily at 0700 and 1700 h. Calf starter and water were offered ad libitum and intakes were recorded daily. Calves were challenged with 108 cfu of sulfamethazine-resistant Salmonella enterica serotype Typhimurium orally on d 14 of the study. Fecal Salmonella shedding was determined on d 14 to 21 (daily), 24, 28, and 35 using selective media. Blood samples were collected on d 0, 7, 14, 16, 18, 21, 24, 28, and 35 and analyzed for hematology; plasma were analyzed for haptoglobin concentrations. All data were reported as CON, SC, and SC+XPC, respectively. Calf starter intake was increased from d 22 to 35 among SC+XPC calves and from d 29 to 35 among SC calves. The SC+XPC calves had a lower neutrophil-to-lymphocyte ratio (0.81, 0.83, and 0.69 ± 0.051) throughout the study. The SC+XPC calves also had lower hematocrits (35.1, 35.3, and 33.4 ± 0.54%) and hemoglobin concentrations (10.8, 10.6, and 10.1 ± 0.16 mg/dL) throughout the study. We found a tendency for calves fed SC and SC+XPC to have more solid fecal scores during the week after the challenge. We observed no treatment or treatment × time differences on plasma haptoglobin concentrations (63, 48, and 60 ± 0.5 µg/mL). No treatment differences were observed in the fecal shedding of the Salmonella; however, we noted a tendency for a treatment difference in the percentage of calves positive for Salmonella present in the ileal tissue at d 21 after the challenge (25, 50, and 60%). Supplementing preweaned Holstein calves with both SC in milk replacer and XPC in calf starter improved starter intake and improved fecal consistency immediately after a mild Salmonella enterica challenge, but more data are needed to further understand how these yeast fermentation products influence the immune responses to Salmonella enterica.

Effects of Saccharomyces cerevisiae fermentation products on dairy calves: Performance and health.

The aim of the study was to evaluate the effects of Saccharomyces cerevisiae fermentation products (SCFP) on performance and health of calves during the first 63 d of age. Sixty Holstein calves (30 males and 30 females) at 2 d of age were blocked by sex and date of birth then randomly assigned within blocks to 1 of 3 treatments. A texturized calf starter was fed ad libitum containing 0 (control), 0.5, or 1% SCFP (Original XPC, Diamond V, Cedar Rapids, IA) of DM. In addition, the supplemented calves were fed 1 g/d SCFP (SmartCare, Diamond V) in milk until d 30. All calves were fed 4 L of colostrum within 1 h of birth and were subsequently fed milk twice daily until weaned at 56 d of age. Male calves were harvested on d 56. Performance and health of weaned female calves were monitored until 63 d of age to determine the effect of preweaning treatment of SCFP on weaning stress. Starter intake, fecal scores, and medical treatments were recorded daily. Body weight measures and blood samples were collected on d 2, 28, 56, and 63. Serum was analyzed for blood urea nitrogen, fatty acids, insulin-like growth factor-1, glucose, and total protein. Oxidative biomarkers and total antioxidant capacity were also evaluated in the serum. Body weight, DMI, blood parameters, and oxidative biomarkers did not differ among treatments. Supplementation of SCFP lowered fecal scores in the pre- and postweaning periods. Saccharomyces cerevisiae fermentation products can be used to reduce the diarrhea in calves grown under normal commercial conditions.

Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test.

Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.