Anti-Insulin Receptor Autoantibodies Are Not Required for Type 2 Diabetes Pathogenesis in NZL/Lt Mice, a New Zealand Obese (NZO)-Derived Mouse Strain

The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte–enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions ofBlymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh6nullmales gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the Blymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers

Large grazers modify effects of aboveground–belowground interactions on small-scale plant community composition

Aboveground and belowground organisms influence plant community composition by local interactions, and their scale of impact may vary from millimeters belowground to kilometers aboveground. However, it still poorly understood how large grazers that select their forage on large spatial scales interact with small-scale aboveground–belowground interactions on plant community heterogeneity. Here, we investigate how cattle (Bos taurus) modify the effects of interactions between yellow meadow ants (Lasius flavus) and European brown hares (Lepus europaeus) on the formation of small-scale heterogeneity in vegetation composition. In the absence of cattle, hares selectively foraged on ant mounds, while under combined grazing by hares and cattle, vertebrate grazing pressure was similar on and off mounds. Ant mounds that were grazed by only hares had a different plant community composition compared to their surroundings: the cover of the grazing-intolerant grass Elytrigia atherica was reduced on ant mounds, whereas the relative cover of the more grazing-tolerant and palatable grass Festuca rubra was enhanced. Combined grazing by hares and cattle, resulted in homogenization of plant community composition on and off ant mounds, with high overall cover of F. rubra. We conclude that hares can respond to local ant–soil–vegetation interactions, because they are small, selective herbivores that make their foraging decisions on a local scale. This results in small-scale plant patches on mounds of yellow meadow ants. In the presence of cattle, which are less selective aboveground herbivores, local plant community patterns triggered by small-scale aboveground–belowground interactions can disappear. Therefore, cattle modify the consequences of aboveground–belowground interactions for small-scale plant community composition

Isolation and Host Range of Bacteriophage with Lytic Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite Decontaminant.

Staphylococcus aureus (SA) is a commensal bacterium and opportunistic pathogen commonly associated with humans and is capable of causing serious disease and death including sepsis, pneumonia, and meningitis. Methicillin-resistant SA (MRSA) isolates are typically resistant to many available antibiotics with the common exception of vancomycin. The presence of vancomycin resistance in some SA isolates combined with the current heavy use of vancomycin to treat MRSA infections indicates that MRSA may achieve broad resistance to vancomycin in the near future. New MRSA treatments are clearly needed. Bacteriophages (phages) are viruses that infect bacteria, commonly resulting in death of the host bacterial cell. Phage therapy entails the use of phage to treat or prevent bacterial infections. In this study, 12 phages were isolated that can replicate in human SA and/or MRSA isolates as a potential way to control these infections. 5 phage were discovered through mitomycin C induction of prophage and 7 others as extracellular viruses. Primary SA strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates by spot testing. Primary isolates were tested for susceptibility to oxacillin in order to determine which were MRSA. Experiments were performed to assess the host range and killing potential of newly discovered phage, and significant reductions in bacterial load were detected. We explored the utility of some phage to decontaminate fomites (glass and cloth) and found a significant reduction in colony forming units of MRSA following phage treatment, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics

Antibiotic resistance is lower in Staphylococcus aureus isolated from antibiotic-free raw meat as compared to conventional raw meat.

The frequent use of antibiotics contributes to antibiotic resistance in bacteria, resulting in an increase in infections that are difficult to treat. Livestock are commonly administered antibiotics in their feed, but there is current interest in raising animals that are only administered antibiotics during active infections. Staphylococcus aureus (SA) is a common pathogen of both humans and livestock raised for human consumption. SA has achieved high levels of antibiotic resistance, but the origins and locations of resistance selection are poorly understood. We determined the prevalence of SA and MRSA in conventional and antibiotic-free (AF) meat products, and also measured rates of antibiotic resistance in these isolates. We isolated SA from raw conventional turkey, chicken, beef, and pork samples and also from AF chicken and turkey samples. We found that SA contamination was common, with an overall prevalence of 22.6% (range of 2.8-30.8%) in conventional meats and 13.0% (range of 12.5-13.2%) in AF poultry meats. MRSA was isolated from 15.7% of conventional raw meats (range of 2.8-20.4%) but not from AF-free meats. The degree of antibiotic resistance in conventional poultry products was significantly higher vs AF poultry products for a number of different antibiotics, and while multi-drug resistant strains were relatively common in conventional meats none were detected in AF meats. The use of antibiotics in livestock contributes to high levels of antibiotic resistance in SA found in meat products. Our results support the use of AF conditions for livestock in order to prevent antibiotic resistance development in SA

SA/MRSA isolates used in these studies.

<p>Isolates were confirmed as SA or MRSA as detailed in Methods. Methicillin resistance was tested by growth on MSA plates in the presence of 2μg/mL oxacillin. ATCC = American Type Culture Collection, NS = human nasal swab, HA = hospital acquired, DH = dog hair, CJ = raw chicken, RB = raw beef, TK = raw turkey.</p><p>SA/MRSA isolates used in these studies.</p

Decontamination of lab coats and glass coverslips.

<p>MRSA samples were inoculated onto either lab coat fabric or glass coverslips and then exposed to either a single phage or a mock phage treatment (sterile phage buffer only). The MOI ranged from 50 to 20,000. Bacteria were recovered and viable bacterial counts were determined by serial dilution and growth on LB agar plates. Results are reported as colony-forming units ml^-1 A) MRSA strain M1 treated with either SEW, M1M4, CJ11 or CJ12 recovered from lab coat fabric. B) MRSA strain M1 treated with either SEW, M1M4, CJ11 or CJ12 recovered from glass coverslips. C) MRSA strain DH1 treated with either SEW, M1M4, CJ11 or CJ12 recovered from lab coat fabric. D) MRSA strain DH1 treated with either SEW, M1M4, CJ11, or CJ12 recovered from glass coverslips. Assays were performed in triplicate; standard error is indicated. * p ≤ 0.05 by unpaired, one-tailed student’s t test when evaluating phage-treated vs mock-treated samples. # p ≤ 0.005 by same student’s t test.</p

Decontamination of lab coats and glass coverslips with a phage cocktail.

<p>MRSA samples were inoculated onto either lab coat fabric or glass coverslips and then exposed to a phage cocktail consisting of SEW, M1M4, CJ11, CJ12, or mock phage treatment (sterile phage buffer only). The combined MOI ranged from 300 to 1,300. Bacteria were recovered from treated materials and viable bacterial counts were determined by serial dilution and growth on LB agar plates. A) MRSA strain M1 and DH1 treated with phage cocktail or mock treatment recovered from lab coat fabric. B) MRSA strain M1 and DH1 treated with phage cocktail or mock treatment recovered from glass coverslips. Assays were performed in triplicate; standard error is indicated. * p ≤ 0.05 by unpaired, one-tailed student’s t test when evaluating phage-treated vs mock-treated samples. # p ≤ 0.005 by same student’s t test.</p

Assessment of host range by lysis of bacterial cultures.

<p>Phage were added to log phase bacterial cultures to assess host range and to determine the killing efficiency of the phages. Optical density was measured at 600nm to quantify cell density of the culture. A) MRSA strain DH1 infected with phage strain SEW across a time course. B) MRSA strain DH1 infected with phage strain CJ11 across a time course. C) A variety of bacterial strains were challenged with different phage and OD₆₀₀ readings were taken at 4h. Results are reported in ΔOD₆₀₀ units which were calculated as the difference between the OD₆₀₀ of mock-treated cultures (sterile phage buffer only) and phage-treated cultures. D) Various combinations of bacterial strains were treated with either single phage strains or combinations phage. In panels C and D, the percent difference in OD₆₀₀ readings (phage-treated divided by mock-treated) is also shown. All experiments were performed in triplicate; standard error is indicated. * p ≤ 0.002 by student’s t test. # p ≤ 0.01 by student’s t test when evaluating phage treated vs mock treated samples.</p

Assessment of host range by spot testing.

<p>Following plaque purification, phages were tested for host range by spot testing on lawns of SA and MRSA isolates. Detection of any lawn clearing is indicated by an X. All testing was performed in triplicate, and virus stocks were serially diluted to confirm presence of phage lysis (discreet plaque formation) rather than bacteriocin activity.</p><p>Assessment of host range by spot testing.</p