Prevalence, antibiotic resistance and molecular characterisation of Staphylococcus aureus in pigs at agricultural fairs in the USA.

Fairs and petting zoos have been associated with outbreaks of zoonotic disease. Previously, the presence of meticillin-resistant Staphylococcus aureus (MRSA) was documented in commercial pigs; therefore, it was hypothesised that antibiotic-resistant S aureus may also occur in pigs exhibited at agricultural fairs. To test this hypothesis, 157 pigs were swabbed at two state fairs in 2008 to 2009. Both nares were sampled and cultures were grown in enrichment broth, then plated onto selective MRSA plates and blood plates. S aureus was confirmed using phenotypic and molecular methods, and was analysed using spa typing, gene-specific polymerase chain reaction and antibiotic susceptibility testing. The presence of S aureus was confirmed in samples collected from pigs exhibited at USA pig shows. Twenty-five of 157 (15.9 per cent) samples were positive for S aureus. Two isolates (8 per cent) were resistant to meticillin; 23/25 (92 per cent), 14/25 (56 per cent) and 15/25 (60 per cent) were resistant to tetracycline, erythromycin and clindamycin, respectively. spa typing revealed multiple isolates of spa type t034 (9/25, 36 per cent) and t337 (7/25, 28 per cent) and singletons of t002, t209, t526, t1236, t1334, t1683, t3075, t5784 and t5883. These results verify the presence of antibiotic-resistant S aureus in pigs exhibited at USA fairs, suggesting that pigs are a potential reservoir for S aureus within this environment.

MRSA in pork production shower facilities: an intervention to reduce occupational exposure.

Methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization has been documented in swine and swine workers. MRSA has also been found in the shower facilities of conventional swine farms. We previously conducted a review of the literature to identify measures used to reduce MRSA prevalence in athletic facilities. In this study, we evaluated those measures for adaptability to the pork production environment. A best practices protocol was developed to reduce MRSA levels in pork production shower facilities and implemented in two conventional swine production systems.

Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) on retail meat in Iowa.

Several recent studies have indicated a high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in retail-available meat. However, few studies have investigated MRSA in meat in the United States. The aim of this study was to determine the presence of Staphylococcus aureus (S. aureus) on meat samples available at retail stores. Samples of fresh raw pork, chicken, beef, and turkey were purchased from 22 food stores throughout Iowa. S. aureus strains were isolated from 27 of 165 samples, giving an overall prevalence of 16.4%. Turkey, pork, chicken, and beef had individual S. aureus prevalence rates of 19.4%, 18.2%, 17.8%, and 6.9%, respectively. Two isolates of MRSA were isolated from pork, giving an overall prevalence of 1.2%. One MRSA isolate was positive for the PVL gene. Common spa types included t034, t337, t008, and t002. These results suggest that MRSA is present on low numbers of retail meat in Iowa.

Activity of HIV-1 integrases recovered from subjects with varied rates of disease progression.

We recently described 102 HIV-1 integrase sequences that were amplified from blood cells or plasma obtained up to 18 years ago from 5 hemophiliacs who later died of AIDS and 5 hemophiliacs subsequently classified as slow or nonprogressors ( J Acquir Immune Defic Syndr Hum Retrovirol 1998;19:99-110). Although the region of the HIV-1 genome that encodes integrase was highly conserved, none of the deduced protein sequences of the patient-derived enzymes matched that of the clade B consensus or standard laboratory integrases. To test the hypothesis that the activity of HIV-1 integrases prevalent within an infected person contributes to the rate of disease progression, we have now expressed and purified these proteins and compared them in various assays. Most of the 75 unique full-length integrase proteins from the 102 clones were enzymatically active. Comparison of proteins derived from samples obtained soon after infection showed that the specificity and extent of viral DNA processing and the amount of DNA joining (the two biologically relevant activities of integrase) did not differ between the two groups of patients. In addition, the relative usage of alternative nucleophiles for processing and the amount of nonspecific nicking catalyzed by the proteins were indistinguishable between the patient groups. Although the patient-derived enzymes often exhibited different patterns of target site preferences compared with the laboratory integrase, there was no correlation with clinical course. Thus, the activities of HIV-1 integrases prevalent within these infected individuals, at least as reflected by standard assays, did not influence or predict the rate of disease progression.

Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection.

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.

Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases.

Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.

Direct evidence for linkage of roan and extension loci in Belgian horses.

A bay roan Brabant Belgian stallion (ERn/ ern) was bred to eight chestnut American Belgian mares (ern/ ern), producing 57 foals. Thirty foals were bay roan, 25 were chestnut, one was bay, and one was chestnut roan. The recombination rate was 0.035 +/- 0.024, indicating fairly close linkage between the roan (Rn) and extension (E) loci.