Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms.

BACKGROUND: Over the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority. RESULTS: All pigs on farm 1 carried LA-MRSA in the nares at all three time points and five pigs on farm 2 at one time point. Nasal quantity varied between 10 and 103 CFU/swab and quantity on the skin between 10 and 102 CFU/swab. In the controlled environment, MRSA was detected in at least one of the nasal samples from each animal. spa type t034 was predominant. cgMLST showed one cluster with minimum allele differences between 0 and 11. CONCLUSIONS: The study shows predominantly low-level carriage (< 103 CFU/swab) of LA-MRSA on farms. In the controlled environment we observed a decline in nasal carriage but constant skin carriage. cgMLST showed that strains of spa type t034 are closely related at the national level.

Phage Treatment Trial to Eradicate LA-MRSA from Healthy Carrier Pigs.

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10-100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined.

Complete Genome Sequences of Two Klebsiella pneumoniae Phages Isolated as Part of an International Effort.

We report the genomic sequences of phages KpCHEMY26 and KpGranit, isolated in Israel during a worldwide effort against a multidrug- and phage-resistant strain of Klebsiella pneumoniae from a patient in Finland. These results demonstrate the importance of an efficient worldwide network for collaborating in personalized therapy for infectious diseases.

Characterization of vB_SauM-fRuSau02, a Twort-Like Bacteriophage Isolated from a Therapeutic Phage Cocktail.

Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.