Enzymes are often required to function in a particular reaction condition by the industrial procedure. In order to identify critical residues affecting the optimum pH of Staphylococcal lipases, chimeric lipases from homologous lipases were generated via a DNA shuffling strategy. Chimeric 1 included mutations of G166S, K212E, T243A, H271Y. Chimeric 2 consisted of substitutions of K212E, T243A, H271Y. Chimeric 3 contained substitutions of K212E, R359L. From the screening results, the pH profiles for chimeric 1 and 2 lipases were shifted from pH 7 to 6. While the pH of chimeric 3 was shifted to 8. It seems the mutation of K212E in chimeric 1 and 2 decreased the pH to 6 by changing the electrostatic potential surface. Furthermore, chimeric 3 showed 10 ˚C improvement in the optimum temperature due to the rigidification of the catalytic loop through the hydrophobic interaction network. Moreover, the substrate specificity of chimeric 1 and 2 was increased towards the longer carbon length chains due to the mutation of T243A adjacent to the lid region through increasing the flexibility of the lid. Current study illustrated that directed evolution successfully modified lipase properties including optimum pH, temperature and substrate specificity through mutations, especially near catalytic and lid regions.

Staphylococci are major causes of infections in mammals. Mammals are colonized by diverse staphylococcal species, often with moderate to strong host specificity, and colonization is a common source of infection. Staphylococcal infections of animals not only are of major importance for animal well-being but have considerable economic consequences, such as in the case of staphylococcal mastitis, which costs billions of dollars annually. Furthermore, pet animals can be temporary carriers of strains infectious to humans. Moreover, antimicrobial resistance is a great concern in livestock infections, as there is considerable antibiotic overuse, and resistant strains can be transferred to humans. With the number of working antibiotics continuously becoming smaller due to the concomitant spread of resistant strains, alternative approaches, such as anti-virulence, are increasingly being investigated to treat staphylococcal infections. For this, understanding the virulence mechanisms of animal staphylococcal pathogens is crucial. While many virulence factors have similar functions in humans as animals, there are increasingly frequent reports of host-specific virulence factors and mechanisms. Furthermore, we are only beginning to understand virulence mechanisms in animal-specific staphylococcal pathogens. This review gives an overview of animal infections caused by staphylococci and our knowledge about the virulence mechanisms involved.

The bacterium Staphylococcus hyicus causes porcine exudative epidermitis in piglets, which represents both health and welfare concerns. Few genome sequences of this pathogen are published. We provide four additional ones to help future genomic analysis of S. hyicus. These are genomes of strains isolated from Canadian swine.

A massive outbreak of exudative epidermitis (EE) occurred on a Western German piglet producing farm with 350 productive sows. Gilts are produced on site. In one group of piglets, more than 50% of suckling and nursery piglets were clinically affected; furthermore, gilts as well as sows showed localized blackish-squamous skin lesions in the neck area. Generalized infection in suckling and nursery piglets resulted in mortality rates of up to 10% per weaning group. Swabs of moist, affected areas of skin taken on the farm in addition to swab and organ samples collected during necropsy were examined via bacterial cultivation. Both Staphylococcus (St.) hyicus and St. chromogenes strains were detected in affected skin lesions, with St. hyicus also present in systemic localizations. Further characterization of the St. hyicus strains identified ExhA and SHETA as toxins involved, and isolates showed resistance to penicillin and aminopenicillin. In the short term, antibiotic treatment with trimethoprim-sulfadiazine of the whole age group combined with individual treatment of severely affected animals as well as washing with an iodine-containing solution improved the clinical signs. In order to reduce the antibiotic use, an autogenous vaccine against the isolated St. hyicus and St. chromogenes strains for gilts and sows was produced and applied as a basic immunization twice before farrowing. In addition, external and internal biosecurity was evaluated and adjusted using an objective questionnaire (Bio-check.UGentTM). The combination of taken measures resulted in a long-term improvement of the overall health status. Several months after the severe EE outbreak, the sporadic occurrence of new EE cases in individual piglets could be controlled by the adjustment of the autogenous vaccine with an additional St. hyicus isolate. The case report illustrates how the combination of continuous monitoring, individual and group antibiotic treatment, biosecurity evaluation, and the use of appropriate immune prophylaxis can improve the clinical picture of EE.
Exsudative Epidermitis (EE) – meist, aber nicht ausschließlich ausgelöst durch Staphylococcus (St.) hyicus – ist ein verbreitetes Problem auf Ferkelerzeugerbetrieben. So auch in einem westdeutschen eigenremontierenden Betrieb mit 350 produktiven Sauen. Beginnend mit schwärzlich-schuppigen Hautläsionen bei einzelnen Saug- und Aufzuchtferkeln sowie Jung- und Altsauen, bestand bereits in der nächsten Absetzgruppe bei etwa 50% der Ferkel die klinische Verdachtsdiagnose der EE. Die Mortalitätsrate im Aufzuchtstall stieg bei betroffenen Ferkelgruppen auf 10%. Sowohl die (histo)pathologische Untersuchung als auch der wiederholte Nachweis von St. hyicus aus Hauttupfern sowie Tupfern aus anderen Organen bestätigten die klinische Verdachtsdiagnose. Neben St. hyicus konnte aus einzelnen Hauttupfern zudem St. chromogenes isoliert werden. In der weiterführenden Charakterisierung der St. hyicus-Stämme wurden ExhA und SHETA als beteiligte Toxine identifiziert, und die Isolate zeigten unter anderem Resistenzen gegen Penicilline und Aminopenicilline. Kurzfristig verbesserte eine bestandsweise antibiotische Therapie mit einem Trimethoprim-Sulfadiazin-haltigen Präparat in Kombination mit der individuellen Behandlung einzelner Tiere und dem Waschen mit einer jodhaltigen Lösung die klinische Symptomatik. Zur dauerhaften Reduktion des Einsatzes antibiotischer Mittel wurde sich für die Herstellung eines bestandsspezifischen Impfstoffs mit den isolierten St. hyicus- und St. chromogenes-Stämmen entschieden, welcher als Mutterschutzvakzine an die Sauen appliziert wurde. Zusätzlich wurde die externe und interne Biosicherheit anhand eines objektiven Fragebogens (Bio-check.UGentTM) evaluiert und angepasst. Die Kombination der Maßnahmen führte zu einer langfristigen Besserung des klinischen Geschehens. Das erneute Auftreten leichter Hautläsionen bei einzelnen Ferkeln konnte mit der Anpassung des bestandsspezifischen Impfstoffes durch neu gewonnene St. hyicus-Isolate zielführend in den Griff bekommen werden. Der Fallbericht verdeutlicht, wie mit der Kombination aus kontinuierlichem Monitoring, Einzeltier- und Gruppentherapie, Evaluierung der Biosicherheit und dem Einsatz einer angemessenen Immunprophylaxe selbst hartnäckige Fälle der EE auf ein Minimum reduziert werden können.

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. IMPORTANCE Skin lesions in cattle associated with feeding by Haematobia fly species are a significant welfare issue in Australia, North and South America, and Europe. The development of these lesions has been attributed to a number of causal factors, but the exact etiology and pathogenesis were unclear. This study characterized Staphylococcus agnetis and Staphylococcus hyicus strains from cattle skin lesions and in vector flies and demonstrated their role in the pathogenesis of these lesions. These findings will aid the development of targeted and more effective treatment and control strategies for lesions associated with fly infestation in cattle.

Staphylococcus hyicus is the causative agent of porcine exudative epidermitis. This disorder affects animals in all producing countries and presents a widespread occurrence in Brazil. This study evaluated strains from a historical collection in order to detect the presence of exfoliative-toxin-encoding genes (SHETB, ExhA, ExhB, ExhC, ExhD), characterize the strains using PFGE, and determine their respective antimicrobial resistance profiles. The results obtained from the evaluation of 77 strains from 1982 to 1987 and 103 strains from 2012 reveal a significant change in resistance profiles between the two periods, especially regarding the antimicrobial classes of fluoroquinolones, amphenicols, lincosamides, and pleuromutilins. The levels of multidrug resistance observed in 2012 were significantly higher than those detected in the 1980s. It was not possible to correlate the resistance profiles and presence of genes encoding toxins with the groups obtained via PFGE. Only 10.5% of the strains were negative for exfoliative toxins, and different combinations of toxins genes were identified. The changes observed in the resistance pattern of this bacterial species over the 30-year period analyzed indicate that S. hyicus could be a useful indicator in resistance monitoring programs in swine production. In a country with animal protein production such as Brazil, the results of this study reinforce the need to establish consistent monitoring programs of antimicrobial resistance in animals, as already implemented in various countries of the world.

Abstract  The pathogenesis of wound infections is largely dependent on adherence mechanisms and toxins. Studies of Staphylococcus intermedins indicate that pyoderma isolates can bind to extracellular matrix proteins exposed in wounds but interpretation of adherence studies is complicated as some organisms possess multiple mechanisms. Studies of blocking and promotion of adherence by antibiotics also show variability. Epidermolytic toxins are recognized in S. aureus and S. hyicus but not in S. intermedins, although a synergohymenotropic toxin has been described and canine syndromes occur where epidermal splitting and S. intermedins infection co-exist. Staphylococcal enterotoxins and toxic shock toxin-1 are recognized as superantigens which cause cytokine release non-specifically, promoting inflammation in wounds. Treatment of infected skin depends largely on antibiotics but drug resistance will limit this; a move towards vaccines and re-examination of the value of antisepsis is occurring. Use of dressings which reduce microbial growth must also be a factor in wound management. Resumen  La patogenesis de las infecciones de heridas depende en gran medida de los mecanismos de adherencia y de las toxinas. Algunos estudios en Staphylococcus intermedius indican que los aislamientos a partir de áreas de pioderma pueden adherirse a proteinas de la matriz extracelular expuesta en las heridas aunque la interpretación de estudios de adherencia es complicado ya que algunos organismos poseen múltiples mecanismos. Los estudios con antibióticos sobre su capacidad de bloqueo y promoción de la adherencia también muestran variabilidad. Se reconocen toxinas epidermolíticas en S. aureus y S. hyicus pero no en S. intermedius, aunque se ha descrito una toxina sinergohimenotrópica y se producen sindromes caninos en áreas de coexistencia de rotura epidérmica y S. intermedius. Las enterotoxinas estafilocócicas y la Toxina-1 de Shock Tóxico son reconocidas como superantígenos que causan liberación inespecífica de citoquinas, promoviendo la inflamación a nivel de las heridas. El tratamiento de la piel infectada depende en gran medida de los antibióticos, aunque esto se encuentra limitado por las resistencias; se está tendiendo a las vacunaciones y a una re-evaluación del valor de la antisepsia. El uso de vendas que reduzcan el crecimiento bacteriano deberia también ser un factor en el manejo de heridas. [Noble, W.C., Lloyd, D.H. Pathogenesis and management of wound infections in domestic animals (Patogenesis y manejo de heridas infectadas en animales domesticos). Veterinary Dermatology 1997; 8: 243-248] Zusammenfassung  Die Pathogenese von Wundinfektionen hängt hauptsächlich von Haftungsmechanismen und Toxinen ab. Studien von Staphylokokkus intermedius weisen nach, dass Isolate von Pyodermien sich an in Wunden exponierte extrazelluläre Matrixproteine binden; die Interpretation dieser Haftungsstudien wird durch multiple Mechanismen einiger Organismen kompliziert. Studien, diese Haftung durch Antibiotika zu blockieren oder zu fördern, zeigen variable Ergebnisse. Epidermolytische Toxine sind für S. aureus und S. hyicus, aber nicht für S. intermedius beschrieben, obwohl von einem synergohymenotropischen Toxin berichtet wird und beim Hund Syndrome auftreten, in denen epidermale Spaltung und S. intermedius Infektion koexistieren. Enterotoxine und Toxischer Schock Toxin 1 von Staphylokokkus werden als Superantigene anerkannt, die nicht spezifische Zytokinfreisetzung verursachen und so die Entzündung in Wunden fördern. Die Behandlung infizierter Haut erfolgt weitgehend durch Antibiotika, aber Resistenzbildung wird deren Verwendung einschränken; im Moment geht die Tendenz mehr in Richtung Impfungen und die Neubewertung von Antisepsis. Die Verwendung von Verbandsmaterialien, die das Wachstum von Mikroorganismen einschränken, muss in der Wundversorgung berücksichtigt werden. [Noble, W.C., Lloyd, D.H. Pathogenesis and management of wound infections in domestic animals (Pathogenese und Versorgung von Wundinfektionen bei Haustieren). Veterinary Dermatology 1997; 8: 243-248] Résumé  La pathogénie des plaies infectées dépend largement de mécanismes d\'adhérence et de toxines. L\'étude de Staphylococcus intermedius montre que des germes de pyodermite peuvent adhérer aux protéines des matrices extracellulaires mises à nu par des blessures mais l\'interprétation des études d\'adhérence est compliquée par le fait que certains germes possèdent de multiples mécanismes. Des études d\'inhibition ou d\'activation d\'adhérence par des antibiotiques démontrent également une variabilité. On connait l\'existence de toxines épidermolytiques chez S. aureus et S. hyicus mais pas chez S. intermedius, bien qu\'une toxine synergohymenotropique ait été décrite, et que des syndrômes où clivage épidermique et infection àStaphylococcus intermedius coexistent aient été décrit chez le chien. II est admis que des entérotoxines staphylococciques et la toxine 1 du choc toxique agissent comme des superantigènes qui provoquent une libération non spécifique de cytokines, provoquant l\'inflammation des plaies. Le traitement des plaies infectées dépend largement des antibiotiques mais est limité par le phénomène d\'antibiorésistance; actuellement, une tendance à reconsidérer la valeur des vaccins et de l\'antisepsie se dessine. L\'utilisation de pansements qui réduisent la prolifération bactérienne peut également être un facteur du traitement des plaies. [Noble, W.C., Lloyd, D.H. Pathogenesis and management of wound infections in domestic animals (Pathogénie et traitement des plaies infectées chez les animaux domestiques). Veterinary Dermatology 1997; 8: 243-248].

A case of generalized exudative epidermitis (EE) is described, which occurred in a very small piglet producing farm in Austria. The antimicrobial treatment prescribed by the herd veterinarian did not improve the clinical problem. Therefore, the University Clinic for Swine intervened in the case. Lab investigations were initiated in which Staphylococcus hyicus (SH) and Staphylococcus aureus (SA), both methicillin-resistant and susceptible strains, could be isolated from the skin of affected piglets. Poor hygiene and management practices were identified as predisposing factors on site. Adaptation of antimicrobial treatment according to results of the in vitro susceptibility testing and the implementation of proper hygiene measures resolved the clinical problem. Here, we describe a fatal coinfection of SH and SA in suckling piglets.

There is an urgent need to explore new antimicrobial agents due to the looming threat of bacteria resistance. Bovine lactoferricin (LfcinB), as a multifunctional peptide, has the potential to be a new active drug in the future. In this study, it aims to investigate the effect of fatty acid conjugation on antimicrobial peptide activity and topical therapeutic efficacy in a mouse model infected with Staphylococcus hyicus. Both Lfcin4 and Lfcin5 were conjugated with the unsaturated fatty acid linoleic acid (18-C) at their N-terminus and modified by acylation at the C-terminus. The derived peptides of Lin-Lf4NH2 and Lin-Lf5NH2 showed better antibacterial activity (MICs of 3.27 to 6.64 μM) than their parent peptides (MICs of 1.83 to 59.57 μM). Lin-Lf4NH2 (63.2%, 5 min) and Lin-Lf5NH2 (35.8%, 5 min) could more rapidly penetrate bacterial membrane than Lf4NH2 (2.34%, 5 min) and Lf5NH2 (1.94%, 5 min), which further confirmed by the laser scanning confocal microscopy (LSCM). Electron microscopy observations showed Lin-Lf4NH2 and Lin-Lf5NH2 disrupted S. hyicus cell membranes and led to the leakage of contents. Furthermore, after treatment with Lin-Lf4NH2 and Lin-Lf5NH2, the abscess symptoms of mice were significantly alleviated; the recovery rate of abscesses scope of Lin-Lf4NH2 (73.25%) and Lin-Lf5NH2 (71.71%) were 38.8 and 37.9-fold higher than that of untreated group (1.89%), respectively, and superior to Lf4NH2 (46.87%) and Lf5NH2 (58.75%). They significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and chemokine (MCP-1) in S. hyicus skin lesions. This study provides evidence that conjugation of a fatty acid to antimicrobial peptides can improve the activity and have potential for topical therapeutic of S. hyicus skin infections. KEY POINTS: • Lin-Lfcin4NH2/Lfcin5NH2 showed stronger antimicrobial activity than parent peptides. • Lin-Lfcin4NH2/Lfcin5NH2 had a more effective ability to destroy bacterial membranes. • Lin-Lfcin4NH2/Lfcin5NH2 showed a topically higher efficacy than parent peptides.

Exudative epidermitis (EE), also known as greasy pig disease, is one of the most frequent skin diseases affecting piglets. Zoonotic infections in human occur. EE is primarily caused by virulent strains of Staphylococcus (S.) hyicus. Generally, antibiotic treatment of this pathogen is prone to decreasing success, due to the incremental development of multiple resistances of bacteria against antibiotics. Once approved, bacteriophages might offer interesting alternatives for environmental sanitation or individualized treatment, subject to the absence of virulence and antimicrobial resistance genes. However, genetic characterization of bacteriophages for S. hyicus has, so far, been missing. Therefore, we investigated a piglet raising farm with a stock problem due to EE. We isolated eleven phages from the environment and wash water of piglets diagnosed with the causative agent of EE, i.e., S. hyicus. The phages were morphologically characterized by electron microscopy, where they appeared Siphoviridae-like. The genomes of two phages were sequenced on a MiSeq instrument (Illumina), resulting in the identification of a new virulent phage, PITT-1 (PMBT8), and a temperate phage, PITT-5 (PMBT9). Sequencing of three host bacteria (S. hyicus) from one single farm revealed the presence of two different strains with genes coding for two different exfoliative toxin genes, i.e., exhA (2 strains) and exhC (1 strain). The exhC-positive S. hyicus strain was only weakly lysed by most lytic phages. The occurrence of different virulent S. hyicus strains in the same outbreak limits the prospects for successful phage treatment and argues for the simultaneous use of multiple and different phages attacking the same host.