Multidrug resistant staphylococci isolated from pigs with exudative epidermitis in North eastern Region of India.

Exudative epidermatitis or greasy pig disease (GPD) is a contagious disease of pig and endemic worldwide caused by toxigenic strains under genus Staphylococcus. The present study reported an outbreak of GPD in Champhai district of Mizoram adjoining to the southern border of Myanmar. A total of 60 samples were collected from 22 clinically affected animals and processed for isolation and identification of Staphylococcus spp. All the isolates were subjected to antimicrobial sensitivity assay, biofilm production assay and detection of virulence genes, biofilm genes and mec genes followed by cloning and sequencing for phylogenetic analysis. A total of 44 staphylococci belonged to four species (S. sciuri, S. aureus,S. lentus, and S. hyicus) were isolated. Majority of the isolates were multidrug resistant with maximum resistance against ampicillin, penicillin including vancomycin. None of the S. hyicus isolates was methicillin resistant (MRSH) but 66·67% isolates were MRSA. By PCR, mecA gene was detected in S. aureus (n = 2), S. sciuri (n = 4) and S. lentus (n = 3). Biofilm associated gene icaD was detected in S. aureus (n = 3), S. sciuri (n = 5), S. hyicus (n = 4) and S. lentus (n = 6). The exfoliative toxin genes (ehxB, shetA and tsst1) were detected in S. hyicus (n = 3) and S. aureus (n = 1) isolates. All the isolates were closely related with the isolates from pigs of China, Germany, Japan and USA. The pathogens might be transmitted through illegal migration of pigs from Myanmar to India.

Genetic and biochemical characterization of hyicin 3682, the first bacteriocin reported for Staphylococcus hyicus.

Hyicin 3682, the first bacteriocin reported for Staphylococcus hyicus, is a BsaCOL variant produced by S. hyicus 3682, a strain isolated from bovine milk. Hyicin 3682 is found in the culture supernatant, is bactericidal and its producing strain exhibits a much broader spectrum of antimicrobial activity than the producing strain of BsaCOL against several Gram-positive bacteria, which include foodborne pathogens, food-spoilage microorganisms and bacterial species of medical and veterinary importance. Sequencing of the genome of S. hyicus 3682 provided the nucleotide sequence of the entire gene cluster involved in hyicin 3682 production, which seems to be located on pRJ109, the single plasmid carried by this strain. This gene cluster is expressed and consists of 8525bp and of eight genes (hyiA, hyiB, hyiC, hyiD, hyiP, hyiF, hyiE and hyiG) encoded on the same DNA strand. The mature lantibiotic exhibits 91% identity to BsaCOL and its molecular mass was found to be ∼26Da higher due to two amino acid substitutions. S. hyicus 3682 proved to be only partially immune to its cognate bacteriocin up to 1024 AU/ml. Therefore, hyicin 3682, the first Bsa variant reported in coagulase-negative staphylococci, does exhibit antimicrobial and siblicidal activities.

A fatal suppurative pneumonia in piglets caused by a pathogenic coagulase-positive strain of Staphylococcus hyicus.

Staphylococcus hyicus is one of the opportunistic pathogens that cause infections to animals. Early studies have demonstrated that S. hyicus is the causative agents of exudative epidermitis in pigs, arthritis in horses and chicken, mastitis in cow, and bacteremia, sepsis and multiple organ failure in humans. Here, we report the isolation and identification of a representative S. hyicus isolate, named JLHN15, from a pig farm with a disease characterized by bacteremia, suppurative pneumonia and fibrinous pericarditis. Our results indicate that JLHN15 is a pathogenic coagulase-positive Staphylococcus. To the best knowledge, this is the first report of S. hyicus causing an infection characterized by suppurative pneumonia and sepsis.

Comparative genomics of toxigenic and non-toxigenic Staphylococcus hyicus.

The most common causative agent of exudative epidermitis (EE) in pigs is Staphylococcus hyicus. S. hyicus can be grouped into toxigenic and non-toxigenic strains based on their ability to cause EE in pigs and specific virulence genes have been identified. A genome wide comparison between non-toxigenic and toxigenic strains has never been performed. In this study, we sequenced eleven toxigenic and six non-toxigenic S. hyicus strains and performed comparative genomic and phylogenetic analysis. Our analyses revealed two genomic regions encoding genes that were predominantly found in toxigenic strains and are predicted to encode for virulence determinants for EE. All toxigenic strains encoded for one of the exfoliative toxins ExhA, ExhB, ExhC, or ExhD. In addition, one of these regions encoded for an ADP-ribosyltransferase (EDIN, epidermal cell differentiation inhibitor) and a novel putative RNase toxin (polymorphic toxin) and was associated with the gene encoding ExhA. A clear differentiation between toxigenic and non-toxigenic strains based on genomic and phylogenetic analyses was not apparent. The results of this study support the observation that exfoliative toxins of S. hyicus and S. aureus are located on genetic elements such as pathogenicity islands, phages, prophages and plasmids.

An investigation of resistance to ß-lactam antimicrobials among staphylococci isolated from pigs with exudative epidermitis.

BACKGROUND: A high proportion of staphylococci isolated from pigs affected with exudative epidermitis were found to be resistant to ß-lactam antimicrobials. The primary objective of this research was to investigate and characterize ß-lactam resistance in Staphylococcus hyicus, Staphylococcus aureus and other staphylococci isolated from these pigs. RESULTS: The antimicrobial resistance patterns of 240 staphylococci isolates were determined by disk diffusion, of which 176 (73.3%) of the isolates were resistant to 3 ß-lactams (penicillin G, ampicillin, and ceftiofur). The presence of mecA gene was identified in 63 staphylococci isolates from skin samples by PCR. The mecA gene was identified in 19 S. aureus, 31 S. hyicus, 9 Staphylococcus chromogenes, 2 Staphylococcus pseudintermedius isolates, and in 1 isolate each of Staphylococcus arlettae, and Staphylococcus cohnii subspecies urealyticus. From SCCmec typing results, the majority (45/63, 71.4%) were shown to be SCCmec type V. One isolate was SCCmec III. Fourteen isolates were detected as mec class A, mec class C or ccr type 5. The ccr complex and mec complex was not detected in 3 isolates of methicillin resistant S. hyicus (MRSH) based on multiplex PCR. Of the 30 isolates of MRSA identified from nasal samples of the pigs, 29 isolates were SCCmec type V and 1 isolate was SCCmec type II. Staphyloccoci isolates that were mecA negative but resistant to ß-lactam antimicrobials were further examined by screening for mecC, however all were negative. Furthermore, the majority of mecA negative ß-lactam resistant staphylococci isolates were susceptible to oxacillin and amoxicillin-clavulanic acid in a double disk diffusion test. CONCLUSIONS: Methicillin resistance can be identified in a variety of staphylococcal species isolated from pigs. In this study there was a great deal of similarity in the SCCmec types between staphylococcal species, suggesting that resistance may be passed from one species of staphylococci to another species of staphylococci. While this has been reported for acquisition of methicillin-resistance from coagulase negative staphylococci to S. aureus, these data suggest that transmission to or from the porcine pathogen S. hyicus may also occur. The identification of methicillin resistance in a variety of staphylococcal species in pigs does raise concerns about the spread of serious multi-drug resistance in food producing animals and warrants further study.

Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk.

The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 10 Brazilian dairy herds and study their capability to produce enterotoxins. Herds were selected based on size and use of milking technology, and farms were visited once during the study. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20 mL) was collected from all CMT-positive quarters. Identification of Staphylococcus spp. was performed using conventional microbiology, and PCR was used to determine the presence of enterotoxin-encoding genes (sea, seb, sec, and sed). Of the 1,318 CMT-positive milk samples, Staphylococcus spp. were isolated from 263 (19.9%). Of these isolates, 135 (51%) were coagulase-positive staphylococci (CPS) and 128 (49%) were coagulase-negative staphylococci (CNS). Eighteen different species of CNS were isolated, among which S. warneri, S. epidermidis and S. hyicus were the most frequent. The distribution of Staphylococcus species was different among herds: S. epidermidis was found in 8 herds, S. warneri was found in 7 herds, and S. hyicus in 6 herds. Some of the CNS species (S. saprophyticus ssp. saprophyticus, S. auricularis, S. capitis, and S. chromogenes) were isolated in only one of the farms. Genes related to production of enterotoxins were found in 66% (n=85) of all CNS and in 35% of the CPS isolates. For both CNS and CPS isolates, the most frequently identified enterotoxin genes were sea, seb, and sec; the prevalence of sea differed between CPS (9.5%) and CNS (35.1%) isolates. Staphylococcus warneri isolates showed a greater percentage of sea than seb, sec, or sed, whereas S. hyicus isolates showed a greater percentage of sea than sec. Over 60% of CNS belonged to 3 major species, which carried 62.2 to 81.3% of the enterotoxin genes. The high prevalence highlights the potential for food poisoning caused by these species. For possible high-risk situations for food poisoning, such as milk produced with total bacterial counts greater than regulatory levels and stored under inappropriate temperatures, monitoring contamination with CNS could be important to protect human health. Because the prevalence of CNS intramammary infections in dairy herds is usually high, and these species can be found in great numbers in bulk milk, identification of risk factors for production of staphylococcal enterotoxins should be considered in future studies.

Larvae of the parasitoid wasp Ampulex compressa sanitize their host, the American cockroach, with a blend of antimicrobials.

Food resources contaminated with spoilage or pathogenic microorganisms pose severe problems to all higher organisms. Here, we describe a food-hygienic strategy of the emerald cockroach wasp Ampulex compressa. The wasp larvae develop on and inside the American cockroach Periplaneta americana, a host that can harbor various putrefactive microbes, as well as human and insect pathogens. From P. americana, we isolated the Gram-negative bacterium Serratia marcescens, which is a potent entomopathogen that can rapidly kill insect larvae. It is also known as a food contaminant and as an opportunistic human pathogen. Using behavioral observations and chemical analyses, we demonstrated that A. compressa larvae impregnate their cockroach hosts from inside with large amounts of an oral secretion containing a blend of γ-lactones and isocoumarins with (R)-(-)-mellein [(R)-(-)-3,4-diydro-8-hydroxy-3-methylisocoumarin] and micromolide [(4R,9Z)-octadec-9-en-4-olide] as dominant components. We fractionated hexane extracts of the secretion and investigated the antimicrobial properties of the fraction containing the lactones and isocoumarins, as well as of synthetic (R)-(-)-mellein and micromolide, against S. marcescens and a Gram-positive bacterium, Staphylococcus hyicus, in broth microdilution assays. The test fraction inhibited growth of both tested bacteria. The activity of the fraction against S. marcescens was explained by (R)-(-)-mellein alone, and the activity against S. hyicus was explained by the combined action of (R)-(-)-mellein and micromolide. Our data suggest that the specific combination of antimicrobials in the larval secretion provides an effective frontline defense against the unpredictable spectrum of microbes that A. compressa larvae may encounter during their development inside their cockroach hosts.

Staphylococcus hyicus bacteremia in a farmer.

Bacteria known in animal infectious diseases can cause challenges in human diagnostic laboratories. We present pitfalls in the identification and susceptibility testing of Staphylococcus hyicus, a pathogen that typically causes exudative epidermitis in pigs. In this case, the coagulase-positive staphylococcus isolated from a septic patient was misidentified as Staphylococcus aureus.

Development of a high-expression system for staphylococcal exfoliative toxin genes.

We constructed a new expression system for staphylococcal exfoliative toxin (ET). The expression vector, pETA-exp2, was constructed based on Bacillus-Escherichia shuttle vector pHY300PLK. The pETA-exp2 vector includes the regulator of the ETA gene (eta), the promoter and Shine-Dalgarno (SD) sequences of eta, a SalI sequence at the end of the signal sequence of eta, a nucleotide sequence encoding mature ETA, an XhoI site, a 6x His sequence just before the stop codon and the end of the transcription sequence of eta. The nucleotide sequences coding for the mature proteins of ETB, ExhA, ExhB, ExhC, ExhD and SHETB were amplified by polymerase chain reaction (PCR) and inserted into pETA-exp2. These recombinant plasmids were transformed into Bacillus megaterium. The major protein in the culture supernatant of the transformant was recombinant ET (rET). The yields of all rETs were high, and all of them showed exfoliative activity in susceptible animals. The antigenicities of rETs and ETs were not distinguishable from each other.

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction.

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.

An IgG-binding protein A homolog in Staphylococcus hyicus.

Shotgun phage display was used to identify a homolog of the IgG-binding protein staphylococcal protein A in Staphylococcus hyicus type strain CCUG 15602/ATCC 11249. This bacterium is the causative agent of exudative epidermitis in pigs and can also cause mastitis in cattle. A protein with similar features as the originally identified protein A in Staphylococcus aureus was described; an YSIRK-type signal peptide, four IgG-binding domains, a putative peptidoglycan-binding domain, and a cell wall anchoring motif (LPXTG) was present. The highest degree of similarity was to a protein A homolog in Staphylococcus pseudintermedius. However, typical Xr polypeptide repeats present in the protein A of S. aureus and S. pseudintermedius could not be identified in the protein A of S. hyicus. The presence of the spa gene in ten porcine and eight bovine clinical isolates of S. hyicus was investigated by PCR. In all isolates, the spa gene could be detected but the amplicons were of two sizes. Sequence analysis of four selected PCR amplicons showed that only three IgG-binding domains were present in the protein A of clinical isolates generating a smaller spa fragment. The finding of spa in S. hyicus contributes to an increased understanding of potential virulence factors in this species.