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.

In vitro antibacterial activity of plant essential oils against Staphylococcus hyicus and Staphylococcus aureus, the causative agents of exudative epidermitis in pigs.

Greasy pig disease or exudative epidermitis, a generalized or localized skin disease affecting piglets, is mainly caused by Staphylococcus hyicus, although other staphylococcal species such as Staphylococcus aureus may also induce disease. Piglets with skin lesions can be treated systemically with antibiotics. However, antimicrobial resistance to ß-lactam antibiotics are now frequently observed in S. hyicus and S. aureus isolates. In this study, the antibacterial activity of plant essential oils as well as their ability to potentiate the effect of several antimicrobial compounds against S. hyicus and S. aureus were investigated with a view to a potential use as skin disinfectants. Among ten essential oils tested, those from cinnamon, thyme, and winter savory were the most active with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values ranging from 0.078 to 0.313% (v/v). Using a fluorescent probe with DNA affinity, it was found that thyme and winter savory oils act, at least in part, by disturbing the bacterial membrane integrity. At concentrations below the MIC, thyme and winter savory oils reduced biofilm formation by S. hyicus. Moreover, a treatment of pre-formed biofilms of S. hyicus with cinnamon or thyme oils significantly decreases its viability. Synergistic interactions between essential oils, more particularly from thyme and winter savory, and penicillin G, chlorhexidine or nisin, were observed. This study supports the therapeutic potential of essential oils as topical therapeutic agents against exudative epidermitis.

Exudative epidermitis of piglets caused by non-toxigenic Staphylococcus sciuri.

Infections with strains of Staphylococcus sciuri are a potential threat to animal and public health, and a cause for considerable concern. We isolated and identified S. sciuri as a pathogen from an acute outbreak of exudative epidermitis in piglets for further genetic identification using experimental infections. The results of this study showed that S. sciuri strain NJ1306 reproduced exudative epidermitis in experimentally infected 5-day-old piglets. The isolated bacteria also caused sudden death in BALB/c mice following intraperitoneal injection with 5×108 CFU of the isolate. The data indicated that strain NJ1306 of S. sciuri was pathogenic to piglets and mice, and the study provided the first known report of clinical lung lesions and endocarditis in piglets due to S. sciuri.

Zinc-resistance gene CzrC identified in methicillin-resistant Staphylococcus hyicus isolated from pigs with exudative epidermitis.

Methicillin-resistant Staphylococcus hyicus (MRSH) was investigated for czrC, a gene conferring zinc-resistance. The czrC gene was identified in 50% (14/28) of MRSH isolates, representing 14 pigs with exudative epidermitis from 8 farms. Newly weaned pigs, which are particularly susceptible to exudative epidermitis, are commonly fed high levels of zinc oxide.

GèneczrCcodant pour la résistance au zinc identifié dansStaphylococcus hyicusrésistant à la méthicilline isolé de porcs atteints de la dermite exsudative du porcelet.Staphylococcus hyicus résistant à la méthicilline (SHRM) a fait l'objet d'une enquête pour détecter czrC, un gène qui confère une résistance au zinc. Le gène czrC a été identifié dans 50 % (14/28) des isolats SHRM, représentant 14 porcs atteints de dermite exsudative du porcelet provenant de 8 fermes. Les porcelets nouvellement sevrés particulièrement sensibles à la dermite exsudative reçoivent couramment des niveaux élevés d'oxyde de zinc.(Traduit par Isabelle Vallières).

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.

Antimicrobial susceptibilities of exfoliative toxigenic and non-toxigenic Staphylococcus hyicus strains in Japan.

Staphylococcus hyicus isolates (n=207), including 150 exfolitative toxigenic and 57 non-toxigenic strains, were examined for their susceptibility to 13 antimicrobial agents by using the dehydrated 96-well MIC panel system. The frequency of their resistance to penicillin and ampicillin was 76.8% (159/207), followed by erythromycin (56%, 116/207), trimethoprim-sulfamethoxazole (28.5%, 59/207), chloramphenicol (24.2%, 50/207), kanamycin (19.8%, 41/207), and doxycycline (1.4%, 3/207). Resistance to chloramphenicol and trimethoprim-sulfamethoxazole was significantly higher in toxigenic strains than non-toxigenic strains (p<0.01), whereas kanamycin and erythromycin resistance was significantly higher in non-toxigenic strains (p<0.01 and <0.05, respectively). Resistance to two or more antimicrobials was observed in 85.5% (177/207) of total strains, with a significantly higher occurrence in toxigenic strains (89.3%, 134/150 vs. 75.4%, 43/57; p<0.05).

Nationwide molecular surveillance of exfoliative toxigenic Staphylococcus hyicus on pig farms across Japan.

We investigated the carriage of Staphylococcus hyicus and their exfoliative toxin genes (exhA, exhB, exhC, exhD, and shetb) among 424 pigs from 38 Japanese pig farms by PCR and characterized the isolates by 16S-23S intergenic spacer region polymorphism analysis. S. hyicus (n=207) were isolated from 17.9% of pigs and 72.5% of them were toxigenic. The isolation rate of toxigenic S. hyicus was four times higher in the pigs with exudative epidermitis than the healthy pigs (87.6% versus 19.6%; p<0.01). Among the toxins, exhA was the most prevalent, being detectable in 49.3% of toxin gene-positive strains. S. hyicus isolates were distributed into nine ITS-PCR types (a-i), with ITS-PCR types d, h, and e being predominant accounting for 70% (145/207) of total isolates or 76% (114/150) of toxin gene-positive strains. This study denotes high prevalence of toxigenic strains among S. hyicus circulating on pig farms in Japan.

A highly pathogenic strain of Staphylococcus sciuri caused fatal exudative epidermitis in piglets.

Staphylococcus sciuri are important human pathogens responsible for endocarditis, peritonitis, septic shock, urinary tract infection, pelvic inflammatory disease and wound infections. However, little information is known regarding the pathogenicity of S. sciuri to animals. From the pericardial fluid of a diseased piglet with exudative epidermitis (EE), we isolated a strain of Staphylococcus in pure culture. Surprisingly, this isolate was a member of S. sciuri rather than S. hyicus as identified by its biochemical traits and also by analysis of 23S ribosomal DNA using Internal Transcribed Spacer PCR. In addition, inoculation of newborn piglets with 1x10(10) CFU of the isolate by oral feeding or intra-muscular injection successfully reproduced EE in piglets, which suggested that the oral intake of the pathogen by the animals is one of the major routes of exposure. These unexpected findings prioritized S. sciuri as important zoonotic agents, which may have ramifications for human medicine.

Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany.

In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n=23) and Germany (n=17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis.

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.

Cloning of swine desmoglein 1 and its direct proteolysis by Staphylococcus hyicus exfoliative toxins isolated from pigs with exudative epidermitis.

Exudative epidermitis (EE) is an acute, often fatal skin disease of piglets caused by Staphylococcus hyicus. Clinical and histopathological manifestations of EE are similar to those of staphylococcal scalded skin syndrome (SSSS), a human blistering skin disease, in which exfoliative toxins produced by Staphylococcus aureus digest the extracellular domains of desmoglein (Dsg) 1 and cause loss of epidermal cell-cell adhesion. The aims of this study were to isolate and characterize cDNA for full length of swine Dsg1, and to determine whether the extracellular domains of swine Dsg1 produced by baculovirus (sDsg1-His) could be digested by four isoforms of exfoliative toxin produced by S. hyicus (ExhA, ExhB, ExhC and ExhD). Nucleotide sequencing revealed that swine Dsg1 cDNA consisted of an open reading frame of 3138 bp, encoding a precursor protein of 1045 amino acids. Deduced amino acid sequence of the swine Dsg1 precursor were highly homologous to corresponding bovine, canine, human and murine sequences. Immunoadsorption assay with a secreted form of sDsg1-His revealed that sDsg1-His specifically absorbs the immunoreactivity of 10 human pemphigus foliaceus sera against swine keratinocyte cell surfaces, suggesting its proper conformation. When sDsg1-His was incubated in vitro with Exhs, all four isoforms of Exh directly digested sDsg1-His into smaller peptides, whereas removal of calcium from sDsg1-His completely inhibited its proteolysis by these four Exhs. Recognition and digestion of calcium-stabilized structure on the extracellular domains of swine Dsg1 by Exhs indicated that EE shares similar molecular pathophysiological mechanisms of intra-epidermal splitting with SSSS in humans.

Production of exfoliative toxin by isolates of Staphylococcus hyicus from different countries.

A total of 218 isolates of Staphylococcus hyicus from pigs in eight countries (Belgium, Croatia, Germany, Japan, Korea, Slovenia, the uk and the usa) and 44 isolates from other animals in Belgium, India, Japan and the usa were examined for the genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD by multiplex pcr. The expression of the toxins was confirmed by immunoblot analysis, using monoclonal or polyclonal antibodies specific for each of the toxins. The porcine isolates were from pigs with exudative epidermitis, pigs with other lesions and from healthy pigs, and one or more of the toxins could be found among the isolates from the pigs in all the countries. Toxigenic strains of S hyicus were isolated from both healthy and diseased pigs, but the chance of isolating toxigenic strains from pigs with exudative epidermitis was greater than from pigs with other lesions or healthy pigs. Of the 44 isolates from other animal species, only one isolate, from a hare from Belgium, produced ExhB, and one isolate, from a cow with mastitis from Japan, produced ExhA.

Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes.

Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S. chromogenes producing exfoliative toxin type B, ExhB, was identified by the use of a multiplex PCR specific for the exfoliative toxins from S. hyicus. The exfoliative toxin from S. chromogenes reacted in immunoblot analysis with polyclonal and monoclonal antibodies specific to ExhB from S. hyicus and had an apparent molecular weight of 30 kDa. Sequencing the gene encoding the exfoliative toxin from S. chromogenes revealed that the molecular weight of the toxin with the signal peptide and the mature toxin was 30,553 and 26,694 Da, respectively. Comparison of the exhB genes from S. chromogenes strain VA654 and S. hyicus strain 1289D-88 showed differences in seven base pairs of the DNA sequences and in two amino acid residues in the deduced amino acid sequences. Pigs were experimentally inoculated with S. chromogenes strain VA654. By clinical observations and histopathological evaluation of the skin alterations, all pigs revealed development of generalized exudative epidermitis. No toxin producing S. hyicus was isolated from the pigs and all ExhB-positive bacterial isolates were identified as S. chromogenes. This confirmed that the disease-causing agent was the inoculated S. chromogenes strain VA654. The results of this study show that S. chromogenes may cause exudative epidermitis in pigs.

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus.

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.

The porcine skin associated T-cell homing chemokine CCL27: molecular cloning and mRNA expression in piglets infected experimentally with Staphylococcus hyicus.

CCL27 (also named CTACK, ALP, ILC and ESkine) is a CC chemokine primarily expressed by keratinocytes of the skin. The cognate receptor of CCL27 named CCR10 (GPR-2), is also expressed in skin-derived cells, and in addition by a subset of peripheral blood T-cells and in a variety of other tissues. In this paper, we report the cloning of porcine CCL27 cDNA and investigation of CCL27 mRNA expression in Staphylococcus hyicus infected piglets. At the protein level, 77 and 74% homology was found to human and mouse CCL27 sequences, respectively. The results of the expression analyses show that CCL27 mRNA is upregulated in the skin of infected piglets and to a lesser extent in piglets recovered from disease and without clinical signs of infection, indicating a role for CCL27 both during inflammation and after recovery from an infection.

Trends in antimicrobial susceptibility in relation to antimicrobial usage and presence of resistance genes in Staphylococcus hyicus isolated from exudative epidermitis in pigs.

From 1996 to 2001 a total of 467 Staphylococcus hyicus isolates from exudative epidermitis (EE) in pigs in Denmark were examined for susceptibility to 13 different antimicrobial agents. The presence of selected genes encoding macrolide (erm(A), erm(B) and erm(C)), penicillin (blaZ), streptogramin (vat, vga, vga(B), vat(B), vat(D) and vat(E)), streptomycin (aadE) and tetracycline resistance (tet(K), tet(L), tet(M) and tet(O)) were determined in selected isolates. The occurrence of erythromycin resistance increased from 33% in 1996 to a maximum of 62% in 1997 and decreased to 26% in 2001. Resistance to sulphametazole increased from 17% in 1996 to 30% in 1998 but has since decreased to 4% in 2001. Resistance to trimethoprim increased to 51% in 1997 and decreased to 21% in 2001. Resistance to tetracycline (21-31%) remained relatively constant during 1996-2000, but increased to 47% in 2001. Resistance to penicillin (54-75%) streptomycin (33-53%) and tetracycline (21-47%) remained relatively constant over the time investigated. All 48 penicillin resistant isolates examined contained the blaZ gene and 40 (85%) of the streptomycin resistant isolates the aadE gene. It was not possible to detect any streptogramin resistance gene in four streptogramin resistant isolates. Of the 55 erythromycin resistant isolates examined, five contained erm(A), 13 erm(B), 35 erm(C) and two both erm(A) and erm(C). The presence of erm(B) was confirmed by hybridization to plasmid profiles in all 13 PCR-positive isolates. Of 52 tetracycline resistant isolates examined, two contained tet(L), 38 tet(K) and 12 both tet(K) and tet(L).

Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis.

Exfoliative toxins of approximately 30 kDa produced by Staphylococcus hyicus strains NCTC 10350, 1289D-88 and 842A-88 were purified and specific polyclonal antisera were raised against each of the toxins. It was shown by immunoblot analysis and ELISA that three exfoliative toxins from S. hyicus were antigenically distinct. The three toxins were designated ExhA, ExhB and ExhC. From 60 diseased pigs, each representing an outbreak of exudative epidermitis, a total of 584 isolates of S. hyicus were phage typed and tested for production of exfoliative toxin. ExhA-, ExhB- and ExhC-producing S. hyicus isolates were found in 12 (20%), 20 (33%) and 11 (18%), respectively, of the 60 pig herds investigated. Production of the different types of exfoliative toxin was predominantly associated with certain phage groups. However, toxin production was found in all of the six phage groups defined by the phage typing system. Some changes in the distribution of isolates between phage groups were observed when the results of this study were compared to previous investigations. In this study two new antigenically distinct exfoliative toxins were isolated and tools for in vitro detection of toxin producing S. hyicus isolates and for further studies on the exfoliative toxins from S. hyicus have been provided.

A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline.

The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene. Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S. aureus. Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Antimicrobial susceptibility of Staphylococcus hyicus isolated from exudative epidermitis in pigs.

Exudative epidermitis or greasy pig syndrome is caused by the coagulase-variable staphylococcal species Staphylococcus hyicus. Treatment of this disease is problematic because of the limited number of antimicrobial agents available for this purpose. Thirteen antimicrobial agents were evaluated for their activities against 100 S. hyicus strains isolated from pigs with exudative epidermitis. Novobiocin was the most active compound tested, with an MIC for 90% of the strains tested (MIC90) of < or = 0.06 microgram/ml. Enrofloxacin, ampicillin, and ceftiofur were the next most active compounds, with MIC90s of 0.25, 0.5, and 1.0 microgram/ml, respectively. However, 41.4% of the 99 strains tested were positive for beta-lactamase production. The MIC90s of erythromycin, tetracycline, and streptomycin were > 32.0 micrograms/ml. Initial testing with sulfadiazine-trimethoprim yielded an MIC90 of > 64.0 micrograms/ml, but subsequent testing with thymidine phosphorylase-supplemented medium yielded an MIC90 of 0.06 microgram/ml. Both lincomycin and spectinomycin were relatively inactive against the S. hyicus strains tested, with MIC90s of > 64.0 and > 128.0 micrograms/ml, respectively. However, the combination of the two compounds at ratios of 1:2 (lincomycin to spectinomycin) and 1:8 were more active, with MIC90s of 16.0 and 4.0 micrograms/ml, respectively. These results indicate that novobiocin and sulfadiazine-trimethoprim were the most active compounds tested against the S. hyicus strains isolated from pigs with exudative epidermitis. Furthermore, the combination of lincomycin and spectinomycin was more active than the individual compounds against the strains tested.