Wound care antiseptics - performance differences against Staphylococcus aureus in biofilm

Background

Staphylococcus aureus is commonly isolated from infected wounds both in animals and humans. It is known to be an excellent biofilm former and biofilms are present in as many as 60% of chronic wounds. Despite that the presence of biofilms in infections are common, antiseptics are usually qualified for in vivo testing according to their effect on planktonic cells. As it is well known that bacteria in biofilms are more tolerant to antiseptics than planktonic bacteria, biofilm infections can be difficult to treat. The aim of the study was to compare three different categories of antiseptics, biguanide (chlorhexidine), quaternary ammonium compound (QAC; Pyrisept) and iodine/iodophores (2% iodine liniment), with regards to efficacy in killing S. aureus in biofilm. If there was observed a difference in efficacy between these antiseptics, a second aim was to find the most effective of the three antiseptics.

Results

Large differences in the bactericidal effect of the different antiseptics against S. aureus in biofilm were observed in the present study. Iodine treatment was found to be the most effective followed by Pyrisept and chlorhexidine.

Conclusions

The bactericidal effect of the different antiseptics used in the present study was found to vary significantly against S. aureus in biofilm. The present study gives valuable knowledge with regards to selecting the antiseptics that are most likely to be successful in treating biofilm infected wounds. This study also contributes to focus attention on the importance of qualifying antiseptics based on results using biofilm bacteria rather than planktonic bacteria.     

Effectiveness of calcium hypochlorite,quaternary ammonium compounds,and sodium hypochlorite in eliminating vegetative cells and spores of Bacillus anthracis surrogate

BackgroundThe spore-forming bacterium Bacillus anthracis causes anthrax, an often-fatal infection in animals. Therefore, a rapid and reliable strategy to decontaminate areas, humans, and livestock from B. anthracis is very critical.ObjectivesThe aim of this study was performed to evaluate the efficacy of sodium hypochlorite, calcium hypochlorite, and quaternary ammonium compound (QAC) sanitizers, which are commonly used in the food industry, to inhibit spores and vegetative cells of B. anthracis surrogate.MethodsWe evaluated the efficacy of sodium hypochlorite, calcium hypochlorite, and a QAC in inhibiting vegetative cells and spores of a B. anthracis surrogate. We treated a 0.1-mL vegetative cell culture or spore solution with 10 mL sanitizer. The samples were serially diluted and cultured.ResultsWe found that 50 ppm sodium hypochlorite (pH 7), 1 ppm calcium hypochlorite, and 1 ppm QAC completely eliminated the cells in vegetative state. Exposure to 3,000 ppm sodium hypochlorite (pH 7) and 300 ppm calcium hypochlorite significantly eliminated the bacterial spores; however, 50,000 ppm QAC could not eliminate all spores.ConclusionsCalcium hypochlorite and QAC showed better performance than sodium hypochlorite in completely eliminating vegetative cells of B. anthracis surrogate. QAC was ineffective against spores of the B. anthracis surrogate. Among the three commercial disinfectants tested, calcium hypochlorite most effectively eliminated both B. anthracis vegetative cells and spores.     

Bovine Mastitis due to Prototheca zopfii: Clinical,Epidemiological and Pathological Aspects in a Brazilian Dairy Herd

The clinical, epidemiological and pathological aspects of protothecal mastitis in a Brazilian dairy herd are described. Prototheca zopfii infection was diagnosed in 11 of 121 milking cows. Clinical mastitis refractory to usual therapy was observed in 7 cows. Several environmental conditions conducive to the growth of Prototheca spp., such as wetness, muddiness and the presence of organic material, were present in the dairy. Improper milking practices and insanitary infusion of the intramammary antibiotics were also observed. Six cows with protothecal mastitis were slaughtered and the affected quarters of each cow were examined by histology and immunohistochemical staining for bovine keratin and P. zopfii. The histological lesions were characterized by interstitial infiltrates of macrophages, plasma cells and lymphocytes; algae were seen in the alveolar lumen and interstitium. The lack of a positive reaction with an antiserum against bovine keratin in the mammary alveolar epithelial layer in some affected areas suggests destruction of milk-producing tissues, which may be related to the low milk production observed. The algal organisms stained positively with a polyclonal antibody against P. zopfii.     

Phenotypic characterization of mastitic Prototheca spp. isolates

Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.     

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.     

Inactivation of avian influenza virus using common detergents and chemicals

Six disinfectant chemicals were tested individually for effectiveness against low pathogenic avian influenza virus (LPAIV) A/H7N2/Chick/MinhMa/04. The tested agents included acetic acid (C2H4O2), citric acid (C6H8O7), calcium hypochlorite (Ca(ClO)2), sodium hypochlorite (NaOCl), a powdered laundry detergent with peroxygen (bleach), and a commercially available iodine/acid disinfectant. Four of the six chemicals, including acetic acid (5%), citric acid (1% and 3%), calcium hypochlorite (750 ppm), and sodium hypochlorite (750 ppm) effectively inactivated LPAIV on hard and nonporous surfaces. The conventional laundry detergent was tested at multiple concentrations and found to be suitable for inactivating LPAIV on hard and nonporous surfaces at 6 g/L. Only citric acid and commercially available iodine/acid disinfectant were found to be effective at inactivating LPAIV on both porous and nonporous surfaces.     

Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Oocysts of Cryptosporidium parvum are resistant to environmental conditions and many disinfectants. A combination of cell culture and quantitative real time PCR (cc–qPCR) is established for evaluation of anticoccidial disinfectants against C. parvum. C. parvum oocysts were treated with disinfectants, washed and oocysts were incubated with HCT-8 cell monolayers in the presence of excystation medium for 3 h. Subsequently, unbound parasites were removed by washing with growing medium and the infected monolayers were further maintained in fresh growing medium for 48 h. Genomic DNA was extracted from each sample and qPCR performed targeting a specific sequence of the 70 kDa heat shock protein gene in order to quantify development. Treatment of oocysts with cresolic disinfectants demonstrated dose dependent reduction of viability of oocysts. More than 98% inactivations were recorded with at least 2% concentration of cresolic disinfectants after 2 h of treatment. Bleach (sodium hypochlorite) at 6% solution induced 92.7% inactivation of C. parvum oocysts after 2 h. Thermally treated oocysts (56 and 70 °C for 20 min) demonstrated complete inactivation, whereas at 38 °C no inactivation was observed. Application of Neopredisan^® 135-1 and Aldecoc^® TGE (4% for 2 h) as recommended according to the current guidelines stipulated by DVG (German Veterinary Society) consistently inactivated more than 99.5% of oocysts. The suggested cc–qPCR method appeared to be suited for standardized testing of inactivation measures, particularly for evaluation of chemical disinfectants and thus cc–qPCR is proposed as an alternative to the established chicken infectivity model for Eimeria tenella for testing anticoccidial disinfectants. A minimum inactivation of 99.5% in cc–qPCR model is claimed as a suitable threshold for certification of chemical products for disinfection of coccidia oocysts.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.     

Dietary intake of iodine‐enriched eggs decreases the incidence of mouse mammary tumors caused by the activated ErbB2 oncogene

Human epigenetic studies suggest that consumption of seaweed prevents mammary cancer, which possibly is explained by iodine daily intake. In this study, we evaluated the efficacy of dietary intake of iodine‐enriched eggs on mammary tumor incidence caused by the expression of activated type ErbB2. Female transgenic mice were divided into three groups, and fed a basic diet, a diet supplemented with ordinary eggs, or with iodine‐enriched eggs. The number of mammary tumors greater than 5 mm in diameter was recorded in mice at 6 months of age. We report that the average number of mammary tumors per mouse was significantly lower in the iodine‐enriched egg‐added diet group than in either the basic diet or ordinary egg diet groups. These results indicate that iodine intake through livestock‐derived products can reduce the incidence of mammary cancers caused by the expression of activated type ErbB2.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.     

Canine superficial bacterial folliculitis: Current understanding of its etiology,diagnosis and treatment

Superficial bacterial folliculitis (SBF) is more common in the dog than other mammalian species. Until recently, a successful outcome in cases of canine SBF was possible by administering a potentiated amoxicillin, a first generation cephalosporin or a potentiated sulfonamide. Unfortunately, this predictable susceptibility has changed, because methicillin resistant Staphylococcus pseudintermedius (MRSP) and Staphylococcus aureus (MRSA) are becoming more prevalent in canine SBF cases. The increasing frequency of multidrug resistance complicates the selection of antimicrobial therapy. Antimicrobial agents that were once rarely used in cases of canine SBF, such as amikacin, rifampicin and chloramphenicol, are becoming the drugs of choice, based on bacterial culture and susceptibility testing. Furthermore, changes in antimicrobial susceptibility have helped to re-emphasize the importance of a multimodal approach to treatment of the disease, including topical therapy. Due to the increasing frequency of identification of highly resistant Staphylococcus spp., topical antimicrobial therapy, including the use of diluted sodium hypochlorite (bleach), is becoming necessary to successfully treat some cases of canine SBF. Other important antiseptics that can be used include chlorhexidine, benzoyl peroxide, ethyl lactate, triclosan and boric acid/acetic acid. This review discusses the diagnostic and therapeutic management of canine SBF, with a special emphasis on treating methicillin resistant staphylococcal infections.     

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Changes in various metabolic parameters in blood and milk during experimental Escherichia coli mastitis for primiparous Holstein dairy cows during early lactation

Background

The objective of this study was to characterize the changes in various metabolic parameters in blood and milk during IMI challenge with Escherichia coli (E. coli) for dairy cows during early lactation. Thirty, healthy primiparous Holstein cows were infused (h = 0) with ~20-40 cfu of live E. coli into one front mammary quarter at ~4-6 wk in lactation. Daily feed intake and milk yield were recorded. At –12, 0, 3, 6, 12, 18, 24, 36, 48, 60, 72, 96, 108, 120, 132, 144, 156, 168, 180 and 192 h relative to challenge rectal temperatures were recorded and quarter foremilk was collected for analysis of shedding of E. coli. Composite milk samples were collected at -180, -132, -84, -36, -12, 12, 24, 36, 48, 60, 72, 84, 96, 132 and 180 h relative to challenge (h = 0) and analyzed for lactate dehydrogenase (LDH), somatic cell count, fat, protein, lactose, citrate, beta-hydroxybutyrate (BHBA), free glucose (fglu), and glucose-6-phosphate (G6P). Blood was collected at -12, 0, 3, 6, 12, 18, 24, 36, 60, 72, 84, 132 and 180 h relative to challenge and analyzed for plasma non-esterified fatty acids (NEFA), BHBA and glucose concentration. A generalized linear mixed model was used to determine the effect of IMI challenge on metabolic responses of cows during early lactation.

Results

By 12 h, E. coli was recovered from challenged quarters and shedding continued through 72 h. Rectal temperature peaked by 12 h post-challenge and returned to pre-challenge values by 36 h post-IMI challenge. Daily feed intake and milk yield decreased (P <0.05) by 1 and 2 d, respectively, after mastitis challenge. Plasma BHBA decreased (12 h; P <0.05) from 0.96 ± 1.1 at 0 h to 0.57 ± 0.64 mmol/L by 18 h whereas concentration of plasma NEFA (18 h) and glucose (24 h) were significantly greater, 11 and 27%, respectively, after challenge. In milk, fglu, lactose, citrate, fat and protein yield were lower whereas yield of BHBA and G6P were higher after challenge when compared to pre-challenge values.

Conclusions

Changes in metabolites in blood and milk were most likely associated with drops in feed intake and milk yield. However, the early rise in plasma NEFA may also signify enhanced adipose tissue lipolysis. Lower concentrations of plasma BHBA may be attributed to an increase transfer into milk after IMI. Decreases in both milk lactose yield and % after challenge may be partly attributed to reduced conversion of fglu to lactose. Rises in G6P yield and concentration in milk after challenge (24 h) may signify increased conversion of fglu to G6P. Results identify changes in various metabolic parameters in blood and milk after IMI challenge with E. coli in dairy cows that may partly explain the partitioning of nutrients and changes in milk components after IMI for cows during early lactation.     

Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.     

The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.