Development of effective antimicrobial cocktails to prevent bacterial contamination of allograft tissues under low temperature conditions.

OBJECTIVES: Prevention of bacterial transmission in recipient patients via allograft decontamination with an antimicrobial cocktail consisting of cefmetazole (cefoxitin), vancomycin, lincomycin and polymyxin B is an important procedure commonly practised in tissue banks. However, some allografts are lost due to the failure of decontamination under low temperature conditions. Here, we aimed to develop new antimicrobial cocktails that exert a high bactericidal activity at 4°C. METHODS: Bacterial species used in this study were selected as major causative pathogens of allograft tissue contamination. The efficacy of the combination of 2 antimicrobial agents was determined by the checkerboard titration method. The bactericidal effects of the new antimicrobial cocktails were evaluated under the same conditions as those used for the storage and preservation of allograft tissues. RESULTS: Among the selected antimicrobial agents, daptomycin exhibited the highest bactericidal activity against methicillin-resistant Staphylococcus aureus under low temperature conditions. The combination of daptomycin + gentamicin and daptomycin + levofloxacin showed a synergistic or additive effect against various bacterial species. The antimicrobial cocktail containing 200 µg/ml of daptomycin, gentamicin and levofloxacin could eradicate ≤104 colony-forming units/ml of methicillin-resistant S. aureus and Enterococcus faecalis, which exhibit a low susceptibility to antimicrobial agents at 4°C for 24 h. CONCLUSIONS: We have developed a new formula for an antimicrobial cocktail to effectively and sufficiently prevent bacterial contamination of allograft tissues under low temperature conditions in vitro.

Specific clones of Staphylococcus lugdunensis may be associated with colon carcinoma.

Staphylococcus lugdunensis produces a tannase with activity that may be associated with the onset of colon carcinoma. To clarify this feature of colon carcinoma-associated S. lugdunensis, we obtained isolates from healthy subjects and patients with colon adenomas and carcinomas and analyzed their genetic backgrounds. In total, 40 S. lugdunensis isolates from 288 rectal swabs collected between 2002 and 2008 were used. These isolates were classified into four groups according to the diseases of the subjects: healthy (n=13), colon carcinoma (n=13), colon adenoma (n=9), and unknown (n=5). The isolates were also classified by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. In addition, an antimicrobial susceptibility test and detection of resistance genes were performed for all isolates. According to the PFGE analysis, 40 isolates could be classified into five groups. Among the groups, carcinoma and colon adenoma patients were significantly more frequently (40.9%) classified into group D (p<0.05), whereas healthy subjects were more frequently (38.5%) classified into group A. All isolates in group D were typed as ST27, which was clearly different than isolates in the other groups. All isolates were susceptible to the antimicrobial agents tested, including ß-lactams, although seven strains produced ß-lactamase. Our data suggest that a specific clone of S. lugdunensis might be associated with colon carcinoma and colon adenoma. This clone showed high susceptibility to many antimicrobial agents. Therefore, eradication therapy may lead to a decreased risk of colon carcinoma.

[Status and problem of first line eradication of H. pylori infection].

This year eradication of H. pylori was applied for not only peptic ulcer but also chronic gastritis on National insurance system. However recently decrease in first line eradication rate of H. pylori using PPI/AC regimen. Certainly eradication rate after 2000 decreased in intention to treat (ITT) and per protocol(PP) compared to that before 2000. This tendency was induced by increase in CAM-resistant H. pylori. But after 2007 eradication rate decreased only in ITT, eradication rate didn't decrease in PP. That tendency was induced by bad compliance and evaluation of the eradication.

PCR detection of Helicobacter pylori in clinical samples.

Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material.

Novel anti-acne actions of nadifloxacin and clindamycin that inhibit the production of sebum, prostaglandin E(2) and promatrix metalloproteinase-2 in hamster sebocytes.

Acne vulgaris is characteristic of excess sebum production and the induction of inflammatory reactions, for example, the augmentation of cytokine, prostaglandin (PG) and matrix metalloproteinase (MMP) production in sebaceous glands and pilosebaceous units. As Propionibacterium acnes is considered to be involved in the aggravation of acne vulgaris, antimicrobial agents have been found to be effective for treating acne leading to the remission of inflammation. However, it is not fully understood whether antimicrobial agents influence sebum production and/or the inflammatory reactions in sebaceous gland cells (sebocytes). In the present study, topical antimicrobial agents such as nadifloxacin (NDFX) and clindamycin (CLDM) decreased the production of triacylglycerols (TG), which are a major component of sebum in insulin-differentiated hamster sebocytes. These antibiotics also suppressed insulin-augmented gene expression and the production of perilipin, by which intracellular lipid droplet formation was concomitantly inhibited. On the other hand, peptidoglycan (PGN) from Gram-positive bacteria dose-dependently increased TG production in hamster sebocytes. The augmented TG production was decreased by treating NDFX or CLDM. Furthermore, NDFX and CLDM inhibited the PGN-augmented PGE(2) production in the sebocytes. Moreover, NDFX, but not CLDM, suppressed the PGN-augmented gene expression and production of pro-MMP-2/progelatinase A in hamster sebocytes. Therefore, these results provide novel evidence that NDFX and CLDM exhibit anti-lipogenesis and anti-inflammatory activities against insulin- or PGN-activated sebocytes which at least partly mimic acne pathology in vitro. Moreover, NDFX for acne therapy is likely to be effective in not only inhibiting microbial proliferation but also in preventing the onset of acne scar formation.

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB.

BACKGROUND AND AIMS: Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87 and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. MATERIALS & METHODS: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. RESULTS: Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. CONCLUSION: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.

Effect of pretreatment with Lactobacillus gasseri OLL2716 on first-line Helicobacter pylori eradication therapy.

BACKGROUND AND AIM: Helicobacter pylori eradication clearly decreases peptic ulcer recurrence rates. H. pylori eradication is achieved in 70-90% of cases, but treatment failures due to poor patient compliance and resistant organisms do occur. Lactobacillus gasseri can suppress both clarithromycin-susceptible and -resistant strains of H. pylori in vitro. The aim of this study was to determine the effect of pretreatment with L. gasseri- containing yogurt on H. pylori eradication. We conducted a randomized, controlled clinical trial in patients with H. pylori infection. METHODS: A total of 229 patients were randomized into either a 1-week triple therapy of rabeprazole (10 mg bid), amoxicillin (750 mg bid), and clarithromycin (200 mg bid) or triple therapy plus L. gasseri-containing yogurt. In the yogurt-plus-triple therapy groups, yogurt containing L. gasseri OLL2716 (112 g) was given twice daily for 4 weeks (3 weeks pretreatment and also 1 week during eradication therapy). Clarithromycin resistance was determined by the detection of a mutation in 23S rRNA using nested polymerase chain reaction and the direct sequencing of DNA from pretreatment feces. H. pylori eradication was diagnosed based on the urea breath test and a stool antigen test after 8 weeks of eradication. RESULTS: The status of H. pylori susceptibility to clarithromycin was successively determined in 188 out of 229 samples. The rate of infection with clarithromycin-resistant strains of H. pylori was 27.1%. Overall eradication (intention to treat/per protocol) was 69.3/74.5% for the triple-only group, and 82.6/85.6% for the yogurt-plus-triple group (P = 0.018/P = 0.041). Eradication of primary clarithromycin-resistant strains tended to be higher for yogurt-plus-triple therapy than triple-only therapy (38.5 vs 28.0%, respectively, P = 0.458). CONCLUSION: This study confirmed that the major cause of treatment failure is resistance to clarithromycin. A 4-week treatment with L. gasseri-containing yogurt improves the efficacy of triple therapy in patients with H. pylori infection.

Susceptibility of Propionibacterium acnes isolated from patients with acne vulgaris to zinc ascorbate and antibiotics.

PURPOSE: The in vitro antimicrobial activity of ascorbic acid derivatives against Propionibacterium acnes was tested either alone or in combination with a variety of antimicrobial agents, and their fractional inhibitory concentration index was determined using checkerboard tests. The antimicrobial effectiveness of zinc ascorbate in the treatment of acne vulgaris, either alone or in combination with antibiotics such as clindamycin that are commonly used in Japan for the treatment of acne vulgaris, was therefore examined. MATERIALS AND METHODS: The antimicrobial susceptibility of 41 strains of clindamycin-sensitive and/or clindamycin-resistant P. acnes isolated from acne vulgaris patients was tested, in comparison with a type strain of P. acnes. RESULTS: Zinc ascorbate showed antimicrobial activity against a type strain of P. acnes and its concentration (0.064%) was sufficiently lower than the normal dose (5%) of other ascorbic acid derivatives. Combinations of zinc ascorbate with clindamycin, erythromycin, and chloramphenicol showed an additive effect, and zinc ascorbate alone effectively inhibited the growth of all P. acnes including clindamycin-resistant strains. CONCLUSION: The results provide novel evidence that the combination of zinc ascorbate and clindamycin is effective for acne vulgaris treatment.

[Antimicrobial activity and frequency of spontaneous gentamicin-resistant mutants in bacteria related skin infections].

Gentamicin is used in an ointment form for the treatment of skin infections. To investigate the effect of gentamicin used as an ointment, the antimicrobial susceptibilities against Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, and Pseudomonas aeruginosa isolated from community and medical settings were studied and compared with other antibacterial agents such as fradiomycin, chloramphenicol, and bacitracin used as active ingredient for each ointment. Gentamicin showed antibacterial activities for all standard bacteria tested, but fradiomycin and chloramphenicol showed no such activities for St. pyogenes and P. aeruginosa, respectively. Bacitracin showed activity for St. pyogenes only. The strains of staphylococci isolated from healthy people were highly susceptible to gentamicin, while 49.3% of the isolates from the patients with skin infections were resistant to gentamicin and 96.4% of the gentamicin-resistant staphylococci carried the aminoglycoside-resistance gene aacA-aphD. The growths of all strains tested, except for two strains of P. aeruginosa, were inhibited by close below 128 µg/ml of gentamicin. Furthermore, the frequencies of spontaneous mutants resistant to gentamicin, fradiomycin, and chloramphenicol were each investigated using S. aureus, S. epidermidis, St. pyogenes, and P. aeruginosa. At doses of more than 32 µg/ml of gentamicin, no resistant mutants in any of bacteria strains tested were obtained. The concentration of gentamicin on the skin was calculated at approximately 895 µg/ml at least when the commercially used 0.1% gentamicin ointment was applied to the skin. Therefore, our study strongly indicates that the gentamicin ointment used has a potency of sufficiently inhibiting the growth of bacteria, including gentamicin-resistant strains, which cause skin infections in the community.

Characterization of enterococcus strains contained in probiotic products.

Probiotics are additives containing live microbes that beneficially affect a host by improving the properties of the host intestinal microflora. Recently, advances in medical treatments have led to increased numbers of immunocompromised patients; some patients contract opportunistic infections of Enterococcus species, which are considered non-pathogenic bacteria. To evaluate the safety of probiotics containing Enterococcus strains, we isolated Enterococcus from six probiotic products and compared the pathogenic genes and antimicrobial susceptibility of the probiotic strains to those of clinical isolates. Our study showed that all Enterococcus strains contained in probiotic products were E. faecium, and no vancomycin-resistant strains were found. In addition, no pathogenic genes, such as ace, agg, gelE, cylM, cylB, cylA, cpd, cob, ccf, efaA(fs), efaA(fm), esp(fs), or esp(fm), were found in the probiotic strains. Pulsed-field gel electrophoresis (PFGE) analysis showed obvious genetic differences between the probiotic strains and the clinical isolates. The data suggested that the probiotic Enterococcus strains were not transmitted to hospitalized patients. Therefore, our results strongly suggest that probiotic products are unlikely agents for causing opportunistic infections.

Augmentation of gene expression and production of promatrix metalloproteinase 2 by Propionibacterium acnes-derived factors in hamster sebocytes and dermal fibroblasts: a possible mechanism for acne scarring.

Aberrant extracellular matrix (ECM) remodeling in sebaceous glands and pilosebaceous units in the skin is associated with scar formation under acne conditions. To investigate the involvement of Propionibacterium acnes (P. acnes), a Gram-positive anaerobic microbial species, in ECM remodeling in sebaceous glands and pilosebaceous units, we examined the effects of P. acnes culture media, formalin-fixed P. acnes, and peptidoglycan (PGN) from Gram-positive bacteria walls on the production of promatrix metalloproteinase 2 (proMMP-2)/progelatinase A in hamster sebocytes and dermal fibroblasts. When hamster sebocytes (1.8×10(5) cells) and dermal fibroblasts (1×10(5) cells) were treated with P. acnes culture media and formalin-fixed P. acnes (corresponding to 1×10(6) and 1×10(7) bacterial cells), the production of proMMP-2 was augmented. In addition, PGN (5-50 µg/ml) dose-dependently augmented the production of proMMP-2 in both cells. Furthermore, the PGN (50 µg/ml)-augmented proMMP-2 production was resulted from an increase of its transcript. In contrast, there were no changes in cell proliferative activity in either the P. acnes or PGN-treated sebocytes and dermal fibroblasts, indicating that the augmented proMMP-2 production was not due to an increase in cell numbers. Therefore, these results provide novel evidence that PGN transcriptionally up-regulates the production of proMMP-2 in hamster sebocytes and dermal fibroblasts. Given an increase in the quantity of Gram-positive bacteria, including P. acnes in acne lesions, the aberrant ECM degradation may progress in sebaceous glands and pilosebaceous units, which is associated with acne scar formation.

Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

Plasmids that carry the multidrug efflux genes qacA and qacB are widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). Although the QacA and QacB proteins are similar to each other, their respective substrate specificities may differ. We investigated the variability and structure-function relationships of QacA and QacB in MRSA isolates. The amino acid sequences of 7 QacA and 25 QacB proteins showed that QacB was present in three variants, designated QacBII, QacBIII, and QacBIV, that were different from the prototypic QacB variant encoded by plasmid pSK23, which was named QacBI, while QacA was present in two variants. When cloned and expressed in S. aureus, the strain carrying qacBIII exhibited higher susceptibility to dyes and decreased susceptibility to norfloxacin and ciprofloxacin compared to strains carrying the other QacB variants. Site-directed mutagenesis experiments revealed that the residue at position 320 in QacB plays an important role in the resistance phenotypes to dyes and fluoroquinolones. Furthermore, the accumulation of norfloxacin and ciprofloxacin in the strain carrying qacBIII was significantly decreased. Our data demonstrate that the plasmid-mediated multidrug efflux pump QacB variant QacBIII confers the capability for fluoroquinolone efflux on S. aureus.

Using the tannase gene to rapidly and simply identify Staphylococcus lugdunensis.

The coagulase-negative Staphylococcus lugdunensis, a bacterium similar to Staphylococcus aureus, produces tannase that degrades tannin. We developed a polymerase chain reaction-based method to rapidly and simply identify this species by detecting the tanA gene for S. lugdunensis tannase.

Analysis of clarithromycin resistance and CagA status in Helicobacter pylori by use of feces from children in Thailand.

The clarithromycin resistance and CagA status of Helicobacter pylori in Thai children were investigated using fecal samples. Of the 284 samples, H. pylori was detected in 120 samples, and the clarithromycin resistance rate was 29.2%. The cagA gene was detected in 59 samples, and only 6.8% of these samples contained the East Asian CagA type.

Involvement of Propionibacterium acnes in the augmentation of lipogenesis in hamster sebaceous glands in vivo and in vitro.

Propionibacterium acnes is considered to be involved in the aggravation of acne vulgaris, but it remains unclear whether P. acnes directly influences lipogenesis in sebaceous glands. In this study, we showed that a culture medium of P. acnes (acnes-CM) and formalin-killed P. acnes (F-acnes) prepared from P. acnes strains, JCM6473 and JCM6425, intracellularly augmented lipid droplet formation and triacylglycerol (TG) synthesis in undifferentiated and insulin-differentiated hamster sebocytes. Acnes-CM and F-acnes prepared from four clinical P. acnes strains elicited the same lipogenesis augmentation. The augmented TG production resulted from an increase in the diacylglycerol acyltransferase activity. Topical application of acnes-CM to the skin of hamster auricles every day for 4 weeks revealed that sebum accumulation was augmented in sebaceous glands and ducts. Furthermore, both acnes-CM and F-acnes increased the production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a cytochrome P450 (CYP)-linked sebaceous lipogenic factor, in differentiated sebocytes. A CYP inhibitor, SKF-525A, decreased the acnes-CM- and F-acnes-augmented production of TG and 15d-PGJ(2). Thus, to our knowledge these results provide previously unreported evidence that P. acnes directly participates in the augmentation of sebaceous lipogenesis through a proposed mechanism in which an increase of 15d-PGJ(2) production through the CYP pathway is closely associated with the enhancement of TG production.

Isolation and identification of a potent antimalarial and antibacterial polyacetylene from Bidens pilosa.

Diseases caused by malaria parasites and pathogenic bacteria were thought to be on the brink of eradication in the 1950-1960s, but they have once again become a serious threat to mankind as a result of the appearance of multidrug resistant strains. The spread of these multidrug resistant organisms has prompted a worldwide search for new classes of effective antimalarial and antibacterial drugs. Natural products have been recognized as highly important candidates for this purpose. Our attention has focused on the herbal plant Bidens pilosa, a weed common throughout the world, as one of the target plants in the search for new active compounds, owing to its empirical use in the treatment of infectious diseases and to pharmaco-chemical studies of its crude extract. We report the isolation of two new compounds of B. pilosa, the linear polyacetylenic diol 1 and its glucoside 2 which have previously been isolated from different plants. Compound 1 exhibited highly potent antimalarial and antibacterial properties in vitro as well as potent antimalarial activity by way of intravenous injection in vivo, thereby representing a promising new class of drugs potentially effective in the treatment of malarial and bacterial diseases. We suspect that discovery of these compounds in B. pilosa in appreciable quantity is because the Fijian tradition of using the fresh plant for extraction rather than the Asian tradition of using dried plants (1 is unstable in the dried state) was followed.

Anti-infectious activity of tryptophan metabolites in the L-tryptophan-L-kynurenine pathway.

To study the anti-infectious effect of a vascular allograft, the antimicrobial activity of tryptophan metabolites mediated by indoleamine 2,3-dioxygenase was determined. The growth of methicillin-resistant Staphylococcus aureus (MRSA) over 10 h in extracts from post-transplantation vascular allograft was significantly slower than that of extracts from non-transplantation vascular allograft regardless of the presence of tryptophan (p<0.05). When the antimicrobial activity of the tryptophan metabolites in the L-tryptophan-L-kynurenine pathway was examined, 3-hydroxy-DL-kynurenine and alpha-picolinic acid had strong antibacterial activity against MRSA, S. epidermidis, Escherichia coli, and multidrug-resistant Pseudomonas aeruginosa, although antimicrobial activities of anthranilic acid, 3-hydroxyanthranilic acid, and quinolinic acid against them were low. The results showed that, of the tested tryptophan metabolites, 3-hydroxy-DL-kynurenine and alpha-picolinic acid contributed to the anti-infectious effects of the allograft by inhibiting of the growth of microorganisms.

Molecular epidemiology and antimicrobial susceptibilities of 273 exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan.

The molecular epidemiology and antimicrobial susceptibilities of 273 Staphylococcus aureus isolates positive for the exfoliative toxin-encoding gene obtained from patients with impetigo in Japan in 2006 were studied. The mecA gene was detected in 74 meticillin-resistant S. aureus (MRSA) and 23 meticillin-susceptible S. aureus (MSSA) isolates. All isolates with the staphylococcal cassette chromosome (SCC) mec were classified into type IV (92.8%, 90/97) or V (7.2%, 7/97). The ET-encoding gene etb was found primarily in strains with mecA (87.7%, 71/81), whilst eta (86.6%, 161/186) was detected mainly in strains without mecA. The chromosomal enterotoxin-encoding gene cluster egc was found in 83.0% of strains with eta, whilst no enterotoxin-encoding gene was detected in strains with only etb. PFGE showed that each strain carrying eta, etb and etd could be classified into distinct groups. The susceptibility profiles of MRSA to antimicrobial agents excluding beta-lactams were similar to those of MSSA. Gentamicin- and clarithromycin-resistant strains were frequently found for both MRSA and MSSA. The aminoglycoside-resistance gene aacA-aphD was detected in 97.3% of MRSA and 85.4% of MSSA. Additionally, the macrolide-resistance gene ermA or ermC was detected in 67.6% of MRSA and 71.4% of MSSA. Therefore, these results suggest that SCCmec types IV or V have spread, particularly in MSSA carrying etb in the community.