Susceptibility of MRSA biofilms to denture-cleansing agents

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen, which is responsible for considerable morbidity and mortality in the United Kingdom. The major reservoir of this organism is thought to be the anterior nares, but there is increasing evidence that this pathogen is present in the oral cavity, particularly in denture wearers. The purpose of this study was to determine whether MRSA, grown as biofilms on denture acrylic resin, could be eradicated using commercially available agents. EMRSA-15 or EMRSA-16 was grown in a model system on the surface of denture acrylic resin for 4, 24 or 120 h before the samples were exposed to a range of disinfectants for time intervals of 1, 5 and 10 min. All of the agents reduced the number of cultivable MRSA bacteria present on the acrylic resin surface at 4 h, with 2% sodium hypochlorite (NaOCl) eliminating MRSA below the level of detection after an exposure of 1 min. However, the established MRSA biofilms (24 and 120 h) were more resistant to killing by the agents, although 2% NaOCl was still able to eradicate all ages of MRSA biofilms within 1 min of exposure.     

Evaluation of characteristics of Pediococcus spp. to be used as a vaginal probiotic

Aims

The aim of our research was to select, identify and characterize an isolate of lactic acid bacteria to be considered as a vaginal probiotic.

Methods and Results

Thirty‐five isolates of Pediococcus spp. showed bacteriocinogenic activity against Listeria monocytogenes and the ability to survive in simulated vaginal fluid (SVF) at pH 4·2. One isolate of Pediococcus spp. was selected and characterized to evaluate its safety before the use as vaginal probiotic. Pediococcus pentosaceus SB83 did not show the presence of virulence factors such as the production of gelatinase, lipase and DNase, haemolytic activity, nor the presence of virulence genes (genes esp, agg, gelE, efaAfm, efaAfs, cylA, cylB and cylM). Pediococcus pentosaceus SB83 was considered sensitive to chloramphenicol, gentamicin, streptomycin, kanamycin, erythromycin and ampicillin. This strain was considered resistant to tetracycline and vancomycin. Pediococcus pentosaceus SB83 was a biofilm producer at different pH values (4·2, 5·5 and 6·5) in SVF and in de Man, Rogosa and Sharpe medium.

Conclusions

The in vitro results provide a basis for the use of P. pentosaceus SB83 as a vaginal probiotic, to prevent colonization by L. monocytogenes in pregnant women.

Significance and Impact of Study

The application of vaginal probiotics could have the potential for preventing vaginal infections and consequently reduce abortion and neonatal infections.     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

Comparative Study of Agar Diffusion Test and the NCCLS Macrobroth Method for In Vitro Susceptibility Testing of Candida spp.

We performed a prospective double-blind study to evaluate the correlation between inhibition zones obtained by a disk-diffusion test, using Neo-sensitabs of fluconazole (Rosco Diagnostica), and the MICs generated by the NCCLS macrobroth dilution assay. Eighty clinical isolates, representing 5 of the clinically relevant species of Candida, were tested simultaneously by both methods. A clear inverse correlation was found between the results obtained by both tests (r = −0.69). In addition, there was high degree of agreement between methods in the identification of susceptible isolates. However, the resistance definition by disk-diffusion test had a positive predictive value of only 17%. Our data support the hypothesis that Rosco Fluconazole Neo-sensitabs have potential as a screening test for the identification of Candida isolates susceptible to fluconazole. Resistant isolates should be further investigated by standardized broth procedures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods and Results:  Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped ⁻ suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes , showing a minimal inhibitory concentration (MIC) of 200 AU ml⁻¹.
Conclusions:  A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance and Impact of the Study:  Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Susceptibility of Pediococcus isolates to antimicrobial compounds in relation to hop-resistance and beer-spoilage

Background  

Though important in the context of food microbiology and as potential pathogens in immuno-compromised humans, bacterial isolates belonging to the genus Pediococcus are best known for their association with contamination of ethanol fermentation processes (beer, wine, or fuel ethanol). Use of antimicrobial compounds (e.g., hop-compounds, Penicillin) by some industries to combat Pediococcus contaminants is long-standing, yet knowledge about the resistance of pediococci to antimicrobial agents is minimal. Here we examined Pediococcus isolates to determine whether antibiotic resistance is associated with resistance to hops, presence of genes known to correlate with beer spoilage, or with ability to grow in beer.     

Synthesis of a new class of antimicrobial agents incorporating the indolin-2-one moiety

New furanone derivatives incorporating the indolin-2-one moiety 3 were prepared via the Perkin reaction of isatins 1 with aroylpropionic acids 2 under conventional conditions or microwave irradiation. A series of functionally heterocyclic derivatives (e.g., pyridazines, pyrroles, and sulfonamides) incorporating the indolin-2-one moiety was achieved via reaction of 3 with different reagents under microwave irradiation conditions. The newly synthesized compounds were characterized on the basis of FTIR, ¹H, ¹³C NMR and mass spectral studies. Some of the new synthesized compounds were screened for antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus), Gram-negative bacteria (Escherichia coli and Shigilla flexneri) and antifungal activity against Aspergillus flavus and Candida albicans. Compound 8 j was equipotent to chloramphenicol in inhibiting the growth of E. coli minimum inhibitory concentration (MIC 2.5 μg/mL). Compound 8j may possibly be used as a lead compound for developing a new antibacterial agents. The antibacterial activity is expressed as the corresponding MIC (μg/mL) values.     

Bioactive glasses as delivery systems for antimicrobial agents

Most biomaterial‐associated infections are caused by opportunistic pathogens and bacteria that are regularly found within the microflora of the implant site. In addition, a biomaterial implant or device remains at risk of infection by hematogenous spread of bacteria disseminated from infections elsewhere in the body or from infected peri‐implant tissue in revision surgery. The resulting infections are frequently accompanied by patient morbidity and discomfort and can lead to surgical replacement of the implant after lengthy, unsuccessful attempts to mitigate infections with antibiotic treatments. Therefore, extensive study is aiming to find new infection‐resistant antimicrobial biomaterials and coatings for implants and devices to effectively reduce the incidence of biomaterial‐associated infections. An overview of the in vitro and in vivo antimicrobial efficacies of the numerous biomaterials currently available is beyond the scope of this review. Herein, we provide a comprehensive review of bioactive glasses as biomaterial delivery systems for antimicrobial agents.     

Tolerance ofStaphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents

During a prospective study of indwelling vascular catheter-related infections, 134 isolates ofStaphylococcus epidermidis were grown from 700 catheter tips.In vitro antimicrobial susceptibility testing of these isolates to oxacillin, vancomycin and ofloxacin was performed using the standard broth microdilution technique. These results were compared to those for the same organisms grown in biofilm before the addition of antimicrobial agents. In 96-well flat bottom microtiter plates, 10⁴–10⁵ colony forming units ofS. epidermidis in 0.1 ml broth were grown for 18 h at 37°C, at which time a biofilm was observed for all isolates. Different concentrations of antimicrobial agents (0.1 ml) were then added to the plates. The plates were incubated for 18 h at 37°C. Since MICs could not be estimated in these plates, all the wells were subcultured after mixing the biofilm with the broth. Minimum bactericidal concentrations (MBCs) were defined as 99.9% reduction in colony forming units. For organisms grown in suspension, 100% of the isolates were susceptible to vancomycin, 81% to ofloxacin and 40% to oxacillin. MBCs of susceptible isolates were within four-fold differences for vancomycin (53%), oxacillin (50%), and ofloxacin (51%). When grown as a biofilm, 78%, 93% and 71% of isolates had MBCs of 2048 g ml^–1 of oxacillin, vancomycin and ofloxacin respectively. These data demonstrate the reduced bactericidal activity of antimicrobial agents againstS. epidermidis in a biofilm and a simple method for its detection in the microbiology laboratory.     

Susceptibility of the Lyme disease spirochete to seven antimicrobial agents.

The antimicrobial susceptibility of five Lyme disease spirochete strains (two human and three tick isolates) was determined. A macrodilution broth technique was used to determine on three separate test occasions the minimal inhibitory concentrations (MICs) of seven antibiotics. The Lyme disease spirochete was most susceptible to erythromycin with a MIC of less than or equal to 0.06 micrograms/ml. The spirochete was also found to be susceptible to minocycline, ampicillin, doxycycline, and tetracycline-HCL with respective mean MICs of less than or equal to 0.13, less than or equal to 0.25, less than or equal to 0.63, and less than or equal to 0.79 micrograms/ml. The spirochete was moderately susceptible to penicillin G with a mean MIC of 0.93 micrograms/ml. All strains were resistant to rifampin at the highest concentration tested (16.0 micrograms/ml).     

Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents

AIMS: The objective of this study was to determine the susceptibility of Arcobacter butzleri isolates to various antimicrobial agents used in the treatment of infectious diseases in humans and animals. METHODS AND RESULTS: Thirty-nine A. butzleri strains isolated from broiler chickens were tested for their susceptibility to 23 antimicrobial agents using a disc diffusion method. All isolates were resistant to aztreonam, cefuroxime sodium, cephalothin, orbenin, oxacillin, penicillin G and trimethoprim/sulphamethoxazol. Of the 39 isolates tested, 26 were also found resistant to amoxycillin, amoxycillin/clavulanic acid and ampicillin. One isolate was resistant to, and four showed intermediate level of resistance to, erythromycin. All isolates were susceptible to amikacin, chloramphenicol, danofloxacin, enrofloxacin, nitrofurantoin, nalidixic acid, tetracyclines and tobramycin. CONCLUSIONS: The majority of the isolates were found resistant to antibiotics commonly used for the treatment of infectious bacterial diseases in humans and animals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that A. butzleri strains vary in their resistance to certain kinds of antibiotics and caution should be taken when choosing a suitable antibiotic for the treatment of disease(s) caused by this organism.     

Pediocin SA-1, an antimicrobial peptide from Pediococcus acidilactici NRRL B5627: production conditions, purification and characterization

Fermentation broths of Pediococcus acidilactici NRRL B5627 exhibited a certain antimicrobial activity due to a bacteriocin produced during early growth and until the stationary phase of growth was reached (at optimum of 60% dissolved oxygen saturation). Its size was determined by electrospray ionization mass spectrometric analysis as 3.660 kDa. N-terminal sequencing showed that the bacteriocin had 19 amino acid residues in the order KYYGXNGVXTXGKHSXVDX. The purified bacteriocin is similar to pediocins isolated by various Pediococci and therefore, it belongs to the class IIa of bacteriocins and is thus designated pediocin SA-1. Sensitivity of the purified pediocin to various storage temperatures and enzyme treatments was examined. Purified pediocin SA-1 is heat stable for up to 60 min at 121 °C. Pediocin SA-1 is inhibitory to several food-borne pathogens and food spoilage bacteria. It appears to be significantly more effective against Listeria spp. compared to pediocin PD-1 produced by P. damnosus. The mode of action of the purified bacteriocin appears to be bactericidal.     

Flow cytometric determination of Enterococcus spp. susceptibility to four antimicrobial agents

Two Enterococcus strains (E. faecalis and E. faecium) isolated from 2 patients in an intensive care unit (blood and drain, respectively) were analyzed for susceptibility to 4 antibiotics (penicillin, vancomycin, gentamicin, streptomycin) by agar dilution standard method (MICs), time-kill and flow cytometry. We compared the data from classical methods of antibiotic susceptibility detection, that are compulsory 24 hrs long and flow cytometry results at 5 and 24 hrs cultivation. The results from both classical and flow cytometric analyses were highly cogent and revealed the fact that flow cytometry is very useful in early diagnosis of bacterial resistance to antibiotics.     

Susceptibility of larvae of the sheep blowfly Lucilia cuprina to various Heterorhabditis spp., Neoaplectana spp., and in undescribed steinernematid (Nematoda)

The susceptibility of third state larvae of the sheep blowfly Lucilia cuprina in sand to 11 species and strains of Heterorhabditis and Neoaplectana and to one species of an undescribed steiner-nematid was tested at various dosages. Larvae were susceptible to all, although far less so to one strain of N. bibionis and to the undescribed steinernematid. Estimates of LD₅₀ and LD₉₀ are presented for each effective strain. Heterorhabditis spp. were able to reproduce in L. cuprina larvae subjected to low dosages of effective-stage juveniles whereas Neoaplectana spp. were not able to reproduce at any dosage. The possibility exists for using Heterorhabditis species as a control agent against L. cuprina larvae after these have left the sheep to pupate in the soil.     

An ATP Bioluminescence Assay Applicable to Rapid Fluconazole Susceptibility Testing of Dermatophytes

An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content-based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high-resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.     

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.     

Diversity and antimicrobial activity of Pseudovibrio spp. from Irish marine sponges

Aims: To evaluate the diversity and antimicrobial activity present among Pseudovibrio spp. isolated from marine sponges. Methods and Results: Seventy‐three bacterial isolates from the marine sponges Polymastia boletiformis, Axinella dissimilis and Haliclona simulans were identified as Pseudovibrio spp. using phylogenetic analysis of 16S rRNA gene sequences. Genetic diversity among these isolates was estimated using random amplification of polymorphic DNA (RAPD), and 33 RAPD types were identified among the 73 Pseudovibrio isolates. These Pseudovibrio spp. were assayed for the production of compounds with antimicrobial activity against various clinically relevant pathogens. Sixty‐two (85%) of the isolates showed activity against at least one of the pathogens tested, including Escherichia coli, Salmonella enterica serotype Typhimurium, methicillin‐resistant Staphylococcus aureus (MRSA), and Clostridium difficile. PCR screens of the Pseudovibrio isolates also revealed the presence of potential antibiotic‐producing polyketide synthase genes. Conclusions: Marine sponges harbour a diverse population of Pseudovibrio spp., the majority of which demonstrate antimicrobial activity. The identification of several different antimicrobial activity spectra suggests that the Pseudovibrio isolates may produce a suite of antimicrobial compounds. Significance and Impact of the Study: This is the first study in which an extended population of Pseudovibrio isolates from marine sponges has been analysed and establishes the little‐studied Pseudovibrio as a potentially important genus in the search for antimicrobial compounds of clinical relevance.