Strains Of Clinical Isolates Research Articles

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

Extended-Spectrum β-Lactamase 생성 Klebsiella pneumoniae 균주의 유전형 검출

임상검체에서 분리되는 그람음성 막대균의 제 3세대 cephalosporin에 대한 내성율의 증가는 임상적으로 심각한 문제가 되고 있다. 3세대 cephalosporin 및 monobactam계 항균제에 대한 내성은 주로 Extended-Spectrum <TEX>${\beta}$</TEX>-Lactamase(ESBL)의 생성에 기인한다. 따라서 ESBL 유전자의 정확한 검출은 병원내의 감염경로 파악을 위한 감시 및 역학조사를 위해 필수적이다. 본 연구는 2012년 2월부터 8월까지 대전, 충남, 충북지역의 대학병원으로부터 ESBL 생성 Klebsiella penumoniae 46균주를 분리하여 Clinical and Laboratory Standards Institute (CLSI)에 따라 ceftazidime (CAZ)과 CAZ/clavulanate (CLA)를 이용한 combination disk test (CDT) 방법에 의해 표현형을 조사하고, 유전형 특이 프라이머를 이용한 multiplex PCR을 수행하여 유전형을 검출하였다. CDT 결과 42균주가 ESBL생성균주로 확인되었다. PCR 결과, 46균주 모두 TEM형이었으며, 37균주는 SHV형, 14균주는 CTX-M형으로 나타났으며 10균주가 TEM, SHV, CTX-M 유전자를 모두 가지고 있었다. Multiplex PCR에 의한 유전형 검출 방법은 임상에서 분리한 ESBL생성 K. penumoniae균주의 감별과 검출에 유용한 방법으로 사료된다. Among Gram-negative pathogens in Korea, the incidence of resistance to third generation cephalosporins is becoming an ever-increasing problem. The production of extended-spectrum <TEX>${\beta}$</TEX>-lactamase (ESBL) is the main mechanism of bacterial resistance to a third-generation cephalosporins and monobactams. Accurate identification of the ESBL genes are necessary for surveillance and epidemiological studies of the mode of transmission in the hospital. This study was conducted to detect the genes encoding ESBL of 46 K. penumoniae isolated from Daejeon, Chungnam and Chungbuk regional university hospitals from February to August in 2012. The phenotypes of the isolated specimens were examined according to the combination disc test (CDT) by the Clinical and Laboratory Standards Institute (CLSI). Forty two ESBL producing K. penumoniae isolates could be detected using ceftazidime (CAZ) discs with and without clavulanate (CLA). By CDT, 42 K. pneumoniae strains were confirmed to be ESBL strains. Genotyping was performed by multiplex PCR with type-specific primers. By PCR analysis, TEM gene in 46 strains, SHV gene in 37 strains and CTX-M genes in 14 strains were identified. Ten isolates did carry genes encoding ESBLs of all types TEM, SHV and CTX-M. The multiplex polymerase chain reaction (PCR) analysis was better to detect and differentiate ESBL producing K. penumoniae strains in clinical isolates.

Anticandidal and Antioxidant Activities of Fractions and Isolated Compounds of Pyrostegia venusta (Ker Gawl.) Miers

The in vitro anticandidal and antioxidant activities of phenolic compounds detected in flower extracts of Pyrostergia venusta were investigated. One flavonoid (quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside) and two phenylpropanoid glycosides (verbascoside and isoverbascoside) were purified by using the HPLC technique. The antimicrobial activity of the extracts was evaluated against five Candida strains (C. albicans, C. krusei ATCC 6258, and clinical isolates strains of Candida sp: C. albicans, C. krusei, C. tropicalis, C. parapsilosis and C. guilhermondii). The antimicrobial and antioxidant activities were confirmed in extracts. Semi-purified fraction and verbascoside showed similar activity to amphotericin B. Acknowledgements: Ouro Fino – Agribusiness

Antimicrobial Assay of Chlorhexidine-Wetted Textile Napkins for Surgical Site Disinfection in Ocular Surgery

Background: As a new intraoperative disinfection method, chlorhexidine-wetted textile napkins have been employed in order to cover the upper and lower eyelid edges, eyelid skin, eyelashes, lid margins and palpebral conjunctiva during phacoemulsification cataract extraction. This study was conducted to compare the antimicrobial activity of textile napkins before and after their use. Methods: This study evaluated 80 textile napkins wetted with 0.02% aqueous solution of chlorhexidine. All textile napkins were divided into groups. The study group consisted of 60 used textile napkins which were collected from 29 patients (30 eyes) at the end of phacoemulsification, and the control group included 20 unused sterile textile napkins. Antimicrobial assay was performed by means of measuring the growth inhibition zones of the standard or clinical isolate strains under the textile napkins on the surface of agar media. Results: The number of textile napkins and the diameter of the growth inhibition zones (mm) in the study group and in the control group relating to gram-positive, gram-negative, and fungi were: 24/31 vs. 8/31, 32/30 vs. 8/30, and 4/30 vs. 4/30. The diameter of the growth inhibition zones of gram-positive bacteria was more than other investigated microorganisms. In the growth inhibition zones, exogenous microorganism colonies were not found. Conclusion: Antimicrobial activity of textile napkins wetted with 0.02% aqueous solution of chlorhexidine against gram-positive bacteria is more than gram-negative bacteria and fungi, and is preserved to the end of the phacoemulsification.

In vitro antimicrobial activities of Acalypha ornate leaf extracts on bacterial and fungal clinical isolates

Traditional medicine practitioners make use of medicines which are plant-derived to treat diseases. These are usually sourced from their local environment with a history of curing many different ailments. Acalypha ornate is one such local plant which is native to the tropics and has been claimed to cure bacterial and fungal infections. This study investigated the in vitro antimicrobial and anti-fungal activities of water and methanol leaf extracts of Acalypha ornata against five clinical bacteria and four fungi isolates. This was done in order to verify its claimed use by natives in traditional medicine for the treatment of bacterial and fungal infections, particularly in children. The water and methanol leaf extracts were found to inhibit the growth of clinical isolate strains of bacteria including Klebsiella pneumonia, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The leaf extracts were also shown to have some antifungal activity. The growth of Trichophyton rubrum and Trichophyton mentagrophytes was reduced. No inhibitory effect was recorded against Aspergillus flavus and Candida albicans. This study provides scientific evidence to support the ethno-medicinal uses of the plant for the treatment of skin infections, therefore merits further studies to isolate the bioactive compounds responsible for the observed activities.

Synthesis and biological evaluation of 5-aryloxypyrazole derivatives bearing a rhodanine-3-aromatic acid as potential antimicrobial agents

Three novel series of 5-aryloxypyrazole derivatives have been synthesized and tested for their antibacterial activity. The majority of the synthesized compounds showed potent inhibitory activity against Gram-positive bacteria Staphylococcus aureus 4220, especially against the strains of multidrug-resistant clinical isolates (MRSA3167/3506 and QRSA3505/3519). Among which compounds IIIb, IIIg and IIIm showed the most potent levels of activity (MIC=1μg/mL) against the multidrug-resistant strains. And cytotoxic activity assay showed that the compounds tested did not affect cell viability on the Human cervical (HeLa) cells at their MICs. The current study therefore suggests that 5-aryloxypyrazoles bearing a rhodanine-3-aromatic acid moiety are promising scaffolds for the development of novel Gram-positive antibacterial agents.

Antibacterial activity of selected metabolites from Chilean lichen species against methicillin-resistant staphylococci

The in vitro antibacterial activities of eight compounds isolated from lichens, collected in several Southern regions of Chile (including Antarctica), were evaluated against methicillin-resistant clinical isolates strains of Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus warneri. The minimum inhibitory concentrations, calculated in microdilution, were ranging from 8 µg mL−1 for sphaerophorin to 1024 µg mL−1 for fumarprotocetraric acid. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Evaluation of Extract of Ficus Exasperata Vahl Root Bark for Antimicrobial Activities Against Some Strains of Clinical Isolates of Bacterial and Fungi

Ficus exasperata Vahl a small tree well known on account of its very rough leaves being used as paper widely spread in all eco-regions of Nigeria. The plant has been ethnobotanically reported to have diverse medicinal uses. It is used in herbal medicine to treat cough, hemorrhoid and lowering high blood pressure. Methanol and Ethylacetate extract of this plant were obtained by maceration and tested for antimicrobial activity using agar diffusion and micro broth method dilution techniques. The extracts were tested against strains of S. aureus, E. coli, B. subtilis, Ps. aeruginosa and fungi including four species of Can- dida. The study showed that extracts of Ficus exasperata root bark exhibited profound antibacterial activity against the typed and clinical isolates. High antifungal activity, particularly against the Candida species was noted as Candida species are implicated in candidiasis and vaginal thrush. Generally, the methanol extract was more effective than the ethylacetate extracts on the test mi- cro-organisms. Minimum Inhibitory Concentration(MIC) of the extract was determined However, Phytochemical analysis of the root bark revealed the presence of saponin and cardiac glycoside which is an indication of medicinal significance of the root bark of Ficus exasperata. This study justifies the ethno pharmacological uses of these medicinal plants for treatment of microbial infections.

Synthesis and antimicrobial evaluation of l-phenylalanine-derived C5-substituted rhodanine and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone

Four novel series of compounds, including the l-phenylalanine-derived C5-substituted rhodanine (6a–q, 7a–j) and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone (9a–e, 11a–e) have been designed, synthesized, characterized, and evaluated for their antibacterial activity. Some of these compounds showed significant antibacterial activity against Gram-positive bacterias, especially against the strains of multidrug-resistant clinical isolates, among which compounds 6c–e, 6g, 6i, 6j and 6q exhibiting high levels of antimicrobial activity against Staphylococcus aureus RN4220 with minimum inhibitory concentration (MIC) values of 2 μg/mL. Compound 6q showed the most potent activity of all of the compounds against all of the test multidrug-resistant clinical isolates tested. Unfortunately, however, none of the compounds were active against Gram-negative bacteria at 64 μg/mL.

Application of broad-spectrum PCR amplification and direct sequencing for identification of the infrequent bacterial cultures from clinical sources, targeting the bacterial 16S rRNA gene with universal primes

Objectives To identify the infrequent strains in clinical isolates by broad-spectrum PCR amplification and direct sequencing targeting the bacterial 16S rRNA gene. Methods Total 48 clinical isolates and 7 false-positive blood culture samples were collected from 7 different hospitals or institutions from December 2010 to September 2011.The bacterial 16S rRNA gene were amplified and sequenced by universal prime sets of 27f-1492r and 27f-1525r，and MicroSeq 500 16S rRNA gene kit.The homology analysis was used by the Basic Local Alignment Search Tool, and comparing to gene sequence of the type strain.provided by the List of Prokaryotic names with Standing in Nomenclature.The criteria for the bacterial identification was interpreted according to the Clinical and Laboratory Standards Institute （CLSI） MM18-A. Results All of the 48 cultured strains were succeeded amplifying and sequencing the targeted 16S rRNA genes.According to the criteria of CLSI MM18-A, total 35 strains were specified to the species level, 11 strains were specified to the genus level, and the other 2 strains were specified to possible novel genus and species.Combining the analysis the sequence of other housekeeping gene with the results of biochemical results, total 42 strains can be specified to the species level, including some clinical important pathogens, such as Streptobacillus, Capnocytophaga, Nocardia, Mycobacterium, Roseomonas and Campylobacter.Two false-positive blood culture samples were managed to amplify 16S rRNA genes and finally identified as Streptococcus pneumoniae.We also identified one novel subspecies of Campylobacter fetus, and some new valid-published species, such as Acinetobacter parvus,Mycobacterium phocaicum,Roseomonas mucosa and Halomonas johnsoniae. Conclusions The 16S rRNA gene sequence based identification has unique advantages over the phenotypic methods.It is universal to almost of all the bacteria, and can provide the genetic classified information. It is very suitable for the clinical infrequent and special bacterial cultures, such as the slow-growing, fastidious, or un-cultured bacteria.（Chin J Lab Med,2012,35:612-619） Key words: RNA，ribosomal，16S; Polymerase chain reaction; Sequence analysis; Bacteriological techniques

The structure of the wall teichoic acid isolated from Enterococcus faecalis strain 12030

Wall teichoic acid (WTA) was isolated from Enterococcus faecalis 12030, a clinical isolate and biofilm-producing strain, and analyzed using compositional chemical methods, nuclear magnetic resonance spectroscopy, and mass spectrometry. The repeating units of WTA were composed of d-glucose, d-galactose, 2-acetamido-2-deoxy-d-galactose, 2-acetamido-2-deoxy-d-glucose, d-ribitol, and phosphate in a molar ratio 1:2:1:1:1:1, and had the structure given below.▪

18S ribosomal DNA based PCR diagnostic assay for Trichomonas vaginalis infection in symptomatic and asymptomatic women in India

ObjectiveTo identify the cases of trichomoniasis in symptomatic and asymptomatic Trichomonas vaginalis (T. vaginalis) infected patients by PCR amplification of hypervariable 18S rRNA gene and to assess the sensitivity of restriction fragment length polymorphism (RFLP) technique for their diagnosis. MethodsWe enrolled 498 women of child bearing age groups, with their pre-informed consent, attending OPD for their routine checkups and STI related problems. Trichomoniasis was diagnosed on the basis of wet mount preparations and PCR with a primer set targeting a well-conserved region in the 18S rRNA genes of T. vaginalis, respectively. Sequencing was done for differentiating the symptomatic and asymptomatic strains of axenic and clinical isolates. ResultsAfter PCR diagnosis T. vaginalis infection was detected in 17 (3.42%) out of 498 clinical isolates. Seventeen axenic and sixteen clinical strains of T. vaginalis tested were successfully detected by PCR yielding a single predicted product of 312 bp in gel electrophoresis followed by restriction digestion with restriction endonuclease HaeIII. After restriction digestion they gave two bands, one of 101 and the other of 211 bp, while there was negative response with DNA from Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. An optimal analytical sensitivity and specificity of one T. vaginalis organism per PCR was achieved. Sequence of symptomatic and asymptomatic strains of axenic and clinical isolates are somewhat differentiated on the basis of point mutations in their 18S rRNA gene. ConclusionsOnly few factors are known to predict symptoms of T. vaginalis infection, although the majority of women are infected with trichomoniasis are reported. Therefore the application of sensitive PCR based diagnosis may be quite useful for routine diagnosis of T. vaginalis strains.

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Bacteriophages ϕMR299-2 and ϕNH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells

ABSTRACT Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107 to 2 × 107 P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients.

Characterization and purification of a bacteriocin from Lactobacillus paracasei subsp. paracasei BMK2005, an intestinal isolate active against multidrug-resistant pathogens

A strain of Lactobacillus paracasei subsp. paracasei BMK2005 isolated from healthy infant faeces has shown a remarkable antibacterial activity against 32 bacterial pathogenic strains of human clinical isolates. Among them, 13 strains belonging to species of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa were resistant to Cefotaxime (CTX) and Ceftazidime (CAZ), and 4 strains of Staphylococcus aureus were resistant to Methicillin (MRSA). This antibacterial activity was attributed to a bacteriocin designated as Paracaseicin A. It was heat-stable up to 120°C for 5 min and active within the pH range of 2-5. Its activity was lost when treated with proteases, which reveals its proteinaceous nature. This bacteriocin was successfully purified only by two steps of reversed phase chromatography. Its molecular mass, determined by mass spectrometry analysis, was 2,462.5 Da. To our knowledge, the present study is the first report on characterization and purification of a bacteriocin, produced by a L. paracasei subsp. paracasei strain exhibiting an antibacterial activity against various multidrug-resistant species of Gram-positive and Gram-negative bacteria, which reveals its potential for use in prevention or treatment of infections caused by multidrug-resistant species especially in cases of antibiotics-associated diarrhea (AAD).

In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔE model methods

The in vitro antimicrobial activities of usnic acid were evaluated in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, were evaluated. A synergistic action was observed in combination with gentamicin, while antagonism was observed with levofloxacin. The combination with erythromycin showed indifference, while variability was observed for clindamycin and oxacillin. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index (FICI) and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings, beside confirm the well known antimicrobial activity of usnic acid, suggest, however, that this substance might be a good candidate for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Molecular Modeling Study of the Carbohydrate Region of the Endotoxin from Burkholderia cenocepacia ET‐12

AbstractIn this work we define the solution conformation of the saccharide of a lipopolysaccharide molecule extracted from a clinical isolate strain of the human opportunistic pathogen Burkholderia cenocepacia. The conformational behavior has been explored by means of NMR spectroscopy, NOE‐based conformational analysis, molecular modeling (MM), and molecular dynamics (MD) studies. The results indicate a remarkable rigidity of the inner core region, ascribable to the structural motifs essential for bacterial surviving, which markedly contributes to the strength, stability, and rigidity of the cellular membrane. On the other hand, the monosaccharide residues extending from the inner core to the outer core region show significant flexibility and can be the target of recognition and binding of endogenous and host‐released proteins. This study sheds light on the three dimensional arrangement of Burkholderia LPS and is an important step toward developing a structure–function relationship analysis of the virulence factor that is responsible for the severe pathogenicity Burkholderia.

Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes.

Evaluation of automated repetitive-sequence–based PCR (DiversiLab) compared to PCR ribotyping for rapid molecular typing of community- and nosocomial-acquired Clostridium difficile

Automated repetitive PCR (rep-PCR; DiversiLab) was compared to PCR ribotyping of the 16S-23S RNA intergenic spacer of Clostridium difficile (CD) as the “gold standard” method for CD typing. PCR products were separated on DiversiLab LabChips (bioMérieux, St. Laurent, Quebec, Canada) utilizing a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) operating the DiversiLab v1.4 assay. Bioanalyzer data were exported to a secure DiversiLab website and analyzed with DiversiLab v3.4 software. Replicability of each method was verified by confirming that the 5 CD reference strains (RS) formed distinct clusters (CD4, CD6, VL0047, VL0013 [ribotype 027], VL0018 [ribotype 001]) by both typing methods. Ninety randomly selected clinical isolates (CS) were analyzed by both methods: 49 from community-acquired and 41 from hospital-acquired cases. A similarity index (SI) of ≥90% was used to define clusters when comparing the known RS cluster to the PCR ribotyping and rep-PCR patterns of CS. Fourteen different PCR-ribotype clusters were identified, but most CS formed 4 major clusters (i.e., CD4 [15/90; 17%], CD6 [17%], 027 [12%], and 001 [9%]). A total of 7 rep-PCR types were identified, but most CS formed 2 major rep-PCR clusters (i.e., CD4 [29/90; 32%] and CD6 [23%]); several PCR ribotypes occurred within a single rep-PCR cluster. Rep-PCR did not distinguish 027 or 001 isolates; i) 027 RS strain did not cluster, ii) eleven 027 CS strains clustered as CD4, iii) no 027 CS strains clustered with the 027 RS, and iv) only 2 001 CS clustered with the RS. Agreement between the PCR-ribotype and rep-PCR clusters only occurred for 35/90 (39%) of the CS using a rep-PCR SI of ≥90%. Rep-PCR time to results was similar, but the annual costs of routinely using this method are 32% higher than PCR ribotyping. Routine use of rep-PCR for CD typing is limited by its lack of definitive separation of the hypertoxigenic 027 or 001 outbreak CD strains.

Anti-Staphylococcus aureus activity of Pisolithus albus from Pune, India

In vitro studies were conducted to investigate the anti-staphylococcal activity of crude extracts of two strains of Pisolithus albus collected from different hosts in the vicinity of Pune University Campus, Pune, India. Organic solvents of increasing polarities were used for extraction of secondary metabolites. In addition, bioactive components of known antimicrobial activity were isolated and tested against thirty strains of clinical isolates of methicillin resistant Staphylococcus aureus. Strong activity was exhibited by ethyl acetate and methanol extracts while aqueous extract showed weak activity. All strains were resistant to the chloroform extract. Maximum effectiveness was recorded for sesquiterpenoids extract (zone diameter: 16.67 to 31.5 mm). Triterpenoids and diterpenoids extracts displayed weak to moderate activity. Polysaccharides fractions IIIa and IIIb were weakly effective while fractions I and II did not show activity. Minimal inhibitory concentration was determined to be in a range of 0.62 to 1.2 mg/ml. Key words: Pisolithus albus, Staphylococcus aureus, methicillin resistant antimicrobial activity.