PubMed Health⌕ Search

Biomedical subjects

M Manafi

Publications and source records attributed to M Manafi.

17 recordsLinked to original sources

Characterization of pathogenic Escherichia coli isolated from humans in Austria: phenotypes, toxin gene types and epidemiology.

One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR. All but one of these isolates contained the eae gene. The EHEC-hly gene could be detected in all E. coli O157 and in 50% of E. coli O26 isolates. Forty-five (40.9%) of the 110 E. coli were positive for both stx(1) and stx(2) genes, 2 (1.8%) isolates were positive for stx(1) and 57 isolates (51.8%) were positive for stx(2) only. Among the 102 stx(2) positive isolates, 14 (13.7%) E. coli O157 contained also the stx(2c) variant gene. No other stx(2) variant was identified. Six clinical isolates (five E. coli O157:H7 and one E. coli O26) did not contain stx genes. Ten non-pathogenic E. coli isolates which were amplified as controls didn't contain any stx and eae gene but two of the ten strains contained the EHEC-hly gene. By their growth on chromogenic media, all but two of 50 E. coli O157 could be differentiated from eight E. coli O26 and 10 non-pathogenic E. coli. Sixty-one of the O157:H7 isolates were further subjected to pulsed-field gel electrophoresis (PFGE) which identified 49 distinguishable patterns. In five cases where contact infection among family members was suspected, indistinguishable PFGE patterns confirmed the epidemiological relatedness of the isolates. Moreover, two PFGE clusters were identified which comprised five and three strains, respectively. These findings indicate the occurrence of both family and diffuse outbreaks of E. coli O157 infections in Austria during recent years and demonstrate the need for molecular subtyping of these pathogens.

Adhesins, Bacterial↗

Comparative evaluation of different chromogenic/fluorogenic media for detecting Escherichia coli O157:H7 in food.

Escherichia coli O157:H7 is a serious and common human pathogen that can cause diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (HUS). This study evaluated the enrichment, detection and confirmation procedures for the isolation of E. coli O157:H7 from raw ground beef and raw drinking milk. The purpose of this investigation was to compare Rainbow Agar O157 (RB; Biolog, Hayward, USA), Biosynth Culture Medium O157:H7 (BCM O157:H7; Biosynth, Staad, Switzerland) and Fluorocult HC (HC; Merck, Darmstadt, Germany) with the conventional Sorbitol MacConkey Agar (SMAC, Merck) using mEC + n (raw ground beef) and mTSB + n (raw milk) enrichment media. Single-path GLISA test (Gold Labeled Immuno Sorbent Assay; Merck) was used as the confirmation test. Growth of 466 strains of gram-negative rods isolated from food samples and 46 known E. coli strains from type culture and other collections (34 E. coli O157:H7 strains and 12 serotypes other than E. coli O157:H7) was examined on the agar media. The E. coli O157:H7 strains could readily be isolated and recognized uniquely by their typical black/grey colonies on RB and blue/black colonies on BCM O157:H7. Examination of the 46 known strains of E. coli reference strains showed false negative results on BCM O157:H7 (3.0%), RB (8.8%), HC (5.9%) and SMAC (5.9%) agars. On BCM O157:H7 no false negative results were found with the typical E. coli O157:H7 (beta-D-glucuronidase and sorbitol negative strains). One of two atypical E. coli O157:H7 strains (beta-D-glucuronidase positive) showed similar colouration to the typical strains and was mis-identified by each of the three media (RB, BCM O157:H7, and SMAC agar media). None of the 60 food samples tested yielded E. coli O157:H7. Examination of the food samples, showed that RB gave the lowest number of false positives. The percentages were RB (2.1%), BCM O157:H7 (3.3%), HC (6.2%), and SMAC (57.3%).

Animals↗

Performance of candida ID, a new chromogenic medium for presumptive identification of Candida species, in comparison to CHROMagar Candida.

Candida ID agar allows identification of Candida albicans and differentiation of other Candida species. In comparison with CHROMagar Candida, we evaluated the performance of this medium directly from 596 clinical specimens. In particular, detection of C. albicans after 24 h of incubation was easier on Candida ID (sensitivity, 96.8%) than on CHROMagar (sensitivity, 49.6%).

Candida↗

New developments in chromogenic and fluorogenic culture media.

This review describes some recent developments in chromogenic and fluorogenic culture media in microbiological diagnostic. The detection of beta-D-glucuronidase (GUD) activity for enumeration of Escherichia coli is well known. E. coli O157:H7 strains are usually GUD-negative and do not ferment sorbitol. These characteristics are used in selective media for these organisms and new chromogenic media are available. Some of the new chromogenic media make the Salmonella diagnostic easier and faster. The use of chromogenic and fluorogenic substrates for detection of beta-D-glucosidase (beta-GLU) activity to differentiate enterococci has received considerable attention and new media are described. Rapid detection of Clostridium perfringens, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus are other application of enzyme detection methods in food and water microbiology.

Chromogenic Compounds↗

Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media.

This study compared the performance of LMX(R) broth (LMX), Chromocult Coliform(R) agar (CC) and Chromocult Coliform agar plus cefsulodin (10 microg ml-1) (CC-CFS), with standard methods multiple tube fermentation (MTF), for the enumeration of total coliforms and Escherichia coli from marine recreational waters. LMX and CC are two media designed to concurrently detect total coliform (TC) bacteria and E. coli by the specific action of beta-galactosidase (total coliforms) and beta-glucuronidase (E. coli). Overall results for the TC test showed that LMX, CC and MTF recovered 2.63, 1.95 and 1.90 times as many TCs as CC-CFS, respectively. Data from the multiple range test showed significant differences (P < 0.05) between TC counts on CC-CFS and LMX. The traditional MTF was less sensitive for E. coli enumeration. However, there was no statistically significant differences between LMX, CC, CC-CFS and the MTF method for E. coli enumeration. Background interference was reduced on CC-CFS and the counts obtained reflected more accurately the number of TCs. Therefore, the contribution of beta-galactosidase positive, non coliform bacteria (Aeromonas spp. and Vibrio spp.) to TC counts should not be neglected.

Bacteriological Techniques↗

Evaluation of CHROMagar Candida for rapid screening of clinical specimens for Candida species.

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei. We evaluated this medium and compared it with a reference medium, Sabouraud glucose agar, for the presumptive identification of yeast species isolated directly on the medium from 1150 clinical specimens. A total of 731 specimens showed no growth, 299 isolates (70.2%) showed growth to the same extent on both media. Forty mixed cultures were detected on both media. More than one isolate was detected in 30 of the tested specimens on either CHROMagar (26 specimens) or Sabouraud glucose agar (four specimens). We found a sensitivity of 98.8% and a specificity of 100% for C. albicans, 66.7% and 99.8% for C. tropicalis, 100% and 100% for C. krusei, and 98% and 95.7% for C. glabrata. Regarding these results, CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of yeasts and better detection of mixed cultures in clinical specimens.

Candida↗

Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic gram-positive cocci.

The contribution of enzyme tests to the accurate and rapid routine identification of gram-positive cocci is introduced. The current taxonomy of the genera of aerobic and facultatively anaerobic cocci based on genotypic and phenotypic characterization is reviewed. The clinical and economic importance of members of these taxa is briefly summarized. Tables summarizing test schemes and kits available for the identification of staphylococci, enterococci, and streptococci on the basis of general requirements, number of tests, number of taxa, test classes, and completion times are discussed. Enzyme tests included in each scheme are compared on the basis of their synthetic moiety. The current understanding of the activity of enzymes important for classification and identification of the major groups, methods of testing, and relevance to the ease and speed of identification are reviewed. Publications describing the use of different identification kits are listed, and overall identification successes and problems are discussed. The relationships between the results of conventional biochemical and rapid enzyme tests are described and considered. The use of synthetic substrates for the detection of glycosidases and peptidases is reviewed, and the advantages of fluorogenic synthetic moieties are discussed. The relevance of enzyme tests to accurate and meaningful rapid routine identification is discussed.

Animals↗

Fluorogenic and chromogenic enzyme substrates in culture media and identification tests.

Rapid detection and identification of microorganisms is extremely important in many fields of applied and research microbiology. In general, fluorogenic and chromogenic substrates have proved to be a powerful tool, utilizing specific enzymatic activities of certain microorganisms, either in parallel with or instead of traditional methods. By incorporation of synthetic fluorogenic or chromogenic substrates into primary selective media, enumeration and detection can be performed directly on the isolation plate. The introduction of many of these media and identification tests has led to improved accuracy and faster detection of target organisms, often reducing the need for isolation of pure cultures and confirmatory tests.

Chromogenic Compounds↗

Evaluation of a new chromogenic agar medium for the identification of urinary tract pathogens.

This study evaluated the performance of CPS ID2 (bioMérieux) compared to that of conventionally used selective agar for the identification of bacteria responsible for urinary tract infections. This medium detects bacterial enzymes using chromogenic substrates. Two hundred and nineteen samples of urine were tested in order to evaluate new CPS ID2 in comparison to blood agar and MacConkey agar. According to our results, the CPS ID2 agar is an easy, rapid and sensitive method for the screening of colonies suspected of being Escherichia coli, reducing the number of biochemical tests needed. Also, enterococci and Proteae can be easily detected. Other micro-organisms require further identification. The greatest value of this medium is the accurate identification of polymicrobial cultures.

Agar↗

Adaptation of two commercially available DNA probes for the detection of E. coli and Staphylococcus aureus to selected fields of dairy hygiene--an exemplary study.

The application of two commercially available colorimetric DNA hybridization tests (GENE-TRAK E. coli and Staphylococcus aureus) to selected aspects of dairy hygiene was investigated. Bacterial isolates of different origin, naturally contaminated cheese varieties, nonfat dry milk, milk concentrates, artificially contaminated milk and raw milks from udder quarters were examined. Based on the observation that the sensitivity of the E. coli DNA probe was comparable to that of the beta-D-glucuronidase-based fluorescence reaction (with 4-methyl-umbelliferyl-beta-D-glucuronide) of E. coli strains in Fluorocult lauryl sulfate broth, a Most Probable Number technique for enumerating E. coli in cheese using the DNA probe was developed. Another specific DNA probe was applied for the detection of S. aureus as a mastitis agent. By using a modified sample preparation, specific diagnosis of this microorganism in milk from udder quarters was enabled within 6 hours. This procedure is recommended to be used in screening tests. Based on the examples presented the potential of these tests in several fields of hygiene was illustrated.

Animals↗

A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae.

A new selective differential agar medium for rapid presumptive identification of Enterobacteriaceae from water and food samples is described (EMX ID agar). By a combination of fluorogenic and chromogenic substrates, the medium detects the presence of beta-D-glucuronidase, beta-D-galactosidase, beta-D-xylosidase, tryptophane deaminase and H2S; additionally, cytochrome-oxidase and indole production can be demonstrated. This medium provides an inexpensive means for simple and rapid presumptive identification of E. coli and coliforms and for the differentiation within the Klebsiella-Enterobacter and the Proteus-Providencia-Morganella group. Furthermore, it allows to distinguish between the H2S-positive Enterobacteriaceae Citrobacter freundii, Salmonella spp., S. arizonae, Edwardsiella, Proteus mirabilis, P. vulgaris and some oxidase-positive bacteria.

Agar↗

Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Methods based on the application of chromogenic and fluorogenic substrates enable specific and rapid detection of a variety of bacterial enzymatic activities. By using these techniques, enzymatic reactions can be examined simultaneously or individually, either directly on the isolation plate or in cell suspensions. For this purpose, various testing principles and test kits for clinical and food microbiology have been introduced successfully during the last few years. In this paper we present a survey of different enzymes of microbial origin that are utilized for microbiological identification and differentiation and the corresponding methods. Particular emphasis is given to the examination of Escherichia coli and the description of the different techniques as used in routine analysis.

Bacteria↗

Fluorogenic and chromogenic substrates--a promising tool in microbiology.

During the last few years the use of fluorogenic and chromogenic substrates for rapid and sensitive detection of bacteria has proved to be a powerful alternative to traditional methods. These sophisticated substrates might find widespread application in, for instance, the assay of clinically important enzymes, flow cytometry, and direct epifluorescent filter technique. Specific enzyme detection offers another approach to differential identification and characterization of viable bacteria from a sample. The use of some chromogenic and fluorogenic substrates specific for bacterial enzymes and their applications to microbial identification is reported. Particular emphasis is given to the examination of Escherichia coli and the description of the different techniques as used in routine analysis.

Bacteria↗

Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria.

Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.

Animals↗

[A combined chromogenic-fluorogenic medium for the simultaneous detection of coliform groups and E. coli in water].

A comparison was made with different chromogenic and fluorogenic substrates, 4-methylumbelliferyl-beta-D-glucuronide (MUG), 4-nitrophenyl-beta-D-glucuronide (PNPG), 4-methylumbelliferyl-beta-D-galactopyranoside (MUGA), 2-nitrophenyl-beta-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL), for the rapid and simultaneous enumeration of total coliforms and E. coli in water samples, based on 2 commercially available culture-media. The combination of the chromogenic compound X-GAL (for detecting coliforms) and of the fluorogenic compound MUG (for detecting E. coli) incorporated either into ECD agar or into lauryl sulfate broth proved to be most useful. The optimum concentration of the X-GAL/MUG supplement was (50 micrograms/ml/70 micrograms/ml) for the solid medium (EMX agar) and (60 micrograms/ml/70 micrograms/ml) for the fluid medium (LMX broth). As a result of the examination of 244 Enterobacteriaceae strains isolated from water samples and clinical material, it was shown that the use of EMX agar (LMX broth) had several advantages over conventional methods. A routine method for the analysis of water samples was proposed involving the EMX agar and the LMX broth.

Chromogenic Compounds↗

[Significance of Yersinia enterocolitica and thermophilic Campylobacter for water hygiene].

The taxonomy of Yersinia enterocolitica as well as their classification methods are still unreliable with respect to their pathogenicity. Therefore, a basis of evaluation was established by using extended biochemical tests (API-20-E, API-50-CH) and electrophoretic methods for identifying strains from human, food and water sources. All strains show great inhomogenicity, which was higher for non-human strains than for those isolated from clinical specimens. Thermophilic Campylobacters are especially prevalent in wild living fowl thus being a potential risk for surface water contamination. The results of the study show that high colony counts were found only during winter, whereas in summer the concentration lay below the infective dose. Furthermore, thermophilic Campylobacters were isolated only from samples that contained a higher number of fecal indicator organisms.

Bacteriological Techniques↗