Numerical Taxonomy Analysis of Bacteria Isolated from the Completed 'Most Probable Numbers' Test for Coliform Bacilli

Lactose-fermenting bacteria were isolated from oyster, water and sediment samples tested to determine the Most Probable Number (MPN) of coliforms present. The completed test series was used to enumerate the total number of coliforms. The strains from the completed tests of the MPN analysis were compared with named reference cultures, for which 100 phenotypic characters were recorded and the taxonomic data analysed by methods of numerical taxonomy. The isolates were recovered in 13 phenetic groups, defined at or above the 80% similarity level. Nine phena were identified as Acinetobacter calcoaceticus, Chromobacterium violaceum, Citrobacter freundii, Erwinia herbicola, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, Serratia liquefaciens , and Ser. marcescens. Of these, Ac. calcoaceticus and C. violaceum were isolated exclusively from oyster tissue samples and strains of the other taxa from both water and oyster samples. Some strains of Esch. coli, Er. herbicola, H. alvei and bacteria that were not Enterobacteriaceae were found to lose the ability to ferment lactose, following storage on laboratory media. Identifications from diagnostic keys, rapid identification systems and IMViC patterns were not always in agreement.     

Identification of enterococci isolated from cow's milk cheese: comparison of the classical methods and the API 20 STREP system (technical note)

A comparison of the results obtained using the classical methods with those of the API 20 Strep system was carried out in identifying 24 enterococci strains isolated from San Simón cow's milk cheese, a traditional Spanish variety. The results of both identification systems coincided exactly in 9 strains (37.5% of the strains studied). In one strain the results obtained using the classical methods did not coincide with those using the API 20 Strep method. 3 strains (12.5%) could not be identified using the API 20 Strep system. However, 11 strains (45%), that remained doubtful between both species E. faecalis and E. faecium on the basis of the classical methods, were identified using the API 20 Strep system. The API 20 Strep system does not include some biochemical tests of importance in identifying of foodborne enterococci and could not identify the atypical strains of Enterococcus. Moreover, this system is adapted to the identification of enterococci of clinical origin and their database does not include some species common in foods. However, it could have an application in combination with the classical methods in order to carry out a reasonably rapid and reliable identification of enterococci related to cheese.     

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65(PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Multi-Biochemical Test System for Distinguishing Enteric and Other Gram-Negative Bacilli

A multi-biochemical test system consisting of nine tests, entitled Enterotube, was evaluated in parallel with conventional tests to determine its value in the identification of enteric and certain other gram-negative bacilli. The 242 bacterial strains studied were from a variety of pathological specimens and from our culture collection. When the results with individual tests represented in both test systems were compared, no discrepancies were noted in the indole test, and one discrepancy was recorded for dextrose. In 7 of 242 hydrogen sulfide tests, 3 of 242 phenylalanine tests, 22 of 242 urease tests, 15 of 242 dulcitol tests, 12 of 242 lactose tests, 27 of 217 lysine decarboxylase tests, and 5 of 242 citrate tests, the Enterotube results were contrary to those obtained with conventional methods. The lysine decarboxylase test in the Enterotube posed a problem of interpretation and readability and is not an acceptable alternative to the conventional methods. Fifteen of the strains studied were incorrectly identified by the Enterotube system and four could not be differentiated from other closely related strains. Salmonella could be identified as to group, whereas Shigella strains were frequently misidentified as Escherichia. The Enterotube method is simple and convenient, and all media are inoculated at once from a single colony.     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.     

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

Aim: To investigate the application of high‐resolution melt (HRM) analysis for rapid species‐level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. Methods and Results: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR‐DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. Conclusion: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. Significance and Impact of the Study: HRM profiling is suggested as an additional approach for identification of DGGE bands.     

74例金黄色葡萄球菌乳腺炎的耐药性分析

目的分析哺乳期乳房脓肿患者的金黄色葡萄球菌的耐药情况,指导临床合理使用抗菌药物。方法将厦门市妇幼保健院乳腺门诊及病房2009年2月至2011年1月抽取的128例哺乳期乳房脓肿患者的乳腺脓液接种于哥伦比亚血平板,置35℃培养箱,过夜孵育18~24 h。革兰阳性球菌,触酶阳性,血浆凝固酶试验(+),必要时采用ATB-Expression细菌鉴定系统确认。用K-B纸片法检测其对苯唑西林等10种抗菌药物的敏感度。结果 128例检出细菌86例,阳性率67.2%,其中检出74株金黄色葡萄球菌(86.0%),其对青霉素、红霉素和克林霉素有较强的耐药性,耐药率分别为94.6%、64.9%和59.5%;耐苯唑西林19株(25.7%),对复方新诺明、磷霉素和庆大霉素较为敏感,对万古霉素、替考拉宁和左旋氧氟沙星100%敏感。结论哺乳期乳房脓肿致病菌主要为金黄色葡萄球菌,耐甲氧西林金黄色葡萄球菌(MRSA)占25.7%,其耐药情况不容忽视,青霉素类、大环内酯类等耐药率较高不能作为经验用药。建议临床使用抗菌药物前及时送检乳腺脓液细菌培养,以药敏结果指导临床合理经济用药。     

Quantitative ethnobotany of two east Timorese cultures

This is the first time aspects of the ethnobotany of East Timor have been reported. The medicinal plant traditions of two distinct East Timorese cultures, the Laklei and Idate, were studied and compared using quantitative ethnobotanical methods. A total of 86 medicinal plant species were identified. The medicinal plant traditions of the Laklei and Idate cultures were compared using Trotter and Logan’s (1986) quantitative “informant agreement ratio.” On average, informant consensus was greater in Laklei, suggesting a medicinal plant tradition that is more defined than in Idate, where informants are more likely to use the same medicinal plants when treating the same usage categories. Furthermore, only 11 of the 86 medicinal plant species documented were used by both cultures, of which only six had similar mentions. These findings have important implications for the understanding of ethnobotany as they demonstrate how relatively closely situated cultural groups can have significantly different traditional knowledge systems.     

一种产邻苯二酚菌株高通量筛选方法

目的：对产邻苯二酚菌株进行筛选。方法：采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果：硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值（495nm）和富集培养基中菌液浊度值（OD630）的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属（Pseudomonas sp.）。结论：硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

Evaluation of the Minitek system for characterization of Bacillus species

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Evaluation of the Minitek system for characterization of Bacillus species.

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Phenotypic identification of non-clinical isolates of Acinetobacter species

Five different phenotypic identification systems were used in attempts to place 164 Acinetobacter strains obtained from a biological nutrient removal plant and 16 reference cultures into the genospecies of Bouvet and Grimont, and Tjernberg and Ursing. Of these strains only four, including two of the reference strains, were identified at a Willcox probability level of >0·95 and a modal likelihood fraction of > 0·0001 by all five systems. These different identification schemes were compared for their usefulness with such non-clinical isolates.     

A frequency matrix for probabilistic identification of some bacilli

A matrix comprising frequencies for positive results for 44 Bacillus taxa for 30 characters has been constructed. The 44 taxa include most of the common species and several clusters of environmental isolates including those described as B. firmus-B. lentus intermediates. The tests, which were chosen for their high diagnostic value, included some of the traditional tests used for identification of bacilli supplemented with a range of sugar fermentations and other characterization tests. The matrix was evaluated by identifying hypothetical median organisms, cluster representatives and a panel of 23 reference strains. All reference strains achieved Willcox probabilities above 0.995. Fifty-eight environmental isolates were also subjected to the 30 tests and identification was attempted. Forty-one strains (70%) achieved a Willcox probability greater than 0.95, which was considered an acceptable identification, and were assigned to 12 taxa. If the SE of taxonomic distance was also considered in the identification score (an acceptable value being less than 7.0), the number of acceptable identifications was reduced to 34 (59%). It was encouraging that bacteria from garden soils identified to the common species such as B. subtilis, B. cereus and B. licheniformis whereas some of the bacteria from an estuarine habitat were identified as species such as B. firmus which are normally identified with that habitat.     

Diagnostic Key to Mycobacteria Encountered in Clinical Laboratories

A diagnostic key has been developed which will permit identification of most mycobacteria encountered in clinical laboratories. The key is based on performance of a few simple tests. The efficiency and accuracy of the key was evaluated in terms of correlation between identifications based on the few tests and those arrived at through application of the techniques of numerical taxonomy, which involves a large battery of tests. Of 679 cultures of mycobacteria other than Mycobacterium tuberculosis, 86.5% were correctly identified by use of the key, and only 1.8% of the cultures were erroneously identified. The remaining cultures required further examination.     

Classification and biological characteristics of coagulase-positive Staphylococci isolated from animals

A total of 165 coagulase-positive staphylococcal strains of different origin (142 S. aureus strains and 23 S. intermedius strains) were subjected to biological typing in accordance with the schemes of Hajek-Marsalek and Meyer-Witte. The former of these schemes permitted to identify 68% and the latter 18% of S. aureus strains. The cultures isolated from swine and chickens had the most uniform composition: 85-86% of the strains belonged to biotype B. 44% of the strains isolated from cows and sheep belonged to biotypes C (ecovars bovis and ovis) and A (ecovar hominis); the rest of the strains could not be identified. 96% of the strains isolated from minks were made up of S. intermedius, more than a half of them belonging to biotype E (ecovar canis). In 80% of S. aureus strains and 48% S. intermedius cultures protein A was detected. Only 9% of S. aureus strains of animal origin were found capable of producing enterotoxins (A-D). The expediency of working out a unified scheme for the biotyping of coagulase-positive staphylococci is discussed.