API ZYM System Research Articles

Enzyme Profile ofLactobacillusStrain GG by a Rapid API ZYM System: A Comparison of Intestinal Bacterial Strains

Lactobacillus GG and eight strains of lactobacilli ( L. acidophilus, L. rhamnosus, L. bulgaricus and L. helviticus ) and other clinical organisms ( Escherichia coli, Peptostreptococcus anaerobius, Bacteroides fragilis and Clostridium difficile ) were compared for their enzyme profiles. Specific activities of 19 hydrolytic enzymes for each strain were determined using the microenzyme API ZYM system. Lactobacillus GG enzyme profile showed high peptidase, chymotrypsin and phosphatase activities, and lower esterase-lipases activity; these enzyme activities were not different from other Lactobacillus strains. β-Glucuronidase activity was not detected in Lactobacillus GG. These results may facilitate identification of some of the biochemical characteristics of Lactobacillus GG, as well as indicate that Lactobacillus GG, as a dairy product, may result in less toxic compounds being released from hydrolytic reaction of β-glucuronidase in the colon. Keywords - Lactobacilli, Intestinal microfiora, Bacterial enzymes, API ZYM

Measurement of Enzymatic Activity in Deep Marine Sediments Using the Semiquantitative APIZym Test System Messung enzymatischer Aktivität in tiefen Meeressedimenten mit Hilfe des APIZym‐Testsystems

AbstractAPIZym strips were used for measuring the activity of 19 enzymatic reactions in marine sediment samples from the continental slope of the Celtic Sea in 135…1630 m depth. The activities of phosphatases were dominant followed by esterases, peptidases, and glycosidases. The activities were influenced by the sediment depth within the cores and the depth of the station. The results indicate that the APIZym system is able to resolve vertical stratification and spatial variability of enzymes possessing activity rates exceeding 10 nmol substrate h−1 cm−3 in sediment but not of those with lower rates which for example would be found in deep‐sea sediments.

Enzyme Profile ofLactobacillusStrain GG by a Rapid API ZYM System: A Comparison of Intestinal Bacterial Strains

Enzyme Profile of<i>Lactobacillus</i>Strain GG by a Rapid API ZYM System: A Comparison of Intestinal Bacterial Strains

Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish

In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates of Vibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD50 ranging from 0.06 to 3.7 μg protein/g fish) and mice (LD50 ranging from 0.02 to 0.43 μg protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP samples were cytotoxic for fish and homoiothermic cell lines. The levels of enzymic and cytotoxic activities were clearly associated with the degree of virulence for fish. Moreover, the enzymic patterns of both live cells and ECP evaluated with the API-ZYM system were very similar among the strains, indicative that most of the activities are associated with exoenzymes. The in vivo and in vitro biological activities were considerably reduced after heat treatment (100°C for 10 min), but not totally lost in the highly virulent strains. Although we have demonstrated that the toxicity of the ECP is not directly associated with their lipopolysaccharides (LPS) content, these compounds could confer some heat-stabilizing effect to the toxic fractions. © 1993 Springer-Verlag New York Inc.

Identification of Pathogenic Fish Bacteria Using the API ZYM System

The API ZYM system was used to identify the bacterial fish pathogens Enterococcus seriolicida, β-hemolytic Streptococcus sp., Renibacterium salmoninarum, Aeromonas salmonicida, A. hydrophila, Pasteurella piscicida, Edwardsiella tarda, Vibrio anguillarum, Pseudomonas fluorescens, Flexibacter columnaris, and F. maritimus. As additional tests, Gram stain and the activities of cytochrome oxidase and catalase were examined. The results of APS ZYM and the additional tests were coded for easy identification. Several biotypes were demonstrated by Streptococcus sp., V. anguillarum, and A. hydrophila. Other bacteria showed uniform profiles. This system can distinguish each fish pathogen from all other bacterial species examined.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

The API ZYM system: a tabulated review from 1977 to date

The ApiZym system is a commercially available test system for the detection of enzymatic profiles of bacteria. This paper reviews published ApiZum studies from 1977 to date. It presents the results of 60 publications which were compiled and tabulated with special emphasis on practical subdivisions to facilitate clinical use. The ApiZym system results to be a rapid and simple performing tool for the discrimination of similar microorganisms.

Biochemical Activities of Bacillus Species Isolated from Flat Sour Evaporated Milk

Forty strains of bacilli were isolated from flat sour evaporated milk and were characterized as Bacillus stearothermophilus (5 strains), Bacillus licheniformis (10 strains), Bacillus coagulans (15 strains), Bacillus macerans (5 strains), and Bacillus subtilis (5 strains). The hydrolases of these strains were examined with the API ZYM system, and the ability of these strains to produce acid in milk incubated at different temperatures was also examined. Esterase, esterase lipase, lipase, valine aminopeptidase, phosphoamidase, β-glucuronidase, and β-glucosidase were the activities found in all strains tested; they were strain-dependent and ranged between 1 and 5 by the API ZYM test. The same strains produced various concentrations of acid in milk. These organisms may be responsible for the flat sour spoilage in evaporated milk.

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.

Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi.

An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae. Similar enzyme profiles were obtained for two of the N. rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates. Centroid-clustering analysis revealed three distinct enzyme profiles.

Isolationin vitro ofStrongwellsea castrans [Fungi: Entomophthorales] a pathogen of adult cabbage root flies,Delia radicum [Dipt.: Anthomyiidae

The entomogenous fungusStrongwellsea castrans was isolatedin vitro for the first time, by incubating conidia projected from infected cabbage root flies (Delia radicum) in a simple, semi-defined liquid medium comprising dextrose, yeast extract and lactalbumin hydrolysate buffered to pH 7. The fungus grew as long unitunicate hyphae. After transfer to a solid nutrient medium, multinucleate hyphal bodies were formed which developed a thick, laminated wall. Neither conidia nor resting spores developed in liquid or on solid media and the fungus survived successive sub-culturing only in liquid media. Using the API-ZYM system, tests on extracts on hyphae ofS. castrans were positive for 11 enzymes but there were no consistent differences in enzyme profiles betweenS. castrans and fungi of the related genusErynia.

The API ZYM system as a tool for typing of Pasteureila multocida strains from humans

33 human clinical isolates of Pasteurella multocida were typed with the API ZYM enzymatic activity test kit. All isolates showed in repeat tests the presence of alkaline phosphatase, acid phosphatase, phosphoamidase, esterase (C 4), esterase lipase (C 8), and leucine arylamidase. 14 isolates exhibited additional enzyme activities, and five different reaction patterns could be recognized among the 33 isolates. The results of these enzyme activity tests are reproducible if parameters such as isolation medium, density of inoculum, and incubation time of the test strips are standardized.

Simple test for differentiation between fresh pork and frozen/thawed pork

Enzyme profile of the exudates from fresh and frozen/thawed pork has been assayed by using a semi-quantitative method (APIZYM). Only three enzymes (esterase-lipase, ß-glucuronidase and α-glucosidase) showed significant differences ( P < 0·01) between fresh and frozen/thawed pork. Different storage conditions were tested (freezing temperature: −10 to −60°C for 13 days and time of storage: 11 to 54 days at −18°C) but no significant differences were found among them. The APIZYM system would constitute a simple method for detecting frozen/thawed pork.

Differentiation of Penicillium griseofulvum Dierckx isolates by enzyme assays and by patulin and griseofulvin analyses

The production of patulin and griseofulvin by 49 different isolates of Penicillium griseofulvum Dierckx was analyzed by high-performance liquid chromatography. Eleven isolates were obtained from pistachio nuts, 37 were obtained from wheat seeds, and 1 was obtained from the American Type Culture Collection. Activities of 19 enzymes were also assayed by the API ZYM system. From these results it may be deduced that there are two different groups among the strains tested which cannot be distinguished by morphological and cultural characteristics. One group of isolates did not produce detectable amounts of patulin and griseofulvin when grown in sucrose-yeast extract and Wickerham media, while enzymatic activities were quantitatively distinct from the other group, which produced patulin and griseofulvin in variable proportions. Leucine arylamidase, phosphoamidase, and beta-D-glucosidase are the main enzymes with differing activities between the two groups. Differences in physiological characteristics among isolates of a single species reveal shortcomings in the classification of the penicillia based only on morphological criteria. Thus, determination of the ability to yield mycotoxins and antibiotics as well as determination of enzymatic activities appear to be very valuable tools in the taxonomy of these fungi and for food toxicology.

Evaluation of the API-ZYM system for identification of Listeria

The API-ZYM system (Analytab Products) was evaluated for potential use for confirming the identity of Listeria isolates. A total of 68 Listeria strains from various sources were assayed using standard procedures. All isolates were positive for esterase (C4), esterase lipase (C8), phosphohydrolase (one exception), β-glucosidase, acid phosphatase and lipase (one exception). Negative responses were observed consistently for valine aminopeptidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-fucosidase and cystine aminopeptidase (one exception). Variable responses were noted for alkaline phosphatase, leucine aminopeptidase, chymotrypsin, α-glulucosidase and N-acetyl glucosaminidase. Comparison of these results with those of other Gram positive species indicated that the API-ZYM system could be used for rapid confirmation of Listeria to the subgenus level.

Enzymatic characterization of Nocardia spp. and related bacteria by API ZYM profile

Characterization of 62 isolates belonging to the genus Nocardia and related bacteria was obtained by using the API ZYM system. The difference in enzymatic profile should allow a relatively efficient, low-cost means to identify aerobic actinomycetes of clinical significance.

Characterization of Enzyme Profiles of Lactobacillus casei Species by a Rapid API ZYM System

Lactobacilli have gained much attention recently due to their strong peptidase and esterase activities, which are of great importance in the accelerated maturation and enzyme modification of cheese. Twenty strains of Lactobacillus casei (ssp. casei, ssp. rhamnosus, and ssp. pseudoplantarum) were compared for their enzyme profiles. Specificity and specific activity studies were carried out on 19 hydrolytic enzymes for each strain using the microenzyme API ZYM system. Typical Lactobacillus casei enzyme profile indicated the presence of weak proteinase and high peptidase and esterase-lipase activities. The API ZYM method has been modified to evaluate and compare closely related Lactobacillus casei strains and also to identify the location of their enzymes (extracellular, cytoplasmic, cell wall, and membrane bound). The peptidase and most of the esterases and carbohydrases were intracellular, but the phosphatases were mostly cell wall and membrane associated. The API ZYM method offers a simple and convenient way for quick assessment of the hydrolytic enzymes of starter strains for both the industrial and research purposes.

Growth of Bifidobacteria and Their Enzyme Profiles

Bifidobacteria of human origin (22 strains) were evaluated for their potential use in yogurt-type products. Strains were grouped according to their growth rate in 12% reconstituted skim milk. Among the strains that grew quickly and produced acid in milk rapidly, the ratio of acetic: lactic acid varied from .4:1.0 to 3.0:1.0 Enzyme profiles of all strains were determined with the API ZYM system. All bifidobacteria possessed α- and β-galactosidases and α-glucosidase activities. A harmful enzyme, β-glucuronidase, was detected in only one strain of bifidobacteria tested. The faster growing strains also possessed high leucine aminopeptidase activity.

Comparison of Zymograms of Some Insect Cell Lines by Means of APIZYM System

Comparison of Zymograms of Some Insect Cell Lines by Means of APIZYM System

Enzymatic profiles of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillae

The enzymatic activities of 39 strains of Erysipelothrix rhusiopathiae and 34 of E tonsillae were determined with the API ZYM system. The profiles of these two species were very similar, differing solely in N-acetyl-beta-glucosaminidase activity. Whereas 90 per cent of strains of E rhusiopathiae exhibited strong activity with N-acetyl-beta-glucosaminidase, positive reactions were observed for this enzyme in only 24 per cent of strains of E tonsillae. These results support previous DNA-DNA hybridisation studies and suggest that E tonsillae is a new species of the genus Erysipelothrix.