Purification of Peptidases of Aspergillus oryzae and Some Properties of the Purified Peptidases

The purification of Aspergillus oryzae peptidases was attempted by the fractional precipitation with acetone, ammonium sulphate, and by starch zone electrophoresis. We, thus, achieved a great success in the separation of dipeptidase free from aminopolypeptidase and proteinase as well as in the separation of aminopolypeptidase free from dipeptidase and proteinase.

The specific activity (C₀) of the former (leucylglycine hydrolysis) was 7000 and that of the latter (leucylglycylglycine hydrolysis) 22000.

The leucylglycine dipeptidase was remarkably activated by Zn⁺⁺, and Co⁺⁺. Some other enzyme properties were also found and are discussed.     

Studies on Peptidases of Aspergillus oryzae

The homogeneity of Aspergillus dipeptidase prepared according to the standard method established by us was ascertained by ultracentrifugation and some characteristic properties of the enzyme was further investigated.

Hydrolysis of various dipeptides by the purified dipetidase was tested in the presence of divalent metal ions such as Co⁺⁺ or Zn⁺⁺, and the characteristics of greatest interest may be enumerated as follows:

1. The homogeneous dipeptidase requires Zn⁺⁺ for activation in the case of the hydrolysis of leucylglycine, leucylalanine leucylleucine, etc.

2. The homogeneous dipeptidase requires Co⁺⁺ for activation in the case of the hydrolysis of glycylleucine, glycylleucine, glycylglycine, glycylphenylalanine, etc.

3. In the case of the hydrolysis of alanylglycine, alanylleucine, valylglycine, etc., this enzyme does not require any metal ions.

     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Production and Some Properties of a New Type of Acid Carboxypeptidase of Penicillium Molds

Among some 38 strains of the genus Penicillium we investigated seven wild-type strains (P. daleae IFO-6087, P. frequentans AHU-8328, P. funiculosum IAM-7013, P. janthinellum IFO-8070, IAM-7026, P. lividum IAM-7200, and P. oxalicum AHU-8336) that were found to be excellent strains for a new type of acid carboxypeptidase production in a surface koji culture at 25 C. The production of acid carboxypeptidase was determined in various culture conditions in a koji culture. The maximum yields of acid carboxypeptidase were obtained by P. janthinellum IFO-8070. Partial purification and isolation of the acid carboxypeptidase from strains of Penicillium were performed with gel filtration on Bio-Gel P-100. Characterization studies indicate that the acid carboxypeptidases from P. daleae IFO-6087, P. funiculosum IAM-7013, P. janthinellum IFO-8070, and P. oxalicum AHU-8336 have some properties similar to those of the enzyme of Aspergillus saitoi with regard to the hydrolysis of several peptides at acidic pH range but have other slightly different properties with regard to stability, pH optima, inhibitors, and molecular weights.     

Purification and properties of dipeptidase from Escherichia coli AJ005

Summary A Co²⁺-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10^–3 M KCN and completely by 10^–3 M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45°C and 50°C, respectively. The apparent Km value was 6.7 × 10^–4 M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.Abbreviations EDTA Ethylenediaminetetraacetate - PCMB pchloromercuribenzoate     

Isolation of Myosin Light Chain Kinase Inhibitors from Microorganisms: Dehydroaltenusin,Altenusin, Atrovenetinone,and Cyclooctasulfur

Dehydroaltenusin, cyciooctasulfur, atrovenetinone, and altenusin were isolated from the culture broths of Penicillium verruculosum IAM-13756, Streptomyces verticil/us subsp. tsukushiensis ATCC-21633, Penicillium sp. SPC-16375, and Penicillium sp. SPC-16524, respectively, as new myosin light chain kinase (MLCK) inhibitors. These compounds inhibited the calmodulin-dependent activity of MLCK with IC₅₀ values of 0.69, 0.86, 3.7, and 340 μM, respectively. Among them, dehydroaltenusin was the best MLCK inhibitor in terms of potency and selectivity examined in the purified enzyme systems.     

Isolation,technological characterization and in vitro probiotic evaluation of Lactococcus strains from traditional Turkish skin bag Tulum cheeses

Purpose

The present study was undertaken to evaluate in vitro prerequisite probiotic and technological characteristics of ten Lactococcus strains isolated from traditional goat skin bags of Tulum cheeses from the Central Taurus mountain range in Turkey.

Methods

All isolates were identified based on the nucleotide sequences of the 16S rRNA gene. Eight isolates belonged to Lactococcus lactis and two belonged to Lactococcus garvieae. Probiotic potential was determined from resistance to acid and bile salt, resistance to gastric and pancreatic juices, resistance to antibiotic, auto-aggregation, co-aggregation, diacetyl, hydrogen peroxide and exopolysaccharide productions. Technological properties were verified by alcohol, NaCl and hydrogen peroxide resistance and temperature tests.

Results

L. lactis NTH7 displayed high growth at all alcohol concentrations while L. lactis NTH4 grew very well even at NaCl concentrations of 10%. All strains showed to some extent resistance to acid and bile. Five strains exhibited desirable survival in gastric juice (pH 2.0), while three strains survived in pancreatic juice (pH 8.0). All Lactococcus isolates were sensitive to ampicillin, chloramphenicol, erythromycin, vancomycin, kanamycin, gentamycin and tetracycline. Also, only L. lactis NTH7 from among the isolates showed resistance against penicillin. L. lactis NTH10 and L. lactis NTH7 had higher auto-aggregation values in comparison with all other strains. All the strains demonstrated a co-aggregation ability against model food pathogens, particularly, L. lactis NTH10 which showed a superior ability with L. monocytogenes. All the ten strains produced H₂O₂ and exopolysaccharide (EPS); however, diacetyl production was detected for only four strains including L. lactis NTH10.

Conclusion

These results demonstrate that the L. lactis NTH10 isolate could be regarded as a favorable probiotic candidate for future in vivo studies.

     

Identification and probiotic properties of lactobacilli isolated from two different fermented beverages

Purpose

Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals.

Methods

Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential.

Results

Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract.

Conclusion

Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

     

Production of Nucleic Acid-Related Substances by Fermentative Processes

1. Suitable agar plate media were selected for isolation of nucleotide producing strains, by salvage synthesis, from natural sources. Since this agar medium contains a high concentration of phosphates, manganese and glucose, it is specific for these bacteria.

2. With this plate medium, 113 bacterial strains accumulating 5′inosinic acid (IMP) or IMP-like substances were isolated effectively from feces of a variety of birds and mammals and from soils.

Some of the strains isolated were recognized to accumulate other nucleotides, purine bases and sugars, such as guanine nucleotides, XMP, xanthine, ribulose or xylnlose, with or without hypoxanthine in the media.

3. Five strains of IMP accumulating bacteria were identified; two were classified as Brevibacteriurm, two as Corynebacterium and one as Arthrobacterium species by taxonomical studies. But their characteristics did not completely coincide with those of bacteria described in Bergey’s manual.

4. One of the IMP producing bacteria isolated, culture No. 21–26, actually consisted of two separate strains, namely No. 21–26–101 and No. 21–26–102. The highest production of IMP or guanine nucleotides was obtained, when each strain was inoculated together to the fermentation medium from each seed culture in the same inoculum size.

5. The nucleotide productions by No. 21–26–101 or No. 21–26–102 with authentic strains were examined by the mixed culture technique. It was found that production of IMP or guanine nucleotides by Brevibacterium ammoniagenes ATCC 6871 was stimulated remarkably in the presence of No. 21–26–102.     

Studies on the Utilization of Hydrocarbons by Microorganisms

During the course of an investigation of the microbial assimilation of aromatic hydrocarbons, several strains were found to produce a large amount of cumic acid from p-cymene.

Five strains, S449B1, B2, B3, B4 and B6, were isolated from soil with the aromatic hydrocarbon substrates. They all assimilated both p-cymene and cumene. The strain S449B3 grew also on p-xylene, and S449B6 on p-xylene, toluene, and ethylbenzene.

They were all shown to be capable of producing an ultraviolet-absorbing substance from p-cymene. This substance was isolated in crystalline form and identified as cumic acid by infrared absorption spectrum and other observations.

The superior strain, S449B6, produced the acid as much as 1000 mg/1 in shaking culture at 30°C after 24 hours. The yields were increased up to approximately 1700 mg/1 after further investigations. Addition of calcium carbonate and considerable agitation were favorable conditions for the acid production.

The taxonomical studies of these strains were carried out, and they were all identified as closely resembling Pseudomonas desmolytica.     

Plant growth enhancement is not a conserved feature in the Caulobacter genus

Aims

Species within the Caulobacter genus have been termed ‘hub species’ in the plant microbiome. To understand these interactions, we assessed the interactions between several Caulobacter strains and a common host plant.

Methods

We identified a set of 11 Caulobacter strains that range in genetic diversity and tested them for their ability to increase the growth of Arabidopsis thaliana. In addition, biochemical assays were employed to determine if these Caulobacter strains produce common plant growth promoting (PGP) biosynthates. To identify potential PGP-related genes, genomic analyses were performed to compare the genomes of PGP Caulobacter strains to those of non-PGP Caulobacter strains.

Results

For the PGP Caulobacter strains, we observed that common PGP biosynthates did not contribute to the observed Caulobacter-mediated plant growth stimulation. Genomic analyses suggested that the genomes of PGP strains maintain similar metabolic pathways compared to those of non-PGP strains, and that common genes related to PGP factors do not explain the PGP mechanisms for the Caulobacter strains we analyzed.

Conclusions

Plant growth enhancement is not a conserved feature in the Caulobacter genus, and some Caulobacter strains even inhibit plant growth. Moreover, common PGP factors do not fully explain Caulobacter-mediated plant growth enhancement.

     

Microbiological,biochemical, physicochemical surface properties and biofilm forming ability of Brettanomyces bruxellensis

Purpose

Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species.

Methods

The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis.

Result

Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.

Conclusion

The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.

     

Purification and Characterization of Dipeptidase Hydrolyzing L-Cysteinylglycine from Radish Cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Safety and probiotic functionality of isolated goat milk lactic acid bacteria

Purpose

Lactic acid bacteria (LAB) are traditionally employed in the food industry. LAB strains from goat milk may also present probiotic potential, and it is fundamental to study the safety and functionality aspects which are desirable for their use in food. The objective of this study was to verify the probiotic potential of lactic bacteria isolated from goat milk.

Methods

The presence of safety-related virulence factors (hemolytic activity, gelatinase production, coagulase, and sensitivity to antibiotics) as well as functionality (exopolysaccharide (EPS) production, proteolytic activity, autoaggregation, gas production, survival in the gastrointestinal tract, and antimicrobial activity against bacteria that impair oral health) were determined.

Result

The selected LAB strains are safe against the evaluated parameters and have characteristics of possible probiotic candidates. Especially L. plantarum (DF60Mi) and Lactococcus lactis (DF04Mi) have potential to be added to foods because they have better resistance to simulated gastrointestinal conditions. In addition, they are isolated with already proven antimicrobial activity against Listeria monocytogenes, an important food-borne pathogen. DF60Mi was able to produce EPS (exopolysaccharides). LS2 and DF4Mi strains, both Lactococcus lactis subsp. lactis, demonstrated antimicrobial activity against S. mutans ATCC 25175, a recurrent microorganism in oral pathologies, mainly caries.

Conclusion

This study provides subsidies for future exploration of the potentialities of these LAB strains for both the development of new functional foods and for application in oral health.

     

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn “catalytic triad.” Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp “catalytic triad.” We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a β-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.     

Isolation and Determination of Yeasts Utilizing Kerosene as a Sole Source of Carbon

In order to produce microbial cell substances from petroleum, 83 strains of kerosene-utilizing yeasts, as a sole source of carbon, were isolated from 37 materials in contact with petroleum in the petroleum refinery. They could be distributed in either of 15 cultural groups with their colony appearances. Fifteen representative strains in 15 cultural groups were served for determination and identified with the following species: Candida tropicalis, 9 strains; C. guilliermondii, 2 strains; C. intermedia, 2 strains; C. pulcherrima, 1 strains; Torulopsis pinus, 1 strain.

In order to clarify what the ability of hydrocarbon utilization means biologically, 46 standard strains were served for test, of which the following 5 strains could utilize kerosene as a sole source of carbon: Candida albicans IAM 4888; C. arborea IAM 4147; C. lipolytica IAM 4947; C. tropicalis IAM 4862 and IAM 4924. Considering the result, the ability of utilizing kerosene would seem to characterize the genus, but it was not evident that it would characterize the species.

C. tropicalis Pk-233 gave the best cell yield among the above strains when kerosene was employed as a sole source of carbon and moreover, in the production of the cells of Pk-233, employing kerosene as a carbon material was compared with employing glucose.     

Investigation of the Physiological,Biochemical and Antifungal Susceptibility Properties of Candida auris

Background

Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences.

Objectives

To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus.

Methods

Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied.

Results

The optimal growth temperatures of all strains were 35–37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to?>?64 µg/mL) and miconazole (MIC 8 to?>?16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1–4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades.

Conclusion

Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

     

Utilization of Polyphenyl and Polyphenyl-related Compounds by Microorganisms. Part I

Two hundreds and fifty eight strains of microorganisms have been isolated from 526 samples (soil, leaf and river water gathered from 17 prefectures) by repeating liquid enrichment culture techniques in the medium containing biphenyl, diphenylmethane, diphenylethane or terphenyl, as the sole source of carbon.

In the course of investigation, several strains were found to produce a large amount of γ-benzoylbutyric acid from biphenyl. Furthermore these strains utilized p-Cl-biphenyl and produced p-Cl-benzoic acid in good yield.

Microorganisms obtained were almost short rod, motile bacteria, and fungi were also found from the screening medium of diphenylethane.     

Studies on Milk Clotting Enzymes Produced by Basidiomycetes

In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.

When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.

On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.

As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.

Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months’ ripening but that those produced by the enzyme of I. lacteus had good quality.     

Updating the Taxonomy of Dermatophytes of the BCCM/IHEM Collection According to the New Standard: A Phylogenetic Approach

Recent taxonomical revisions based on multilocus gene sequencing have provided some clarifications to dermatophyte (Arthrodermataceae) family tree. These changes promoted us to investigate the impact of the changed nomenclature of the dermatophyte strains in the BCCM/IHEM fungal collection, which contains strains of all dermatophyte genera except for Ctenomyces. For 688 strains from this collection, both internal transcribed spacer region (ITS) and partial β-tubulin (BT) sequences were aligned and a multilocus phylogenetic tree was constructed. The ITS?+?BT phylogentic tree was able to distinguish the genera Arthroderma, Lophophyton, Microsporum, Paraphyton, Nannizzia and Trichophyton with high certainty. Epidermophyton, which is widely considered as a well-defined genus with E. floccosum as the only representative, fell within the Nannizzia clade, whereas the phylogenetic analysis, based on the ITS region alone, differentiates Epidermophyton from Nannizzia as a separate genus. Re-identification and reclassification of many strains in the collection have had a profound impact on the composition of the BCCM/IHEM dermatophyte collection. The biggest change is the decline of prevalence of Arthroderma strains; starting with 103 strains, only 22 strains remain in the genus after reassessment. Most Arthroderma strains were reclassified into Trichophyton, with A. benhamiae and A. vanbreuseghemii leaving the genus. The amount of Microsporum strains also dropped significantly with most of these strains being reclassified into the genera Paraphyton and Nannizzia.