Isolation and Characterization of Thermophilic Bacteria from a Hot Spring in the State of Hidalgo, Mexico, and Geochemical Analysis of the Thermal Water.

Hot springs worldwide can be a source of extremophilic microorganisms of biotechnological interest. In this study, samplings of a hot spring in Hidalgo, Mexico, were conducted to isolate, identify, and characterize morphologically, biochemically, and molecularly those bacterial strains with potential industrial applications. In addition, a physicochemical and geochemical examination of the hot spring was conducted to fully understand the study region and its potential connection to the strains discovered. The hot spring was classified as sulfate-calcic according to the Piper Diagram; the hydrogeochemical analysis showed the possible interactions between minerals and water. Eighteen bacterial strains were isolated with optimal growth temperatures from 50 to 55 °C. All strains are Gram-positive, the majority having a rod shape, and one a round shape, and 17 produce endospores. Hydrolysis tests on cellulose, pectin, and xylan agar plates demonstrated enzymatic activity in some of the strains. Molecular identification through the 16S rDNA gene allowed classification of 17 strains within the Phylum Firmicutes and one within Deinococcus-Thermus. The bacterial strains were associated with the genera Anoxybacillus, Bacillus, Anerunibacillus, Paenibacillus, and Deinococcus, indicating a diversity of bacterial strains with potential industrial applications.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production.

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

Biooxidation of hydrogen sulfide to sulfur by moderate thermophilic acidophilic bacteria.

The copper industry utilizes significant amounts of sulfuric acid in its processes, generating sulfate as waste. While sulfate-reducing bacteria can remove sulfate, it produces hydrogen sulfide (H2S) as a byproduct. This study examined the capability of a consortium consisting of Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus to partially oxidize H2S to S° at a temperature of 45 °C. A fixed-bed bioreactor, with glass rings as support material and sodium thiosulfate as a model electron donor, was inoculated with the consortium. Formation of biofilms was crucial to maintain the bioreactor's steady state, despite high flow rates. Afterward, the electron donor was changed to H2S. When the bioreactor was operated continuously and with high aeration, H2S was fully oxidized to SO42-. However, under conditions of low aeration and at a concentration of 0.26 g/L of H2S, the consortium was able to oxidize H2S to S° with a 13% yield. S° was discovered attached to the glass rings and jarosite. The results indicate that the consortium could oxidize H2S to S° with a 13% yield under low aeration and at a concentration of 0.26 g/L of H2S. The findings highlight the capability of a Sulfobacillus consortium to convert H2S into S°, providing a potential solution for addressing environmental and safety issues associated with sulfate waste generated by the mining industry.

Bacterial diversity in water from Xifeng Hot Spring in China.

The Xifeng Hot Spring is one of the eight largest hot springs in China, which is rich in radon gas and sulphur in karst scenery. Little is known about the microbiota structure in the spring. The water was collected from three sites containing the outlet of spring water discharge site (OWD), spring pool for tourist (SPT) and sewage effluent pool (SEP) in the Xifeng Hot Spring and further analyzed by culture-independent technique and culture-dependent method. A total of 57 phyla were identified from the water samples. The dominate phyla at OWD was Bacteroidetes (46.93%), while it was Proteobacteria in both sites of SEP and SPT with relative richness of 61.9% and 94.9%, respectively. Two bacteria, Deinococcus and Hymenobacter, that confirmed to be radiation-resistant, seven sulphur bacteria and three thermophilic bacteria were detected from Xifeng Hot Spring. Furthermore, it was found that genus Flavobacterium was susceptible to environmental change with abundance of 11 ~ 2825 times higher in OWD than the other two groups. Compared bacteria from the OWD group with that from 14 hot springs in six countries, total 94 unique genera bacteria were found out from the Xifeng Hot Spring including four thiometabolism-related bacteria (Propionispira, Desulforegula, Desulfobacter and Desulfococcus) and the thermophilic bacterium (Symbiobacterium). Using microbial culturing and isolation technology, sixteen strains were isolated from the water samples of three sites. The diversity of microbiota was abundant and variable along with the niche changed in conditions and surroundings. It indicated that numbers of valuable bacteria resources could be explored from the special surroundings of Xifeng Hot Spring.

Recombinant expression and characterization of a new laccase, bioinformatically identified, from the Antarctic thermophilic bacterium Geobacillus sp. ID17.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Physicochemical and bacterial changes during composting of vegetable and animal-derived agro-industrial wastes.

This study investigates the impact of different agro-industrial organic wastes (i.e., sugarcane filter cake, poultry litter, and chicken manure) on the bacterial community and their relationship with physicochemical attributes during composting. Integrative analysis was performed by combining high-throughput sequencing and environmental data to decipher changes in the waste microbiome. The results revealed that animal-derived compost stabilized more carbon and mineralized a more organic nitrogen than vegetable-derived compost. Composting enhanced bacterial diversity and turned the bacterial community structure similar among all wastes, reducing Firmicutes abundance in animal-derived wastes. Potential biomarkers indicating compost maturation were Proteobacteria and Bacteroidota phyla, Chryseolinea genus and Rhizobiales order. The waste source influenced the final physicochemical attributes, whereas composting enhanced the complexity of the microbial community in the order of poultry litter > filter cake > chicken manure. Therefore, composted wastes, mainly the animal-derived ones, seem to present more sustainable attributes for agricultural use, despite their losses of C, N, and S.

Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

Research trends and strategies for the improvement of anaerobic digestion of food waste in psychrophilic temperatures conditions.

The organic fraction of municipal solid waste is mainly composed of food waste (FW), and traditional disposal practices for this fraction are generally considered to have negative environmental and economic impacts. However, the organic characteristics of this fraction could also be exploited through the anaerobic digestion of FW (FW-AD), which represents unique advantages, including the reduction of the area required for final disposal and environmental pollution and the same time the generation of renewable energy (mainly methane gas), and a by-product for agricultural use (digestate) due to its high nutrient content. Although approximately 88% of the world's population resides in areas with temperatures below 8 °C, psychrophilic conditions (temperatures below 20 °C) have hardly been studied, while mesophilic (66%) and thermophilic (27%) ranges were found to be more common than psychrophilic FW-AD (7%). The latter condition could decrease microbial activity and organic matter removal, which could affect biogas production and even make AD unfeasible. To improve the efficiency of the psychrophilic FW-AD process, there are strategies such as: measurement of physical properties as particle size, rheological characteristics (viscosity, consistency index and substrate behavior index), density and humidity, bioaugmentation and co-digestion with other substrates, use of inocula with psychrophilic methanogenic communities, reactor heating and modification of reactor configurations. However, these variables have hardly been studied in the context of psychrophilic conditions and future research should focus on evaluating the influence of these variables on FW-AD under psychrophilic conditions. Through a bibliometric analysis, this paper has described and analyzed the FW-AD process, with a focus on the psychrophilic conditions (<20 °C) so as to identify advances and future research trends, as well as determine strategies toward improving the anaerobic process under low temperature conditions.

Virulence factors of foodborne pathogen Campylobacterjejuni.

Campylobacter jejuni is a highly frequent cause of gastrointestinal foodborne disease in humans throughout the world. Disease outcomes vary from mild to severe diarrhea, and in rare cases the Guillain-Barré syndrome or reactive arthritis can develop as a post-infection complication. Transmission to humans usually occurs via the consumption of a range of foods, especially those associated with the consumption of raw or undercooked poultry meat, unpasteurized milk, and water-based environmental sources. When associated to food or water ingestion, the C. jejuni enters the human host intestine via the oral route and colonizes the distal ileum and colon. When it adheres and colonizes the intestinal cell surfaces, the C. jejuni is expected to express several putative virulence factors, which cause damage to the intestine either directly, by cell invasion and/or production of toxin(s), or indirectly, by triggering inflammatory responses. This review article highlights various C. jejuni characteristics - such as motility and chemotaxis - that contribute to the biological fitness of the pathogen, as well as factors involved in human host cell adhesion and invasion, and their potential role in the development of the disease. We have analyzed and critically discussed nearly 180 scientific articles covering the latest improvements in the field.

One waste and two products: choosing the best operational temperature and hydraulic retention time to recover hydrogen or 1,3-propanediol from glycerol fermentation.

This study aimed to compare the production of hydrogen and 1,3-propanediol from crude glycerol (10 g/L) in mesophilic (30 °C) and thermophilic (55 °C) anaerobic fluidized bed reactors, namely AFBR30 °C and AFBR55 °C, respectively, at hydraulic retention times (HRT) reduced from 8 to 1 h. In AFBR30 °C, the absence or low hydrogen yields can be attributed to the production of 1,3-propanediol (maximum of 651 mmol/mol glycerol), and the formation of caproic acid (maximum of 1097 mg/L) at HRTs between 8 and 2 h. In AFBR55 °C, the hydrogen yield of 1.20 mol H2/mol glycerol consumed was observed at the HRT of 1 h. The maximum yield of 1,3-propanediol in AFBR55 °C was equal to 804 mmol/mol glycerol at the HRT of 6 h and was concomitant with the production of hydrogen (0.87 mol H2/mol glycerol consumed) and butyric acid (1447 mg/L).

Increased Malbranchea pulchella ß-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs.

ß-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, ß-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2 ) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the ß-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy.

Composting of swine production chain wastes with addition of crude glycerin: organic matter degradation kinetics, functional groups, and carboxylic acids.

Little is known about the effect of adding crude glycerin (CG) as a carbon source during the composting of agro-industrial residues, such as those generated in the swine production chain, especially concerning the impact on organic matter humification. Therefore, the aim of this work was to study the effect of adding crude glycerin during the composting of organic swine waste, using appropriate analyses to determine the degree of maturation of the organic material. The experiment was performed using composters constructed from pallets. The variables considered were temperature, mass, volume, organic matter, functional groups, carboxylic acids, pH, electrical conductivity, total organic carbon, total Kjeldahl nitrogen, total phosphorus, potassium, basal respiration, and germination index. For all the CG concentrations tested, thermophilic temperatures were reached, while higher amounts of CG (4.5 and 6.0%) maintained temperatures above 55 °C for longer periods (28 days). Fourier transform infrared spectroscopy analysis showed the presence of an aromatic stretching vibration signal at 1620 cm-1, confirming mineralization of the organic matter, while the decrease of carboxylic acids at the end of the composting period indicated stabilization. The organic composts presented high nutrient contents and absence of toxicity, indicating that they could be safely used in agriculture.

Statistical optimization of cellulases by Talaromyces thermophilus utilizing Saccharum spontaneum, a novel substrate

BACKGROUND: At present, cellulases are the most important enzymes worldwide, and their demand has been increasing in the industrial sector owing to their notable hydrolysis capability. RESULTS: In the present study, contrary to conventional techniques, three physical parameters were statistically optimized for the production of cellulase by thermophilic fungi by using response surface methodology (RSM). Among all the tested thermophilic strains, the best cellulase producing fungus was identified as Talaromyces thermophilus ­ both morphologically and molecularly through 5.8S/ITS rDNA sequencing. The central composite design (CCD) was used to evaluate the interactive effect of the significant factors. The CCD was applied by considering incubation period, pH, and temperature as the model factors for the present investigation. A second-order quadratic model and response surface method revealed that the independent variables including pH 6, temperature 50 C, and incubation period 72 h significantly influenced the production of cellulases. The analysis of variance (ANOVA) indicated that the established model was significant (P 0.05) and showed the high adequacy of the model. The actual and predicted values of CMCase and FPase activity showed good agreement with each other and also confirmed the validity of the designed model. CONCLUSIONS: We believe the present findings to be the first report on cellulase production by exploiting Kans grass (Saccharum spontaneum) as a substrate through response surface methodology by using thermophilic fungus, Talaromyces thermophilus.

Biofilm-forming ability of poultry Campylobacter jejuni strains in the presence and absence of Pseudomonas aeruginosa.

The aims of this study were to evaluate the ability of Campylobacter jejuni isolated from a poultry slaughterhouse to form biofilm in the presence and absence of Pseudomonas aeruginosa, and the effect of surface (stainless steel, polystyrene), temperature (7, 25, and 42 °C), and oxygen concentration (microaerophilic and aerobic conditions) on the formation of biofilm. The genes ahpC, cadF, clpP, dnaJ, docA, flaA, flaB, katA, kpsM, luxS, racR, and sodB, related to biofilm formation by C. jejuni, were also investigated. All isolates formed biofilm on stainless steel and on polystyrene, in both aerobic and microaerophilic atmospheres, including temperatures not optimal for C. jejuni growth (7 and 25 °C), and biofilm also was formed in the presence of P. aeruginosa. In dual-species biofilm on stainless steel, biofilm formation was 2-6 log CFU·cm-2 higher at 7 °C for all isolates, in comparison with monospecies biofilm. Ten genes (ahpC, cadF, clpP, dnaJ, docA, flaA, flaB, luxS, racR, and sodB) were detected in all isolates, but katA and kpsM were found in four and six isolates, respectively. The results obtained are of concern because the poultry C. jejuni isolates form biofilm in different conditions, which is enhanced in the presence of other biofilm formers, such as P. aeruginosa.

Thermophilic anaerobic digestion of winery effluents in a two-stage process and the effect of the feeding frequency on methane production.

This investigation evaluates a two-stage process to treat highly concentrated winery effluents, including a thermophilic methanogenic stage. The inoculum adaptation, the effect of the organic loading rates on both stages, and the methanogenic reactor's feeding frequency on the process performance were studied. An active thermophilic inoculum was obtained by a one-step temperature increase from 35 to 55 °C. The application of organic loads above 120 kg COD m-3 d-1 in the acidogenic stage ensured the highest acetic acid concentration, while methane production rates as high as 7.1 Nm3 CH4 m-3 d-1 and a yield of 348 L CH4 kg-1 COD were obtained in the thermophilic methanogenic stage using an organic loading rate of 29.9 kg COD m-3 d-1. However, a lower removal of organic matter was observed under that condition. Lower feeding frequencies improved methane productivity and yield, suggesting that this parameter is a useful process optimization tool.

Comparison of antimicrobial resistance in thermophilic Campylobacter strains isolated from conventional production and backyard poultry flocks.

1. The aim of this study was to compare the resistance pattern of thermophilic Campylobacter spp. isolated from conventional production (n = 34) and backyard poultry flocks (n = 36) from Rio de Janeiro State, Brazil. The disc diffusion method and statistical tests were used for investigation and analysis of the resistance pattern of Campylobacter spp. isolated from different rearing systems.2. Antimicrobial resistance percentages to amoxycillin with clavulanic acid (AMC), ampicillin (AMP), ceftiofur (CTF), ciprofloxacin (CIP), enrofloxacin (ENO), erythromycin (ERI), gentamicin (GEN) and tetracycline (TET) were 32.4%, 44.1%, 67.6%, 97.1%, 82.4%, 26.5%, 5.9% and 38.2% in conventional production flocks respectively, while the backyard flock's resistance levels were 0.0%, 13.9%, 69.4%, 100.0%, 91.7%, 5.6%, 0.0% and 16.7%, respectively.3. Campylobacter spp. from conventional poultry production was more resistant to AMC, AMO, ERI and TET (P > 0.05) when compared to strains from backyard poultry. A higher frequency of resistance to fluoroquinolones (FLQ), CIP and ENO, was observed in strains from both systems, demonstrating the spread of resistant strains among poultry production environments.

Comparison of Single-Step Methods to Enrich Lipase Concentrations in Bacterial Cell Lysates

Abstract Lipases are currently used in food technology for the modification of fats and oils. The thermal stability of lipase is an essential characteristic for this application. This study compares four individual single-step methods (heat treatment, ethanol precipitation, ammonium sulfate precipitation, and size-exclusion chromatography) to enrich lipase concentrations from thermophilic bacterial (Geobacillius stearothermophilus and Anoxybacillus flavithermus) cell lysates. SDS-PAGE and size exclusion chromatography were used to determine the molecular weights of the lipases and the enrichment efficiencies were determined using specific enzyme activities. The molecular weight of G. stearothermophilus lipase was approximately 42 kDa, and approximately 33 kDa for A. flavithermus lipase. For each organism, ethanol precipitation resulted in the highest enrichment fold, followed by ammonium sulfate precipitation, gel filtration and heat treatment respectively. The highest yields for G. stearothermophilus lipase were obtained with ammonium sulfate precipitation, followed by get filtration, and ethanol precipitation respectively. The highest yields for A. flavithermus lipase were obtained from heat treatment followed by ammonium sulfate precipitation, gel filtration and ethanol precipitation respectively. Ethanol precipitation and heat treatment are simple methods for enzyme enrichment from cell lysates and can result in high enzyme yields with moderate enrichment folds compared to complex multi-step purification methods.

The Microbial Composition in Circumneutral Thermal Springs from Chignahuapan, Puebla, Mexico Reveals the Presence of Particular Sulfur-Oxidizing Bacterial and Viral Communities.

Terrestrial thermal springs are widely distributed globally, and these springs harbor a broad diversity of organisms of biotechnological interest. In Mexico, few studies exploring this kind of environment have been described. In this work, we explore the microbial community in Chignahuapan hot springs, which provides clues to understand these ecosystems' diversity. We assessed the diversity of the microorganism communities in a hot spring environment with a metagenomic shotgun approach. Besides identifying similarities and differences with other ecosystems, we achieved a systematic comparison against 11 metagenomic samples from diverse localities. The Chignahuapan hot springs show a particular prevalence of sulfur-oxidizing bacteria from the genera Rhodococcus, Thermomonas, Thiomonas, Acinetobacter, Sulfurovum, and Bacillus, highlighting those that are different from other recovered bacterial populations in circumneutral hot springs environments around the world. The co-occurrence analysis of the bacteria and viruses in these environments revealed that within the Rhodococcus, Thiomonas, Thermonas, and Bacillus genera, the Chignahuapan samples have specific species of bacteria with a particular abundance, such as Rhodococcus erytropholis. The viruses in the circumneutral hot springs present bacteriophages within the order Caudovirales (Siphoviridae, Myoviridae, and Podoviridae), but the family of Herelleviridae was the most abundant in Chignahuapan samples. Furthermore, viral auxiliary metabolic genes were identified, many of which contribute mainly to the metabolism of cofactors and vitamins as well as carbohydrate metabolism. Nevertheless, the viruses and bacteria present in the circumneutral environments contribute to the sulfur cycle. This work represents an exhaustive characterization of a community structure in samples collected from hot springs in Mexico and opens opportunities to identify organisms of biotechnological interest.

Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst.

Cladribine (2-chloro-2'-deoxy-ß-d-adenosine) is a 2'-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite. The immobilized biocatalyst (T. alba-Ac-Bent 1.00 %), was able to biosynthesize cladribine with a conversion of 89 % in 1 h of reaction and retains its activity for more than 270 reuses without significantly activity loss, showing better operational stability and mechanical properties than the natural matrix. A microscale assay using the developed system, could allow the production of at least 181 mg of cladribine in successive bioprocesses.

Stenamoeba dejonckheerei sp. nov., a Free-Living Amoeba Isolated from a Thermal Spring.

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species.