Clostridium Isolates Research Articles

The frequency of bacterial isolation of <i>Clostridium</i> spp. and their associations in various forms of clostridiosis in cattle

The article presents the results of isolation of Clostridium spp. bacteria and their associations from cattle with different clinical forms of clostridiosis. From 2016 to 2023, 910 samples of biomaterial collected from cows, heifers and calves under 6 months of age, as well as from stillborn calves and aborted fetuses in case of reproductive pathologies in cows were investigated by bacteriological methods. The following species play a major role in the etiology of bovine clostridiosis: C. perfringens, C. septicum, C. novyi (C. oedematiens), C. histolyticum and C. sordellii. C. histolyticum (65.2%), C. septicum (45.6), C. perfringens (29.7) and C. sporogenes (26.1) were isolated most frequently from cows and heifers, while C. sordellii (1.7) and C. novyi (0.9%) were isolated less frequently. Clostridium spp. bacteria of six species were isolated from animals in enterotoxemia and enteritis, and five species in vaginitis and endometritis. In other clinical forms of the disease, bacteria of four species were isolated from cows and heifers. The spectrum of bacteria involved in the development of abomasitis, enteritis and enterotoxemia in calves was similar and represented by four bacterial species: C. histolyticum, C. septicum, C. perfringens and C. sporogenes. Bacteria of five species were isolated from the calves with septicaemic form of the disease. The peculiarities of clinical manifestation and the course of clostridiosis in cattle depended on the species composition of pathogens and their associations. Diseases caused by Clostridium spp. bacteria were acute or subacute, characterized by visible lesions of organs and tissues in a few hours after the animal showed signs of disease and in most cases ended in death.

Development of real-time PCR assay for quantitative detection of Clostridium septicum

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.

Evaluation of bacterial load and Multi-drug Antibacterial resistant Salmonella and Clostridium isolates from contaminated tools used in butcheries around Ibadan

Evaluation of bacterial load and Multi-drug Antibacterial resistant Salmonella and Clostridium isolates from contaminated tools used in butcheries around Ibadan

Antimicrobial susceptibility testing of anaerobic bacteria causing bacteremia: A 13-year (2010–2022) retrospective study in a tertiary hospital

Infections from anaerobic microorganisms result from breached mucosal barriers, posing a significant mortality risk. A retrospective study at Hospital Universitario La Paz (Madrid) from 2010 to 2022 analyzed 491 (6.17 %) anaerobic bacteremia cases out of 7956 significant bacteremia cases among 171,833 blood culture requests. Bacteroides fragilis was the most frequently isolated species (28.3 %), followed by Clostridium perfringens (13.6 %). B. fragilis showed good susceptibility to amoxicillin/ clavulanic acid (86 %), piperacillin/tazobactam (86 %), and metronidazole (87.7 %). In general, non-fragilis Bacteroides species showed low susceptibility to penicillin (7 %), amoxicillin (17.5 %), and clindamycin (64.9 %). Of our 13 non-perfringens Clostridium isolates, four exhibited resistance to penicillin and four showed resistance to clindamycin. Lactobacillus species were highly susceptible to antibiotics tested. Prevotella spp. showed low susceptibility to penicillin (20 %), amoxicillin (20 %), and clindamycin (40 %). The study contributes valuable data for monitoring and improving anaerobic bacteremia treatment.

Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses.Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g.In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene.Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

BIOLOGICAL PROPERTIES OF CL. PERFRINGENS STRAINS

the biological properties of isolated clostridium were studied, clostridium isolates were identified by cultural morphological properties.

Study of the inhibitory effect of Lactobacillus strains and lysozyme on growth of Clostridium spp. responsible for cheese late blowing defect

Three strains of Lactobacillus were previously isolated from dairy products, Lactobacillus casei 26, Lactobacillus delbrueckii subsp. bulgaricus 76 and L. casei 95. Anti-costridial activities of these strains were evaluated against a collection of Clostridium isolates, belonging to species responsible for late blowing defect in cheese. The three strains of Lactobacillus inhibited the growth of all Clostridium isolates analyzed. The inhibitory effect of cell-free supernatant of each of these Lactobacillus strains combined with that of commercial strain L. casei BAL C, or with a solution of lysozyme, were compared by disc diffusion assay. Mixtures of cell-free Lactobacillus supernatants and lysozyme exhibited higher inhibitory activity than the supernatants and lysozyme solution separately (P &lt; 0.05). Additionally, Lactobacillus strains were resistant to lysozyme concentrations usually used during cheese making process.

Determination of Type and Molecular Identity of Clostridium perfringens Isolated from Patients with Multiple Sclerosis (MS)

Background &amp; Objectives: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Clostridium perfringens ε-toxin (ETX) can cause emerging complications of MS due to its tendency to the blood-brain barrier. This study aimed to determine the presence of toxin-producing genes in intestinal C.perfringens in patients with multiple sclerosis.&#x0D; Materials &amp; Methods: Stool samples were taken from 70 MS patients and 70 without MS individuals. The samples were enriched in cooked meat media, and cultural and biochemical methods separated Clostridium isolates. PCR tested these isolates to identify C. perfringens species. In addition, the presence of alpha, beta, epsilon and iota toxin-producing genes was evaluated in all isolates by Multiplex PCR.&#x0D; Results: Isolates containing the etx gene were observed in 10 patients, while no itxA gene was identified in any isolates. The results showed that isolates in 8 patients were type D. Also, the gene encoding toxin type D was identified in 2 isolates obtained from the control group.&#x0D; Conclusion: Our findings indicated the high frequency of C. Perfringens in MS patients. In the studied samples with clinical presentations, most of these organisms were type D bacteria that produce ε-toxin.

A Family Outbreak of Type E Botulism Caused by Contaminated Vacuum-Packed Ambient-Stored Chili Chicken Feet in Zhangjiakou, China.

The epidemiological investigation and laboratory-based confirmation were performed on samples from a family botulism outbreak in Zhangjiakou, Hebei province, China. Forty-four samples, including 14 samples (leftover food, and swabs taken of both food packaging bags and dishes, and serum and vomitus of the victims) related to outbreak and 30 causative food products after outbreak, were collected and analyzed. Isolation, bacterial identification, toxin detection, and whole-genome sequencing of Clostridium spp. cultured from the latter samples and animal assays were performed. Mice injected with the cultures of the leftover chili chicken feet, together with the inner layer of its packaging bag, the plate for serving it, and supernatant of two patients' serum that demonstrated the typical signs of botulism. The polyvalent anti-botulinum neurotoxin (BoNT) and the monovalent anti-BoNT/E exhibited protective effects when administered to mice. Three Clostridium botulinum cultures were obtained and verified to be positive for BoNT/E. The whole genome analysis of the isolates revealed that the classic bont/e gene orfX cluster was found to be located on the chromosomes of all three isolates. Single nucleotide polymorphism analysis suggested that these might be from the same source. Our findings indicated that this botulism outbreak occurred following the ingestion of vacuum-packed chili chicken feet contaminated with BoNT/E produced by C. botulinum.

A simple method for the isolation of cold-tolerant Clostridium spp. from meat samples

Pure isolates of Clostridium spp. are required when further investigations are needed, such as the characterisation of the colonies, sporulation and germination, biochemical analysis, growth rate and growth conditions, toxin production, spoilage potential and genomic profile. However, the isolation of cold-tolerant Clostridium spp. from suspicious meat samples is challenging due to their slow growth rates and overgrowth with less fastidious meat microbiota. Therefore, this study aimed to establish a practical method for the isolation of psychrophilic and psychrotolerant Clostridium spp. Primarily, meat drip samples (n = 3), enriched in Peptone Yeast Glucose Starch (PYGS) broth, were heated with different temperatures and durations, before they were subcultured on Columbia blood agar (CBA). The treatment procedure of heating samples at 80 °C for 5 min was evaluated as effective and was further validated using pure cultures of lactic acid bacteria (n = 5), bacteria belonging to the family of Enterobacteriaceae (n = 5), and cold-tolerant Clostridium spp. (n = 34). Almost all other meat microbiota was inactivated by heating at 80 °C for 5 min, while 28 strains of cold-tolerant Clostridium spp. survived. Finally, the treatment procedure was applied with drip of meat samples (n = 41) previously tested positive for 49 cold-tolerant Clostridium spp. using specific multiplex qPCR. All meat drip samples were enriched in PYGS broth by incubating them at 4 °C for 3 weeks, to ensure growth of Clostridium spp. before the enrichments were proceeded to heat treatment and to culturing on CBA. A total of 35 (71 %) from 49 Clostridium spp. strains were isolated using this culturing procedure. The accuracy of the recovery rates obtained from two replicates was 68 %. The method of detection and isolation applied in this study is easy and resulted in a high isolation rate of cold-tolerant Clostridium spp.

Culture and genome-based analysis of four soil Clostridium isolates reveal their potential for antimicrobial production

BackgroundSoil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods.ResultsConditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes.ConclusionsThe presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.

111 Pathogenic Bacterial Levels in US Silages

Abstract Fresh forage crops have traditionally been preserved by fermentation, either based on the spontaneous fermentation by epiphytic lactic acid bacterial populations or driven by the addition of lactic acid bacteria inoculant cultures. Although this method of preservation is effective at retarding the spoilage and pathogenic bacteria it does not eliminate these organisms from the forage. Spore-forming and other bacteria are often able to survive the low pH and organic acid levels in the fermented forages and outgrow during feed-out and in the animal. The purpose of this research was to understand the levels and diversity of pathogens in silage systems as an important prerequisite to develop effective ways to control them. From February 2016 to December of 2019, 866 corn silage samples and 577 haylage samples were collected from 457 US dairy farms across 27 states to assess the pathogenic levels in silage. Results indicate the clostridia levels in the haylage samples averaged 9.4 x 103 cfu/g with a range of less than 10 cfu/g to 4.1 x 106 cfu/g. Corn silage had a significantly lower level of clostridia (p&amp;lt;0.05) averaging 2.3 x 103 cfu/g. Clostridium perfringens was the dominant species identified in both haylage and corn silage and accounted for more than 60% of all the clostridia isolates. The other major clostridia species identified were C. beijerinckii, Paraclostridium bifermentens and C. butyricum. E. coli levels were also determined in the haylage and corn silage samples. In haylage, the average E. coli level was 2.1 x 104 cfu/g and ranged from less than 10 cfu/g to 2.5 x 106 cfu/g and were significantly higher (p&amp;lt;0.05) than levels detected in corn silage (average of 5.4 x 101 cfu/g). Further work is underway to determine the source of these pathogens and methods of controlling through the fermentation process.

Comparative genomics of Clostridium species associated with vacuum-packed meat spoilage

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

Modulating microbiome metabolites in vivo

A new genetic tool enables mechanistic dissection of microbiome–host interactions

Epidemiological and genetic characterization of Clostridium butyricum cultured from neonatal cases of necrotizing enterocolitis in China.

Laboratory-based characterization and traceback of Clostridium butyricum isolates linked to outbreak cases of neonatal necrotizing enterocolitis (NEC) in a hospital in China. In total, 37 samples were collected during the NEC outbreak. Classical bacteriological methods were applied to isolate and identify Clostridium spp. Meanwhile, 24 samples collected after an outbreak were similarly tested. All Clostridium isolates were identified to species level as either C. butyricum or C. sporogenes. These isolates were subsequently subtyped using pulsed-field gel electrophoresis (PFGE). Genomic DNA was purified from 2 representative C. butyricum isolates and sequenced to completion. Of 37 samples collected during the NEC outbreak, 17 (45.95%) were positive for Clostridium spp. One species, C. butyricum, was cultured from 10 samples. Another species cultured from 2 other samples was identified as C. sporogenes. Both of these species were cocultured from 5 samples. Pulsotyping showed that the 15 C. butyricum and the 7 C. sporogenes isolates produced indistinguishable DNA profiles. No NEC cases were reported after disinfection following the outbreak, and all samples collected after the outbreak were negative for Clostridium spp. Whole-genome sequencing (WGS) indicated that sialidase, hemolysin, and enterotoxin virulence factors were located on the chromosomes of 2 C. butyricum isolates. The outbreak of NEC was epidemiologically linked to C. butyricum contamination within the hospital. This is the first report of an NEC outbreak associated with C. butyricum infection in China.

Bacteria producing antimicrobials against Clostridium difficile isolated from human stool

Clostridium difficile infection (CDI) is a common cause of morbidity and mortality in hospitalized patients worldwide. The major problem facing current treatment is multiple recurrences, prompting the need for alternative therapies. In this study we isolated bacterial species, from Egyptian individuals’ stool, with antimicrobial activity against clinical isolates of C. difficile and tried to examine the nature of the produced antimicrobials. In vitro antibacterial activity against C. difficile was initially screened in 123 fecal samples cultures using an agar overlay method. The isolates with antimicrobial activity against C. difficile in addition to Clostridium isolates were identified using partial 16S rDNA gene sequencing analysis. The isolates acting against C. difficile belonged to Lactobacillus, Enterococcus and Clostridium genera. The concentrated cell-free supernatants (CFSs) from these bacterial isolates were examined for antimicrobial activity against C. difficile growth by broth dilution method. 10 x concentrated CFSs of five isolates showed inhibition for C. difficile growth which was significantly different (p < 0.001) from control. Lactobacillus agilis T99A and Clostridium butyricum T58A isolates were selected for further evaluation of the produced antimicrobials. The antimicrobial activity of 10x CFSs of the two isolates was stable after enzymatic treatment with proteinase K or heating treatments up to 90 °C or neutralizing pH. The spectrum of activity of the two isolates was evaluated using different gram-positive and gram-negative bacterial species and did not show antimicrobial activity against these species. Our results showed two unconventional bacterial isolates: L. agilis T99A and C. butyricum T58A producing extracellular thermo stable antimicrobial agents against C. difficile clinical isolates.

Isolation of Different Clostridium Isolates for Acetone-Butanol-Ethanol (ABE) Production from Sargassum sp.

Macroalgae with several species are an abundant, carbon-neutral renewable resource and rich in carbohydrates which make it suitable for biobutanol production. Recently, due to advantages of biobutanol as a liquid biofuel, it can be used as a substitute for gasoline and diesel. Many researches mentioned that, different Clostridium species are able to use fermentable sugars for production of biobutanol from different biomasses through ABE (Acetone- Butanol- Ethanol) fermentation process. In this study the (Sargassum sp.) was used as carbon source for biobutanol production. Thirty-three anaerobic mesophilic isolates were isolated on RCM medium from five soil cultivated with different crops. Only thirteen spore forming, mesophilic, anaerobic Clostridium isolates used for the fermentation process. 100 g/L from Sargassum sp were hydrolyzed by sulfuric acid (6 % (v/v)) followed by thermal pretreatment at 121°C for 20 minutes to produce total reducing sugars (24.151 ± 0.273 g/L), which were fermented by Clostridium isolates. The most promising isolate (HG1) which produce the highest butanol level, acetone and ethanol (3.743, 1.401 and 1.031 g/l) respectively was identified according to Butanol dehydrogenases (bdhA) gene analysis as Clostridium acetobutylicum. This paper emphasizes the importance of (Sargassum sp.) as a renewable feedstock for the biobutanol production.

Features of bone destruction due to the collagenase activity of bacteria of the Bacillus and Clostridium genera

The purpose of work was to study the activity of collagenase biosynthesis by bacilli and clostridia isolated from the bone microflora in a pure culture at different periods of bone decomposition and at different values of acidity of the medium and the temperature of cultivation. The comparative collagenase activity of the strains of Bacillus mycoides, Bacillus subtilis, Clostridium putrificum, Clostridium sporogenes found in microflora of bone tissue was determined. The catalytic activity of collagenases was evaluated according to the modified method of agar blocks by the diameter of precipitation zone as a result of diffusion of enzyme into an agar medium with collagen. Within 6 months of experiment, the dynamics of changes in the number of collagenolytic strains was monitored. For all isolated Bacillus and Clostridium isolates, a steady increase in collagenase synthesis was observed throughout the study period, with the exception of Bac. subtilis, which is probably due to the acidity of medium, which did not correspond to the optimal pH range for this species. The optimum temperature for manifestation of the maximum collagenolytic activity of bacilli was 40 °C, and for clostridia - 30 °C. The study of proteolytically active species involved in the destruction of bone collagen is promising for the purposes of forensic examination. The use of bacterial enzymes in forensic practice remains a difficult task, however, the use of their substrate specificity can significantly expand the evidence base of the examinations.

Isolation and toxin gene detection of Clostridium (Clostridioides) difficile from traditional and commercial quail farms and packed quail meat for market supply - Short communication.

Clostridium (Clostridioides) difficile is a Gram-positive anaerobic rod-shaped bacterium and the main cause of nosocomial diarrhoea in humans. In recent years, the transmission of C. difficile from environmental reservoirs (e.g. food) to humans has become a major focus of research. The aim of this study was to investigate the prevalence and corresponding toxin genes of C. difficile in faecal samples and meat of quails. Thirty samples of packed quail meat in Mashhad, Iran and 500 faecal samples (pooled to n = 5) were collected on quail farms in the Northeastern Khorasan region for further investigation. Of 100 pooled quail faecal samples 10% showed cultural growth of C. difficile. In meat samples two out of 30 specimens (7%) showed cultural growth. In six of ten isolates from faecal samples toxin genes (tcdB and tcdA) were present, while four isolates harboured no toxin genes. However, in meat isolates no toxin genes were present. Mutations in the tcdC gene were not detected, indicating that 'hypervirulent' strains such as RT027 and RT078 were not present. The data suggest that quail and quail products might hold a potential for the spread of C. difficile.

Full length obtains of hydA and phylogenetic analysis of bio-hydrogen production new species of Clostridium based on efficient hydA degenerate primers

In fermentative bio-hydrogen production, bacteria from genus Clostridium are considered to be efficient candidates. Studies on bacteria isolation and functional gene analysis of Clostridium make contribution to the H2 production efficiency improvement. In this study, six degenerate primers were designed based on [FeFe]-hydrogenase (hydA) gene sequences from representative biohydrogen producers of Clostridium, and employed to obtain the hydA gene open reading frames (ORFs) of C. guangxiense and C. neuense. Their phylogenetic relationships were also evaluated with hydA gene partial sequences. The ORFs of hydA gene from strains C. guangxiense ZGM211T and C. neuense G1T were obtained with the same length of 1719 bp and theoretical pI 8.09, but different stop codons of TAA and TAG, and Mw of 63.03 and 63.13 kDa, respectively. The phylogenetic tree constructed with hydA partial gene sequences was very similar to that of full-length 16S rRNA gene. It more accurately reflected the phylogenetic relationship of C. guangxiense, C. neuense and related species, and suggested that the hydA gene could be an assistant or reference to 16S rRNA gene in the phylogenetic relationship investigation of bio-hydrogen producers within genus Clostridium.