The antibiotic susceptibility pattern of gas gangrene-forming Clostridium spp. clinical isolates from South-Eastern Hungary.

Introduction:Clostridium perfringens and other gas gangrene-forming clostridia are commensals of the human gut and vaginal microbiota, but can cause serious or even fatal infections. As there are relatively few published studies on antibiotic susceptibility of these bacteria, we decided to perform a 10-year retrospective study in a South-Eastern Hungarian clinical centre.Methods: A total of 372 gas gangrene-forming Clostridium spp. were isolated from clinically relevant samples and identified with rapid ID 32A (bioMérieux, France) and MALDI-TOF MS (Bruker Daltinics, Germany) methods. Antibiotic susceptibility was determined with E-tests.Results: We identified 313 C. perfringens, 20 C. septicum, 10 C. sordellii, 10 C. sporogenes, 9 C. tertium, 6 C. bifermentans, 4 C. histolyticum isolates. In C. perfringens isolates, the rate of penicillin resistance was 2.6% and the rate of clindamycin resistance 3.8%. Penicillin resistance was found in 6.8% and clindamycin resistance in 8.5% of the non-perfringens Clostridium spp. isolates.Conclusion: The antibiotic susceptibility of C. perfringens isolates was in good agreement with previous publications. The rates of resistance to penicillin and clindamycin were very low. The resistance rates of non-perfringens Clostridium spp. isolates were higher than those of C. perfringens strains, but lower than those published in the literature.

Paraclostridium is the Main Genus of Anaerobic Bacteria Isolated from New Species of the Marine Sponge Plakina in the Brazilian Southeast Coast.

Despite the broad assessment of sponge bacterial diversity through cultivation-independent and dependent strategies, the knowledge focusing on cultivable anaerobes from this holobiont is still incipient. Plakina is a genus with the highest number of described species from the smallest of poriferan classes, Homoscleromorpha. The Brazilian Atlantic coast has been presenting itself as a hotspot for the discovery of new plakinidae species, with initial surveys just now concerning to characterize their microbiome. The current study aimed to isolate and identify strict anaerobes from recently described species of Plakina collected at the coast of Cabo Frio, RJ. Samples of four sympatric morphotypes of Plakina cyanorosea and Plakina cabofriense were collected on the coast of Cabo Frio, RJ. Using five different culture media, a total of 93 bacterial isolates were recovered, among which 60 were strict anaerobes and, ultimately, 34 remaining viable. A total of 76.5% from these strains were mostly identified as Clostridium bifermentans by mass spectrometry and 82.4% identified by 16S rRNA sequencing, almost all of them affiliated to the genus Paraclostridium, and with one isolate identified as Clostridium butyricum by both techniques. None of the anaerobic bacteria exhibited antimicrobial activity by the adopted screening test. The present work highlights not only the need for cultivation and characterization of the anaerobic microbiota from marine sponges but also adds the existing scarce knowledge of culturable bacterial communities from Homoscleromorph sponges from Brazilian coast.

Brain abscess and cervical lymphadenitis due to Paraclostridium bifermentans: A report of two cases.

Paraclostridium bifermentans (current nomenclature of Clostridium bifermentans since 2016) is a gram-positive, spore-forming anaerobic bacterium. Here, we describe two cases associated with this organism. The first, primarily a case of tubercular brain abscess where P. bifermentans was isolated as part of a polymicrobial flora, following a neurosurgical procedure for the same and the second, a case of cervical lymphadenitis from which it was isolated as the sole causative agent. There are only a few reported cases of P. bifermentans in literature and these cases illustrate the widening spectrum of infections related to it.

Evaluating new bio-hydrogen producers: Clostridium perfringens strain JJC, Clostridium bifermentans strain WYM and Clostridium sp. strain Ade.TY.

Three newly discovered H2 producing bacteria namely Clostridium perfringens strain JJC, Clostridium bifermentans strain WYM and Clostridium sp. strain Ade.TY originated from landfill leachate sludge have demonstrated highly efficient H2 production. The maximum H2 production attained from these isolates are in the descending order of strain C. perfringens strain JJC > C. bifermentans strain WYM > Clostridium sp. strain Ade.TY with yield of 4.68 ± 0.12, 3.29 ± 0.11, and 2.87 ± 0.10 mol H2/mol glucose, respectively. The result has broken the conventional theoretical yield of 4 mol H2/mol glucose. These isolates were thermodynamically favourable with Gibbs free energy between -33 and -35 kJ/mol (under process conditions: pH 6, 37 °C and 5 g/L glucose). All three isolates favour butyrate pathway for H2 production with the ratio of acetate and butyrate of 0.77, 0.65 and 0.80 for strain JJC, WYM and Ade.TY, respectively. This study reported provides a new insight on the potential of unique bacteria in H2 production.

Influence of 6 different intestinal bacteria on Beltsville Small White turkey semen.

The turkey industry relies totally on artificial insemination to continue and improve production. If something compromises the insemination process, such as contaminated semen, it becomes a detrimental loss to the industry. Bacteria have been found in broiler breeder males to reduce sperm motility. The Sperm Quality Index (SQI) is a quick method to determine avian sperm motility using the sperm quality analyzer (SQA). Therefore, the objective of this study was to determine if bacteria have an effect on turkey sperm motility using the SQA. For the experiment, one mL of pooled neat semen was collected from Beltsville Small White Turkey toms. Six intestinal bacteria, Bifidobacterium animalis, Campylobacter jejuni, Clostridium bifermentans, Escherichia coli, Lactobacillus acidophilus, and Salmonella enterica were grown overnight. For each bacterium, 4 treatments were made that consisted of exposing pooled semen to either saline, sterile broth, an overnight culture of each individual bacterium, or a centrifuged pellet of each bacterium re-suspended in saline. The experiment was repeated 3 times. Once the semen was exposed to the respective treatment, a portion was pulled into a capillary tube and placed into the SQA to obtain the SQI. Each treatment was evaluated at zero, 10, and 20 min creating a completely randomized design with a split plot over time. A pH reading also was taken at each time point. The results indicated that all broths containing bacteria immediately reduced turkey sperm motility. Sperm became practically immotile in overnight cultures of Clostridium, Bifidobacterium, or Lactobacillus However, there was a time by treatment interaction in the SQI for Campylobacter, Clostridium, E. coli, Salmonella, and Lactobacillus The pH of semen decreased upon exposure to Bifidobacterium and Lactobacillus No difference in pH was found when semen was exposed to E. coli, Campylobacter, Salmonella, or Clostridium treatments. In conclusion, the results reveal when turkey semen is exposed to different bacteria, sperm motility is immediately reduced, which could be possible from bacterial attachment or bacterial by-products providing an undesirable environment for sperm.

Hypothetical glycerol pathways of newly isolated strains capable of 1,3-propanediol production.

Study presented here demonstrates the ability of three newly isolated strains, obtained from environmental probes (manure, bottom sediment, and food waste) and identified as Clostridium bifermentans, Clostridium butyricum, and Hafnia alvei, to synthesize 1,3-propanediol (1,3-PD), organic acids (such as lactic, acetic, fumaric, succinic, and butyric acids), and ethanol from glycerol. The production of 1,3-PD as well as the glycerol pathways in C. bifermentans and H. alvei cells have not been investigated and described yet by others. Moreover, there is no data in the available literature on the products of glycerol utilization by H. alvei and there is only some incoherent data (mainly from the first half of the twentieth century) about the ability of C. bifermentans to carry out glycerol degradation. Additionally, this study presents complete hypothetical glycerol pathways and the basic fermentation kinetic parameters (such as yield and productivity) for both strains as well as for the newly isolated C. butyricum strain.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria.

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

The cry toxin operon of Clostridium bifermentans subsp. malaysia is highly toxic to Aedes Larval Mosquitoes.

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.

Effects of OLRs and HRTs on hydrogen production from high salinity substrate by halophilic hydrogen producing bacterium (HHPB).

The effects of hydraulic retention time (HRT) and organic loading rate (OLR) on hydrogen production were investigated with glucose medium containing 2% NaCl. Halophilic hydrogen producing bacterium (HHPB) Clostridium bifermentans 3AT-ma, which can survive under high salt conditions, was used. Sponge media were used as 20% of working volume. The OLR and HRT were varied in 10-60 g-glucose/L-reactor/day and 24-6h. With OLR of 20 g-glucose/L/day, shorter HRT resulted in higher hydrogen producing rate and yield. When the OLR was increased from 20 to 60 g-glucose/L-reactor/day at HRT 6h, the hydrogen production rate increased, while the hydrogen production yield decreased due to the increase and accumulation of volatile fatty acids. Biohydrogen production was possible from the salinity substrate using HHPB, and the maximum hydrogen production yield was 1.1 mol-H2/mol-glucose with optimal conditions of OLR of 20 g-glucose/L/day and HRT of 12h.

Isolation process of industrially useful Clostridium bifermentans from natural samples.

A selective isolation procedure of clostridial strains from natural samples able to convert glycerol to 1,3-propanediol (1,3-PD) and organic acids was investigated. The modified PY medium of high concentration of NaHCO(3) was shown to be highly selective for Clostridium bifermentans. Obtained isolates produced mainly 1,3-PD, lactic, acetic, and formic acids from glycerol.

A Case of Clostridium bifermentans Bacteremia in a Patient with Myelodysplastic Syndrome

Cases of anaerobic bacteremia are rare, and the clinical impact of clostridial bacteremia remains to be clarified. Previous clinical reports have suggested that C. bifermentans is less virulent than other Clostridia species. This microorganism has occasionally been reported to cause septic arthritis, necrotizing pneumonia with empyema, brain abscesses, endocarditis, and metastatic osteomyelitis. Herein, we report on a case of C. bifermentans bacteremia in a patient with myelodysplastic syndrome in South Korea.

GAC adsorption filters as barriers for viruses, bacteria and protozoan (oo)cysts in water treatment.

Granular Activated Carbon (GAC) adsorption filtration is commonly used in drinking water treatment to remove NOM and micro-pollutants and on base of the process conditions a certain capacity to eliminate pathogenic micro-organisms was expected. The experiences with the mandatory quantitative microbial risk assessment of Dutch drinking water revealed a lack of knowledge on the elimination capacity of this process for pathogens. The objective of the current study was to determine the capacity of GAC filtration to remove MS2, Escherichia coli and spores of Clostridium bifermentans as process indicators for pathogens and more directly of (oo)cysts of Cryptosporidium parvum and Giardia lamblia. Challenge tests with fresh and loaded GAC were performed in pilot plant GAC filters supplied with pre-treated surface water at a contact time which was half of the contact time of the full-scale GAC filters. MS2 phages were not removed and the removal of E. coli and the anaerobic spores was limited ranging from < or =0.1-1.1 log. The (oo)cysts of C. parvum and G. lamblia, however, were removed significantly (1.3-2.7 log). On base of the results of the experiments and the filtration conditions the removal of the indicator bacteria and (oo)cysts was largely attributed to attachment. The model of the Colloid Filtration Theory was used to describe the removal of the dosed biocolloids in the GAC filters, but the results demonstrated that there is a lack of quantitative knowledge about the influence of collector characteristics on the two major CFT parameters, the single collector and the sticking efficiency.

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1.

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.

Complete dechlorination of tetrachloroethylene by use of an anaerobic Clostridium bifermentans DPH-1 and zero-valent iron.

A laboratory test was conducted to examine the combined effect of an anaerobic Clostridium bifermentans DPH-1 and addition of zero-valent iron (Fe0) on the reductive dechlorination of tetrachloroethylene (PCE). In addition, the dechlorination of cis-1,2-dichloroethylene (cDCE) produced from PCE was examined using Fe0. The cDCE produced was completely dechlorinated to non-toxic end products, mostly, ethylene by a subsequent chemical reductive process. Production of ethylene was dramatically increased with increase of initial cDCE concentration in the range of 10.3 microM to 928 microM (1.0-90 mg l(-1)) and the velocity constant was calculated to be 0.38 day(-1). On the other hand, the combined use of strain DPH-1 and Fe0 showed the most significant effect on the initial PCE dechlorination, but cohesion of Fe0 was found to inhibit the dechlorination rate of PCE. It is thought that phosphoric acid iron contained in a medium forms film on the surface of iron particle, so oxidation of iron is inhibited.

Analysis of protection afforded by a Clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases C.

The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the alpha-toxin. Previously, mice have been protected against challenge with heterologous alpha-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa(247-370) or alpha-toxoid) of the alpha-toxin. In this study, we have determined the ability of the alpha-toxoid to protect against the lethal effects of a divergent C. perfringens alpha-toxin and against the PLCs of C. absonum or C. bifermentans, species which have been isolated from cases of clostridial myonecrosis. Protection against the C. perfringens alpha-toxin variant, the C. absonum alpha-toxin or the C. bifermentans PLC was elicited by immunisation with the alpha-toxoid in vivo.

Source tracking aerosols released from land-applied class B biosolids during high-wind events.

DNA-based microbial source tracking (MST) methods were developed and used to specifically and sensitively track the unintended aerosolization of land-applied, anaerobically digested sewage sludge (biosolids) during high-wind events. Culture and phylogenetic analyses of bulk biosolids provided a basis for the development of three different MST methods. They included (i) culture- and 16S rRNA gene-based identification of Clostridium bifermentans, (ii) direct PCR amplification and sequencing of the 16S rRNA gene for an uncultured bacterium of the class Chloroflexi that is commonly present in anaerobically digested biosolids, and (iii) direct PCR amplification of a 16S rRNA gene of the phylum Euryarchaeota coupled with terminal restriction fragment length polymorphism to distinguish terminal fragments that are unique to biosolid-specific microorganisms. Each method was first validated with a broad group of bulk biosolids and soil samples to confirm the target's exclusive presence in biosolids and absence in soils. Positive responses were observed in 100% of bulk biosolid samples and in less than 11% of the bulk soils tested. Next, a sampling campaign was conducted in which all three methods were applied to aerosol samples taken upwind and downwind of fields that had recently been land applied with biosolids. When average wind speeds were greater than 5 m/s, source tracking results confirmed the presence of biosolids in 56% of the downwind samples versus 3% of the upwind samples. During these high-wind events, the biosolid concentration in downwind aerosols was between 0.1 and 2 microg/m3. The application of DNA-based source tracking to aerosol samples has confirmed that wind is a possible mechanism for the aerosolization and off-site transport of land-applied biosolids.

Transport of Clostridium bifermentans DPH-1 through the laboratory column can be explained by two-region model for bioremediation.

Transport of Clostridium bifermentans DPH-1 was characterized from laboratory-made column tests using fully saturated Toyoura sand. The conventional transport models were fitted to the column test results to investigate the applicability of prediction and assessment of bacterial transport in actual subsurface or ground water. Laboratory column tests confirmed that the transport characteristics of Clostridium bifermentans DPH-1 in activating tetrachloroethylene dechlorination could be described by mobile-immobile two-region model. The parameters of two-region model i.e. peclet number, retardation factor, fraction rate of mobile water and stanton number were characterized by fitting results. These parameters were also justified by a verification experiment. Two-region model parameters suggested that bacterial injection into the ground at a large concentration is difficult for rehabilitation of widely dispersed contaminated ground water.

Recovery of anaerobic bacteria from wounds after lawn-mower injuries.

Accidental injury while using lawn mowers can cause serious infectious complications in the injured extremity. Anaerobic bacteria were rarely recovered from this infection. Two children who sustained injury in their foot by a lawn mower developed severe wound infection. Culture of the wound from 1 patient had heavy growth of Clostridium bifermentans and Peptostreptococcus magnus, and the culture from the other child grew Clostridium perfringens. Antimicrobial therapy directed at the pathogens and vigorous surgical irrigation and debridement led to complete recovery from the infection. This report illustrates the recovery of anaerobic bacteria from children that had wound infection after lawn-mower injury.