Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor

A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 108). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30–35% and 25–35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20–25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15–20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.
Introduction
Many industrial processes, such as metal smelting, flue gas scrubbing and mining generate sulfate-rich wastewater (Lens et al. 1998). These wastewaters usually do not contain any organic carbon, and the addition of external electron donor is required for their treatment. These waste streams cause a range of problems associated with the now well-established anaerobic treatment of these wastewaters, such as a decrease in methane production (Colleran et al. 1995), sulfide toxicity (O’Flaherty and Colleran 2000), foul smell (Lens et al. 2001) and corrosion (Vincke et al. 2001). Of late, bioreactor processes have been developed for treating these sulfate and metal-rich wastewaters. This technology developed by the company Paques BV in The Netherlands (Pol et al. 2001) makes use of the dissimilatory sulfate-reducing capacity of sulfate-reducing bacteria (SRB) to simultaneously remove sulfate and metals in the form of metal sulfides. A typical application is a two-step process that includes dissimilatory sulfate reduction to sulfide (Gibson 1990) as the first step. Subsequently, the sulfide will be bound by heavy metals, such as cobalt, lead, nickel and zinc and precipitated as metal sulfide (Colleran et al. 1995). The second step is the biological oxidation of the remaining sulfide to insoluble elemental sulfur, which is either recovered by sedimentation or a small portion converted to H2SO4 and re-used to neutralise the alkalinity generated because of sulfide production in the first step.
Several full-scale sulfate removal bioreactors are currently in operation. The dominant sulfidogenic communities in these bioreactors are heterotrophic SRB belonging to the family Desulfovibrionaceae (Kaksonen et al. 2004; van Houten et al. 2006). The genus Desulfovibrio represents a phylogenetically coherent group; all species incompletely oxidise lactate to acetate but can utilise hydrogen, formate and ethanol as well (Widdel and Bak 1992).
In an engineered system fed with a single nutritional source and kept under constant operational parameters such as pH, temperature and salinity, competition among species might tend to reduce the species and sub-species diversity according to the principle of competitive exclusion (Gause 1934). However, physiologically competing species can co-exist if they occupy different niches, whereby each species uses distinct parts of the resource base. In the present study, we investigated the co-existence of physiologically similar hydrogenotrophic SRB in a full-scale sulfidogenic bioreactor treating sulfate-rich wastewater using a combination of cultivation and molecular techniques. The reactor was fed with ethanol as carbon and energy source. SRB isolated in pure culture were characterised genetically and physiologically. Micro-diversity among the dominant culturable isolates was assessed by repetitive enterobacterial palindromic polymerase chain reaction (rep-PCR) (Versalovic et al. 1994). Whole-cell hybridisation with fluorescently labelled general and specific probes was used for SRB community characterisation and for the estimation of the relative abundance of the different SRB populations in the reactor.
Materials and methods
Sludge source
Granular sludge was obtained from an upflow anaerobic sludge bed (UASB) reactor, treating sulfate-rich wastewater from a chemical plant located in Emmen, The Netherlands. The wastewater fed to the reactor did not contain any organic compound, so ethanol was added as an external electron donor and carbon source. The reactor had a volume of 300 m3 and a feed rate of 60–75 m3 h−1 The ratio between the amount of electron donor added and the amount of sulfate in the reactor was around 0.4 kg/kg. Sulfide produced in the reactor was either converted to elemental sulfur through biological sulfide oxidation or precipitated with toxic metals. The reactor was operated at a temperature of 30°C and a pH of 7.0–7.5. The concentration of sulfate in the wastewater was approximately 1,500 mg/l and ca. 100 mg/l in the effluent.
Culture media and isolation of SRB
A basal bicarbonate-buffered and sulfide-reduced medium was used for the enumeration and isolation procedures. The mineral medium contained per litre of distilled water: 0.2 g KH2PO4, 0.25 g NH4Cl, 0.5 g KCl, 0.1 g CaCl2·2H2O, 0.4 g MgCl2·6H2O, 1.0 g NaCl and 0.5 ml of a resazurin solution (0.5 mg ml−1) as a redox indicator. The medium was supplemented with (per litre) the following: 30 ml 1 M NaHCO3 solution, 1 ml of a vitamin solution, 1 ml of trace element solution with ethylenediamine tetraacetic acid (EDTA; Widdel and Bak 1992) and 0.1 g of yeast extract. As a reducing agent, 7.5 ml l−1 of 0.2 M Na2S·9H20 was added. Either lactate or ethanol (20 mM) was used as an electron donor and sulfate (10 mM) as electron acceptor. Enumeration of the potentially dominant heterotrophic sulfate reducers was performed by serial dilutions in Hungate tubes. The Hungate tubes were incubated at 30°C in the dark for 7 weeks. The highest positive dilution tubes that showed growth were selected for further isolation. Growth was assayed by measuring sulfide production photometrically (Cord-Ruwisch 1985).
Pure cultures were obtained by repeated transfer in agar shake tubes (Widdel and Pfennig 1984). Purity of the isolates was checked by microscopic observation and further confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments (Teske et al. 1996).
Phenotypic characterisation
Substrate utilisation by the isolated strains was determined in duplicate in the same bicarbonate-buffered medium as used for enumeration and isolation but without yeast extract. Growth was tested in 100-ml serum bottles closed with butyl rubber stoppers and aluminium crimp seals. The inoculum size was 1% (v/v). The cultures were incubated at 30°C for 5 weeks in the dark. Growth was determined by substrate consumption or product formation as well as by checking for increase in optical density at 660 nm (OD660). The following substrates were tested as electron donors in the presence of 10 mM sulfate: 10 mM each of pyruvate, fumarate, butyrate, formate, propanol, ethanol, methanol, serine and cysteine; 5 mM each of malate, glycolate and glycerol; 2.5 mM of benzoate; and 0.5% (w/v) of casamino acids. Sulfite, thiosulfate and 2% (w/v) elemental sulfur were tested as electron acceptors in the presence of lactate as electron donors.
For measuring catalase activity, the fully grown isolates were centrifuged at 13,000 × g. The cell pellets were re-suspended on glass slides with a drop of 3% (v/v) H2O2, bubbles indicated the presence of catalase. Detection of desulfoviridin was performed according to Postgate (1959). Gram staining was performed as previously described (Bartholomew 1962).
Analytical methods
Acetate and other volatile fatty acids were analysed either by gas chromatography (GC) or high performance liquid chromatography (HPLC). For GC, a Chromopack 9001, equipped with a flame ionisation detector and a fused-silica capillary column 15 × 0.53 mm HP-Innowax, was used. The column temperature was 120°C. The temperature of the injector and detector were 180 and 200°C, respectively. Helium was used as a carrier gas. An Aminex HPX-87H column from Bio-Rad (T = 60°C) coupled to a UV and a RI detector was used for HPLC; phosphoric acid (0.05 M) was used as an eluent. Sulfide was measured quantitatively by a colorimetric assay (Cline 1969).
DNA extraction
Genomic DNA was isolated from the bacterial cultures using the Ultra Clean Soil DNA extraction kit (MOBIO Laboratories, California) according to the manufacturer’s protocol. The quality of the extracted DNA was examined on a 1% (w/v) agarose gel and the amount quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies, Delaware). Extracted DNA was stored at −20°C until subsequent use in different PCR reactions.
PCR amplification and DGGE of 16S rRNA genes
For DGGE analysis, amplification of partial 16S rRNA gene was carried out using the primers 341F-GC and 907R as described by (Schäfer and Muyzer 2001), while primers GM3 and GM4 (Muyzer et al. 1995) were used to amplify the nearly complete 16S rRNA gene for sequencing and subsequent phylogenetic analysis. PCR amplification and DGGE was performed as described previously (Schäfer and Muyzer 2001). The quality of the PCR products was examined on 1% (w/v) agarose gel, and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies).
DNA sequencing and phylogenetic analysis
The nearly complete 16S rRNA gene fragments, obtained from the strains L3 and L7, were purified using the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany). Purified PCR products were sequenced by the company BaseClear (Leiden, The Netherlands). The DNA sequences of about 1,400 bp were first compared to the sequences deposited in public databases using the NCBI BLAST search tool (http://www.ncbi.nlm.nih.gov/BLAST; McGinnis and Madden 2004). Subsequently, the sequences were imported into the ARB software programme (Ludwig et al. 2004) and aligned using the automatic aligner function. The alignment was further corrected manually, and a phylogenetic tree was constructed using the neighbour-joining algorithm with Felsenstein correction.
rep-PCR fingerprinting
The genetic diversity of the isolates was analysed by rep-PCR (Versalovic et al. 1994) using the primer GTG5 (5O′-gTggTggTggTggTg-3′). The amplification reaction was performed as previously described (Foti et al. 2006). A 1-kb size marker and 600 ng of the PCR product were loaded onto a 1.5% (w/v) agarose gel containing 0.5 × TAE-buffer (200 mM Tris-acetate, 0.5 mM EDTA, pH 8). The electrophoresis was performed for 14 h in a cold room at a constant voltage of 65 V. The gel was subsequently stained with ethidium bromide (0.5 μg/ml) and photographed under UV illumination using the GelDoc UV Transilluminator (Bio-Rad, Hercules, CA).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis
Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein (in cell-free extract obtained by sonication) was performed on 12% polyacrylamide gels according to Laemmli (1970). The gels were stained with Coomassie Brilliant Blue and photographed using visible light.
Design of oligonucleotide probes
Specific oligonucleotide probes for the 16S rRNA of the two strains were designed using the Probe Design tool of the ARB software package (Ludwig et al. 2004). The requirements for designing the probes included high specificity, with no organism outside the intended target group having 100% similarity within the target sequence, and the target sequence being located within the high accessibility region of 16S rRNA molecule as suggested previously (Behrens et al. 2003). The probes were named with a number that indicates the position of the first base in the target sequence (by Escherichia coli numbering). The oligonucleotides used for in situ hybridisation are given in Table 1.
Table 116S rRNA-targeted oligonucleotide probes used in this studyProbe nameaTarget organismsProbe sequence (5′–3′)ReferenceEUB338_IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. (1990)EUB338_IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. (1999)EUB338_IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. (1999)ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann (1991)SRB385Most DeltaproteobacteriaCGG CGT CGC TGC GTC AGGAmann et al. (1990)SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. (1996)DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. (1992)DSBA1017Desulfobacca acetoxidansGTT GCC AGG CAC CCC CATDar et al. (2007)DSV119Desulfovibrio strain L3GGC AGA TCA TCC ACG CGTThis studyDSV139Desulfovibrio strain L7CGC TGT TAT CCC GAT CACThis studyaEUB338 is a combination of EUB338_I, EUB338_II and EUB338_III.
Whole cell hybridisation
Cells from strains L3 and L7 and from the original reactor were fixed, washed and spotted onto Teflon-coated multi-well microscopic slides as described previously (Dar et al. 2007). Hybridisation was carried out according to the protocol described by Manz et al. (1992) using a formamide concentration of 35% (v/v). Quantification of the hybridised cells was performed as described previously (Neef et al. 1996). The hybridised cells were analysed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualised with general probes for bacteria (EUB338 I, II and III), SRB (SRB385 and SRB385Db) or with the general DNA stain DAPI (4′, 6′ -diamidino-2-phenylindole). In addition, a general probe specific for members of the domain Archaea (ARCH915) was used. The hybridisation experiments were done in duplicate using different fluorochromes for each probe. Different microscopic fields on each slide were analysed to confirm the results. Hybridisation stringencies of the newly designed probes were determined by performing hybridisations with increasing formamide concentrations as described previously (Manz et al. 1992) using target organism(s) and non-target organism displaying three mismatches within the target region.
Sequence accession numbers
The nearly complete rRNA gene sequences of strains L3 and L7 have been deposited in GenBank under accession nos. EF055876 and EF055877, respectively.
Results
Reactor performance
The amount of ethanol dosed to the reactor was continuous and limited to the amount required for sulfate reduction according to the net stoichiometric reaction for sulfidogenic oxidation of ethanol and its major degradation intermediate, acetate. The equations are as follows:
The sulfate removal efficiency at the time of sampling was more than 93%. Less than 1 mg of acetate was estimated in the effluent and no significant biogas (CH4) production from the oxidation of organic source was observed at the time of sampling.
Enumeration and isolation of SRB
The maximum number of culturable SRB observed with lactate or ethanol as substrate in the serial dilution tubes was in the order of 108 cells ml−1. The tubes with growth in the highest dilution were used for isolation in agar shake tubes. In total, 26 strains were obtained in pure culture, 17 on lactate and nine on ethanol as substrate. Sub-cultivation of the isolated strains was carried out on lactate. Apart from microscopic observation, the purity of the isolated strains was confirmed by single bands obtained from the DGGE of partial 16S rRNA genes amplified from the strains. The DGGE results of the 26 isolated strains identified two melting types (results not shown).
Genomic fingerprinting
The isolated strains were subjected to genomic fingerprinting, i.e. rep-PCR, to resolve any higher degree of genetic diversity among the strains. The rep-PCR profiles of the strains (Fig. 1) revealed the presence of two distinct genotypes with no micro-diversity among the strains. Furthermore, the whole cell protein profile of representative strains of the two groups, grown on the same substrate (i.e. lactate and sulfate) and under similar conditions of pH and temperature, gave only two distinct profiles (Fig. 2), confirming the presence of two genotypes. Two isolates, strain L3 and L7, were chosen as representatives of the two genotypes and were subjected to a more detailed phylogenetic and phenotypic analysis.
Fig. 1rep-PCR patterns of different strains isolated from the full-scale sulfidogenic bioreactor. M is the molecular weight markerFig. 2Whole cell protein profile of different strains isolated from the full-scale sulfidogenic bioreactor. M is the molecular weight marker. The arrows depict protein bands that are expressed in one or the other genotype
Phylogenetic analysis
A similarity check using the NCBI BLAST search tool of the nearly complete 16S rRNA gene sequences obtained from strain L3 and L7 indicated 98% sequence similarity to Desulfovibrio strain SB1 and 99% sequence similarity to Desulfovibrio mexicoense, respectively. The phylogenetic affiliation of the obtained sequences is presented in Fig. 3. A neighbour-joining tree based on nearly complete 16S rRNA gene sequences was generated, confirming the close affiliation of the isolated strains L3 and L7 to Desulfovibrio strain SB1 and Desulfovibrio mexicoense, respectively.
Fig. 3Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing the phylogenetic affiliation of the two isolated SRB, strain L3 and L7. The sequence of Desulfobacter postgatei was used as an outgroup but was pruned from the tree. Dots on the nodes indicate bootstrap values of 90% or higher (1,000 replicates).The bar indicates 1% sequence difference
Phenotypic characterisation of the strains
All isolates had a vibrio to spiral cell morphology. Two distinct groups could be identified based on size and motility. One group of cells related to Desulfovibrio strain SB1 was motile, and their size ranged from 4–7 μm in length and 1 μm in width. The other group was non-motile, and the size was 1–2 μm in length and 0.5 μm in width.
Table 2 shows the substrate utilisation patterns of the two isolates. Both strains used lactate, ethanol, pyruvate, glycerol and casamino acids as electron donors. Hydrogen and formate could only be used as substrates in the presence of acetate as carbon source. The organic substrates were incompletely oxidised to acetate. Acetate, propionate, butyrate, glycolate and methanol could not be used as electron donors by both strains. Fumarate and malate were used by strain L3 but not by strain L7, while serine and cysteine were utilised by strain L7 but not by strain L3. Sulfate, thiosulfate and sulfite were used as electron acceptors by both strains. In addition, strain L7 could also use elemental sulfur as an electron acceptor.
Table 2Phenotypic characterisation of strains L3 and L7CharacteristicsStrain L3Strain L7Cell morphologyVibrioVibrioMotility+−Desulfoviridin++Catalase++Gram staining−−Electron donors H2 plus acetate++ Pyruvate++ Lactate++ Acetate−− Propionate−− Fumarate+− Butyrate−− Formate++ Propanol−− Ethanol++ Methanol−− Serine−+ Cysteine−+ Malate+− Glycolate−− Glycerol++ Benzoate−− Casamino acids++Electron acceptors Thiosulfate++ Sulfite++ Elemental sulfur−+
Whole cell hybridisation
After isolation of the most abundant culturable SRB, whole cell hybridisation with specific oligonucleotide probes was performed to estimate the abundance of strains L3 and L7 in the original sludge sample. The specificity of the designed oligonucleotide probes (DSV119 and DSV139) was verified using growing cells of strain L3 and Desulfovibrio gigas for probe DSV119 and cells of strain L7 and Desulfovibrio mexicoense for probe DSV139. Strain L3 served as a non-target species for L7 and vice versa. A formamide concentration of 35% (v/v) was found stringent enough to discriminate between the two strains (Fig. 4a). In addition, probes specific for cells of Desulfobulbus (i.e. DSR660) and Desulfobacca acetoxidans (i.e. DSBA1017), which we had detected previously in a similar sulfidogenic reactor (Dar et al. 2007), were also used.
Fig. 4a Mixture of cells of strain L3 and L7 hybridised with probe Dsv139 labelled with Cy5 (blue), probe Dsv119 labelled with Cy3 (red) and probe SRB385 labelled with Fluos (green). b Sludge sample from a full-scale sulfidogenic bioreactor hybridised with probe Dsv139 labelled with Cy3 (red), probe Dsv119 labelled with Cy5 (blue) and SRB385 labelled with Fluos (green). c Sludge sample hybridised with probe Dsv139 labelled with Cy5 (blue), probe Dsv119 labelled with Cy3 (red) and EUB338 labelled with Fluos (green). d Sludge sample hybridised with probe DSR660 labelled with Fluos (green), probe SRB385 labelled with Cy3 (red) and EUB338 labelled with Cy5 (blue). e Sludge sample hybridised with probe SRB385 labelled with Fluos (green), probe DSBA1017 labelled with Cy3 (red) and probe EUB338 labelled with Cy5 (blue). f Sludge sample hybridised with probe Arch915 labelled with Fluos (green), probe SRB385 labelled with Cy3 (red) and EUB338 labelled with Cy5 (blue). Bar is 20μm
The relative percentage of cells that hybridised with probe DSV119, which is specific for strain L3, was 30–35% of the total SRB385-positive cells and 15–20% of the EUB338-positive cells (Table 3; Fig. 4b,c). The probe specific for strain L7, i.e. DSV139, was detected between 25–35% of the SRB385-positive cell and 10–20% of the EUB338-positive cells (Table 3; Fig. 4b,c). The Desulfobulbus and Desulfobacca-specific probes also gave a positive signal, but these SRB were somewhat less abundant than Desulfovibrio positive cells. The Desulfobulbus-specific probe, DSR660, targeted approximately 20–25% of the total SRB cells and 10–15% of the total bacterial cells (Table 3; Fig. 4d). The Desulfobacca acetoxidans-specific probe DSBA1017 targeted approximately 15–20% of the total SRB cells and 8–10% of bacterial cells (Table 3; Fig. 4e). The percentage of cells that hybridised with probe specific for Archaea (probe ARCH915) was less than 0.1% of the total DAPI stained cells (Table 3; Fig. 4f).
Table 3Relative abundance of SRB and ArchaeaaProbeSRB385EUB338DAPIDSV11930–3515–2012–16DSV13925–3510–208–16DSR66020–2510–158–12DSBA101715–208–106–8ARCH915––<0.1aPercentage of positive cells relative to those detected by a mixture of probes SRB385 and SRB385Db (SRB385), a mixture of probes EUB338 I, II and III (EUB338) and to cells stained with the DNA stain DAPI.
Discussion
Based on the observed sulfate removal efficiency (>93%) and the amount of ethanol dosed to the reactor, it may be assumed that a major part of electron flow from the substrate is scavenged by the SRB. The presence of less than a milligram of acetate in the effluent stream with no significant production of CH4 points to the oxidation of ethanol mainly through sulfate reduction, although fermentation of ethanol through acetogenesis cannot be ruled out. The metabolic products of fermentation and acetogenesis, mainly acetate and hydrogen, may serve as substrates for methanogens and sulfate reducers; however, under high sulfate concentrations in the reactor, hydrogen and acetate would be more readily used by hydrogenotrophic and acetate-utilising sulfate reducers, respectively, because of more favourable substrate affinity (Ks) values of the SRB for these substrates (Stams et al. 2005). Fermentation of ethanol with propionate as the major reduced end product has also been described in the literature (Schink 1984; Tholozan et al. 1992); and under such fermentation, propionate will serve as an energy source for the members of the genus Desulfobulbus among the SRB.
Genetic diversity among the dominant culturable isolates
Compared to high micro-diversity among phylogenetically similar strains detected in sediments (Sass et al. 1998; Wieringa et al. 2000; Klepac-Ceraj et al. 2004), no such micro-diversity was observed within the 26 strains isolated from the bioreactor. The chemical complexity of sediments with steep gradients of substrate concentrations, redox potential and pH may give rise to a number of physico-chemical and depth-defined micro-niches, resulting in the evolution of co-existing but genetically distinct sub-populations (Gray et al. 1999). In addition, Torsvik et al. (2002) suggested structural complexity of sediments being an important factor that allows nutritional partitioning, creating numerous niches that, in turn, results in genetic diversification of populations. From the 26 strains isolated, the ones that had identical 16S rRNA gene sequences were also identical by genomic fingerprinting (Fig. 1) and whole cell protein electrophoresis (Fig. 2). Based on the rep- and protein profiles, two genotypes could be identified among the 26 strains isolated. The absence of micro-diversity among the dominant culturable Desulfovibrio populations suggests the existence of a few ecological niches in the reactor. The long-term operation of the reactor under stable operational parameters, like constant temperature, pH, salinity etc., combined with the selection pressure because of nutritional limitation (i.e. the use of a single energy source), might be the reason for absence of micro-diversity. In addition, the upward flow of wastewater through the reactor results in frequent mixing, which, in turn, might prevent the creation of micro-habitats that are assumed to be important for the evolution of genetically distinct sub-populations (Torsvik et al. 2002).
Although the viable cell count of SRB observed (108 cell ml−1) was comparable to the previous studies (Vester and Ingvorsen 1998; Oude Elferink et al. 1999; Roest et al. 2005; van Houten 2006), it cannot be ruled out that potential biases associated with culture-based enumeration techniques might have underestimated the overall SRB population diversity.
Phenotypic and phylogenetic characterisation of the dominant culturable isolates
The physiological characteristics (Table 2) of strain L3 and L7 indicated that they are sulfate reducers. The two isolates oxidised lactate and ethanol incompletely to acetate in the presence of sulfate as electron acceptor. Both used H2 and formate in the presence of acetate as carbon source. These metabolic traits, in addition to the presence of desulfoviridin and a typical vibrio-shaped morphology, suggested that they are the members of the genus Desulfovibrio (Widdel and Bak 1992). Desulfovibrio are Gram-negative sulfate reducers, most of which oxidise their substrates incompletely to acetate (Widdel and Bak 1992). Previous studies have demonstrated the dominance of Desulfovibrio species in freshwater sediments (Sass et al. 1998), in oil wells (Voordouw et al. 1996) and in several wastewater treatment plants (Santegoeds et al. 1998; Dar et al. 2005). 16S rRNA sequence analysis of the two strains, L3 and L7, confirmed their affiliation to other members of the genus Desulfovibrio (Fig. 3),with Desulfovibrio strain SB1 and Desulfovibrio mexicoense as closest relatives, respectively. Desulfovibrio strain SB1, a mesophilic, Gram-negative SRB was isolated from anaerobic sludge of a gas lift reactor-treating sulfate and zinc-rich wastewater (van Houten 2006); while Desulfovibrio mexicoense was isolated from a UASB digester-treating wastewater from a cheese-manufacturing factory in Mexico (Hernandez-Eugenio et al. 2000).
Co-existing SRB populations in the reactor
After isolation of the most abundant culturable SRB, i.e. the motile strain L3 and non-motile strain L7, whole cell hybridisation using fluorescently labelled oligonucleotide probes was performed to search for their specific signals in the fixed sludge sample. Hybridisation results not only confirmed the presence of the two isolates but also gave an estimate of their abundance relative to the total SRB population and to the overall bacterial community present (Table 3; Fig. 4). Fluorescence in situ hybridisation (FISH) results indicated that the two isolates indeed made up a major part of the hydrogenotrophic SRB community present in the reactor. Cells detected by probe Dsv119, specific for the motile strain L3, appeared as single cells or as chains of three to four cells, while cells detected by probe Dsv139, specific for the non-motile strain L7, appeared as individual cells or as loose aggregates.
According to the ecological principle of competitive exclusion, co-existence of physiologically related populations in the same habitat can be understood if they occupy distinct ecological niches (Gause 1934). Although a number of similarities can be drawn between the strains, L3 and L7, from their substrate utilisation profiles (Table 2), the phenotypic differences together with differences in the range of substrates used by the two isolates may allow them to adapt to slightly different niches within the reactor. The two isolates differed in as many as five physiological properties, which are the use of malate, formate, serine and cysteine as energy sources and sulfur as an electron acceptor (Table 2). The use of elemental sulfur in particular by strain L7 will give it a selective advantage over strain L3 in those places in the reactor where elemental sulfur might be available because of the re-circulation fluid from the aerobic reactor; whereas the motility of strain L3 will confer a competitive advantage to it over strain L7. Potential benefits of motility may include increased efficiency of nutrient acquisition and avoidance of toxic substances (An et al. 2006). The presence of Desulfobulbus-like SRB besides the members of Desulfovibrio in the same habitat could be explained because of the ability of Desulfobulbus to use the fermentation product propionate as an energy source as well; furthermore, under limiting sulfate concentrations, Desulfobulbus competes more successfully for ethanol than other sulfate reducers by its ability to ferment ethanol (Laanbroek et al. 1982). The simultaneous presence of Desulfovibrio and Desulfobulbus in wastewater treatment systems has often been reported in the literature (Nanninga and Gottschal 1987; Raskin et al. 1995; Okabe et al. 2003).
Among the complete oxidisers, the probe specific to Desulfobacca acetoxidans gave a positive signal, suggesting their dominance among the acetotrophic sulfate reducers. Desulfobacca acetoxidans, first isolated from a sulfidogenic bioreactor (Oude Elferink et al. 1999), is a Gram-negative SRB that can utilise acetate as the only source of organic carbon and electron donor. Kaksonen et al. (2004) also found Desulfobacca acetoxidans-like SRB in their lab-scale fluidised-bed reactors that were fed with a single electron donor, i.e. lactate or ethanol.
In summary, this study demonstrated the presence of a consortium of four sulfate-reducing populations in the reactor maintained on a single substrate (ethanol). This is in contrast to the findings of Kaksonen et al. (2004) who reported a relatively more diverse consortium of SRB in a fluidised-bed reactor fed with ethanol or lactate as the only energy source, using clone libraries and DGGE as molecular methods for microbial characterisation. The observed difference in the extent of diversity might be due to the difference in the molecular methods employed. The general probes like DSR660, used during FISH analysis, cannot discriminate among the different species, and the probes like DSV119, DSV139 and DSBA1017 target only the specific populations. A likely complete oxidation of ethanol through sulfate reduction might thus be assumed to be taking place through a combined effort of hydrogenotrophic sulfate reducers (i.e. the motile strain L3, the non-motile strain L7 and Desulfobulbus spp.) oxidising ethanol incompletely to acetate and Desulfobacca acetoxidans oxidising acetate completely to CO2. Genetic diversity analysis of the most dominant culturable Desulfovibrio populations suggests that long-term stable operation of a reactor with constant operational parameters and frequent mixing might result in the absence of micro-diversity.

sulfate-reducing bacteria
ecological niches
microbial ecology


The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS.
Introduction
Demyelinating diseases, such as multiple sclerosis (MS), the major demyelinating disease of humans affects not only young adults, but also children. It is the leading cause of neurological deficit in North America and Western Europe [1]. It is characterized by the patchy destruction of the myelin sheath around the axon and is, therefore, classified as a primary demyelinating disease [2]. The reason for this patchy destruction is not known. The MS plaque is characterized by a focal area of myelin destruction associated with astroglial scar formation. These lesions are scattered throughout the CNS with a predilection for optic nerves, brain stem, spinal cord, and periventricular white matter [3]. Perivascular lymphocytic infiltration with macrophages, microglial cells and astrocytes is a common feature. More recently, it has been recognized that extensive oligodendrocyte apoptosis and myelin macrophages are found in MS tissue, suggesting a primary neurodegenerative mechanism, followed by the autoimmune response as a secondary event [4]. In this type of apoptosis, the cardinal feature is damage to the most distal part of the oligodendiocyte process in the periaxonal region [5]. The chemical changes in myelin that precede this destruction are under intensive investigation. In the present overview, we suggest that enzymatic changes induced in myelin basic protein (MBP) may represent an important factor.
The cause of MS is unknown. Two major theories have emerged: an autoimmune or a neurodegenerative cause. The autoimmune etiology is considered to arise from sensitization of T-cells in the periphery by a mechanism of molecular mimicry, usually involving a viral antigen with peptide sequences similar to those found in myelin proteins, especially MBP, a candidate autoantigen in MS. The neurodegenerative hypothesis involves metabolic changes in the myelin constituents, which destabilize the membrane architecture, resulting in myelin degradation. Since the myelin proteins are primarily responsible for maintaining the structure of the membrane we focused our attention on myelin proteins, in particular, modifications that are found on MBP.
Myelin basic protein is an unusual protein. It is a single protein chain consisting of 170 amino acids. Since it does not contain cysteine, it cannot form disulfide bonds. It is a flexible structure, which adapts to environmental conditions, by changing conformation. It is a member of a group of proteins considered to be intrinsically disordered and, therefore, have considerable conformational flexibility (reviewed extensively in the contribution of Harauz and Musse in this issue). A further unusual feature of MBP is the large number and variety of post-translational modifications, such as N-terminal acylation, GTP- and ADP-ribose binding sites, deamidation, methylated arginine, methionine sulphoxide, phosphorylation, and deimination of arginyl residues. Citrullination occurs on six sites in the 18.5 kDa MBP from humans, while phosphorylation is found on ten sites at least. These are summarized in Fig. 1.
Fig. 1Post-translational modification of MBP. All post-translational modifications, except methylation of Arg107 (human sequence), decrease the positive charge. The known modifications are acylation of the N-terminus, GTP binding, ADP-ribosylation (if R25 is not citrullinated), deamidation, methionine sulphoxide, mono, and dimethylation of Arg107, phosphorylation at Ser/Thr residues and Tyr and deimination of arginyl residues (six sites in the 18.5 kDa MBP from normal brain). The structure, shown as a hairpin, occurs at the tri-proline region at the site of the bend. The figure was adapted and modified from Moscarello (1990, Myelin basic protein: a dynamically changing structure. In: Dynamic interactions of myelin proteins, George Hashim and Mario Moscarello (eds) Prog Clin Bio Res, vol 336)
One of the modifications, in which we are particularly interested, involves the conversion of peptide-bound arginine to peptide bound citrulline, an enzymatic reaction called deimination. The conversion of arginine to citrulline involves the release of ammonia. For each arginine converted to citrulline, one positive charge is lost from the protein. We first demonstrated that citrulline was present in a crude MBP fraction from myelin [6]. We then purified the citrulline containing MBP on CM52 cation exchange columns essentially as described earlier [7–9]. From these columns, we obtained a number of fractions, all of which were MBP. Fractionation of the various MBPs was shown to be dependent on the net charge of the highly modified MBP, resulting from phosphorylation, deamidation, C-terminal arginine loss, and others (Fig. 1). The citrullinated MBP was recovered in the flow-through volume and required further purification by high-performance liquid chromatography (HPLC). The purified protein was sequenced and the arginyl residues that were deiminated were identified. These were R25, R31, R122, R130, R159, and R169 [10]. This decreased positive charge affected the ability of the protein to interact with and organize lipid vesicles. Using liquid X-ray diffraction, we were able to show that the citrullinated MBP (termed “C-8” for convenience) was unable to organize lipid bilayers into compact multilayers, which was readily achieved by the non-citrullinated charge isomer (termed C-1, so-called because it bound most tightly to the CM52 column) [11]. At this time, we speculated that this failure to organize bilayers into compact multilayers, may contribute to myelin instability as seen in MS. These data demonstrated that the citrullinated MBP had a limited ability to organize and compact the lipid bilayers.
The citrullinated MBP was isolated from white matter of MS brain and compared to that isolated from normal brain. We reported that the relative amount of the citrullinated MBP was increased in MBP isolated from MS brain. Whereas, it accounted for 20% of the MBP isolated from normal brain, a threefold increase was found in chronic MS. Thus, the ratio of citrullinated MBP/non-citrullinated MBP was 0.82 in the normal and it was 2.45 in the MS MBP [12]. In a single case of Marburg’s disease (a fulminating form of MS), 80–90% of the MBP was citrullinated, with a citrullinated MBP/non-citrullinated MBP ratio of 6.7 [13, 14]. Therefore, MBP in Marburg’s type of MS was almost totally deiminated. Citrullination of MBP has been reported to diminish charge density, which diminishes its interaction with lipids, making it more vulnerable to proteolytic attack [15].
The effects of deimination on protein structure and protein-lipid interactions in myelin are diverse and have been thoroughly reviewed [16]. Briefly, deimination of MBP alters the 3D structure of MBP, producing a more open conformation. The more open conformation of MBP increases its susceptibility to proteases such as cathepsin D, a myelin associated protease. MBP containing six citrullinyl residues is digested four times faster than non-deiminated MBP. The MBP containing 18 citrullinyl residues was digested 45 times faster than MBP from normal brain. These data support the view that deimination of MBP not only decreases the positive charge on the molecule, but also creates a more open structure [17].
These perturbations in structure affect the interactions of the protein with the lipid bilayer and decrease the compaction of the multilayer structure of myelin. The deiminated protein had a reduced ability to bind and aggregate lipid vesicles and the highly deiminated form of MBP caused fragmentation of lipid vesicles [18], a process which may have implications for myelin breakdown in MS.
Electrostatic interactions between the negatively charged phospholipids and the positively charged amino acids (Arg and Lys) are the origin of attraction of MBP to the lipid. MBP is postulated to work as a glue for adjacent bilayers [19, 20] by binding to the polar head groups. This mechanism of interaction is interfered with by deimination of arginyl residues, since the resulting citrulline carries a neutral charge.
The conversion of arginine→citrulline in proteins is carried out by the enzyme peptidyl arginine deiminase (PAD). Of the five known PAD enzymes, PAD 2 is the one involved in the deimination of MBP. Since our studies on the deiminated MBP in humans was obtained from autopsy material, the question of the origins of the citrullinated protein, as possibly artefactual, was addressed. To resolve this issue, we explored the temporal relationship between the appearance of the citrullinated protein and the up-regulation of the PAD 2 enzyme, which could only be done in a relevant animal model. Such a model was available to us. It is a transgenic model, containing 70 copies of the transgene for the myelin proteolipid protein DM20. This mouse is normal for the first 3 months of its life and then demyelinates spontaneously. We measured PAD enzyme and citrullinated MBP. We found that PAD 2 was up-regulated at 2 months of age and citrullinated MBP at 3 months of age, suggesting a precursor-product type relationship [21]. We postulated that up-regulation of PAD 2 was the primary response. To substantiate this hypothesis, a thorough study was carried out on PAD 2 regulation.
In this regard, we have reported that paclitaxel (Taxol) inhibited the activity of purified PAD 2 isolated from bovine brain in an in vitro assay [22]. In in vivo studies, we demonstrated that paclitaxel attenuated clinical disease in the spontaneously demyelinating mouse model described above [23]. In a recently submitted manuscript, we extended our Taxol studies further by demonstrating that the addition of the methyl donor, vitamin B12, suppressed both immune and non-immune demyelinating disease in mice (F. G. Mastronardi et al. 2006, submitted data), better than paclitaxel alone. These data suggest that drugs, which target PAD 2, in the presence of a methyl donor, such as vitamin B12, may be beneficial in demyelinating disease.
Another PAD enzyme, PAD 4, has been implicated in the pathogenesis of psoriasis [24], rheumatoid arthritis [25, 26] and MS (F. G. Mastronardi et al. 2006, submitted data). PAD 4 is different from other PAD enzymes, because the gene contains a nuclear localization signal (PPAKKKST) [27], not present in any of the other PAD enzymes. Translocation of the enzyme from the cytoplasm to the nucleus, has been shown to involve the cytokine tumor necrosis factor α (TNFα). The substrate for PAD 4 in the nucleus is histone (F. G. Mastronardi et al. 2006, submitted data). Deimination of arginyl residues of histones decreases the positive charge on histone, which compromises its ability to interact with DNA, possibly resulting in apoptosis of oligodendrocytes. On the other hand, deacetylation of histones would increase the positive charge on histone. Therefore, these two processes, the deimination of arginyl residues and the deacetylation of lysyl residues of histones affect the positive charge on histone in opposite ways, and these two processes may have a regulatory function.
Since we determined that up-regulation of PAD 2, preceded demyelination in the animal model, we searched for the mechanism responsible. An examination of the nucleotide sequence of the PAD 2 promoter revealed that it was high in CpG sequences. CpG sequences are important, because cytosine is a target for methylation by DNA methyltransferase, which produces 5-methylcytosine. Methylation of the promoter region of a gene silences the gene, whereas demethylation increases transcription.
Accordingly, we isolated DNA from white matter obtained from normal individuals and patients who died with MS. The DNA was treated with bisulphite to convert cytosine into uracil. The methylcytosine is not affected by bisulphite. We found that methylcytosine accounted for less than 4% of the cytosines in the PAD 2 promoter in MS (n = 17), whereas in the normal, the methylcytosine in the PAD 2 promoter was 12% (n = 8). Therefore, the PAD 2 promoter was hypomethylated in the DNA from MS white matter samples. DNA was also obtained from thymus of the same MS patients. The amount of methylcytosine was the same in both MS and normal samples. Therefore, the hypomethylation found in MS appeared to be tissue specific. When we examined other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s, the amounts of methylcytosine were the same as normal (Mastronardi et al. 2006 in preparation). Therefore, the hypomethylation of the PAD 2 promoter was unique to MS brain. This hypomethylation of the promoter of PAD 2 may account for the increase in PAD 2 in MS brain (summarized in Fig. 2).
Fig. 2Deimination pathway in the pathogenesis of demyelinating disease. Normal myelin is shown on the left. The promoter of the PAD 2 gene contains methylated cytosines (closed lollipops) along its CpG island, which regulates its transcription. Some MBPcit is made under normal conditions by the deimination of arginyl residues “C” on MBP. The amount of MBPcit is not enough to destabilize the bilayer. During demyelination, the promoter of PAD 2 is demethylated (open lollipops) at cytosines in the CpG island. This demethylated promoter increases transcription of PAD 2, which increases the amount of MBPcit in myelin with subsequent destabilization of the myelin. Degradation of myelin protein fragments, in particular MBPcit fragments, become available to sensitize T-cells in the periphery, initiating the immune response
The removal of methyl groups from methylcytosine in CpG sequences is carried out by a DNA demethylase. We proposed that up-regulation of the demethylase may account for the hypomethylation state of the PAD 2 promoter in MS. When we measured the total demethylase activity in brain homogenates from normal and MS white matter, using a synthetic methylcytosine-guanosine (CpG) substrate [28] we demonstrated that the enzyme in NAWM from MS brain was increased twofold over normal, suggesting the DNA demethylase may have an important role in the hypomethylation of the PAD 2 promoter. Therefore, the hypomethylated state of the PAD 2 promoter may involve up-regulation of the DNA demethylase at least in part.
As a result of the work summarized in this overview, we postulate the following sequence of events during the pathogenesis of MS. The citrullinated MBP, because of its decreased positive charge, prevents proper compaction of the bilayer, so that the multilamellar structure is less stable. Since PAD 2 has been shown to be present in myelin by immunogold electron microscopy (unpublished results), the conversion of non-citrullinated to citrullinated MBP can occur locally in myelin. Preliminary results have shown that immunogold labeling of PAD 2 in mouse optic nerve was not random, but was found in clusters in the axon itself and in the myelin sheath. In the latter, the clusters were found mainly at the junction of the myelin sheath and the axon (periaxonal) with some gold particles distributed in the myelin sheath. The presence of the PAD 2 enzyme at these sites suggests that the citrullinated MBP would be formed in these clusters in myelin, eventually resulting in plaques.
When we examined the extent of labeling in the optic nerve of the spontaneously demyelinating transgenic mouse, we found a threefold increase in the number of gold particles. This increase in PAD 2 labeling demonstrated that increased amounts of PAD 2 were present in the transgenic mouse, accounting for an increase in the citrullinated MBP. Since citrullinated MBP is present in the normal myelin sheath (20% of the total MBP), citrullinated MBP can be tolerated to this extent. However, increased amounts as found in chronic MS (threefold), and in fulminating MS (six to sevenfold), cannot be accommodated in a compact myelin sheath, resulting in destabilization.
Peptidyl arginine deiminase 2 has been reported recently to be elevated in primary open-angle glaucoma, where it was considered responsible for the deimination of several optic nerve proteins, in addition to MBP. Concomitant with this protein deimination, a decrease in arginine methylation was also observed [29]. Therefore, the PAD 2 enzymes have important roles in several nervous system diseases.
Oligodendrocyte apoptosis has been reported in MS white matter in the absence of lymphocytes or myelin phagocytes [4]. The major feature of this type of injury is damage to the most distal part of the oligodendrocyte process (periaxonal), which results in clumping of proteins [5], followed by apoptosis. The possible role of the PAD 2 enzyme in this process has not yet been demonstrated. If it can be established that protein deimination at sites of PAD 2 clusters does occur, it may suggest that demyelination begins in the periaxonal myelin sheath and subsequently moves outward. Although this hypothesis is speculative, it warrants investigation. The increased amount of PAD 2 in MS white matter probably reflects a greater production of the enzyme. Since the PAD 2 promoter is rich in CpG islands, we showed that the promoter was hypomethylated. These studies are ongoing in our laboratory. They suggest immediately that the reversal of the hypomethylated state may be a therapeutic target in MS.
The importance of methylation in a therapeutic strategy of MS has been suggested by us recently [(F. G. Mastronardi et al. 2006, submitted data), 30]. In these studies, we demonstrated that vitamin B12 in combination with either interferon β or paclitaxel, effectively ameliorated demyelinating disease in both EAE and non-immune demyelinating disease. In a detailed mass spectrophotometric study of the various components of MBP, we showed arginine methylation was decreased in most of the components of the MBPs from MS material [31], supporting the view that defects in methylation may involve protein methylation as well. The methylation process requires Vitamin B12, which transfers its methyl group to homocyteine synthesizing methionine, which is then converted to S-adenosytmethionine, the methyl donor in all biological methylation reactions, including methylation of cytosine in DNA to methylcytosine. These studies with the preliminary studies reported above suggest an important role for methylation/demethylation reactions in the pathogenesis of MS.

citrulline
multiple sclerosis
myelin
peptidyl arginine deiminase
demyelination
apoptosis


Toxoplasmosis, Cytomegalovirus, Listeriosis, and Preconception Care

Toxoplasma gondii (T. gondii), cytomegalovirus (CMV), and Listeria monocytogenes (L. monocytogenes) can all negatively affect pregnancy outcomes. Preconception counseling about such effects can reduce the risks posed by these pathogens. Informing women of childbearing age about these pathogens and how to prevent their negative effects can help women make informed decisions about prevention. This brief summarizes some basic information about these infections and provides some web sites and articles for further information about how to prevent them within the context of preconception care.
Toxoplasmosis is a disease caused by Toxoplasma gondii (T. gondii), a protozoan parasite mainly transmitted to humans via three routes: a) ingestion of raw or undercooked contaminated meat; b) exposure to T. gondii oocysts (a form of the organism passed in cat feces), through cat litter or soil (e.g., from gardening or unwashed fruits or vegetables), or contaminated water; and c) congenital in which maternal infection is passed transplacentally via blood to the fetus [1]. Congenital infection leads to stillbirth and severe neurological illness in some instances, although the majority of infected newborns are asymptomatic at birth and some develop sequelae such as mental retardation, blindness, and epilepsy later in life [2]. Extrapolation from regional studies suggests that ∼400–4,000 cases of congenital toxoplasmosis occur each year in the United States [2].
Adults with normal immune function who are infected with T. gondii are usually asymptomatic or have self-limited symptoms (e.g., fever, malaise, and lymphadenopathy) [1]. Once infected, these individuals usually develop an immune response against toxoplasmosis [3, 4]. A recent study based on the National Health and Nutrition Survey conducted from 1988–1994 (NHANES III) reported that, among women aged 15–44 years, seroprevalence of T. gondii antibodies was 15%, suggesting that ∼85% of women of childbearing age are susceptible to T. gondii infection [5].
Three principal interventions are presently used to reduce morbidity and mortality from congenital toxoplasmosis: a) education about how to prevent infection (especially during pregnancy); b) prenatal and newborn screening to identify and treat congenital infection; and c) animal rearing and production methods designed to reduce T. gondii contamination of meat. Of the three, education about how to prevent infection is applicable to adolescents and women in the preconception period. Education programs during pregnancy have been associated with improved knowledge and behavior and a reduction in infection rates [6–9]. Toxoplasma infection can be prevented by one or more of the following: a) cooking meat to a sufficient temperature to kill Toxoplasma; b) peeling or thoroughly washing fruits and vegetables before eating; c) cleaning cooking surfaces and utensils after they have contacted raw meat, poultry, or unwashed fruits or vegetables; d) pregnant women avoiding changing cat litter or using gloves, then washing hands thoroughly; and e) not feeding raw or undercooked meat to cats and keeping cats inside to prevent acquisition of Toxoplasma by eating infected prey [2].
Although prenatal and newborn screening programs have been evaluated, they are controversial because of the lack of proven efficacy of treatment, side effects of treatment, and potential complications of invasive procedures such as amniocentesis to evaluate fetal infection [10–23]. Animal rearing and production methods to reduce T. gondii contamination of meat are effective and have been associated with a reduction in the prevalence of T. gondii in important meat sources such as pork [24]; however the need for improvement in producers’ knowledge and production practices is acknowledged [25]. Education about toxoplasmosis is an important component of preconception care that can be integrated with information about other diseases that affect women.
Human cytomegalovirus (CMV) is the largest DNA virus belonging to the herpesvirus family. Humans are the only reservoirs for the human herpesviruses, and they can transmit these agents through direct contact with infected blood, tissues, bodily fluids, feces, and fomites [26]. A pregnant woman infected with CMV can transmit this virus to her unborn fetus, which can cause damage to the central nervous system, hematopoietic system, kidneys, endocrine glands, gastrointestinal tract, lungs, and liver. Long-term sequelae include cerebral palsy, mental retardation, and hearing loss [26, 27]. The birth prevalence rate of congenital CMV infection varies between ∼0.6–1.5% [28–30] in the United States, making it the most commonly transmitted virus in utero [26, 31] and a major cause of cerebral palsy, mental retardation, and hearing loss among children [27, 31].
CMV infects almost all humans at some point in their lives. Adults with normal immune function infected with CMV are usually asymptomatic or might experience mild flu-like symptoms, or even mononucleosis with symptoms such as malaise, persistent fever, myalgia, and cervical lympadenopathy [26, 31]. Once a human is infected, the virus passes into a latent state [26]. Although the virus can be reactivated, it is usually kept under control, because adults with normal immune function usually retain lifelong immunity against CMV.
Primary CMV infection of women during pregnancy or periconceptionally results in transmission to the fetus transplacentally in ∼30–40% of maternal infections [26, 32–34]. Preexisting maternal immunity strongly reduces the risk of transmission to the fetus [34]. However, approximately 10–15% of newborns with congenital CMV will be symptomatic and from ∼6–25% of those born without symptoms will develop late sequelae [35].
The most common means for women to be infected with CMV is by exposure to toddlers who shed large amounts of the virus in their saliva and urine for many months following their first (usually asymptomatic) infection [26, 36–40]. Daycare providers and pregnant women who have a toddler of their own are at high risk for infection [41–46]. Sexual transmission, blood transfusion, and organ transplantation are other means by which CMV is transmitted [26]. It is important to counsel all women about safe sex practices.
Currently, there are no vaccines available for preventing CMV infection, although some promising advances have been made [47, 48]. It is, therefore, extremely important to provide women of childbearing age with information about how they can prevent CMV infection before conception. The most effective means for preventing CMV infection is handwashing [26, 49–53]. Education about careful hygiene and frequent handwashing, especially after contact with the saliva and urine of young children, and careful disposal of diapers, tissues, and other contaminated items can reduce the transmission of CMV. Avoiding sharing drinking glasses and eating utensils with young children can also prevent transmission. This is especially true for women who work in daycare settings and for those who are pregnant [26, 49–54].
As some studies suggest that CMV infection prior to conception can result in congenital CMV infection [26, 30, 55], counseling all women of childbearing age about how to prevent transmission preconceptionally could significantly reduce the incidence of congenital CMV infection.
Listeriosis is an invasive foodborne infection caused by the motile, gram-positive bacterium Listeria monocytogenes (L. monocytogenes). The disease affects primarily pregnant women, newborns, and adults with weakened immune systems [56]. Maternal infection during pregnancy is usually a self-limited, nonspecific acute febrile illness of the third trimester. In contrast to the maternal illness, fetal and neonatal infection is severe and frequently fatal. Infection in the fetus and newborn occurs by transplacental transmission or possibly from exposure to L. monocytogenes in the perinatal period. The effects of intrauterine infection on the fetus and neonate include preterm labor, amnionitis, spontaneous abortion, still birth, and early-onset of the neonatal sepsis syndrome, evident at birth or shortly thereafter. Late-onset neonatal disease is likely due to infection at or around the time of birth, and presents as meningitis at one to several weeks of age [57].
Listeriosis is a rare disease; the incidence rate in 10 states participating in the Foodborne Diseases Active Surveillance Network (FoodNet) was 2.7 cases per 1,000,000 population in the year 2004 [58]. However, the incidence rate in susceptible subgroups is much higher. The rate among neonates younger than 28 days of age in FoodNet sites was 52.8 per 100,000 population in the year 2000 (CDC unpublished data). More importantly, listeriosis has a very high case fatality rate (20–30% in neonates) and is responsible for an estimated 500 deaths each year in the United States [59].
The food items implicated in outbreaks of listeriosis include ready-to-eat meats, such as turkey delicatessen meat [60], meat paté [61], pork tongue in jelly [62], hot dogs [63], and dairy products made from unpasteurized milk, in particular fresh soft cheeses [64].
Primary prevention for listeriosis focuses on improvements in food processing and on consumer education. Substantial efforts by the food industry and food regulatory agencies have been directed toward reducing the likelihood that high risk foods will be contaminated with L. monocytogenes [65]. Despite this, pregnant women, immunocompromised persons, and the elderly should be advised to avoid paté, fresh soft cheeses made from unpasteurized milk and to cook ready-to-eat foods such as hotdogs, delicatessen meats, and left over foods until steaming [66]. Information regarding these foods at high risk of contamination with Listeria can be incorporated into preconception care dietary recommendations.
For more information
CDC web sites
Toxoplasmosis: http://www.cdc.gov/ncidod/dpd/parasites/ toxoplasmosis/default.htm
CMV: http://www.cdc.gov/cmv
Listeria: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ listeriosis_g.htm
Publications for practitioners
Disclaimer: These references are included for information only. CDC has no control over the information in these articles. Views and opinions of these organizations are not necessarily those of CDC, the Department of Health and Human Services (HHS), or the U.S. Public Health Service (PHS).
American College of Obstetricians and Gynecologists. Perinatal viral and parasitic infections. ACOG Practice Bulletin 2000;20.
Brundage, SC. Preconception health care. American Family Physician. 2002 June; 65(12):2507–14.
Hanlin RB. Congenital infections and preconception counseling. J S C Med Assoc. 2002 Oct;98(6):277–80.

toxoplasmosis
cytomegalovirus
listeriosis
preconception care
listeria
infection


Acute success and short-term follow-up of catheter ablation of isthmus-dependent atrial flutter; a comparison of 8 mm tip radiofrequency and cryothermy catheters

Objectives To compare the acute success and short-term follow-up of ablation of atrial flutter using 8 mm tip radiofrequency (RF) and cryocatheters.
Introduction
Atrial flutter is a common arrhythmia [1], difficult to suppress with medication [2], and is associated with significant symptoms. Since it was first proposed [3–5], ablation for atrial flutter has increasingly been used for its therapy, especially since induction of bidirectional cavo-tricuspid isthmus block was shown to be associated with better immediate outcomes and lower recurrence rates [6–11]. Failure to successfully ablate atrial flutter in the long-term may be due to particular anatomic problems [12–14], poor catheter stability in this region, and incorrect interpretation of isthmus block. Important developments in the field of atrial flutter ablation have been a better understanding of the anatomy of the isthmus, refinements of the definition of bidirectional isthmus block, and the arrival of new catheter technology.
Numerous studies have compared different energy types, different catheter tip sizes and different energy settings [15–19], as well as the use of advanced cardiac mapping systems [20]. The preferred tools now are 8 mm, or irrigated tip catheters [21]. From a recent meta-analysis it would appear that these two technologies are equally effective with acute success quoted at 84% and 85% for the primary catheter chosen before changeover, whereafter final success rates of up to 99% can be achieved [22]. Not all studies have this high success rate and the preponderance of studies on ablation of atrial flutter suggest that these high success rates are not repeated in all centers [23]. Also of importance is a clinical recurrence rate, in the face of acute success, of at least 5 to 12% when using radiofrequency [19, 24, 25]. Disadvantages of radiofrequency (RF) energy include pain, overheating with “popping”, char formation and risk to coronary arteries.
Cryoablation is a relatively recent addition to the transvenous ablation armamentarium and has been shown to be comparable to RF for some arrhythmias. It may have some advantages over RF, especially as regards discomfort during ablations [17, 26]. The development of 6 and 8 mm tip cryocatheters has increased the interest in this technology for atrial flutter ablation. As far as we are aware there have been no randomized comparative studies published comparing 8 mm tip cryocatheters with 8 mm tip RF catheters.
Methods
Patient population
Consecutive patients with ongoing symptoms and documented atrial flutter with or without fibrillation were included. At least one recent episode of atrial flutter (within the last 6 months) was documented on a 12 lead ECG and was suggestive for isthmus dependency. Patients with drug-induced flutter (and prior AF) could be included, and had their drug therapy continued after ablation. Patients were excluded if they had undergone a previous flutter ablation, if thrombus was present in the atria, or after previous cardiac surgery for valvular or congenital heart disease. Baseline investigations included a standard echocardiogram, a simple questionnaire asking about clinical wellbeing and a subjective assessment of the arrhythmia burden both in terms of duration and frequency. The study was approved by the ethics committee of our institution. All patients signed written informed consent.
Assessment before ablation
Antiarrhythmic drugs, except for AV nodal slowing agents, and amiodarone, were discontinued at least five half-lives prior to ablation. Patients were studied in the fasting, post-absorptive state. A coronary sinus (CS) catheter was inserted through the left subclavian vein, and a multipolar circular right atrial catheter with alternating 2–10–2 mm interelectrode distance was positioned from the right groin, with the tip positioned immediately lateral to the planned position of the isthmus line and anterior to the crista terminalis. Heparin 100 U/kg was given and a further 5 U/kg given if the procedure lasted longer than 180 min.
If the patient was in sinus rhythm, isthmus conduction was confirmed by pacing. If flutter was present, entrainment was performed to confirm isthmus dependence, and the patient left in flutter. If AF was present, the patient was cardioverted after a transoesophageal echocardiogram, and then isthmus conduction confirmed. Absence of isthmus conduction or non-isthmus dependence was not seen in the selected patients. No induction of arrhythmia was attempted if patients had sinus rhythm.
Radiological assessment of the right atrial isthmus was made in a right anterior oblique (RAO) 30°, and a left anterior oblique (LAO) 45° view (each with 40 cc at 18 cc/s). Angiograms were acquired digitally to allow for post-hoc analysis. The treating physician was able to view the angiographic findings to optimize the planned ablation line. The isthmus length was assessed from the inferior hinge point of the tricuspid valve to the IVC at the end of atrial diastole (the frame before opening of the tricuspid valve) [14]. Morphology was assessed visually as to the presence of a Eustachian valve or a recess, as well as to the general shape i.e. flat or concave.
Ablation procedure
The catheters were a 9Fr 8 mm tip catheter (FreezorMax, Cryocath Technologies Inc, Kirkland, Canada) with a cryoconsole for the cryoablation group, and a 7Fr 8 mm tip single sensor catheter (EPT Blazer II, Boston Scientific, Natick, MA, USA) with an EPT-1000XP generator for the radiofrequency group. A large curve was initially selected in both groups, with change out of catheter curve during the study only as necessary. Applications of −75°, for 4 min were given with cryothermy, and applications of 60 Watt, for 60 s, targeted at 60°C for RF. Lines were made with discrete applications between the tricuspid valve and the inferior vena cava at an approximately 6 o’clock position in LAO 45°, unless otherwise dictated. If termination of atrial flutter occurred, or if the patient was in sinus rhythm, continuous pacing from the proximal coronary sinus was employed to continually assess isthmus conduction. After the first line, a new assessment of conduction was performed. If conduction over the isthmus remained present, gaps were sought. If there was still isthmus conduction, a slightly more medial or lateral line was made. In no patient was an attempt made to perform a septal ablation line. Final assessment of acute block was confirmed after 30 min waiting.
The end point for successful ablation was induction of complete bidirectional isthmus block, defined as the presence of reversal of activation on the lateral and septal wall when pacing the CS os and low lateral RA, the presence of widely split potentials along the isthmus line, by activation mapping across the isthmus, and by differential pacing. All 4 were required before calling the ablation successful. In the case where bidirectional block was not achieved, ablation was stopped when no large, sharp signals could be identified over a broad area of the isthmus.
As pain perception was assessed, sedation was standardized. Before venous puncture 5 mg of diazepam was given intravenously, and repeated at the patient’s request. Fentanyl 50 μg intravenously was given when the patient requested pain control and the physician considered this necessary. This was repeated as needed. Dosages of both diazepam and fentanyl were recorded.
In the initial 40 patients creatine kinase (CK) and CK-MB were taken before the procedure, 2 and 24 h after the start of the procedure. For the final 22 patients the laboratory had changed the measurement to CK mass. We then modified the protocol to measure CK-MB mass, Troponin T, and Myoglobin at 4 and 24 h after the start of the procedure.
No crossover, other than in catheter curve, was allowed in an attempt to remove any possible bias. Change over to an irrigated tip ablation catheter was also not allowed. In patients in whom no block could be induced, a repeat procedure was scheduled not earlier than 6 weeks after the initial ablation, at the physicians’ discretion. The choice of energy source at that time was at the physicians’ discretion.
Patients were all questioned with regard to pain perception using a visual analogue score, where patients are shown a line from 0 to 10, where 0 is no pain, and 10 is the highest pain level imaginable, and were asked to point to the position on the line where their pain level during ablation was.
Follow-up
All patients received an event-recorder for the first 6 weeks after the procedure and were requested to send at least daily strips as well as strips made during symptoms. Patients visited the outpatient clinic 6 weeks after the procedure. After this period all patients were asked to report symptoms and if these were present were given a further event monitor until documentation of symptoms was obtained. If at all possible a 12 lead ECG was also obtained. A second assessment with a questionnaire was performed after 3 months, again asking a question about general clinical well being and also symptom burden in regard to duration and frequency. Clinical files were followed up after 9 months.
Statistical analysis
For patients in whom another ablation was performed (AVNRT in two, and pulmonary vein ablation in six others) procedure and fluoroscopy times were limited to the flutter approach, which was done first, including 30 min waiting time. Biomarker assessment was not done in these patients. Continuous variables are expressed as mean ± standard deviation, with medians as necessary. Parametric and non-parametric tests were used where appropriate. A p-value of < 0.05 was considered significant.
Results
Patient data
62 patients were included as planned, with clinical characteristics as outlined in Table 1. There were no significant differences in any of the parameters between the group assigned to radiofrequency (RF group) versus that assigned to cryotherapy (cryo group). The large number of patients with prior AF is due to the fact that we had initially taken a decision to perform isthmus ablation first in all patients with AF who had shown typical atrial flutter on any 12-lead ECG, prior to performing a left atrial procedure, initially in a separate procedure and only later in the same session.
Table 1Demographics AllCryoRFp valueNumber623230Age (years ± SD)56 ± 1055 ± 1156 ± 9NSMale/female27/527/528/2NSAtrial fibrillation history47 (76%)25 (78%)22 (73%)NSCryo Cryoablation; NS not significant; RF radiofrequency; SD standard deviation
Angiographic data
Right atrial angiography was not performed in 4 patients because of mild renal dysfunction or allergy to contrast material. The angiogram was of insufficient quality in eight others.
The mean isthmus length was 35.2 ± 14.6 mm and its topography was assessed as being flat or only mildly concave in 28, markedly concave in 19 and showed a pit or aneurysm in 10. A clear Eustachian valve was seen on six angiograms.
Ablation data
Assessment of acute results showed bidirectional isthmus block, using the criteria mentioned, in 47 of 62 patients (76%). This was in 25 of 30 patients (83%) of the RF group and in 22 of 32 (69%) of the cryo group (NS). In 1 patient in the RF group the procedure was terminated because of recurrent AF with early recurrence and inability to assess isthmus block. This patient was taken as a failure which was confirmed at the time of a subsequent AF ablation. Procedural data for all patients are given in Table 2.
Table 2Procedure data and recurrent arrhythmias AllCryoRFp valueNumber623230Application number22 ± 1318 ± 1025 ± 160.05Ablation time (s)2,742 ± 1,9303,792 ± 1,9001,459 ± 950<0.001Acute success47 (76%)22 (69%)25 (83%)NSSingle line47 (76%)25 (78%)22 (73%)NS2 lines9 (15%)2 (6%)7 (24%)0.0733 lines4 (6%)3 (9%)1 (3%)NSReversal of block during application7 (11%)6 (19%)1 (3%)0,091Reversal of block during 30 min6 (10%)1 (3%)5 (7%)NSIsthmus length (mm)35 ± 1535 ± 1736 ± 11NSSheath usage7 (11%)0 (0%)7 (23%)<0.005Recurrent arrhythmiasFlutter (typical) After success1 (2%)0 (0%)1 (4%)NS After failure11 (73%)7 (70%)4 (50%)NSFlutter (atypical)1 (2%)0 (0%)1(3%)NSAtrial tachycardia5 (8%)2 (6%)3 (10%)NSAtrial fibrillation28 (45%)13 (41%)15 (50%)NSThe numbers are given with the standard deviation.Cryo Cryoablation; NS not significant; RF radiofrequency
In the successful patients the number of applications to ensure block in the whole group was 17 ± 11. It was 23 ± 13 in the RF group and 12 ± 6 in the cryo group (p < 0.005). Total ablation time was 1,283 ± 777 s and 2,905 ± 1,245 s (p = 0.0001). In those in whom bidirectional block could not be achieved, the total number of applications was 33 ± 14. For RF and cryo the values were 39 ± 21 applications with a total time of 2,724 ± 1,102 s vs 29 ± 8 applications with a time of 5,873 ± 1,337 s (p < 0.03 and < 0.0011, respectively).
In 22 of 30 in the RF group and 25 of 32 in the cryo group, a single line at approximately 6 o’clock was drawn. In the RF group two lines were drawn in seven, and three in one. In the cryo group two lines were made in four, and three lines in three. The need to draw more than one line was associated with failure of the procedure in four of eight in the RF group and five of seven in the cryo group (NS).
Short lived block, either recurring during the application or immediately thereafter, occurred in one patient in the RF group and in six in the cryo group, with a trend to statistical significance (p = 0.091), with it being a predictor of failure if it occurred more than three times. In five patients in the RF group and one patient in the cryo group, conduction recurred later during a waiting period (with a median of 15 min) requiring further applications (NS). In only one patient in the RF group was late recurrence associated with failure to induce bidirectional block. In no patient in whom bidirectional block was present at the end of the 30 min waiting period did isoprenaline change this. The average power applied in the RF group was 52 ± 6 W.
In the RF group the signal was significantly diminished at the end of each application whether isthmus block was present or not, while there tended to be preservation of signals on the cryoablation catheter after ablation across the isthmus until block occurred. Only then were low voltage signals seen.
Failures were not significantly associated with length of the isthmus (39.2 ± 23.5 vs 34 ± 9.0 mm in success, although there was a trend to this (p = 0.12) There was no significant difference in anatomy between the two groups.
Procedure data and complications
The overall procedure time was 160 ± 49 min, with no difference between 170 ± 48 min in the RF group and 151 ± 49 min in the cryo group. Overall fluoroscopy times were 28 ± 14 min, with a difference between 33 ± 15 min in the RF group and 23 ± 11 min in the cryo group (p < 0.02).
Change of catheter curve occurred in one patient in the RF group from large to standard curve. An SL1 sheath (Daig, Minnetonka, MN, USA) was used in seven patients in the RF group for stability, while no sheath usage occurred in the cryo group (p = 0.005). During the procedure, six patients required cardioversion for induced AF, four in the RF group and two in the cryo group (NS). There were two small pericardial effusions seen on echocardiography without further significance (one in each group).
For the initial 40 patients there was a significantly higher peak CK and CK-MB after cryo (Table 3). This remained so after 24 h. For the last 22 patients we observed the same for CK-MB mass, but not for Troponin T (Table 3).
Table 3Biomarkers after catheter ablation AllCryoRFp valueProcedure valuesCK (U/l)141 ± 96184 ± 10296 ± 600.02CK-MB (U/l)27 ± 1636 ± 1718 ± 80.011CK-MB mass (μg/l)18 ± 2133 ± 244.4 ± 1.20.004Troponin T (μg/l)0.49 ± 0.320.54 ± 0.380.39 ± 0.27NSValues after 24 hrsCK (U/l)264 ± 245289 ± 173136 ± 740.022CK-MB (U/l)37 ± 2851 ± 3018 ± 60.011CK-MB mass (μg/l)8.4 ± 7.312 ± 105.16 ± 3.30.07Troponin T (μg/l)0.44 ± 0.300.54 ± 0.360.38 ± 0.29NSThe numbers are given with the standard deviation.CK Creatine kinase; Cryo cryoablation; NS not significant; RF radiofrequency
In assessing the level of comfort during the procedure and the pain experienced by patients, similar numbers from both groups assessed sedation as adequate (67% for RF and 63% for cryo). The pain scores given at the end of the procedure were not significantly different (42.9 ± 24.0 for RF and 43.7 ± 15.8 for cryo). Diazepam was given as standard at the beginning of the procedure and as necessary thereafter for discomfort related to having to lie still for protracted periods. The usage of diazepam was statistically similar in both groups (7.4 ± 3.4 mg in the RF group and 8.0 ± 3.1 mg in the cryo group). However, a significantly higher usage of fentanyl in the RF group was observed (70.0 ± 44.9 μg vs 10.0 ± 22.1 μg; p < 0.01).
Follow-up results
Patients were followed up for between 90 and 411 days (138 ± 81 days, median 90 days), which was similar in both groups (p = ns).
A total of 6 patients were taking no antiarrhythmic medication prior to ablation and this increased to 13 post isthmus ablation.
Recurrent arrhythmias were frequent in both groups of patients. ECG documented recurrent flutter occurred only in one patient from the RF group. A further procedure confirmed recovery of isthmus conduction. This recurrence was seen at 14 months post ablation, whereas he had previously had monthly episodes of flutter. One other patient had symptoms with an apparently non-isthmus dependent flutter documented on ECG on day 1 post ablation and not since, and elected not to have a further procedure. We cannot exclude asymptomatic arrhythmias but at least during the first 6 weeks, patients sent in daily event monitor transmissions, which one would hope would have captured at least some asymptomatic recurrences, particularly of atrial flutter.
In 11 of 15 patients in whom the initial procedure failed, a redo procedure was performed after the elected period of 6 weeks because of documented recurrent flutter. The redo procedure required a small number of point touch ups in five patients (three in the RF group and two in the cryo group), while more extensive diffuse isthmus applications were required in six (one in the RF group and five in the cryo group). In those undergoing repeat ablation, success was achieved in all and during follow up no recurrent atrial flutter was noted.
Previously undocumented atrial tachycardias were also seen in both groups (three in the RF group, and two in the cryo group). ECG documented AF recurrence occurred in 28 patients, at a similar rate in both groups (15 in the RF group and 13 in the cryo group). The likelihood of asymptomatic recurrences is probably higher for atrial fibrillation but was not the primary focus of this study.
Discussion
Acute success
This study showed an acute success rate of 83% for an 8 mm tip RF catheter vs. 69% for an 8 mm tip cryocatheter. This non significant difference is clearly concerning. However, the cryoablation group required significantly less applications to achieve success, with a similar procedural duration, significantly lower fluoroscopy time, and with a much lower requirement for analgesia with fentanyl. Arrhythmia recurrences in the initially successful patients were similar with a very low flutter recurrence rate.
The acute success rate with 8 mm tip RF compares favorably with that found in the meta-analysis of Da Costa et al. [22]. However, the acute results for cryotherapy are lower than in previously published studies. The shorter fluoroscopy time using cryotherapy is related to cryoadherence during applications [26]. With the catheter firmly attached, no fluoroscopy to check position is required. This is also reflected in the fact that no long sheaths were necessary. A lower requirement for analgesia has previously been described and this study confirms this finding [17, 26].
Difficulties during ablation
Recurrence during or shortly after ablation occurring more commonly in the cryo group probably relates to a reversible cooling effect at the periphery of the ice ball which recovers during or shortly after termination of ablation, while the central lesion acutely formed is more permanent. The more common recurrence during the waiting period after RF ablation probably relates to a longer reversal time of the acute RF effects (either edema or temperature effect).
Cryoablation for flutter
Cryoablation may have some advantages over RF in addition to those mentioned above such as less thrombogenicity [27], and maintenance of tissue architecture with homogenous well delineated lesions [28]. A 10Fr, 6.5 mm tip catheter showed acute success of 94–100% and 6 month recurrence rates of 0–25% [17, 29, 30]. A system with 7Fr, 6 mm tip catheters showed success in 87% to 88% using a septal line, without symptomatic recurrences, but with resumption of isthmus conduction at repeat study at 6 months in 30–35% [31, 32]. Using a 9Fr, 8 mm tip increased success rates to100% with symptomatic recurrence of 0% to 10% and recurrent conduction at 1 to 3 months in 19% to 32% [32–34]. There is some discussion as to whether 3, 4 or 8 min of ablation are needed for adequate lesion formation [33, 35].
Higher peak CK levels, confirmed by the high CK-mass in the last patients, suggest that the damage caused by cryotherapy may be more extensive despite the lower number of applications. Several explanations are possible. The first is that the CK levels may be underestimated after RF, which denatures proteins in another way than cryotherapy. Troponin T levels are more accurate in estimation of myocardial damage, but they also tended to be higher, be it not significantly. This may suggest that lesions are equal with both approaches.
Future developments
Newer data suggest that ablation per point guided by the maximal voltage may be a useful technique, but this has not been confirmed for cryotherapy [36].
Study limitations
This study was a single centre, largely single operator study involving a relatively limited number of patients with a high population of AF patients, making follow-up rather complicated. In present practice and later in this series, patients with the combination are often treated in one session, which still leaves physicians the choice whether cryo should be used for flutter, following its use for the pulmonary veins [37]. We only studied the acute and short-term success, while flutter recurrence may happen at more than 4 months of follow-up. While we attempted to exclude asymptomatic recurrences especially in the first 6 weeks, we cannot exclude the possibility of these, especially of AF and also late after the ablation. Isthmus conduction recovery is also possible in asymptomatic cases. In the RF group we limited the power to 60 W to ensure safety and this may be too conservative. Further, in our practice routine use of long sheaths was discouraged. At the time of this study use of an 8 mm tip with RF was considered as effective as irrigated tip ablation, so we elected to use this approach for the RF group. More recent studies have shown higher success rates than we obtained and have also suggested alternatives in the approach. These include the use of irrigated tip catheters and long sheaths as well as other technical issues and newer techniques such as maximal voltage guided ablation. A higher success rate for RF would clearly have been even more prejudicial to cryotherapy in this study. We were also extremely critical in our assessment of isthmus conduction, using multiple criteria. Angiograms were also performed in fixed views and not based on other catheter positions as has been suggested by others [14].
Conclusion
Acute success with cryotherapy for atrial flutter ablation, while non-statistically less effective in this study, requires fewer applications and is associated with a significantly lower requirement for pain relief. While our acute results for both cryotherapy and RF may not be as high as those in some comparable studies the recurrence rate was only 2.5% in the RF group, with no clinical recurrence in the cryo group. While cryotherapy cannot perhaps be advocated as first line therapy, it may be useful in certain circumstances. Certainly, if cryoablation is performed for AF, and if the isthmus needs to be ablated, cryotherapy might be used as well [37].

catheter ablation
atrial flutter
radiofrequency
cryothermy
arrhythmia
atrial fibrillation