Keywords

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Taxonomy, Historical and Current

Sutterellaceae (Sut.te.rel.la’ce.ae. N.L. fem. n. Sutterella type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Sutterellaceae, the Sutterella family).

The genus Sutterella was first described by Wexler et al. (1996) and was placed within the family Alcaligenaceae in the second edition of Bergey’s Manual of Systematic Bacteriology (Wexler 2005). The genus Parasutterella was described by Nagai et al. (2009) as the closest neighbor of genus Sutterella. Morotomi et al. (2011) proposed the new family Sutterellaceae to accommodate these two genera after finding that the genera Sutterella and Parasutterella formed a separate line of descent within the order Burkholderiales. In their research, this lineage could not be associated with any of the four known families (Alcaligenaceae, Burkholderiaceae, Comamonadaceae, and Oxalobacteraceae) in the order Burkholderiales (Fig. 38.1 ). The rationale for the new family was based both on the distinct phylogenetic positions and on the biological and biochemical differences of the genera Sutterella and Parasutterella from known genera in the family Alcaligenaceae, which is phylogenetically the nearest neighboring family of the Sutterellaceae. All known members of the genera Sutterella and Parasutterella are oxidase- and catalase negative, and no aerobic growth has been observed, although some species may grow in a microaerophilic atmosphere. In contrast, species in the Alcaligenaceae are oxidase- and catalase positive, and can generally grow aerobically (Table 38.1 ). All type strains of the Sutterellaceae contain C18:1ω9c (32–68 % of the total) and C16:0 (9–23 %) as the predominant fatty acids (Tables 38.1 and 38.2 ). However, C18:1ω9c has not been reported to be a major component in any species of the family Alcaligenaceae (Table 38.1 ). The major respiratory quinone of the members of the Sutterellaceae is methylmenaquinone-5 (MMK-5) or MMK-6, whereas ubiqinones have not been detected (Tables 38.1 and 38.3 ). In contrast, species of the family Alcaligenaceae have, in general, been characterized by the presence of ubiquinone-8 (Q-8) as the major isoprenoid quinone (Table 38.1 ; Fletcher et al. 1987; Oyaizu-Masuchi and Komagata 1988).

Fig. 38.1
figure 1figure 1

Phylogenetic reconstruction of the family Sutterellaceae based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. The sequence datasets and alignments were used according to the All-Species Living Tree Project (LTP) database (Yarza et al., 2010; http://www.arb-silva.de/projects/living-tree). The tree topology was stabilized with the use of a representative set of nearly 750 high-quality type-strain sequences proportionally distributed among the different bacterial and archaeal phyla. In addition, a 40% maximum-frequency filter was applied in order to remove hyper variable positions and potentially misplaced bases from the alignment. Scale bar indicates estimated sequence divergence. This tree was provided by Dr. Raul Muñoz of the Instituto Mediterráneo de Estudios Avanzados

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Table 38.1 Differential characteristics of members of the family Sutterellaceae and those of the phylogenetically nearest (Fig. 38.1 ) family, the Alcaligenaceae
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Table 38.2 Fatty acid compositions of the type strains of the genera Sutterella and Parasutterella
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Table 38.3 Isoprenoid quinone compositions of the type strains of the genera Sutterella and Parasutterella
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Molecular Analyses

Phylogeny

On the basis of 16S rRNA gene sequence similarities, members of the family Sutterellaceae are members of the order Burkholderiales, with the nearest neighboring family being the Alcaligenaceae (Fig. 38.1 ). The phylogenetic distance between type species of the family is relatively high. The 16S rRNA gene sequence of the type strain of Sutterella wadsworthensis shares 94.6 % similarities with the sequences of Sutterella parvirubra and Sutterella stercoricanis. The type strains of S. parvirubra and S. stercoricanis share 94.7 % similarity. The type strains of the two Parasutterella species, Parasutterella excrementihominis and Parasutterella secunda, share 90.0 % similarity. In contrast, similarities between members of the Sutterellaceae and type species of the neighboring family Alcaligenaceae range between 88.3 % and 90.9 %. Thus, all species described so far can be unambiguously identified by their 16S rRNA gene sequence (Morotomi et al. 2011).

Genome

Sutterella parvirubra YIT 11816T, P. excrementihominis YIT 11859T, and four strains of S. wadsworthensis (HGA0223, HGP1, 3_1_45B, and 2_1_59BFAA) were selected for inclusion in the catalog of reference genomes by the Human Microbiome Project (http://commonfund.nih.gov/hmp/) and, at the time of writing, the assembled and annotated genomic sequences of three strains (S. parvirubra YIT 11816T, P. excrementihominis YIT 11859T, and S. wadsworthensis 3_1_45B) have been submitted to the GenBank/EMBL/DDBJ databases. The genome size, G+C content, number of predicted protein-encoding genes, and number of predicted rRNA and tRNA genes of these strains are listed in Table 38.4 . Details are available from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

Table 38.4 Basic genome statistics for strains of the family Sutterellaceae
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DNA–DNA Hybridization

Sutterella wadsworthensis WAL 9799T did not undergo DNA–DNA hybridization with DNA from either Campylobacter gracilis or other Campylobacter species (Campylobacter rectus, Campylobacter curvus, Campylobacter consisus, Campylobacter sputorum, and Campylobacter showae), which cannot be differentiated phenotypically (Wexler et al. 1996). The degree of the hybridization between S. wadsworthensis CCUG 42229T (= WAL 9799T) and S. stercoricanis 5BAC4T was 35 %, which is low enough to be consistent with separate species status (Greetham et al. 2004). No hybridization studies have been done for the Parasutterella species, as the 16S rRNA gene sequence similarity between the type strains of the respective species was only 90.0 % that indicates separate genospecies (Morotomi et al. 2011).

Matrix-associated laser desorption/ionization—time of flight mass spectrometry (MALDI-TOF MS).

Eleven strains of S. wadsworthensis, including the type strain, were analyzed by MALDI-TOF MS, and the results included a dominant peak at approximately 9,400 Da that was common to all S. wadsworthensis strains (Mukhopadhya et al. 2011). No mass spectrometry data is available for any other species in the Sutterellaceae.

Neither riboprinting nor ribotyping analyses are available for any member of the Sutterellaceae.

Phenotypic Analyses

Members of the Sutterellaceae are strictly anaerobic or microaerophilic, non-motile, non-spore-forming, Gram-negative rods or coccobacilli. Biochemically, they are largely unreactive and asaccharolytic.

Differential characteristics of members of the family Sutterellaceae and those of the phylogenetically nearest family, the Alcaligenaceae, are listed in Table 38.1 . All type strains of the genera Sutterella and Parasutterella cannot grow under aerobic conditions, and are oxidase- and catalase-negative (Table 38.1 ). In addition, they are negative for urease and indole production and for hydrolysis of gelatin and aesculin (Greetham et al. 2004; Sakon et al. 2008; Nagai et al. 2009; Morotomi et al. 2011 and unpublished). In contrast, species of the family Alcaligenaceae have, in general, the opposite characteristics (Table 38.1 ). All type strains of the Sutterellaceae contain C18:1ω9c (32–68 %) and C16:0 (9–23 %) as the predominant fatty acids, and MMK-5 or MMK-6 as the major respiratory quinone (Tables 38.1 and 38.2 ). The typical fragmentation of a ubiquinone ring nucleus at mole peak of m/z = 197 was not detected in these strains, indicating that ubiquinones are not present in the known strains of the genera Sutterella and Parasutterella (Morotomi et al. 2011). In contrast, species of the family Alcaligenaceae are characterized by the presence of ubiquinone-8 (Q-8) as the major isoprenoid quinone, and fatty acids other than C18:1ω9c are the major cellular fatty acids (Table 38.1 ).

All type strains of the genera Sutterella and Parasutterella do not utilize the following API 20A substrates: L-arabinose, D-cellobiose, glucose, glycerol, lactose, maltose, D-mannitol, D-mannose, D-melezitose, D-raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, D-trehalose, and D-xylose (Greetham et al. 2004; Sakon et al. 2008; Nagai et al. 2009; Morotomi et al. 2011 and unpublished). They are negative for the following API ZYM and API rapid ID 32 A reactions: N-acetyl-β-glucosaminidase, α-arabinosidase, chymotrypsin, cystine arylamidase, fermentation of mannose, fermentation of raffinose, α-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, glutamyl glutamic acid arylamidase, histidine arylamidase, indole production, lipase (C14), α-mannosidase, 6-phosphate β-galactosidase, proline arylamidase, pyroglutamic acid arylamidase, trypsin, urease, and valine arylamidase (Greetham et al. 2004; Sakon et al. 2008; Nagai et al. 2009; Morotomi et al. 2011 and unpublished). Diagnostic phenotypic differences for the fatty acid compositions of the type strains of the genera Sutterella and Parasutterella are listed in Table 38.2 .

The fatty acid and isoprenoid quinone compositions of the type strains of the genera Sutterella and Parasutterella are listed in Tables 38.2 and 38.3 , respectively. Table 38.5 provides additional phenotypic details.

Table 38.5 Diagnostic phenotypic differences for the type strains of the genera Sutterella and Parasutterella
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Sutterella Wexler, Reeves, Summanen, Molitoris, Mcteague, Duncan, Wilson, and Finegold 1996a, 257VP

Sut.ter.el’la. M.L. dim. Fem. n. Sutterella named in memory of Vera Sutter, respected colleague and director of the Wadsworth Anaerobe Laboratory for 20 years.

The data described for Sutterella species in this section are from Sakon et al. (2008), Greetham et al. (2004), Wexler et al. (1996), Wexler (2005).

Cells of Sutterella species are straight rods (S. wadsworthensis and S. stercoricanis), 0.5–1.0 μm × 1.0–3.0 μm, or are coccoid to coccobacillary (S. parvirubra), approximately 0.4–1.0 μm × 0.4–2.0 μm. They grow under anaerobic conditions or in a microaerophilic atmosphere. Colonies of S. wadsworthensis on Brucella blood agar with 5 % lysed sheep blood, 1 μg/mL Vitamin K1, 1 μg/mL hemin, and 1 % w/w formate/fumarate after 48 h at 37 °C in anaerobic chamber are circular, entire, convex, yellow to brown, translucent to opaque, 1–1.5 mm in diameter. Colonies of S. parvirubra on GAM agar after 48 h anaerobic incubation are 0.2–1.1 mm in diameter, circular, flat, and translucent. No culture data has been reported for S. stercoricanis in the literature. Sutterella wadsworthensis and S. stercoricanis are resistant to 20 % (v/v) bile; no data are available for S. parvirubra. Other biological and biochemical characteristics of the type strains of the genus Sutterella are listed in Tables 38.1 – 38.3 and 38.5 .

The G + C values for DNA of the species determined by HPLC for S. parvirubra YIT 11816T and S. stercoricanis 5BAC4T are 64.4 and 60.0 mol%, respectively. This value for S. parvirubra YIT 11816T (64.4 mol%) is slightly lower than that determined by the genome analysis (65.3 mol%, Table 38.4 ). The value for S. wadsworthensis 3_1_45B determined by genome analysis is 55.1 mol% (Table 38.4 ), and that for the type strain has not been reported.

The type species is S. wadsworthensis.

The type strains are S. parvirubra YIT 11816T (= DSM 19354T = JCM 14724T); S. stercoricanis 5BAC4T (= CCUG 47620T = CIP 108024T); S. wadsworthensis WAL 9799T (= ATCC 51579T = CCUG 42229T = CIP 104799T = DSM 14016T).

Parasutterella Nagai, Morotomi, Sakon, and Tanaka 2009, 1795VP

Pa.ra.sut.te.rel’la. Gr. prep. para besides, next to; N.L. fem. n. Sutterella name of a bacterial genus; N.L. fem. n. Parasutterella a genus similar to Sutterella.

The data described for Parasutterella species in this section are from Nagai et al. (2009) and Morotomi et al. (2011).

Cells of Parasutterella species are cocci to coccobacilli, 0.4–1.3 μm × 0.6–2.6 μm. Colonies on modified GAM agar after 4 days of anaerobic incubation at 37 °C are translucent to beige, circular, convex, and pinpoint in size. Growth in peptone-yeast extract broth (Holdeman et al. 1977) is weak, producing no visible turbidity, and no short-chain fatty acids are detected as an end product of metabolism. Addition of glucose, lactate, or succinate does not enhance growth or result in the production of short-chain fatty acids. Other biological and biochemical characteristics of the type strains of the genus Parasutterella are listed in Tables 38.1 – 38.3 and 38.5 .

The G+C of DNA of the species determined by HPLC for P. excrementihominis YIT 11859T and P. secunda YIT 12071T are 49.8 and 48.2 mol%, respectively. The value for P. excrementihominis YIT 11859T (49.8 mol%) is slightly higher than that determined by genome analysis (48.1 mol%, Table 38.4 ).

The type species is P. excrementihominis.

The type strains are P. excrementihominis YIT 11859T (= DSM 21040T = JCM 15078T) and P. secunda YIT 12071T (= DSM 22575T = JCM 16078T).

Isolation, Enrichment, and Maintenance Procedures

Sutterella wadsworthensis grows under anaerobic conditions or in a microaerophilic atmosphere of 2 % or 6 % oxygen. The type strain of S. stercoricanis grows in a microaerophilic atmosphere of 2 % oxygen but not at 6 % oxygen, or under anaerobic conditions. Growth of the type strains of S. parvirubra, P. excrementihominis, and P. secunda was only observed under strict anaerobic conditions (Wexler et al. 1996; Wexler 2005; Greetham et al. 2004; Sakon et al. 2008; Nagai et al. 2009; Morotomi et al. 2011).

Members of the family Sutterellaceae are asaccharolytic and their colonies on agar plates are very small, ranging from pinpoint in size to 1.5 mm in diameter. Therefore, the main problem in isolating these organisms from samples of feces or intestinal contents is the exclusion of the dominant intestinal microbiota, which cover large areas of the isolation plates.

Sutterella wadsworthensis is isolated on Brucella blood agar with 5 % lysed sheep blood, 1 μg/mL Vitamin K1, 1 μg/mL hemin, and 1 % w/w formate/fumarate, and is mainly obtained from the intestinal tract and from infections of gastrointestinal origin (Wexler 2005).

Sutterella parvirubra YIT 11816T was isolated from the feces of a healthy human adult on a medium 10 (Caldwell and Bryant 1966) agar plate supplemented with 40 mM succinic acid as the sole carbon source, from which the other basal carbon sources (a mixture of glucose, cellobiose, soluble starch, and volatile fatty acids) had been excluded (Sakon et al. 2008).

Sutterella stercoricanis 5BAC4T was isolated from the feces of a healthy male Labrador Retriever dog on bacteroides agar (Holdeman et al. 1977) by Greetham et al. (2004).

Parasutterella excrementihominis YIT 11859T was isolated from the feces of a healthy human adult on anaerobe basal agar (Oxoid), pH 6.0 (Nagai et al. 2009).

Parasutterella secunda YIT 12071T was isolated from the feces of a healthy human adult on modified Gifu anaerobic agar (GAM; Nissui Pharmaceutical) supplemented with oxacillin (4 μg/mL; Sigma) (Morotomi et al. 2011).

Some growth media suitable for cultivation of strains of the family Sutterellaceae and their compositions are shown in the websites of Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (http://www.dsmz.de/) and Japan Collection of Microorganisms (http://www.jcm.riken.jp/JCM/JCM_Home_J.shtm).

The strains are generally maintained in anaerobic medium as broths or agar slants at 4 °C for a few days. Medium-term maintenance is as suspensions in 20 % v/v glycerol, 20 % w/v skim milk, or 10 % w/v skim milk supplemented with 1 % w/v sodium glutamate at −70 °C. Long-term preservation is by lyophilization.

Ecology

Sutterella wadsworthensis was first reported when performing biochemical characterization and susceptibility testing of Campylobacter gracilis–like clinical isolates from patients with diverse infections of the gastrointestinal tract (Wexler et al. 1996). Although a potential role of S. wadsworthensis in human gastrointestinal diseases has been documented in the past (Wexler et al. 1996; Molitoris et al. 1997), recent evaluation of the colonic mucosal isolates of this species from patients with inflammatory bowel disease has led to the conclusion that this species is probably a commensal; S. wadsworthensis was detected in 83.8 % of adult patients with ulcerative colitis as opposed to 86.1 % of the control subjects (Mukhopadhya et al. 2011). This study also indicated that S. wadsworthensis adheres closely to the mucosal lining and is thus more likely to be detected in biopsy samples than in feces.

Recently improved sequencing technology has led to the deposition of a large number of uncultured bacterial clones in the GenBank/EMBL/DDBJ public databases. There is evidence that S. wadsworthensis occurs in human feces as a common member of the human indigenous microflora, because many uncultured bacteria with highly similar 16S rRNA gene sequences (>98.7 % identity, the threshold proposed for distinguishing species by Stackebrandt and Ebers (2006) have been reported in these databases. These uncultured bacteria have been identified in fecal samples and intestinal biopsy samples from apparently healthy subjects from different countries, suggesting that S. wadsworthensis is a normal inhabitant of the human intestinal microbiota.

As described above, S. wadsworthensis is not associated with inflammatory bowel disease, but its presence has been reported in ileal mucosal biopsy samples from children with autism and gastrointestinal dysfunction (AUT-GI) by Williams et al. (2012). They reported that the 16S rRNA gene sequences of either S. wadsworthensis or S. stercoricanis were found in 12 of 23 AUT-GI children but in none of 9 control children with GI but not autism. Further investigations of the microbiome are needed in larger cohorts of patients with AUT-GI compared to the control GI groups, as well as in patients with AUT but without GI manifestations and in normally developing children with no GI disturbances.

The two other Sutterella species, S. parvirubra and S. stercoricanis, were isolated as novel species of the genus from healthy human feces (Sakon et al. 2008) and from the feces of a healthy Labrador Retriever dog (Greetham et al. 2004), respectively. Although there are no subsequent reports of the isolation of these species, a number of uncultured bacteria with highly similar 16S rRNA gene sequences have been deposited in the GenBank/EMBL/DDBJ databases. For example, the most similar 16S rRNA gene sequences (99.8–99.9 % similarity) to the type strain of S. parvirubra were derived from studies of uncultured bacteria from human intestinal mucosal biopsies (accession nos. FJ507106, FJ507078, and FJ506786; Walker et al. 2011). In contrast, the most similar 16S rRNA gene sequence for S. stercoricanis (99.2 % similarity) was detected in the feces of dhole (Cuon alpinus, a species of canid native to southern and southeastern Asia). This sequence (accession no. JN559525) is a direct submission by Zhang et al. (Unpublished). Williams et al. (2012) reported detecting S. stercoricanis 16S rRNA gene sequences in ileal mucosal biopsy specimens from patients diagnosed with AUT-GI symptoms, although it remains unclear whether this species contributes to the disease or is simply a normal component of the human intestinal microbiota.

The genus Parasutterella contains two species, P. excrementihominis (Nagai et al. 2009) and P. secunda (Morotomi et al. 2011). These species were isolated from the feces of healthy human subjects and each was described based on a single strain. There are no subsequent reports of isolation of these species, but a number of uncultured bacteria with highly similar 16S rRNA gene sequences have been deposited in the GenBank/EMBL/DDBJ databases. For P. excrementihominis, 200 clones (as of August 2012) with similar 16S rRNA gene sequence (>98.7 % similarity) have been derived from feces, intestinal contents, and mucosal biopsies of healthy human subjects and of patients with gastrointestinal diseases (e.g., ulcerative colitis, Crohn’s disease, and Clostridium difficile–associated diarrhea); from feces of the black lemur (Eulemur macaco), the brown rat (Rattus norvegicus), the wolf (Canis lupus), and cattle (Bos taurus); and from human skin samples and mattress dust. Based on these similar sources of isolation and the similar 16S rRNA gene sequences, P. excrementihominis is presumably common in the intestines of humans and other animals. For P. secunda, nine clones with a similar 16S rRNA gene sequence (>98.7 %) have been derived from human feces and the intestinal contents of turkeys and cattle.

Overall, all these data suggest that members of the family Sutterellaceae are common inhabitants of the intestines of humans and various animals.

Pathogenicity, Clinical Relevance

In addition to the details presented in the Ecology section, there is limited information on the antibiotic sensitivity of this family. Most strains of S. wadsworthensis (>95 %) are susceptible to amoxicillin/clavulanate, ticarcillin/clavulanate, cefoxitin, ceftriaxone, and clindamycin, and 85–95 % of the strains are susceptible to piperacillin, piperacillin/tazobactam, ceftizoxime, ciprofloxacin, trovafloxacin, azithromycin, clarithromycin, erythromycin, and roxithromycin (Wexler 2005). Strains of S. wadsworthensis (8 strains) were susceptible to kanamycin and colistin, but were resistant to vancomycin (Warren et al. 2005).

No information on antibiotic sensitivity and resistance is available for other species of the genera Sutterella and Parasutterella.