A Sarcina bacterium linked to lethal disease in sanctuary chimpanzees in Sierra Leone.

Human and animal infections with bacteria of the genus Sarcina (family Clostridiaceae) are associated with gastric dilation and emphysematous gastritis. However, the potential roles of sarcinae as commensals or pathogens remain unclear. Here, we investigate a lethal disease of unknown etiology that affects sanctuary chimpanzees (Pan troglodytes verus) in Sierra Leone. The disease, which we have named "epizootic neurologic and gastroenteric syndrome" (ENGS), is characterized by neurologic and gastrointestinal signs and results in death of the animals, even after medical treatment. Using a case-control study design, we show that ENGS is strongly associated with Sarcina infection. The microorganism is distinct from Sarcina ventriculi and other known members of its genus, based on bacterial morphology and growth characteristics. Whole-genome sequencing confirms this distinction and reveals the presence of genetic features that may account for the unusual virulence of the bacterium. Therefore, we propose that this organism be considered the representative of a new species, named "Candidatus Sarcina troglodytae". Our results suggest that a heretofore unrecognized complex of related sarcinae likely exists, some of which may be highly virulent. However, the potential role of "Ca. S. troglodytae" in the etiology of ENGS, alone or in combination with other factors, remains a topic for future research.

Sarcina organisms in the gastrointestinal tract: a clinicopathologic and molecular study.

Sarcina organisms were first observed in and recorded from the stomach contents of a patient suffering from vomiting by John Goodsir in 1842. Since that time, their fine structure, phylogenetic classification, and biochemical characteristics have been described. Although numerous cases of fatal disease have been attributed to this organism in the veterinary literature, only a few human cases have been documented. As a result, whether this organism causes disease in humans has not been definitively established. We report the clinicopathologic findings in a series of 5 patients with Sarcina-like organisms identified in upper gastrointestinal endoscopic biopsies with molecular confirmation. Our findings have shown that the organism is most commonly found in patients with a history of gastric outlet obstruction or delayed gastric emptying. Although many of the patients do not demonstrate direct mucosal injury from the organism, the presence of a concurrent gastric ulcer puts the patient at increased risk for complications such as emphysematous gastritis or perforation. The finding of Sarcina organisms should prompt further investigation for functional causes of gastric outlet obstruction and delayed gastric emptying, such as occult malignancy.

Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis.

Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.

Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli.

Sarcina ventriculi grows in a remarkable range of mesophilic environments from pH 2 to pH 10. During growth in acidic environments, where acetate is toxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation. PDC is rare in bacteria and absent in animals, although it is widely distributed in the plant kingdom. The pdc gene from S. ventriculi is the first to be cloned and characterized from a Gram-positive bacterium. In Escherichia coli, the recombinant pdc gene from S. ventriculi was poorly expressed due to differences in codon usage that are typical of low-G+C organisms. Expression was improved by the addition of supplemental codon genes and this facilitated the 136-fold purification of the recombinant enzyme as a homo-tetramer of 58 kDa subunits. Unlike Zymomonas mobilis PDC, which exhibits Michaelis-Menten kinetics, S. ventriculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs. Amino acid residues involved in the allosteric site for pyruvate in fungal PDCs were conserved in S. ventriculi PDC, consistent with a conservation of mechanism. Cluster analysis of deduced amino acid sequences confirmed that S. ventriculi PDC is quite distant from Z. mobilis PDC and plant PDCs. S. ventriculi PDC appears to have diverged very early from a common ancestor which included most fungal PDCs and eubacterial indole-3-pyruvate decarboxylases. These results suggest that the S. ventriculi pdc gene is quite ancient in origin, in contrast to the Z. mobilis pdc, which may have originated by horizontal transfer from higher plants.

Sarcina -like bacteria, Clostridium fallax and Clostridium sordellii in lambs with abomasal bloat, haemorrhage and ulcers.

A study of abomasal disease in lambs aged 2-5 weeks, made during the period 1993-1998, included 67 cases and 45 non-affected controls. Gross pathological findings included various combinations of bloat, haemorrhage and ulcers in the abomasum. Sarcina -like bacteria were found in sections and smears from the abomasum of 79% (53/67) of the cases. From one case, a lamb with abomasal bloat, the anaerobic "packet"-forming Sarcina ventriculi was cultivated from the abomasal contents and identified by biochemical reactions and sequencing of the 16S rRNA gene. Sarcina -like bacteria were observed microscopically in specimens from 94% (44/47) of the lambs with abomasal gas and in 45% (9/20) of those with ulcers or haemorrhage or both but little gas. On culture, abomasal contents from 41 cases yielded Clostridium fallax from 16 (39%) and Clostridium sordellii from eight (20%); abomasal cultures from 30 control lambs were negative for the three bacterial species. Quantitative cultivation, carried out on abomasal contents from live lambs and lambs dead

Phylogenetic placement of Sarcina ventriculi and Sarcina maxima within group I Clostridium, a possible problem for future revision of the genus Clostridium. Request for an opinion.

The 16S rRNA gene sequences of Sarcina ventriculi DSM 286T (T = type strain) and Sarcina maxima DSM 316T were determined. Phylogenetic analysis revealed that these two species are closely related to each other and belong to group I Clostridium (sensu Johnson and Francis). The implications of these phylogenetic findings for future revision of the genus Clostridium are discussed.

Cloning and expression of midecamycin 4"-acylase gene in spiramycin producing strains.

A recombinant plasmid p66B containing the midecamycin 4"-acylase gene was obtained by cloning this gene into plasmid vetor pIJ680 from the primary clone pCN6C5, presumably harboring the midecamycin biosynthetic gene. The expression of the midecamycin 4"-acylase gene (p66B) in spiramycin producing strains resulted mainly in the production of 4"-isovalerylspiramycin. Another positive clone pCN10F5 was discovered from the genomic library of S. mycarofacians 1748 by probing with p66B DNA BamHI-BamHI 2.3kb fragment. A BamHI-BamHI 8.0kb homologous region on pCN10F5 was determined by Southern hybridization and was subcloned into plasmids pWHM3 and pIJ680. Recombinant plasmids pWF5 and p6F5 with molecular size about 15.2kb and 13.3kb, respectively, were obtained. Transformation of spiramycin producing strains with these plasmids resulted in the production of two major components. Based on their physicochemical properties and spectral evidences, component I was identified as 4"-propionylspiramycin III, and component II as 4"-propionylspiramycin II. Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the spiramycin producing strain. Only pCN10F5 clone was identified from the genomic library of S. mycarofaciens 1748 when the 4"-isovaleryltransferase gene of carbomycin producing strain S. thermotolerans was used as a probe in colony hybridization. It suggests that there is a difference between the 4"-acyltransferase genes in the pCN6C5 and pCN10F5 clones.