Evaluation of different methods for the detection of Clostridium perfringens phosphatases

Eisgruber, H.; Geppert, P.; Sperner, Brigitte; Stolle, A.

doi:10.1016/s0168-1605(02)00244-1

Cited by 11 publications

(33 citation statements)

References 13 publications

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“…The presence of a similar-sized antigenic band in the clinical strains suggests the ubiquitous nature of the enzyme in C. perfringens isolates, a property noted by other investigators interested in the clinical utility of this enzyme in C. perfringens identification (7,42).…”

Section: Resultsmentioning

confidence: 71%

“…CppA is of particular importance, as it serves as a clinically reliable and sensitive enzyme marker to differentiate C. perfringens from other clostridial species (42). The rapid conversion of pNPP to p-nitrophenol by cell-associated acid phosphatase from a colony of obligately anaerobic gram-positive rods is indicative in 95 of 100 isolates of the presence of C. perfringens (1,7,42).…”

Section: Discussionmentioning

confidence: 99%

“…This lack of information is notable. A growing cadre of investigators (1,7) has recognized the clinical utility of acid phosphatase as a biochemical marker for the identification of C. perfringens isolated from water and other sources and its use in the differentiation of C. perfringens from ϳ95% of all currently recognized clostridial species in the genus (42).…”

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confidence: 99%

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Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

Reilly¹,

Chance²,

Calcutt³

et al. 2009

Appl Environ Microbiol

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Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ϳ31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3 and 5 nucleoside monophosphates at pH 6. Calculated K m s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 mol of P i /s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.Clostridium perfringens is a ubiquitous gram-positive, endospore-forming anaerobic bacterium. While found in soil, the organism is often a constituent of the intestinal flora and is capable of causing severe gastrointestinal and histotoxic infections in humans and animals. As a species, C. perfringens is a very heterogeneous group of organisms with respect to metabolic by-products produced in vitro and pathogenic potential (25). Although the literature is replete with descriptions of the immunological, bio-and physicochemical attributes of C. perfringens-encoded toxins, as well as molecular and morphological details of sporulation, detailed analysis of other constituent enzymes, including those involved in acquisition and regulation of inorganic phosphate, has received relatively little attention. This lack of information is notable. A growing cadre of investigators (1, 7) has recognized the clinical utility of acid phosphatase as a biochemical marker for the identification of C. perfringens isolated from water and other sources and its use in the differentiation of C. perfringens from ϳ95% of all currently recognized clostridial species in the genus (42).Acid phosphatases (EC 3.1.3.2) are ubiquitous and catalyze, at an acidic pH, the transfer of phosphoryl groups from phosphomonoesters to water (48). These enzymes play essential roles in the generation, acquisition, and mobilization of inorganic phosphate and critical roles in phosphoryl relay systems intimately involved in signal transduction pathways in both prokaryotes and eukaryotes. A subgroup of phosphatases has been designated nonspecific acid phosphatases (NSAPs) by Thaller and coworkers (45) and includes those bacterial polyspecific phosphatases that are secreted across the cytoplasmic membrane and exhibit optimum catalytic activity at an acidic pH (41). NSAPs are recognized as a distinct group of phosphatases and categorized into three classes (46). The class C enzyme...

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Section: Resultsmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

Reilly¹,

Chance²,

Calcutt³

et al. 2009

Appl Environ Microbiol

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“…Since C. perfringens has been found to possess acid phosphatase activity in acidic conditions, a method to confirm the organism's presence was developed by measuring its acid phosphatase using different phosphatase reagents (Ueno et al, 1970). Since then, improved methods based on acid phosphatase detection have been adapted as routine protocols to detect C. perfringens (Eisgruber et al, 2003). Although the acid phosphatase activity has long been employed as a characteristic of C. perfringens that differentiates it from other clostridial species, there are still few reports about the gene(s) that contribute to the acid phosphatase profile.…”

Section: Introductionmentioning

confidence: 99%

Genetic and biochemical analysis of a class C non-specific acid phosphatase (NSAP) of Clostridium perfringens

et al. 2010

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Clostridium perfringens, a Gram-positive anaerobe, is a human pathogen that causes gas gangrene in muscle tissues. Its ability to grow and survive in the host is believed to be due to the production of numerous enzymes that enable the organism to obtain essential nutrients from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode a non-specific acid phosphatase in C. perfringens. Multiple alignments of the amino acid sequence showed that CPE0201 shares two signature motifs that belong to a class C acid phosphatase family. Expression of CPE0201 was shown to be positively regulated by the global VirR/VirS-VR-RNA regulatory cascade at the transcriptional level. To determine the acid phosphatase activity of the CPE0201-encoded protein, cloning, expression, purification and several biochemical characterizations were carried out. The optimum pH for activity of the CPE0201 enzyme was 4.8, and its V max and K m were 3.08 nmol ml−1 min−1 and 2.84 mM, respectively, with p-nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when Na2HPO4 was added to the medium. The enzyme appears to be associated with the surface of the cell, where it may function to acquire inorganic phosphate from organic phosphomonoesters in acidic conditions, which could play an important role in the survival and growth of C. perfringens in the host tissue.

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“…perfringens based on acid phosphatase activity. The phosphatase releases naphtyl from 1-naphtyl phosphate which forms an azo dye with diazonium o-dianisidine (Eisgruber et al, 2003). Sartory et al (2006) stated, that the test for acid phosphatase is a reliable alternative method for confirming presumptive C .…”

Section: Introductionmentioning

confidence: 99%

Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

Paier

Sommer

Haider

et al. 2011

Journal of Microbiological Methods

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Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method.

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Evaluation of different methods for the detection of Clostridium perfringens phosphatases

Cited by 11 publications

References 13 publications

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

Genetic and biochemical analysis of a class C non-specific acid phosphatase (NSAP) of Clostridium perfringens

Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

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