Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery ofClostridium difficile

doi:10.1053/jhin.1999.0253

Journal of Hospital Infection

Volume 44, Issue 1, January 2000, Pages 65-69

https://doi.org/10.1053/jhin.1999.0253 Get rights and content

Abstract

Clostridium difficile is an increasingly prevalent nosocomial pathogen. Environmental contamination by spores is believed to be a major factor propagating the spread of C. difficile. Various approaches including the use of bile salts have been described to enhance the recovery of C. difficile from clinical and environmental specimens. We found that lysozyme (5 mg/L) incorporated into a selective medium containing bile salts significantly increased the recovery of C. difficile from swabs of 197 environmental sites (11% versus 24% samples positive, P< 0·01). Furthermore, in a separate series of experiments additional use of cooked meat broth enrichment significantly enhanced the recovery of C. difficile (35% versus 45%, P = 0·009). Conversely, we found that pre-exposure to alkaline thioglycollate did not improve the yield of C. difficile. Lysozyme incorporation markedly increases the recovery of C. difficile from environmental samples probably by stimulation of spore germination. Our findings suggest that previous attempts to determine the level of environmental C. difficile contamination have markedly underestimated the true prevalence of this pathogen.

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Cited by (59)

Approaches to the detection of Clostridioides difficile in the healthcare environment
2019, Journal of Hospital Infection
Citation Excerpt :
Some authors examined plates for growth after 48 h [19,23,25,30,33] while others incubated plates for 48 h, reviewed the plates and re-incubated them for a further 72 h if culture-negative before deciding if there was no growth [26,29,32]. The duration of incubation of the enrichment broth when enrichment was used was 48 h [22,33]. Both the European Society of Clinical Microbiology and Infectious Diseases' (2014) and the Infectious Diseases Society of America's guidelines (2018) on the management of CDI support the use of PCR (and other nucleic acid amplification techniques) or an enzyme immunoassay for the detection of glutamate dehydrogenase (GDH) as the first step of a multi-step algorithm for the laboratory diagnosis of CDI [38,39].
Clostridioides difficile, a spore-forming bacillus, is a major cause of healthcare-associated infection, and can survive for prolonged periods in the inanimate environment. Environmental sampling to detect C. difficile is not routine but may be undertaken as part of outbreak management and during research projects. We conducted a literature search covering the period between 1980 and 2018 to review methods for the detection of this pathogen in the environment. There are many acceptable sampling methods used for environmental screening, including contact plates, cotton swabs, flocked swabs and sponges. Most recent studies suggest that sponges are the most effective method of sampling and have the added benefit of being capable of sampling larger and curved areas. Culture methods are the most common laboratory method of detecting C. difficile from environmental samples. However, the results are variable depending on the type of agar used and the turnaround times can be long. Molecular methods such as real-time polymerase chain reaction (RT-PCR), although more commonly used to detect C. difficile from faecal specimens, has been used with varying degrees of success in environmental sampling. Further studies are needed to determine whether molecular techniques could offer a more reliable, faster method of environmental sampling, giving infection prevention and control teams more reassurance that patients are being placed in adequately decontaminated hospital environments.
Clostridium difficile infection in pregnant and postpartum women in 2 hospitals and a review of literature
2019, American Journal of Infection Control
Healthcare-associated Clostridium difficile infection (CDI) in pregnant/postpartum women is underreported, especially outside of North America. We report a cluster of cases in 2 neighboring secondary care hospitals in South-East England. The objective of this study was to identify the epidemiology and risk factors for infection.
An investigation into a cluster of cases of confirmed CDI in pregnant/postpartum women was performed over a 12-month period, from June 2016 to June 2017.
Eleven cases, in 10 patients, were identified, including 1 patient who had a relapse. Eight of 10 patients developed symptoms after hospital discharge. All patients had received broad-spectrum antibiotics prior to CDI onset. Environmental vectors, such as labor room mattresses, that were found difficult to effectively decontaminate after heavy contamination with blood, feces, and other body fluids may have been possible reservoirs. An infection control care bundle was successful in preventing further cases.
Antibiotic use and exposure to the organism in a contaminated labor room environment are likely risk factors for healthcare-associated CDI in postpartum women. Active surveillance is necessary to prevent these infections, as these cases often present after hospital discharge.
Development and validation of a new protocol for detecting and recovering clostridium difficile from meat samples
2018, Journal of Food Protection
There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.
Investigating the effect of supplementation on Clostridium difficile spore recovery in two solid agars
2018, Anaerobe
A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine & l-phenylalanine) on C. difficile spore outgrowth were investigated.
Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine).
CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ± 0.02 vs 0.12 ± 0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine.
We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.
Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting
2014, Anaerobe
Citation Excerpt :
However, semi-quantification of positive broths showed that CCMB-TAL gave an improved recovery rate over CCFB (Table 1). This observation seems to confirm previous studies finding that the presence of lysozyme increases the recovery rate of C. difficile [14]. Subsequently, lysozyme was added to CCFB to see if its addition improved recovery in this medium.
Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock.
Evaluation of the illumigene C. difficile assay for toxigenic Clostridium difficile detection: A prospective study of 302 consecutive clinical fecal samples
2014, Diagnostic Microbiology and Infectious Disease
Toxigenic Clostridium difficile is a major pathogen causing nosocomial diarrhea. Consequently, rapid detection of toxigenic C. difficile is very important in clinical laboratories. The illumigene C. difficile DNA amplification assay (illumigene; Meridian Bioscience, Inc.) is a rapid method that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification. In the present study, we evaluated the diagnostic performance of the illumigene assay using 302 consecutive stool specimens in comparison with the VIDAS C. difficile A&B enzyme-linked fluorescent immunoassay (VIDAS-CDAB; bioMérieux). Toxigenic culture was used as the reference method. The sensitivity, specificity, positive predictive value, and negative predictive value of the illumigene assay were 88.1%, 96.7%, 86.7%, and 97.1%, respectively, while those of the VIDAS-CDAB assay were 40.4%, 98.8%, 87.5%, and 88.5%, respectively. It is of note that use of a combination of the illumigene and VIDAS-CDAB assays did not improve any of the 4 evaluated parameters (88.1%, 95.5%, 82.5%, and 97.1%, respectively). The illumigene assay showed limits of detection of 250 and 11,467 CFU/mL for ATCC 9688 (tcdA+, tcdB+, cdtB−) and ATCC 43598 (tcdA−, tcdB+, cdtB−) reference strains, respectively, and there was no cross-reactivity with 8 frequently isolated bacterial species. In conclusion, the illumigene assay might be a useful method for rapid detection of toxigenic C. difficile in clinical laboratories. Additionally, the VIDAS-CDAB assay seems unnecessary if the illumigene assay is used.

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^f1: Corresponding author: Dr Mark H. Wilcox, Department of Microbiology, University of Leeds, Leeds, LS2 9JT.

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Journal of Hospital Infection

Abstract

References (27)

Incidence and impact of Clostridium difficile infection in the UK, 1993–1996

J Hosp Infect

Financial burden of hospital-acquired Clostridum difficile infection

J Hosp Infect

Clostridium difficile infection: responses, relapses and reinfections

J Hosp Infect

Evaluation of symptom recurrence in Clostridium difficile infection – relapse or reinfection?

J Hosp Infect

Clinical and molecular epidemiology of sporadic and clustered cases of nosocomial Clostridium difficile diarrhea

Am J Med

Comparison of Cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance of hospitalized patients

Diagn Microbiol Infect Dis

Clostridium difficile in district general hospitals

J Hosp Infect

Commun Dis Rep CDR Wkly

Contamination of a hospital environment by Clostridium difficile

Curr Microbiol

Am J Med

Initiation of germination of Clostridium difficile spores by Iysozyme. [French] Comptes Rendus Hebdomadaires des Séances de I’ Academie des Sciences – D:

Sciences Naturelles

Role of the laboratory in investigations of Clostridium difficile diarrhea

Clin Infect Dis

Sebald M. Lysozyme-dependent germination of spores of Clostridium perfringens ATCC 3624 after heat treatment [French]

Cited by (59)

Approaches to the detection of Clostridioides difficile in the healthcare environment

Clostridium difficile infection in pregnant and postpartum women in 2 hospitals and a review of literature

Development and validation of a new protocol for detecting and recovering clostridium difficile from meat samples

Investigating the effect of supplementation on Clostridium difficile spore recovery in two solid agars

Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting

Evaluation of the illumigene C. difficile assay for toxigenic Clostridium difficile detection: A prospective study of 302 consecutive clinical fecal samples

Regular ArticleValue of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery ofClostridium difficile

Abstract

J Hosp Infect

J Hosp Infect

J Hosp Infect

J Hosp Infect

Am J Med

Diagn Microbiol Infect Dis

J Hosp Infect

Commun Dis Rep CDR Wkly

Contamination of a hospital environment by Clostridium difficile

Curr Microbiol

Am J Med

Initiation of germination of Clostridium difficile spores by Iysozyme. [French] Comptes Rendus Hebdomadaires des Séances de I’ Academie des Sciences – D:

Sciences Naturelles

Role of the laboratory in investigations of Clostridium difficile diarrhea

Clin Infect Dis

Sebald M. Lysozyme-dependent germination of spores of Clostridium perfringens ATCC 3624 after heat treatment [French]

Regular Article
Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery ofClostridium difficile