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  • 1 University of Liège, Belgium
  • 2 UniversitéCatholique de Louvain, Belgium
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Despite increasing interest in the bacterium, the methodology for Clostridium difficile recovery has not yet been standardized. Cycloserine–cefoxitin fructose taurocholate (CCFT) has historically been the most used medium for C. difficile isolation from human, animal, environmental, and food samples, and presumptive identification is usually based on colony morphologies. However, CCFT is not totally selective. This study describes the recovery of 24 bacteria species belonging to 10 different genera other than C. difficile, present in the environment and foods of a retirement establishment that were not inhibited in the C. difficile selective medium. These findings provide insight for further environmental and food studies as well as for the isolation of C. difficile on supplemented CCFT.

Abstract

Despite increasing interest in the bacterium, the methodology for Clostridium difficile recovery has not yet been standardized. Cycloserine–cefoxitin fructose taurocholate (CCFT) has historically been the most used medium for C. difficile isolation from human, animal, environmental, and food samples, and presumptive identification is usually based on colony morphologies. However, CCFT is not totally selective. This study describes the recovery of 24 bacteria species belonging to 10 different genera other than C. difficile, present in the environment and foods of a retirement establishment that were not inhibited in the C. difficile selective medium. These findings provide insight for further environmental and food studies as well as for the isolation of C. difficile on supplemented CCFT.

Introduction

Many studies have reported changes in the epidemiology of Clostridium difficile and its presence in foods, animals, and the environment [1, 2]. Interest in these types of C. difficile samples continues to expand and the possibility of zoonotic and food transmission of the bacterium is still the main focus of several research reports [3]. However, an isolation procedure for research purposes has not yet been standardized. In recent years, a large number of studies have focused on the improvement of differential media and culture methods [4–6], including ethanol shock, sample enrichment in a selective broth, or the use of chromogenic and other pre-made agars. However, pre-made agars are expensive and thus unaffordable for many research groups. Furthermore, they are used for the clinical recovery of C. difficile from faecal samples and not for the semi-quantification of viable spores [7]. Since it was first proposed by George et al. [8], cycloserine–cefoxitin fructose (CCF) has been the most commonly used medium for C. difficile isolation from human, animal, environmental, and food samples. The addition of taurocholate, desoxycholate or cholate has also been shown to induce germination of C. difficile spores when they are incorporated in CCF [6, 9]. Other modifications to improve this media have been proposed; Delmée et al. [10] included cefotaxime instead of cefoxitin, which increases the sensitivity and specificity of the medium.

Few studies have focused on the identification of other bacterial species growing in CCF. George et al. [8] reported the growing of Lactobacillus spp., unidentified yeast and unidentified anaerobic Gram-negative rods on CCF. Only one further study [11] described other Clostridium colonies growing in cycloserine–cefoxitin fructose taurocholate (CCFT), including Clostridium sporogenes, Clostridium cadaveris, Clostridium perfringens, Clostridium bifermentans, and Clostridium septicum.

The objective of this study was to identify by comparative 16S ribosomal DNA sequence analysis the spectrum of bacteria cultured on CCFT, using surface and food samples. The growth of isolates was also tested in modified CCFT medium (with cefotaxime) and strains were further characterized for susceptibility to two selective agents, cefotaxime and cycloserine.

Materials and Methods

This study was conducted over 4 months, from March to June 2013, and included 188 food samples and 246 surface samples [12]. The meals sampled were composed of raw and/or cooked ingredients, according to the daily menu. Every Friday morning, samples from the week were transported to the laboratory for immediate analysis. The food preparation date, analysis date, quantity, and ingredients for each sample were recorded. Samples from surfaces were taken on two different occasions with a 65-day interval between them. A variety of areas (total area of approximately 100 cm2) were swabbed before or after routine cleaning, including residents’ rooms and other common areas [12].

Culture was performed on CCFT as described previously [12] in an anaerobic workstation (LedTechno, Heusden-Zolder, Belgium) at 37 °C. Colonies other than those with the characteristic morphology of C. difficile were then subcultured on Columbia agar plates with 5% horse blood (Biomérieux, Marcy-l’Étoile, France). Total DNA was harvested from a single colony and extracted as described previously [13]. Molecular identification of bacteria by 16S ribosomal DNA sequence analysis was performed using the primers and conditions described by Simpson et al. [14]. Sequencing and product purification were performed as described previously [15]. Following sequencing, consensus sequences were created using the Geneious program (http://www.geneious.com). The genus and species of each consensus sequence were deduced from a comparison with the non-redundant nucleotide database (http://blast.ncbi.nlm.nih.gov) using the basic local alignment search tool. A 99% identity was used as a threshold for species identification [16].

All the isolated strains were subcultured on modified-CCFT agar to include the selective agents cycloserine (400 µg/mL) and cefotaxime (3.6 μg/mL). After incubation for 48 h in an anaerobic atmosphere at 37 °C, the plates were examined to verify bacterial growth in the modified medium. In addition, all the isolates were tested for susceptibility to cycloserine and cefotaxime antimicrobials. The test was performed by paper disc diffusion according to the French Society of Microbiology (FSM) (www.sfm-microbiologie.org) guidelines. For cefotaxime, the test was performed with a 30-μg standard disc (Becton-Dickinson, Erembodegem, Belgium). For cycloserine, as commercial standard discs are not available, the test was adapted to the protocol as described previously by Mith et al. [17] with a final concentration of 120 μg of cycloserine in the disc. The plates were incubated for 48 h in an anaerobic workstation. The antibacterial activity was evaluated by measuring the diameter of inhibitory zones in millimeters using Top Craft digital callipers (Globaltronics GmbH & Co. KG, Germany). Means were then calculated from the results of three determinations. The entire tests were performed in duplicate. Bacteroides fragilis ATCL 25285 was tested as a quality control.

Results and Discussion

Ethanol shock was not used in the course of this study, nor was alcohol selection of microorganisms conducted; we can therefore describe a wider range of species capable of growing in this medium. On the other hand, for both food and surface samples, no colony growing was observed in more than half of the plates analyzed. For surface samples, these findings may indicate that the nursing home had a good-implemented clean program to control not only the spread of C. difficile, but also other bacteria. For food samples, it is probable that cooking removes the microbial load of the raw foods and that there are also a good hygiene food handling procedures. Furthermore, cultured colonies were observed in low numbers, which facilitated the identification of different morphologies despite not having used the ethanol shock step.

From food samples, a total of 59 strains were isolated and identified by 16S rDNA sequencing analysis. Results revealed a total of 7 bacterial genera comprising 20 different species. The bacteria most frequently isolated belonged to the genera Lactobacillus, Clostridium, and Enterococcus. Within these, the dominant species were identified as Lactobacillus rhamnosus (n = 6), Enterococcus faecium (n = 5), and Enterococcus faecalis (n = 5) (Table I). C. sporogenes (n = 12) was the most common clostridia isolated. In agreement with the results of this study, Limbago et al. [8] reported a total of 13 isolates identified as C. sporogenes obtained from ground beef and ground turkey after culture on C. difficile selective medium.

Table I.

16S rDNA sequencing identification of bacteria growing on the CCFAT medium isolated from food samples after CCFT enrichment

Isolated bacteriumTotal number of isolatesSample weeksaNumber of isolates/weekbSamples composed of one or more raw ingredientsSample composed of cooked ingredients only
GenusClostridium
Clostridium baratii129/03101
Clostridium butyricum210/052c11
Clostridium orbiscindens124/05101
Clostridium sporogenes1222/034c13
26/04202
03/05211
17/05101
24/05110
07/06101
28/06101
Clostridium subterminale422/03101
05/042c02
12/04101
GenusEnterococcus
Enterococcus casseliflavus307/06110
14/06101
28/06110
Enterococcus durans329/03211
31/05101
Enterococcus faecalis529/03101
10/052c11
20/06101
28/06110
Enterococcus faecium524/05101
14/06101
20/06110
28/06202
Enterococcus gallinarum114/06101
GenusLactobacillus
Lactobacillus sakei329/03101
12/04110
28/06101
Lactobacillus salivarius128/03110
Lactobacillus rhamnosus629/03101
05/04101
12/04101
19/04101
10/05101
24/05101
Lactobacillus casei219/04110
31/05110
Lactobacillus graminis117/05110
GenusPaenibacillus
Paenibacillus lautus124/05101
GenusPediococcus
Pediococcus pentosaceaus503/05101
10/05110
17/05220
28/06101
Pediococcus acidilactici110/05110
Genus Propionobacterium
Propionobacterium acnes122/03101
GenusWeisella
Weisella viridescens114/06101

The date refer to the Friday on which samples from the proceeding week were collected and transported to the laboratory for immediate analysis.

Number of the different bacterial species obtained from food samples in each week of sampling.

Two isolates were from food prepared on the same day but in different services.

For environmental surfaces, a total of eight different bacterial species were identified. Most of these species have been previously observed as able to survive for months on surfaces [18]. E. faecalis (n = 26) and Eggerthella lenta (n = 14) were the most frequently isolated bacteria from the areas sampled. Regarding the genus Clostridium, only one isolate (Clostridium tertium) was obtained. Other species identified were E. faecium (n = 2), Staphylococcus haemolyticus (n = 2), Staphylococcus capitis (n = 1), Pediococcus pentosaceus (n = 2), and Finegoldia magna (n = 2) (Table II).

Table II.

Different bacteria from the nursing home environment isolated on CCFT

Sampling areaNumber of samplesIsolated bacteriumNumber of isolatesSpecific information regarding the isolate area
Kitchen
External kitchen doorknobs4--
Internal Kitchen doorknobs4--
Refrigerators handles2--
Cover of the food warmer (bain marie)2Eggerthella lenta1
Kitchen cutting board for meat2--
Kitchen cutting board for vegetables2Eggerthella lenta1
Slicer machine2Pediococcus pentosaceus1
Oven handle2--
Touch control kitchen faucet4Eggerthella lenta1
Meal delivery carts (for rooms and canteen)14Enterococcus faecalis1Carts for canteen
Trays (for rooms and canteen)8Enterococcus faecalis1Tray for canteen
Kitchen wall2
Kitchen floor2
Kitchen staff bathroom and locker room
External doorknobs9Eggerthella lenta1
Internal doorknobs9Clostridium tertium1Toilet internal doorknob
Toilet seat4Eggerthella lenta2a
Cistern flush button2
Paper towel dispenser2Eggerthella lenta1
Shower controls2
Sink faucet2
Soap dispenser2
Towel bar2
Control knob (radiator)2
Bathroom wall2
Bathroom floor2Eggerthella lenta1
Light switch2Eggerthella lenta1
Residents’ rooms
External doorknobs8Eggerthella lenta1Room E
Internal doorknobs8Enterococcus faecium1Room F
Enterococcus faecalis1Room D
Bedside8Finegoldia magna1Room D
Enterococcus faecalis1Room F
Eggerthella lenta1Room E
Bed8Finegoldia magna1Room D
Enterococcus faecalis2Room G/B
Invalid chair1Enterococcus faecalis1Room D
Room wall8Enterococcus faecalis1Room F
Room floor8Enterococcus faecalis4Room D/E/F/B
Private bathrooms
External doorknobs8Enterococcus faecalis2Room D/0
Staphylococcus haemolyticus1Room E
Internal doorknobs8Staphylococcus haemolyticus1Room D
Sink faucet8Enterococcus faecalis1Room A
Staphylococcus capitis1Room E
Eggerthella lenta1Room E
Cistern flush button8Enterococcus faecalis2Room D/C
Toilet brush handle8
Toilet seats8Enterococcus faecalis1Room D
Toilet support bar6Eggerthella lenta1Room F
Towel8Enterococcus faecalis4Room D/G/0/A
Chamber pot1Enterococcus faecalis1Room D
Bathroom wall8Eggerthella lenta1Room E
Bathroom floor8Enterococcus faecalis1Room E
Common areas
Couch2
Coffee table2
Elevator control panels12Enterococcus faecalis2a
Pediococcus pentosaceus1
Staircase railings4Enterococcus faecium1
Hall wall2
Hall floor2Enterococcus faecalis1

Note: Sampling before cleaning: rooms 0, C, E, F; sampling after cleaning: rooms A, B, D, G; rooms with residents tested positive for C. difficile at the time of sampling: D, E.

One isolate from each sampling day.

In this study, all the described strains isolated from food and surface samples were able to grow in CCFT in the same culture conditions established for C. difficile recovery. The estimated concentration in the researcher-prepared agar of D-cycloserine was 400 µg/mL and 3.6 μg/mL for cefoxitin (with an average 20 mL of CCFT per plate). In the modified-CCFAT, which included the selective agents’ cefotaxime and cycloserine in the same concentrations, all the isolated strains were also able to grow, except the only strain identified as Weisella viridescens.

Previously reported data describe a C. difficile minimal inhibitory concentration ≥1,024 µg/mL for D-cycloserine in 16 different strains of C. difficile[8]. However, in the available antibiotic management guidelines, there are no disk breakpoints or critical concentrations for this drug. In relation to cefotaxime, according to the FSM, the sensitivity and resistant zone diameters proposed are ≥21 mm and <15 mm, and the critical concentrations for susceptibility and resistance are ≤4 mg/L and >32 mg/L for strict anaerobes. However, it must be taken into account that these values refer only to therapeutic breakpoints.

For most of the isolated strains, the observed zone of inhibition was lower or equal to the size of the C. difficile inhibition zone. Results obtained from a D-cycloserine disc diffusion test (120 μg/disc) showed that for all the isolates belonging to the genus Clostridium, Pediococcus, Propionibacterium, Staphylococcus, and Paenibacillus, no inhibition zone was present in the plate. For the genus Lactobacillus, no inhibition zone was observed for any of the isolates except Lactobacillus graminis and Lactobacillus salivarius, for which zones of 22.7 mm and 28.3 mm in diameter were, respectively, detected. Regarding the genus Enterococcus, all the species studied displayed an inhibition diameter between 13 mm and 16 mm except Enterococcus gallinarum, which had a maximum diameter of 22 mm. E. lenta showed a diameter of 29.6 mm, while F. magna had a diameter of 26 mm. For cefotaxime (30 μg/disc), the results were more heterogeneous. Isolates belonging to the genus Lactobacillus, including L. rhamnosus and L. graminis showed full resistance to cefotaxime (no inhibition zone), while two other species of this genus, Lactobacillus sakei and Lactobacillus casei, had diameters of 19 mm and 20.5 mm, respectively. Regarding the genus Clostridium, most of the species showed an inhibition zone ≥10 mm and ≤32 mm (Clostridium orbiscidens 31.3 mm; C. sporogenes 20.6 mm; Clostrodium baratii 15.6 mm; and Clostridium butyricum 11.8 mm). Only three species, C. tertium, Clostridium subterminale, and C. difficile presented full resistance to the drug. Most of the isolates belonging to the genus Enterococcus showed resistance (no inhibition zone) with only the E. gallinarum strain presenting an inhibition zone, 16.6 mm in diameter. S. capitis and S. haemolyticus also showed full resistance to cefotaxime (no inhibition zone). Other strains like Paenibacillus lautus, Propionobacterium acnes, F. magna, and E. lenta had diameters of 18.6 mm, 23.2 mm, 22.9 mm, and 22.4 mm, respectively. While P. pentosaceus had a diameter of 16.6 mm, Pediococcus acidilactici showed no inhibition zone in the plate, indicating full resistance.

A total of 70 out of the 188 samples analyzed (70.7%) were composed entirely of cooked ingredients, while 55 (29.3%) contained one or more raw ingredients, such as lettuce, tomato, mushroom, or raw meat. These percentages may explain why only 20 strains were isolated from raw food (mostly from fresh vegetables), while 40 strains were isolated from cooked food samples (all of them are composed of meat or fish as the main ingredient). Samples were frozen before analysis, which may affect the survival of some of the bacterial groups [19]. Regarding fresh vegetables, they can harbor large and diverse populations of bacteria. A previous study [20] demonstrated significant differences in bacterial community structure dependent upon the type of vegetables involved, and also treatments undertaken in the course of production. In this context, several factors could play a role in the lower recovery of strains from raw food samples. Methods of cleaning and sanitizing vegetables can cause a significant reduction in the total plate count [21]. Dominant taxa in vegetables belong to aerobic groups, like Pseudomonas, Xantomonas, or other non-Enterobacteriaceae species; therefore, they are not detectable under the anaerobic culture conditions of this study [20, 22]. Regarding cooked foods, most of the bacteria found were classified in genus Clostridium, Enterococcus, and Lactobacillus. Several studies have addressed the survival of Clostridium spores in extreme conditions in the environment. While freezing temperatures seem to have little impact on the viability of most of the spores, their viability at different temperatures varies by species. For example, viable spores of C. sporogenes and C. butyricum can survive temperatures of 100 °C for hours [23, 24]. Enterococci have shown an important heat resistance and, depending on the isolates and species, they can survive pasteurization temperatures [25]. Some species of Lactobacillus have also been shown to have the potential to survive pasteurization. However, their resistance depends on genetic variations among strains, the physiological status of the cells and other environmental factors [26, 27]. Therefore, it is not surprising that these groups of bacteria (specially Clostridium and Enterococcus) were isolated more frequently from samples comprising fish or meat (even if they were cooked) as contamination with this faecal species would have occurred more frequently in slaughterhouse conditions compared to contamination in the environment.

On the other hand, the use of antimicrobial agents in animal production has caused an increase in the resistance of Enterobaceriaceae and other bacterial families, with higher production of β-lactamases, which hydrolyze the β-lactam ring and inactivate the β-lactams [28]. The results are a high prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in meat products. While the connection between ESBL-producing bacteria in food animals, retail meats, and humans has been suggested previously [25], a few publications describe ESBL resistance in bacteria from vegetables, or identify which species were detected in which vegetable types [22]. In this study, we selected a final cefoxitin (CCFT) and cefotaxime (modified CCFT) concentration of 3.6 μg/mL. The epidemiological cut-off value (ECOFF) available for cefoxitin ranges between 4 μg/mL (Staphylococcus aureus) and 8 μg/mL (Escherichia coli, Klebsiella spp., Salmonella spp., and Staphylococcus spp.). The epidemiological cut-off value available for cefotaxime varies between ≤0.25 μg/mL (E. coli, Klebsiella spp., and Streptococcus spp.), 0.5 μg/mL (Citrobacter spp., Enterobacter spp., and Streptococcus spp.), 1 μg/mL (Yersinia enterocolitica and Serratia spp.), 2–4 μg/mL (Staphylococcus spp.), and 32 μg/mL (Pseudomonas aeuroginosa). Most of the strains selected in this study have probably already acquired resistance (at least to cefotaxime); therefore, it is not surprising that twice as many isolates were obtained from cooked foods, including in most of the cases meats.

As in the case of meats and Enterobacteriaceae, several fermented foods have recently been suggested as potential vehicles for the exchange of antibiotic resistance genes between acid lactic bacteria and other pathogens in the gastrointestinal tract [29]. As most of the Lactobacillus species isolated in this study presented resistance to both of the drugs, it will be interesting to determine in the course of future studies whether the resistance of these strains results from an intrinsic mechanism or are due to genes encoding possible transferable resistance determinants.

In relation to the surface samples, the species belonging to genus Enterococcus, including E. faecalis and E. faecium, were frequently isolated from different swabs (kitchen, residents’ rooms, private bathrooms, and common areas). These species have been commonly found in clinical samples [18, 30] and observed to persist between 5 days and 4 months in hospital environments and on other inanimate surfaces [31, 32]. In this nursing home, residents’ rooms are cleaned and disinfected daily using bleach-based disinfectants (sodium hypochlorite 10%). Automated gaseous decontamination of residents’ rooms (stabilized hydrogen peroxide 6%) is also performed weekly; isolates from bathroom walls and bathroom floors were only obtained when sampling was performed before cleaning routine. Doorknobs, bedsides, cistern buttons, toilet seats, and chamber pots were found contaminated after cleaning in only one resident’s room (D), which may indicate less effort and time spent on cleaning this room. Beds were also found to be contaminated after being cleaned in three different rooms, but in each case, isolates were obtained from the beds of dependent residents. The dependent classification was used for residents who were confined to bed; this means that at the moment of cleaning and at the moment of sampling the residents were present in the bed, which hinders cleaning procedures and also favors rapid recontamination of the sample surface. E. faecalis was isolated from the armrest of one invalid chair. This chair was in the resident’s room; however, it was not treated as a part of the cleaning routine. Room walls and room floors were most frequently contaminated before cleaning was performed. The only floor (room D) that was contaminated after cleaning was also from a dependent resident receiving nursing assistance with the continuous circulation of the nursing staff a likely source of the floor contamination. It should be noted that this room (D) was inhabited by a patient diagnosed with C. difficile infection (CDI) 9 days before the study began and positive for the bacterium at the moment of sampling. For residents suffering from CDI, the protocol implementing by the healthcare facility prescribes the automated gaseous decontamination of the room every day. However, in this specific case, the critical health status of the patient required continuous monitoring by the nurses and medical assistants, resulting in the constant movement of medical personnel around the room. Therefore, although special measures were taken by the staff (double gloving if manipulating faeces, constant disinfection of hands), automated gaseous decontamination was not possible, at least before surface sampling was performed. The flow of personnel in this room could also have contributed to the fact that this was the room most contaminated after cleaning. There was one other resident positive for C. difficile at the moment of sampling (room E), however, while the bacterium was detected in their faeces, CDI was not diagnosed and therefore special protocols of disinfection were not applied.

Besides Enterococcus, E. lenta was the most commonly bacterium isolated. E. lenta is an anaerobic Gram-positive non-sporulating bacteria poorly studied due to difficulties with phenotypic identification. It is recognized as a part of the normal human intestinal microbiome but it has been also associated with gastrointestinal infections. A recent study identified E. lenta in 33 patients suffering intra-abdominal pathology with a median age of 68 years [33]. In relation to elderly people and gut microbiota, decreased microbial diversity is correlated with increased age. Furthermore, individuals living in short- or long-term residential facilities have been shown to have less diversity in microbiota than those living in the community [34]. It seems that long-term residential subjects have a higher proportion of Bacteroidetes in their gut, whereas elderly people in the community have a higher proportion of Firmicutes [35]. Reductions in some clostridia or bifidobacteria species and proliferation of opportunistic bacteria such as E. faecalis were also reported in hospitalized elderly patients [35]. In this study, only one isolate obtained from the internal doorknobs of the kitchen staff bathroom was identified as C. tertium. These findings could suggest that Clostridium species were sub-dominant in faecal microbiota of these elderly residents, and explains why other species present in higher proportions and resistant to the selective agents used in the medium were more commonly isolated.

In conclusion, this study focuses on the identification of bacteria growing on selective media (CCFT and modified CCFT). These C. difficile home-made culture media have a relatively low cost but offer high sensitivity for research purposes. Data reported provide the identification of the spectrum of bacteria growing on CCFT, which could also help further environmental screening studies in nursing homes and other healthcare environments.

References

  • 1.

    Rodriguez-Palacios, A., Borgmann, S., Kline, T. R., LeJeune, J. T.: Clostridium difficile in foods and animals: History and measures to reduce exposure. Anim Health Res Rev 14, 1129 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Vindigni, S. M., Surawicz, C. M.: C. difficile infection: Changing epidemiology and management paradigms. Clin Transl Gastroenterol 6, e99 (2015).

  • 3.

    Bauer, M. P., Kuijper, E. J.: Potential sources of Clostridium difficile in human infection. Infect Dis Clin North Am 29, 2935 (2015).

  • 4.

    Foster, N. F., Riley, T. V.: Improved recovery of Clostridium difficile spores with the incorporation of synthetic taurocholate in cycloserine-cefoxitin-fructose agar (CCFA). Pathology 44, 454456 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Boseiwaga, L. V., Foster, N. F., Thean, S. K., Squire, M. M., Riley, T. V., Carson, K. C.: Comparison of ChromID Clostridium difficile agar cycloserine-cefoxitin-fructoseagar for the recovery of Clostridium difficile. Pathology 45, 495500 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Tyrrell, K. L., Citron, D. M., Leoncio, E. S., Merriam, C. V., Goldsrein, E. J. C.: Evaluation of cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate and lysozyme for recovery of Clostridium difficile isolates from faecal samples. J Clin Microbiol 51, 30943096 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Lister, M., Stevenson, E., Heeg, D., Minton, N. P., Kuehne, S. A.: Comparison of culture based methods for the isolation of Clostridium difficile from stools samples in a research setting. Anaerobe 28, 226229 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    George, W. L., Sutter, V. L., Citron, D., Finegold, S. M.: Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9, 214219 (1979).

    • Search Google Scholar
    • Export Citation
  • 9.

    Wilson, K. H.: Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J Clin Microbiol 18, 10171019 (1983).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Delmée, M., Wauters, G.: Rôle de Clostridium difficile dans les diarrhéessurvenant après antibiothérapy: Etude de 87 cas. [The role of Clostridium difficile in diarrhea appearing after antibiotic treatment: A study of 87 cases]. Acta Clin Belg 36, 187184 (1981).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Limbago, B., Thompson, A. D., Greene, S. A., MacCannell, D., MacGowan, C. E., Jolbitado, B., Hardin, H. D., Estes, S. R., Weese, J. S., Songer, J. G., Gould, L. H.: Development of a consensus method for culture of Clostridium difficile from meat and its use in a survey of U.S. retail meats. Food Microbiol 32, 448451 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Rodriguez, C., Korsak, N., Taminiau, B., Avesani, V., Van Broeck, J., Brach, P., Delmée, M., Daube, G.: Clostridium difficile from food and surface samples in a Belgian nursing home: An unlike source of contamination. Anaerobe 32, 8789 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Rodriguez, C., Taminiau, B., Van Broeck, J., Avesani, V., Delmée, M., Daube, G.: Clostridium difficile in young farm animals and slaughter animals in Belgium. Anaerobe 18, 621625 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Simpson, P. J., Stanton, C., Filzgerald, G. F., Ross, R. P.: Genomic diversity and relatedness of bifidobacteria isolated from a porcine caecum. J Bacteriol 185, 25712581 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Rodriguez, C., Taminiau, B., Avesani, V., Van Broeck, J., Delmée, M., Daube, G.: Multilocussequence typing analysis and antibiotic resistance of Clostridium difficile strains isolated from retail meat and humans in Belgium. Food Microbiol 42, 166171 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Kim, M., Oh, H. S., Park, S. C., Chun, J.: Towards a taxonomy coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64, 346354 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Mith, H., Duré, R., Delcenserie, V., Daube, G., Clinquart, A.: Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Sci Nutr 2, 403416 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    D’azevedo, P. A., Dias, C. A. G., Teixeira, L. M.: Genetic diversity and antimicrobial resistance of enterococal isolates from southern region of Brazil. Rev Inst Med Trop Sao Paulo 48, 1116 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Alexander, D. C., Tittiger, F.: Bacteriological studies of meat pies and frozen prepared dinners. Can J Comp Med 35, 511 (1971).

  • 20.

    Leff, J. W., Fierer, N.: Bacterial communities associated with the surfaces of fresh fruits and vegetables. PLoS One 8, e59310 (2013).

  • 21.

    Pinto, L., Ippolito, A., Baruzzi, F.: Control of spoiler Pseudomonas spp. on fresh cut vegetables by neutral electrolyzed water. Food Microbiol 50, 102108 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Van Hoek, A. H. A. M., Veenman, C., Van Overbeek, W. M., Lynch, G., de Roda Husman, A. M., Blaak, H.: Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables. Int J Food Microbiol 204, 18 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Morton, R. D., Scott, W., Bernard, D. T., Wiley, R. C.: Effect of heat and pH on toxigenic Clostridium butyricum. J Food Sci 55, 17251727 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Mah, J. H., Kang, D. H., Tang, J.: Comparison of viability and heat resistance of Clostridium sporogenes stored at different temperatures. J Food Sci 74, M2327 (2009).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    McAuley, M. C., Gobius, K. S., Britz, M. L., Craven, H. M.: Heat resistance of thermoduric enterococci isolated from milk. Int J Food Microbiol 154, 162168 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Jordan, K. N., Cogan, T. M.: Heat resistance of Lactobacillus spp. isolated from cheddar cheese. Lett Appl Microbiol 29, 136140 (1999).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Christiansen, P., Waagner Nielsen, E., Vogensen, F. K., Brogren, C. H., Ardo, Y.: Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk. Int Dairy J 16, 11961204 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Kluytmans, J. A., Overdevest, I. T., Willemsen, I., Klytmans-van-den Bergh, M. F., van der Zwaluw, K., Heck, M., Rijnsburger, M., Vandenbroucke-Gravis, C. M., Savelkout, P. H., Johnston, B. D., Gordon, D., Johnson, J. R.: Extended-spectrum β-lactamase-producing Escherichia coli from retail chicken meat and humans: Comparison of strains, plasmids, resistance genes and virulence factors. Clin Infect Dis 56, 478487 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Van Reenen, C. A., Dicks, L. M. : Horizontal gene transfer amongst probiotic lactic acid bacteria and other intestinal microbiota: What are the possibilities? A review. Arch Microbiol 193, 15768 (2011).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Fisher, K., Phillips, C.: The ecology, epidemiology and virulence of Enterococcus. Microbiology 155, 174957 (2009).

  • 31.

    Kramer, A, Schwebke, I, Kampf, G.: How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect Dis 6, 130 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Gardinier, B. J., Tai, A. Y., Kotsanas, D., Francis, M., Roberts, S. A., Ballard, S. A., Junckerstorff, R. K., Korman, T. M.: Eggerthella lentabacteremia: Clinical and microbiological characteristics. J Clin Micro 53, 626635 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Claesson, M. J., Jeffery, I. B., Conde, S., Power, S. E., O’Connor, E. M., Cusack, S., Harris, H. M., Coakley, M., Lakshminarayanan, B., O’sullivan, O., Filzgerald, G. F., Deane, J., O’Connor, M., Harnedy, N., O’Connor, K., O’Mahony, D., Van Sideren, D., Wallace, M., Brennan, L., Stanton, C., Marchesis, J. R., Fitzgerald, A. P., Shanahan, F., Hill, C., Ross, R. P., O’Toole, P. W.: Gut microbiota composition correlates with diet and health in the elderly. Nature 9, 178184 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Zapata, H. J., Quagliarello, V. J.: The microbiota and microbiome in aging: Potential implications in health and age related diseased. J Am Geriatr Soc 63, 776781 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Bartosch, S., Fite, A., Macfarlane, G. T., McMurdo, M. E.: Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol 70, 35753581 (2004).

    • Crossref
    • Search Google Scholar
    • Export Citation

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  • 1.

    Rodriguez-Palacios, A., Borgmann, S., Kline, T. R., LeJeune, J. T.: Clostridium difficile in foods and animals: History and measures to reduce exposure. Anim Health Res Rev 14, 1129 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Vindigni, S. M., Surawicz, C. M.: C. difficile infection: Changing epidemiology and management paradigms. Clin Transl Gastroenterol 6, e99 (2015).

  • 3.

    Bauer, M. P., Kuijper, E. J.: Potential sources of Clostridium difficile in human infection. Infect Dis Clin North Am 29, 2935 (2015).

  • 4.

    Foster, N. F., Riley, T. V.: Improved recovery of Clostridium difficile spores with the incorporation of synthetic taurocholate in cycloserine-cefoxitin-fructose agar (CCFA). Pathology 44, 454456 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Boseiwaga, L. V., Foster, N. F., Thean, S. K., Squire, M. M., Riley, T. V., Carson, K. C.: Comparison of ChromID Clostridium difficile agar cycloserine-cefoxitin-fructoseagar for the recovery of Clostridium difficile. Pathology 45, 495500 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Tyrrell, K. L., Citron, D. M., Leoncio, E. S., Merriam, C. V., Goldsrein, E. J. C.: Evaluation of cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate and lysozyme for recovery of Clostridium difficile isolates from faecal samples. J Clin Microbiol 51, 30943096 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Lister, M., Stevenson, E., Heeg, D., Minton, N. P., Kuehne, S. A.: Comparison of culture based methods for the isolation of Clostridium difficile from stools samples in a research setting. Anaerobe 28, 226229 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    George, W. L., Sutter, V. L., Citron, D., Finegold, S. M.: Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9, 214219 (1979).

    • Search Google Scholar
    • Export Citation
  • 9.

    Wilson, K. H.: Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J Clin Microbiol 18, 10171019 (1983).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Delmée, M., Wauters, G.: Rôle de Clostridium difficile dans les diarrhéessurvenant après antibiothérapy: Etude de 87 cas. [The role of Clostridium difficile in diarrhea appearing after antibiotic treatment: A study of 87 cases]. Acta Clin Belg 36, 187184 (1981).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Limbago, B., Thompson, A. D., Greene, S. A., MacCannell, D., MacGowan, C. E., Jolbitado, B., Hardin, H. D., Estes, S. R., Weese, J. S., Songer, J. G., Gould, L. H.: Development of a consensus method for culture of Clostridium difficile from meat and its use in a survey of U.S. retail meats. Food Microbiol 32, 448451 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Rodriguez, C., Korsak, N., Taminiau, B., Avesani, V., Van Broeck, J., Brach, P., Delmée, M., Daube, G.: Clostridium difficile from food and surface samples in a Belgian nursing home: An unlike source of contamination. Anaerobe 32, 8789 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Rodriguez, C., Taminiau, B., Van Broeck, J., Avesani, V., Delmée, M., Daube, G.: Clostridium difficile in young farm animals and slaughter animals in Belgium. Anaerobe 18, 621625 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Simpson, P. J., Stanton, C., Filzgerald, G. F., Ross, R. P.: Genomic diversity and relatedness of bifidobacteria isolated from a porcine caecum. J Bacteriol 185, 25712581 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Rodriguez, C., Taminiau, B., Avesani, V., Van Broeck, J., Delmée, M., Daube, G.: Multilocussequence typing analysis and antibiotic resistance of Clostridium difficile strains isolated from retail meat and humans in Belgium. Food Microbiol 42, 166171 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Kim, M., Oh, H. S., Park, S. C., Chun, J.: Towards a taxonomy coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64, 346354 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Mith, H., Duré, R., Delcenserie, V., Daube, G., Clinquart, A.: Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Sci Nutr 2, 403416 (2014).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    D’azevedo, P. A., Dias, C. A. G., Teixeira, L. M.: Genetic diversity and antimicrobial resistance of enterococal isolates from southern region of Brazil. Rev Inst Med Trop Sao Paulo 48, 1116 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Alexander, D. C., Tittiger, F.: Bacteriological studies of meat pies and frozen prepared dinners. Can J Comp Med 35, 511 (1971).

  • 20.

    Leff, J. W., Fierer, N.: Bacterial communities associated with the surfaces of fresh fruits and vegetables. PLoS One 8, e59310 (2013).

  • 21.

    Pinto, L., Ippolito, A., Baruzzi, F.: Control of spoiler Pseudomonas spp. on fresh cut vegetables by neutral electrolyzed water. Food Microbiol 50, 102108 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Van Hoek, A. H. A. M., Veenman, C., Van Overbeek, W. M., Lynch, G., de Roda Husman, A. M., Blaak, H.: Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables. Int J Food Microbiol 204, 18 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Morton, R. D., Scott, W., Bernard, D. T., Wiley, R. C.: Effect of heat and pH on toxigenic Clostridium butyricum. J Food Sci 55, 17251727 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Mah, J. H., Kang, D. H., Tang, J.: Comparison of viability and heat resistance of Clostridium sporogenes stored at different temperatures. J Food Sci 74, M2327 (2009).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    McAuley, M. C., Gobius, K. S., Britz, M. L., Craven, H. M.: Heat resistance of thermoduric enterococci isolated from milk. Int J Food Microbiol 154, 162168 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Jordan, K. N., Cogan, T. M.: Heat resistance of Lactobacillus spp. isolated from cheddar cheese. Lett Appl Microbiol 29, 136140 (1999).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Christiansen, P., Waagner Nielsen, E., Vogensen, F. K., Brogren, C. H., Ardo, Y.: Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk. Int Dairy J 16, 11961204 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Kluytmans, J. A., Overdevest, I. T., Willemsen, I., Klytmans-van-den Bergh, M. F., van der Zwaluw, K., Heck, M., Rijnsburger, M., Vandenbroucke-Gravis, C. M., Savelkout, P. H., Johnston, B. D., Gordon, D., Johnson, J. R.: Extended-spectrum β-lactamase-producing Escherichia coli from retail chicken meat and humans: Comparison of strains, plasmids, resistance genes and virulence factors. Clin Infect Dis 56, 478487 (2013).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Van Reenen, C. A., Dicks, L. M. : Horizontal gene transfer amongst probiotic lactic acid bacteria and other intestinal microbiota: What are the possibilities? A review. Arch Microbiol 193, 15768 (2011).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Fisher, K., Phillips, C.: The ecology, epidemiology and virulence of Enterococcus. Microbiology 155, 174957 (2009).

  • 31.

    Kramer, A, Schwebke, I, Kampf, G.: How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect Dis 6, 130 (2006).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Gardinier, B. J., Tai, A. Y., Kotsanas, D., Francis, M., Roberts, S. A., Ballard, S. A., Junckerstorff, R. K., Korman, T. M.: Eggerthella lentabacteremia: Clinical and microbiological characteristics. J Clin Micro 53, 626635 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Claesson, M. J., Jeffery, I. B., Conde, S., Power, S. E., O’Connor, E. M., Cusack, S., Harris, H. M., Coakley, M., Lakshminarayanan, B., O’sullivan, O., Filzgerald, G. F., Deane, J., O’Connor, M., Harnedy, N., O’Connor, K., O’Mahony, D., Van Sideren, D., Wallace, M., Brennan, L., Stanton, C., Marchesis, J. R., Fitzgerald, A. P., Shanahan, F., Hill, C., Ross, R. P., O’Toole, P. W.: Gut microbiota composition correlates with diet and health in the elderly. Nature 9, 178184 (2012).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Zapata, H. J., Quagliarello, V. J.: The microbiota and microbiome in aging: Potential implications in health and age related diseased. J Am Geriatr Soc 63, 776781 (2015).

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Bartosch, S., Fite, A., Macfarlane, G. T., McMurdo, M. E.: Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol 70, 35753581 (2004).

    • Crossref
    • Search Google Scholar
    • Export Citation

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