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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g−1) were incubated overnight (18 h) at 37 °C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3·5 Salmonella ml−1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0·99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.

Salmonella spp. remain amongst the most important food-borne pathogens worldwide. In recent years there has been a marked increase in food-borne salmonellosis with outbreaks being reported in several countries including Spain, Italy, England and America (Smyth et al. 1989; Bean & Griffin 1990; Galbraith 1990; Rodrigue et al. 1990; Fantasia et al. 1991). Outbreaks have been linked to a wide range of foods including poultry, eggs, beef, fish, dairy products and chocolate (Fontaine et al. 1978; Gill et al. 1983; Tacket et al. 1985; Cartwright & Evans 1988; Cowden et al. 1989; Thomas 1989; Kapperud et al. 1990; Izat et al. 1991).

Currently available cultural methods for the detection of Salmonella in food are time consuming, taking up to 4–6 d to detect the pathogen, and are generally unsuitable for use in an industrial based laboratory. This is clearly unsatisfactory and a range of alternative rapid methods has been developed (Tietjen & Fung 1995). Approaches to rapid methodology for detection of Salmonella and other pathogens of concern to the food industry include electrical methods (impedance and conductance) and methods based on immunological and DNA hybridization techniques. While these tests are considerably faster than the traditional plate count methods, they still rely on lengthy enrichment (48 h) to increase very low initial Salmonella numbers to detectable levels. A novel rapid method for the detection of Listeria monocytogenes has been reported which takes 20 h to carry out (Sheridan et al. 1997). This method involved an overnight enrichment in buffered peptone water (BPW), isolation of the L. monocytogenes by attachment to a polycarbonate membrane immersed in the culture, followed by detection of the pathogen using immunofluorescent microscopy. The basic principles of this method would appear to have the potential to be applied to other pathogens of concern to the food industry, such as Salmonella.

It is already widely reported in the literature that Salmonella can attach to a wide range of inert surfaces such as stainless steel, glass and polymeric substrates, and to biological surfaces such as skin, muscle and cell membranes (Dickson & Daniels 1991; Mafu et al. 1991; Absolom et al. 1983). This suggests that a surface adhesion membrane extraction system may prove suitable for isolation of this pathogen.

The purpose of this study was to develop a rapid method for the detection of Salmonella based on an 18 h enrichment step, isolation by membrane surface adhesion and detection by immunofluorescence microscopy. This paper describes the application of this novel rapid technique to inoculated meat samples and it's correlation with a traditional cultural method. This novel rapid method was also applied to a small number of commercial meat samples to investigate the presence of naturally occurring Salmonella.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Meat samples

Beef samples were obtained from the on-site abattoir at The National Food Centre. Diaphragm meat (900 g) was used in inoculation experiments and was prepared by aseptically removing the excess fat tissue, cutting the meat and mincing through a sterile 5 mm steel plate (Crypto Peerless Mincer, model EB 12F, London, UK). All beef samples used in inoculation experiments were screened for the presence of naturally occurring Salmonella spp. using the traditional cultural method described below.

In addition, commercial minced beef (n = 20) and chicken samples (n = 80) were purchased from local retail outlets and examined for the presence of naturally occurring Salmonella.

Cultural detection of Salmonella

This method involved incubating 25 g of suspect sample in 225 ml BPW for 24 h at 37 °C. A 0·1 ml aliquot was transferred to 10 ml Rappaport Vassiliadis (RV, Oxoid) and incubated for a further 24 h at 42 °C. The sample was then streaked onto plates containing brilliant green agar (BGA, Oxoid) and mannitol lysine crystal violet brilliant green agar (MCLB, Oxoid), and incubated for 24 h at 37 °C. Suspect Salmonella colonies were confirmed using a series of biochemical tests (Anonymous 1993). Confirmed Salmonella spp. were subsequently serotyped using a series of slide agglutinations with specific antisera by the Central Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin.

Mutant Salmonella enteritidis

A mutant antibiotic-resistant strain of Salm. enteritidis (NTCC 5765) was used in these studies in order to facilitate recovery and enumeration of the pathogen from meat samples containing high levels of competitive flora.

The double mutant used in this study was prepared by the method of de Blackburn & Davies 1994). In this procedure, an isolate of Salm. enteritidis (NCTC 5765) was sequentially incubated in the presence of nalidixic acid (100 g ml−1) and streptomycin sulphate (2000 g ml−1). Double resistant mutants were recovered on nutrient agar containing nalidixic acid (50 g ml−1) and streptomycin sulphate (1000 g ml−1). The mutant was maintained on Protect beads (Technical Service Consultants Ltd, Heywood, UK) and stored at −20 °C, and was streaked at regular intervals onto Tryptone Soya Agar (TSA, Oxoid) supplemented with antibiotics to ensure viability.

Salm. enteritidis inoculum suspension

The double mutant was streaked onto TSA supplemented with nalidixic acid and streptomycin sulphate and incubated overnight at 37 °C. A single colony from the agar plate was inoculated into 10·0 ml Brain Heart Infusion broth (BHI, Oxoid) and incubated overnight at 37 °C. The number of Salm. enteritidis ml−1 suspension was determined using a membrane filtration epifluorescent technique described by Sheridan et al. (1997).The suspension was subsequently serially diluted in 0·1% peptone water (Oxoid) (9·0 ml) to give a desired final concentration of Salm. enteritidis (log 3·50 cfu ml−1).

Enrichment studies

Broth and meat cultures.

Experiments were carried out to determine the most suitable enrichment system for Salm. enteritidis.

Broth cultures were prepared by inoculating 225 ml non-selective broth (BPW) or selective enrichment broth (RV) with 5·0 ml Salm. enteritidis suspension (as described above) to produce a final concentration of approximately log10 2·0 cfu ml−1 broth.

Meat culture systems were prepared by adding Salm. enteritidis suspension (5·0 ml) to minced beef (25 g) in foil trays (20 cm × 10 cm), which had been previously sterilized by flooding with alcohol, and sterilized by flaming. The inoculated meat was thoroughly mixed using a sterile spatula and added to BPW or RV (225 ml) in a sterile flask giving a final concentration of approximately log10 2·00 cfu ml−1 culture.

Generation of growth curves.

Broth and meat cultures prepared from BPW or RV were incubated in a water-bath at 37 °C ± 0·2 for 10 h. At intervals (0, 2, 4, 6, 8 and 10 h) during incubation, aliquots of 1·0 and 0·1 ml were withdrawn from cultures and diluted as required in 0·1% peptone water (9·0 ml). Aliquots (0·1 ml) were plated onto BGA containing nalidixic acid (0·025 g/500 ml−1) and streptomycin sulphate (0·5 g/500 ml−1). All plates were incubated overnight at 37 °C and typical Salm. enteritidis colonies counted. Growth curves were preformed in dulplicate and repeated five times.

Buffered peptone water was found to be the most suitable enrichment broth as it allowed the greatest rate of cell growth, and was therefore used in subsequent rapid method experiments.

Rapid method

Anti-Salmonella antibody.

A fluorescein isothiocyanate (FITC) labelled anti-mouse conjugated antibody was obtained from Biogenesis Ltd (Poole, UK). This antibody contains mixed group antigens for Salm. enteritidis, Salm. typhimurium and Salm. heidelberg and is specific for the Salmonella O and H antigens. The antibody was diluted 1/50 in a 1% solution of skimmed milk powder (Marvel, Stafford, UK) containing 0·1% Tween-80 (Sigma), dispensed into 5·0 ml aliquots and stored at −20 °C.

Previous experimental trials using immunofluorescent microscopy concluded that this antibody did not react with other common meat microflora including Escherichia coli, Brochothrix thermosphacta, Pseudomonas aeruginosa, Ps. fragi, Staphylococcus aureus, Listeria monocytogenes and Yersinia enterocolitica.

Isolation of Salmonella by surface adhesion.

Broth and meat culture systems were inoculated with Salm. enteritidis (mutant strain) to an initial level of log10 2·0 cfu ml−1 as previously described. The cultures were incubated at 37 °C until Salm. enteritidis reached a concentration of approximately log10 6·00 ml−1 (5 h).

A series of experiments was carried out to determine whether Salm. enteritidis could be isolated from these enrichment broths by surface adhesion to a polycarbonate membrane (Sheridan et al. 1997), and to determine the length of immersion time required to allow attachment. The enriched broth or meat cultures (100 ml) were decanted into a staining trough designed to hold glass slides. A membrane/slide assembly was prepared which involved completely immersing a glass microscope slide (76 mm × 32 mm) in a 100 ml molten bacteriological agar (1%, Oxoid) for 10 s. The glass slide was removed and a polycarbonate membrane (0·6 WP language English (U.S.)μWP language English (U.K.)m pore size, 25 mm in diameter, Poretics, Livermore, CA, USA) was placed on the surface of the slide. The membrane became fixed to the slide during solidification of the agar. Membrane/slide assemblies (n = 20) were immersed in the staining trough containing the enriched broths and duplicate slides were removed aseptically at intervals of 2, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min. Polycarbonate membranes were detached from the glass slides using sterile forceps.

Enumeration of bacteria.

The bacteria adhering to the membrane were enumerated by rinse counts or by immunofluorescence.

(i) Rinse counts. One of each set of duplicate polycarbonate membranes with adherent bacteria was individually placed in 10 ml volumes of 0·1% peptone water and shaken for 1–2 min (Sheridan et al. 1997). The vigorous shaking facilitated bacterial detachment and resuspension of the bacteria into the peptone water. The suspension was serially diluted; 0·1 ml aliquots were then plated out onto BGA containing nalidixic acid and streptomycin sulphate and incubated at 37 °C for 24 h.

(ii) Immunofluorescence microscopy. The second membrane of each duplicate set of polycarbonate membranes was placed on a vacuum manifold and washed under vacuum with 30 ml phosphate-buffered saline (PBS) (Oxoid) containing 0·1% Tween (Merck). The washed membrane was placed in a sterile Petri dish, flooded with 0·5 ml fluorescein conjugated anti-Salmonella antibody and incubated at 37 °C for a further 30 min. The membrane was rewashed under vacuum using 30 ml PBS with 0·1% Tween to remove any unbound antibody. The membrane was dried under vacuum and mounted in oil on a glass slide. A Nikon Optiphot microscope (Nikon, Japan) fitted with an epifluorescent attachment, a 100 W mercury vapour light source, a filter with wavelength range of 350–400 nm and a 60× oil immersion plan objective was used to examine the membrane. Under these conditions, Salmonella cells were clearly visible as hollow cells surrounded by a fluorescent green ‘halo’. The antibody-labelled cells in 20 fields of vision were counted and multiplied by a working factor of 1·25 (Sheridan et al. 1998) to determine the population density of Salmonella per mm2 of membrane.

The results of this study showed that Salm. enteritidis could be isolated from the enrichment broth by surface adhesion to a membrane immersed for 15 min, and these data were used in subsequent studies.

Inoculated culture studies.

The detection limit of the rapid assay and the extent to which it correlated with traditional assay techniques was determined as follows. Broth and meat culture systems inoculated with the mutant strain of Salm. enteritidis to a final concentration of approximately log10 2·0 cfu ml−1 were prepared as previously described, and incubated in a water-bath at 37 °C (±0·2 °C) for up to 8 h. After 0, 2, 4, 6 and 8 h incubation, Salm. enteritidis numbers in broth and meat cultures were estimated by the traditional plate count, rinse count and immunofluorescent microscopy.

Retail samples (naturally contaminated).

Samples of retail minced beef (n = 20) and chicken pieces (n = 80) were examined for the presence of Salmonella using the rapid method and traditional cultural techniques. Traditional cultural detection of Salmonella was carried out as described above. For the rapid count, a 100 ml aliquot of an overnight enrichment culture (BPW) was withdrawn and an SAIF count was carried out as described.

Statistical analysis

In this study, the growth characteristics of interest were the growth rate and the duration of the lag phase. Regression analysis was used to analyse the data and calculate these growth kinetics using the same approach as Duffy et al. (1994).

Linear regression analysis was also used to determine the relationship between (a) enrichment time and the numbers of Salm. enteritidis (traditional plate counts) in the culture, and (b) enrichment time and numbers of Salm. enteritidis adhering to the membranes as determined by rinse and immunofluorescence count, and Salm. enteritidis numbers (cfu ml−1) from traditional plate counts and the surface adhesion immunofluorescent method (cfu mm2).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Enrichment media for the rapid method

Preliminary experiments in this study examined the growth kinetics of Salm. enteritidis in RV and BPW for broth and meat systems. No differences were noted in broth systems but in meat cultures, a significantly higher growth rate (P < 0·001) and shorter lag phase (P < 0·025) were recorded in BPW (Table 1). Overall, the results indicated that BPW is a more suitable enrichment medium for use in conjunction with a rapid method.

Table 1.  Growth kinetics of Salmonella enteritidis in BPW and RV media in broth and meat systems
Lag Phase
BrothCulture typeGrowth rate (cfu ml−1 h−1)S.E.(h)S.E.DF
  1. s.e., Standard error of mean.

  2. DF, degrees of freedom.

BPWBroth0·0100·0010·680·1526
RVBroth0·0080·0011·290·4612
BPWMeat0·0150·0011·520·2616
RVMeat0·0090·0012·630·3720

Immersion times and the numbers of Salm. enteritidis adhering to the membrane

Salmonella enteritidis was found to adhere to the membrane when it was immersed in a culture system. Table 2 shows the mean numbers of Salm. enteritidis attached to the membrane after different immersion times as determined by rinse and SAIF counts. Adherence of Salm. enteritidis to the membrane was rapid during the first 15 min of immersion, with the net number attaching to the membrane continually increasing. Between 15 and 60 min, increase in numbers observed was not statistically significant (P < 0·5). Similar results were recorded for broth cultures (data not shown).

Table 2.  Relationship between immersion time and the number of Salmonella enteritidis adhering to membranes in meat culture systems determined by a rinse count and a surface adhesion immunofluorescent (SAIF) count
Mean membrane counts (log10 cfu mm2)
Immersion time (min)RinseS.E.ImmunofluorescentS.E.
  1. All samples contained log10 6 cfu ml−1 of enrichment broth.

  2. Mean of six samples.

  3. s.e., Standard error of means.

  4. Degrees of freedom = 5.

53·620·203·510·26
104·010·223·710·23
154·370·274·320·22
204·420·194·340·25
254·400·234·330·26
304·590·224·300·30
404·450·254·360·27
504·370·274·390·28
604·250·264·310·24

Enrichment times and the numbers of Salm. enteritidis in broth and meat systems

Table 3 presents data showing the increase in numbers of Salm. enteritidis over a period of 8 h in broth and meat systems determined by traditional plate counts and membrane counts (rinse and immunofluorescent). Using regression analysis, the numbers of Salm. enteritidis determined by plate counts was shown to increase linearly with time in both broth (r2 = 0·99, rsd = ± 0·19) and meat cultures (r2 = 0·99, rsd (residual standard deviation) = ± 0·18). Similar numbers of Salm. enteritidis were detected on the immersed membranes by immunofluoresence and rinse counts. The numbers of Salm. enteritidis were found to increase linearly with enrichment time in both broth (r2 = 0·98, rsd = ± 0·23) and meat cultures (r2 = 0·99, rsd = ± 0·23).

Table 3.  Relationship between enrichment time and number of Salmonella enteritidis in broth and meat culture systems determined using traditional plate counts and membrane counts (rinse and immunofluorescent)
Mean membrane counts(log10 cfu mm2)
Enrichment time (h)Plate counts (log10 cfu ml−1)RinseImmunofluorescent
Broth culture
03·000·100·10
24·110·911·11
44·922·112·44
66·443·313·45
87·204·224·19
Meat culture
03·650·180·09
24·441·101·13
45·952·462·56
66·523·863·47
87·564·414·17

A good correlation was recorded (r2 = 0·99, rsd = ± 0·18) between traditional plate counts and the numbers detected by the surface adhesion immunofluorescent (SAIF) counts (Fig. 1). These data show that a minimum detection level of log10 3·5 cfu ml−1 was necessary in the enrichment sample to allow the detection of Salm. enteritidis on the membrane by the SAIF technique.

image

Figure 1. Relationship between the number of Salmonella enteritidis in inoculated meat cultures as determined by surface adhesion immunofluorescent counts (SAIF) (log10 cfu mm2) and traditional plate counts (log10 cfu ml−1)

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Retail samples

A total of 100 retail samples were tested for the presence of naturally occurring Salmonella. The SAIF method detected the presence of Salmonella in 31 of the 100 samples examined; 29 poultry (36%) and two minced beef (10%) samples were found to be contaminated. The traditional method also identified these 31 positive samples and four lactose fermentors which were not Salmonella spp. In addition, one group C Salmonella was identified using both methods but this was not typable. The most prevalent Salmonella serotype was Salm. bredeney, accounting for 74% of isolates (Table 4).

Table 4.  Serotyping of Salmonella spp. isolated from retail meat and poultry samples
Salm. enteritidisSalm. typhimuriumSalm. bredeneySalm. indianaSalm. infantisSalm. kentucky
  1. One Group C Salmonella could not be typed.

  2. Four lactose fermenters falsely identified using standard protocol.

3123211

Table 5 shows data obtained from the 31 samples which were positive for Salmonella as determined by (a) traditional plate counts at time 0 and time 18 and (b) SAIF counts at T18. From these data, it is possible to establish that the initial numbers of Salmonella naturally present in the retail samples examined varied from very low levels of contamination to log10 2·18 cfu g−1. After an enrichment period of 18 h, the levels of Salmonella in these samples increased to Ðlog10 4·18 cfu ml−1. This was previously ascertained to be above the minimum level of detection of the SAIF method.

Table 5.  Number of Salmonella at time 0 and after enrichment (18 h) as determined by a traditional plate count and a surface adhesion immunofluorescent (SAIF) count for retail samples
Sample no.Plate count at To (log10 cfu g−1)Plate count at T18 (log10 cfu ml−1)SAIF counts at T18 (log10 cfu mm2)
11·735·331·83
21·605·212·08
30·894·401·30
41·455·802·57
51·305·181·56
61·185·111·82
70·704·851·62
80·785·231·72
91·165·722·03
101·274·811·46
110·844·621·27
120·784·951·33
131·204·931·46
140·844·521·30
151·704·481·10
161·684·611·35
171·314·651·18
180·994·551·40
191·034·591·00
201·545·420·87
210·814·181·59
221·024·981·10
231·315·091·40
241·255·251·70
251·495·191·76
260·784·390·99
270·304·311·00
281·585·301·98
291·254·871·30
302·186·362·85
311·455·882·00

By substituting the data from the 31 positive retail samples into the regression equation developed for inoculated samples (Fig. 1), it was possible to predict Salmonella plate counts from the SAIF counts. Figure 2 shows the excellent correlation (r2 = 0·83) between the actual plate count and the predicted plate counts.

image

Figure 2. Relationship between the number of Salmonella in enriched retail poultry samples as determined by experimental and predicted plate counts (log10 cfu ml−1)

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

This study found that the use of a selective media containing inhibitory agents offered no advantages in terms of an enrichment step as part of a rapid technique. In addition, using a non-selective broth such as BPW allows the recovery of injured cells (Bailey & Cox 1992), which is of particular importance in the analyses of processed foods.

The second phase of this study showed that Salm. enteritidis could be isolated from an enriched culture using a surface adhesion technique. The investigation found that maximum attachment of Salm. enteritidis to the polycarbonate membrane occurred after 15 min immersion in the culture, while immersion for an additional time of up to 60 min did not result in a significant increase in Salm. enteritidis attachment. This is similar to the attachment rates recorded for other pathogens, including L. monocytogenes and Yersinia enterocolitica, to a polycarbonate membrane in an enriched culture (Sheridan et al. 1997, 1998).

In this study, similar attachment rates were recorded for Salm. enteritidis enriched in broth and meat culture systems, indicating that the attachment of the pathogen was not affected by the presence of naturally occurring meat microflora. Other workers have also reported that attachment of one bacterial species was unaffected by other species present (Farber & Idziak 1984; Sheridan et al. 1997). Adherence is mainly dependent on physiochemical properties, including the structure of the attachment surface and environmental conditions such as pH (Dickson & Koohmaraie 1989; Mafu et al. 1991; Duffy & Sheridan 1997a).

The surface adhesion-based extraction of Salmonella gives a rapid and clean deposit of the bacteria onto the membrane surface from which they can then be detected using FITC-labelled antibodies. The membrane surface adhesion technique and immunomagnetic beads for the isolation of L. monocytogenes from enrichment broths were compared and the surface adhesion method was reportedly quicker while achieving similar extraction rates (Duffy et al. 1997b). It is assumed that a similar scenario would be recorded for the isolation of Salmonella.

Studies using inoculated meat determined that a level of log10 3·50 cfu ml−1 of Salm. enteritidis was necessary for detection by the SAIF technique. Data generated in the early part of this study on the modelling of Salmonella growth in BPW predicted that if very low initial levels of Salm. enteritidis (1 cfu 25 g−1) were present in the sample, an enrichment period of 18 h would be sufficient to allow the pathogen to reach a detectable level. Retail samples naturally contaminated with Salmonella spp. have been shown in this study to contain levels of approximately <log10 2·18 cfu g−1. Therefore, a shorter enrichment period might be possible for many samples. This enrichment period is considerably shorter than the 48 h enrichment period needed for commercial ELISA and DNA based technologies, which require a detection level of 105–106 cfu ml−1 target cells in the enriched sample (Foster et al. 1992; Felsine et al. 1993).

In inoculated meat samples, an excellent correlation was reported between Salm. enteritidis traditional plate counts and the numbers detected by the SAIF (r2 = 0·99). This indicates that it would be possible to predict accurately numbers of Salmonella in the enrichment broth from those detected by the SAIF technique. Predictive modelling techniques could then be applied to estimate the initial level of contamination in the product. This type of information would be invaluable in the development and application of risk assessment models (Buchanan & Whiting 1996).

Correlation of the SAIF assay with traditional methods was carried out using a small amount of naturally contaminated retail samples. A total of 31 samples tested positive for Salmonella by both methods, with no false positives or negatives being recorded for the rapid technique. After an 18 h enrichment, experimental plate counts obtained compared very favourably with predicted plate counts as determined from the SAIF method.

Salmonella bredeney was the most commonly isolated serotype in this study. No published data are available regarding the most prevalent Salmonella serotypes in foods in Ireland. However, clinical reports from the southern regions of the country noted that Salm. typhimurium was the most frequently isolated serotype, followed by Salm. enteritidis (Anonymous 1997 Infoscan. Communicable Disease Report. Salmonella. Quarterly Reports, 7, 1–4.

et al. 1995). The main serotypes responsible for outbreaks of salmonellosis in other countries in recent years have been Salm. typhimurium, Salm. enteritidis and Salm. virchow (Joseph, C.A. & Palmer, S.R. 1989Arroyo & Arroyo 1995).

In conclusion, the SAIF technique is easy to perform, ensuring rapid isolation and identification of Salmonella from enriched samples. It requires minimal time and labour and would have considerable potential for on-line use in an HACCP system in an industrial based laboratory where the rapidity of a result is vital.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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