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Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration... Ann Microbiol (2013) 63:179–185 DOI 10.1007/s13213-012-0459-y ORIGINAL ARTICLE Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples Shui-lian Bi & Lei Shi & He Yan & He-cheng Meng Received: 23 November 2011 /Accepted: 23 March 2012 /Published online: 20 April 2012 Springer-Verlag and the University of Milan 2012 Introduction Abstract We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched Campylobacter spp. have become the most common cause in Bolton broth and Preston broth, followed by plating on of acute human bacterial diarrhea in most industrialized Skirrow, mCCDA, and blood agar (a membrane filter on its countries, outnumbering the cases caused by Salmonella. surface) media, respectively. Thirty-nine of 99 samples were Although the infection is usually self-limiting, the serious positive and 71 Campylobacter were isolated by one or sequelae such as Guillain-Barré syndrome and reactive ar- more methods. More than one species of Campylobacter thritis could occur (Zilbauer et al. 2008). Within the genus were obtained in 8 (20.5 %) of 39 positive samples and two Campylobacter, C. jejuni and C. coli account for the major- genotypes were yielded on the same medium (11 samples, ity of human infections. Poultry and poultry products are 28.2 %) by pulsed-field gel electrophoresis (PFGE) geno- considered primary sources of human campylobacteriosis typing. Enrichment by Preston broth was significantly better and play important roles in disease transmission (Neimann than by Bolton broth (P<0.05). Moreover, the latter failed to et al. 2003; Wingstrand et al. 2006; Humphrey et al. 2007). detect Campylobacter jejuni strains. Skirrow medium was Present culture methods for the detection of Campylo- significantly less efficient than mCCDA medium and mem- bacter spp. in food and fecal samples involve plating onto brane filtration method (P<0.05). Overall, the combination selective media and incubation under microaerophilic con- of PC (primary enrichment in Preston broth, followed by ditions with or without preceding enrichment. Enrichment selective enrichment on mCCDA agar), PF (primary enrich- procedure is an important part of the technique, as this ment in Preston broth, followed by membrane filtration should provide a better recovery of Campylobacter than culture onto blood agar), and BF (primary enrichment in direct plating (Fricke et al. 1983). Two effective enrichment Bolton broth, followed by membrane filtration culture onto broths are Bolton broth, which is recommended by ISO blood agar) methods provided the optimum isolation rate of 10272–1 and recommended in protocols produced by the Campylobacter spp. U.S. Food and Drug Administration for the recovery of Campylobacter from wide range of sample types, and Pres- . . Keywords Campylobacter spp. Skirrow medium ton broth, which has been widely used to isolate Campylo- MCCDA medium Membrane filtration bacter from foodstuffs (Baggerman and Koster 1992; Uyttendaele and Debevere 1996; Nielsen and Nielsen 1999; Pearson et al. 2000). Plating media can be categorized S.-l. Bi (*) into blood-based or blood-free types. A blood-containing School of Food Science, Guangdong Pharmaceutical University, selective agar was developed by Skirrow (1977) and is still Zhongshan, Guangdong Province, People’s Republic of China one of the most widely used today. Modified charcoal cefo- e-mail: shuilianbi@foxmail.com perazone deoxycholate agar (mCCDA) is the only widely : : L. Shi H. Yan H.-c. Meng used plating medium without blood. This is useful to avoid College of Light Industry and Food Sciences, the disadvantages of blood, which is expensive, has a short South China University of Technology, shelf life, and can be easily contaminated. Membrane filtration Guangzhou, Guangdong Province, People’s Republic of China 180 Ann Microbiol (2013) 63:179–185 is a physical method based on the principle that Campylobac- with supplement SR155E) agars, respectively. The plates ter and related genera can pass to the antibiotic-free medium were incubated at 37 °C in a microaerobic atmosphere for through the pores of a membrane laid on the medium surface, 48 h. whereas other organisms are held back (Piersimoni et al. 1995). Culture using membrane filtration This study will compare the efficacy of the different separation methods and which combination of broths and A 0.45-μm pore size cellulose triacetate membrane (Advan- media would obtain the optimal isolation rate of Campylo- tec, Tokyo, Japan) was placed on the surface of a blood agar bacter spp. Three different solid media (Skirrow, mCCDA, plate. Six to eight drops of Bolton and Preston culture and membrane filtration onto blood agar) will be used to samples were delivered with a Pasteur pipette onto the isolate the Campylobacter species from enriched samples of surface of the membrane. The filter was left to stand for raw chicken, duck, pork, and beef in Bolton and Preston 30-40 min at 37 °C until all the fluid had passed through and broths. PFGE analysis will be utilized for genotyping of all was then discarded. Afterwards, the plates were incubated the colonies of Campylobacter spp. under microaerobic conditions at 37 °C for 48 h. Campylobacter identification Materials and methods Suspect colonies were picked as long as the Campylo- Samples bacter-like colonies seemed different (with a metal sheen or not, bigger or smaller, flat or claw, etc.) from A total of 99 raw meat samples (including 9 beef, 11 pork, a plate and were identified phenotypically on the basis 52 chicken, and 27 duck) were randomly obtained from of Gram stain morphology, cytochrome oxidase produc- different local butcher shops located in Guangzhou, China, tion, hydrolysis of hippurate and indoxyl acetate, catalase between January and April 2009. The samples were trans- activity, and growth patterns at 25 and 42 °C. DNA extraction ferred to the laboratory in a cold-storage container on ice and was performed with the DNeasy tissue kit (Qiagen, Hilden, processed for Campylobacter species within 2 h of collection. Germany) according to the manufacturer’s instructions. The phenotypical identification results were confirmed by Cam- Isolation and identification of Campylobacter from meat pylobacter (cdt gene) PCR detection kit (TaKaRa, Tokyo, samples Japan). The confirmed colonies were subtyped by PFGE as described below, and if their genotypes were identical and Enrichment culture isolated from the same plate, only one was employed. On the other hand, all the colonies were employed if their genotypes Briefly, 10 g were transferred aseptically into a sterile filter were different. bag and a 10 ml volume of 0.9% (w/v) saline was poured into this bag. After the bag was stomached for 30 s using a PFGE analysis of isolates BagMixer lab blender 400 (Interscience, Saint Nom, France), 1 ml homogenized solution was then transferred Agarose plugs with embedded chromosomal DNA were to 4 ml of Bolton [Oxoid Bolton broth base CM983 with prepared for PFGE as described by Ribot et al. (2001). Plugs supplement SR183E and 5 % (v/v) lysed horse blood] and were digested with 40 U SmaI enzyme (TaKaRa) at 30 °C Preston [Oxoid Nutrient broth no. 2 CM67 with supplement for 4 h. The restriction fragments were separated by elec- SR117E, SR232E and 5 % (v/v) lysed horse blood] enrich- trophoresis in 0.5×Tris-borate-EDTA buffer at 14 °C for ment broths, respectively. All enrichments were incubated at 18 h using a Chef Mapper XA electrophoresis system 37 °C for 24 h under microaerobic atmosphere conditions (Bio-Rad, Hercules, CA, USA) with pulse times of 6.36– (5 % O ,10%CO and 85 % N ) using a multi-gas 35.38 s. After electrophoresis, the gels were stained in 2 2 2 incubator MCO-5 M (Sanyo, Tokyo, Japan). ethidium bromide (1 μg/ml), and DNA bands were visual- ized with Gel Doc 1000 (Bio-Rad). Selective culture Statistical analyses Following incubation for 24 h for Bolton and Preston broths, one loopful of approximately 10 μl culture sample Significance was estimated by MacNemar’s test for paired was streaked onto Skirrow [Oxoid blood agar base CM271 samples and chi-square test using the SAS software version with supplement SR69E and 5 % (v/v) lysed horse blood] 8.2 (SAS Institute, Cary, NC, USA). For all tests, P≤0.05 was considered significant. and mCCDA (Oxoid blood-free selective agar base CM739 Ann Microbiol (2013) 63:179–185 181 Results total, the sensitivity of Preston enrichment was better than Bolton enrichment (P<0.05). There was significant (P< Of 99 samples tested, 39 (39.4 %) samples were positive for 0.05) difference between the Bolton enrichment-based pro- Campylobacter spp. by at least one procedure used. For the tocols. In this study, 60 (84.5 %) of Campylobacter strains 79 poultry samples, Campylobacter were isolated from 21 were only isolated by one procedure, 10 (14.1 %) strains by out of 52 chicken samples (40.4 %) and 15 out of 27 duck both methods and 1 (1.4 %) strains by three procedures samples (55.6 %), indicating a high incidence of Campylo- (Table 3). bacter in retail poultry contrasting with lower detection rates When comparing the sensitivity rates of samples with the in beef and pork samples. Twenty-one and 49 isolates of C. six procedures, the results of 56.4 % with PC, 56.4 % with PF jejuni and C. coli were isolated from 14 and 33 meat and 30.8 % with BF were observed. The sensitivity of samples samples, respectively. Campylobacter fetus was found in from BS and PS was only 10.3 % (Table 4). No single method only one chicken sample and none of the C. jejuni isolates could detect all positive samples. The highest sensitivity rates originated from beef and pork samples. The results of Cam- were found when the combination of BF, PC, and PF methods pylobacter spp. by phenotypical identification were consis- were used. The rate was significantly higher than PC or PF tent with those obtained by PCR. The number of the isolates method alone (P<0.05); however, compared to the other was much higher than the number of positive samples combinations of three methods, no statistical significant dif- (Table 1). ference was found for the combination of BF, PC, and PF In the 39 positive samples, by using only one separation methods. This combination yielded 38 positive samples and method, 19 (48.7 %) samples were detected positive for 64 Compylobacter isolates, being 97.4 % of the total positive Campylobacter, and only one Campylobacter strain was samples and 90.1 % of the total isolates obtained. isolated from each sample. With the applying of two or more procedures, 18 (46.2 %) samples were found positive Discussion for 16 C. jejuni and 31 C. coli; in other 2 positive samples, we obtained 2 C. coli with BF method (primary enrichment Generally, the PCR identification algorithm and biochemical in Bolton broth, followed by membrane filtration culture onto blood agar) and 2 C. jejuni with PS method (primary tests are to the species level. This is an advantage over the culture methods, where identification is only to the genus enrichment in Preston broth, followed by selective enrich- ment on Skirrow agar). There were 8 of 39 positive samples level. Considering the possibility exists of duplicate colonies, we suggest the genotyping of PFGE for Campylobacter iso- isolated two or more species of Campylobacter, which all lates. There have been many studies comparing different the C. jejuni were isolated based on Preston enrichment selective media and detection methods of Campylobacter broth (Table 2). (Arzate Barbosa et al. 1999; Paulsen et al. 2005); however, Of the 71 Campylobacter isolates, it was obvious that 31 were detected using the PC (primary enrichment in Preston the authors did not take PFGE or any other genotying method for analysis. It was difficult to make certain of the accuracy of broth, followed by selective enrichment on mCCDA agar), compared with 4, 15, 7, and 26 by BS (primary enrichment the results. As in many other studies regarding food, poultry meat in Bolton broth, followed by selective enrichment on Skir- row medium), BF, PS, and PF (primary enrichment in Pres- (chicken and duck) samples were particularly contaminated. In this study, however, the percentage regarding chicken was ton broth, followed by membrane filtration culture onto blood agar), respectively. No Campylobacter spp. was lowerthanduckand C. jejuni found in all samples was significantly lower than C. coli (P<0.05). The two results obtained by BC (primary enrichment in Bolton broth, fol- contrasted with the findings of other studies (Federighi et al. lowed by selective enrichment on mCCDA agar) and no C. 1999; Borck et al. 2002). jejuni isolates were found based on Bolton enrichment. In Table 1 Detection of Campylo- Sample type No. of samples No. of positive No. of genotyped isolates bacter species in various raw samples meat samples C. jejuni C. coli C. fetus Beef 9 1 0 2 0 Pork 11 2 0 3 0 a Chicken 52 21 12 27 1 If PFGE genotypes of Cam- pylobacter isolated from the Duck 27 15 9 17 0 same sample were identical, only Total 99 39 21 49 1 one was employed. 182 Ann Microbiol (2013) 63:179–185 Table 2 The number of Cam- Sample Origin Species No. of isolates by each of method No. of C. jejuni No. of C. coli pylobacter isolates and the no. genotypes by genotypes by number of their PFGE type PFGE PFGE BS BC BF PS PC PF given by the 20 samples which yielded more than one species 1 Beef C. coli 11 2 or genotypes 2 Pork C. coli 22 3 Chicken C. jejuni 11 C. coli 22 4 Chicken C. coli 12 1 3 5 Chicken C. coli 12 1 2 6 Chicken C. jejuni 21 2 7 Chicken C. coli111 3 8 Chicken C. coli 11 2 9 Chicken C. jejuni 11 C. coli 21 3 10 Chicken C. jejuni 12 2 C. coli11 1 11 Chicken C. jejuni 21 3 C. coli11 1 12 Chicken C. jejuni 11 1 C. coli 11 C. fetus 1 13 Duck C. jejuni 11 C. coli 11 14 Duck C. coli 12 2 15 Duck C. coli111 3 16 Duck C. coli 11 1 17 Duck C. jejuni 11 C. coli 11 2 18 Duck C. jejuni 22 19 Duck C. jejuni 11 2 a 20 Duck C. jejuni 11 2 If PFGE genotypes of Cam- pylobacter isolated from the C. coli 12 1 2 same plate were identical, only Total 4 0 9 7 26 18 18 33 one was employed. In the previous studies, one species of Campylobacter with two different genotypes of C. jejuni or C. coli on one isolated on different media has been reported (Bolton et al. medium. It was obvious that our findings differed from those 1988; Endtz et al. 1991). Different colonies picked from the of previous studies in three aspects: (1) increased isolation same medium were stated clearly only in a few studies. rate; (2) isolated from meat (from patient previously); and (3) Goossens et al. (1986)and Borcketal. (2002) isolated to the best of our knowledge, this is the first time that more two species of Campylobacter (C. jejuni and C. coli)in2 than two Campylobacter species have been isolated from one (1.59 %) of 126 positive samples and 2 (1.61 %) of 124 plate of a chicken sample. positive samples, respectively. Of the 43 specimens found Conventional enrichment cultures, e.g., ISO 10272-1, positive for Campylobacter on the membrane filter, one have been widely used to isolate Campylobacter from com- (2.3 %) yielded two species (C. jejuni and C. coli) and two plex sample types. Normally, Bolton broth is favored, as it (4.7 %) yielded two different biotypes (Piersimoni et al. has been shown to be effective in the isolation of Campylo- 1995). Instead, more than one species of Campylobacter bacter from poultry-related samples (Baylis et al. 2000). were obtained in 8 (20.6 %) of 39 positive samples in our Preston broth, a common choice of enrichment medium in study (C. jejuni and C. coli in 7 samples, and C. jejuni, C. isolating thermophilic Campylobacter, is poor in supporting coli,and C. fetus in 1 sample). On the other hand, 11 the growth of C. coli. It has also been shown by Ng et al. (28.2 %) Campylobacter-positive samples were obtained (1985) that some C. coli strains are strongly inhibited by the Ann Microbiol (2013) 63:179–185 183 Table 3 Comparison of the number of Campylobacter spp. isolated by preventing contaminants. Although the BC method did not the use of six different methods in 99 raw meat samples show a positive effect on the isolation of Campylobacter,the highest recovery of Campylobacter spp. was observed by Method No. of Campylobacter isolates using the PC (or PF) method. The results of this study were C. jejuni C. coli C. fetus Total consistent with several previous studies showing that mCCDA was an extremely good medium for the isolation of BS 0 4 0 4 Campylobacter species (Gun-Munro et al. 1987; Endtz et al. BC 0 0 0 0 1991). BF 0 15 0 15 Antimicrobials in Campylobacter-selective media were PS 5 2 0 7 inhibitory to the growth of some strains of C. jejuni and C. PC 12 19 0 31 coli (Ng et al. 1985). Antibiotic-sensitive Campylobacter PF 7 18 1 26 strains have been isolated from human feces by the use of membrane filtration onto antibiotic-free agar, and the more If PFGE genotypes of Campylobacter isolated from the same sample by using one method were identical, only one was employed. unusual Campylobacter spp. have been grown satisfactorily only when the membrane filtration method was used (Steele combination of antibiotics present in Preston broth. In this and McDermott 1984; Lastovica 2006). In this study, the study, however, Preston broth had a significantly higher membrane filtration technique appeared to have the excel- isolation rate of Campylobacter spp. than Bolton broth. lent abilities to isolate Campylobacter spp., while the use of Preston broth was also better in recovering C. coli compared small Petri dishes makes it cheap and easy to perform. By with Bolton broth. With Bolton enrichment broth, C. jejuni the use of Bolton enrichment broth, the membrane filtration strains were not obtained. method was significantly superior to mCCDA and Skirrow. As two different selective media (Skirrow and Based on Preston enrichment Bolton, the membrane filtra- mCCDA) contained different antimicrobials were used tion technique had better recovery of Campylobacter spp. for culturing Campylobacter, the Cefoperazone used in than Skirrow and equal to mCCDA. In addition, besides C. mCCDA as a selective agent had been shown excellent jejuni and C. coli, one C. fetus strain was isolated by using suppressive properties of enteric flora (Goossens et al. the membrane filtration method, this result agreed with 1986; Gun-Munro et al. 1987). This study showed that Goossens et al. (1986) who found the membrane filtration Skirrow was the least selective medium, being poorly method not only raised the isolation rate of C. jejuni and C. selective of existing growth of contaminants. Compared coli but also resulted in the isolation of less common Cam- with Skirrow, mCCDAwas a superior medium for reducing or pylobacter spp. Table 4 The number of Campylobacter and positive samples by single method or a combination of two or three methods Method BS BF PS PC PF Campy Sample Campy Sample Campy Sample Campy Sample Campy Sample BS 4 4 –– – – –– – – BF –– 15 12 –– – – –– PS –– – – 74 –– – – PC –– – – – – 31 22 –– PF – – –– – – –– 26 22 BS + BF 19 16 –– – – –– – – BS + PS 11 8 –– – – –– – – BS + PC 33 22 –– – – –– – – BS + PF 30 22 –– – – –– – – BF + PS 26 20 22 16 –– – – –– BF + PC 47 29 45 29 –– – – –– BF + PF 44 32 40 32 –– – – –– PS+PC 38 2450 3136 24 –– – – PS+PF 37 2447 3433 24 –– – – PC+PF 52 3164 3855 3250 31 –– Campylobacter isolates. If PFGE genotypes of Campylobacter isolated from the same plate were identical, only one was employed. 184 Ann Microbiol (2013) 63:179–185 Baylis CL, MacPhee S, Martin KW, Humphrey TJ, Betts RP (2000) As far as the isolation of Campylobacter with six proce- Comparison of three enrichment media for the isolation of Cam- dures was concerned, although both PC and PF methods pylobacter spp. from foods. J Appl Microbiol 89:884–891 gave the best results, the isolation rates of both methods Bolton FJ, Hutchinson DN, Parker G (1988) Reassessment of were not high. As we have observed, the highest isolation selective agars and filtration techniques for isolation of Cam- pylobacter species from feces. Eur J Clin Microbiol 7:155– rate of Campylobacter was obtained when PC, PF, and BF were combined. This rate was significantly higher than that Borck B, Stryhn H, Ersbùll AK, Pedersen K (2002) Thermophilic obtained when the PC or PF method was used alone. Campylobacter spp. in turkey samples: evaluation of two auto- It was noteworthy that, for each method, the true isolation mated enzyme immunoassays and conventional microbiological techniques. J Appl Microbiol 92:574–582 rate of Campylobacter spp. may be underestimated because Endtz HP, Ruijs GJ, Zwinderman AH, Reijden TVD, Biever M, Mouton of the limitations of culture methods as follows: RP (1991) Comparison of six media, including a semisolid agar, for the isolation of various Campylobacter species from stool speci- (a) To simplify subsequent experiments and optimal recov- mens. J Clin Microbiol 29:1007–1010 ery of unusual Campylobacter species, incubation of C. Federighi M, Magras C, Pilet MF, Woodward D, Johnson W, Jugiau F, jejuni was at 37 °C. When C. jejuni isolates were Jouve JL (1999) Incidence of thermotolerant Campylobacter in obtained from foodstuffs using enrichment at a single foods assessed by NF ISO 10272 standards: results of a two-year study. Food Microbiol 16:195–204 temperature, certain genotypes may escape detection and Fricke BCR, Girdwood RWA, Munro D (1983) A comparison of proce- frustrate epidemiological investigations (Scates et al. dures for the isolation of campylobacters from seagull faeces. J Hyg 2003). Therefore, to obtain a wide range of types, it will 91:445–450 be necessary to enrich samples at both 37 and 42 °C. Goossens H, Boeck MD, Coignau H, Vlaes L, Vanden BC, Butzler JP (1986) Modified selective medium for isolation (b) A number of competing flora identified to be Proteus of Campylobacter spp. from feces: comparison with Preston mirabilis were present on Skirrow, mCCDA, and blood medium, a blood-free medium, and a filtration system. J Clin media. The spread phenomenon of Proteus mirabilis Microbiol 24:840–843 Gun-Munro J, Rennie RP, Thornley JH, Richardson HL, Hodge on the media interfered with the isolation of Campylo- D, Lynch J (1987) Laboratory and clinical evaluation of isolation bacter. Whether there were other factors contributing media for Campylobacter jejuni. J Clin Microbiol 25:2274– to these needs to be investigated. Humphrey TJ, O'Brien S, Madsen M (2007) Campylobacters as zoo- In conclusion, the isolation of Campylobacter spp. notic pathogens: a food production perspective. Int J Food Micro- was highly dependent on the different combination of biol 117:237–257 enrichment broths and selective media (or filter system) Lastovica AJ (2006) Emerging Campylobacter spp.: The tip of the iceberg. Clin Microbiol News 28:49–55 used in the laboratory. Compared with Skirrow, Neimann J, Engberg J, Molbak K, Wegener HC (2003) A case control mCCDA medium and the membrane filter technique study of risk factors for sporadic Campylobacter infections in (using Preston enrichment broth) were superior methods Denmark. Epidemiol Infect 130:353–366 for routine use. Skirrow was an unacceptable medium Ng LK, Stiles ME, Taylor DE (1985) Inhibition of Campylobacter coli and Campylobacter jejuni by antibiotics used in selective growth for the routine monitoring of Campylobacter from meat media. J Clin Microbiol 22:510–514 samples. The combination of the PC, PF, and BF methods Nielsen EM, Nielsen NL (1999) Serotypes and typability of Campylo- will result in maximal isolation rates of Campylobacter spp. In bacter jejuni and Campylobacter coli isolated from poultry prod- addition, it is very important that PFGE analysis was applied ucts. Int J Food Microbiol 46:199–205 Paulsen P, Kanzler P, Hilbert F, Mayrhofer S, Baumgartnerb S, Smuldersa to Campylobacter isolates. FJM (2005) Comparison of three methods for detecting Campylo- bacter spp. in chilled or frozen meat. Int J Food Microbiol 103:229– Acknowledgments This work was supported by the National Natu- Pearson AD, Greenwood MH, Donaldson J, Healing TD, Jones DM, ral Science Foundation of China (20877028), the Science Foundation Shahamat MR, Feltham KA, Colwell RR (2000) Continuous of the Ministry of Education of China (706046) and the Fundamental source outbreak of campylobacteriosis traced to chicken. J Food Research Funds for the Central Universities (2012ZZ0083). Protect 63:309–314 Piersimoni C, Bornigia S, Curzi L, De Sio G (1995) Comparison of two selective media and a membrane filter technique for isolation of Campylobacter species from diarrhoeal stools. Eur J Clin Microbiol 14:539–542 References Ribot EM, Fitzgerald C, Kubota K, Swaminathan B, Barret TJ (2001) Rapid pulsed-field gel electrophoresis protocol for subtyping of Arzate Barbosa P, Garcia Gonzalez R, Ponce Nava E, Nachamkin I Campylobacter jejuni. J Clin Microbiol 5:1889–1894 (1999) Comparison of two selective media for the isolation of Scates P, Moran L, Madden RH (2003) Effect of incubation tempera- Campylobacter species from a pediatric population in Mexico. ture on isolation of Campylobacter jejuni genotypes from food- Diagn Microbiol Infect Dis 34:329–332 stuffs enriched in Preston broth. Appl Environ Microbiol 69:4658– Baggerman WI, Koster T (1992) A comparison of enrichment and membrane filtration methods for the isolation of Campylobacter Skirrow MB (1977) Campylobacter enteritis: a“new”disease. Br Med J from fresh and frozen foods. Food Microbiol 9:87–94 2:9–11 Ann Microbiol (2013) 63:179–185 185 Steele TW, McDermott SN (1984) The use of membrane filters applied Wingstrand A, Neimann J, Engberg J, Nielsen EM, Gerner-Smidt directly to the surface of agar plates for the isolation of Campylo- P, Wegener HC, Molbak K (2006) Fresh chicken as main risk bacter jejuni from faeces. Pathology 16:263–265 factor for campylobacteriosis, Denmark. Emerg Infect Dis Uyttendaele M, Debevere J (1996) Evaluation of Preston medium for 12:280–285 detection of Campylobacter jejuni in vitro and in artificially and Zilbauer M, Dorrell N, Wren B, Bajaj-Elliott M (2008) Campylobacter naturally contaminated poultry products. Food Microbiol 13:115– jejuni-mediated disease pathogenesis: an update. Trans R Soc 122 Trop Med Hyg 102:123–129 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

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Springer Journals
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Copyright © 2012 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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10.1007/s13213-012-0459-y
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Abstract

Ann Microbiol (2013) 63:179–185 DOI 10.1007/s13213-012-0459-y ORIGINAL ARTICLE Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples Shui-lian Bi & Lei Shi & He Yan & He-cheng Meng Received: 23 November 2011 /Accepted: 23 March 2012 /Published online: 20 April 2012 Springer-Verlag and the University of Milan 2012 Introduction Abstract We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched Campylobacter spp. have become the most common cause in Bolton broth and Preston broth, followed by plating on of acute human bacterial diarrhea in most industrialized Skirrow, mCCDA, and blood agar (a membrane filter on its countries, outnumbering the cases caused by Salmonella. surface) media, respectively. Thirty-nine of 99 samples were Although the infection is usually self-limiting, the serious positive and 71 Campylobacter were isolated by one or sequelae such as Guillain-Barré syndrome and reactive ar- more methods. More than one species of Campylobacter thritis could occur (Zilbauer et al. 2008). Within the genus were obtained in 8 (20.5 %) of 39 positive samples and two Campylobacter, C. jejuni and C. coli account for the major- genotypes were yielded on the same medium (11 samples, ity of human infections. Poultry and poultry products are 28.2 %) by pulsed-field gel electrophoresis (PFGE) geno- considered primary sources of human campylobacteriosis typing. Enrichment by Preston broth was significantly better and play important roles in disease transmission (Neimann than by Bolton broth (P<0.05). Moreover, the latter failed to et al. 2003; Wingstrand et al. 2006; Humphrey et al. 2007). detect Campylobacter jejuni strains. Skirrow medium was Present culture methods for the detection of Campylo- significantly less efficient than mCCDA medium and mem- bacter spp. in food and fecal samples involve plating onto brane filtration method (P<0.05). Overall, the combination selective media and incubation under microaerophilic con- of PC (primary enrichment in Preston broth, followed by ditions with or without preceding enrichment. Enrichment selective enrichment on mCCDA agar), PF (primary enrich- procedure is an important part of the technique, as this ment in Preston broth, followed by membrane filtration should provide a better recovery of Campylobacter than culture onto blood agar), and BF (primary enrichment in direct plating (Fricke et al. 1983). Two effective enrichment Bolton broth, followed by membrane filtration culture onto broths are Bolton broth, which is recommended by ISO blood agar) methods provided the optimum isolation rate of 10272–1 and recommended in protocols produced by the Campylobacter spp. U.S. Food and Drug Administration for the recovery of Campylobacter from wide range of sample types, and Pres- . . Keywords Campylobacter spp. Skirrow medium ton broth, which has been widely used to isolate Campylo- MCCDA medium Membrane filtration bacter from foodstuffs (Baggerman and Koster 1992; Uyttendaele and Debevere 1996; Nielsen and Nielsen 1999; Pearson et al. 2000). Plating media can be categorized S.-l. Bi (*) into blood-based or blood-free types. A blood-containing School of Food Science, Guangdong Pharmaceutical University, selective agar was developed by Skirrow (1977) and is still Zhongshan, Guangdong Province, People’s Republic of China one of the most widely used today. Modified charcoal cefo- e-mail: shuilianbi@foxmail.com perazone deoxycholate agar (mCCDA) is the only widely : : L. Shi H. Yan H.-c. Meng used plating medium without blood. This is useful to avoid College of Light Industry and Food Sciences, the disadvantages of blood, which is expensive, has a short South China University of Technology, shelf life, and can be easily contaminated. Membrane filtration Guangzhou, Guangdong Province, People’s Republic of China 180 Ann Microbiol (2013) 63:179–185 is a physical method based on the principle that Campylobac- with supplement SR155E) agars, respectively. The plates ter and related genera can pass to the antibiotic-free medium were incubated at 37 °C in a microaerobic atmosphere for through the pores of a membrane laid on the medium surface, 48 h. whereas other organisms are held back (Piersimoni et al. 1995). Culture using membrane filtration This study will compare the efficacy of the different separation methods and which combination of broths and A 0.45-μm pore size cellulose triacetate membrane (Advan- media would obtain the optimal isolation rate of Campylo- tec, Tokyo, Japan) was placed on the surface of a blood agar bacter spp. Three different solid media (Skirrow, mCCDA, plate. Six to eight drops of Bolton and Preston culture and membrane filtration onto blood agar) will be used to samples were delivered with a Pasteur pipette onto the isolate the Campylobacter species from enriched samples of surface of the membrane. The filter was left to stand for raw chicken, duck, pork, and beef in Bolton and Preston 30-40 min at 37 °C until all the fluid had passed through and broths. PFGE analysis will be utilized for genotyping of all was then discarded. Afterwards, the plates were incubated the colonies of Campylobacter spp. under microaerobic conditions at 37 °C for 48 h. Campylobacter identification Materials and methods Suspect colonies were picked as long as the Campylo- Samples bacter-like colonies seemed different (with a metal sheen or not, bigger or smaller, flat or claw, etc.) from A total of 99 raw meat samples (including 9 beef, 11 pork, a plate and were identified phenotypically on the basis 52 chicken, and 27 duck) were randomly obtained from of Gram stain morphology, cytochrome oxidase produc- different local butcher shops located in Guangzhou, China, tion, hydrolysis of hippurate and indoxyl acetate, catalase between January and April 2009. The samples were trans- activity, and growth patterns at 25 and 42 °C. DNA extraction ferred to the laboratory in a cold-storage container on ice and was performed with the DNeasy tissue kit (Qiagen, Hilden, processed for Campylobacter species within 2 h of collection. Germany) according to the manufacturer’s instructions. The phenotypical identification results were confirmed by Cam- Isolation and identification of Campylobacter from meat pylobacter (cdt gene) PCR detection kit (TaKaRa, Tokyo, samples Japan). The confirmed colonies were subtyped by PFGE as described below, and if their genotypes were identical and Enrichment culture isolated from the same plate, only one was employed. On the other hand, all the colonies were employed if their genotypes Briefly, 10 g were transferred aseptically into a sterile filter were different. bag and a 10 ml volume of 0.9% (w/v) saline was poured into this bag. After the bag was stomached for 30 s using a PFGE analysis of isolates BagMixer lab blender 400 (Interscience, Saint Nom, France), 1 ml homogenized solution was then transferred Agarose plugs with embedded chromosomal DNA were to 4 ml of Bolton [Oxoid Bolton broth base CM983 with prepared for PFGE as described by Ribot et al. (2001). Plugs supplement SR183E and 5 % (v/v) lysed horse blood] and were digested with 40 U SmaI enzyme (TaKaRa) at 30 °C Preston [Oxoid Nutrient broth no. 2 CM67 with supplement for 4 h. The restriction fragments were separated by elec- SR117E, SR232E and 5 % (v/v) lysed horse blood] enrich- trophoresis in 0.5×Tris-borate-EDTA buffer at 14 °C for ment broths, respectively. All enrichments were incubated at 18 h using a Chef Mapper XA electrophoresis system 37 °C for 24 h under microaerobic atmosphere conditions (Bio-Rad, Hercules, CA, USA) with pulse times of 6.36– (5 % O ,10%CO and 85 % N ) using a multi-gas 35.38 s. After electrophoresis, the gels were stained in 2 2 2 incubator MCO-5 M (Sanyo, Tokyo, Japan). ethidium bromide (1 μg/ml), and DNA bands were visual- ized with Gel Doc 1000 (Bio-Rad). Selective culture Statistical analyses Following incubation for 24 h for Bolton and Preston broths, one loopful of approximately 10 μl culture sample Significance was estimated by MacNemar’s test for paired was streaked onto Skirrow [Oxoid blood agar base CM271 samples and chi-square test using the SAS software version with supplement SR69E and 5 % (v/v) lysed horse blood] 8.2 (SAS Institute, Cary, NC, USA). For all tests, P≤0.05 was considered significant. and mCCDA (Oxoid blood-free selective agar base CM739 Ann Microbiol (2013) 63:179–185 181 Results total, the sensitivity of Preston enrichment was better than Bolton enrichment (P<0.05). There was significant (P< Of 99 samples tested, 39 (39.4 %) samples were positive for 0.05) difference between the Bolton enrichment-based pro- Campylobacter spp. by at least one procedure used. For the tocols. In this study, 60 (84.5 %) of Campylobacter strains 79 poultry samples, Campylobacter were isolated from 21 were only isolated by one procedure, 10 (14.1 %) strains by out of 52 chicken samples (40.4 %) and 15 out of 27 duck both methods and 1 (1.4 %) strains by three procedures samples (55.6 %), indicating a high incidence of Campylo- (Table 3). bacter in retail poultry contrasting with lower detection rates When comparing the sensitivity rates of samples with the in beef and pork samples. Twenty-one and 49 isolates of C. six procedures, the results of 56.4 % with PC, 56.4 % with PF jejuni and C. coli were isolated from 14 and 33 meat and 30.8 % with BF were observed. The sensitivity of samples samples, respectively. Campylobacter fetus was found in from BS and PS was only 10.3 % (Table 4). No single method only one chicken sample and none of the C. jejuni isolates could detect all positive samples. The highest sensitivity rates originated from beef and pork samples. The results of Cam- were found when the combination of BF, PC, and PF methods pylobacter spp. by phenotypical identification were consis- were used. The rate was significantly higher than PC or PF tent with those obtained by PCR. The number of the isolates method alone (P<0.05); however, compared to the other was much higher than the number of positive samples combinations of three methods, no statistical significant dif- (Table 1). ference was found for the combination of BF, PC, and PF In the 39 positive samples, by using only one separation methods. This combination yielded 38 positive samples and method, 19 (48.7 %) samples were detected positive for 64 Compylobacter isolates, being 97.4 % of the total positive Campylobacter, and only one Campylobacter strain was samples and 90.1 % of the total isolates obtained. isolated from each sample. With the applying of two or more procedures, 18 (46.2 %) samples were found positive Discussion for 16 C. jejuni and 31 C. coli; in other 2 positive samples, we obtained 2 C. coli with BF method (primary enrichment Generally, the PCR identification algorithm and biochemical in Bolton broth, followed by membrane filtration culture onto blood agar) and 2 C. jejuni with PS method (primary tests are to the species level. This is an advantage over the culture methods, where identification is only to the genus enrichment in Preston broth, followed by selective enrich- ment on Skirrow agar). There were 8 of 39 positive samples level. Considering the possibility exists of duplicate colonies, we suggest the genotyping of PFGE for Campylobacter iso- isolated two or more species of Campylobacter, which all lates. There have been many studies comparing different the C. jejuni were isolated based on Preston enrichment selective media and detection methods of Campylobacter broth (Table 2). (Arzate Barbosa et al. 1999; Paulsen et al. 2005); however, Of the 71 Campylobacter isolates, it was obvious that 31 were detected using the PC (primary enrichment in Preston the authors did not take PFGE or any other genotying method for analysis. It was difficult to make certain of the accuracy of broth, followed by selective enrichment on mCCDA agar), compared with 4, 15, 7, and 26 by BS (primary enrichment the results. As in many other studies regarding food, poultry meat in Bolton broth, followed by selective enrichment on Skir- row medium), BF, PS, and PF (primary enrichment in Pres- (chicken and duck) samples were particularly contaminated. In this study, however, the percentage regarding chicken was ton broth, followed by membrane filtration culture onto blood agar), respectively. No Campylobacter spp. was lowerthanduckand C. jejuni found in all samples was significantly lower than C. coli (P<0.05). The two results obtained by BC (primary enrichment in Bolton broth, fol- contrasted with the findings of other studies (Federighi et al. lowed by selective enrichment on mCCDA agar) and no C. 1999; Borck et al. 2002). jejuni isolates were found based on Bolton enrichment. In Table 1 Detection of Campylo- Sample type No. of samples No. of positive No. of genotyped isolates bacter species in various raw samples meat samples C. jejuni C. coli C. fetus Beef 9 1 0 2 0 Pork 11 2 0 3 0 a Chicken 52 21 12 27 1 If PFGE genotypes of Cam- pylobacter isolated from the Duck 27 15 9 17 0 same sample were identical, only Total 99 39 21 49 1 one was employed. 182 Ann Microbiol (2013) 63:179–185 Table 2 The number of Cam- Sample Origin Species No. of isolates by each of method No. of C. jejuni No. of C. coli pylobacter isolates and the no. genotypes by genotypes by number of their PFGE type PFGE PFGE BS BC BF PS PC PF given by the 20 samples which yielded more than one species 1 Beef C. coli 11 2 or genotypes 2 Pork C. coli 22 3 Chicken C. jejuni 11 C. coli 22 4 Chicken C. coli 12 1 3 5 Chicken C. coli 12 1 2 6 Chicken C. jejuni 21 2 7 Chicken C. coli111 3 8 Chicken C. coli 11 2 9 Chicken C. jejuni 11 C. coli 21 3 10 Chicken C. jejuni 12 2 C. coli11 1 11 Chicken C. jejuni 21 3 C. coli11 1 12 Chicken C. jejuni 11 1 C. coli 11 C. fetus 1 13 Duck C. jejuni 11 C. coli 11 14 Duck C. coli 12 2 15 Duck C. coli111 3 16 Duck C. coli 11 1 17 Duck C. jejuni 11 C. coli 11 2 18 Duck C. jejuni 22 19 Duck C. jejuni 11 2 a 20 Duck C. jejuni 11 2 If PFGE genotypes of Cam- pylobacter isolated from the C. coli 12 1 2 same plate were identical, only Total 4 0 9 7 26 18 18 33 one was employed. In the previous studies, one species of Campylobacter with two different genotypes of C. jejuni or C. coli on one isolated on different media has been reported (Bolton et al. medium. It was obvious that our findings differed from those 1988; Endtz et al. 1991). Different colonies picked from the of previous studies in three aspects: (1) increased isolation same medium were stated clearly only in a few studies. rate; (2) isolated from meat (from patient previously); and (3) Goossens et al. (1986)and Borcketal. (2002) isolated to the best of our knowledge, this is the first time that more two species of Campylobacter (C. jejuni and C. coli)in2 than two Campylobacter species have been isolated from one (1.59 %) of 126 positive samples and 2 (1.61 %) of 124 plate of a chicken sample. positive samples, respectively. Of the 43 specimens found Conventional enrichment cultures, e.g., ISO 10272-1, positive for Campylobacter on the membrane filter, one have been widely used to isolate Campylobacter from com- (2.3 %) yielded two species (C. jejuni and C. coli) and two plex sample types. Normally, Bolton broth is favored, as it (4.7 %) yielded two different biotypes (Piersimoni et al. has been shown to be effective in the isolation of Campylo- 1995). Instead, more than one species of Campylobacter bacter from poultry-related samples (Baylis et al. 2000). were obtained in 8 (20.6 %) of 39 positive samples in our Preston broth, a common choice of enrichment medium in study (C. jejuni and C. coli in 7 samples, and C. jejuni, C. isolating thermophilic Campylobacter, is poor in supporting coli,and C. fetus in 1 sample). On the other hand, 11 the growth of C. coli. It has also been shown by Ng et al. (28.2 %) Campylobacter-positive samples were obtained (1985) that some C. coli strains are strongly inhibited by the Ann Microbiol (2013) 63:179–185 183 Table 3 Comparison of the number of Campylobacter spp. isolated by preventing contaminants. Although the BC method did not the use of six different methods in 99 raw meat samples show a positive effect on the isolation of Campylobacter,the highest recovery of Campylobacter spp. was observed by Method No. of Campylobacter isolates using the PC (or PF) method. The results of this study were C. jejuni C. coli C. fetus Total consistent with several previous studies showing that mCCDA was an extremely good medium for the isolation of BS 0 4 0 4 Campylobacter species (Gun-Munro et al. 1987; Endtz et al. BC 0 0 0 0 1991). BF 0 15 0 15 Antimicrobials in Campylobacter-selective media were PS 5 2 0 7 inhibitory to the growth of some strains of C. jejuni and C. PC 12 19 0 31 coli (Ng et al. 1985). Antibiotic-sensitive Campylobacter PF 7 18 1 26 strains have been isolated from human feces by the use of membrane filtration onto antibiotic-free agar, and the more If PFGE genotypes of Campylobacter isolated from the same sample by using one method were identical, only one was employed. unusual Campylobacter spp. have been grown satisfactorily only when the membrane filtration method was used (Steele combination of antibiotics present in Preston broth. In this and McDermott 1984; Lastovica 2006). In this study, the study, however, Preston broth had a significantly higher membrane filtration technique appeared to have the excel- isolation rate of Campylobacter spp. than Bolton broth. lent abilities to isolate Campylobacter spp., while the use of Preston broth was also better in recovering C. coli compared small Petri dishes makes it cheap and easy to perform. By with Bolton broth. With Bolton enrichment broth, C. jejuni the use of Bolton enrichment broth, the membrane filtration strains were not obtained. method was significantly superior to mCCDA and Skirrow. As two different selective media (Skirrow and Based on Preston enrichment Bolton, the membrane filtra- mCCDA) contained different antimicrobials were used tion technique had better recovery of Campylobacter spp. for culturing Campylobacter, the Cefoperazone used in than Skirrow and equal to mCCDA. In addition, besides C. mCCDA as a selective agent had been shown excellent jejuni and C. coli, one C. fetus strain was isolated by using suppressive properties of enteric flora (Goossens et al. the membrane filtration method, this result agreed with 1986; Gun-Munro et al. 1987). This study showed that Goossens et al. (1986) who found the membrane filtration Skirrow was the least selective medium, being poorly method not only raised the isolation rate of C. jejuni and C. selective of existing growth of contaminants. Compared coli but also resulted in the isolation of less common Cam- with Skirrow, mCCDAwas a superior medium for reducing or pylobacter spp. Table 4 The number of Campylobacter and positive samples by single method or a combination of two or three methods Method BS BF PS PC PF Campy Sample Campy Sample Campy Sample Campy Sample Campy Sample BS 4 4 –– – – –– – – BF –– 15 12 –– – – –– PS –– – – 74 –– – – PC –– – – – – 31 22 –– PF – – –– – – –– 26 22 BS + BF 19 16 –– – – –– – – BS + PS 11 8 –– – – –– – – BS + PC 33 22 –– – – –– – – BS + PF 30 22 –– – – –– – – BF + PS 26 20 22 16 –– – – –– BF + PC 47 29 45 29 –– – – –– BF + PF 44 32 40 32 –– – – –– PS+PC 38 2450 3136 24 –– – – PS+PF 37 2447 3433 24 –– – – PC+PF 52 3164 3855 3250 31 –– Campylobacter isolates. 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Annals of Microbiology – Springer Journals

Published: Apr 20, 2012

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