Predominant enterobacteria on modified-atmosphere packaged meat and poultry

Food Microbiology 34 (2013) 252e258

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Predominant enterobacteria on modified-atmosphere packaged meat and poultry Elina Säde*, Anna Murros, Johanna Björkroth Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, 00014 Helsinki, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2012 Received in revised form 11 October 2012 Accepted 23 October 2012 Available online 11 November 2012

Enterobacteria on modified-atmosphere (MA) packaged meat (n ¼ 54) and poultry (n ¼ 32) products were enumerated, and 899 isolates were picked and ribotyped. For identification, 16S rRNA genes of representative strains were sequenced and analyzed. Altogether 54 (60%) of the samples contained enterobacteria >104 CFU/g. In 34% of the poultry samples, enterobacteria counts were >106 CFU/g suggesting that enterobacteria may contribute to spoilage of MA packaged poultry. The enterobacteria identified were predominantly Hafnia spp. (40%) and Serratia spp. (42%) with Hafnia alvei, Hafnia paralvei, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia proteamaculans, and Serratia quinivorans being the species identified. In addition, 6% of the isolates were identified as Rahnella spp., 3% as Yersinia spp., and 1% as Buttiauxella spp. Percentage distributions of the predominant genera in different products showed that 89% of the Serratia spp. were from products packaged under a high-O2 MA containing CO2 (25e35%), whereas most (76%) isolates of Hafnia originated from anaerobically packaged red meat and poultry. These findings suggest that the gas mixture used for MA packaging influence the selection of enterobacteria growing on meat and poultry. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Enterobacteria Meat Modified-atmosphere packaged Ribotyping Spoilage

1. Introduction Raw meat and poultry are highly perishable commodities which readily support the microbial growth even when stored under chill temperatures. The preservation effect is enhanced when chilling is combined with a packaging system modifying the gas atmosphere surrounding the meat. The use of CO2-enriched modified atmosphere (MA) has a selective effect on growth of many microbes developing on MA packaged meat. While high CO2 levels suppress the growth of aerobic meat spoilage bacteria, such as Pseudomonas spp., psychrotrophic facultative anaerobic bacteria less sensitive to CO2 grow to predominate in the microbial community. Therefore, on MA packaged meat, lactic acid bacteria and Brochothrix thermosphacta often constitute the major part of the microbial community. In addition, Enterobacteriaceae (enterobacteria) commonly occur, but their numbers usually remain low relative to those of lactic acid bacteria. However, enterobacteria may play a key role in meat spoilage due to their ability to metabolize amino acids to malodorous volatile compounds such as foul-smelling diamines and sulfuric compounds (Baylis, 2006; Borch et al., 1996; Garcia-Lopez et al., 1998; Samelis, 2006). Furthermore, as the family Enterobacteriaceae includes human pathogens, their

* Corresponding author. Tel.: þ358 9 19157118; fax: þ358 9 19157101. E-mail address: elina.sade@helsinki.fi (E. Säde). 0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2012.10.007

presence may compromise the safety of meat, particularly if meat is improperly cooked or handled. Enterobacteria numbers and the species growing on chilled, packaged raw meat and poultry have been reported (Ercolini et al., 2006, 2010, 2011; Doulgeraki et al., 2011; Lindberg et al., 1998; Nieminen et al., 2012; Pennacchia et al., 2011) and reviewed (Baylis, 2006; Borch et al., 1996; Doulgeraki et al., 2012; Samelis, 2006; Stanbridge and Davies, 1998). According to the literature, a range of enterobacteria may grow on chilled meats with Hafnia alvei, Pantoea (Enterobacter) agglomerans, Rahnella spp., Serratia spp., and Yersinia enterocolitica being those frequently reported at the end of chilled storage. Among these meat-associated enterobacteria, H. alvei, Serratia liquefaciens and P. agglomerans have also been implicated in meat spoilage, and reported to cause various defects such as objectionable odors (Dainty et al., 1989; Garcia-Lopez et al., 1998; Stanbridge and Davies, 1998), gaseous distension of vacuum packages (Brightwell et al., 2007; Chaves et al., 2012), and green discoloration (Dainty et al., 1989; Stanbridge and Davies, 1998). The data available indicate that the pH of meat, and storage and packaging conditions affect the growth and species diversity of enterobacteria, as well as the spoilage potential of enterobacteria species on meat and poultry (Borch et al., 1996; Doulgeraki et al., 2012; Stanbridge and Davies, 1998). The aim of this study was to identify the predominant enterobacteria on various raw meat and poultry products in commercial MA packages, and to evaluate the role of enterobacteria in spoilage

E. Säde et al. / Food Microbiology 34 (2013) 252e258

of these products. For that purpose, we enumerated enterobacteria from meat and poultry products, and characterized the predominant isolates by ribotyping. Subsequently, we sequenced the 16S rRNA gene for strains representing the common ribotype patterns and conducted sequence similarity searches to assign the closest matches to each unique sequence. 2. Materials and methods 2.1. Meat and poultry products MA packages of raw red meat (beef, pork) and poultry (broiler, turkey) products were purchased from retail stores or obtained directly from the meat processors. The products were grouped in five categories: i) beef and pork, such as unmarinated steaks and strips (n ¼ 14); ii) marinated beef and pork, such as steaks and loins (n ¼ 9); iii) minced meat including minced beef and mixtures of ground beef and pork (n ¼ 31); iv) poultry, including boneless and skinless broiler fillets and turkey thigh meat (n ¼ 16); and v) marinated poultry, including broiler leg cuts, turkey thigh meat and skinless broiler fillets (n ¼ 16). Twenty of the thirty-two packages of minced meat were also included in a previous study (Nieminen et al., 2011). The packages were stored at 6  C, and sampled on their labeled use-by date or one to four days after this date; or, in the case of marinated beef and pork products, the products were sampled at the end of the expected storage life (12e17 d after packaging). Information about gas mixtures used for packaging was obtained from the packers. The red meat products, including minced meat, were packaged under high oxygen atmospheres containing O2 (65e70%) and CO2 (25e35%), with <10% residual air. The marinated beef and pork products and all poultry products were packaged under mixtures of CO2 (35e70%) and N2. 2.2. Microbiological analysis, pH and culture conditions 2.2.1. Colony counts, sample pH and enterobacteria isolates Products were sampled (25 g), homogenized, and diluted and the pH of each meat homogenate was measured as described before (Vihavainen et al., 2008). Enterobacteria were enumerated on pour plates of violet red bile glucose (VRBG) agar (Lab M, Bury, UK) incubated at 30  C for 48 h. For each sample, 10e15 colonies were picked from the plates containing 10e150 CFU, and subcultured, at 30  C for 24e48 h, on plates of Colombia blood agar base (Oxoid, Basingstoke, UK) supplemented with 5% bovine blood. Colonies were tested for their oxidase reactions using Microbact Oxidase strips (Oxoid). Oxidase-negative colonies were assumed to be enterobacteria, and were counted as such. Samples were classified into one of three categories on the basis of enterobacteria counts: as low (<104 CFU/g), moderate (104e 106 CFU/g), and high (>106 CFU/g). The classification reflects the expected impacts of the numbers of enterobacteria in each range of meat spoilage. 2.2.2. Reference strains To compare the ribotyping fingerprints of meat-derived isolates with those of enterobacteria reference strains, the following strains were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ): Buttiauxella gaviniae DSM 9393T, H. alvei DSM 30097T, Hafnia paralvei DSM 23255, Obesumbacter proteus DSM 2777T, Rahnella aquatilis DSM 4594TT, Serratia fonticola DSM 4576T, Serratia grimesii DSM 30063T, S. liquefaciens DSM 4487T, Serratia quinivorans DSM 4597T, Serratia proteamaculans DSM 4543T, Y. enterocolitica DSM 13030T, Yersinia intermedia DSM 18517T, and Yersinia nurmii DSM 22296T.

253

2.3. Culture conditions, and DNA extraction for ribotyping and sequencing Strains were grown at 30  C on blood agar plates and stored in bead vials (Protect; Technical Service Consultants, Lancashire, UK) at 80  C. A single colony was cultured in 10 ml of tryptic soy broth (Difco laboratories, Sparks, MD, USA) at 30  C for 18e24 h, and the cells from 1.5 ml were pelleted by centrifugation. DNA was extracted by the guanidium thiocyanate method described by Pitcher et al. (1989) with the addition of 0.6 mg/ml proteinase K (Finzymes, Espoo, Finland) to the cell suspension buffer. 2.4. HindIII ribotyping with 16S and 23S rRNA gene targeting probes 2.4.1. Procedure The references strains and meat-derived isolates were subjected for ribotyping. Ribotyping was performed as described previously (Vihavainen et al., 2008) using five probes (Regnault et al., 1997) targeting the 16S and 23S rRNA encoding genes. Briefly, HindIII e digested DNA fragments were resolved by agarose gel electrophoresis, and blotted onto a nylon membrane. Southern blots were hybridized with digoxigenin-labeled probes, and the labeled fragments were detected and visualized to reveal a ribotyping fingerprint. 2.4.2. Cluster analysis of ribotyping patterns Ribotyping patterns were analyzed using the BioNumerics software (version 5.10; Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis of patterns was performed using the Dice similarity coefficient and the unweighted pair group method with arithmetic mean (UPGMA) method. In the resulting dendrogram, a ribotype was defined as either an isolate that gave a unique pattern, or a group of isolates that gave indistinguishable patterns. The reproducibility of the ribotyping patterns was confirmed by repeated running of DNA samples from isolates and reference strains. 2.5. 16S rRNA gene sequencing and sequence-based identification One to ten representative strains of those ribotypes of which there were five or more meat-derived isolates were subjected for sequence analysis. The almost complete 16S rRNA gene was amplified, and the amplicons were purified as described earlier (Murros-Kontiainen et al., 2011). Sequencing was performed at the Institute of Biotechnology (Helsinki, Finland) using Applied Biosystems BigDye chemistry. Four primers were used for sequencing: the same primers used for PCR amplification as well as the primers pD and pEr of Edwards et al. (1989). Isolates were classified by submitting each unique sequence to the SeqMatch tool (Cole et al., 2005; Cole et al., 2009) available at the Ribosomal Database Project’s (RDP-II) web site (http://rdp.cme. msu.edu; release 10, update 28). The sequence identity scores yielded were used to assign the closest relative among the type strains and other well-characterized reference strains available at the RDP database. The unique 16S rRNA gene sequences were deposited in GenBank (GenBank IDs: JX162032eJX162086). 3. Results 3.1. Enterobacteria counts on meat and poultry products Counts of enterobacteria varied within samples of the same products category as well as between product categories (Table 1).

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E. Säde et al. / Food Microbiology 34 (2013) 252e258

Table 1 Counts of enterobacteria (CFU/g) and pH of modified-atmosphere packaged meat and poultry products sampled at the end of their shelf-life. Product category (n)

Beef and pork (14)a Beef and pork, marinated (9) Beef and pork, minced (31) Broiler and turkey (16) Broiler and turkey, marinated (16)

Range of counts <104

104e106

>106

4b 8 15

8 1 15 9 5

2

7

1 7 4

Mean pH (SD) 5.48 5.14 5.78 6.07 5.60

(0.20) (0.14) (0.15) (0.20) (0.34)

a

No. samples analyzed. No of samples yielding respective count of oxidase-negative colonies on violetred bile glucose (VRBG) agar. b

The majority (25 of 32; 78%) of the MA packaged poultry products carried moderate to high levels of enterobacteria. In 11 (34%) of the poultry samples the numbers of enterobacteria were >106 CFU/g, including three samples where counts exceeded 107 CFU/g. In contrast, enterobacteria counts on MA packaged marinated beef and pork cuts were often low (Table 1), with counts being <600 CFU/g in 5 of 9 samples. 3.2. Ribotyping of meat-derived enterobacteria Ribotyping of 899 presumptive meat-derived enterobacteria isolates differentiated them into 69 ribotypes. Of these ribotypes, 41 were each represented by five or more isolates. These frequent ribotypes were 93% of the isolates. Fig. 1 presents the frequent ribotyping patterns and the number of isolates of each frequent ribotype. The ribotyping patterns of reference strain are included in the dendrogram. The other meat-associated ribotypes which were each represented by less than five isolates were considered to be present in insignificant numbers. These were excluded from further analyses and are referred to as “unassigned”. Table 2 summarizes the distributions of frequent and unassigned ribotypes in meat and poultry products. Many of the frequent ribotypes, such as ribotypes 12, 33, 34, 38 and 39, were associated with both raw and marinated red meat and poultry products. 3.3. Identification of enterobacteria based on 16S rRNA gene sequencing and ribotyping We obtained high quality (<1% base-call ambiguities), 16S rRNA gene sequences for 81 of 95 meat-derived isolates subjected for sequencing. The remaining 14 isolates that were sequenced were discarded due to poor quality or too short 16S rRNA gene sequence data. In total, these 81 isolates yielded 51 unique sequences. These isolates belonged to the genera Buttiauxella, Hafnia, Rahnella, Serratia and Yersinia (Table 3). All queried sequences showed high (>0.950) sequence identity scores with either the 16S sequences of type strains or other well-characterized strains in the RDP II database. In general, species assignments based on sequences of the 16S rRNA gene were in agreement with the ribotyping results, with strains of the same ribotype being assigned to the same species by sequencing (Table 3). Furthermore, the reference strains gave ribotyping patterns identical or similar ribotyping patterns to those of isolates assigned to the same species or genus (Fig. 1). 3.4. Predominant enterobacteria on MA packaged meat and poultry H. alvei or H. paralvei were common enterobacteria species on commercial, MA packaged beef, pork and poultry products, with nearly 40% (356 of 899) of the isolates being identified as Hafnia

spp. (Fig. 2). In addition, 33% of all isolates were identified as S. liquefaciens (187 of 899) and S. quinivorans (107 of 899). The relative distributions of genera in different product categories varied (Fig. 2). Hafnia spp. were frequently recovered from poultry and marinated beef and pork, whereas nearly 90% (333 of 376) of the isolates identified as Serratia spp. originated from minced meat or unmarinated beef and pork. 4. Discussion Some Enterobacteriaceae are a meat safety concern but others are of commercial importance due to their ability to grow and spoil meat during refrigerated storage. The results of this study indicate that enterobacteria were frequently present in high numbers on MA packaged poultry products at the end of their shelf-life, and suggest that enterobacteria may contribute to spoilage of poultry products. The relatively high enterobacteria counts found are consistent with earlier studies of MA packaged broiler meat which found that enterobacteria often reach levels >106 CFU/g during refrigerated storage (Nieminen et al., 2012; Smolander et al., 2004). Although, enterobacteria probably were not the predominant fraction of the bacterial community (Björkroth et al., 2005; Nieminen et al., 2012; Smolander et al., 2004), the counts of enterobacteria on the retail poultry products approached levels of 107 CFU/g where spoilage by enterobacteria can occur (Samelis, 2006; Stanbridge and Davies, 1998). The relatively high enterobacteria counts on poultry products as compared to products of other categories may have been related to the higher initial pH of poultry muscle. In contrast to poultry and unmarinated beef and pork, the numbers of enterobacteria were low on samples of marinated beef and pork with inherent low pH. This is in agreement with earlier studies reporting that on meat of low pH or treated with acetic acid, a common ingredient of meat marinade, growth of enterobacteria is inhibited, particularly when the meat is stored under an anoxic modified atmosphere and at refrigeration temperatures (Garcia-Lopez et al., 1998; Gill and Newton, 1982; Nieminen et al., 2012). However, since the present study included only nine samples of marinated red meat, further studies are necessary to clarify whether enterobacteria on marinated beef and pork products are inhibited. Alternatively, the higher enterobacteria counts on poultry detected in the present study may have arisen from higher and more variable initial bacterial contamination of poultry meat (Garcia-Lopez et al., 1998; Samelis, 2006). Nevertheless, others have shown that although the initial enterobacteria contamination of MA packaged poultry was low (102 CFU/g), enterobacteria may grow to levels of 106e107 CFU/ g during refrigerated storage (Björkroth et al., 2005; Nieminen et al., 2012; Smolander et al., 2004). Enterobacteria recovered from refrigerated, MA packaged meat and poultry mainly belong to the genera Buttiauxella, Hafnia, Rahnella, Serratia and Yersinia. These species have been recovered from various sources associated with animals and farm environment (Baylis, 2006; Grimont and Grimont, 2006; Janda, 2006). Consequently, these organisms may enter the meat processing chain, and are frequent contaminants of carcasses and meat processing surfaces (Baylis, 2006; Garcia-Lopez et al., 1998; Stiles and Ng, 1981). Their common occurrence in the meat and poultry processing chains likely explains the finding that enterobacteria strains with indistinguishable ribotypes were often recovered from products of various producers and of different meat animals. Comparison of relative proportions of enterobacteria species from different product revealed, that the majority (76%) of isolates identified as Hafnia spp. originated from anaerobically packaged beef, pork and poultry. In contrast, Serratia spp. were frequent on beef, pork and minced meat packaged under a high-O2 MA. These

Pattern similarity% 20 40 60 80

100

HindIII ribotyping pattern

Strain

Ribotype

n

Nearest neighbor (16S rRNA gene sequencing)

1

6

S. proteamaculans DSM 4543 T

2

66

S. quinivorans DSM 4597 T

EBR2-n EBR1-e S. proteamaculans DSM

4543 T

ENUB8

3

28

S. proteamaculans DSM 4543 T

JL15g

4

35

S. quinivorans DSM 4597T

Kapa3d

5

13

S. grimesii DSM 30063T

S. quinivorans DSM 4597 T JL7g

6

6

S. quinivorans DSM 4597 T

JL8n

7

6

S. proteamaculans DSM 4543 T

8

5

B. gaviniae DSM 9393 T

Sisu4i

9

24

S. grimesii DSM 30063 T

Poky3a

10

11

S. liquefaciens CIP 103238 T

11

52

S. liquefaciens CIP 103238 T

JL1-h

12

110

S. liquefaciens CIP 103238 T

JLG1-h

13

9

S. liquefaciens CIP 103238 T

JLG2-l

14

5

S. liquefaciens CIP 103238 T

EBM1-j

15

5

Rahnella genomosp. 2, 2 WMR58

ARM5a-a

16

9

Y. enterocolitica DSM 13030 T

17

9

Y. enterocolitica DSM 13030 T

18

9

Y. intermedia ATCC 29909 T

ABF6a-c

19

8

Y. nurmii DMS 22296 T

ARM7b-b

20

5

S. fonticola DSM 4576 T

21

5

Rahnella genomosp. 2, WMR58

22

5

Y. enterocolitica DSM 13030 T

JL5a

23

29

Rahnella genomosp. 3, DSM 30078

JL9m

24

8

Rahnella genomosp. 3, DSM 30078

B. gaviniae DSM 9393 T JLA6-c S. grimesii DSM 30063 T

Poky4i S. liquefaciens DSM

4487 T

ARM5a-e Y. intermedia DSM

18517 T

APN6b-b Y. nurmii DSM 22296 T

S. fonticola DSM 4576 T JLA8-d Y. enterocolitica DSM 13030 T JL10a R. aquatilis DSM 4594 T

JLA5-g

25

7

Rahnella genomosp. 3, DSM 30078

JL14f

26

5

B. gaviniae DSM 9393 T

APN6a-b

27

9

H. alvei ATCC 29926

NALE4e

28

6

H. alvei ATCC 29926

NAFIM3a-j

29

6

H. alvei ATCC 29926

ARM3a-a

30

35

H. alvei ATCC 29926

NALE6f

31

5

H. paralvei ATCC 29927 T

APN3b-d

32

17

H. alvei ATCC 29926

ARM6b-b

33

10

H. paralvei ATCC 29927 T

ARM6b-e

34

18

H. alvei ATCC 29926

AFN4b-c

35

49

H. paralvei DSM 23255 T

ARM7a-c

36

19

H. paralvei ATCC 29972 T

NAFIM3B-a

37

7

H. alvei ATCC 29926

38

20

H. alvei ATCC 29926

39

137

H. paralvei CCUG 429

APM5b-c

40

9

H. alvei ATCC 29926

JL14k

41

9

H. alvei ATCC 29926

H. alvei DSM 30163 T NAFIM3A-h H. paralvei DSM 23255 APN6a-d O. proteus DSM 2777 T

Fig. 1. UPGMA dendrogram depicting the similarities among the frequent ribotyping profiles obtained for enterobacteria isolates and ribotyping profiles of closely related reference strains. Ribotype designation, number (n) of isolates of each ribotype, and the nearest neighbor (as defined based on 16S rRNA gene sequence analysis of representative strains) is indicated on the right. B. ¼ Buttiauxella; H. ¼ Hafnia; O. ¼ Obesumbacterium; R. ¼ Rahnella; S. ¼ Serratia; and Y. Yersinia.

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E. Säde et al. / Food Microbiology 34 (2013) 252e258

Table 2 Distributions of ribotypes among enterobacteria isolates from meat and poultry products. Ribotype

Pork and beef Meat only (n ¼ 138)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Unassigned a

13 7

6 7 26 18 6 2

Broiler and turkey

Minced (n ¼ 377) 3 58 11 35 5 4 3 5 15 3 24 81 3 3

Marinated (n ¼ 84)

Meat only (n ¼ 158) 3 7 4

2 2 3

2

6

5

4 1 1

8 4

3 5 5 25 8 5 5

3

1 3

1 3 3 4

1 1

35 2 3

6 25 1 1 21

7 10 29 6 2

1

Nearest neighborb

Ribotype (strains seq./ unique seq.)a

GenBank ID

Strain

Scorec

GenBank ID

1 (1/1) 2 (2/2)

JX162032 JX162033 JX162034 JX162035 JX162036 JX162037 JX162038 JX162039 JX162040 JX162041 JX162042 JX162043 JX162044 JX162045 JX162046 JX162047 JX162048 JX162049 JX162050 JX162051 JX162052 JX162053 JX162054 JX162055 FJ717339 JX162056 JX162057 JX162058 JX162059 JX162060 JX162061 JX162062 JX162063 JX162064 JX162065 JX162066 JX162067 JX162068 JX162069 JX162070 JX162071 JX162072 JX162073 JX162074 JX162075 JX162076 JX162077 JX162078 JX162079 JX162080 JX162081 JX162082 JX162083 JX162084 JX162085 JX162086

S. proteamaculans DSM 4543T S. quinivorans DSM 4597T

0.995 0.984 0.980 0.989 0.985 0.979 0.987 0.966 0.970 1.000 0.996 0.992 0.994 0.987 0.980 0.993 0.983 0.992 0.986 0.984 0.995 0.995 0.980 0.975 0.986 0.988 0.968 1.000 0.989 0.987 0.987 0.982 0.951 0.993 0.974 0.986 0.980 0.991 0.984 0.983 0.986 0.984 0.999 0.999 0.999 0.999 0.989 0.989 0.984 0.986 0.986 0.999 0.993 0.994 0.989 0.985

AJ233434 AJ233435

4 5 6 7 8 9

(1/1) (1/1) (1/1) (1/1) (1/1) (2/2)

10 (1/1) 11 (3/2) 12 (5/2)

1 5

1

2 6 1 4

Query sequence

3 (3/2)

1 1

1

1 4

1

1 2 3

3 8 1

4

Marinated (n ¼ 142)

Table 3 Nearest neighbor of unique 16S rRNA gene sequences of frequent ribotypes as defined by the highest sequence match among 16S rRNA gene sequences available in Ribosomal Database Project II.

2 1

13 14 15 16 17 18

(1/1) (1/1) (1/1) (2/1) (1/1) (2/2)

19 20 21 22 23

(1/1) (1/1) (1/1) (1/1) (3/2) (1/1) (1/1) (1/1) (3/2)

2 18

16

7 2 5 31 7

7 4 6 17 11

24 25 26 27

1 27 2

3 21 6

28 (2/1) 29 (1/1) 30 (5/2)

9

28

31 (1/1) 32 (2/2)

Number of isolates ribotyped.

findings suggest that the gas mixture used in the MA affects the selection of enterobacteria. Others, too, have described similar patterns of predominance and reported that high-O2 MA together with chilled storage temperature selected for Serratia spp. on meat, whereas H. alvei was the predominant enterobacteria on meat stored under anaerobic conditions (Doulgeraki et al., 2011; Stanbridge and Davies, 1998), or under aerobic MA conditions at abusive temperatures (Doulgeraki et al., 2011). Such findings led to the speculations that H. alvei was susceptible to oxygen levels applied for MA packing of beef and pork (Stanbridge and Davies, 1998), and that this sensitivity was induced at chilled temperatures (Doulgeraki et al., 2011). However, due to lack of systematic and comparative data on the growth responses of enterobacteria to meat characteristics and storage conditions, the impact of these factors on the selection of species in a specific meat system remain uncertain. Currently, sequence analysis of a specific region of 16S rRNA gene is widely used for identification of bacteria on meats, and the advantages of this approach are widely recognized (Doulgeraki et al., 2012). In a recent study we noticed that partial 16S rRNA gene sequences lacked phylogenetic resolution for differentiation

33 (1/1) 34 (1/1) 35 (5/3)

36 (3/2) 37 (1/1 38 (1/1) 39 (12/2) 40 (2/2) 41 (1/1)

S. proteamaculans DSM 4543T S. quinivorans DSM 4597T S. grimesii DSM 30063T S. quinivorans DSM 4597T S. proteamaculans DSM 4543T B. gaviniae DSM 9393T S. grimesii DSM 30063T S. liquefaciens CIP 103238T S. liquefaciens CIP 103238T S. liquefaciens CIP 103238T T

S. liquefaciens CIP 103238 S. liquefaciens CIP 103238T R. genomosp. 2, WMR58 Y. enterocolitica DSM 13030T Y. enterocolitica DSM 13030T Y. intermedia ATCC 29909T Y. nurmii DMS 22296T S. fonticola DSM 4576T R. genomosp. 2, WMR58 Y. enterocolitica DSM 13030T R. genomosp.3 DSM 30078 R. genomosp.3 DSM 30078 R. genomosp.3 DSM 30078 B. gaviniae DSM 9393T H. alvei ATCC 29926 H. alvei ATCC 29926 H. alvei ATCC 29926 H. alvei ATCC 29926 H. paralvei ATCC 29927T H. alvei ATCC 29926 H. H. H. H. H. H.

paralvei ATCC 29927T alvei ATCC 29926 paralvei ATCC 29927T paralvei CCUG 429 paralvei ATCC 29927T alvei ATCC 29926

H. H. H. H. H.

alvei ATCC 29926 alvei ATCC 29926 paralvei ATCC 29927T paralvei CCUG 429 alvei ATCC 29926

H. alvei ATCC 29926

AJ233434 AJ233435 AJ233430 AJ233435 AJ233434 AJ233403 AJ233430 AJ306725 AJ306725 AJ306725 AJ306725 AJ306725 AM160791 FJ717344 FJ717344 AF36638 FJ717338 AJ233429 AM160791 FJ717344 U90758 U90758 U90758 AJ233403 FM179942 FM179942 FM179942 FM179942 FM179943 FM179942 FM179943 FM179943 FM179943 FM179944 FM179943 FM179942 FM179942 FM179942 FM179943 FM179944 FM179942 FM179942

a

No. strains sequenced representing corresponding ribotype/no. of unique sequences obtained for corresponding ribotype. b Type strains, and well-characterized, cultured isolates were considered. S. ¼ Serratia, R. ¼ Rahnella, B. ¼ Buttiauxella, Y. ¼ Yersinia, and H. ¼ Hafnia. c s_ab score; sequence identity score, the number of unique 7-base oligomers shared between the query sequence and a given RDP sequence divided by the lowest number of unique oligos in either of the two sequences.

of meat-associated enterobacteria (Nieminen et al., 2012). Thus, in the present study, we analyzed almost complete 16S rRNA gene sequences covering several variable regions. Nonetheless, many queried sequences, namely those assigned to Buttiauxella spp.,

E. Säde et al. / Food Microbiology 34 (2013) 252e258

257

100% 90% 80%

Percentage distribution

70% 60% 50% 40% 30% 20% 10% 0%

Minced meat (n=377)

Buttiauxella spp.

Raw meat (n=138)

Hafnia spp.

Marinated meat (n=84)

Rahnella spp.

Raw poultry (n=158)

Serratia spp.

Yersinia spp.

Marinated poultry (n=142)

Unassigned

Fig. 2. Numbers of enterobacteria isolates of different genera and their percentage distributions in modified-atmosphere packaged raw and marinated red meat and poultry products. Total number of isolates identified from different product categories is indicated in parenthesis. Identifications were based on HindIII-ribotyping and 16S rRNA gene sequence analysis.

Hafnia spp. or Serratia spp. showed relatively high sequence identity scores with two or more type strains. It is, therefore, possible that some of the species level assignments we made based on the nearest neighbor search were inaccurate, and failed to reflect the exact phylogenetic position of these strains. These findings concerning the limited resolution of full-length 16S rRNA gene sequences are consistent with those of others (Dauga, 2002; Huys et al., 2010; Spröer et al., 1999), and confirm that other methods, such as sequence analyses of housekeeping genes, are required to discriminate between closely related species of enterobacteria. The findings of the present study show the need for better targeted methods for identifying meat-associated enterobacteria and for investigation of their roles and interactions in the spoilage bacteria communities of meat and poultry. The strains recovered in the present study, particularly those from poultry, represent potential meat spoilage bacteria capable of adapting and competing in MA packaged meats, and could serve as model organisms for further studies of meat spoilage. Acknowledgments We thank H. Lundström, E. Merivirta and H. Niinivirta for technical assistance. This research was part of the ELVIRA program funded by the Academy of Finland and TEKES (the Finnish Funding Agency for Technology and Innovation). The authors are part of the Center of Excellence in Microbial Food Safety Research (MiFoSa), Academy of Finland. References Baylis, C.L., 2006. Enterobacteriaceae. In: Blackburn, C.W. (Ed.), Food Spoilage Microorganisms. Woodhead Publishing Limited, Cambridge, UK, pp. 624e667. Björkroth, J., Ristiniemi, M., Vandamme, P., Korkeala, H., 2005. Enterococcus species dominating in fresh modified-atmosphere-packaged, marinated broiler legs are overgrown by Carnobacterium and Lactobacillus species during storage at 6  C. International Journal of Food Microbiology 97, 267e276.

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