Complementary Antibacterial Effects of Bacteriocins andOrganic Acids as Revealed by Comparative Analysis ofCarnobacterium spp. from Meat

Peipei Zhang,a Michael Gänzle,b,c Xianqin Yanga,b

aAgriculture and Agri-Food Canada, Lacombe, Alberta, CanadabDepartment of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, CanadacCollege of Bioengineering and Food Science, Hubei University of Technology, Wuhan, People’s Republic of China

ABSTRACT Carnobacterium maltaromaticum and Carnobacterium divergens are oftenpredominant in the microbiota of vacuum-packaged (VP) meats after prolongedstorage at chiller temperatures, and more so in recent studies. We investigated theantibacterial activities of C. maltaromaticum and C. divergens (n � 31) from VP meatsby phenotypic characterization and genomic analysis. Five strains showed antibacte-rial activities against Gram-positive bacteria in a spot-lawn assay, with C. maltaro-maticum strains having an intergeneric and C. divergens strains an intrageneric inhi-bition spectrum. This inhibitory activity is correlated with the production ofpredicted bacteriocins, including carnobacteriocin B2 and carnolysin for C. maltaro-maticum and divergicin A for C. divergens. The supernatants of both species culturedin meat juice medium under anaerobic conditions retarded the growth of mostGram-positive and Gram-negative bacteria in broth assay in a strain-dependent manner.C. maltaromaticum and C. divergens produced formate and acetate but not lactate underVP meat-relevant conditions. The relative inhibitory activity by Carnobacterium strainswas significantly correlated (P � 0.05) to the production of both acids. Genomic analysisrevealed the presence of genes required for respiration in both species. In addition, twoclusters of C. divergens have an average nucleotide identity below the cutoff value forspecies delineation and thus should be considered to be two subspecies. In conclusion,both bacteriocins and organic acids are factors contributing significantly to the antibac-terial activity of C. maltaromaticum and C. divergens under VP meat-relevant conditions.A few Carnobacterium strains can be explored as protective cultures to extend the shelflife and improve the safety of VP meats.

IMPORTANCE The results of this study demonstrated that both bacteriocins and or-ganic acids are important factors contributing to the antibacterial activities of Carnobac-terium from vacuum-packaged (VP) meats. This study demonstrated that formate andacetate are the key organic acids produced by Carnobacterium and demonstrated theirassociation with the inhibitory activity of carnobacteria under VP meat-relevant storageconditions. The role of lactate, on the other hand, may not be as important as previ-ously believed in the antimicrobial activities of Carnobacterium spp. on chilled VP meats.These findings advance our understanding of the physiology of Carnobacterium spp. tobetter explore their biopreservative properties for chilled VP meats.

KEYWORDS Carnobacterium, bacteriocins, formate, genome analysis, heme uptake,vacuum-packaged meat

Carnobacterium divergens and Carnobacterium maltaromaticum are psychrotrophiclactic acid bacteria (LAB). Both species are major components of the microbiota of

refrigerated foods, including seafood, meats, and dairy products, especially at later

Citation Zhang P, Gänzle M, Yang X. 2019.Complementary antibacterial effects ofbacteriocins and organic acids as revealedby comparative analysis of Carnobacteriumspp. from meat. Appl Environ Microbiol85:e01227-19. https://doi.org/10.1128/AEM.01227-19.

Editor Christopher A. Elkins, Centers forDisease Control and Prevention

© Crown copyright 2019. The government ofAustralia, Canada, or the UK (“the Crown”) ownsthe copyright interests of authors who aregovernment employees. The Crown Copyrightis not transferable.

Address correspondence to Xianqin Yang,xianqin.yang@canada.ca.

Received 3 June 2019Accepted 24 July 2019

Accepted manuscript posted online 9August 2019Published

FOOD MICROBIOLOGY

crossm

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 1Applied and Environmental Microbiology

1 October 2019

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

stages of storage (1, 2). Comparison of the spoilage-related activities of 45 C. maltaro-maticum strains in air-stored and vacuum-packaged (VP) beef demonstrated that allthese strains produce volatile organic compounds in a strain-independent manner;however, the volatile molecules produced by these strains are of low sensory impact,and their contribution to the spoilage of meat under either condition is negligible (3).The predominance of carnobacteria on food may be achieved by inhibiting the foodmicrobiota through production of antimicrobial compounds. Not surprisingly, carno-bacteria have been explored as protective cultures to inhibit pathogens and spoilageorganisms for meat, seafood, and dairy products (1, 2). C. maltaromaticum strain CB1has been approved for use in several countries, including the United States, Canada,Mexico, Colombia, and Costa Rica (4, 5).

The effect of carnobacteria on their companion meat microbiota relates to thestrain-dependent production of antibacterial compounds, including bacteriocins andorganic acids. Bacteriocins are ribosomally synthesized antimicrobial peptides and areclassified on the basis of structural properties into three major groups, classes I, II, andIII (6–8). Class I bacteriocins are small (�5 kDa), posttranslationally modified peptides;class I bacteriocins particularly include lantibiotics containing the unusual amino acidlanthionine (Lan). Their mode of action varies with their structure (9). Class II bacterio-cins are also �5 kDa but are unmodified peptides that typically permeabilize bacterialcell membranes. They are further divided into four groups, IIa to IId, depending on theirstructures. Both class I and class II bacteriocins are thermostable. Class III comprisesbacteriolysins, which are larger than 30 kDa and thermolabile (6, 7). Class IIa bacterio-cins, divercin V41 and divergicin M35, and class IId, divergicin A, have been isolatedfrom various C. divergens strains (10–12). Compared to C. divergens, a larger number ofbacteriocins have been reported for C. maltaromaticum, including class I (carnolysin),class IIa (carnobacteriocin B2 and BM1, piscicolin 126 and CS526, and maltaricin CPN),and class IId (carnobacteriocin X) (13–20).

Organic acid metabolism by carnobacteria and their role in the microbial ecology ofmeat and meat products are not as well understood as bacteriocin production. Carno-bacteria are homofermentative lactic acid bacteria that metabolize glucose via theEmbden-Meyerhof pathway to pyruvate as a central metabolic intermediate (21–24).Pyruvate is further converted to the alternative end product lactate or acetate, ethanol,formate, and CO2; respiratory metabolism in the presence of oxygen can additionallyinfluence the type and concentration of organic acids that are produced (21–24).Organic acid and bacteriocin production by carnobacteria is not only strain dependentbut is influenced by the condition under which they are cultured (21–23, 25, 26). Thequest to isolate and characterize new strains continues to draw attention from the researchcommunity, in part at least, due to the strain-dependent nature of the antibacterialactivities of carnobacteria.

A few recent reports have demonstrated that Carnobacterium spp., particularly C.maltaromaticum, are associated with extremely long storage life of VP beef (27–29).However, the exact mechanisms by which they contribute to shelf life are not wellunderstood, as information on the production of either bacteriocins or organic acids byand the antibacterial activities of these strains/species under VP meat-relevant anaer-obic conditions is largely unavailable. Our previous research reported draft genomes of31 Carnobacterium strains recovered from VP beef and pork and divided them into 11(I to XI) phylogenetic groups by core genome alignment (30). The objective of thepresent study was to characterize these strains by phenotypic analysis under anaerobicconditions and genomic analysis with a focus on their antibacterial properties, as wellas their phylogenetic positioning in relation to the 18 C. maltaromaticum and 5 C.divergens strains of which genomes are publicly available.

RESULTSPhylogenetic analysis. Strains of C. divergens and C. maltaromaticum of which the

genomes were available in GenBank were clustered into two distinct groups (Fig. 1).Within the C. maltaromaticum cluster, strains isolated from diseased sharks formed a

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 2

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

clade of mostly clonal strains. C. maltaromaticum strains (A1, A5, A7, and A14) isolatedfrom chilled VP beef in Alberta clustered with strains from poultry, milk, or VP meat. The32 C. divergens strains in the phylogenetic tree were also clustered into two majorgroups, without any apparent pattern between clustering and source of isolation.

The orthologous average nucleotide identity (ANI) between strains of C. divergensand C. maltaromaticum was 74.3 to 75.2% (see Table S1 in the supplemental material).

FIG 1 Phylogenetic tree of the sequenced genomes of C. maltaromaticum and C. divergens. The tree was constructed based on the alignment of concatenatednucleic acid sequences of eight housekeeping genes (ack, dnaB, dnaK, ftsZ, greA, gyrA, ileS, and polA) of the Carnobacterium strains and E. faecalis V583. Thevalue shown on each node is the local bootstrap support value of 1,000 replicates. The scale bar represents the number of substitutions per site. Thevisualization of the tree was improved by truncating long branches, with the length of each truncated branch labeled.

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 3

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

The intraspecies ANI was 96.6 to �99.9% for C. maltaromaticum and 93.3 to �99.9% forC. divergens. An ANI of 95 to 96% has been suggested for species delineation (31); thetwo clusters of C. divergens share an intercluster ANI of 93.3 to 93.9%, which maysuggest that they should be classified as distinct species or subspecies.

Genome characterization and pangenome analysis. The size of the assembled C.maltaromaticum and C. divergens draft genomes was 3.50 to 3.53 and 2.62 to 2.88 Mbp,respectively (Table S2). The GC content was 34.9 to 35.5% and 34.3 to 34.5%, respec-tively. The number of RNA-encoding genes ranged from 45 to 61 in C. divergens andfrom 57 to 71 in C. maltaromaticum.

The pangenomes of the C. maltaromaticum (n � 4) and C. divergens (n � 27) strainshad 1,087 (30.3%) and 1,867 (48.9%) accessory genes, respectively (Fig. S1A). Thecategories each accounting for �3% of the accessory genomes in both species were G(carbohydrate transport and metabolism), K (transcription), L (replication, recombina-tion, and repair), M (cell wall/membrane/envelope biogenesis), V (defense mechanism),W (extracellular structures), and genes with unassigned functions or bacteriophagegenes (Fig. S1B). C. maltaromaticum had larger fractions of G, K, L, M, V, and W in theaccessory genome than C. divergens. A CRISPR gene cluster was identified in thegenomes of all strains of C. divergens groups IV and V (Table S2). Prophage genes,important contributors to genomic plasticity, were found in all strains. However, anintact prophage region was only present in the genomes of C. divergens group IV, V, VI(except B7), IX, and XI strains.

Functional genome analysis. To assess the potential contribution of bacteriocinproduction to meat preservation, genes encoding putative bacteriocins were identifiedin the strains of C. divergens and C. maltaromaticum (Table 1). All four C. maltaromati-cum genomes include the carnobacteriocin BM1/B1 structural gene, with 100% identityto the reference peptide sequence. Putative encoding genes for both subunits ofcarnolysins were found in groups I and II C. maltaromaticum. For C. maltaromaticum A5and A14, a peptide with 98.5% similarity to carnobacteriocin B2 was also predicted. Thedifference in sequence was caused by a substitution of Asn (N) with Tyr (Y) before the

TABLE 1 Predicted bacteriocins in Carnobacterium spp. isolated from vacuum-packaged meat

Bacteriocin

% identity between query and reference peptide sequences

C. maltaromaticumgroups and strains C. divergens groups and strains

I II III IV V VI VII VIII IX X XI

A7b

A5,b

A14 A1b A10b

B1,b B6, C5,C7, C10,b

C14B7,b B8,C6,b C16 C13b

B3,b C1,C12,b C17 C8b

B5,b C3, C4,C9,b C11,C15

A2,b A4,A8, A12

Class IIaCarnobacteriocin

BM1/B1100 100 100 –c – – – – – – –

Carnobacteriocin B2/Carnocin CP52

– 98.5 – – – – – – – – –

Class IId – – – – – 100 – – 100 – –Divergicin A – – – – – 100 – – 100 – –

Class I (lantibiotics)Carnolysina

Subunit A1 100 100 – – – – – – – – –Subunit A2 100 100 – – – – 46.8 – – – –

Cerecidin – – – – – – 36.7 – – – –Cytolysin

Large unit – – – – – – – – – – –Small unit – – – – – – 34.9 – – – –

aCarnolysin is a two-peptide lantibiotic.bThe strain was selected for evaluation of inhibitory activity using the spot-lawn and broth assays.c–, the bacteriocin-encoding gene was not found in the genome(s).

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 4

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

signature motif YGNGV of carnobacteriocin B2 (Fig. S2). Production of divergicin A waspredicted for strains of C. divergens groups VI and IX. In addition, a lanthipeptide genecluster with genes encoding homologues of carnolysin subunit A2, cerecidin, andcytolysin small subunit was predicted for C. divergens C13 (Fig. S3). The respectiveidentities to reference sequences were 46.8, 36.7, and 34.9% (Table 1; Fig. S4).

The potential of strains to convert carbohydrates to organic acids was assessed byanalysis of key genes involved in carbohydrate metabolism and respiration. Genesencoding key enzymes for the homofermentive pathway for both hexose (fructose-bisphosphate aldolase, EC 4.1.2.13; phosphofructokinase, EC 2.7.1.11) and pentose(transaldolase, EC 2.2.1.2; transketolase, EC 2.2.1.1) were present in all genomes (Fig. 2).The gene encoding 2-dehydro-3-deoxyphosphogluconate aldolase (EC 4.1.2.14), thekey enzyme of the Entner-Doudorof pathway, was also identified. Phosphoketolase (EC4.1.2.9), the key enzyme in the phosphoketolase pathway, was absent in all carnobac-teria (Fig. 2). Pyruvate formate-lyase (EC 2.3.1.54) was found in both C. maltaromaticumand C. divergens. Genes for four enzymes involved in pyruvate metabolism to lactate,

FIG 2 The presence/absence of genes encoding proteins involved in different biological pathways in Carnobacterium genomes. Pathways are differentiated bycolor, with the intensity of shading increased with the increasing number of genes. The numbers indicate copies of genes. The abbreviations for carbohydratemetabolism are FBA, fructose-bisphosphate aldolase (EC 4.1.2.13); PFK, phosphofructokinase (EC 2.7.1.11); PK, phosphoketolase (EC4.1.2.9); TAK, transketolase(EC 2.2.1.1); TAL, transaldolase (EC 2.2.1.2); EDA, 2-dehydro-3-deoxyphosphogluconate aldolase (EC 4.1.2.14); PFL, pyruvate formate-lyase (EC 2.3.1.54); LDH,lactate dehydrogenase (EC 1.1.1.27/28); PDH, pyruvate dehydrogenase alpha- and beta-unit (EC 1.2.4.1); PTA, phosphotransacetylase (EC 2.3.1.8); ACK, acetatekinase (EC 2.7.2.1); LactAldDH, lactaldehyde dehydrogenase (EC 1.2.1.22); and G/D-DHA, glycerol/diol dehydratase (EC 4.2.1.28/30). The abbreviations for therespiration system are NADH_DH, NADH dehydrogenase (EC 1.6.99.3) and CydABCD, cytochrome d ubiquinol oxidase (EC 1.10.3). menF (EC 5.4.4.2), menD (EC2.2.1.9), menH (EC 4.2.99.20), menC (EC 4.2.1.113), menE (EC 6.2.1.26), menB (EC 4.1.3.36), menA (EC 2.5.1.74), and ubiE (EC 2.1.1.-) are enzymes for biosynthesisof menaquinone (electron transporter). IsdA to IsdI are proteins involved in the iron surface determinant (Isd) heme uptake pathway.

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 5

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

acetate, and CO2, including lactate dehydrogenase (EC 1.1.1.27/28), pyruvate dehydro-genase (EC 1.2.4.1), phosphotransacetylase (EC 2.3.1.8), and acetate kinase (EC 2.7.2.1)(24), were all found in all strains. C. divergens and C. maltaromaticum differed withrespect to the number of genes coding for L-lactate or D-lactate dehydrogenases (Fig.2). The genes encoding two key enzymes for lactate-to-propionate and propanol,lactaldehyde dehydrogenase (EC 1.2.1.22), and glycerol/diol dehydratase (EC 4.2.1.28)(32, 33), were not found in any of the 31 strains, indicating the inability of C. maltaro-maticum and C. divergens to produce propionate or propanol. The genes encoding keyenzymes involved in respiration were found in all 31 strains (Fig. 2). IsdA to IsdI mediateheme uptake by Staphylococcus aureus (34). Homologous genes of IsdA, C, E, F, and G/Iwere present in all 31 Carnobacterium genomes. However, isdB, D, and H were notfound in any of the genomes.

Putative genes encoding components of importance to acid tolerance in LAB,including ATP synthase (F1F0), arginine deiminase (EC 3.5.3.6), glutamate decarboxylase(EC 4.1.1.15), and Na�/H� antiporter, were found in all 31 genomes. Other potentialacid resistance components, such as ornithine decarboxylase (EC 4.1.1.17), glutaminase(EC 3.5.1.2), agmatine deiminase, and urease, were not found in any of the genomes.Similarly, the enzyme responsible for production of biogenic amines, histidine decar-boxylase (EC 4.1.1.22), was not found in any of the genomes, but tyrosine decarboxylase(EC 4.1.1.25) was found in all 31 Carnobacterium genomes. A putative glycogenbiosynthesis cluster was found in the strains of C. divergens group XI but not in any ofthe other strains. Putative tetracycline resistance genes were found in most Carnobac-terium strains except for A7, A5, A14, B7, and C13, but genes encoding aminoglycoside,�-lactam, colistin, or fluoroquinolone resistance were absent in the 31 genomes. Inaddition, among strains with putative tetracycline resistance, the genetic determinanttet(M) was found in group III, IV, and V strains, while tet(S) was found in other groups;mobile elements were found in the flanking region of tet(M)/tet(S) of all these strains.

Antibacterial activity determined by spot-lawn assay and broth assay. Tocorrelate the potential of carnobacteria for production of organic acids and bacteriocinswith their inhibitory activity, inhibition of meat spoilage-associated bacteria and patho-gens by 15 Carnobacterium strains was assessed in two assays, a spot-lawn assay anda broth assay. In the spot-lawn assay, C. maltaromaticum A7 (group I) and A5 (group II)showed the largest inhibition spectrum, with inhibition observed for most Gram-positive indicator bacteria, including Listeria monocytogenes, Lactobacillus sakei, Leu-conostoc gelidum, Enterococcus spp., C. maltaromaticum, and C. divergens (Table S3). A5resulted in larger inhibition zones (1.3 to 4.2 mm) than A7 (0.5 to 2.9 mm). C. divergensgroups VI (B7 and C6) and IX (C8) had identical inhibition spectra and were inhibitoryagainst all C. maltaromaticum strains and C. divergens A10, B3, C12, and A2. Theinhibition of these C. divergens strains was stronger on C. divergens (3.1 to 5.1 mm) thanon C. maltaromaticum (0.7 to 1.8 mm). Mutual inhibition was found between C. mal-taromaticum A7 and A5 and C. divergens B7, C6, and C8 (Table S3 and Fig. S5). However,the inhibitory spectrum of strains B7, C6, and C8 was limited to Carnobacterium spp.None of the 15 Carnobacterium strains produced an inhibition zone against any of theGram-negative bacteria tested. Group III, IV, V, VII, VIII, X, and XI Carnobacterium spp. didnot show any inhibition by spot-lawn assay against any of the 45 indicator bacteria(Table S3). In short, the inhibitory activity of carnobacteria as determined by thespot-lawn assay (Table S3) matched their potential to produce bacteriocins except forstrains A1 and C13 (Table 1).

In broth assay, the cell-free supernatants of all 15 strains of Carnobacterium spp.retarded the growth of indicator bacteria (Table S4 and Fig. S6). Growth inhibition wasobserved for all combinations except for C. divergens B7 against Hafnia alvei S1, C6against Rahnella sp. S8, B5 against Serratia liquefaciens, and C9 against H. alvei S1. Themean inhibition strength of combinations of effector and indicator bacteria showinginhibition zones in the spot-lawn assay was significantly larger than that of combina-tions which did not show inhibition zones in the spot-lawn assay (P � 0.05; Fig. 3).

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 6

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

To investigate the contribution to inhibition by factors other than bacteriocins, thecombinations not showing inhibition in the spot-lawn assay were further analyzed.Significant differences were found among the Carnobacterium strains (P � 0.05), withstrains C13 (VII) and B3 (VIII) showing the largest and smallest mean inhibitionstrengths, respectively (Fig. 4). The overall response of indicator bacteria to inhibitorycompounds in the cell-free supernatants of Carnobacterium was also strain dependent(Fig. 5).

Production of organic acids by Carnobacterium spp. The metabolism of strains ofCarnobacterium spp. reduced the pH of meat juice medium (MJM) (pH 5.93 � 0.02) by0.55 to 0.73 (Fig. 6A), indicating acid production. The concentration of glucose in thesupernatants of these cultures was reduced by 1.75 to 2.66 mM compared to blank MJMwhich contained 5.83 � 0.24 mM glucose (Fig. 6B). Concentrations of formate andacetate in the supernatants were increased by 3.32 to 4.64 mM and 1.61 to 2.21 mM,respectively, compared to blank MJM (Fig. 6C and D). The concentration of lactic acid,propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, or caproic acidin the supernatants was not significantly different from their respective concentrationsin blank MJM (data not shown). These data correspond well with the metaboliteconcentrations that are expected for homofermentative metabolism of glucose viaglycolysis and pyruvate formate lyase.

For the combinations of effector and inhibitor bacteria that did not show inhibitionin the spot-lawn assay, the relative inhibition strength determined in the broth assaywas significantly correlated (P � 0.05) with the protonated formic and acetic acids inthe supernatant of Carnobacterium MJM cultures (Table 2). The pH was correlated withthe production of both formate and acetate (P � 0.05).

DISCUSSION

This study investigated the antibacterial potential of Carnobacterium spp. recoveredfrom VP meats via both comparative genomic analysis and phenotypic characterization.The use of carnobacteria as biopreservatives and their role in meat spoilage arecontroversial. One contributor to this controversy is that carnobacteria were oftenfound to be a sizable component of the microbiota of spoiled products, and all major

FIG 3 Inhibition strength of cell-free supernatants against indicator bacteria determined by broth assay ofCarnobacterium strains that did not (NO; 620 combinations) or did (YES; 55 combinations) show inhibition inthe spot-lawn assay. Boxes indicate data within the first and third quartile (interquartile range [IQR]), centralhorizontal lines indicate medians, � marks indicate means, and whiskers indicate the lowest data point within1.5 IQR of the first quartile and the highest data point within 1.5 IQR of the third quartile.

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 7

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

components were regarded as spoilage bacteria, in particular, in earlier storage lifestudies. Investigation of the spoilage-related activities of 45 C. maltaromaticum strainson meat revealed that all these strains produced organic volatile compounds; however,their contribution to spoilage was negligible (3). Another factor contributing to thecontroversial role of carnobacteria is that the by-products generated by carnobacteriawhen growing on meat are not only species dependent, but also strain dependent (1,2, 23). The inter- and intraspecies differences found in this study in bacteriocin andorganic acid production provide insight into mechanisms by which certain strains of C.maltaromaticum extend the storage life of VP meat (28, 30).

The functional genome analysis of this study was based on the draft genomesequences of 31 Carnobacterium strains with a sequencing depth that is sufficient forphylogenetic analysis and for identification of genes coding for bacteriocin productionand carbohydrate metabolism and other metabolic properties that relate to their use asbiopreservatives (35, 36).

Carnobacterium spp. tolerate acid conditions, with C. divergens being more tolerantthan C. maltaromaticum, in particular, group XI C. divergens (30). Genomic analyses didnot reveal any difference with respect to genes related to acid resistance. However,group XI C. divergens differs from other strains with respect to the presence of aglycogen biosynthesis cluster. A comparable glycogen biosynthesis cluster not onlymediated growth of Lactobacillus acidophilus in the absence of glucose, but also relatedto the bile resistance of this organism (37).

Transferable antibiotic resistance genes should be absent in cultures that areintentionally used in food production; in addition, decarboxylation of histamine andtyramine to biogenic amines is undesirable (38, 39). The 31 Carnobacterium genomesencoded tyrosine decarboxylase but not histamine decarboxylase, matching pheno-typic data (2). Food and storage condition-related tyramine production should thus beexamined prior to using these strains as bioprotective cultures in food. Information onantibiotic resistance genes in carnobacteria is scarce. None of the 31 Carnobacterium

FIG 4 Inhibition strength of cell-free supernatants of Carnobacterium strains against indicator bacteriadetermined using broth assay. The combinations of Carnobacterium spp. and indicator bacteria thatshowed a visible inhibition zone in the spot-lawn assay were not included in the analysis. The differencebetween means is significant (P � 0.05) if two strains do not share a letter. Boxes indicate data within thefirst and third quartile (interquartile range [IQR]), central horizontal lines indicate medians, � marksindicate means, and whiskers indicate the lowest data point within 1.5 IQR of the first quartile and thehighest data point within 1.5 IQR of the third quartile.

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 8

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

strains harbor genes for aminoglycoside, �-lactam, colistin, or fluoroquinolone resis-tance. Tetracycline is the most commonly used antibiotic in beef production in Canada,and its usage has been linked to increased prevalence of resistant Escherichia coli ofbovine origin (40). The prevalence of tetracycline resistance genes in most Carnobac-terium strains investigated in this study may relate to their bovine origin (41).

Many LAB respire in the presence of heme and oxygen (42), with menaquinonebeing required for some species (42–44). Heme-dependent oxygen consumption hasbeen noted for some strains of C. maltaromaticum (25, 45) and contributed to meatspoilage by L. gelidum subsp. gasicomitatum (46). Genes encoding all components ofthe respiration chain were found in all 31 strains examined in this study. The hemo-globin and heme uptake system in the strains, however, was incomplete, with ho-mologs for uptake of hemoglobin and haptoglobin missing (34, 47). This suggests thatcarnobacteria are equipped with heme uptake systems, while hemoglobin or hapto-globin may not be used. The cytochrome bd oxidase works at low oxygen concentra-tions and improves stationary-phase survival of Lactococcus lactis by reducing oxidativeand acid stress (48). A low level of oxygen is present on the surface of VP meat, and theheme-based respiration of Carnobacterium spp. may then contribute to their compet-itiveness through a growth advantage and reducing stress (46).

Genome analysis predicted production of several bacteriocins, particularly by thosestrains of Carnobacterium that were most inhibitory in the spot-lawn assay. Thepredicted carnobacteriocin B2 in C. maltaromaticum group II strains (A5 and A14) hasan N2Y mutation next to the conserved YGNGV motif at the N terminus. Substitutionsin the conserved motif or the C terminus of carnobacteriocin B2 render the bacteriocininactive (49), but an active N2Y variant of carnobacteriocin B2 has been reported (15).C. maltaromaticum A1 harbors genes for structural (cbnBM1) and immunity (cbiBM1)

FIG 5 Mean inhibition strength of Carnobacterium on the growth of 45 indicator bacteria. The combinations of Carnobacterium spp. and target bacteria whichshowed inhibition zones in the spot-lawn assay were not included in the statistics. Error bars represent standard errors of mean. Means with different lettersare significantly different (P � 0.05).

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 9

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

proteins of carnobacteriocin BM1 (CbnBM1), an antilisterial class IIa bacteriocin, but thisstrain did not show any antibacterial activity by the spot-lawn assay. In C. maltaromati-cum LV17B, carnobacteriocin BM1 production is dependent on the plasmid-borne genecluster cbnKRTD (47, 50), which is absent in C. maltaromaticum A1. The lack ofantibacterial activity by C. divergens C13 in the spot-lawn assay is likely due to themissing structural genes for the carnolysin subunit A1 or cytolysin large subunits, whichare required for an active bacteriocin (51–54). The inhibitory spectrum of the strainsexamined in this study is in agreement with previous reports that the inhibitoryspectrum of carnolysin and carnobacteriocin B2 is relatively broad, while divergicin A

FIG 6 Analysis of the supernatants of Carnobacterium spp. cultured in meat juice medium at 2°C for 10 days under anaerobic conditions. (A) pH. (B) Decreasein glucose concentration compared to blank medium. (C) Increase in acetic acid concentration compared to blank medium. (D) Increase in formic acidconcentration compared to blank medium. Error bars represent standard errors of mean of two independent replicates, each with four technical replicates. Thedifference between means is significant (P � 0.05) if two strains do not share a letter.

TABLE 2 Correlation between IS, production of formate or acetate by Carnobacterium, and the pH of the spent medium when culturedin MJMa

Dependent variable and Pearsoncorrelation test parameter pH

Total formicacid

Total aceticacid

Undissociatedformic acid

Undissociatedacetic acid

ISP value 0.085 0.087 0.136 0.042 0.047Correlation coefficient –0.459 0.457 0.403 0.531 0.521

pHP value \ 0.003 0.028 \ \Correlation coefficient \ –0.716 –0.565 \ \

aIS refers to the inhibition strength of Carnobacterium spp. determined by broth assay for effector-indicator combinations which did not produce any visible inhibitionzone in the spot-lawn assay. Text in bold indicates analysis where a statistical difference was observed. \, the correlation was not tested.

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 10

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

inhibits only strains of the same genus. Taken together, the antimicrobial activitiesobserved in the spot-lawn assay are well explained by the genome analysis of putativegenes involved in producing bacteriocins.

In contrast to bacteriocins, organic acids at low concentrations are often bacterio-static rather than bactericidal, and their effect is not readily detected in the spot-lawnassay (55). Therefore, a broth assay in MJM was performed. The growth of mostGram-positive and Gram-negative bacteria was retarded by the cell-free supernatantsof C. maltaromaticum and C. divergens, including those strains that did not havepotential for bacteriocin production. This suggests that bacteriocin-less strains alsohave potential for shaping the microbial community structure on VP meats. In partic-ular, the inhibition of Gram-negative spoilage organisms was not related to bacteriocinformation but was entirely dependent on the formation of organic acids. Interestingly,all 15 effector strains were self-inhibitory in the broth assay, and often, the strongestinhibition was observed for intraspecific or intrageneric combinations, which could bea strategy for total population control in a closed system (56).

Lactate is the primary metabolite of Carnobacterium from glucose when strains aregrown at high substrate concentrations and without the addition of lactate (21, 57, 58).In contrast, we observed formation of acetate and formate as the major or onlymetabolites in MJM that contained low concentrations of glucose but 40 mM lactate.Similarly, C. maltaromaticum and C. divergens produced acetate but not lactate duringstorage of modified-atmosphere-packaged shrimp at 5°C (23). The genomes of carno-bacteria encoded a comparable set of genes related to carbohydrate metabolism;accordingly, quantitative but not qualitative differences were observed in the metab-olite pattern of different strains. Under carbohydrate-limiting conditions, homofermen-tative LAB metabolize pyruvate via pyruvate formate-lyase or pyruvate dehydrogenaseto formate/CO2, acetate, and ethanol as an alternative to lactate formation (24), withpyruvate formate-lyase being preferentially used under anaerobic conditions at near-neutral pH (59, 60). In addition, downregulation of L-lactate dehydrogenase and up-regulation of pyruvate formate-lyase in response to low pH and high lactate concen-trations were observed in Lactococcus lactis (61). The lack of lactate production byCarnobacterium spp. observed in this study could then be caused by feedback inhibi-tion of lactate dehydrogenase by the relatively high concentration of lactate in MJM.Our data on the relative amounts of formate, acetate, and lactate produced underanaerobic conditions in MJM, along with the previous observation of a lack of lactateproduction by Carnobacterium in modified-atmosphere-packaged shrimp, suggest thatlactate is not the primary organic acid produced by Carnobacterium when growing onmuscle food, where the concentration of naturally occurring lactate is high. Instead,formate and acetate are produced, and they both contribute to the antibacterial activityof Carnobacterium spp.

In conclusion, this study shows that both C. maltaromaticum and C. divergens,especially the former species, can be explored as protective cultures to improve meatquality and safety due to their potential of producing bacteriocins and organic acids, inparticular, formate and acetate. To date, investigations of the use of lactic acid bacteriaas biopreservatives focused on bacteriocin-producing strains. However, owing to thecomplementary inhibitory spectra and modes of action of bacteriocins and organicacids, both types of metabolites should be exploited for meat preservation.

MATERIALS AND METHODSBacterial strains. Four C. maltaromaticum and 27 C. divergens strains, covering 11 phylogenetic

groups from the culture collection of the Lacombe Research and Development Centre, were included inthis study (30) (Table S2). These strains had been recovered from VP beef and pork primal cuts from threedifferent abattoirs after storage at –1.5 and 2°C for up to 180 days. Isolates recovered from the same meatsample and with matching genomes were regarded as clonal isolates of the same strain, and isolatesfrom different samples or with different genome sequences were regarded as different strains (30). All31 strains were included for genomic analysis, and 15 strains (effector strains) were selected for furtherphenotypic characterization in relation to their biopreservative activities against 45 indicator bacterialstrains. These 45 indicator strains included 17 foodborne pathogens, including Listeria monocytogenes,various serotypes of Shiga toxin-producing E. coli, and Salmonella enterica serovar Typhimurium, 13

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 11

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

spoilage bacteria recovered from meat, including Hafnia, Rahnella, Serratia, Lactobacillus, Leuconostoc,and Enterococcus strains, and the 15 effector strains of Carnobacterium spp. (Tables S3 and S4).

Phylogenetic analysis. Genomes of the 31 VP meat Carnobacterium strains from our culturecollection were sequenced using Illumina MiSeq PE 250-bp technology with a read depth of �50 for eachgenome (30). SPAdes was used for assembly, and contigs of �1,000 bp were removed. The assembleddraft genomes contained 9 to 68 contigs (Table S2). The draft genomes were annotated using RASTversion 2.0 (62). The overall similarity between any two genome sequences was calculated using anorthologous average nucleotide identity tool (OAT) (https://www.ezbiocloud.net/tools/orthoani) (63).The phylogenetic positions of these 31 VP meat Carnobacterium strains in relation to other strains ofthese two species were determined. Briefly, all additional genomes of C. maltaromaticum (n � 18) and C.divergens (n � 5) currently available in GenBank were downloaded (Table S5). The sequences of eighthousekeeping genes (41) encoding acetate kinase (ack), replicative DNA helicase (dnaB), molecularchaperone (dnaK), cell division protein (ftsZ), transcription elongation factor (greA), DNA gyrase subunitA (gyrA), isoleucine-tRNA ligase (ileS), and DNA polymerase (polA) were extracted from all the aboveCarnobacterium genomes (n � 54) and aligned using MUSCLE (Codons) in MEGA X (64). The alignmentsof each gene were concatenated using Geneious version 11.0.5 (65). A neighbor-joining tree wasconstructed using the Kimura 2-parameter model in MEGA X, including transitions and transversions. Thevariation rate among sites was modeled with a Gamma distribution. The tree was rooted againstEnterococcus faecalis V583, and the branch support was assessed with 1,000 bootstrap replicates.

Pangenome analysis. Protein sequences (.faa file) produced from RAST for the 31 VP meat C.maltaromaticum and C. divergens genomes were compiled on the species level. The sequences were thenclustered using USEARCH version 10.0.240 at an identity threshold of 0.6 (66, 67). The seed sequenceswere parsed using a Python script to produce a pangenome file showing the presence/absence of genesfor C. maltaromaticum and C. divergens (67). Protein sequences shorter than 50 amino acids wereremoved prior to downstream analysis. The function of the seed protein sequences of all clusters waspredicted using the Web CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi)with the following settings: database, COG (clusters of orthologous groups of proteins); expected valuethreshold, 0.01; composition-corrected scoring turned on; maximum number of hits, 500.

Functional genome analysis. Clustered regularly interspaced short palindromic repeats (CRISPRs)and prophage genes were identified for each genome using CRISPR Recognition Tool (CRT) version 1.1(68) and PHASTER (http://phaster.ca/), respectively. For each genome, putative bacteriocin open readingframes (ORFs) were identified using the online server Bagel 4 (http://bagel4.molgenrug.nl/index.php).Antibiotic resistance genes were searched for each genome using the ResFinder 3.0 server hosted by theCenter for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/ResFinder/) (69). Enzyme/protein-encoding genes involved in carbohydrate metabolism, respiration system, and acid resistance weresearched in the pangenome files created above. To investigate genes encoding the proteins required forthe uptake of heme and glycogen biosynthesis, a local database containing protein sequences of 31Carnobacterium genomes was searched using BLAST� (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast�/2.7.1/), with reference genes listed in Table S6. An E value of �1e�5 was used as the cutoffthreshold for BLAST (67, 70). Matched protein sequences were subjected to InterPro (https://www.ebi.ac.uk/interpro/search/sequence-search) to examine them for the presence of functional domains.

Preparation of inoculum. Anaerobic meat juice medium (MJM) was prepared and made anaerobicfollowing the protocol described by Yang et al. (71), with external glycogen being omitted. The 15Carnobacterium effector strains (Table S2) were grown in half-strength brain heart infusion broth (Oxoid,Mississauga, ON, Canada) at 25°C for 24 h, and 0.1 ml of the overnight cultures was inoculated in 7 mlof anaerobic MJM and incubated at 2 � 0.5°C until stationary phase (10 days). Growth was assessed byoptical density at 600 nm (OD600) using a cell density meter (model 40; Thermo Fisher Scientific, Ottawa,ON, Canada). Indicator microorganisms were grown in half-strength brain heart infusion broth at 25°C for24 h. Cultures of the effector and indicator microorganisms thus prepared were used as inoculumthroughout the study unless otherwise stated.

Antibacterial activity of Carnobacterium spp. Antibacterial activities of the 15 Carnobacteriumeffector strains were assessed by spot-lawn agar assay and broth assay (55). For the spot-lawn assay, 20�l of each indicator brain heart infusion culture was plated on tryptone soya agar (Oxoid) as a lawn bya spiral plater (Eddy Jet 2; Neutec, Farmingdale, NY, USA). Two aliquots (technical replicates) of 10 �l ofeach Carnobacterium effector strain grown in anaerobic MJM were then spotted onto the lawn of eachplate, and two independent plates were used for each combination of effector and indicator strains. Afterair drying for 10 min, the agar plates were placed in an anaerobic jar. Anaerobic conditions were createdusing an atmosphere generation sachet (AnaeroGen; Thermo Fisher Scientific, Ottawa, ON, Canada). Afterincubation at 15°C for 5 days, the plates were photographed, and the diameter of the inhibition zone wasmeasured using the software Image J (https://imagej.nih.gov/ij/).

For the broth assay, cell-free supernatant (CFS) of each Carnobacterium effector strain was preparedby filtrating the anaerobic MJM culture through a 0.2-�m pore-sized syringe filter (Nalgene; ThermoFisher Scientific, Ottawa, ON, Canada). The brain heart infusion culture of each indicator strain wasadjusted to OD600 of 0.2 to 0.25 (108 CFU/ml). The adjusted bacterial suspension was further diluted usingMJM to approximately 105 CFU/ml, and 100 �l of the dilution was added to wells of a 96-well microtiterplate, each containing 100 �l of CFS (treatment), phosphate-buffered saline (buffer, pH 5.98 � 0.07,control; Mediatech, Inc., Manassas, VA, USA) or MJM (control). The relatively low initial indicator level wasintended for exploring the potential of these strains as biocontrol measures for fresh meat, where theinitial level of contamination is, in general, less than 4 log CFU/cm2 (72). Each treatment or control hadduplicate wells. The microtiter plates were incubated at 15°C under anaerobic conditions as before. The

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 12

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

OD600 was measured every 24 h for up to 5 days using a plater reader (POLARstar; Omega, BMG Labtech,Germany). The OD data were calibrated by subtracting the background values of the blank medium. Thetime to reach OD600 0.05 (T0.05), approximately 7 log CFU/ml, was then calculated for each well. For theindicator bacteria in wells containing CFS which did not reach OD600 0.05, the T0.05 was recorded as 120h, the duration of the incubation. The effect of CFS on the growth of an indicator strain was regardedas inhibitory if the T0.05 of treatment (TCFS) was larger than that of both MJM (TMJM) and buffer (TBuffer)controls. The inhibition strength was then calculated as (TCFS – TMJM)/TMJM (55, 73).

Analysis of supernatants of effector Carnobacterium strains. The CFS of each Carnobacteriumeffector strain grown in anaerobic MJM for 10 days was analyzed for pH, glucose, and organic acids, withuninoculated MJM included as controls. Two independent replicates were carried out for each measure-ment. The pH was measured using a portable pH meter (Accumet; Thermo Fisher Scientific, Ottawa, ON,Canada). Concentrations of glucose and lactate were determined using a glucose (HK) assay kit (Sigma,Oakville, ON, Canada) and a lactate colorimetric assay kit II (BioVision, Milpitas, CA, USA), respectively,following the manufacturers’ instructions. Formate was determined using an enzymatic method de-scribed previously with slight modification (74). Briefly, 300 �l of each CFS was mixed with 1.5 ml of 5.4%trichloroacetic acid (Thermo Fisher Scientific) and incubated at 0°C for 5 min. The acidified samples werecentrifuged at 8,000 � g for 10 min at 4°C to remove proteins. Then, 600 �l of the supernatant wasneutralized with 60 �l of 3M potassium carbonate (Sigma) and kept at 4°C for 16 h. For the enzymaticreaction, 240 �l of the treated sample or formate standard was mixed with 1.2 ml of phosphate buffer(pH 7.5; Hardy Diagnostics, Santa Maria, CA, USA), 30 �l of 0.05 M �-NAD (�-NAD sodium salt fromSaccharomyces cerevisiae; Sigma), and 30 �l of formate dehydrogenase (20 units/ml; Sigma). The mixturewas added into wells of a 96-well microtiter plate with 200 �l for each well and four wells for each sampleor standard. The plate was incubated at 37°C for 20 min, and absorbance at 340 nm (A340) was measuredusing the microtiter plate reader. The concentration of formate in each sample was calculated accordingto A340, and a standard curve was generated using known concentrations of formate. The detection limitfor glucose, lactate, and formate was 0.1 mM, 0.06 mM, and 0.3 mM, respectively. Concentrations ofacetate, propionate, isobutyrate, butyrate, isovalerate, valerate, and caproate were determined using gaschromatography at the Department of Agricultural, Food, and Nutritional Science, University of Alberta,Edmonton, Canada, using conditions described previously (71).

Statistical analysis. Analysis of variance (ANOVA) was performed using the general linear model(GLM) procedure in SAS version 9.4 (SAS Institute, Cary, NC) to compare the inhibition strength (brothassay) between the combinations of Carnobacterium spp. and indicator bacteria which produced aninhibition zone in the spot-lawn assay and those that did not. For the combinations that did not showa visible inhibition zone, ANOVA was performed to compare the inhibition strength of 15 Carnobacteriumstrains against 45 indicator bacteria. The difference in pH of spent medium, glucose consumption, andacetate and formate production among Carnobacterium strains was also assessed using ANOVA. Undis-sociated formic acid (UndisFA) and acetic acid (UndisAA) were each calculated as UndisFA � FA/[1 � 10(pH - 3.75)] and UndisAA � AA/[1 � 10(pH - 4.76)], respectively. The Pearson method was used toexamine the correlation between the inhibition strength or pH of CFS of 15 Carnobacterium strains andproduction of formate, acetate, UndisFA, or UndisAA by them. A significance level of 0.05 was used forall statistical analyses.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.01227-19.SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB.

ACKNOWLEDGMENTSWe acknowledge the funding support from Agriculture and Agri-Food Canada

(AAFC; A-1603).Technical support from Katie Petrella is appreciated. We also thank Yuanyao Chen

and Arun Kommadath of AAFC, Tamsyn Stanborough of the Commonwealth Scientificand Industrial Research Organisation of Australia, Igor Makunin of the University ofQueensland, and Min Yue of Zhejiang University for their helpful suggestions on dataanalysis.

REFERENCES1. Laursen BG, Bay L, Cleenwerck I, Vancanneyt M, Swings J, Dalgaard P,

Leisner JJ. 2005. Carnobacterium divergens and Carnobacterium maltaro-maticum as spoilers or protective cultures in meat and seafood: pheno-typic and genotypic characterization. Syst Appl Microbiol 28:151–164.https://doi.org/10.1016/j.syapm.2004.12.001.

2. Leisner JJ, Laursen BG, Prevost H, Drider D, Dalgaard P. 2007. Carno-bacterium: positive and negative effects in the environment and in

foods. FEMS Microbiol Rev 31:592– 613. https://doi.org/10.1111/j.1574-6976.2007.00080.x.

3. Casaburi A, Nasi A, Ferrocino I, Di Monaco R, Mauriello G, Villani F,Ercolini D. 2011. Spoilage-related activity of Carnobacterium maltaro-maticum strains in air-stored and vacuum-packed meat. Appl EnvironMicrobiol 77:7382–7393. https://doi.org/10.1128/AEM.05304-11.

4. Koné AP, Zea JMV, Gagné D, Cinq-Mars D, Guay F, Saucier L. 2018.

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 13

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

Application of Carnobacterium maltaromaticum as a feed additive forweaned rabbits to improve meat microbial quality and safety. Meat Sci135:174 –188. https://doi.org/10.1016/j.meatsci.2017.09.017.

5. Health Canada. 2010. Information document on Health Canada’s pro-posal to amend the food and drug regulations to permit the use of amicrobiological preparation of Carnobacterium maltaromaticum strainCB1 in certain ready-to-eat meat and poultry products. Accessed 10November 2018. https://www.canada.ca/en/health-canada/services/food-nutrition/public-involvement-partnerships/use-microbiological-preparation-carnobacterium-maltaromaticum-strain-certain-ready-meat-poultry-products.html.

6. Cotter PD, Hill C, Ross RP. 2005. Bacteriocins: developing innate immu-nity for food. Nat Rev Microbiol 3:777–788. https://doi.org/10.1038/nrmicro1273.

7. Cotter PD, Ross RP, Hill C. 2013. Bacteriocins: a viable alternative toantibiotics? Nat Rev Microbiol 11:95–105. https://doi.org/10.1038/nrmicro2937.

8. Alvarez-Sieiro P, Montalbán-López M, Mu D, Kuipers OP. 2016. Bacterio-cins of lactic acid bacteria: extending the family. Appl Microbiol Biotech-nol 100:2939 –2951. https://doi.org/10.1007/s00253-016-7343-9.

9. McAuliffe O, Ross RP, Hill C. 2001. Lantibiotics: structure, biosynthesisand mode of action. FEMS Microbiol Rev 25:285–308. https://doi.org/10.1111/j.1574-6976.2001.tb00579.x.

10. Metivier A, Pilet M-F, Dousset X, Sorokine O, Anglade P, Zagorec M, PiardJ-C, Marlon D, Cenatiempo Y, Fremaux C. 1998. Divercin V41, a newbacteriocin with two disulphide bonds produced by Carnobacteriumdivergens V41: primary structure and genomic organization. Microbiol-ogy 144:2837–2844. https://doi.org/10.1099/00221287-144-10-2837.

11. Tahiri I, Desbiens M, Benech R, Kheadr E, Lacroix C, Thibault S, Ouellet D,Fliss I. 2004. Purification, characterization and amino acid sequencing ofdivergicin M35: a novel class IIa bacteriocin produced by Carnobacteriumdivergens M35. Int J Food Microbiol 97:123–136. https://doi.org/10.1016/j.ijfoodmicro.2004.04.013.

12. Worobo RW, Van Belkum MJ, Sailer M, Roy KL, Vederas JC, Stiles ME.1995. A signal peptide secretion-dependent bacteriocin from Carnobac-terium divergens. J Bacteriol 177:3143–3149. https://doi.org/10.1128/jb.177.11.3143-3149.1995.

13. Herbin S, Mathieu F, Brule F, Branlant C, Lefebvre G, Lebrihi A. 1997.Characteristics and genetic determinants of bacteriocin activities pro-duced by Carnobacterium piscicola CP5 isolated from cheese. Curr Mi-crobiol 35:319 –326. https://doi.org/10.1007/s002849900262.

14. Quadri LE, Sailer M, Roy KL, Vederas JC, Stiles ME. 1994. Chemical andgenetic characterization of bacteriocins produced by Carnobacteriumpiscicola LV17B. J Biol Chem 269:12204 –12211.

15. Tulini FL, Lohans CT, Bordon KC, Zheng J, Arantes EC, Vederas JC, DeMartinis EC. 2014. Purification and characterization of antimicrobialpeptides from fish isolate Carnobacterium maltaromaticum C2: carno-bacteriocin X and carnolysins A1 and A2. Int J Food Microbiol 173:81– 88.https://doi.org/10.1016/j.ijfoodmicro.2013.12.019.

16. Bhugaloo-Vial P, Dousset X, Metivier A, Sorokine O, Anglade P, BoyavalP, Marion D. 1996. Purification and amino acid sequences of piscicocinsV1a and V1b, two class IIa bacteriocins secreted by Carnobacteriumpiscicola V1 that display significantly different levels of specific inhibitoryactivity. Appl Environ Microbiol 62:4410 – 4416.

17. Martin-Visscher LA, van Belkum MJ, Garneau-Tsodikova S, Whittal RM,Zheng J, McMullen LM, Vederas JC. 2008. Isolation and characterizationof carnocyclin A, a novel circular bacteriocin produced by Carnobacte-rium maltaromaticum UAL307. Appl Environ Microbiol 74:4756 – 4763.https://doi.org/10.1128/AEM.00817-08.

18. Yamazaki K, Suzuki M, Kawai Y, Inoue N, Montville TJ. 2005. Purificationand characterization of a novel class IIa bacteriocin, piscicocin CS526,from surimi-associated Carnobacterium piscicola CS526. Appl EnvironMicrobiol 71:554 –557. https://doi.org/10.1128/AEM.71.1.554-557.2005.

19. Jack RW, Wan J, Gordon J, Harmark K, Davidson BE, Hillier AJ, WettenhallRE, Hickey MW, Coventry MJ. 1996. Characterization of the chemical andantimicrobial properties of piscicolin 126, a bacteriocin produced byCarnobacterium piscicola JG126. Appl Environ Microbiol 62:2897–2903.

20. Hammi I, Delalande F, Belkhou R, Marchioni E, Cianferani S, Ennahar S.2016. Maltaricin CPN, a new class IIa bacteriocin produced by Carnobac-terium maltaromaticum CPN isolated from mould-ripened cheese. J ApplMicrobiol 121:1268 –1274. https://doi.org/10.1111/jam.13248.

21. De Bruyn IN, Holzapfel WH, Visser L, Louw AI. 1988. Glucose metabolismby Lactobacillus divergens. J Gen Microbiol 134:2103–2109. https://doi.org/10.1099/00221287-134-8-2103.

22. De Bruyn IN, Louw AI, Visser L, Holzapfel WH. 1987. Lactobacillus diver-gens is a homofermentative organism. Syst Appl Microbiol 9:173–175.https://doi.org/10.1016/S0723-2020(87)80018-8.

23. Laursen BG, Leisner JJ, Dalgaard P. 2006. Carnobacterium species: effectof metabolic activity and interaction with Brochothrix thermosphacta onsensory characteristics of modified atmosphere packed shrimp. J AgricFood Chem 54:3604 –3611. https://doi.org/10.1021/jf053017f.

24. Gänzle MG. 2015. Lactic metabolism revisited: metabolism of lactic acidbacteria in food fermentations and food spoilage. Curr Opin Food Sci2:106 –117. https://doi.org/10.1016/j.cofs.2015.03.001.

25. Borch E, Molin G. 1989. The aerobic growth and product formation ofLactobacillus, Leuconostoc, Brochothrix, and Carnobacterium in batchcultures. Appl Microbiol Biotechnol 30:81– 88.

26. Brillet-Viel A, Pilet M-F, Courcoux P, Prévost H, Leroi F. 2016. Optimiza-tion of growth and bacteriocin activity of the food bioprotective Carno-bacterium divergens V41 in an animal origin protein free medium. FrontMarine Sci 3. https://doi.org/10.3389/fmars.2016.00128.

27. Didimo Imazaki PH, Maia Danielski G, Daube G, Ernlund Freitas deMacedo R, Clinquart A. 2017. In vitro evaluation of the competing effectof Carnobacterium maltaromaticum isolated from vacuum-packed beefwith long shelf-life against three major food pathogens. Abstr Interna-tional Congress of Meat Science and Technology, Cork, Ireland. http://hdl.handle.net/2268/221877.

28. Youssef MK, Gill CO, Tran F, Yang X. 2014. Unusual compositions ofmicroflora of vacuum-packaged beef primal cuts of very long storagelife. J Food Prot 77:2161–2167. https://doi.org/10.4315/0362-028X.JFP-14-190.

29. Kaur M, Bowman JP, Porteus B, Dann AL, Tamplin M. 2017. Effect ofabattoir and cut on variations in microbial communities of vacuum-packaged beef. Meat Sci 131:34 –39. https://doi.org/10.1016/j.meatsci.2017.04.021.

30. Zhang P, Badoni M, Ganzle M, Yang X. 2018. Growth of Carnobacteriumspp. isolated from chilled vacuum-packaged meat under relevant acidicconditions. Int J Food Microbiol 286:120 –127. https://doi.org/10.1016/j.ijfoodmicro.2018.07.032.

31. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard forthe prokaryotic species definition. Proc Natl Acad Sci U S A 106:19126 –19131. https://doi.org/10.1073/pnas.0906412106.

32. Oude Elferink SJ, Krooneman J, Gottschal JC, Spoelstra SF, Faber F,Driehuis F. 2001. Anaerobic conversion of lactic acid to acetic acid and1,2-propanediol by Lactobacillus buchneri. Appl Environ Microbiol 67:125–132. https://doi.org/10.1128/AEM.67.1.125-132.2001.

33. Dishisha T, Pereyra LP, Pyo SH, Britton RA, Hatti-Kaul R. 2014. Fluxanalysis of the Lactobacillus reuteri propanediol-utilization pathway forproduction of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and1,3-propanediol from glycerol. Microb Cell Fact 13:76. https://doi.org/10.1186/1475-2859-13-76.

34. Contreras H, Chim N, Credali A, Goulding CW. 2014. Heme uptake inbacterial pathogens. Curr Opin Chem Biol 19:34 – 41. https://doi.org/10.1016/j.cbpa.2013.12.014.

35. Desai A, Marwah VS, Yadav A, Jha V, Dhaygude K, Bangar U, Kulkarni V,Jere A. 2013. Identification of optimum sequencing depth especially forde novo genome assembly of small genomes using next generationsequencing data. PLoS One 8:e60204. https://doi.org/10.1371/journal.pone.0060204.

36. Illumina. 2009. De novo assembly using Illumina reads. https://www.illumina.com/Documents/products/technotes/technote_denovo_assembly_ecoli.pdf. Accessed 17 May 2019.

37. Goh YJ, Klaenhammer TR. 2013. A functional glycogen biosynthesispathway in Lactobacillus acidophilus: expression and analysis of theglg operon. Mol Microbiol 89:1187–1200. https://doi.org/10.1111/mmi.12338.

38. Barbieri F, Montanari C, Gardini F, Tabanelli G. 2019. Biogenic amineproduction by lactic acid bacteria: a review. Foods 8:17. https://doi.org/10.3390/foods8010017.

39. Laulund S, Wind A, Derkx PMF, Zuliani V. 2017. Regulatory and safetyrequirements for food cultures. Microorganisms 5:28. https://doi.org/10.3390/microorganisms5020028.

40. Cameron A, McAllister TA. 2016. Antimicrobial usage and resistance inbeef production. J Anim Sci Biotechnol 7:68. https://doi.org/10.1186/s40104-016-0127-3.

41. Iskandar CF, Borges F, Taminiau B, Daube G, Zagorec M, Remenant B,Leisner JJ, Hansen MA, Sorensen SJ, Mangavel C, Cailliez-Grimal C,Revol-Junelles AM. 2017. Comparative genomic analysis reveals ecolog-

Zhang et al. Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 14

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

ical differentiation in the genus Carnobacterium. Front Microbiol 8:357.https://doi.org/10.3389/fmicb.2017.00357.

42. Pedersen MB, Gaudu P, Lechardeur D, Petit MA, Gruss A. 2012. Aerobicrespiration metabolism in lactic acid bacteria and uses in biotechnology.Annu Rev Food Sci Technol 3:37–58. https://doi.org/10.1146/annurev-food-022811-101255.

43. Brooijmans R, Smit B, Santos F, van Riel J, de Vos WM, Hugenholtz J.2009. Heme and menaquinone induced electron transport in lactic acidbacteria. Microb Cell Fact 8:28. https://doi.org/10.1186/1475-2859-8-28.

44. Yamamoto Y, Poyart C, Trieu-Cuot P, Lamberet G, Gruss A, Gaudu P.2005. Respiration metabolism of group B Streptococcus is activated byenvironmental haem and quinone and contributes to virulence. MolMicrobiol 56:525–534. https://doi.org/10.1111/j.1365-2958.2005.04555.x.

45. Meisel J, Wolf G, Hammes WP. 1994. Heme-dependent cytochromeformation in Lactobacillus maltaromicus. Syst Appl Microbiol 17:20 –23.https://doi.org/10.1016/S0723-2020(11)80026-3.

46. Jääskeläinen E, Johansson P, Kostiainen O, Nieminen T, Schmidt G, Somer-vuo P, Mohsina M, Vanninen P, Auvinen P, Björkroth J. 2013. Significance ofheme-based respiration in meat spoilage caused by Leuconostoc gasicomi-tatum. Appl Environ Microbiol 79:1078–1085. https://doi.org/10.1128/AEM.02943-12.

47. Quadri LE, Kleerebezem M, Kuipers OP, de Vos WM, Roy KL, Vederas JC,Stiles ME. 1997. Characterization of a locus from Carnobacterium pisci-cola LV17B involved in bacteriocin production and immunity: evidencefor global inducer-mediated transcriptional regulation. J Bacteriol 179:6163– 6171. https://doi.org/10.1128/jb.179.19.6163-6171.1997.

48. Rezaïki L, Cesselin B, Yamamoto Y, Vido K, Van West E, Gaudu P, Gruss A.2004. Respiration metabolism reduces oxidative and acid stress to im-prove long-term survival of Lactococcus lactis. Mol Microbiol 53:1331–1342. https://doi.org/10.1111/j.1365-2958.2004.04217.x.

49. Quadri LEN, Yan LZ, Stiles ME, Vederas JC. 1997. Effect of amino acidsubstitutions on the activity of carnobacteriocin B2: overproduction ofthe antimicrobial peptide, its engineered variants, and its precursor inEscherichia coli. J Biol Chem 272:3384 –3388. https://doi.org/10.1074/jbc.272.6.3384.

50. Quadri LE, Sailer M, Terebiznik MR, Roy KL, Vederas JC, Stiles ME. 1995.Characterization of the protein conferring immunity to the antimicrobialpeptide carnobacteriocin B2 and expression of carnobacteriocins B2 andBM1. J Bacteriol 177:1144 –1151. https://doi.org/10.1128/jb.177.5.1144-1151.1995.

51. Zhang L, Teng K, Wang J, Zhang Z, Zhang J, Sun S, Li L, Yang X, ZhongJ. 2018. CerR, a single-domain regulatory protein of the Luxr family,promotes cerecidin production and immunity in Bacillus cereus. ApplEnviron Microbiol 84:e02245-17. https://doi.org/10.1128/AEM.02245-17.

52. Wang J, Zhang L, Teng K, Sun S, Sun Z, Zhong J. 2014. Cerecidins, novellantibiotics from Bacillus cereus with potent antimicrobial activity. ApplEnviron Microbiol 80:2633–2643. https://doi.org/10.1128/AEM.03751-13.

53. Haas W, Gilmore MS. 1999. Molecular nature of a novel bacterial toxin:the cytolysin of Enterococcus faecalis. Med Microbiol Immunol 187:183–190. https://doi.org/10.1007/s004300050091.

54. Lohans CT, Li JL, Vederas JC. 2014. Structure and biosynthesis of carno-lysin, a homologue of enterococcal cytolysin with D-amino acids. J AmChem Soc 136:13150 –13153. https://doi.org/10.1021/ja5070813.

55. Zhang P, Baranyi J, Tamplin M. 2015. Interstrain interactions betweenbacteria isolated from vacuum-packaged refrigerated beef. Appl EnvironMicrobiol 81:2753–2761. https://doi.org/10.1128/AEM.03933-14.

56. Kelvin Lee KW, Hoong Yam JK, Mukherjee M, Periasamy S, Steinberg PD,Kjelleberg S, Rice SA. 2016. Interspecific diversity reduces and function-ally substitutes for intraspecific variation in biofilm communities. ISME J10:846 – 857. https://doi.org/10.1038/ismej.2015.159.

57. Hiu S, Holt R, Sriranganathan N, Seidler R, Fryer J. 1984. Lactobacilluspiscicola, a new species from salmonid fish. Int J Syst Evol Microbiol34:393– 400. https://doi.org/10.1099/00207713-34-4-393.

58. Holzapfel WH, Gerber ES. 1983. Lactobacillus divergens sp. nov., a new

heterofermentative Lactobacillus species producing L(�)-lactate. Syst ApplMicrobiol 4:522–534. https://doi.org/10.1016/S0723-2020(83)80010-1.

59. Wagner N, Tran QH, Richter H, Selzer PM, Unden G. 2005. Pyruvatefermentation by Oenococcus oeni and Leuconostoc mesenteroides androle of pyruvate dehydrogenase in anaerobic fermentation. Appl EnvironMicrobiol 71:4966 – 4971. https://doi.org/10.1128/AEM.71.9.4966-4971.2005.

60. Thomas TD, Ellwood DC, Longyear VM. 1979. Change from homo- toheterolactic fermentation by Streptococcus lactis resulting from glucoselimitation in anaerobic chemostat cultures. J Bacteriol 138:109 –117.

61. Wu H, Zhao Y, Du Y, Miao S, Liu J, Li Y, Caiyin Q, Qiao J. 2018. Quantitativeproteomics of Lactococcus lactis F44 under cross-stress of low pH andlactate. J Dairy Sci 101:6872–6884. https://doi.org/10.3168/jds.2018-14594.

62. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K,Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL,Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD,Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008.The RAST server: Rapid Annotations using Subsystems Technology. BMCGenomics 9:75. https://doi.org/10.1186/1471-2164-9-75.

63. Lee I, Ouk Kim Y, Park S-C, Chun J. 2016. OrthoANI: an improved algorithmand software for calculating average nucleotide identity. Int J Syst EvolMicrobiol 66:1100–1103. https://doi.org/10.1099/ijsem.0.000760.

64. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: MolecularEvolutionary Genetics Analysis across computing platforms. Mol BiolEvol 35:1547–1549. https://doi.org/10.1093/molbev/msy096.

65. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S,Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, MeintjesP, Drummond A. 2012. Geneious Basic: an integrated and extendabledesktop software platform for the organization and analysis of sequencedata. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199.

66. Edgar RC. 2010. Search and clustering orders of magnitude faster thanBLAST. Bioinformatics 26:2460–2461. https://doi.org/10.1093/bioinformatics/btq461.

67. Stanborough T, Fegan N, Powell SM, Tamplin M, Chandry PS. 2017.Insight into the genome of Brochothrix thermosphacta, a problematicmeat spoilage bacterium. Appl Environ Microbiol 83:e02786-16. https://doi.org/10.1128/AEM.02786-16.

68. Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, Hugen-holtz P. 2007. CRISPR Recognition Tool (CRT): a tool for automaticdetection of clustered regularly interspaced palindromic repeats. BMCBioinformatics 8:209. https://doi.org/10.1186/1471-2105-8-209.

69. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O,Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobialresistance genes. J Antimicrob Chemother 67:2640 –2644. https://doi.org/10.1093/jac/dks261.

70. Zheng J, Ruan L, Sun M, Ganzle M. 2015. A genomic view of lactobacilliand pediococci demonstrates that phylogeny matches ecology andphysiology. Appl Environ Microbiol 81:7233–7243. https://doi.org/10.1128/AEM.02116-15.

71. Yang X, Balamurugan S, Gill CO. 2009. Substrate utilization by Clostrid-ium estertheticum cultivated in meat juice medium. Int J Food Microbiol128:501–505. https://doi.org/10.1016/j.ijfoodmicro.2008.10.024.

72. Yang X. 2016. Microbial ecology of beef carcasses and beef products, p442– 462. In Anderson de Souza SA (ed), Quantitative microbiology infood processing. John Wiley & Sons, Ltd., Chichester, UK. https://doi.org/10.1002/9781118823071.ch22.

73. Héquet A, Laffitte V, Brocail E, Aucher W, Cenatiempo Y, Frère J, FremauxC, Berjeaud JM. 2009. Development of a new method for the detectionof lactic acid bacteria capable of protecting ham against Enterobacteri-aceae. Lett Appl Microbiol 48:668 – 674. https://doi.org/10.1111/j.1472-765X.2009.02590.x.

74. Perez PF, De Antoni GL, Añon MC. 1990. An enzymatic method for deter-mination of formate production by Streptococcus thermophilus. J Dairy Sci73:2697–2701. https://doi.org/10.3168/jds.S0022-0302(90)78954-0.

Comparative Analysis of Carnobacterium spp. from Meats Applied and Environmental Microbiology

October 2019 Volume 85 Issue 20 e01227-19 aem.asm.org 15

on Novem

ber 12, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from