Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes ( hdc A) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus , oligonucleotides unique to the hdc A genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc œnos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. œnos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdc A gene products of Gram-positive bacteria.
The amplification products of the hdc A genes from Lact. buchneri and Leuc. œnos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdc A gene.
In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.     

Cloning and sequencing of the histidine decarboxylase genes of gram-negative,histamine-producing bacteria and their application in detection and identification of these organisms in fish

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.     

Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.     

Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP)

Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276?bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10?c.f.u.?ml(-1). Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index?=?0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates.     

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis.     

一种利用RT-HPLC分析乳酸菌产生物胺的方法

具有脱羧酶活性的乳酸菌可通过氨基酸的脱羧反应产生具有潜在安全风险的生物胺。本文利用脱羧酶培养基初步筛查61株乳酸菌产生物胺情况,再通过RT-HPLC法测定其在发酵液和发酵乳中的生物胺含量。用10%的三氯乙酸提取样品中的生物胺,采用苯甲酰氯衍生处理后,以甲醇/水为流动相,进行梯度洗脱,流速0.8mL/min,紫外检测器波长为254nm。结果显示,组胺和酪胺得到良好的分离,在给定的浓度范围内呈现良好的线性关系(R20.995)。在发酵液和发酵乳中添加生物胺混合标准溶液,平均回收率为97.92%-101.14%,相对偏差RSD5%。结果表明,发酵液与发酵乳中生物胺的RT-HPLC法,是一种快捷、稳定、灵敏度高的检测方法,其与脱羧酶培养基法结合可以准确地实现对乳酸菌产生物胺的评价。     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

A novel multiplex PCR/RFLP assay for the identification of Streptococcus bovis/Streptococcus equinus complex members from dairy microbial communities based on the 16S rRNA gene

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises pathogenic species associated with different degrees with human infections but also spontaneously fermented dairy products. We aimed therefore at developing a specific identification assay for the SBSEC targeting the 16S rRNA gene comprising a multiplex PCR followed by a differentiating restriction fragment length polymorphisms (RFLP). The multiplex PCR assay was positively applied on 200 SBSEC isolates including reference strains. The assay did not yield false-positive amplifications with strains of closely related bacteria and isolates of non-SBSEC streptococci, lactococci, enterococci, and other genera of dairy origin. The downstream RFLP using MseI and XbaI enabled further discrimination of Streptococcus infantarius/S.?bovis (biotype II.1) from Streptococcus gallolyticus (biotype I and II.2)/Streptococcus alactolyticus and S.?equinus. Furthermore, the newly developed primers can be used directly for Sanger sequencing. Conclusively, this novel PCR/RFLP assay is applicable in the complex dairy microbial communities and provides an important tool to assess the prevalence of members of the SBSEC in dairy products.     

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR

Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65 degrees C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65 degrees C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri , since Naegleriae ( N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini ) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, algae, y, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri .     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.     

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.     

产生物胺乳酸菌的筛查与检测

目的对上海市场上食品和药品中分离出的20株乳杆菌,13株链球菌,3株乳球菌和3株肠球菌的产生物胺能力进行检测,以揭示其潜在的安全性问题。方法检测的生物胺共包括6种：分别为酪胺、精胺、尸胺、组胺、腐胺和色胺。利用添加了前体氨基酸的氨基酸脱羧酶筛选培养基对各菌株的产胺能力进行初筛,通过培养基中指示剂的颜色变化判定产胺能力。结果在检测的39株菌中,8株菌具有产酪胺的能力,7株菌具有产精胺的能力,1株菌具有产组胺的能力,1株菌具有产腐胺的能力。尤其是精胺和酪胺的产量较为引人关注。结论生物胺的危害水平取决于个体解毒的能力,但在筛选食品药品用菌株时应运用规范的方法来检测其产生物胺的能力,以保障相应食品药品的安全问题。     

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.     

Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a

Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.     

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.