Viability and Diversity of the Microbial Cultures Available in Retail Kombucha Beverages in the USA.

Kombucha is a two-stage fermented sweetened tea beverage that uses yeast and lactic acid bacteria (LAB) to convert sugars into ethanol and lactate and acetic acid bacteria (AAB) to oxidize ethanol to acetate. Its popularity as a beverage grew from claims of health benefits derived from this vibrant microbial bioconversion. While recent studies have shed light on the diversity of cultures in Kombucha fermentation, there is limited information on the diversity, and especially viability, of cultures in retail beverages that advertise the presence of Kombucha and probiotic cultures. In this study, 12 Kombucha beverages produced by different manufacturers throughout the US were purchased and microbially characterized. Eight of the beverages contained viable Kombucha cultures, while 3 of the remaining 4 had viable Bacillus cultures as added probiotics. Amplicon profiling revealed that all contained Kombucha yeast and bacteria cells. The dominant yeasts detected were Lachancea cidri (10/12), Brettanomyces (9/12), Malassezia (6/12), and Saccharomyces (5/12). Dominant LAB included Liquorilactobacillus and Oenococcus oeni, and AAB were Komagataeibacter, Gluconobacter, and Acetobacter. One beverage had a significant amount of Zymomonas mobilis, an ethanol-producing bacterium from Agave cactus. While Kombucha beverages differ in the types and viability of cultures, all except one beverage contained detectable viable cells.

Development of an endolysin enzyme and its cell wall-binding domain protein and their applications for biocontrol and rapid detection of Clostridium perfringens in food.

Clostridium perfringens is a well-known pathogen that causes food-borne illnesses. Although bacteriophages can be effective natural food preservatives, phage endolysin and cell wall-binding domain (CBD) provide useful materials for lysis of C. perfringens and rapid detection. The genome of phage CPAS-15 consists of 51.8-kb double-stranded circular DNA with 78 open reading frames, including an endolysin gene. The apparent absence of a virulence factor or toxin gene suggests its safety in food applications. C. perfringens endolysin (LysCPAS15) inhibits host cells by up to a 3-log reduction in 2 h, and enhanced green fluorescent protein (EGFP)-fused CBD protein (EGFP-LysCPAS15_CBD1) detects C. perfringens within 5 min. Both exhibit broader host range spectra and higher stabilities than a bacteriophage. Tests in milk show the same host lysis and specific detection activities, with no hindrance effect from food matrices, indicating that endolysin and its CBD can provide food extended protection from C. perfringens contamination.

Use of Lactococcus lactis as a production system for peptides and enzymes encoded by a Lantibiotic gene cluster from Bifidobacterium longum.

Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic, but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production, genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters, its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack of DNA binding motifs did not support this, and one current analysis suggested a high likelihood of it interacting with LanD. Therefore, gene lanR1 together with lanADMIT were codon optimized and synthesized. Those genes were then cloned into an efficient dual-plasmid nisin-controlled expression system in L. lactis. The addition of the lanR1 gene exhibited toxicity in E. coli, specifically causing a shorter cell size as observed by SEM. No toxicity was observed in L. lactis. While this production system did not result in the production of a bioactive lantibiotic by L. lactis, it did successfully produce all the peptides and enzymes encoded by the original lantibiotic gene cluster from B. longum, as confirmed by LC-MS. This will now facilitate efforts into determining the proper conditions required for these enzymes to produce a bioactive lantibiotic.

Thermus and the Pink Discoloration Defect in Cheese.

A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch's postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.

Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese.

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses.

BACKGROUND: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. RESULTS: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. CONCLUSIONS: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.

Temporal and spatial differences in microbial composition during the manufacture of a continental-type cheese.

We sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine-salted continental-type cheese in cheeses produced early and late in the production day. Differences in the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas, and Bifidobacterium, not routinely associated with a continental-type cheese produced from pasteurized milk, were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high-throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.

Diabetes and renal disease: who does what?

Care of patients with diabetes and chronic kidney disease (CKD) in the UK is divided between primary care, diabetologists and nephrology. In a retrospective analysis, we examined the distribution of care provision for patients with diabetes and CKD. Nephrology services see a minority of diabetic patients with CKD, but they see the majority of those with an estimated glomerular filtration rate (eGFR) of <30 ml/min. Of those followed in nephrology, 70% showed no evidence of progressive renal dysfunction. The nephrology cohort were significantly younger that those seen by primary care physicians or diabetologists. Half of the patients with diabetes and CKD seen in either the primary care and diabetology cohorts, with no nephrology input, had a rate of fall of eGFR of >5 ml/min/yr. This suggests that older age might deter referral to nephrology, which is based predominantly on CKD stage. This results in a significant proportion of patients with stable renal function being seen by nephrology, and in the under-referral of a large cohort of patients with progressive CKD.

Nucleic acid-based approaches to investigate microbial-related cheese quality defects.

The microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.

Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria.

Bifidobacteria are widely used as probiotics and have attracted increasing research interest worldwide. However, molecular techniques are still very scarce mainly due to the low efficiencies and strain-specific electroporation protocols that have been developed. Bacterial conjugation enables the transfer of genetic material among a relatively wide range of organisms and with virtually no size limitation. A conjugation protocol was developed based on the RP4 conjugative machinery in the Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100 % transfer frequency occurring when the ratio was 10(5) : 1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. In some cases the copy number of this resident plasmid was too low to observe via gel electrophoresis, but it could be detected by Southern hybridization. Plasmid curing resulted in a strain, DJO10A-W3, that had lost both plasmids and this showed a one-log increase in conjugation efficiency due to the lack of plasmid incompatibility. In conclusion, this novel conjugative gene transfer protocol can be used for the introduction of genetic material (without size restriction) into Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.

Contribution of the Actinobacteria to the growing diversity of lantibiotics.

Currently, 76 lantibiotics have been described; the vast majority being produced by members of the Firmicute phylum of bacteria. There is a growing number being identified from the Actinobacteria phylum and some of these exhibit novel modifications leading to an increased functional diversity among lantibiotics. In this review, we discuss the currently characterized lantibiotics highlighting the expanding diversity provided by those from the Actinobacteria. This increased diversity has the potential to expand lantibiotic applications as antimicrobials in foods and pharmaceuticals. In addition, a phylogenetic classification system based on the full prepropeptide sequences showed remarkable consistency with current classification systems and may provide a more rapid and convenient means for classifying lantibiotics.

Effect of a synbiotic yogurt on levels of fecal bifidobacteria, clostridia, and enterobacteria.

While ingestion of synbiotic yogurts containing Bifidobacterium animalis subsp. lactis and inulin is increasing, their effect on certain microbial groups in the human intestine is unclear. To further investigate this, a large-scale, crossover-design, placebo-controlled study was utilized to evaluate the effect of a synbiotic yogurt containing B. animalis subsp. lactis Bb-12 and inulin on the human intestinal bifidobacteria, clostridia, and enterobacteria. Fecal samples were collected at 14 time points from 46 volunteers who completed the study, and changes in the intestinal bacterial levels were monitored using real-time PCR. Strain Bb-12 could not be detected in feces after 2 weeks of washout. A live/dead PCR procedure indicated that the Bb-12 strain detected in the fecal samples was alive. A significant increase (P < 0.001) in the total bifidobacterial numbers was seen in both groups of subjects during the final washout period compared to the prefeeding period. This increase in total bifidobacteria corresponded with a significant decrease (P < 0.05) in numbers of clostridia but not enterobacteria. No significant differences in numbers of bifidobacteria, clostridia, or enterobacteria were observed between the probiotic and placebo groups during any of the feeding periods. However, subgrouping subjects based on lower initial bifidobacterial numbers or higher initial clostridial numbers did show corresponding significant differences between the synbiotic yogurt and placebo groups. This was not observed for a subgroup with higher initial enterobacterial numbers. While this synbiotic yogurt can increase bifidobacterial numbers and decrease clostridial numbers (but not enterobacterial numbers) in some individuals, it cannot modulate these microbial groups in the majority of individuals.

Transcription analysis of a lantibiotic gene cluster from Bifidobacterium longum DJO10A.

Bifidobacterium longum DJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of the lanA gene was undertaken. Comparative microarray analysis of broth and agar cultures of B. longum DJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed that lanA gene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures of B. longum DJO10A induced lanA gene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintained lanA expression. The expression of lanA was log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis of lanA revealed a 284-bp 5' untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp -75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of this lanA gene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.

Constructivist learning of anatomy: gaining knowledge by creating anatomical casts.

Educators are encouraged to provide inquiry-based, collaborative, and problem solving activities that enhance learning and promote curiosity, skepticism, objectivity, and the use of scientific reasoning. Making anatomical casts or models by injecting solidifying substances into organs is an example of a constructivist activity for achieving these goals. This report describes a student-implemented protocol for making postmortem anatomical casts of the bronchial tree and coronary arteries of rats using Silastic® sealant. The teacher facilitated this process by asking leading questions to guide the students toward the development of their own conclusions. This relatively simple and inexpensive procedure has important applications for the constructivist approach to study cardiovascular and respiratory morphology.

Genomic insights into bifidobacteria.

Since the discovery in 1899 of bifidobacteria as numerically dominant microbes in the feces of breast-fed infants, there have been numerous studies addressing their role in modulating gut microflora as well as their other potential health benefits. Because of this, they are frequently incorporated into foods as probiotic cultures. An understanding of their full interactions with intestinal microbes and the host is needed to scientifically validate any health benefits they may afford. Recently, the genome sequences of nine strains representing four species of Bifidobacterium became available. A comparative genome analysis of these genomes reveals a likely efficient capacity to adapt to their habitats, with B. longum subsp. infantis exhibiting more genomic potential to utilize human milk oligosaccharides, consistent with its habitat in the infant gut. Conversely, B. longum subsp. longum exhibits a higher genomic potential for utilization of plant-derived complex carbohydrates and polyols, consistent with its habitat in an adult gut. An intriguing observation is the loss of much of this genome potential when strains are adapted to pure culture environments, as highlighted by the genomes of B. animalis subsp. lactis strains, which exhibit the least potential for a gut habitat and are believed to have evolved from the B. animalis species during adaptation to dairy fermentation environments.

Genomics can advance the potential for probiotic cultures to improve liver and overall health.

The concept of probiotics has evolved immensely since it was first proposed a century ago. There are numerous potential health benefits attributed to certain probiotic bacteria, from preventing gastrointestinal (GI) infections to stimulating the immune system. Recent evidence is now quite compelling for a role of probiotics in enhancing liver health. Liver injury is on the rise worldwide with non-alcohol fatty liver disease (NAFLD) the fastest rising liver problem, due largely to the rise in obesity and type II diabetes. A damaged liver can progress to more serious conditions such as steatohepatitis and cirrhosis, and the intestinal microflora are believed to play a large role in this progression. When the intestinal microbial flora is high in facultative microbes, particularly the Enterobacteriaceae, and low in anaerobes such as bifidobacteria, higher levels of ammonia, endotoxins and other compounds enter the blood stream. This results in direct liver damage and also indirectly from pro-inflammatory cytokines such as TNF-alpha. Probiotics have been shown to modulate the intestinal microflora and decrease the urease producing gram negatives and increase the anaerobic population. While results have been obtained with current probiotic strains, more effective strains could be obtained if all the characteristics bacteria use to survive and compete successfully in the intestine were known. The genomics era is now providing the tools to more effectively understand probiotic interactions in the intestine. This will lead to a new generation of exciting probiotics in the future.

Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector.

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.

Identification of a nisI promoter within the nisABCTIP operon that may enable establishment of nisin immunity prior to induction of the operon via signal transduction.

Certain strains of Lactococcus lactis produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons: nisABTCIP, encoding production and immunity (nisI); nisRK, encoding regulation; and nisFEG, also involved in immunity. Transcription of nisABTCIP and nisFEG requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both Lactococcus and Enterococcus backgrounds revealed that nisI mRNA was present under conditions when no nisA transcription was occurring, suggesting an internal promoter within the operon. The nisA transcript was significantly more stable than nisI, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from nisI. Fusing this region to a lacZ gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the nisI promoter was mapped at bp 123 upstream of the nisI translation start codon. Ordered 5' deletions revealed that transcription activation depended on sequences located up to bp -234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this nisI promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.