Wednesday, October 7, 2009
W. Florian Fricke, Patrick F. McDermott, Mark K. Mammel, Shaohua Zhao, Timothy J. Johnson, David A. Rasko, Paula J. Fedorka-Cray, Adriana Pedroso, Jean M. Whichard,
J. Eugene LeClerc, David G. White, Thomas A. Cebula, and Jacques Ravel
Salmonella enterica is a common cause of food –borne gastroenteritis. This combined with the rise of multidrug resistant S. enterica isolates is a grave medical concern. The S enterica subsp. enterica serovar Kentucky is the most common serotype found in chickens [1,2]. Moreover this serotype is often found to be resistant to antibiotics such as tetracycline and streptomycin . The goal of the study was to find clues to the development of multi-drug resistance in S. Kentucky. The authors analyzed the complete sequences of the 3 large plasmids (pCVM29188_146, pCVM29188_101, pCVM29188_46) that they isolated from S. Kentucky CVM29188. Only the two large plasmids (pCVM29188_146 and pCVM29188_101) were found to carry antibiotic resistance genes. Thus, genes coding for resistance to aminoglycosides (strAB) and tetracyclins (tetRA) were found on pCVM29188_146 and those coding for resistance to cephalosporins (bla CMY-2) were found on pCVM29188_101. Both resistance plasmids (pCVM29188_101 and pCVM29188_146) in this study were found to have intact transfer regions, while the smaller plasmid pCVM29188_46 (46kb) did not have any transfer genes. Sequence similarity of the replication and transfer genes to other plasmids suggest that plasmid pCVM29188_101 may belong to the IncI1 group while plasmid pCVM29188_46 may belong to the IncFII group. The backbone of plasmid pCVM29188_146 is very similar to two plasmids isolated from avian pathogenic E. coli strains and also have the same virulence factors. The plasmid pCVM29188_46 has little similarity to other plasmids and has a lot of hypothetical proteins. They next conducted mating experiments with plasmids pCVM29188_146 and pCVM29188_101 and showed their transfer to two strains of Salmonella and a strain of E. coli. Next they wanted to test the abundance of the virulence genes on other isolates of S. Kentucky from meat, clinical and agricultural sources. So they screened 287 S. Kentucky isolates for the presence of virulence genes by PCR with primers specific for the 5 loci of pCVM29188_146 that were responsible for encoding virulence factors. They found that 64% of all S. Kentucky strains tested positive for the presence of at least one locus associated with virulence. The association was even stronger among the S. Kentucky strains that were isolated from chicken. This however was not the case for the 6 other Salmonella serovars that were isolated from chicken Moreover, all S. Kentucky strains that had at least one virulence locus of Pcvm29188_146 also had resistance to tetracycline and a subgroup of these had resistance to streptomycin. This suggests that a strong association may exist between S. Kentucky and plasmids like pCVM29188_146 that encode both virulence factors and resistance to antibiotics such as tetracycline and streptomycin. One explanation that the authors offer for this association is that pathogenic E. coli strains bearing virulence plasmids may have encountered resistance plasmids, leading to integration of the resistance genes into the virulence plasmid. This new plasmid could have transferred into a Salmonella strain such as the S. Kentucky commonly found in chickens. They do acknowledge that this does not explain why other Salmonella strains isolated from chicken do not have this plasmid type. The second explanation is that virulence plasmids may help S. Kentucky in coping with stress or other enterobacteria and hence, the association. This again does not explain why the association is only seen in S. Kentucky isolated from chicken.
This is an interesting study of plasmids from a strain of bacterium that has medical relevance to us. It is indeed surprising that there is such a clear association of the virulence and antibiotic resistance encoding plasmid with the S. Kentucky strain isolated from chicken. Their explanations for the association seem a little weak.
1. FDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS). Retail meat annual report, 2006. FDA, Bethesda MD. http://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM073302.pdf
2. USDA. 2008. National Antimicrobial Resistance Monitoring System for Enteric
Bacteria (NARMS). Veterinary isolates final report, slaughter isolates,
2006. USDA, Washington, DC. http://www.ars.usda.gov/sp2UserFiles/Place/66120508/NARMS/narms_2006/NARMS2006.pdf.
University of Idaho
Monday, October 5, 2009
Mastura Akhtar, Helmut Hirt, Ludek Zurek
Vectors have largely aided the spread of microorganisms. Vectors move bacteria from one place to another as they themselves go about their life cycle. The movements of a house fly would be a prime example of this type of vector. However, a role of the vector not considered as often is its role as a habitat for a bacterium itself. During transport, or as a permanent environment, bacteria encounter a unique combination of other bacteria and nutrients that only the vector could assemble. This provides for a microenvironment that can play a key role in the evolution of and dissemination of traits beneficial to bacterial species. While inside the fly these traits can be transferred horizontally through conjugation, transduction and transformation. This study considers how plasmid mediated horizontal gene transfer in the gut of a house fly can mediate tetracycline resistance to transfer between bacterial species.
Two strains of enterocci, bacteria normally residing in the gastrointestinal tract, were selected for donor and recipient. The donor contained the tetM gene to identify transformants using selective media. Flies were separated into two groups, one that received the donor first, via infected food supply, and the other received the recipient strain first. After twelve hours flies were given food source containing the opposite strain for one hour. Each group was then subdivided so that while flies were checked for the presence of donor, recipient and transformants over the next five days and half would have their eating appendage sterilized and half would not.
Results showed that regardless of whether the donor or recipient was introduced first, both groups established concentrations of donor and recipient cells that were similar. The was also no statistical difference in rate of gene transfer between the two groups. Concentration of donor and recipient cells in the digestive tract was similar to that of the surface sterilized, suggesting that observed donor, recipient and transformants were localized to the gut.
Transformants began to be detected 24 hours after both stains were combined. Their presence was screened for using selective media. Groups of flies were sterilized at the surface to eliminate the possibility of surface contamination and transformation outside the vector. Portions of the food supplied to the flies were periodically screened for transformants with very little occurrence, suggesting this did not play a key role. However, it is possible that transfer is taking place on the eating appendage. The conditions in which intestinal gene transfer are best suited are not well understood. The final possible explanation could be that transformants could be the product of high plasmid transfer rate and subsequent rapid clonal expansion of transformants.
Horizontal transfer in a vector could lead to the spread of various genes and provide a unique microenvironment for evolution. The house fly’s unique combination of contact with decaying organic matter and food provides ample opportunity for transfer of traits between bacteria evolved to live in harsh environments to those that are common in food. This potential introduces a need to better understand horizontal gene transfer and evolution in microenvironments that can directly affect humans.
University of Idaho
Friday, September 25, 2009
Schmitt I, Lumbsch HT (2009) PLoS ONE 4(2): e4437. doi:10.1371/journal.pone.0004437
Until recently, the studies of gene transfer have been mostly focused on prokaryotes, and the process of gene transfer is assumed to be of limited significance to eukaryotes. The availability of diverse eukaryotic genome sequence data is dramatically changing our views on the important role gene transfer can play in eukaryotic evolution. The rapid increase in fungal sequence data has promoted this kingdom to the forefront of comparative genomics. As a result, interkingdom HGT became a hot topic in recent years. Whereas there is very few documented evidence for interkingdom HGT, but they did happen. Here, we will present an ancient interkingdom HGT event between bacteria and fungi.
The targeted gene discussed here is the polyketide synthase (PKSs) genes, which involved in antibiotic and mycotoxin production. Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms.
Bacteria and fungi commonly harbor a group of PKSs that consists of a single protein complex carrying all catalytic sites (typeI PKS). In this paper, the authors are focusing on a clade of fungal type I PKSs gene which is closely related to bacterial PKSs. Since 6-methylsalicylic acid synthase (6-MSAS) was the first PKS in this group to be characterized, this clade is also termed as‘‘6-MSAS-type PKS’’. The lichenized fungi, which are characterized by a sophisticated vegetative morphology and a rich polyketide metabolism, were selected as the research materials in this study. The total genomic DNA of the lichenized fungi, which collected from 12 different countries, were extracted, and then the KS domain of fungal 6MSAS-type PKS genes were amplified by a degenerate primer pair, LC3 and LC5c. The amplified fragments were cloned and sequenced, and then all sequences were subjected to BLAST searches. The alignment was analyzed in a Bayesian phylogenetic framework using MrBayes 3.1. The tree resulting from this analysis was used to determine the PKS clades most closely related to the fungal 6-MSAS group. To evaluate potential problems with outgroup selection, three alignments including different outgroups were compared.
As a result, 24 6-MSA synthase sequence tags from lichen-forming fungi were generated. The results from comparative phylogenetics support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. In the Discussion, the authors inferred that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase. Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi.
This study revealed the phylogenetic origin of the enigmatic fungal 6-MSAS-type PKS biosynthetic gene using comparative analysis. The results provide statistical support to the hypothesis that this PKS was transferred from an actinobacterial source into ascomycete fungi during an ancient HGT event. They also report the finding of 6-MSAS-type PKS genes in a variety of lichen-forming fungi, and speculate about the possible role of lichen symbionts in the evolution of this gene. Overall, this paper added solid evidence to the fact of interkingdom HGT.
Hui Li Ph.D University of Idaho
Friday, September 18, 2009
Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture.
S. Pignato, M. A. Coniglio, G. Faro, F. X. Weill and G. Giammanco
Journal of Water and Health Vol 07 No 2 pp 251–258
In presented paper authors pointed out that the guidelines for cleaning water used for irrigation requires treatments to remove pathogens that can cause enteric infections for crop consumers, producers and handlers. According to the microbiological guidelines for safe use of wastewater in agriculture developed by the World Health Organization(WHO) less than 0.1 intestinal nematode eggs must be detected in 1 litre, while up to 1,000 faecal coliform bacteria per 100 ml can be tolerated for unrestricted irrigation. In the United States, much stricter wastewater quality standards for irrigation are recommended by the Environmental Protection Agency but, lacking federal standards for the quality of reclaimed water, individual states have developed guidelines mainly based on the daily monitoring of faecal coliform bacteria on a single, 100-ml sample, assuming a predictive relationship between indicator microorganisms and pathogen presence.
Although wastewater treatments proved to be effective in eliminating Salmonella spp. and in reaching WHO microbiological standards for safe use of wastewater in agriculture, they were ineffective in reducing significantly the frequency of plasmid-mediated multiple antibiotic resistance in surviving E. coli.
It was shown that 22.71%, 19.41%, 16.84% and 14.28% out of 273 isolates were resistant respectively to ampicillin, tetracycline, sulfamethoxazole, and streptomycin. Some other antibiotic resistant strains were detected at low frequency (trimetoprim – 9.15%; nalidixic acid – 8%; chloramphenicol – 5.12% and kanamycin – 2.93%). Also multiple antibiotic resistance was present in 24.17% of the isolates. Antibiotic resistance was detected to be transferred by conjugation from 54% resistant strains. Three different plasmids with the sizes of 125kb, 54kb and 60kb were isolated from those strains. Also some other mobile elements like class 1 integrons were detected in resistant strains.
Since multiple antibiotic-resistant bacteria carrying integrons and conjugative R plasmids can constitute a reservoir of antibiotic-resistance genes in wastewater reclaimed for irrigation, risks for public health should be considered. Bacterial strains carrying R plasmids and integrons could contaminate crops irrigated with reclaimed wastewater and transfer their resistances to the consumers’ intestinal bacteria. So we should remember to wash every vegetables and fruits before we will eat them…
Friday, September 4, 2009
Rembrandt J. F. Haft, John E. Mittler, and Beth Traxler
The ISME Journal (2009) 3, 761-769
When it comes to their relationship with their hosts it can sometimes be difficult to define what plasmids are. They often encode beneficial traits that can be useful, or even vitally necessary, for their host bacteria. A bacterium that finds itself in the gut of a patient taking antibiotics, for instance, may require plasmid-encoded resistance in order to survive. However, despite the potential usefulness of a given plasmid, plasmid carriage also comes with certain costs. In certain environments the same plasmid that used to be essential for survival can become a burden to its host due to the energy required of the host for plasmid maintenance and upkeep. In such circumstances plasmids can be viewed as parasitic; they need the host to survive but only confer costs, not benefits, to the host. Many plasmids have therefore developed clever ways with which to ensure their survival in bacterial populations, even in the absence of external selective pressures for plasmid maintenance (e.g. the presence of antibiotics).
It’s easy to imagine that in the absence of such selective pressures plasmid-free bacteria would out-compete plasmid-bearing cells. Through vertical inheritance alone this would ensure the eventual loss of plasmids from a mixed population of plasmid-bearing and free cells. One tool that most plasmids have to combat this loss is the ability to pass copies of themselves to neighboring cells via the horizontal gene transfer mechanism of conjugation. Conjugation allows plasmids to infect new hosts such that even in the absence of selection plasmids can survive in a population, even as they reduce the fitness of their hosts. Yet many plasmids have developed systems that inhibit their own horizontal transfer. The authors of this paper used a combination of mathematical models and laboratory experiments to determine when and why a plasmid might benefit in repressing, rather than promoting, their own conjugation.
What the authors predicted in their models (and confirmed in their experiments) is that by reducing transfer frequency plasmids can also reduce the cost that they’re imposing on their hosts. While plasmids that don’t repress conjugation will spread through a bacterial population more quickly on their own than plasmids with functioning repression systems, when both types of plasmids are present the latter will eventually take become dominant. This is because in limiting their horizontal transfer they give the competitive edge to their hosts, which will be more fit and grow faster than the bacteria harboring the plasmids that transfer more frequently, and consequently these transfer-limited plasmids are spread mainly through vertical transfer.
This paper not only elucidates the mystery of the persistence of transfer repression systems in plasmids, but also has broader applications to parasitic strategies in general. The authors point out that for many parasites being slightly less virulent gives some parasites a competitive advantage over those that kill their host before they are able to spread to a new one. The mathematical model that they developed could therefore be applied to a much large scope of systems, of which plasmid transfer is just one.
Bahl MI, Hansen LH, Sorensen SJ. (2007). Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett 266: 250-6.
De Gelder L, Ponciano JM, Joyce P, Top EM. (2007). Stability of a promiscuous plasmid in
different hosts: no guarantee for a long-term relationship. Microbiology 153: 452-63.
Dionisio F. (2005). Plasmids survive despite their cost and male-specific-phages due to
heterogeneity of bacterial populations. Evolution Ecol Res 7: 1-19.
Freter R, Freter RR, Brickner H. (1983). Experimental and mathematical models of
Escherichia coli plasmid transfer in vitro and in vivo. Infect Immun 39: 60-84.
Kerr B, Neuhauser C, Bohannan BJ, Dean AM. (2006). Local migration promotes competitive
restraint in a host-pathogen 'tragedy of the commons'. Nature 442: 75-8.
Kover PX, Clay K. (1998). Trade-off between virulence and vertical transmission and the
maintenance of a virulent plant pathogen. Am Nat 152: 165-175.
Turner PE, Cooper VS, Lenski RE. (1998). Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. Evolution 52: 315-329.
University of Idaho
Friday, August 21, 2009
Niamh Toomey, Áine Monaghan, Séamus Fanning, Declan J. Bolton
Journal of Microbiological Methods, 2009, 77: 23–28
The most common laboratory techniques used to assess HGT in vitro include: plate, filter and broth mating protocols. While there is a general acceptance that higher transfer frequencies occur when using solid-phase mating mediums (since conjugation requires mating cells to be in close contact with each other), few studies have experimentally compared HGT techniques, with a view to standardizing these approaches, thereby providing meaningful direct comparisons.
In this study, plate, filter and broth mating techniques were assessed and compared by using a taxonomically diverse group, Lactic acid bacteria (LAB). LAB is a Gram-positive, catalase-negative group, which share the capacity to ferment sugars into lactic acid. Due to their aerotolerant anaerobic nature they are found in a variety of different environments. LAB also has the potential to act as resistance gene reservoirs with the ability to transfer these genes in a range of different environments; including transfer to pathogenic species. Genes conferring resistance to antimicrobials such as tetracycline, erythromycin, streptomycin, chloramphenicol, and vancomycin, have been found in LAB isolated from foods. These resistance genes were found to be located on transferable elements including plasmids and conjugative transposons. In present study, one L. lactis strain with the broad-host range plasmid pAMβ-1 [containing an erythromycin resistance marker, erm(B)], and two L. lactis strains with the conjugative transposon Tn916 [expressing a tetracycline resistance gene, tet(M)] were used as the donor strains, along with a marked (Strr, Rifr) L. lactis strain as recipient.
The plate mating technique used in this study is almost the same with that used in TopLab, except that following overnight incubation and scraping of the bacterial spots, additional medium was used to wash the plate for accurately calculating the transfer frequency. The filter mating technique is exactly the same with that we used. The broth mating method is just to add equal volume donor culture and recipient culture into one tube, and then following the overnight incubation, directly dilute the mixed culture and spread onto selective plates. Transconjugants were confirmed as the true transconjugants but not the reverted mutants by using antibiotic selection, E-tests to determine MICs, PCR assays to detect the corresponding marker genes, DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), and Southern blotting.
Based on the results of transfer frequency, the general trend is plate > filter > broth. In addition, in most cases, there is no significant difference between plate mating and filter mating. Effects of different pH, varying from 6.0 to 8.0, on the transfer rate were also detected. The pH of medium between 6.0 and 7.0 had no significant effect on transfer rate, while at pH 8.0 the conjugation was completely inhibited.
As the author said, this paper is the first study to examine the influence of mating protocols on both plasmid and transposon conjugal transfer between lactococcal strains. The results provide the detailed information about which system should be recommended in future LAB HGT studies.
However, some of the other research works are not in agreement with the results from this study. Both Lampkowska et al. (2008) and Langella et al. (1996) suggested that the filter facilitates a greater degree of donor-recipient contact than the plate method. Therefore, a question rises up. Need we optimize the mating system (including the mating techniques and environmental factors, such as temperature, pH) when we deal with every different genus, such as Pseudomonas, Agrobacterium, and Cupriavidus, which currently used in our lab? If it does, then a great amount of work we should do. Do you think it is necessary? Feel free to take part in the discussion. It is a Blog, and there should be some discussion here.
Lampkowska, J., Feld, L., Monaghan, A., Toomey, N., Schjørring, S., Jacobsen, B., van der Voet, H., Andersen, S.R., Bolton, D., Aarts, H., Krogfelt, K.A., Wilcks, A., Bardowski, J., 2008. A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species. Int. J. Food Microbiol. 127, 172–175.
Langella, P., Zagorec, M., Ehrlich, S.D., Morel-deville, F., 1996. Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake. FEMS Microbiol. Lett. 139, 51–56.
Hui Li, PhD
University of Idaho
Sunday, August 16, 2009
The defective prophage pool of Escherichia coli O157: Prophage-prophage interactions potentiate horizontal transfer of virulence determinants
Bacteriophage is one of the major genetic elements promoting horizontal gene transfer between bacteria. Enterohemorrhagic Escherichia coli O157:H7 (Sakai isolate) contains eighteen prophages in its genome (Ohnishi et al. 1999). Two of the eighteen prophages carry Shiga toxin gene clusters; stx1AB and stx2AB; these gene products kill eukaryotic cells by inhibiting protein synthesis. Interestingly, all prophages in O157 have mutations in genes that encode basic phage functions such as site-specific recombination, replication, cell lysis, or structural proteins constituting the head and tail. Evidence obtained by genome analysis suggests that each prophage is defective. Thus, one might think this strain is not very problematic. But, it may be not true.
To assess the role of defective prophages in horizontal gene transfer of virulence determinants, the authors analyzed each basic function of the prophages in O157: excision from chromosome, phage DNA amplification in response to DNA damage, ability to be released as phage particles, and infection of new recipient cells.
Among eighteen prophages, eight prophages including the stx gene-containing phages were found to be released as phage particles, and among them four were able to infect both or either of the two E. coli recipient strains tested. These results strongly suggest that prophages in O157 are complementing defective functions with each other to make practically transferable phages.
Importantly, the phage particle produced often contains a chimeric DNA fragment made of two different prophages' fragments, indicating the recombination of prophages in host cells. Although the newly generated phage DNA were not extensively sequenced in this study, it is possible that some phage fragments reconstituted intact, functional phage through recombination.
This paper nicely explains the way bacteriophages live and propagate in a host. I think this paper is a good example of a post-genomic sequence study. In this era, we can understand how mobile genetic elements are acting in a population by combining sequence analysis and standard genetic experiments. I suspect that plasmids and transposons are also repeating cycles of inactivation and activation as shown for these phages. They will never be extinguished as long as similar mobile genetic elements exist in the world.
Ohnishi M. and Tanaka C. et al., (1991) DNA Res. 6: 361-368
Posted by H. Yano, Univerisity of Idaho
Friday, July 31, 2009
This is jet another paper describing plasmid transfer and their role in so called bioaugmentation in natural kind of environments.
Bioaugmentation involves the addition of exogenous micro-organisms that have the ability of degrading the compound of interest. Using this process, new biodegradation pathways can be added, which inherently improve the metabolic conversion of the contaminants. The main problem of this method is that the laboratory strains used to introduce new genetic traits usually cannot grow in natural environmental conditions. This means that bioaugmentation can fail due to the poor establishment and=or survival of new strains under field environmental conditions. The second process used for enhancing natural biodegradation capabilities is biostimulation which consists of adding nutrients (e.g., carbon, nitrogen, electron acceptor, etc.) to promote the growth of indigenous microorganisms. This method is preferred over bioaugmentation because no additional bacterial strains are introduced but it requires the presence of indigenous microorganisms, which are capable of breaking down the contaminant of interest. To overcome the limitations associated with biostimulation and bioaugmentation, it might be possible to combine these two strategies into a technique called genetic bioaugmentation. This technique would consist of adding bacteria with the needed genes and inducing their horizontal gene transfer (HGT) to indigenous bacterial species utilizing natural prokaryotic adaptation mechanisms.
In this paper authors described pWWO plasmid transfer from Pseudomonas putida strain to bacterial population present in activated sludge. The pWWO plasmid is well known for its toluene degradation capability. The plasmid DNA was tagged with transposone containing gfp gene driven by lac promoter. In the donor strain which overproduce LacI repressor gfp is not expressed. In other bacterial hosts gfp is expressed and gives bright green fluorescence which can be measured. Using flow cytometry authors showed that plasmid was actually transferred into bacterial community of activated sludge with number of bacteria expressing GFP protein up to 6%. They showed that the highest number of transconjugants is reached at the third day of experiment and it depends on the donor to recipient ratio with the best results at the 1:20 ratio. Authors tried to connect the plasmid transfer with the actual capability of activated sludge to toluene degradation. This experiment did not really work as the level of toluene degradation remained unchanged during the time of the experiment.
The paper stays in a main stream of microbial ecology and engineering facing the problem of global pollutions and waists utilization. It is very important to use new methods for detecting plasmid transfer in natural environment. Authors were facing the same problem as in previously published papers that there are some limitations in using fluorescent proteins as a plasmid infection marker. The true plasmid bearing cells are not known until gfp gene is expressed and protein is properly matured to give strong fluorescence, which can be detected by flow cytometry. Authors cannot also link the presence of TOL phenotype (toluene degradation) with the number of plasmid bearing cells as the sludge microbial community shows already high toluene degradation activity.
Perhaps, it is not a very “big” paper, but it is a small step toward understanding how the plasmid transfer occurs in natural environment and how can we use this to save our polluted world.
Jaroslaw Krol, PhD