El bacteriòfag lambda, un model de decisió genètica

El bacteriòfag lambda (fag λ) és un virus que, com indica el seu nom (bacteriòfag, ‘que menja bacteris') infecta el bacteri intestinal Escherichia coli. El fag λ s'ha utilitzat històricament com a organisme model per a l'estudi de la multiplicació de virus i, sobretot, per comprendre la regulació gènica. Per regulació gènica entenem on i quan han de funcionar els gens, decisió cabdal que fa que, per exemple, les cèl·lules de la nostra sang siguin molt diferents de les de l'ull, tot i dur els mateixos gens: la diferència és quins es troben actius. El fag λ és, doncs, el paradigma per entendre la regulació gènica, ja que té dos cicles de vida diferents, anomenats cicles lític i lisogènic; per triar quin manarà s'utilitza un sistema senzill (i alhora complex) de regulació genètica. Si el fag tria el cicle lític, es multiplica dins del bacteri fins a lisar-lo i alliberar els fags descendents. Si, en canvi, tria el cicle lisogènic, el DNA del fag s'integra dins del DNA del bacteri i roman latent (profag), com un fragment més de genoma bacterià. Això no mata el bacteri, que continua dividint-se. El DNA del fag es replica juntament amb el DNA del bacteri, que expressa els seus gens i els del fag. La lisogènia pot mantenir-se estable o revertir envers el cicle lític si hi ha certs factors ambientals que afectin la viabilitat del bacteri. El fag λ s'ha utilitzat també com a eina en biologia molecular, entre altres aplicacions, per produir DNA recombinant, aprofitant les capacitats dels seus enzims d'integrar fragments de DNA dins del genoma bacterià.The bacteriophage lambda. The bacteriophage lambda (phage λ) is a virus that infects the intestinal bacteria Escherichia coli. Phage λ has been historically used as a model microorganism on the study of virus multiplication, and moreover for the genetic regulation of its multiplicative cycles. Phage λ has two different multiplicative pathways, called lytic and lysogenic cycles, and can choose among both cycles by a complex system of genetic regulation. If the lytic cycle is selected, λ multiplies within the bacteria generating new phages. The new phages release outside the bacteria by lysis, which causes the death of the cell. If the lysogenic cycle is selected, the phage DNA is integrated within the bacterial DNA and remains in a latent state (prophage) as a part of the bacterial genome. This cycle does not kill the bacterial cell, that can continue its replication together with the replication of the phage DNA. This allows the bacterium to express its own genes and those of the phage. Lysogeny can remain stable or revert towards the lytic cycle, if there are certain environmental factors that affect viability of the bacteria. Phage λ has also been applied as a tool for diverse molecular biology applications. Among others, the production of recombinant DNA, using the abilities of their enzymes to integrate DNA fragments within the bacterial genome

Phages in the human body

Bacteriophages, viruses that infect bacteria, have re-emerged as powerful regulators of bacterial populations in natural ecosystems. Phages invade the human body, just as they do other natural environments, to such an extent that they are the most numerous group in the human virome. This was only revealed in recent metagenomic studies, despite the fact that the presence of phages in the human body was reported decades ago. The influence of the presence of phages in humans has yet to be evaluated; but as in marine environments, a clear role in the regulation of bacterial populations could be envisaged, that might have an impact on human health. Moreover, phages are excellent vehicles of genetic transfer, and they contribute to the evolution of bacterial cells in the human body by spreading and acquiring DNA horizontally. The abundance of phages in the human body does not pass unnoticed and the immune system reacts to them, although it is not clear to what extent. Finally, the presence of phages in human samples, which most of the time is not considered, can influence and bias microbiological and molecular results; and, in view of the evidences, some studies suggest that more attention needs to be paid to their interference

Bacteriophages in clinical samples can interfere with microbiological diagnostic tools

Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer

Modulation of Enterohaemorrhagic Escherichia coli Survival and Virulence in the human Gastrointestinal Tract

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen responsible for human diseases ranging from diarrhoea to life-threatening complications. Survival of the pathogen and modulation of virulence gene expression along the human gastrointestinal tract (GIT) are key features in bacterial pathogenesis, but remain poorly described, due to a paucity of relevant model systems. This review will provide an overview of the in vitro and in vivo studies investigating the effect of abiotic (e.g., gastric acid, bile, low oxygen concentration or fluid shear) and biotic (e.g., gut microbiota, short chain fatty acids or host hormones) parameters of the human gut on EHEC survival and/or virulence (especially in relation with motility, adhesion and toxin production). Despite their relevance, these studies display important limitations considering the complexity of the human digestive environment. These include the evaluation of only one single digestive parameter at a time, lack of dynamic flux and compartmentalization, and the absence of a complex human gut microbiota. In a last part of the review, we will discuss how dynamic multi-compartmental in vitro models of the human gut represent a novel platform for elucidating spatial and temporal modulation of EHEC survival and virulence along the GIT, and provide new insights into EHEC pathogenesis

Bacteriophages and Diffusion of β-lactamase Genes

We evaluated the presence of various β-lactamase genes within the bacteriophages in sewage. Results showed the occurrence of phage particles carrying sequences of blaOXA-2, blaPSE-1 or blaPSE-4 and blaPSE-type genes. Phages may contribute to the spread of some β-lactamase genes

Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.This work was supported by the Spanish Ministerio de Ciencia e Innovación (PID2020-113355GB-I00), the Agencia Estatal de Investigación (AEI) and the European regional fund (ERF). The study was partially supported by the Generalitat de Catalunya (2017SGR170). PB-P has a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BES-2017-081296), SM-C has a grant from Colciencias (Republic of Colombia) and LR-R is lecturer of the Serra-Hunter program, Generalitat de Catalunya. MDR-B has a Margarita Salas fellowship from the Spanish Ministerio de Universidades

Infectious phage particles packaging antibiotic resistance genes found in meat products and chicken feces

Bacteriophages can package part of their host's genetic material, including antibiotic resistance genes (ARGs), contributing to a rapid dissemination of resistances among bacteria. Phage particles containing ARGs were evaluated in meat, pork, beef and chicken minced meat, and ham and mortadella, purchased in local retailer. Ten ARGs (blaTEM, blaCTX-M-1, blaCTX-M-9, blaOXA-48, blaVIM, qnrA, qnrS, mecA, armA and sul1) were analyzed by qPCR in the phage DNA fraction. The genes were quantified, before and after propagation experiments in Escherichia coli, to evaluate the ability of ARG-carrying phage particles to infect and propagate in a bacterial host. According to microbiological parameters, all samples were acceptable for consumption. ARGs were detected in most of the samples after particle propagation indicating that at least part of the isolated phage particles were infectious, being sul1the most abundant ARG in all the matrices followed by β-lactamase genes. ARGs were also found in the phage DNA fraction of thirty-seven archive chicken cecal samples, confirming chicken fecal microbiota as an important ARG reservoir and the plausible origin of the particles found in meat. Phages are vehicles for gene transmission in meat that should not be underestimated as a risk factor in the global crisis of antibiotic resistance.info:eu-repo/semantics/publishedVersio

Infectious phage particles packaging antibiotic resistance genes found in meat products and chicken feces

This work was supported by the Spanish Ministerio de Innovación y Ciencia (AGL2016-75536-P), the Agencia Estatal de Investigación (AEI) and the European regional fund (ERF), the Generalitat de Catalunya (2017SGR170) and the Centre de Referència en Biotecnologia (XeRBa). CERCA Programme from the Generalitat de Catalunya is also acknowledged. M.B.-J. has a grant from COLCIENCIAS (Republic of Colombia). P.B.-P. has a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BES-2017-081296). L.R.-R. is supported by the Beatriu de Pinos postdoctoral programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge.Bacteriophages can package part of their host's genetic material, including antibiotic resistance genes (ARGs), contributing to a rapid dissemination of resistances among bacteria. Phage particles containing ARGs were evaluated in meat, pork, beef and chicken minced meat, and ham and mortadella, purchased in local retailer. Ten ARGs (bla, bla, bla, bla, bla, qnrA, qnrS, mecA, armA and sul1) were analyzed by qPCR in the phage DNA fraction. The genes were quantified, before and after propagation experiments in Escherichia coli, to evaluate the ability of ARG-carrying phage particles to infect and propagate in a bacterial host. According to microbiological parameters, all samples were acceptable for consumption. ARGs were detected in most of the samples after particle propagation indicating that at least part of the isolated phage particles were infectious, being sul1the most abundant ARG in all the matrices followed by β-lactamase genes. ARGs were also found in the phage DNA fraction of thirty-seven archive chicken cecal samples, confirming chicken fecal microbiota as an important ARG reservoir and the plausible origin of the particles found in meat. Phages are vehicles for gene transmission in meat that should not be underestimated as a risk factor in the global crisis of antibiotic resistance

Chicken liver is a potential reservoir of bacteriophages and phage-derived particles containing antibiotic resistance genes

Poultry meat production is one of the most important agri-food industries in the world. The selective pressure exerted by widespread prophylactic or therapeutic use of antibiotics in intensive chicken farming favours the development of drug resistance in bacterial populations. Chicken liver, closely connected with the intestinal tract, has been directly involved in food-borne infections and found to be contaminated with pathogenic bacteria, including Campylobacter and Salmonella. In this study, 74 chicken livers, divided into sterile and non-sterile groups, were analysed, not only for microbial indicators but also for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Both bacteria and phages were detected in liver tissues, including those dissected under sterile conditions. The phages were able to infect Escherichia coli and showed a Siphovirus morphology. The chicken livers contained from 10 3 to 10 6 phage particles per g, which carried a range of ARGs (bla , bla , sul1, qnrA, armA and tetW) detected by qPCR. The presence of phages in chicken liver, mostly infecting E. coli, was confirmed by metagenomic analysis, although this technique was not sufficiently sensitive to identify ARGs. In addition, ARG-carrying phages were detected in chicken faeces by qPCR in a previous study of the group. Comparison of the viromes of faeces and liver showed a strong coincidence of species, which suggests that the phages found in the liver originate in faeces. These findings suggests that phages, like bacteria, can translocate from the gut to the liver, which may therefore constitute a potential reservoir of antibiotic resistance genes. Phage particles carrying antibiotic resistant genes have been found in chicken livers. Metagenomic analysis of chicken liver viromes (PL) and chicken faeces viromes (HP) suggests that these phage particles could translocate from chicken gut to the liver