Unravelling the consequences of the bacteriophages in human samples.

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

Comparison of Commensal and Clinical Isolates for Diversity of Plasmids in Escherichia coli and Klebsiella pneumoniae.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

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 ß-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.

Faecal phageome of healthy individuals: presence of antibiotic resistance genes and variations caused by ciprofloxacin treatment.

OBJECTIVES: Antimicrobial resistance genes (ARGs) can be transferred by means of mobile genetic elements, which play a critical role in the dissemination of resistance in the bacterial community. ARG transmission within mobile genetic elements has been reported in plasmids and transposons but less frequently in bacteriophages. Here, the bacteriophage fraction of seven human faecal samples was purified and deep-sequenced to detect the presence of ARGs in the phage particles. METHODS: Seven faecal samples (five from healthy individuals and two from a patient before and after receiving ciprofloxacin treatment) were used to extract phage DNA, which was purified and then sequenced in a MiSeq (Illumina). Generated reads were checked for quality and assembled, and then the generated contigs analysed with Kraken, PHASTER, VirSorter and Prokka. Some genes were also validated by quantitative PCR. RESULTS: Analysis of the purified phage DNA by Kraken identified from 4 to 266 viruses in the samples. The viral fraction corresponded mainly to the order Caudovirales, including phages from the Siphoviridae and Myoviridae families. Bacterial genes associated with antimicrobial resistance were detected in the viral DNA, as confirmed by quantitative PCR. Higher densities of ARG-carrying phage particles were observed in the post- versus pre-ciprofloxacin treatment sample. CONCLUSIONS: The finding of ARGs in phage particles supports the description of phages as mobile elements contributing to the dissemination of bacterial antibiotic resistance and suggests ciprofloxacin treatment may play a role in the release of ARG-carrying particles, thereby increasing resistance.

Detection of Bacteriophage Particles Containing Antibiotic Resistance Genes in the Sputum of Cystic Fibrosis Patients.

Cystic fibrosis (CF) is a chronic disease in which the bacterial colonization of the lung is linked to an excessive inflammatory response that leads to respiratory failure. The microbiology of CF is complex. Staphylococcus aureus is the first bacterium to colonize the lungs in 30% of pediatric CF patients, and 80% of adult patients develop a chronic Pseudomonas aeruginosa infection, but other microorganisms can also be found. The use of antibiotics is essential to treat the disease, but antibiotic performance is compromised by resistance mechanisms. Among various mechanisms of transfer of antibiotic resistance genes (ARGs), the recently been reported bacteriophages are the least explored in clinical settings. To determine the role of phages in CF as mobile genetic elements (MGEs) carrying ARGs, we evaluated their presence in 71 CF patients. 71 sputum samples taken from these patients were screened for eight ARGs (blaTEM, blaCTX-M-1-group, blaCTX-M-9-group, blaOXA-48, blaVIM, mecA, qnrA, and qnrS) in the bacteriophage DNA fraction. The phages found were also purified and observed by electron microscopy. 32.4% of CF patients harbored ARGs in phage DNA. ß-lactamase genes, particularly blaVIM and blaTEM, were the most prevalent and abundant, whereas mecA, qnrA, and qnrS were very rare. Siphoviridae phage particles capable of infecting P. aeruginosa and Klebsiella pneumoniae were detected in CF sputum. Phage particles harboring ARGs were found to be abundant in the lungs of both CF patients and healthy individuals and could contribute to the colonization of multiresistant strains.

Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil.

Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48, blaVIM, mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were ß-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered.

Antibiotic resistance genes in phage particles isolated from human faeces and induced from clinical bacterial isolates.

Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48, qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with blaTEM and blaCTX-M-9 group being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with blaTEM, blaCTX-M-1 group, blaCTX-M-9 group and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 1010 gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles.

Carbapenemase-producing enterobacteriaceae recovered from a Spanish river ecosystem.

The increasing resistance to carbapenems is an alarming threat in the fight against multiresistant bacteria. The dissemination properties of antimicrobial resistance genes are supported by their detection in a diverse population of bacteria, including strains isolated from the environment. The objective of this study was to investigate the presence of carbapenemase-producing Enterobacteriaceae (CPE) collected from a river ecosystem in the Barcelona metropolitan area (Spain). Identification of ß-lactamases and other resistance determinants was determined as was the antimicrobial susceptibility profile. Moreover, screening of virulence factors, plasmid addiction systems, plasmid partition systems and replicon typing was performed. The results identified 8 isolates belonging to different species (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica). The most prevalent enzyme was KPC-2 (n = 6), followed by VIM-1 (n = 2) and IMI-2 (n = 1), whereas no OXA-48-type was detected. In addition, one strain was positive for both KPC-2 and VIM-1 enzymes. All the carbapenemase-encoding plasmids carried at least one plasmid addiction or partition system, being vagCD and parAB the most frequently detected, respectively. E. coli and K. pneumoniae isolates carried a low number of virulence-associated factors and none of the detected clones has previously been identified in the clinical setting. These findings support the high dissemination potential of the carbapanemase-encoding genes and reinforce the idea that the environment is another reservoir that may play an important role in the capture, selection and dissemination of carbapenem resistance genes.

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.

Comparative analysis of the Shiga toxin converting bacteriophage first detected in Shigella sonnei.

Here we report the first complete nucleotide sequence of a Shiga toxin (Stx) converting phage from a Shigella sonnei clinical isolate that harbors stx1 operon, first identified in the chromosome of Shigella dysenteriae type 1. The phage named Shigella phage 75/02 Stx displayed Podoviridae morphology. It proved to be transferable to Escherichia coli K-12 strains, and cytotoxicity of the lysogenized strains was demonstrated in Vero cell cultures. Genomic analysis revealed that the prophage genome is circular and its size is 60,875 nt that corresponds to 76 ORFs. The genome of Shigella phage 75/02 Stx shows a great degree of mosaic structure and its architecture is related to lambdoid phages. All the deduced proteins, including the 37 hypothetical proteins showed significant homologies to Stx phage proteins present in databases. The phage uniformly inserted into the ynfG oxidoreductase gene framed by phage integrase and antirepressor genes in parental S. sonnei and in the three lysogenized K-12 strains C600, DH5α and MG1655. The Stx1 prophage proved to be stable in its bacterial hosts and remained inducible.

Transfer of antibiotic-resistance genes via phage-related mobile elements.

Antibiotic resistance is a major concern for society because it threatens the effective prevention of infectious diseases. While some bacterial strains display intrinsic resistance, others achieve antibiotic resistance by mutation, by the recombination of foreign DNA into the chromosome or by horizontal gene acquisition. In many cases, these three mechanisms operate together. Several mobile genetic elements (MGEs) have been reported to mobilize different types of resistance genes and despite sharing common features, they are often considered and studied separately. Bacteriophages and phage-related particles have recently been highlighted as MGEs that transfer antibiotic resistance. This review focuses on phages, phage-related elements and on composite MGEs (phages-MGEs) involved in antibiotic resistance mobility. We review common features of these elements, rather than differences, and provide a broad overview of the antibiotic resistance transfer mechanisms observed in nature, which is a necessary first step to controlling them.