Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis

Araujo, Rosa; Muniesa, Maite; Méndez, J.; Puig, Anna; Queralt, Nuria; Lucena, F.; Jofre, Joan

doi:10.1016/s0166-0934(01)00261-0

Cited by 29 publications

(22 citation statements)

References 7 publications

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“…In these experiments, organisms were grown in the absence or presence of subinhibitory concentrations of each antibiotic. 1 and stx 2 , presumably representing phage-coated DNA, appeared in the supernatants of the two stx 1 ϩ /stx 2 ϩ strains. malB amplicons were not elicited from the supernatant, so the stx sequences in the supernatant cannot be attributed simply to bacterial lysis.…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, identical nonsynonymous A3G SNPs 108 nucleotides from the 5Ј border of each octamer in the two ⌬18,895 truncated bacteriophages that we sequenced suggest that the SNPs and the truncations in these strains did not arise independently. Thus, even though the most straightforward mechanism for acquiring or losing stx 1 would be the simple exchange of the octamer for stx 1 and contiguous DNA, or vice versa, the presence of this SNP suggests that at least one of the borders of recombination leading to the acquisition or loss of stx 1 is distal to at least one of the octamer's borders. Indeed, such instructive SNPs in rfbE strongly suggest interlineage cotransfer of large chromosomal segments between E. coli O157:H16 strain 13A81 and E. coli O157:H Ϫ strain 3584-91 (46).…”

Section: Discussionmentioning

confidence: 99%

“…Stx2 has 56% identity to Stx1 (36). The stx 1 and stx 2 A and B subunit genes exist as tandem open reading frames (ORFs) in the central portion of lambdoid bacteriophages in E. coli O157:H7 (19). In Sakai and EDL933, the two E. coli O157:H7 strains that have been completely sequenced, the bacteriophage that encodes Stx1 is integrated into yehV (57), which encodes a protein that positively regulates curli expression (7), and is flanked by duplications of CCCTGT CACGTTACGCGCGTG.…”

mentioning

confidence: 99%

“…Subsequently, sorbitolfermenting stx 2 ϩ E. coli O157 (11,15) separated from this lineage. After these two lineages diverged, E. coli O157:H7 acquired stx 1 (11), presumably via acquisition of the bacteriophage that contained this gene, and lost the ability to ferment sorbitol, while the sorbitol-fermenting stx 2 ϩ E. coli O157 evolved into nonmotile E. coli O157:H Ϫ . Most non-O157:H7 STEC strains associated with human diseases are distantly related to E. coli O157:H7 and form a clade termed the STEC 2 (or enterohemorrhagic E. coli 2) group (54).…”

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confidence: 99%

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Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

2003

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As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H ؊ presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx 1 (stx 1 -negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx 2 bacteriophage usually occupies wrbA in stx 1 ؉ /stx 2 ؉ E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx 1 -negative/stx 2 ؉ E. coli O157:H7 strains, the presumed progenitors of stx 1 ؉ /stx 2 ؉ E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx 2 bacteriophage insertion sites), suggest that stx 1 acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.Shiga toxins 1 and 2 (Stx1 and Stx2) are cardinal virulence factors of Escherichia coli O157:H7. Stx1 is nearly identical to Stx, the principal extracellular cytotoxin of Shigella dysenteriae serotype 1 (8). Stx2 has 56% identity to Stx1 (36). The stx 1 and stx 2 A and B subunit genes exist as tandem open reading frames (ORFs) in the central portion of lambdoid bacteriophages in E. coli O157:H7 (19). In Sakai and EDL933, the two E. coli O157:H7 strains that have been completely sequenced, the bacteriophage that encodes Stx1 is integrated into yehV (57), which encodes a protein that positively regulates curli expression (7), and is flanked by duplications of CCCTGT CACGTTACGCGCGTG. The bacteriophage that encodes Stx2 is integrated into wrbA (32, 40), which encodes a novel multimeric flavodoxin-like protein (13), and is flanked by duplications of GACATATTGAAAC. Almost all E. coli O157:H7 strains possess stx 2 , and approximately three-quarters contain, in addition, stx 1 (strains lacking stx 1 are referred to hereafter as stx 1 -negative strains) (23,38,45). Most human non-O157:H7 Stx-producing E. coli (STEC) strains possess stx 1 but lack stx 2 (5, 6, 28, 51). Except for an Stx2...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

2003

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“…fragilis strain RYC2056 and bacteriophage B40-8, that infect strain HSP40 of Bact. fragilis, were determined by the double agar layer method as described elsewhere (Araujo et al 2001;Anon. 2001).…”

Section: Bacteriophage Enumerationmentioning

confidence: 99%

Removal and inactivation of indicator bacteriophages in fresh waters

et al. 2002

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Aims:The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. Methods and Results: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. Conclusions: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. Significance and Impact of the Study: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.

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Isolation of Bacteriophages of the Anaerobic Bacteria Bacteroides

García-Aljaro

Muniesa

Jofre

2017

Methods in Molecular Biology

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Here we describe the detection, enumeration, and isolation of bacteriophages infecting Bacteroides. The method is based on the infection of Bacteroides host strains and the production of visible plaques in a confluent lawn of the host strain using the double-layer agar method. This is a straightforward methodology that can be applied for the detection, enumeration and isolation of bacteriophages for other anaerobic bacteria, using an appropriate host strain and culture conditions. In the case of bacteriophages of Bacteroides the results can be obtained in less than 24 h, although the time could vary depending on the growth rate of the host strain.

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Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis

Cited by 29 publications

References 7 publications

Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

Removal and inactivation of indicator bacteriophages in fresh waters

Isolation of Bacteriophages of the Anaerobic Bacteria Bacteroides

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