CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids.

Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.

Transmissible drug resistance among E. coli strains isolated from pig and human specimens

Resistance factors, the transmissible genetic elements which confer drug resistance on members of the Enterobacteriaceae, are frequently found in isolates from human specimens. It was suggested that farm animals which receive antibiotics might harbour these R factors and infect the human community by way of the farm workers who handle them. In this study, a total of 252 faecal specimens were collected - 152 from pigs and employees on a farm using antibiotics, and 100 from pigs and employees on control farms using no antibiotics. An E. coli culture was selected from each specimen and examined for resistance to: Ampicillin, Sulphonamide, Streptomysin, Chloramphenicol, Tetracycline, Nalidixic Acid. Resistance cultures were further examined for the ability to transfer drug resistance to sensitive bacteria. 1. 110 faecal specimens were collected from the test pigs which had been given antibiotics. All the specimens yielded multiply resistance E. coli of which 94 (86 percent) were able to transfer all or part of their resistance pattern to a suitable sensitive recipient. 2. 42 humans in contact with the test pigs were examined in the same manner. 86 percent of the isoaltes were resistant to one or more of the drugs and 48 percent transferred drug resistance. 3. Specimens were collected from 72 pigs in the control group. 68 (94 percent) contained drug resistant E. coli of which 16 (22 percent) transferred their resistance. 4. 20 out of 28 humans (i.e. 72 percent) in the control group yielded resistant E. coli and six (21 percent) transferred their resistance. The results indicate that: I. Those pigs exposed to antibiotics harboured coliforms with R factors more frequently than pigs not exposed to antibiotics. II. The control human group also had a lower occurrence of transmissible drug resistance than the group who worked with the antibiotic-treated pigs. III. The relatives of the farm employees excreted more transmissible drug resistant strain (43 percent) than the human control group (21 percent). IV. The highest incidence of transmissible drug resistant strains was among individuals who were taking antibiotics at the same time the specimens were collected. Pigs which had not been given antibiotics for some months showed a lower yield of transmissible drug resistant strains than those currently taking antibiotics at the time the specimens were collected. V. The humans from the test farm, although they were not currently taking antibiotics, yielded drug resistant strains at a rate comparable to the test pigs. However, only 48 percent of these transferred drug resistance, compared with 86 percent of the test pigs. VI. Drug resistant bacteria were found in the majority of specimens from all groups, but the ability to transfer these resistances varied widely (Summary)