E. U. Poluektova

Antibiotic resistance of potential probiotic bacteria of the genus Lactobacillus from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.

Conjugative transfer of the large plasmid p19 in various Bacillus subtilis strains

Cryptic conjugative plasmid p19 from the environmental Bacillus subtilis strain 19 was labeled with the cat gene conferring resistance to chloramphenicol. The resulting plasmid, p19cat, was used to estimate the transfer frequency, to study the dynamics of plasmid transfer, and to detect some specific features of conjugation between various B. subtilis strains.

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.

Molecular Analysis of Some Genes from Plasmid p19 of the Bacillus subtilis 19 Soil Strain Involved in Conjugation

Two fragments of conjugative plasmid p19 (95 kb) from the Bacillus subtilis 19 soil strain were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1–ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the B. subtilis 72 soil strain and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.

Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the East European Plain

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.

The structure of the transposable genetic element ISBsu2 from the cryptic plasmid p1516 of a soil Bacillus subtilis strain and the presence of homologues of this element in the chromosomes of various Bacillus subtilis strains

A cryptic plasmid from a soil strain of Bacillus subtiliswas found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.

[Soil strain of Bacillus subtilis harboring a large plasmid that mediates high-frequency conjugal mobilization].

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.

Characterization of the transfer-related tra region of the conjugative plasmid p19 from a Bacillus subtilis soil strain

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.

The Presence of the Transposable Element ISBsu2 from a Cryptic Plasmid in Chromosomes of Some Bacillus subtilis Strains

No transposable elements had been found before this study in the typical laboratory strain 168 of Bacillus subtilis , whose complete nucleotide sequence has been determined. In this study, we describe the IS-element ISBsu2 discovered in the plasmid of B. subtilis soil strain 1516. It has been shown that this sequence or its homologs are present in the chromosomes of some strains of this bacterium. These data are the first example of the presence of the same IS-element both in the plasmid and in the chromosomes of some B. subtilis strains. Approximately 30‐40% of B. subtilis strains and related bacilli isolated from soil and other sources contain small plasmids (6‐10 kb long). As a rule, these are cryptic plasmids: they do not contain antibiotic resistance genes and may be detected only on the electrophoregrams of bacterial lysates [1‐3]. When analyzing numerous soil strains of B. subtilis , we found a strain (named B. subtilis 1516) that contained two plasmids of different lengths. After plating this strain to obtain separate colonies, approximately half of them carried an 11.3-kb plasmid, p1516L, and the others carried a smaller (9.9-kb) plasmid, p1516S. We discovered that one of these colonies contained both plasmids. We assumed that this colony consisted of mixed-type cells or its cells carried both plasmids. The treatment of DNA of these plasmids with various restriction endonucleases showed that their structures were similar. The only difference was that the p1516L plasmid contained a 1.4-kb fragment [4]. We assumed that the characteristics of plasmid composition of this B. subtilis strain were determined by a transposition of a transposableelement-like DNA fragment. Indeed, the restriction maps of both plasmids showed that p1516L contained an insert that was absent in p1516S. We determined the nucleotide sequences of this insert and plasmid DNA regions flanking the insert. The length of this insert was 1383 bp. At the boundary between the insert and the plasmid DNA, we detected direct repeats approximately 7 bp long (GTCCTTG). One of these repeats was contained in the insert; the other, in the plasmid DNA. We also detected two pairs of completely inverted 10-member repeats (ACGGTTCCTT‐AAGGAACCGT and TGGGGGAAAT‐ ATTTCCCCCA) located closer to the center of the insert. Using the BLAST software [5], we determined that the amino acid sequence corresponding to the nucleotide sequence of the insert was homologous to transposases of some microorganisms. The greatest homology was exhibited by the hypothetic transposases from Yersinia enterocolitica and Escherichia coli (approximately 40%) and transposase IS1554 from Mycobacterium tuberculosis (33%). In addition, unidentified fragments of Enterococcus faecalis and Enterococcus faecium genomes also shared much homology (up to 70%) with the insert. However, the insert did not carry a whole reading frame, because we detected multiple translational frameshifts. Thus, the structure of this fragment of the p1516L plasmid is typical of IS-elements (the presence of direct and inverted repeats at the boundaries and homology of the central region with some transposases of microorganisms). However, it is most likely that the gene encoding transposase is currently functionally inactive, i.e., it is a pseudogene. We named this novel IS sequence ISBsu2 and deposited it in the GenBank database (accession number AY099458).