The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species

The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51–53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.

ANTIBIOFILM FORMATION ACTIVITY, RESISTANT GENES PROFILING AND DETECTION OF VIRULENCE FACTORS OF TOXIGENIC Vibrio cholerae ISOLATES FROM KISUMU COUNTY, KENYA

ABSTRACTIntroductionVibrio cholerae can switch between motile and biofilm lifestyles with some of its strains forming biofilms in addition to production of various virulence traits and possessing antimicrobial resistance traits. This study is aim to show antibiofilm formation activity, resistant genes profiling and detection of virulence factors of toxigenic vibrio cholerae isolates from Kisumu County.MethodologyA total of 119 Vibrio cholerae O1, biotype El Tor isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on TCBS and PCR assay carried out using standard procedures. Biofilm assay tests and detection of virulence factors were also done by use of standard procedures.ResultsOf the 101 confirmed vibrio cholerae isolates, 80.2% possessed the cholera toxin gene (ctxA) whereas 19.8% did not. Analysis of the toxR gene revealed that 98.0% harbored the toxR gene and only 2.0% did not. It was also revealed that 80.2% harbored the class I integron (inDS gene) while 19.8% did not, 93.1% were confirmed to possess the SXT integrating conjugative element (ICE) while 7.0% did not. The tetracycline resistance gene was present in 96.0% of the isolates. In 7 isolants strains which were resistance to common used antibiotics were screened for biofilm formation. Three of the strains (04/17-07, 06/17-14, and 05/17-03) failed to form biofilm while four strains namely 03/17-16, 02/17-09, 04/17-13 and P. aeruginosa ATCC 10145 as a positive control formed biofilms. In addition, out of those 7 isolants 71.42% produced protease, 85.71% produced phospholipases, 71.42% of isolates has the ability to produce lipase and 100% were able to produce the haemolysin.ConclusionAn understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

Antibiotic resistance features in Klebsiella pneumoniae and Escherichia coli strains isolated from hospital and community acquired urinary tract infections

A total number of 35 strains (n=23 of K. pneumoniae and n=12 of E.coli) were isolated in May 2017 from patients with UTI, hospitalized in the National Institute for Cardiovascular Diseases Prof. C.C. Iliescu and from community infections (CA) diagnosed in Central Reference Synevo-Medicover Laboratory from Bucharest. The hospital strains were identified by BD Phoenix and the CA ones by mass spectrometry using MALDI Biotyper. The antibiotic susceptibility was determined by agar disk diffusion (CLSI, 2017) and automated methods (BD Phoenix and Vitek II system). For molecular characterization, all strains were analyzed be using PCR amplification. The investigated strains revealed the presence of tetracycline resistance gene, i.e. tet(A) (67% in E. coli and 45% of K. pneumoniae strains), tet(D) (8% of E. coli and 5% of K. pneumoniae strains), carbapenemase genes (blaOXA-48 in 40% of the K. pneumoniae strains); blaTEM (25% of E. coli strains and 10% of K. pneumoniae strains).

Identification of a novel tetracycline resistance gene, tet(63), located on a multiresistance plasmid from Staphylococcus aureus

Objectives To identify and characterize a novel tetracycline resistance gene on a multiresistance plasmid from Staphylococcus aureus SA01 of chicken origin. Methods MICs were determined by broth microdilution according to CLSI recommendations. The whole genome sequence of S. aureus SA01 was determined via Illumina HiSeq and Oxford Nanopore platforms followed by a hybrid assembly. The new tet gene was cloned and expressed in S. aureus. The functionality of the corresponding protein as an efflux pump was tested by efflux pump inhibition assays. Results A novel tetracycline resistance gene, tet(63), was identified on a plasmid in S. aureus SA01. The cloned tet(63) gene was functionally expressed in S. aureus and shown to confer resistance to tetracycline and doxycycline, and a slightly elevated MIC of minocycline. The tet(63) gene encodes a 459 amino acid efflux protein of the major facilitator superfamily that consists of 14 predicted transmembrane helices. The results of efflux pump inhibitor assays confirmed the function of Tet(63) as an efflux protein. The deduced amino acid sequence of the Tet(63) protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). The plasmid pSA01-tet, on which tet(63) was located, had a size of 25664 bp and also carried the resistance genes aadD, aacA-aphD and erm(C). Conclusions A novel tetracycline resistance gene, tet(63), was identified in S. aureus. Its location on a multiresistance plasmid might support the co-selection of tet(63) under the selective pressure imposed by the use of macrolides, lincosamides and aminoglycosides.

Early Inoculation of Microbial Suspension in Suckling Piglets Affects the Transmission of Maternal Microbiota and the Associated Antibiotic Resistance Genes

Antibiotic resistance of microbes thriving in the animal gut is a growing concern for public health as it may serve as a hidden reservoir for antibiotic resistance genes (ARGs). We compared 16 control piglets to 24 piglets fed for 3 weeks with S1 or S2 fecal suspensions from two sows that were not exposed to antibiotics for at least 6 months: the first suspension decreased the erythromycin resistance gene ermB and the aminoglycoside phosphotransferase gene conferring resistance to kanamycine (aphA3), while the second decreased the tetracycline resistance gene tetL, with an unexpected increase in ARGs. Using 16S RNA sequencing, we identified microbial species that are likely to carry ARGs, such as the lincosamide nucleotidyltransferase lnuB, the cephalosporinase cepA, and the tetracycline resistance genes tetG and tetM, as well as microbes that never co-exist with the tetracycline resistance gene tetQ, the erythromycin resistance gene ermG and aphA3. Since 73% of the microbes detected in the sows were not detected in the piglets at weaning, a neutral model was applied to estimate whether a microbial species is more important than chance would predict. This model confirmed that force-feeding modifies the dynamics of gut colonization. In conclusion, early inoculation of gut microbes is an interesting possibility to stimulate gut microbiota towards a desirable state in pig production, but more work is needed to be able to predict which communities should be used.