Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938

ABSTRACT The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of β-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known β-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The β-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

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In vitro evalution of antibiotic resistance of Lactobacillus bulgaricus strains isolated from traditional dairy products

Czech Journal of Food Sciences ◽

10.17221/136/2018-cjfs ◽

2019 ◽

Vol 37 (No. 1) ◽

pp. 36-43

Author(s):

Huiling Guo ◽

Bilige Menghe

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Fermented Milk ◽

Lactobacillus Bulgaricus ◽

Dilution Method ◽

Resistance Transfer ◽

Traditional Dairy Products

The antimicrobial susceptibility of 20 Lb. bulgaricus isolates from traditional fermented milk-originated was assessed and then determined the ability to transfer antibiotic resistance genes to other bacteria. The minimum inhibitory concentration of each strain was determined using a standardized dilution method. All the tested strains were found to be susceptible to gentamicin, erythromycin, clindamycin, neomycin, tetracycline, linezolid, chloramphenicol, rifampicin, and quinupristin/dalfopristin, while their susceptibilities to kanamycin, ciprofloxacin, streptomycin, trimethoprim, ampicillin, and vancomycin varied. Polymerase chain reaction (PCR) was used to check whether specific antibiotic resistance genes were present in these Lb. bulgaricus. We detected the rpoB, erm(B), aadA, bla, cat and vanX. Finally, a filter mating assay was applied to investigate the transferability of these resistance markers; and we observe no antibiotic resistance transfer between bacteria. This work demonstrates a low risk of lateral transfer of the antibiotic resistance gene of Lb. bulgaricus.

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Metagenomics Analysis Reveals the Microbial Communities, Antimicrobial Resistance Gene Diversity and Potential Pathogen Transmission Risk of Two Different Landfills in China

Diversity ◽

10.3390/d13060230 ◽

2021 ◽

Vol 13 (6) ◽

pp. 230

Author(s):

Shan Wan ◽

Min Xia ◽

Jie Tao ◽

Yanjun Pang ◽

Fugen Yu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Microbial Communities ◽

Resistance Gene ◽

Resistance Genes ◽

Gene Diversity ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Pathogen Transmission ◽

Transmission Risk ◽

Antimicrobial Resistance Gene

In this study, we used a metagenomic approach to analyze microbial communities, antibiotic resistance gene diversity, and human pathogenic bacterium composition in two typical landfills in China. Results showed that the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in the two landfills, and archaea and fungi were also detected. The genera Methanoculleus, Lysobacter, and Pseudomonas were predominantly present in all samples. sul2, sul1, tetX, and adeF were the four most abundant antibiotic resistance genes. Sixty-nine bacterial pathogens were identified from the two landfills, with Klebsiella pneumoniae, Bordetella pertussis, Pseudomonas aeruginosa, and Bacillus cereus as the major pathogenic microorganisms, indicating the existence of potential environmental risk in landfills. In addition, KEGG pathway analysis indicated the presence of antibiotic resistance genes typically associated with human antibiotic resistance bacterial strains. These results provide insights into the risk of pathogens in landfills, which is important for controlling the potential secondary transmission of pathogens and reducing workers’ health risk during landfill excavation.

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Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.730046 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cui Li ◽

Yulong Wang ◽

Yufeng Gao ◽

Chao Li ◽

Boheng Ma ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Direct Repeat ◽

Epidemiological Data ◽

Multidrug Resistant ◽

Typing Method ◽

Crispr Locus ◽

Research Areas

Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (blaTEM, blaOXA, and blaCTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.

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Genome Assessment of Antibiotic Resistance Genes in Probiotic Bacteria and a Literature Review

10.21203/rs.3.rs-254514/v1 ◽

2021 ◽

Author(s):

Mehdi Fatahi-Bafghi ◽

Sara Naseri ◽

Ali Alizehi

Keyword(s):

Antibiotic Resistance ◽

Genome Sequence ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Antibiotic Resistance Genes ◽

Probiotic Bacteria ◽

Whole Genome Sequence ◽

Probiotic Properties ◽

Antibiotic Resistant ◽

To Come

Abstract Having various clinical applications, probiotic bacteria are currently used in the diet. There are reports of antibiotic resistance genes (ARGs) in these bacteria that can be transferred to other microflora and pathogenic bacteria. The aim of the study is to examine whole-genome sequence analysis in bacteria with probiotic properties. Moreover, this study follows existing issues about the importance and presence of ARGs in these bacteria the dangers of which may affect human health in the years to come. In the present study, 126 complete probiotic bacterial genomes were collected and analysed for ARGs. The results of the study shows there are various antibiotic resistant genes of in these bacteria some of which can be transmitted to other bacteria. We propose microorganisms be applied as a probiotic element in various types of products, antibiogram be conducted for a large number of antibiotics and analysis of complete genome sequence for ARGs prediction.

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Characterization of antibiotic resistance genes in the species of the rumen microbiota

Nature Communications ◽

10.1038/s41467-019-13118-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Yasmin Neves Vieira Sabino ◽

Mateus Ferreira Santana ◽

Linda Boniface Oyama ◽

Fernanda Godoy Santos ◽

Ana Júlia Silva Moreira ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Animal Health ◽

Antibiotic Resistance Genes ◽

Tetracycline Resistance ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Ruminal Bacteria ◽

Genes Encoding

AbstractInfections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

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Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo

Applied and Environmental Microbiology ◽

10.1128/aem.00470-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6352-6360 ◽

Cited By ~ 25

Author(s):

Joanna Boguslawska ◽

Joanna Zycka-Krzesinska ◽

Andrea Wilcks ◽

Jacek Bardowski

Keyword(s):

Antibiotic Resistance ◽

Lactococcus Lactis ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Tetracycline Resistance ◽

Raw Milk ◽

M Gene ◽

Transfer Resistance

ABSTRACT Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10−5 to 10−7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.

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Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.184.15.4259-4269.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4259-4269 ◽

Cited By ~ 172

Author(s):

John W. Beaber ◽

Bianca Hochhut ◽

Matthew K. Waldor

Keyword(s):

Antibiotic Resistance ◽

Vibrio Cholerae ◽

Resistance Genes ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Amino Acid Sequences ◽

Conjugation System ◽

Conjugative Transposons ◽

The One ◽

Functional Analyses

ABSTRACT SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian Vibrio cholerae. We have determined the 100-kb DNA sequence of SXT. This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources. We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision. SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid. More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility. Two SXT loci, designated setC and setD, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer. Another locus, designated setR, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.

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Molecular Analysis of Antibiotic Resistance Gene Clusters in Vibrio cholerae O139 and O1 SXT Constins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.11.2991-3000.2001 ◽

2001 ◽

Vol 45 (11) ◽

pp. 2991-3000 ◽

Cited By ~ 210

Author(s):

Bianca Hochhut ◽

Yasmin Lotfi ◽

Didier Mazel ◽

Shah M. Faruque ◽

Roger Woodgate ◽

...

Keyword(s):

Antibiotic Resistance ◽

Vibrio Cholerae ◽

Resistance Gene ◽

Resistance Genes ◽

Dna Sequences ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Gene Clusters ◽

Vibrio Cholerae O139 ◽

El Tor

ABSTRACT Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXTMO10, these genes are clustered within a composite transposon-like structure found near the element's 5′ end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXTET, does not contain the same resistance genes as SXTMO10. In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

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Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

10.1101/678573 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marinelle Rodrigues ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Kelli L. Palmer ◽

Breck A. Duerkop

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Conjugative Plasmid ◽

Antibiotic Resistant ◽

Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

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Longitudinal assessment of antibiotic resistance gene profiles from the infant gut microbiome

10.21203/rs.2.18486/v1 ◽

2019 ◽

Author(s):

Evelyn Loo ◽

Amanda Zain ◽

Gaik Chin Yap ◽

Rikky W Purbojati ◽

Daniela I Drautz-Moses ◽

...

Keyword(s):

Antibiotic Resistance ◽

Cohort Study ◽

Resistance Gene ◽

Resistance Genes ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Longitudinal Cohort Study ◽

First Year ◽

Beta Lactam ◽

First Year Of Life

Abstract Background: The rapid spread of multidrug- resistant pathogenic bacteria is a worldwide public health concern. Given the high carriage rate of extended spectrum beta-lactamase (ESBL)- producing Enterobacteriaceae in Asia, we aimed to evaluate community prevalence and dynamics by studying the longitudinal changes in antibiotic resistance gene (ARG) profiles and prevalence of ESBL-producing E coli and K. pneumoniae in the intestinal microbiome of infants participating in the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) study, a longitudinal cohort study of pregnant women and their infants. Methods: We analysed the antibiotic resistance genes profile in the first year of life among 75 infants who had stool samples collected at multiple timepoints using metagenomics. Results: The mean number of ARGs per infant increased with age. The most common ARGs identified confer resistance to aminoglycoside, beta-lactam, macrolide and tetracycline antibiotics; all infants harboured these antibiotic resistance genes at some point in the first year of life. Few ARGs persisted throughout the first year of life. Beta-lactam resistant Escherichia coli and Klebsiella pneumoniae were detected in 4 (5.3%) and 32 (42.7%) of subjects respectively. Conclusion: In this longitudinal cohort study of healthy infants living in a region with high endemic antibacterial resistance, we demonstrate that majority of the infants harboured a number of antibiotic resistance genes in their gut and showed that the infant gut resistome is diverse and dynamic over the first year of life.

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