Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.     

Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression.

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.     

Homology among tet determinants in conjugative elements of streptococci.

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes.     

Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.     

Anaerobic central metabolic pathways active during polyhydroxyalkanoate production in uncultured cluster 1 Defluviicoccus enriched in activated sludge communities

We report the isolation and characterization of an unusual strain of Streptococcus salivarius , 3C30, displaying both the macrolide–lincosamide–streptogramin B and the tetracycline resistance phenotypes. It harbours the mef (E), erm (B), and tet (M) genes carried by different genetic elements. The genetic element carrying mef (E), named mega, was investigated by long PCR and sequencing, while the presence of the Tn3872-like element, carrying tet (M) and erm (B), was demonstrated by sequencing of both the int-xis-Tn and the fragment between the two resistance genes. In strain 3C30 the mega element is 5388 bp in size and its nucleotide sequence is identical to that of the element described previously in S. salivarius , with the exception of a 912 bp deletion at the left end. The composite Tn3872-like element appeared to be nonconjugative while the mega element was transferred by conjugation to Streptococcus pneumoniae . It was, however, impossible to transfer it again from these transconjugants to other strains. In addition, only in the 3C30 strain did mega form circular structures, as identified by real-time PCR. In conclusion, we found a clinical strain of S. salivarius carrying both mega and Tn3872-like genetic elements. Mega is transferable by conjugation to S. pneumoniae but it is not transferable again from the transconjugants, suggesting a possible mobilization by recombinases of the coresident Tn3872-like transposon.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Tetracycline resistance mediated by tet(W), tet(M), and tet(O) genes of Bifidobacterium isolates from humans

MICs of tetracyclines were determined for 86 human Bifidobacterium isolates and three environmental strains. The tet(O) gene was found to be absent in these isolates. tet(W) and tet(M) were found in 26 and 7%, respectively, of the Bifidobacterium isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of tet(W) and/or tet(M).     

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet (X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet (X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet (X) gene.
Methods and Results:  Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet (X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet (X) gene revealed a mobilizable transposon-like element (Tn 6031 ) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet (X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet (X) gene using three different recipient strains and numerous experimental conditions.
Conclusions:  This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet (X) gene.
Significance and Impact of the Study:  This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.     

Nucleotide sequence analysis of the class G tetracycline resistance determinant from Vibrio anguillarum.

The nucleotide sequence of the class G tetracycline resistance determinant previously isolated from Vibrio anguillarum has been determined. Two open reading frames of divergent polarity were identified. A resistance gene (tet A) encodes a protein of 393 amino acid residues (deduced molecular mass of 40.9 kDa), and a repressor gene (tet R) encodes a protein consisting of 210 amino acids with a calculated molecular mass of 23.6 kDa. Based on the deduced amino acid sequences, the proteins of tet A(G) and tet R(G) are about 60% homologous with those of RP1/Tn1721 (class A) and pSC101/pBR322 (class C), and about 50% homologous with Tn10 (class B). The relationship of the tet (G) sequence to five known tetracycline resistance determinants (class A to E) is discussed.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

Detection of a common and persistent tet(L)-carrying plasmid in chicken-waste-impacted farm soil

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tc(r)) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tc(r) soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period.     

Incidence of antibiotic resistance and virulence determinants among Enterococcus italicus isolates from dairy products

The main objective of this work was to investigate the biosafety of Enterococcus italicus, a recently described enterococcal species widely diffused in dairy products. For this purpose, the antibiotic susceptibility and the incidence of virulence factors among 30 E. italicus isolates originating mainly from different Italian cheeses were tested. Although not all 30 isolates showed unique genotypes, PCR fingerprinting evidenced a notable genotypic diversity among the E. italicus collection under study. All isolates were susceptible to vancomycin, gentamicin, erythromycin, ampicillin, chloramphenicol and bacitracin. Five isolates corresponding to three unique genotypes exhibited phenotypic resistance to tetracycline with MICs ranging from 64-256mug/ml. By PCR-based detection, the genetic basis of the Tet(R) phenotype in these strains was linked to the tet(S) gene whereas detection of tet(L) and tet(M) genes and the integrase element int of the Tn916/Tn1545 family of transposons were negative. Likewise, none of the strains appeared to contain any of the tested virulence genes gelE, asaI, cpd, agg, cylA, cylB, cylM, ace and hyl(Efm). The results of this study warrant further research into the environmental dissemination of Tet(R)E. italicus and into the potential transferability of its tet(S) genes.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

Identification of a truncated, but functionally active tet(H) tetracycline resistance gene in Pasteurella aerogenes and Pasteurella multocida

Molecular analysis of Pasteurella isolates of animal origin for plasmid-encoded tetracycline resistance genes identified a common tet(H)-carrying plasmid of 5.5 kbp in a single isolate of Pasteurella aerogenes and six isolates of Pasteurella multocida. This plasmid carried a truncated Tn5706 element in which one of the IS elements, IS1596, was lost completely and of the other, IS1597, only a relic of 84 bp was left. Sequencing of the resistance gene region and the flanking areas revealed the presence of a deletion in the 3' end of the tet(H) gene which shortened the tet(H) reading frame by 24 bp. The amino acid sequence of the respective TetH protein comprised only 392 amino acids. Despite this deletion, the tet(H) gene conferred high level tetracycline resistance not only to the original Pasteurella isolates but also to the respective Escherichia coli JM107 and C600 transformants as confirmed by MIC determination. The deletion was probably the result from recombinational events. Two possible recombination sites involved in the deletion of tet(H) and that of IS1597 were identified. Macrorestriction analysis of the Pasteurella isolates carrying plasmid pPAT1 confirmed horizontal and vertical transfer of this plasmid.     

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens . Donor strains were postulated to carry two Tc^R genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet (O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible Tc^R gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet (W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet (W) genes with almost identical sequences are also shown to be present in Tc^R strains of B. fibrisolvens from Australian sheep and in Tc^R strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus . This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet (W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus .