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).     

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.     

Diverse Tetracycline Resistance Genotypes of Megasphaera elsdenii Strains Selectively Cultured from Swine Feces

A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 μg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.     

Tetracycline resistance genes acquired at birth

Newborns acquire their first microbiota at birth. Maternal vaginal or skin bacteria colonize newborns delivered vaginally or by C-section, respectively (Dominguez-Bello et al. 2010 #884). We aimed to determine differences in the presence of four tetracycline (tet) resistance genes, in the microbes of ten newborns and in the mouth and vagina of their mothers, at the time of birth. DNA was amplified by PCR with primers specific for [tet(M), tet(O), tet(Q), and tet(W)]. Maternal vaginas harbored all four tet resistance genes, but most commonly tet(M) and tet(O) (63 and 38 %, respectively). Genes coding for tet resistance differed by birth mode, with 50 % of vaginally delivered babies had tet(M) and tet(O) and 16 and 13 % of infants born by C-section had tet(O) and tet(W), respectively. Newborns acquire antibiotic resistance genes at birth, and the resistance gene profile varies by mode of delivery.     

Quorum Sensing and Production of Autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica Serovar Typhimurium in Foods

Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that Campylobacter jejuni and Campylobacter coli possessed luxS, which is responsible for autoinducer-2 (AI-2) production. Using a Vibrio harveyi luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by C. coli, C. jejuni, Salmonella enterica serovar Typhimurium, and Escherichia coli O157:H7 under different conditions.     

Conjugative chromosomal gene transfer in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet ^r ) and lincomycin (lin ^r ) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative p19cat plasmid producing a hybrid plasmid. Insertion frequency was 10^?4?10^?5. Then genes tet ^r and lin ^r were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F′ and R′ plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Occurrence of Tetracycline Resistance Genes in Aquaculture Facilities with Varying Use of Oxytetracycline

The contribution of human activities to environmental reservoirs of antibiotic resistance is poorly understood. The purpose of this study was to determine if oxytetracycline (OTC) use in aquaculture facilities increased the detection frequency (i.e., prevalence) of tetracycline resistance (tet^R) genes relative to facilities with no recent OTC treatment. We used polymerase chain reaction to screen water and sediment from four noncommercial fish farms in northwestern Wisconsin for the presence of ten tet^R determinants: tet(A), tet(B), tet(D), tet(E), tet(G), tet(M), tet(O), tet(Q), tet(S), and tet(W). Water from farms with recent OTC use had significantly higher tet^R detection frequencies than did water from farms without recent OTC use, with prevalence in raceways and rearing ponds of farms with recent OTC use exceeding by more than twofold that of farms not using OTC. Effluent from all farms, regardless of treatment regime, had higher tet^R detection frequencies than their corresponding influent for all genes, but the specific combinations of tet^R genes detected in a sample were not different from their corresponding influent. Although OTC use was associated with the increased occurrence and diversity of tet^R genes in water samples, it was not found to relate to tet^R gene occurrence in sediment samples. Sediment samples from facilities with no recent OTC use had significantly higher frequencies of tet^R gene detection than did samples from facilities with recent OTC use. All of the tet^R genes were detected in both the medicated and nonmedicated feed samples analyzed in this study. These findings suggest that both OTC treatment in aquaculture facilities and the farms themselves may be sources of tet^R gene introduction to the environment. To our knowledge, this is the first study to use genotypic and cultivation-independent methods to examine tet^R gene occurrence associated with OTC use in aquaculture.     

Transposition of IS1181 in the genomes of Staphylococcus and Listeria

The recombinant plasmid pIP1713 was constructed to analyse the transpositional activity of the insertion sequence IS1181 in Staphylococcus aureus RN4220, Staphylococcus carnosus TM300 and Listeria monocytogenes EGD. This 11.3-kb plasmid contains two genetically different elements: (i) a pE194ts-derived replicon, the ermC gene of which confers resistance to erythromycin in Gram-positive bacteria of several species, and (ii) a copy of IS1181, cloned from S. aureus BM3121, in which the tetracycline resistance gene, tet(T), has been inserted between the transposase-encoded gene and the downstream inverted repeat. When introduced by electroporation into the three bacterial hosts, pIP1713 delivered IS1181Ωtet(T) to various chromosomal sites. Cointegrate structures between pIP1713 and the host chromosome were occasionally detected. Transposition was associated with 8-bp repeats at the insertion sites. IS1181Ωtet(T) could be used for random mutagenesis in Gram-positive bacteria.     

Effects of Ceftiofur and Chlortetracycline Treatment Strategies on Antimicrobial Susceptibility and on tet(A), tet(B), and bla CMY-2 Resistance Genes among E. coli Isolated from the Feces of Feedlot Cattle

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla _CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (n = 289, median = 6; 95% CI = 4–8) compared with the tet(B) gene (n = 208, median = 3; 95% CI = 3–4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.     

Seasonal Variability in Airborne Biotic Contaminants in Swine Confinement Buildings

Little is known about the seasonal dynamics of biotic contaminants in swine confinement buildings (SCBs). The biotic contaminants of seven SCBs were monitored during one visit in the winter and one during the summer. Paired-end Illumina sequencing of the 16S rRNA gene, V3 region, was used to examine seasonal shifts in bacterial community composition and diversity. The abundances of 16S rRNA genes and six tetracycline resistance genes (tetB, tetH, tetZ, tetO, tetQ, and tetW) were also quantified using real-time PCR. Bacterial abundances, community composition and diversity all showed strong seasonal patterns defined by winter peaks in abundance and diversity. Microclimatic variables of SCBs, particularly air speed, PM2.5 and total suspended particles (TSP) were found significantly correlated to abundances, community composition, and diversity of bacterial bioaerosols. Seasonal fluctuations were also observed for four tetracycline resistance genes, tetH, tetO, tetQ, and tetW. The frequency of occurrences of these resistance genes were significantly higher in samples collected during winter and was also significantly correlated with air speed, PM2.5 and TSP. Overall, our results indicate that biotic contaminants in SCBs exhibit seasonal trends, and these could be associated with the microclimatic variables of SCBs. The correlations established in the current study could be helpful in establishing better management strategies to minimize the potential health impacts on both livestock and humans working in this environment.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc^r) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc^r genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc^r genes was quantified by real-time quantitative PCR. To confirm the Tc^r gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc^r genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc^r genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.     

Escherichia coli Clone Sonnei (Shigella sonnei) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.     

Antibiotic resistance in commensal intestinal microflora

The susceptibility toward antibiotics was determined by disc and MIC methods in Lactobacillus and, for comparison, in Escherichia coli strains isolated from cloacal swabs of broiler chickens derived from various farms in Slovakia. The occurrence of acquired tetracycline resistance in E. coli and lactobacilli isolated from the same sample was similar. The presence of tet(M), tet(S), tet(L) and ermB genes was demonstrated in lactobacilli while the tet(M) gene was not detected in E. coli.     

Antimicrobial resistance,virulence genes and PFGE-profiling of Escherichia coli isolates from South Korean cattle farms

To estimate the prevalence of Escherichia coli with potential pathogenicity in cattle farm in South Korea, a total of 290 E. coli isolates were isolated from cattle farms over a period of 2 years in South Korea. These were examined for phenotypic and genotypic characteristics including antimicrobial susceptibility, serotype, and gene profiles of virulence and antimicrobial resistance. The most dominant virulence gene was f17 (26.2%), followed by stx2 (15.9%), ehxA (11.0%), stx1 (8.3%), eae (5.2%), and sta (4.1%). Some shiga-toxin producing E. coli isolates possessed eae (15.9%). All isolates except for one showed resistance to one or more antimicrobials, with 152 isolates exhibiting multidrug-resistance. The most prevalent resistance phenotype detected was streptomycin (63.1%), followed by tetracycline (54.5%), neomycin (40.3%), cephalothin (32.8%), amoxicillin (30.0%), ampicillin (29.7%), and sulphamethoxazole/trimethoprim (16.6%). The associated resistance determinants detected were strA-strB (39.0%), tet(E) (80.0%), tet(A) (27.6%), aac(3)-IV (33.1%), aphA1 (21.4%), bla _TEM (23.8%), and sul2 (22.1%). When investigated by O serotyping and PFGE molecular subtyping, the high degree of diversity was exhibited in E. coli isolates. These results suggest that E. coli isolates from South Korean cattle farms are significantly diverse in terms of virulence and antimicrobial resistance. In conclusion, the gastroinstestinal flora of cattle could be a significant reservoir of diverse virulence and antimicrobial resistance determinants, which is potentially hazardous to public health.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Quantification of Tetracycline Resistance Genes in Feedlot Lagoons by Real-Time PCR

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tc^r) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tc^r gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tc^r genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log₁₀ of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r² = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 μg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tc^r genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tc^r gene numbers in environmental and other samples.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.