Application of Genomic Epidemiology of Pathogens to Farmed Yellowtail Fish Mycobacteriosis in Kyushu, Japan.

To investigate mycobacterial cases of farmed yellowtail fish in coastal areas of western Japan (Kagoshima, Kyushu), where aquaculture fisheries are active, Mycobacterium pseudoshottsii, the causative agent, was isolated from six neighboring fishing ports in 2012 and 2013. A phylogenetic ana-lysis revealed that the strains isolated from one fishing port were closely related to those isolated from other regions of Japan, suggesting the nationwide spread of a single strain. However, strains from Japan were phylogenetically distinct from those from the Mediterranean and the United States; therefore, worldwide transmission was not observed based on the limited data obtained on the strains exami-ned in this study. The present results demonstrate that a bacterial genomic ana-lysis of infected cases, a mole-cular epidemiology strategy for public health, provides useful data for estimating the prevalence and transmission pathways of M. pseudoshottsii in farmed fish. A bacterial genome ana-lysis of strains, such as that performed herein, may play an important role in monitoring the prevalence of this pathogen in fish farms and possible epidemics in the future as a result of international traffic, logistics, and trade in fisheries.

Mobile class A ß-lactamase gene bla GMA-1.

IMPORTANCE: Despite increasing reports, class A ß-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family ß-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A ß-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in ß-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.

Retrospective identification of pathogenic mycobacterial species in fish: Mycobacterium pseudoshottsii YM-3, isolated from a yellowtail fish in 1986 in Kochi, Japan.

The complete genome sequence of mycobacterial strain YM-3, isolated from cultured yellowtail in 1986, was determined. The strain was Mycobacterium pseudoshottsii, a closely related subspecies of Mycobacterium marinum, so the strain was isolated earlier than the first report of the subspecies in 2005.

Host range of strand-biased circularizing integrative elements: a new class of mobile DNA elements nesting in Gammaproteobacteria.

BACKGROUND: The strand-biased circularizing integrative elements (SEs) are putatively non-mobilizable integrative elements for transmitting antimicrobial resistance genes. The transposition mode and the prevalence of SEs in prokaryotes remain vague. RESULTS: To corroborate the transposition mode and the prevalence of SEs, hypothetical transposition intermediates of an SE were searched for in genomic DNA fractions of an SE host. Then, the SE core genes were defined based on gene knockout experiments, and the synteny blocks of their distant homologs were searched for in the RefSeq complete genome sequence database using PSI-BLAST. A genomic DNA fractionation experiment revealed that SE copies are present in a double-stranded nicked circular form in vivo. Operonic structure of three conserved coding sequences (intA, tfp, intB) and srap located at the left end of SEs were identified as essential for attL × attR recombination. The synteny blocks of tfp and srap homologs were detected in 3.6% of the replicons of Gammaproteobacteria but not in other taxa, implying that SE movement is host-dependent. SEs have been discovered most frequently in the orders Vibrionales (19% of replicons), Pseudomonadales (18%), Alteromonadales (17%), and Aeromonadales (12%). Genomic comparisons revealed 35 new SE members with identifiable termini. SEs are present at 1 to 2 copies per replicon and have a median length of 15.7 kb. Three newly identified SE members carry antimicrobial resistance genes, like tmexCD-toprJ, mcr-9, and blaGMA-1. Further experiments validated that three new SE members possess the strand-biased attL × attR recombination activity. CONCLUSIONS: This study suggested that transposition intermediates of SEs are double-stranded circular DNA. The main hosts of SEs are a subset of free-living Gammaproteobacteria; this represents a rather narrow host range compared to those of mobile DNA element groups discovered to date. As the host range, genetic organization, and movements are unique among the mobile DNA elements, SEs provide a new model system for host-mobile DNA element coevolution studies.

Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis.

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a ß-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.

Genetic Environment Surrounding blaOXA-55-like in Clinical Isolates of Shewanella algae Clade and Enhanced Expression of blaOXA-55-like in a Carbapenem-Resistant Isolate.

Although Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D ß-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of blaOXA-55-like transcription and ß-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various Shewanella spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of blaOXA-55-like transcription and ß-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings. IMPORTANCEShewanella spp., which are known to carry chromosomally located blaOXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, S. chilikensis, and S. carassii); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of blaOXA-55-like transcription and ß-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

Characterization of Prototheca CYP51/ERG11 as a possible target for therapeutic drugs.

Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. LAY SUMMARY: Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca's CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

Development of biliary stent applying the antibacterial activity of silver: A literature review.

BACKGROUND: Endoscopic transpapillary stenting is commonly performed in patients with obstructive jaundice caused by a biliary stricture. Although the plastic stent (PS) is widely used for biliary drainage because of the low-cost and easy procedure, patency is short after placement in the bile duct because of the small diameter. Dysfunction of PS is primarily caused by biliary sludge that forms as a result of bacterial adhesion and subsequent biofilm formation on the inner surface of the stent. It is well known that silver ions have excellent antibacterial activity against a wide range of microorganisms. OBJECTIVE: This review provides an overview and perspective of the significance of silver-coated biliary stents. METHODS: We collected literature regarding silver-coated biliary stents, reviewed the current research/development status and discussed their possible usefulness. RESULTS: To date, several in vivo/vitro studies evaluated the patency of silver-blended or silver-coated biliary stents. These studies suggested that the silver coating on a PS was likely to prolong the patency period. CONCLUSION: The development of biliary stents using silver is expected to prolong stent patency and prevent frequent stent replacement.

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

The novel mef(C)-mph(G) macrolide resistance genes are conveyed in the environment on various vectors.

BACKGROUND: The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. METHODS: Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)-mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. RESULTS: The mef(C)-mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)-mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10-4-10-5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (<10-9). CONCLUSIONS: These results suggest that mef(C)-mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)-mph(G).

Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community.

Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted "pAQU group." The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the "pAQU group" plasmids may play an important role in dissemination of ARGs in the marine environment.

Novel conjugative transferable multiple drug resistance plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from marine aquaculture environment.

The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae, isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA, suggesting a new replicon, and contained seven drug resistance genes: bla(CARB-9)-like, floR, mph(A)-like, mef(A)-like, sul2, tet(M) and tet(B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA. Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOB(H12), a sub-group of the MOB(H) plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment.

Differences of genetic diversity and antibiotics susceptibility of Pseudomonas aeruginosa isolated from hospital, river and coastal seawater.

Pseudomonas aeruginosa is an opportunistic pathogen, and ubiquitously found in natural environments. However, details on difference between clinical and environmental isolates have not been reported enough. In this study, we defined existence of marine specific genogroup and different drug susceptibility among isolates from clinical, river and coastal seawaters. Pseudomonas aeruginosa were isolated by using cetrimide kanamycin nalidixic acid agar media and incubation at 42°C, which was specific selection method of this bacterium from the natural aquatic samples. Pulse field gel electrophoresis analysis showed that the levels of genetic variation within P. aeruginosa were different among environmental sites. Pulse field gel electrophoresis also showed a lower diversity within P. aeruginosa in the coastal waters; and coastal strains isolated different sampling points were positioned closely in the same cluster. Most of the aquatic isolates were sensitive to most of the drugs tested and 'intermediate' to panipenem on the contrast to the clinical isolates, suggesting that the clinical use of antibiotics affect significantly to the emergence of the drug-resistant P. aeruginosa.

Occurrence of two genotypes of tetracycline (TC) resistance gene tet(M) in the TC-resistant bacteria in marine sediments of Japan.

The tetracycline (TC) resistance gene tet(M) was monitored in bacteria isolated from Japanese coastal and off-shore marine sediments. The high rate of occurrence of TC resistant (TC(r)) bacteria (120 microg mL(-1) TC) was observed at frequency ranges between 0.0-0.08% in Tokyo Bay, 1.67-1.82% in Sagami Bay and 0.0-4.35% in the open Pacific Ocean. The tet(M) gene was PCR amplified from the TC(r) isolates, showing 127 of 209 isolates (60.8%) as positive. The rate of occurrence of tet(M) was between 32.0-96.0%, 21.1 -28.0% and 0.0-83.3% in the isolates from Tokyo Bay, Sagami Bay and the open Pacific Ocean, respectively. The tet(M) positive isolates belonged to 4 orders of bacteria. Bacillales was the most dominant order (121 strains) among tet(M) possessing bacteria, followed by Actinomycetales (three strains), Flavobacteriales (one strain) and Pseudomonadales (one strain). This indicates that tet(M) is present in various bacterial species and suggests that marine sediments are a natural reservoir of the tet(M) gene. Nucleotide sequence of the tet(M) revealed that two genotypes of tet(M) were found in the bacteria. The two genotypes were placed in genetically distant branches of the phylogenetic tree, suggesting that the two tet(M)s have different origins.

Detection of the sul1, sul2, and sul3 genes in sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam.

To assess the presence and distribution of the sul genes (sul1, sul2, and sul3) and plasmids in human-mediated environments of north Vietnam, we examined a total of 127 sulfonamide-resistant (SR) bacterial isolates from four shrimp ponds (HNAQs), a city canal (HNCs) and three fish ponds that received wastewater directly from swine farms (HNPs). Results from the SR isolates revealed that sul genes were most frequently detected in the HNPs (92.0%), followed by HNCs (72.0%), and the HNAQs (43.0%). Among the sul genes detected, sul1 was the most prevalent gene in all three environments (57.0, 33.0 and 60.0% in HNPs, HNAQs, and HNCs, respectively) followed by sul2 (51.0, 19.0, and 20.0%, respectively) and sul3 (14.0, 6.0, and 8.0%, respectively). All combinations of paired different sul genes were detected, with the combination between sul1 and sul2 being the most frequent in all three environments (20.0, 8.0, and 8.0% in HNPs, HNAQs, and HNCs, respectively). The combination of three sul genes was detected at low frequencies (2-3%) in the HNPs and HNAQs, and was absent in the HNCs. The sul genes were more frequently located on the chromosome than on plasmids. The identification of SR isolates positive for the sul genes and plasmids showed that Acinetobacter was the most dominant. Our study revealed that the sul genes were common in SR bacteria from the aquatic environments we examined from northern Vietnam. Wastewater from swine farms might be "hot spots" of the sul genes and plasmids and may be reservoirs for the exchange of the sul genes among bacteria.

Molecular evidence for the ancient origin of the ribosomal protection protein that mediates tetracycline resistance in bacteria.

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1alpha. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1alpha. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1alpha, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1alpha clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs' extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment.

The diversity of multi-drug resistance profiles in tetracycline-resistant Vibrio species isolated from coastal sediments and seawater.

In this study we examined the multi-drug resistance profiles of the tetracycline (TC) resistant genus Vibrio to determine its susceptibility to two beta-lactams, ampicillin (ABPC), and mecillinam (MPC), as well as to macrolide, erythromycin (EM). The results showed various patterns of resistance among strains that were isolated from very close geographical areas during the same year, suggesting diverse patterns of drug resistance in environmental bacteria from this area. In addition, the cross-resistance patterns suggested that the resistance determinants among Vibrio spp. are acquired differently within the sediment and seawater environments.

Degradation of tributyltin in microcosm using Mekong River sediment.

The degradation of tributyltin (TBT) and changes of bacterial number and community structures were investigated in microcosms using the sediment collected from the Mekong River, Vietnam. Concentrations of TBT in sediments were less than 0.62 ng/g (dry wt), lower than those reported from other areas. TBT-resistant bacteria were found in the three sampling sites, and the occurrence rates were 11-16% out of the total viable count. In this microcosm experiment, initial concentration of TBT [1.0-1.4 microg/g (dry wt)] decreased to 0.6 microg/g (dry wt) during 150 days, whereas that in the control microcosm with autoclaved sediment did not change, indicating that Mekong River sediment contains high TBT-degrading activity by microorganisms. The occurrence of TBT-resistant bacteria and the bacterial community structures monitored by denaturing gradient gel electrophoresis were almost the same between test and control groups, indicating that the addition of TBT had little influence on microbial community structure. Mekong River sediment seems to have a stable microbial community against TBT pollution.

16S rRNA mutations that confer tetracycline resistance in Helicobacter pylori decrease drug binding in Escherichia coli ribosomes.

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.