Absence Of Antibiotic Selection Research Articles

PPSX: A novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters

A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the λ cos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36 kb. The inclusion of a lacZα multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non- E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.

Enhanced protein production by engineered zinc finger proteins

Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to approximately 500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells.

A novel high-copy plasmid, pEC, compatible with commonly used Escherichia coli cloning and expression vectors

A 66164-bp cryptic plasmid, pEIB1, was isolated from strain Vibrio anguillarum MVM425 and sequenced. A plasmid carrying a 1089-bp fragment, containing the minimal replication region of pEIB1, a kanamycin-resistance marker and an L-arabinose promoter, designated pEC, was maintained as a high copy plasmid in E. coli and stably inherited in the absence of antibiotic selection. Significantly, pEC was compatible with the widely used ColE1, pSC101 and p15A replicons making it a useful tool for a dual-plasmid expression system.

Class 1 Integrons Potentially Predating the Association with Tn402-Like Transposition Genes Are Present in a Sediment Microbial Community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."

Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 x 10(7) CFU mug of DNA(-1) in F. tularensis LVS, Francisella novicida U112, and E. coli DH5alpha. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

Improved Efficiency of a Salmonella -Based Vaccine against Human Papillomavirus Type 16 Virus-Like Particles Achieved by Using a Codon-Optimized Version of L1

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

The Acquired Immune System: A Vantage from Beneath

The Acquired Immune System: A Vantage from Beneath

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l −1 (OD 600∼60) and the production of 700 mg l −1 of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l −1 of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l −1 with E. coli as the production host.

Construction of a genetically engineered microorganism for CO 2 fixation using a Rhodopseudomonas/ Escherichia coli shuttle vector

The CO 2 fixation ability of Rhodopseudomonas palustris DH was enhanced by introducing the recombinant plasmid pMG-CBBM containing the form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene ( cbbM) isolated from Rps. palustris NO. 7. Sequencing of a 3.0-kb PstI fragment containing the cbbM gene revealed an open reading frame encoding 461 amino acids, homologous to known cbbM genes, with a ribosome binding site upstream of cbbM and a terminator downstream of cbbM, without promoter. pMG-CBBM, a Rhodopseudomonas/ Escherichia coli shuttle expression plasmid, was derived from the Rhodopseudomonas/ E. coli shuttle cloning vector pMG105, by inserting the promoter of the pckA gene and the cbbM gene into its multiple cloning site. Plasmid pMG-CBBM was transformed into Rps. palustris DH by electroporation, and was stably maintained when transformants were grown either photoheterotrophically or photolithoautotrophically in the absence of antibiotics. This is the first report of an expression plasmid containing a Rps. palustris-specific promoter that allows stable expression of a foreign gene in the absence of antibiotic selection.

Effect of in situ expression of human interleukin-6 on antibody responses against Salmonella typhimurium antigens

In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyA s). Through stable introduction of a second hIL-6-HlyA s expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyA s secretion efficiencies were increased by at least 10-fold. As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did plasmids interfere in their stabilities. Increased hIL-6-HlyA s expression did not adversely interfere with bacterial growth. Comparative immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses. In mice immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain. Higher sera antibody titers were accompanied by increased numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer’s patches, respectively. These data suggest that systemic antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses.

In vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var. Typhimurium

This study examined the ability of different plasmid vectors encoding H C fragment, the non-toxic binding portion of tetanus toxin, to be stably retained by Salmonella enterica var. Typhimurium ( Salmonella typhimurium) vaccine strain BRD509 and, upon immunisation, to induce an antibody response against the carried antigen. The H C fragment expression cassette containing the transcription/translation signals, H C fragment open reading frame and the downstream TrpA terminator, was excised from pTETtac4 and incorporated into the plasmids pIC20H, pBR322, pACYC184 and pRSF1010. The resulting constructs were transferred into attenuated S. typhimurium, BRD509, and the level of H C fragment expression was examined by Western blot analysis. The relative stability of each plasmid in S. typhimurium was determined in vitro in the absence of antibiotic selection, and in vivo following immunisation. The ability of each H C fragment-expressing strain to induce lipopolysaccharide- and tetanus toxoid-specific antibody responses was assayed by an enzyme-linked immunosorbent assay. These studies showed that all the vaccine vector constructs, except the S. typhimurium carrying the expression vector based on pIC20H, were able to elicit a high titre immune response. The level of tetanus toxoid-specific antibody induced by S. typhimurium directly correlated with the level of in vitro and in vivo stability of the H C fragment expression plasmid carried by the bacterium, and not with an increased copy number of the parent plasmid vector.

Restoration of cytoadherence to an adherence-deficient mutant of Mycoplasma arthritidis by genetic complementation.

Mycoplasma arthritidis causes a severe septic arthritis in rats under natural and experimental conditions. An earlier study implicated a membrane lipoprotein designated MAA1 in cytadherence of M. arthritidis. In addition, a spontaneous adherence-deficient mutant was shown to contain a nonsense mutation in the gene encoding MAA1, resulting in production of a truncated product, MAA1Delta. In the present study, a wild-type maa1 gene carried on transposon Tn4001T was introduced into the low-adherence mutant by polyethylene glycol-mediated transformation. The presence of the tranposon and the wild-type maa1 gene in the chromosome of transformants was confirmed by PCR and Southern hybridization. The latter procedure also confirmed that each transformant contained a single copy of the transposon. Western immunoblotting showed that transformants produced both wild-type MAA1 and MAA1Delta, indicating that the introduced wild-type maa1 gene was functional. This phenotype was stably maintained after multiple subcultures even in the absence of antibiotic selection. Finally, transformants were shown to adhere to rat L-2 lung cells in culture at wild-type levels, providing confirmation for an important role for MAA1 in adherence.

Transformation of Rhodococcus rhodnii, a symbiont of the Chagas disease vector Rhodnius prolixus, with integrative elements of the L1 mycobacteriophage

Elimination of vector populations through the use of insecticides is the principal means of controlling Chagas disease. Because of the limitations of insecticide use, we have been developing a new potential method of control, to be used in conjunction with insecticide programs, a method which utilizes genetically modified symbiotic bacteria. These transformed bacteria can express anti-parasitic agents in the gut of the bug where the trypanosomes also are found. Previous studies have shown that it is possible to transform Rhodococcus rhodnii with a shuttle plasmid that contains the gene for cecropin A, an insect anti-microbial peptide. The bacteria expressed this peptide and reduced or eliminated the number of trypanosomes in the bug Rhodnius prolixus [Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3274]. In an effort to improve efficacy and transformation stability, we have begun using plasmids that contain integrative elements from the L1 mycobacteriophage to insert DNA into the genome of the bacterium. The integrative plasmid pBP5 contains the attachment site ( attP) and integrase gene ( int) of the L1 mycobacteriophage, an antibiotic resistance gene and the lacZ gene. After transforming R. rhodnii with pBP5, nine positive clones were obtained and six different insertions sites were identified. In each clone, the integrative plasmid is inserted only once, the lacZ gene is expressed intensely and, all clones but one, remained stable for 100 generations of culture in the absence of antibiotic selection. In addition, the construct remains stable throughout the life cycle of the bug. These data demonstrate that L1 mycobacteriophage integrative plasmids are significantly more stable than episomally located plasmids used in previous studies and will be greatly beneficial for use in the transformation of symbiotic bacteria of Chagas disease vectors.

Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli– Bartonella spp. shuttle cloning vector

We report herein the isolation and molecular characterization of pBGR1, the first native plasmid isolated from the genus Bartonella. Cloning and sequencing revealed a 2725-base pair (bp) cryptic plasmid comprising two open reading frames of considerable length, which were designated rep and mob. The regions containing rep and mob are separated by 140-bp inverted repeat sequences and display a difference in G + C content from one another. A 1435-bp SacI– BclI fragment containing the rep gene is sufficient to mediate replication in the species Bartonella henselae and Bartonella tribocorum, while this replicon does not appear to be functional in Escherichia coli. The Rep protein of 190 amino acids (aa) shares homology to putative replication proteins of cryptic plasmids of Gram-negative origin, which form a subgroup of the rolling-circle replication proteins of the pSN2 plasmid superfamily of Gram-positive bacteria. The Mob protein of 333 aa is related to mobilization proteins of several cryptic plasmids and is associated with a conserved recombination site A. The tra functions of RP4 can mobilize pBGR1 derivatives in a mob-dependent manner. Mobilizable pBGR1-based E. coli–Bartonella spp. shuttle vectors were constructed and were shown to be maintained in B. tribocorum during in vivo passage in a rat model in the absence of antibiotic selection. The small size and stability of these shuttle cloning vectors should render them particularly valuable for genetic studies in Bartonella spp.

Stability, persistence, and evolution of plasmid-encoded VanA glycopeptide resistance in enterococci in the absence of antibiotic selection in vitro and in gnotobiotic mice.

Long-term persistence of VanA glycopeptide-resistant enterococci (GRE) has been observed in the absence of antibiotic selection. In the present study, we examined fitness parameters of a glycopeptide-susceptible Enterococcus faecium parent strain and its plasmid-mediated, VanA-resistant derivative before and after 1,000 generations in serial transfer broth cultures with or without antibiotic selection. With the exception of the vanA-containing plasmid, the strains were otherwise isogenic. The stability of the plasmid-encoded vanA resistance determinant was also investigated in vitro and in gnotobiotic mice. Competition experiments revealed that GRE with newly acquired VanA resistance had a 4% reduction in fitness relative to their susceptible parental counterpart. The relative difference in competitive fitness between resistant and susceptible strains was not significantly changed after 1,000 generations. Environmental adaptation was observed in all strains and exceeded the biological cost of resistance. Thus, the evolved VanA-resistant E. faecium populations out-numbered their unevolved ancestral susceptible E. faecium strain in mixed cultures, but remained less competitive than the evolved parent. The glycopeptide resistance determinant was similarly stably maintained during long-term colonization in gnotobiotic mice without antibiotic selection. In vivo vanA plasmid transfer was observed. The results suggest that environmental adaptation, in vivo gene transfer, and plasmid maintenance system(s) favor long-term VanA GRE persistence without antibiotic selection and compensate for the biological costs of possessing the resistance genes.

Introduction of a plasmid-encoded phoA gene for constitutive overproduction of alkaline phosphatase in three subsurface Pseudomonas isolates

Abstract Three bacterial isolates, Pseudomonas fluorescens F1, Pseudomonas rhodesiae R1 and Pseudomonas veronii V1 were genetically modified by introduction of a plasmid, pJH123, with a phoA hybrid gene that directed constitutive overproduction of the enzyme alkaline phosphatase. The presence of the plasmid in the bacterial hosts elevated extracytoplasmic alkaline phosphatase production from 100- to 820-fold. The growth and survival of the plasmid-bearing hosts in sterilized soil slurries was comparable to parental control strains. In the absence of antibiotic selection, pJH123 was maintained in two of the three hosts (P. fluorescens F1 and P. veronii V1) during incubation in minimal medium. The effects of the genetically enhanced pseudomonads on the liberation of inorganic phosphate (PO(4) (3-)) were determined in sterilized soil slurries following the addition of an organophosphorus compound, glycerol-3-phosphate. A significant accumulation of PO(4) (3-) was measured in soil slurries amended with 10 mM glycerol-3-phosphate and any of the three phosphatase-enhanced pseudomonad isolates. In contrast, soil slurries containing unmodified parental strains did not exhibit significant PO(4) (3-) accumulation. Two of the three enhanced phosphate-liberating strains released sufficient PO(4) (3-) that cell-free supernatants from sterilized soil slurry incubations removed significant amounts of uranium (as much as 69%) from solution.

Introduction of a plasmid-encoded phoA gene for constitutive overproduction of alkaline phosphatase in three subsurface Pseudomonas isolates

Three bacterial isolates, Pseudomonas fluorescens F1, Pseudomonas rhodesiae R1 and Pseudomonas veronii V1 were genetically modified by introduction of a plasmid, pJH123, with a phoA hybrid gene that directed constitutive overproduction of the enzyme alkaline phosphatase. The presence of the plasmid in the bacterial hosts elevated extracytoplasmic alkaline phosphatase production from 100- to 820-fold. The growth and survival of the plasmid-bearing hosts in sterilized soil slurries was comparable to parental control strains. In the absence of antibiotic selection, pJH123 was maintained in two of the three hosts ( P. fluorescens F1 and P. veronii V1) during incubation in minimal medium. The effects of the genetically enhanced pseudomonads on the liberation of inorganic phosphate (PO 4 3−) were determined in sterilized soil slurries following the addition of an organophosphorus compound, glycerol-3-phosphate. A significant accumulation of PO 4 3− was measured in soil slurries amended with 10 mM glycerol-3-phosphate and any of the three phosphatase-enhanced pseudomonad isolates. In contrast, soil slurries containing unmodified parental strains did not exhibit significant PO 4 3− accumulation. Two of the three enhanced phosphate-liberating strains released sufficient PO 4 3− that cell-free supernatants from sterilized soil slurry incubations removed significant amounts of uranium (as much as 69%) from solution.

Suitability of Anabaena PCC7120 expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis for biotechnological application.

We present evidence that Anabaena PCC7120 (A.7120) strains expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis (Bti) have a strong potential for biotechnological application. Characterization of two 4-year-old recombinant A.7120 clones constructed previously in our laboratory [clone 7 and clone 11, each carrying three Bti genes (cry4Aa, cry11Aa, and p20)] revealed three facts. First, the Bti genes were stable in A.7120 even in the absence of antibiotic selection when the genes were integrated in the chromosome (in clone 11); and the genes were also stable as plasmid-borne constructs (in clone 7), provided the cultures were maintained under continued selection. Second, clone 7 (kept under selection) and clone 11 (either kept or not kept under selection) continued to be mosquitocidal through 4 years of culture. Third, growth of the recombinant clones was comparable to the wild type under optimal growth conditions, indicating that growth was not compromised by the expression of toxin genes. These results clear the way for the development of mass production techniques for A.7120 strains expressing Bti toxin genes.

Efficient, long-term transgene expression in Xenopus laevis dermal melanophores.

Xenopus laevis dermal melanophores provide an excellent model system for the investigation of complex cellular processes. Specifically, the expression of exogenous genes in Xenopus melanophores is the basis of recombinant bioassays for the study of receptor-ligand interactions. However, due to their slow rate of cell division and to the relatively low efficiency of current transfection protocols, long-term expression of exogenous genes and the generation of stable melanophore cell lines remains problematic. In this report we demonstrate the efficient, long-term expression of two exogenous proteins, the enhanced green fluorescent protein (EGFP) and the human CD4 (hCD4) cell surface receptor, following stable introduction into Xenopus melanophores via an HIV-1 based vector. Transduction of melanophores with the EGFP expression vector resulted in up to 80% EGFP+ cells. After 1 year in continuous culture in the absence of antibiotic selection, more than 60% of the cells remained EGFP+. Furthermore, we demonstrate the expression of hCD4 melanophores for over 9 months in continuous culture in the absence of antibiotic selection. Our results indicate that lentivirus vectors provide an efficient means of introducing genetic information into Xenopus melanophores, resulting in sustained levels of gene expression. The significance of this gene transfer system for the study of cellular signal transduction pathways is discussed.

Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon.

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.