Identification of the novel B*27:144 allele in an Irish Individual.

The sequence of HLA-B*27:144 differs from HLA-B*27:05:02 by one nucleotide change at position 506.

Effect of subinhibitory concentrations of four commonly used biocides on the conjugative transfer of Tn916 in Bacillus subtilis.

OBJECTIVES: Large amounts of biocides are used to reduce and control bacterial growth in the healthcare sector, food production and agriculture. This work explores the effect of subinhibitory concentrations of four commonly used biocides (ethanol, hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite) on the conjugative transposition of the mobile genetic element Tn916. METHODS: Conjugation assays were carried out between Bacillus subtilis strains. The donor containing Tn916 was pre-exposed to subinhibitory concentrations of each biocide for a defined length of time, which was determined by an analysis of the transcriptional response of the promoter upstream of tet(M) using ß-glucuronidase reporter assays. RESULTS: Ethanol significantly (P = 0.01) increased the transfer of Tn916 by 5-fold, whereas hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite did not significantly affect the transfer frequency. CONCLUSIONS: These results suggest that exposure to subinhibitory concentrations of ethanol may induce the transfer of Tn916-like elements and any resistance genes they contain.

Tetracycline resistance genes and mobile genetic elements from the oral metagenome.

Tetracycline resistance genes are common in the human oral cavity. However, a complete understanding of tetracycline resistance and the vectors responsible for spread of resistance requires that we understand the contribution of organisms that cannot be cultivated in the laboratory. To do this, metagenomic approaches have been applied and this has allowed the isolation of novel tetracycline resistance genes and mobile genetic elements.

Determination of the genetic support for tet(W) in oral bacteria.

The DNA sequence flanking a tet(W) gene in an oral Rothia sp. was determined. The gene was linked to two different transposases, and these were flanked by two almost identical mef (macrolide efflux) genes. This structure was found in 4 out of 20 tet(W)-containing oral bacteria investigated.

Potential role of Veillonella spp. as a reservoir of transferable tetracycline resistance in the oral cavity.

Twelve out of 96 Veillonella spp. isolated from oral samples harbored tetracycline resistance genes. The most common resistance gene was tet(M). A tet(M)-positive Veillonella dispar strain was shown to transfer a Tn916-like element to four Streptococcus spp. by conjugation at a frequency of 5.2 x 10(-6) to 4.5 x 10(-5) per recipient.

Antibiotic resistance in the cultivable plaque microbiota of children from different ethnic groups.

The presence of ampicillin-, penicillin-, erythromycin- and tetracycline-resistant bacteria in the dental plaque of White, South Asian and Japanese children was investigated. There was a high prevalence of antibiotic-resistant bacteria in children from diverse ethnic groups. The median percentage of the cultivable plaque microbiota that was resistant to tetracycline was greater in South Asian (2.9%, range 0.1-17.5%) and Japanese (7.7%, range 1.3-56.2%) children than in White children (0.7%, range 0-5.6%), suggesting that ethnic differences exist in the oral load of tetracycline-resistant bacteria (P<0.01). Multiresistant bacteria were frequently isolated, with 42% of isolates exhibiting resistance to two or more antibiotics. This study has demonstrated that antibiotic-resistant bacteria can be readily isolated from the plaque microbiota of children from different ethnic groups.

Isolation of silver- and antibiotic-resistant Enterobacter cloacae from teeth.

Antibiotic-resistant bacteria pose a serious threat to human health; hence the mechanisms that lead to their selection need to be investigated. One possible mechanism is that the silver and mercury in amalgam dental restorations may select for bacteria that contain heavy metal and antibiotic-resistance determinants, leading to the spread of these resistances, particularly if they are contained on the same mobile genetic element. The incidence of silver-resistant bacteria on teeth is investigated in this work. Two silver-resistant Enterobacter cloacae isolates were isolated from infected teeth containing dental restorations. Both isolates were also resistant to ampicillin, erythromycin, and clindamycin. The silE gene, which is encoded on the silver resistance operon, has been sequenced from both isolates. Results suggest that the silver resistance operon is encoded on plasmid DNA.

Effect of amoxicillin use on oral microbiota in young children.

Dental plaque samples from 40 children were screened for the presence of bacteria resistant to amoxicillin. Fifteen children had used amoxicillin and 25 had not used any antibiotic in the 3 months prior to sample collection. All (100%) of the children harbored amoxicillin-resistant oral bacteria. The median percentage of the total cultivable oral microbiota resistant to amoxicillin was 2.4% (range, 0.1 to 14.3%) in children without amoxicillin use and 10.9% (range, 0.8 to 97.3%) in children with amoxicillin use, with the latter value being significantly higher (P < 0.01). A total of 224 amoxicillin-resistant bacteria were isolated and comprised three main genera: Haemophilus spp., Streptococcus spp., and Veillonella spp. The biodiversity of the amoxicillin-resistant microbiota was similar among the isolates from children with and without previous antibiotic use. The amoxicillin MIC at which 90% of the isolates were inhibited for isolates from children who had used amoxicillin in the previous 3 months was higher (64 mg liter(-1)) than that obtained for the isolates from subjects who had not used antibiotics (16 mg liter(-1)). The majority of the amoxicillin-resistant isolates (65%) were also resistant to at least one of the three antibiotics tested (penicillin, erythromycin, and tetracycline), with resistance to penicillin (51% of isolates) being the most frequently encountered. However, significantly more (P < 0.05) of the amoxicillin-resistant isolates from subjects with previous amoxicillin use were also resistant to erythromycin. This study has demonstrated that a diverse collection of amoxicillin-resistant bacteria is present in the oral cavity and that the number, proportions, MICs, and resistance to erythromycin can significantly increase with amoxicillin use.

Genetic basis of erythromycin resistance in oral bacteria.

We determined the prevalence of erythromycin-resistant bacteria in the oral cavity and identified mef and erm(B) as the most common resistance determinants. In addition, we demonstrate the genetic linkage, on various Tn1545-like conjugative transposons, between erythromycin and tetracycline resistance in a number of isolates.

Novel tetracycline resistance determinant from the oral metagenome.

A major drawback of most studies on how bacteria become resistant to antibiotics is that they concentrate mainly on bacteria that can be cultivated in the laboratory. In the present study, we cloned part of the oral metagenome and isolated a novel tetracycline resistance gene, tet(37), which inactivates tetracycline.

Prevalence of tetracycline resistance genes in oral bacteria.

Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.

Prevalence, proportions, and identities of antibiotic-resistant bacteria in the oral microflora of healthy children.

The aims of this study were to determine the prevalence, proportions and identities of oral bacteria resistant to six antibiotics in 35 children (4-5 years old) who had not received antibiotics during the previous 3 months. Ampicillin-, penicillin-, erythromycin-, and tetracycline-resistant bacteria were harbored by 35 (100%), 34 (97%), 35 (100%), and 34 (97%) children, respectively. None of the children harbored metronidazole-resistant anaerobic bacteria or Gram-positive vancomycin-resistant bacteria. The median percentage of the oral microflora resistant to each of the antibiotics was ampicillin 1% (range 0.1-23), erythromycin 13% (1-45), penicillin 1% (0-14), and tetracycline 2% (0-88). A total of 432 antibiotic-resistant isolates were recovered that comprised 18 genera and 47 species. Ampicillin resistance was widely distributed throughout different genera and species, whereas tetracycline resistance was predominately found in the streptococci. Multiresistant bacteria were frequently isolated with 28% of isolates exhibiting resistance to two or more antibiotics. Veillonella spp., traditionally considered susceptible to penicillin and ampicillin, were found frequently to be resistant to these two antibiotics. This study demonstrates that a diverse collection of antibiotic-resistant pathogenic, opportunistic, and nonpathogenic bacteria can be readily isolated from, and in some subjects dominate, the oral microflora of primary school children in the absence of recently administered antibiotics.

Composition and antibiotic resistance profile of microcosm dental plaques before and after exposure to tetracycline.

The aim of this study was to investigate the effects of tetracycline administration on the viability and antibiotic resistance profiles of microcosm dental plaques. A constant depth film fermenter was used to generate multi-species biofilms, which were grown for 216 h before tetracycline was added. The composition of the microcosm plaques was determined by viable counting on selective and non-selective media. The prevalence of antibiotic-resistant organisms was determined on antibiotic-containing media. Before administration of tetracycline, the biofilms had a total viable anaerobic count of 7 x 10(7) cfu per biofilm. They contained 7% lactobacilli, 19% streptococci and 2% Actinomyces spp. Immediately after pulsing with tetracycline, the composition of the biofilms changed and they consisted of 30% lactobacilli, 1.5% streptococci and 3% Actinomyces spp., with a total anaerobic count of 1 x 10(7) cfu per biofilm. The pre-valence and composition of the antibiotic-resistant microflora changed dramatically after the addition of tetracycline, with the proportion of the microflora displaying resistance to tetracycline increasing from 6% to 45%. Corresponding changes in the proportions of the microflora displaying resistance to other antibiotics were as follows: 5-28% for erythromycin, 1-5% for vancomycin and 0.4-3% for ampicillin. The results of this study have shown that the addition of tetracycline to microcosm dental plaques alters their composition and enriches for bacteria resistant to tetracycline and other unrelated agents.

Prevalence and antibiotic resistance profile of mercury-resistant oral bacteria from children with and without mercury amalgam fillings.

Genes encoding resistance to mercury and to antibiotics are often carried on the same mobile genetic element and so it is possible that mercury-containing dental materials may select for bacteria resistant to mercury and to antibiotics. The main aim of this study was to determine whether the prevalence of Hg-resistant oral bacteria was greater in children with mercury amalgam fillings than in those without. A secondary aim was to determine whether the Hg-resistant isolates were also antibiotic resistant. Bacteria in dental plaque and saliva from 41 children with amalgam fillings and 42 children without such fillings were screened for mercury resistance by cultivation on a HgCl(2)-containing medium. Surviving organisms were identified and their susceptibility to mercury and to several antibiotics was determined. Seventy-eight per cent and 74% of children in the amalgam group and amalgam-free group, respectively, harboured Hg-resistant bacteria; this difference was not statistically significant. Nor was there any significant difference between the groups in terms of the proportions of Hg-resistant bacteria in the oral microflora of the children. Of Hg-resistant bacteria, 88% and 92% from the amalgam group and the amalgam-free group, respectively, were streptococci; 41% and 33% were resistant to at least one antibiotic, most frequently tetracycline. The results of this study show that there was no significant difference between children with amalgam fillings and those without such fillings with regard to the prevalence, or the proportion, of Hg-resistant bacteria in their oral microflora. The study also found that Hg-resistant bacteria were common in children regardless of whether or not they had amalgam fillings and that many of these organisms were also resistant to antibiotics.

Mechanism of integration and excision in conjugative transposons.

Translocation of conjugative transposons proceeds via excision of the element to generate a circular molecule that can then integrate into a new site, which can be in the same or a different cell. This review summarises some of the different mechanisms used for excision and integration of conjugative transposons.

Transfer of TN916-like elements in microcosm dental plaques.

Microcosm dental plaques were grown from an inoculum of human saliva in a constant-depth film fermentor. The inoculum contained four tetracycline-resistant streptococcal species, each of which contained a Tn916-like element. This element was shown to transfer to other streptococci both in filter-mating experiments and within the biofilms in the fermentor.

Characterization of a cell surface protein of Clostridium difficile with adhesive properties.

Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60 degrees C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains of C. difficile representing 11 serogroups showed that the 3' portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase in Escherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of C. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia.