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.

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.All of the tetracycline resistance genes that have been investigated so far have been isolated from cultivable microflora. A major limitation of this approach is that a large proportion of the microflora cannot be cultivated in the laboratory (4, 5). To begin to tackle this problem, we devised an approach that did not require the bacteria to be cultured: cloning of the oral metagenome. DNA was extracted from the bacteria present in saliva and dental plaque samples and cloned into expression libraries in Escherichia coli. We have detected a new tetracycline resistance gene, demonstrating the effectiveness of this approach in isolating antibiotic resistance genes from oral microflora.Dental plaque and saliva samples were collected from 20 healthy adults who had not received antibiotics during the previous 3 months.Bacteria were harvested by centrifugation (3,500 ϫ g, 10 min, 4°C) from the saliva and plaque specimens. The resulting bacterial pellet was washed gently in sterile saline. Two aliquots of the cells were prepared, and DNA was extracted from one of these aliquots by using the Puregene gram-positive DNA isolation protocol and from the other by using the Puregene gram-negative DNA isolation protocol (Gentra Systems) according to the manufacturer's instructions. The extracted DNA was subsequently pooled. To prepare the DNA for library construction, 0.2 ml of DNA (at a concentration of 250 ng/ml) was sonicated for 5 s on ice at 80% power by using an ultrasonic homogenizer (IKA-WERKE). The ends of the DNA were repaired by treating them with 2 U of mung bean nuclease (Promega)/mg in a final volume of 100 l at 37°C for 1 h to produce blunt ends. The resulting DNA fragments were separated by agarose gel electrophoresis, and fragments between 800 and 3,000 bp were cut from the gel and purified by using the Qiagen agarose purification kit. To generate 3Ј-A overhangs prior to cloning into a TOPO-XL vector, the DNA fragments were incubated at 75°C for 1 h with 1 U of Taq DNA polymerase (Bioline)/mg in a final volume of 100 l in the presence of 2 mM dATP in 1ϫ Taq buffer. Prior to ligation into the vector, the DNA was treated by using the Qiagen PCR purification kit. Ligation of DNA into TOPO-XL and subsequent transformation into E. coli TOP10 cells were performed according to the manufacturer's protocol. Plasmid DNA was isolated from E. coli by using the Qiagen Miniprep kit.The library was screened on Luria-Bertani agar plates containing tetracycline at a concentration of 5 g/ml. Plasmid DNA from antibiotic-resistant clones was used to retransform E. coli to confirm that antibiotic resistance was encoded on the insert DNA.To detect the tetracycline resistance genes encoding ribosomal protection proteins [...

Studies of the prevalence and identity of genes encoding resistance to antibiotics in a microbial community are usually carried out on only the cultivable members of the community. However, it is possible to include the as-yet-uncultivable organisms present by adopting a metagenomic approach to such studies. In this investigation, four metagenomic libraries of the oral microbiota were prepared from three groups of 20 adult humans and screened for antibiotic-resistant clones. Clones resistant to tetracycline and amoxycillin were present in all four libraries while gentamicin-resistant clones were found in three of the libraries. The genes encoding tetracycline resistance in the clones were identified and found to be tet(M), tet(O), tet(Q), tet(W), tet37 and tet(A). However, only the first three of these were detected in all three groups of individuals investigated.

Summary Hospital-acquired infections are on the rise and are a substantial cause of clinical and financial burden for healthcare systems. While infection control plays a major role in curtailing the spread of outbreak organisms, it is not always successful. One organism of particular concern is Acinetobacter baumannii , due to both its persistence in the hospital setting and its ability to acquire antibiotic resistance. A. baumannii has emerged as a nosocomial pathogen that exhibits high levels of resistance to antibiotics, and remains resilient against traditional cleaning measures with resistance to Colistin increasingly reported. Given the magnitude and costs associated with hospital acquired infections, and the increase in multidrug-resistant organisms, it is worth re-evaluating our current approaches and looking for alternatives or adjuncts to traditional antibiotics therapies. The aims of this review are to look at how this organism is spread within the hospital setting, discuss current treatment modalities, and propose alternative methods of outbreak management.