Tetracycline-resistant bacteria and ribosomal protection protein genes in soils from selected agricultural fields and livestock farms

Antibiotic resistance in soil environment has eminently been compared and studied between agricultural and pristine soils, and the role of concentrated animal feeding operations has markedly been recognized as one of the major sources of antibiotic resistance. This study described the tetracycline resistance in small-scale farms in pursuit of presenting its possible role and contribution to the persistence of antibiotic resistance in the environment. Results of the study would render additional information on the occurrence of the ribosomal protection protein (RPP) tet genes among the isolated bacteria from the selected agricultural soils. Four tetracycline resistance and RPP genes were determined in two different agricultural soil settings. Both the culture and molecular method were used to determine and measure tetracycline resistance in soils from arable land and animal house. Results revealed a significantly higher number of culturable antibiotic-resistant bacteria in animal houses than arable lands which was suggestive of higher antibiotic resistance in areas where there was direct administration of the antibiotics. However, quantification of the gene copy numbers in the agricultural soils indicated a different result. Higher gene copy number of tetO was determined in one animal house (IAH-3), while the two other tet genes tetQ and tetW were found to be higher in arable lands. Of the total 110 bacterial isolates, tetW gene was frequently detected, while tetO gene was absent in any of the culturable bacterial isolates. Principal component analysis of occurrence and gene copy number of RPP tet genes tetO, tetQ, and tetW also revealed highest abundance of RPP tet genes in the manure and arable soils. Another important highlight of this study was the similarity of the RPP tet genes detected in the isolated bacteria from the agricultural soils to the identified RPP tet genes among pathogenic bacteria. Some of the tetracycline-resistant bacterial isolates were also multidrug resistant as it displayed resistance to tetracycline, erythromycin, and streptomycin using disk diffusion testing.

WGS of Commensal Neisseria Reveals Acquisition of a New Ribosomal Protection Protein (MsrD) as a Possible Explanation for High Level Azithromycin Resistance in Belgium

In this study, we characterized all oropharyngeal and anorectal isolates of Neisseria spp. in a cohort of men who have sex with men. This resulted in a panel of pathogenic Neisseria (N. gonorrhoeae [n = 5] and N. meningitidis [n = 5]) and nonpathogenic Neisseria (N. subflava [n = 11], N. mucosa [n = 3] and N. oralis [n = 2]). A high proportion of strains in this panel were resistant to azithromycin (18/26) and ceftriaxone (3/26). Whole genome sequencing (WGS) of these strains identified numerous mutations that are known to confer reduced susceptibility to azithromycin and ceftriaxone in N. gonorrhoeae. The presence or absence of these known mutations did not explain the high level resistance to azithromycin (>256 mg/L) in the nonpathogenic isolates (8/16). After screening for antimicrobial resistance (AMR) genes, we found a ribosomal protection protein, Msr(D), in these highly azithromycin resistant nonpathogenic strains. The complete integration site originated from Streptococcus pneumoniae and is associated with high level resistance to azithromycin in many other bacterial species. This novel AMR resistance mechanism to azithromycin in nonpathogenic Neisseria could be a public health concern if it were to be transmitted to pathogenic Neisseria. This study demonstrates the utility of WGS-based surveillance of nonpathogenic Neisseria.

Emergence of a Novel tet(L) Variant in Campylobacter spp. of Chicken Origin in China

ABSTRACTTetracyclines are widely used in veterinary medicine and food animal production. Campylobacter members are major foodborne pathogens, and their resistance to tetracycline has been widely reported in different countries. To date, Tet(O), a ribosomal protection protein, is the only confirmed Tet resistance determinant in Campylobacter spp. Here, we reported the detection and characterization of a novel Tet resistance element in Campylobacter spp. of chicken origin. This gene is identified to be a variant of tet(L), which encodes an efflux pump for Tet resistance. The variant was detected in 14 of the 82 tetracycline-resistant Campylobacter isolates collected from chickens in Henan, China. Cloning of the tet(L) variant into tetracycline-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to tetracycline and doxycycline. In addition, this tet(L) variant elevated the MIC (4-fold increase) of tigecycline in the heterologous Escherichia coli host. Sequencing analysis indicated the tet(L) variant was located within a multidrug-resistance genomic island (MDRGI) containing tet(L) variant IS1216E-ORF1-fexA-Δtnp-IS1216E-tet(O)-tnpV-repA. This MDRGI is inserted into conserved gene potB on the chromosome. Multilocus sequence type (MLST) analysis revealed that both clonal expansion and horizontal transfer were involved in the dissemination of the tet(L) variant. These findings reveal the emergence of a new Tet resistance determinant in Campylobacter spp., which may facilitate their adaptation to the antimicrobial selection pressure in chickens.

Mechanism of Action of the Novel Aminomethylcycline Antibiotic Omadacycline

ABSTRACTOmadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistantStaphylococcus aureus(MRSA), β-hemolytic streptococci, penicillin-resistantStreptococcus pneumoniae,Haemophilus influenzae, andLegionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is activein vitroin the presence of the ribosomal protection protein Tet(O).

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 – >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.

Prevalence of Tetracycline Resistance Genes in Oral Bacteria

ABSTRACT 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.