Oxytetracycline Resistance Research Articles

In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.

Unveiling antibiotic resistance dynamics in single and two-stage anaerobic digestion of dairy cow manure: Implications for environmental health

This study investigated antibiotic-resistant bacteria (ARB) survival during mesophilic anaerobic digestion (AD) of dairy cow manure with cefazolin (CEZ) antibiotic. Comparing single- and two-stage AD systems, we assessed ARB removal rates, biogas yield, volatile fatty acid concentration, and microbial populations. The results revealed that in the two-stage AD system, the methanogenic phase (MP) exhibited the highest removal rates of CEZ-resistant (CEZ-r) and oxytetracycline-resistant (OTC-r) bacteria at 9–16 % and 69–72 %, respectively. However, the MP had a higher proportion of ARB among the total culturable bacteria (24–30 %) compared to the acidogenic phase (AP) and single-stage AD. CEZ negatively impacted ARB removal rates and biogas production in both systems, with a more pronounced effect in single-stage AD. Biogas yield in the two-stage AD ranged from 337.7–385.6 mL/gVS, which was 10–20 % higher than that of the single-stage AD, regardless of CEZ addition. The reduction in biogas production due to CEZ was primarily attributed to the suppression of VFA production. As for microbial populations, the changes in the percentages of Firmicutes, Bacteroidetes, and Proteobacteria were likely related to the variations in ARB numbers, and the abundance of Methanosaetaceae significantly influenced biogas production.

Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

Selection of antibiotic resistance in pigs after exposure to feed cross-contaminated with oxytetracycline

Due to possible cross-contamination of animal feedstuff with antibiotics, food-producing animals may be exposed to undesirable low concentrations of antimicrobials. These sub-therapeutic levels of antibiotics can lead to the selection of resistant bacteria in the animal gut. The goal of this study was to assess, through analysis of the faeces of treated and control pigs, the risk of resistant E. coli being selected after daily exposure for three weeks to feed contaminated with oxytetracycline at 1% of the therapeutic dose. Liquid Chromatography coupled to tandem Mass Spectrometry was used to determine the oxytetracycline concentrations in faecal samples. In the treated group, concentrations were in the range of 4481.9 – 8671.2 µg/kg. In the control group, these concentrations were either below the method’s limit of quantification or up to 60.5 µg/kg. After a transient increase in resistance in both groups, microbiological analysis showed that the treated group had a significantly higher oxytetracycline resistance rate by the end of the study than the control group (p < 0.001). Furthermore, the treated animals were found to select co-resistances to nalidixic acid and ampicillin. Finally, at tolerated antibiotic contamination levels of feed, the treated group had a higher proportion of multidrug-resistant isolates at the end of the study than the control one (p < 0.05). The present study demonstrates that, at the tolerated contamination rates, both antimicrobial resistance and multidrug-resistant bacteria can be selected and evidenced in the gut microbiota.

Oxytetracycline-resistant heterotrophic fecal bacteria from pigs of Kabalasan, Baybay City, Leyte, Philippines

The excessive use of oxytetracycline for the cost-efficient production of swine can increase the development of antibiotic resistance among pathogenic bacteria. This study was conducted to isolate and identify putative cultivable heterotrophic bacteria from pig feces and evaluate their resistance to oxytetracycline. Composite fecal samples were serially diluted and plated using Tryptic Soy Agar (TSA). Distinct colonies were selected and grown on TSA plates and the pure cultures were subjected to limited screening for morphological and physiological characteristics. Representative isolates were then identified and subjected to oxytetracycline susceptibility assay through the disk diffusion method. Thirteen out of 15 selected colonies from TSA plates were successfully cultured and grouped into eight groups based on the isolates' morphological and physiological characteristics. Of the eight representative isolates, three were identified as putative Escherichia coli, two were Staphylococcus aureus strains, Staphylococcus vitulinus, Bacillus cereus and Proteus mirabilis. Furthermore, S. vitulinus and S. aureus strains exhibited susceptibility to oxytetracycline. Meanwhile, isolates, P. mirabilis and the three strains of E. coli showed the highest resistance while B. cereus showed the least. These oxytetracycline-resistant isolates can be used as test organisms to determine the antibacterial effect of novel bioactive compounds or as potential sources of oxytetracycline resistance genes that could be used as selection markers in transgenic experiments.

Prevalence of Escherichia coli isolated from oropharynx and trachea of clinically sick poultry and antimicrobial resistance pattern of the strains isolated

The study was carried out to investigate the isolation of Escherichia coli from tracheal and oropharyngeal swab of clinically sick chickens. The antibiotic susceptibility patterns of the isolates to several antimicrobials were determined with a striking emphasis on oxytetracycline. The PCR technique was applied to detect tetA, tetB, and tetC in the tetracycline-resistant isolates. The isolates were initially screened for their resistance patterns against 6 antimicrobials of six different groups using the disc diffusion technique. The results showed that 41% tracheal, 51% oropharyngeal, and 34% samples from both sites were E. coli positive respectively. Antimicrobial resistance profiling of the isolates revealed that all the isolates were resistant to oxytetracycline and sulphamethoxazole-trimethoprim, and also 90 %, 82.9%, 63.4%, and 39% resistant to ciprofloxacin, amoxicillin, gentamicin, and colistin respectively. Notably, 82.9% isolates (95% CI 68.4%–91.8%) showed resistance to ≥3 groups of antimicrobials that means these were multi-drug resistant. Among the tetracycline-resistant isolates, 85.4% (95% CI 71.2%–93.5%), 29.3% (7.5%–44.6%), and 7.3% (1.8% - 20.1) were positive for tetA, tetB, and tetC genes respectively. The frequency of the isolation of E. coli is greater in oropharyngeal than tracheal and both kinds of samples. Commercial poultry with E. coli strains has acquired extensive resistance to oxytetracycline. This study suggests a possible association between the tetA gene and oxytetracycline resistance in E. coli isolates, but further investigations like knockdown, whole-genome sequencing, and rescue experiments are needed to establish a direct causal relationship.

Oxytetracycline-resistant Paenibacillus larvae identified in commercial beekeeping operations in Saskatchewan using pooled honey sampling.

American foulbrood (AFB) is an infectious disease of honey bee brood caused by the endospore-forming bacterium Paenibacillus larvae. P. larvae spores are resilient in the environment, thus colonies with clinical signs of AFB are often destroyed by burning to eradicate the causative agent. To prevent outbreaks of AFB, oxytetracycline metaphylaxis is widely used in North America, resulting in sustained selective pressure for oxytetracycline resistance in P. larvae. To determine if antimicrobial resistance (AMR) is present among P. larvae isolates from commercial beekeeping operations in Saskatchewan, Canada, we performed antimicrobial susceptibility testing of 718 P. larvae samples cultured from pooled, extracted honey collected from 52 beekeepers over a 2-y period, 2019 and 2020. We found that 65 of 718 (9%) P. larvae samples collected from 8 beekeepers were resistant to oxytetracycline with minimum inhibitory concentration (MIC) values of 64-256 µg/mL. Eight of 718 (1%) samples from 4 beekeepers had intermediate resistance to oxytetracycline (MIC: 4-8 µg/mL). Susceptibility testing for tylosin and lincomycin indicated that P. larvae in Saskatchewan continue to be susceptible to these antimicrobials (tylosin MIC: <1 µg/mL, lincomycin MIC: ≤2 µg/mL). Most oxytetracycline-resistant P. larvae samples were identified in northeastern Saskatchewan. Whole-genome sequence analysis identified the P. larvae-specific plasmid pMA67 with tetracycline-resistance gene tet(L) in 9 of 11 oxytetracycline-resistant P. larvae isolates sequenced. Our results highlight the advantage of using pooled, extracted honey as a surveillance tool for monitoring AMR in P. larvae.

Oxytetracycline resistance of Melissococcus plutonius strains in Japan

European foulbrood caused by the Gram-positive lanceolate coccus, Melissococcus plutonius, is a major bacterial infection of honey bee brood. Oxytetracycline (OTC) has been used to combat this disease in some countries. Although OTC resistance was not previously reported despite its long-term use, the first case was finally detected in an M. plutonius strain isolated in Canada in 2019. In Japan, OTC has not been approved for use in the control of any honey bee diseases. However, after testing 77 M. plutonius strains isolated in Japan between the 1980s and 2010s, we identified four OTC-resistant strains with a minimum inhibitory concentration of OTC on agar medium of 16 µg/ml. The four strains were isolated in 2008 (1 strain), 2013 (1 strain), and 2015 (2 strains); i.e., before the Canadian OTC-resistant isolate. These results provide insights into the mechanisms underlying the development of OTC-resistant M. plutonius.

Genomics of Tenacibaculum Species in British Columbia, Canada.

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore's MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

Insight into the microbial and genetic response of anammox biomass to broad range concentrations of different antibiotics: Linking performance and mechanism

Antibiotics have become emerging pollutants occurring in wastewater, influencing the activity of microorganisms responsible for wastewater treatment. Moreover, the potential application of the anammox process in the treatment of antibiotic-containing wastewater has paid much attention. A common antibiotic, OTC (oxytetracycline), CIP (ciprofloxacin), and CLA (clarithromycin) are recognized to be monitored in wastewater due to their relatively high concentration and hazardous impact on the environment. However, their effect on the anammox process remains unknown. Therefore, this paper presents the study concerning the long-term effects of a successive concentration of three antibiotics (OTC, CIP, CLA) on the anammox process, with special emphasis on treatment efficiency, resistance mechanism, bacteria cell morphology, and activated sludge community structure. It is worth noting that the influence of a successive concentration of CIP and CLA has been studied for the first time. Results revealed that anammox community could adapt to CIP, OTC, and CLA at low concentrations (<1 mg L-1), while the high concentration of antibiotics (100 mg L-1) reduced the nitrogen removal rate (NRR) by 27 % (OTC), 30 % (CIP), and 56 % (CLA). Community structure analysis showed that the abundance of Planctomycetes increased with the increase of CIP and CLA concentration while decreasing under OTC stress. On contrary, other nitrogen-cycle bacteria (e.g., Nitrospira) contributed to the nitrogen removal, especially during antibiotic suppression. The abundance of corresponding ARGs (OTC resistance gens: tetX, tetC, tetW, CIP resistance gens: qnrB4, qnrS, CLA resistance gens: mphA) generally increased under antibiotic suppression. In addition, co-occurrence analysis showed that anammox bacteria might participate in the transfer of macrolide resistance genes. The findings of this study are essential in understanding the mechanisms of three antibiotics commonly occurring in wastewater during the anammox process. Moreover, the results have implications for using the anammox process for antibiotic-containing wastewater treatment and provide the operational guidance for stable conducting of the anammox process under antibiotics suppression.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017–2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTCR) Xap strains were found in 2017 and 2020 from four orchards about 20–270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 μg/mL OTC, while one strain, R1, grew on ≤250 μg/mL OTC. Genome sequence analysis of four representative OTCR strains revealed a 14–20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTCR Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 μg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 μg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 μg/mL OTC. However, in the R1 strain resistant to ≤250 μg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.

Subinhibitory Levels of Oxytetracycline in Earthworm Meal Significantly Boost Resistance-Mutation Rates in Bacillus spp. within the Gut of Eisenia fetida

Aims: To estimate changes in the number of oxytetracycline -resistant strains, a dynamic population of Bacillus in the gut of Eisenia fetida cultured in processed-cow-dung (PrCD) supplemented with subinhibitory amounts of oxytetracycline, was studied.&#x0D; Study Design: Sterile water containing oxytetracycline was sprayed over PrCD at concentration of 0.012 µg per mg dehydrated PrCD per spraying. The number of oxytetracycline-resistant Bacillus spp. microbiota of E. fetida’s gut was compared to the number of oxytetracycline-resistant Bacillus spp. in its feed (PrCD). For this purpose, Luria Agar (LA) and Bacillus Agar (BA) plates amended with or without oxytetracycline were used.&#x0D; Place and Duration of Study: The study was carried out in the Omics Laboratory of Department of Biotechnology, University of North Bengal situated in Darjeeling district of West Bengal, India during 2017-18.&#x0D; Methodology: E. fetida gut content and PrCD samples were collected on different days of the experiment for bacteriological analysis. Dilution plating on LA yielded the total number of cultivable heterotrophic bacteria. LA plates amended with oxytetracycline (15 µg/ml) were used to acquire the fraction of heterotrophic bacteria resistant to oxytetracycline. BA plates were also utilized to obtain the fraction of resistant Bacillus spp. population. The frequency of mutation was determined using a conventional formula.&#x0D; Results: In the gut of E. fetida reared in PrCD supplemented with sub-inhibitory concentration of oxytetracycline, a rise in the oxytetracycline-resistant Bacillus population was observed. On day 1, the frequency of oxytetracycline-resistant Bacillus spp. (5×10-8) matched the spontaneous mutation frequency, however higher frequencies on days 2 and 7 (1.6×10-4 and 3.5 x 10-6 respectively) suggested significant dissemination of oxytetracycline resistance in the gut environment.&#x0D; Conclusion: Subinhibitory oxytetracycline concentrations in earthworm diet had a significant effect on mutation rates, showing that evolutionary forces on the gut microbiota may be determining their responses to antibiotic stress.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt

BackgroundThe Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow’s milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA).ResultsThe majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans.ConclusionsThe STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

Prevalence and Risk Factors Associated With Antimicrobial Resistance in Bacteria Related to Bovine Respiratory Disease-A Broad Cross-Sectional Study of Beef Cattle at Entry Into Canadian Feedlots.

A broad, cross-sectional study of beef cattle at entry into Canadian feedlots investigated the prevalence and epidemiology of antimicrobial resistance (AMR) in Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, bacterial members of the bovine respiratory disease (BRD) complex. Upon feedlot arrival and before antimicrobials were administered at the feedlot, deep nasopharyngeal swabs were collected from 2,824 feedlot cattle in southern and central Alberta, Canada. Data on the date of feedlot arrival, cattle type (beef, dairy), sex (heifer, bull, steer), weight (kg), age class (calf, yearling), source (ranch direct, auction barn, backgrounding operations), risk of developing BRD (high, low), and weather conditions at arrival (temperature, precipitation, and estimated wind speed) were obtained. Mannheimia haemolytica, P. multocida, and H. somni isolates with multidrug-resistant (MDR) profiles associated with the presence of integrative and conjugative elements were isolated more often from dairy-type than from beef-type cattle. Our results showed that beef-type cattle from backgrounding operations presented higher odds of AMR bacteria as compared to auction-derived calves. Oxytetracycline resistance was the most frequently observed resistance across all Pasteurellaceae species and cattle types. Mycoplasma bovis exhibited high macrolide minimum inhibitory concentrations in both cattle types. Whether these MDR isolates establish and persist within the feedlot environment, requires further evaluation.

First Survey on the Presence and Distribution of Oxytetracycline-Resistance Genes in Anaplasma Species.

Anaplasma sp. is an obligatory intracellular Gram-negative tick-transmitted bacterial pathogen of humans and animals. Oxytetracycline and chlortetracycline are the drugs of choice for treating domestic animals with acute anaplasmosis. Lack of documented information about oxytetracycline resistance in Anaplasma species in the world was the scope of this study to screen by PCR for the detection of the oxytetracycline-resistance genes in Anaplasma species from infected cattle and sheep in the Northwest and Southwest of Iran. Total of 100 cattle and sheep blood samples collected from 2 provinces in the Northwest and 1 province in the Southwest of Iran were tested microscopically by the Giemsa staining examination and confirmed by PCR. Then the presence of two different oxytetracycline-resistance genes (otrA, and otrB) was detected by PCR in positive samples. The results showed that 60% of Anaplasma-infected samples were identified to have an otrA-resistance gene, and 26.67% had an otrB-resistance gene. The coexistence of two oxytetracycline-resistance determinants was encountered in 13.33% of the isolates. The significant difference in the frequency of otr genes was found among three Anaplasma species (A. marginale, A. centrale and A. ovis), and among three studied regions in Iran (p < 0.05). The identified sequences were submitted to the GenBank and deposited under accession numbers MN880729 and MN895439 for otrB and otrA genes. This study, for the first time, indicated the oxytetracycline-resistance genes in the three most prevalent Anaplasma species in ruminants. This finding helps to select an appropriate treatment strategy for eradication of anaplasmosis.

Novel Transposon Tn6433 Variants Accelerate the Dissemination of tet(E) in Aeromonas in an Aerobic Biofilm Reactor under Oxytetracycline Stresses.

Little is known about the mechanisms that disseminate antibiotic resistance genes (ARGs) in wastewater microbial communities under antibiotic stress. The role of horizontal transfer mechanisms in dissemination of ARGs in an aerobic biofilm reactor under incremental oxytetracycline doses from 0 to 50 mg/L was studied. Aeromonas strains were the most common culturable bacteria in the reactor, with tet(E) as the most prevalent ARGs (73.3%) being possibly responsible for the oxytetracycline resistance phenotype. Genomic sequencing demonstrated that tet(E) was mainly carried by a Tn3 family transposon named Tn6433, whose incidence increased from 14.6% to 75.0% across the treatments. Tn6433 carrying tet(E) was initially detected in Aeromonas chromosomes at an oxytetracycline dose of 1 mg/L but subsequently detected on plasmids pAeca1-a variants (pAeca1-a, pAeca1-b, and pAeme6) and pAeca2 under higher oxytetracycline stress. The core region of the Tn6433-tet(E) structure was highly conserved, consisting of a transposition and resolution module, a class 1 integron, core passenger genes, and a Tn1722/Tn501-like transposon. Such a structure was found on both the chromosome and plasmids, suggesting that Tn6433 mediated the transposition of tet(E) from the chromosome to plasmid pAeca2 under increasing stresses. Bacteria carrying the transferable plasmid pAeca1-a were dominant in high antibiotic treatments, suggesting that Tn6433 disseminated tet(E), conferring selective advantages to recipients of this ARG.

Distribution of oxytetracycline resistance genes in E. coli isolated from pigeon faecal samples

Tetracycline- resistant bacteria have emerged due to the selective pressure of antimicrobial use. The aim of this study was to determine the prevalence of oxytetracycline resistance of Escherichia coli from pigeon faecal samples. All strains were examined for the presence and types of oxytetracycline resistance determinants using disc diffusion and polymerase chain reaction methods. Of 50 faecal E. coli isolates, 30 (60%) were resistant to oxytetracycline. Polymerase chain reaction analyses indicated that the most common genes found in these isolates were tetB (43.3%) and tetA (30%). Only 10% and 3.3% of the isolates contained otrA and otrB, respectively. In conclusion, our findings suggest that oxytetracycline-resistant strains of E. coli are disseminated in pigeons.

Analysis of single nucleotide polymorphisms (SNPs) associated with antibiotic resistance genes in Chilean Piscirickettsia salmonis strains.

The aetiological agent of Piscirickettsiosis is Piscirickettsia salmonis, a Gram-negative intracellular pathogen, and high doses of antibiotics have regularly been employed to treat this infection. Seven florfenicol and/or oxytetracycline resistance genes (tet pump, tetE, Tclor/flor, Tbcr, TfloR, ompF and mdtN) were identified in strains by in silico genome analyses. Later, the number of single nucleotide polymorphisms (SNPs) and its relationship with the resistance to these antibiotics were identified and analysed, using the original LF-89 strain as reference. Trials to determine and compare the minimum inhibitory concentration (MIC) of oxytetracycline and florfenicol in each strain, as well as to quantify the gPCR transcripts levels in the selected genes, were performed. Therefore, variations in the resistance to both antibiotics were observed, where the strain with fewer SNPs showed the highest susceptibility. Consistently, the in silico 3D analyses of proteins encoded by the selected genes revealed structural changes, evident in the sequences with the highest number of SNPs. These results showed that the bacterial resistance to oxytetracycline was mainly linked to the presence of SNPs in relevant sites, antibiotic resistance genes and an OmpF porin, leading to important changes in the protein structure.

Antimicrobial resistance and genotypic profiles of Salmonella Saintpaul isolated along beef processing and distribution continuum

Salmonella Saintpaul (SSa) is increasingly reported from food and foodborne outbreak cases. Pulsed field gel electrophoresis (PFGE) is used for screening and tracking of Salmonella infections. Widespread use of antimicrobial agents in humans and food animals could result in antimicrobial resistant Salmonella serotypes. The aim of this study was to characterize S. Saintpaul (n = 28) isolated from various sampling locations at abattoir and meat processing plant lines in Ethiopia for phenotypic antimicrobial resistance and genotypic diversity, and to track its transfer routes. Sampling location, steps and occasions were considered for each isolate description. Antimicrobial sensitivity testing was performed against seven different antimicrobial agents using disc diffusion method. PFGE with XbaI® enzymatic genomic digestion with BioNumerics® analysis was used for genotypic diversity. Of all the isolates tested, only 17.9% were pan susceptible, and 82.1% were resistant to at least one and at most to three antimicrobials. All isolates were susceptible to gentamycin, trimethoprim-sulfamethoxazol and trimethoprim. Resistance to oxytetracycline (82.2%) was predominant followed by 3.6% resistance to each of chloramphenicol, neomycin and polymyxin B. PFGE analysis revealed three distinguishable clusters of pulsotypes but the majority of the isolates (25/28) belonged to cluster-I (SSaX1-4) pulsotype. Indistinguishable/similar cluster of (SSaX 1-4) isolates among and between sampling location, steps and occasions were observed. Majorities of S. Saintpaul (88%) in the cluster-I pulsotype were resistant to oxytetracycline. Our study indicated that oxytetracycline resistance is very common among the S. Saintpaul isolates studied; and the isolates were diverse with similar resistance profiles within the same genomic pulsotypes. Transfer of S. Saintpaul within, between and across sampling locations, during the same or different occasion were determined from SSaX 1-4 pulsotype while cluster-II (SSaX5) indicates transfer from abattoir to butchery. The unique isolate in cluster-III (SSaX6) shows the presence of other possible source of S. Saintpaul for the beef chain contamination.