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BACKGROUND: The obligate intracellular bacterial family Chlamydiaceae comprises a number of different species that cause disease in various vertebrate hosts including humans. Chlamydia suis, primarily found in the gastrointestinal tract of pigs, is the only species of the Chlamydiaceae family to have naturally gained tetracycline resistance (TetR), through a genomic island (Tet-island), integrated into the middle of chromosomal invasin-like gene inv. Previous studies have hypothesised that the uptake of the Tet-island from a host outside the Chlamydiaceae family was a unique event, followed by spread among C. suis through homologous recombination. In vitro recombination studies have shown that Tet-island exchange between C. suis strains is possible. Our aim in this study was to gain a deeper understanding of the interclade recombination of the Tet-island, among currently circulating C. suis field strains compared to in vitro-generated recombinants, using published whole genome sequences of C. suis field strains (n = 35) and in vitro-generated recombinants (n = 63). RESULTS: We found that the phylogeny of inv better reflected the phylogeny of the Tet-island than that of the whole genome, supporting recombination rather than site-specific insertion as the means of transfer. There were considerable differences between the distribution of recombinations within in vitro-generated strains compared to that within the field strains. These differences are likely because in vitro-generated recombinants were selected for a tetracycline and rifamycin/rifampicin resistant background, leading to the largest peak of recombination across the Tet-island. Finally, we found that interclade recombinations across the Tet-island were more variable in length downstream of the Tet-island than upstream. CONCLUSIONS: Our study supports the hypothesis that the occurrence of TetR strains in both clades of C. suis came about through interclade recombination after a single ancestral horizontal gene transfer event.

Simple Summary: Chlamydia spp. are obligate intracellular bacteria mostly known to cause fertility problems and trachoma in humans but also cause a variety of diseases of major interest in animals. Since some veterinary chlamydial species are known to be zoonotic, it is important to monitor the entire chlamydial outcomes worldwide in humans and animals. Pig farmers and veterinarians are in close contact with swine chlamydia every day. What is not yet fully known is the clinical role that chlamydia plays in swine health, especially in the context of fertility problems. There are suspicions that chlamydia in the genital tract of sows may play a role in infertility, but there is currently no evidence of this. This survey was conducted among veterinarians who diagnose daily in pig farms and must make decisions regarding treatment and prevention. Practitioners are calling for significant action and support from the scientific community because their decisions on diagnosis, therapy, and prevention are often based on intuition rather than scientifically founded knowledge, due to the lack of scientifically validated guidance on control strategies including treatment length, treatment dose, and disinfection procedures. This would not only be important for providing adequate veterinary care to the sows but also in terms of the One Health principle. Background: Although it is generally accepted that Chlamydia spp. can induce fertility problems in sows, many questions concerning diagnostic, therapeutic, and prophylactic approaches remain unanswered. The aim of this study was to gather information about how swine practitioners deal with these knowledge gaps. Methods: Austrian swine practitioners were anonymously asked for their assessment of chlamydiosis using the Lime Survey program. Results: PCR tests are used for molecular chlamydia detection from abortion material (64.3%), vaginal swabs (40.5%), and the genital tract (postmortem slaughterhouse samples) (9.5%). Treatment mainly includes tetracyclines (95.2%), occasionally macrolides (2.4%) or other (not mentioned) antibiotic groups (7.1%). Treatment periods vary greatly, from 5 to 10 days (23.8%), from 11 to 15 days (47.6%), from 16 to 21 days (11.9%), and >21 days (9.5%). Prophylactic measures include hygiene improvement, rodent control, and herd treatments with tetracyclines. Conclusions: Serological methods are perceived as inadequate, and there is a general need for better guidance on interpreting results. Knowledge about the increased incidence of tetracycline-resistant Chlamydia suis strains is limited. Weighing up economic interests, feasibility, and animal welfare plays an important role. Further research to improve diagnosis and prevention of chlamydia-related fertility diseases in sows is urgently needed. [ABSTRACT FROM AUTHOR]

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of cooccurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans. [ABSTRACT FROM AUTHOR]

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.

ABSTRACT Drug efflux systems have recently been recognized as a significant mechanism responsible for multidrug resistance in bacteria. In this study, we described the identification and characterization of a new chromosomally encoded efflux pump (SA00565) in Staphylococcus aureus. SA00565, which belongs to the drug/metabolite transporter (DMT) superfamily, was predicted to be a 10-transmembrane segment transporter. To evaluate the role of sa00565 in resistance, we generated sa00565 gene deletion mutant (Δsa00565) and assessed its susceptibility to 35 different antibiotic treatments. Our results demonstrated that the Δsa00565 mutant exhibited reduced resistance to tetracycline and doxycycline, with 64-fold and 12-fold decreased MICs, respectively. The mechanism of SA00565-mediated tetracycline resistance was demonstrated that SA00565 possesses the capability to efficiently extrud intracellular tetracycline into the environment. The efflux activity of SA00565 was further validated using EtBr accumulation and efflux assays. In summary, our study uncovered a previously unknown function of a DMT family transporter, which serves as a tetracycline efflux pump, thereby contributing to tetracycline resistance in S. aureus.IMPORTANCEIn this study, we addressed the significance of drug efflux systems in multidrug resistance of Staphylococcus aureus, focusing on the unexplored efflux pump SA00565 in the drug/metabolite transporter (DMT) superfamily. Through phylogenetic analysis, gene knockout, and overexpression experiments, we identified the role of SA00565 in antibiotic resistance. The Δsa00565 mutant showed increased susceptibility to tetracycline and doxycycline in disk diffusion assays, with significantly lower MICs compared to the WT. Remarkably, intracellular tetracycline concentration in the mutant was two- to threefold higher, indicating SA00565 actively eliminates intracellular tetracycline. Our findings emphasize the pivotal contribution of SA00565 to tetracycline antibiotic resistance in S. aureus, shedding light on its functional attributes within the DMT superfamily and providing valuable insights for combating multidrug resistance.

Background: Although it is generally accepted that Chlamydia spp. can induce fertility problems in sows, many questions concerning diagnostic, therapeutic, and prophylactic approaches remain unanswered. The aim of this study was to gather information about how swine practitioners deal with these knowledge gaps. Methods: Austrian swine practitioners were anonymously asked for their assessment of chlamydiosis using the Lime Survey program. Results: PCR tests are used for molecular chlamydia detection from abortion material (64.3%), vaginal swabs (40.5%), and the genital tract (postmortem slaughterhouse samples) (9.5%). Treatment mainly includes tetracyclines (95.2%), occasionally macrolides (2.4%) or other (not mentioned) antibiotic groups (7.1%). Treatment periods vary greatly, from 5 to 10 days (23.8%), from 11 to 15 days (47.6%), from 16 to 21 days (11.9%), and >21 days (9.5%). Prophylactic measures include hygiene improvement, rodent control, and herd treatments with tetracyclines. Conclusions: Serological methods are perceived as inadequate, and there is a general need for better guidance on interpreting results. Knowledge about the increased incidence of tetracycline-resistant Chlamydia suis strains is limited. Weighing up economic interests, feasibility, and animal welfare plays an important role. Further research to improve diagnosis and prevention of chlamydia-related fertility diseases in sows is urgently needed.

The dissemination of antimicrobial-resistant bacteria through environment is a major health concern for public health. Pathogenic bacteria in natural environment can mediate the transfer of antimicrobial-resistant genes via horizontal gene transfer to naturally occurring bacteria in the soil. Bhargavaea beijingensis is a Gram-negative bacterium that is commonly found in soil and water. In recent years, there has been an emergence of antibiotic-resistant strains of environmental bacteria, which pose a significant threat to human health. One mechanism of antibiotic resistance in bacteria is through the acquisition of plasmids, which can carry genes that confer resistance to various antibiotics. In this study, a novel plasmid of repUS12 replicon type was identified in the strain PS04 of B. beijingensis, which carried the ermT and tet(L) genes, encoding resistance to macrolides, lincosamides, and tetracycline. The plasmid was found to be the first of its kind in B. beijingensis and was thought to have been acquired through horizontal gene transfer. The emergence of plasmid-mediated resistance in B. beijingensis highlights the need for continued surveillance and monitoring of antibiotic resistance in environmental bacteria. [ABSTRACT FROM AUTHOR]

Acinetobacter spp. and other non-fermenting Gram-negative bacteria (NFGNB) represent an important group of opportunistic pathogens due to their propensity for multiple, intrinsic, or acquired antimicrobial resistance (AMR). Antimicrobial resistant bacteria and their genes can spread to the environment through livestock manure. This study investigated the effects of fresh manure from dairy cows under antibiotic prophylaxis on the antibiotic resistome and AMR hosts in microcosms using pasture soil. We specifically focused on culturable Acinetobacter spp. and other NFGNB using CHROMagar Acinetobacter. We conducted two 28-days incubation experiments to simulate natural deposition of fresh manure on pasture soil and evaluated the effects on antibiotic resistance genes (ARGs) and bacterial hosts through shotgun metagenomics. We found that manure application altered the abundance and composition of ARGs and their bacterial hosts, and that the effects depended on the soil source. Manure enriched the antibiotic resistome of bacteria only in the soil where native bacteria had a low abundance of ARGs. Our study highlights the role of native soil bacteria in modulating the consequences of manure deposition on soil and confirms the potential of culturable Acinetobacter spp. and other NFGNB to accumulate AMR in pasture soil receiving fresh manure. [ABSTRACT FROM AUTHOR]

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Background and Aim: Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Materials and Methods: : Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. Results: tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). Conclusion: The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health.

Background and Objectives: An increase in the antibiotic resistance of Shigella isolates has caused major global challenges in antimicrobial therapy. Knowledge of local antibiotic resistance trends is essential for selecting appropriate antibiotic treatment regimens. This study aimed to evaluate the frequency of efflux-mediated tetracycline resistance (tet) and plasmid-mediated quinolone resistance (qnr) genes among Shigella isolates. Materials and Methods: This survey investigated 91 Shigella isolates, obtained from children with acute diarrhea. The isolates were identified using standard biochemical tests and confirmed by polymerase chain reaction (PCR) assay. Besides, the susceptibility of isolates to six selected antibiotics was assessed by the disk diffusion method. All tetracycline-resistant and nalidixic acid and ciprofloxacin resistant strains were screened for tet and qnr genes by a multiplex PCR assay. Results: According to the results of antibiotic susceptibility tests, the highest level of antibiotic resistance was related to tetracycline (80.2%) and doxycycline (78.1%), respectively. All isolates were sensitive to tigecycline. The PCR results showed that 40.6%, 3.1%, 21.8%, 61.6% and 28.7% of the isolates carried qnrA, qnrB, qnrS, tetA, and tetB genes, respectively. None of the isolates contained tetC and tetD genes. Conclusion: The current findings revealed that tetA and qnrA genes might play a key role in conferring tetracycline and quinolone resistance. [ABSTRACT FROM AUTHOR]

Gemella is a catalase-negative, facultative anaerobic, Gram-positive coccus that is commensal in humans but can become opportunistic and cause severe infectious diseases, such as infective endocarditis. Few studies have tested the antimicrobial susceptibility of Gemella. We tested its antimicrobial susceptibility to 27 drugs and defined the resistant genes using PCR in 58 Gemella strains, including 52 clinical isolates and six type strains. The type strains and clinical isolates included 22 G. morbillorum, 18 G. haemolysans (GH) group (genetically indistinguishable from G. haemolysans and G. parahaemolysans), 13 G. taiwanensis, three G. sanguinis, and two G. bergeri. No strain was resistant to beta-lactams and vancomycin. In total, 6/22 (27.3%) G. morbillorum strains were erythromycin- and clindamycin-resistant ermB-positive, whereas 4/18 (22.2%) in the GH group, 7/13 (53.8%) G. taiwanensis, and 1/3 (33.3%) of the G. sanguinis strains were erythromycin-non-susceptible mefE- or mefA-positive and clindamycin-susceptible. The MIC90 of minocycline and the ratios of tetM-positive strains varied across the different species—G. morbillorum: 2 µg/mL and 27.3% (6/22); GH group: 8 µg/mL and 27.8% (5/18); G. taiwanensis: 8 µg/mL and 46.2% (6/13), respectively. Levofloxacin resistance was significantly higher in G. taiwanensis (9/13 69.2%) than in G. morbillorum (2/22 9.1%). Levofloxacin resistance was associated with a substitution at serine 83 for leucine, phenylalanine, or tyrosine in GyrA. The mechanisms of resistance to erythromycin and clindamycin differed across Gemella species. In addition, the rate of susceptibility to levofloxacin differed across Gemella sp., and the quinolone resistance mechanism was caused by mutations in GyrA alone. [ABSTRACT FROM AUTHOR]

Background and Aim: Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Materials and Methods: Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. Results: tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). Conclusion: The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health. [ABSTRACT FROM AUTHOR]

In this study, a culture-independent approach was applied to compare the microbiome composition and the abundance of the antimicrobial resistance genes (ARGs) aadA2 for aminoglycosides, tet(A), tet(B), tet(K), and tet(M) for tetracyclines, and mcr-1 for colistin in broiler litter samples collected from conventional and antibiotic-free flocks located in Central Italy. A total of 13 flocks and 26 litter samples, collected at the beginning and at the end of each rearing cycle, were submitted to 16s rRNA sequence analysis and quantitative PCR for targeted ARGs. Firmicutes resulted in the dominant phylum in both groups of flocks, and within it, the Clostridia and Bacilli classes showed a similar distribution. Conversely, in antibiotic-free flocks, a higher frequency of Actinobacteria class and Clostridiaceae, Lactobacillaceae, Corynebacteriaceae families were reported, while in the conventional group, routinely treated with antibiotics for therapeutic purposes, the Bacteroidia class and the Enterobacteriaceae and Bacillaceae families were predominant. All investigated samples were found to be positive for at least one ARG, with the mean values of aadA2 and tet(A) the highest in conventional flocks by a significant margin. The results suggest that antibiotic use can influence the frequency of resistance determinants and the microbial community in poultry flocks, even though other environmental factors should also be investigated more deeply in order to identify additional drivers of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

The global concern over antimicrobial resistance (AMR) and its impact on human health is evident, with approximately 4.95 million annual deaths attributed to antibiotic resistance. Regions with inadequate water, sanitation, and hygiene face challenges in responding to AMR threats. Enteric bacteria, particularly E. coli, are common agents linked to AMR-related deaths (23% of cases). Culture-based methods for detecting tetracycline-resistant E. coli may be of practical value for AMR monitoring in limited resource environments. This study evaluated the ColiGlow™ method with tetracycline for classifying tetracycline-resistant E. coli. A total of 61 surface water samples from Kentucky, USA (2020–2022), provided 61 presumed E. coli isolates, of which 28 isolates were obtained from tetracycline-treated media. Species identification and tetracycline resistance evaluation were performed. It was found that 82% of isolates were E. coli, and 18% were other species; 97% were identified as E. coli when using the API20E identification system. The MicroScan system yielded Enterobacter cloacae false positives in 20% of isolates. Adding tetracycline to ColiGlow increased the odds of isolating tetracycline-resistant E. coli 18-fold. Tetracycline-treated samples yielded 100% tetracycline-resistant E. coli when the total E. coli densities were within the enumeration range of the method. ColiGlow with tetracycline shows promise for monitoring tetracycline-resistant E. coli in natural waters and potentially aiding AMR surveillance in resource-limited settings among other environments. [ABSTRACT FROM AUTHOR]

Background and Aim: Slaughterhouses and their effluents could serve as a "hotspot" for the occurrence and distribution of antibiotic-resistant bacteria in the environment. This study aimed to understand the distribution of tetracycline resistance genes in Escherichia coli isolated from the floor surface and effluent samples of pig slaughterhouses in Banten Province, Indonesia. Materials and Methods: Ten samples, each from floor surface swabs and effluents, were collected from 10 pig slaughterhouses in Banten Province. Escherichia coli strains were isolated and identified by referring to the protocol of the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli from the WHO (2021). Quantitative real-time polymerase chain reaction (qPCR) was used to detect the tet genes. Results: The tetA, tetB, tetC, tetM, tetO, and tetX genes were distributed in the isolates from the floor surface samples, and the tetA, tetC, tetE, tetM, tetO, and tetX genes were distributed in the isolates from the effluent samples. The tetO gene (60%) was the most dominant gene in the isolates from floor surface samples, while the tetA gene was the dominant one in the isolates from the effluent samples (50%). The tetA + tetO gene combination was the dominant pattern (15%) in the E. coli isolates. Conclusion: The high prevalence and diversity of the tet genes in floor surface and effluent samples from pig slaughterhouses in Banten Province indicated that the transmission of the tet genes had occurred from pigs to the environment; thus, this situation should be considered a serious threat to public health.

Zoonotic transmission is likely a pathway for antibiotic resistance. Data from a randomized trial of pediatric antibiotic administration were secondarily evaluated to determine if poultry ownership was significantly associated with the presence of gut genetic antibiotic resistance determinants among 118 children in Burkina Faso. Antimicrobial resistance (AMR) determinants were classified using DNA sequencing. We measured the relationship between genetic resistance determinants and chicken ownership using a logistic regression model adjusted for confounding variables. Children in households reporting poultry ownership had four times the odds of tetracycline resistance determinants in the gut compared with those without household poultry (odds ratio [OR]: 4.08, 95% CI: 1.08-15.44, P = 0.04). There was no statistically significant difference found for other antibiotic classes. Understanding the origins of antibiotic resistance may help spur the development of interventions to combat the global AMR crisis.

Background and Objectives: An increase in the antibiotic resistance of Shigella isolates has caused major global challenges in antimicrobial therapy. Knowledge of local antibiotic resistance trends is essential for selecting appropriate antibiotic treatment regimens. This study aimed to evaluate the frequency of efflux-mediated tetracycline resistance (tet) and plasmid-mediated quinolone resistance (qnr) genes among Shigella isolates. Materials and Methods: This survey investigated 91 Shigella isolates, obtained from children with acute diarrhea. The isolates were identified using standard biochemical tests and confirmed by polymerase chain reaction (PCR) assay. Besides, the susceptibility of isolates to six selected antibiotics was assessed by the disk diffusion method. All tetracycline-resistant and nalidixic acid and ciprofloxacin resistant strains were screened for tet and qnr genes by a multiplex PCR assay. Results: According to the results of antibiotic susceptibility tests, the highest level of antibiotic resistance was related to tetracycline (80.2%) and doxycycline (78.1%), respectively. All isolates were sensitive to tigecycline. The PCR results showed that 40.6%, 3.1%, 21.8%, 61.6% and 28.7% of the isolates carried qnrA, qnrB, qnrS, tetA, and tetB genes, respectively. None of the isolates contained tetC and tetD genes. Conclusion: The current findings revealed that tetA and qnrA genes might play a key role in conferring tetracycline and quinolone resistance.

The impact of in-feed use of tylosin in feedlot cattle on Gram-negative foodborne bacteria is unknown. We evaluated the effect of continuous in-feed tylosin use on the concentration and prevalence of tetracycline-resistant (TETr)-, third-generation cephalosporin-resistant (3GCr)-, and extended-spectrum β-lactamase–producing (ESBLs) E. coli in feedlot cattle. A cohort of weaned calves (10 animals/group) were randomized to receive a feed ration with or without tylosin. Fecal samples, regularly collected over the entire feeding period, and pen surface and feed samples, collected at the end of the feeding period, were cultured on selective media. Enumeration and binary outcomes were analyzed by mixed effects linear regression or logistic regression, respectively, using treatment and days on feed as fixed factors, and animal ID as a random variable. Tylosin supplementation did not affect the fecal concentrations of TETr E. coli or fecal prevalence of 3GCr E. coli. However, cattle in the tylosin group were 1.5 times more likely (Odds ratio = 1.5: 95% confidence interval: 1.1–2.0) to harbor ESBLs E. coli than the control cattle. Regardless of tylosin treatment, fecal concentrations of TETr E. coli and the prevalence of 3GCr- and ESBLs-E. coli increased over time. Tylosin-supplemented feed did not affect the prevalence of TETr E. coli; 3GCr and ESBLs-E. coli were not detected from the feed samples. Most of the 3GCr- and ESBLs-E. coli isolates carried the blaCTX-M-15 gene, widely detected among ESBLs-E. coli human isolates. In summary, although in-feed tylosin use in feedlot cattle did not select for TETr- and 3GCr-E. coli, it increased the likelihood of detecting ESBL-producing E. coli. Furthermore, the study indicated that the feedlot production setting gradually increases the levels of E. coli resistant to the critically and/or important antibiotics for public health, indicating an increased risk of their dissemination beyond the feedlot environment.

Lactic acid bacteria are used in industry as starter cultures or probiotics. In the report published by the European Food Safety Authority in 2021, it was emphasized that risk assessment should be carried out on the whole genome sequences of bacteria to be used in foods. Therefore, resistance gene research in lactic acid bacteria has gained importance. Because antibiotic resistance genes are likely to be transmitted to pathogenic bacteria in the intestinal tract or are likely to be ingested through foods containing lactic acid bacteria. Therefore, in this study, four lactic acid bacteria (Lentilactobacillus buchneri Egmn17, Levilactobacillus brevis Atlas17, Levilactobacillus namurensis Ozge01, Lactiplantibacillus plantarum Gmze16) isolated from different fermented foods and Lactiplantibacillus plantarum, a probiotic bacterium, were used. In the study, tetracycline, which has the most common antibiotic resistance among lactic acid bacteria, was selected. It was determined that 3 bacteria were moderately resistant (zone diameter 15-18 mm) (299v, Gmze16 and Egmn17) to tetracycline antibiotic and 2 bacteria were susceptible (zone diameter >19 mm) (Atlas17 and Ozge01). As a result of examining the whole genome sequences of lactic acid bacteria, it was seen that intermediate resistant bacteria have tetracyclinedependent antimicrobial resistance (AMR) genes tetA (MFS efflux pump) and tetO (ribosomal protection protein). In Levilactobacillus brevis Atlas17, while TetA protein was present, MT change was observed in the 322nd amino acid sequence. In addition, the fact that the probiotic bacteria Lactiplantibacillus plantarum 299v has resistance genes also increases the risk of transferring these genes to pathogens in the intestine. Species that are susceptible to the phenotype, such as Levilactobacillus brevis Atlas17, which has been observed to have the tetA gene, are also likely to be able to pass it on to other bacteria when they have silent resistance genes. Therefore, it is important to examine genotype and phenotype together. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance. Methods: Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform. Results: The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes. Conclusions: Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDITet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.

Background and Aim: Slaughterhouses and their effluents could serve as a “hotspot” for the occurrence and distribution of antibiotic-resistant bacteria in the environment. This study aimed to understand the distribution of tetracycline resistance genes in Escherichia coli isolated from the floor surface and effluent samples of pig slaughterhouses in Banten Province, Indonesia. Materials and Methods: Ten samples, each from floor surface swabs and effluents, were collected from 10 pig slaughterhouses in Banten Province. Escherichia coli strains were isolated and identified by referring to the protocol of the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli from the WHO (2021). Quantitative real-time polymerase chain reaction (qPCR) was used to detect the tet genes. Results: The tetA, tetB, tetC, tetM, tetO, and tetX genes were distributed in the isolates from the floor surface samples, and the tetA, tetC, tetE, tetM, tetO, and tetX genes were distributed in the isolates from the effluent samples. The tetO gene (60%) was the most dominant gene in the isolates from floor surface samples, while the tetA gene was the dominant one in the isolates from the effluent samples (50%). The tetA + tetO gene combination was the dominant pattern (15%) in the E. coli isolates. Conclusion: The high prevalence and diversity of the tet genesin floor surface and effluent samples from pig slaughterhouses in Banten Province indicated that the transmission of the tet genes had occurred from pigs to the environment; thus, this situation should be considered a serious threat to public health. [ABSTRACT FROM AUTHOR]

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the "One Health" approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene blaOXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments. [ABSTRACT FROM AUTHOR]

The rearing of chicken in India has undergone a drastic change from backyard production to commercial intensive farming. This has led to the use of antibiotics in therapy, metaphylaxis and as growth promoters. Escherichia coli are commensals that inhabit the gut of man and animals. The detection of virulent pathotypes of E. coli in chicken is a huge threat to human health. The present study assessed 200 retail chicken sold in central Kerala for the presence of virulent E. coli and studied the tetracycline susceptibility followed by detection of tetA and tetB gene. E. coli was detected in 64 per cent of the samples. The virulence genes, eaeA and aggR were detected in 52.2 and 3.9 per cent of the E. coli isolates, respectively. Tetracycline resistance by antibiotic susceptibility testing (ABST) was found in 30.84 per cent of the virulent isolates. The tetracycline efflux pump protein coding genes, tetA and tetB were detected in 56.67 and 25 per cent, respectively. The detection of drug resistant bacteria is a threat to public health as tetracycline is classified as a highly important antibiotic in human medicine.

The ecological state of Lake Sevan, the largest drinking water reservoir for the South Caucasus, formed under the influence of climatic and social changes. This study assesses the bacteriological quality of water in the rivers of the Lake Sevan basin and tetracycline-resistant bacteria isolated from fish and people living near the rivers of the Lake Sevan basin in Armenia in autumn 2019 and spring 2020. No differences have been shown for the tetracycline resistance of the investigated E. coli isolated from the human gut and the Masrik, Argichi, and Gavaraget Rivers. Horizontal gel electrophoresis revealed the same plasmid bands in most of the investigated E. coli with the same tetracycline resistance from the different sources of the Argichi River (obtained from people/fish/water sources where the fish were caught). The results also showed that most of the waters carried Edwardsiella spp. Erwinia spp. Morganella spp. and Proteus spp. in addition to E. coli ; the coliform index did not exceed the standard level of 5 × 104 CFU mL-1 there. These findings highlight the importance of multidisciplinary studies of bacteria from "interacting" ecosystems, which might serve as a basis for the suggestion of microbial antibiotic resistance as another indicator of water pollution. [ABSTRACT FROM AUTHOR]

This study aimed to estimate the antimicrobial resistance (AMR) patterns and tetracycline-resistant gene profiles of Escherichia coli (E. coli) from broiler meat and livers sourced from live bird markets (LBMs) and supermarkets (SMs) in Chattogram, Bangladesh. In total, 405 samples were collected from SMs and LBMs, comprising muscle (n = 215) and liver (n = 190) samples. Disc diffusion tests were used to determine antimicrobial susceptibility profiles. PCR was used to identify E. coli and tetracycline-resistant genes. Over half (57%) of the chicken product samples were positive for E. coli. The AMR profiling of these isolates showed that the highest prevalence of resistance was against sulphamethoxazole–trimethoprim (89%), followed by tetracycline (87%), ampicillin (83%), and ciprofloxacin (61%). Among the antimicrobials listed by the World Health Organization as critically important, E. coli isolates were found to be resistant to cephalexin (37%), gentamicin (32%), and colistin sulfate (21%). A large proportion of E. coli demonstrated multi-drug resistance (MDR). Most (84%) of the tetracycline-resistant isolates encoded tetA. Of the remaining isolates, 0.5% encoded tetC, 6.0% encoded two genes, and 3.6% of isolates were tetD, which was newly identified by this study in Bangladesh. Broiler products in Bangladesh are frequently contaminated with multi-drug-resistant E. coli, with differential carriage of tetracycline genes. The prevalence of tetracycline resistance among E. coli indicates a concern for poultry health and welfare regarding the management of colibacillosis. It also indicates growing public health risks of AMR among broiler-associated pathogens, which can be transferred to humans via the food chain. Appropriate control measures should be developed and implemented, focused on the rational use of antimicrobials in poultry farming systems, to mitigate risk from this drug-resistant zoonotic pathogen from foods of animal origin and to protect public health. [ABSTRACT FROM AUTHOR]

There have been efforts dedicating to investigating the effect of high levels of oxytetracycline (OTC) on tetracycline (TC) resistance among zoonotic pathogen Aeromonas hydrophila (AH), an antibiotic-resistant bacterial indicator. However, the dynamic behavior of TC-resistant AH in response to environmentally relevant OTC concentrations at a population-level has not been fully understood. Here we developed a bacterial population dynamic model to quantify TC-resistant AH posed by OTC-dependent resistance selection pressure. The key environmental factors known as water temperature, water activity, and pH were incorporated into model to produce pattern-oriented simulation outcomes. We estimated resistance acquisition number (R0) and showed that R0 was >1 at water temperature

Treatment of food-producing animals with antimicrobial drugs (AMD) is controversial because of concerns regarding promotion of antimicrobial resistance (AMR). To investigate this concern, resistance genes in metagenomic bovine fecal samples during a clinical trial were analyzed to assess the impacts of treatment on beef feedlot cattle resistomes. Four groups of cattle were exposed, using a 2-by-2 factorial design, to different regimens of antimicrobial treatment. Injections of ceftiofur crystalline-free acid (a third-generation cephalosporin) were used to treat all cattle in treatment pens or only a single animal, and either chlortetracycline was included in the feed of all cattle in a pen or the feed was untreated. On days 0 and 26, respectively, pre- and posttrial fecal samples were collected, and resistance genes were characterized using shotgun metagenomics. Treatment with ceftiofur was not associated with changes to β-lactam resistance genes. However, cattle fed chlortetracycline had a significant increase in relative abundance of tetracycline resistance genes. There was also an increase of an AMR class not administered during the study, which is a possible indicator of coselection of resistance genes. Samples analyzed in this study had previously been evaluated by culture characterization (Escherichia coli and Salmonella) and quantitative PCR (qPCR) of metagenomic fecal DNA, which allowed comparison of results with this study. In the majority of samples, genes that were selectively enriched through culture and qPCR were not identified through shotgun metagenomic sequencing in this study, suggesting that changes previously documented did not reflect changes affecting the majority of bacterial genetic elements found in the predominant fecal resistome.IMPORTANCE Despite significant concerns about public health implications of AMR in relation to use of AMD in food animals, there are many unknowns about the long- and short-term impact of common uses of AMD for treatment, control, and prevention of disease. Additionally, questions commonly arise regarding how to best measure and quantify AMR genes in relation to public health risks and how to determine which genes are most important. These data provide an introductory view of the utility of using shotgun metagenomic sequencing data as an outcome for clinical trials evaluating the impact of using AMD in food animals.

Omadacycline was tested against 125 isolates recovered from infected cat and dog bites in humans. Its activity was similar to that of other compounds in the tetracycline class, and it was active against strains exhibiting tetracycline resistance. Against anaerobic isolates, resistance to tetracyclines was more prominent and omadacycline was the most active of the group. All isolates had omadacycline MICs of

Cow-calf production plays a significant role in the beef production chain. However, bacteria in these systems are not typically monitored for antimicrobial resistance (AMR). We determined the baseline level of AMR in fecal bacteria collected from preweaned calves prior to feedlot entry and evaluated the effects of type of graze and age on AMR occurrence. Two grazing experiments (16 cow-calf pairs each) were conducted on tall fescue or wheat. Fecal samples were cultured for the detection of tetracycline-resistant (TETr), third-generation cephalosporin–resistant (3GCr), and extended-spectrum β-lactamase (ESBL)–producing Escherichia coli. Isolates were characterized for resistance to other antibiotics and resistance mechanisms. Concentrations (P < 0.001) and prevalence (P = 0.007) of TETrE. coli isolates were significantly higher in the calves (5.1 log CFU/g and 93%, respectively) than in the cows (4.4 log CFU/g and 80%, respectively). Wheat grazing did not affect TETr isolates phenotypically; however, it significantly expanded (P = 0.005) the resistant population carrying tet(A) over that carrying tet(B). Fecal prevalence of 3GCr and ESBL-producing isolates was 31.3 and 3.4%, respectively, with no significant effects of age (P = 0.340) or wheat grazing (P = 0.597). All 3GCr and ESBL-producing isolates were multidrug resistant (resistant to at least three antimicrobial classes). 3GCr isolates were positive for blaCMY-2 (73%) or blaCTX-M (27%), and blaCTX-M-15 was the most prevalent gene (94%, n = 17) among the CTX-M–positive isolates. Wheat grazing significantly expanded (P < 0.001) the 3GCr population carrying blaCTX-M and reduced the population carrying blaCMY-2. Five of the seven ESBL-producing isolates were positive for blaCTX-M. Our study revealed age-dependent occurrence of TETrE. coli and that wheat grazing expanded the resistant population carrying certain resistance genes. Cow-calf production is a significant reservoir for antibiotic-resistant bacteria of significant public health importance such as 3GCr and CTX-M ESBL-producing E. coli. TETrE. coli isolates were age dependent and widespread in the cow-calf population. 3GCrE. coli isolates were found in 31% of animals. ESBL-producing E. coli isolates were detected in the cow-calf production system. Wheat grazing modifies resistant populations by favoring certain genes over others. Cow-calf production can promote bacteria resistant to medically important antibiotics. [ABSTRACT FROM AUTHOR]

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h21 versus 0.15 h21) led to higher conjugation frequencies (1024 versus 1026 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework. [ABSTRACT FROM AUTHOR]

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus , has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis , in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 μg/mL, respectively. Baicalein at 64 μg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 μg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.

The global concern over antimicrobial resistance (AMR) and its impact on human health is evident, with approximately 4.95 million annual deaths attributed to antibiotic resistance. Regions with inadequate water, sanitation, and hygiene face challenges in responding to AMR threats. Enteric bacteria, particularly E. coli, are common agents linked to AMR-related deaths (23% of cases). Culture-based methods for detecting tetracycline-resistant E. coli may be of practical value for AMR monitoring in limited resource environments. This study evaluated the ColiGlow™ method with tetracycline for classifying tetracycline-resistant E. coli. A total of 61 surface water samples from Kentucky, USA (2020–2022), provided 61 presumed E. coli isolates, of which 28 isolates were obtained from tetracycline-treated media. Species identification and tetracycline resistance evaluation were performed. It was found that 82% of isolates were E. coli, and 18% were other species; 97% were identified as E. coli when using the API20E identification system. The MicroScan system yielded Enterobacter cloacae false positives in 20% of isolates. Adding tetracycline to ColiGlow increased the odds of isolating tetracycline-resistant E. coli 18-fold. Tetracycline-treated samples yielded 100% tetracycline-resistant E. coli when the total E. coli densities were within the enumeration range of the method. ColiGlow with tetracycline shows promise for monitoring tetracycline-resistant E. coli in natural waters and potentially aiding AMR surveillance in resource-limited settings among other environments.

The study was conducted to evaluate the rate of multidrug resistant Staphylococcus aureus (MDRSA) and to determine the tetracycline resistant genetic determinants in the MDRSA. 2004 different clinical specimens were subjected to bacteriological investigation following standard protocol. 266 S. aureus were isolated and identified which were subjected to antimicrobial susceptibility testing using modified Kirby-Bauer’s disc diffusion method following CLSI guidelines. Both the genomic and plasmid DNA extraction was done and quantified using Nanodrop. Conventional Polymerase Chain Reaction was carried out for the amplification of tetK and tetM genes using specific primers. The rate of S. aureus was found as 266 (13.3%). The rate of MDRSA was found 95 (35.7%). Among the 95 MDRSA isolates, the organism resistant to tetracycline with disc diffusion was found to be 26 (9.8 %). 18 isolates had antibiotic resistance genes either tetK or tetM. 2 samples with tetracycline resistant isolates displayed both the tet K and tet M genes Out of 26 tetracycline-resistant MDRSA isolates, 46.15% (12/26) were positive for tetK, and 23.07% (6/26) were detected to harbor tetM .Two types of determinants tet K and tet M has been recorded significantly. [ABSTRACT FROM AUTHOR]

Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydiaceae species that infect humans, a matter of public health importance.

BackgroundAntimicrobial resistance (AMR) is a growing and significant threat to public health on a global scale. Escherichia coli comprises Gram-negative, fecal-borne pathogenic and commensal bacteria that are frequently associated with antibiotic resistance. AMR E. coli can be ingested via food, water and direct contact with fecal contamination.MethodsWe estimated the prevalence of AMR Escherichia coli from select drinking water sources in northern Tanzania. Water samples (n = 155) were collected and plated onto Hi-Crome E. coli and MacConkey agar. Presumptive E. coli were confirmed by using a uidA PCR assay. Antibiotic susceptibility breakpoint assays were used to determine the resistance patterns of each isolate for 10 antibiotics. Isolates were also characterized by select PCR genotyping and macro-restriction digest assays.ResultsE. coli was isolated from 71 % of the water samples, and of the 1819 E. coli tested, 46.9 % were resistant to one or more antibiotics. Resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim was significantly higher (15-30 %) compared to other tested antibiotics (0-6 %; P

Tetracycline resistance (TetR) has been evidenced as a good phenotypic marker for detection of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex CC398. The aim of this study was to characterise a collection of 95 TetR-MRSA isolates, not belonging to the lineage CC398, that were obtained in a previous multicentre study, to detect other MRSA clonal complexes that could be associated with this phenotypic TetR marker. The TetR-MRSA isolates were recovered from 20 Spanish hospitals during 2016 and they were characterised to determine their antimicrobial resistance and virulence phenotypes/genotypes as well as the presence of the immune evasion cluster (IEC). A high proportion of isolates belonging to the CC1 lineage (46%) were observed, as well as to the CC5, CC8 and CC45 lineages (11% each one). Thirty-two different spa-types were identified, being predominantly CC1-t127 (40%) and CC45-t1081 (11%). The IEC system (with the gene scn as marker) was present in 73% of isolates and 16% produced the Panton Valentine leucocidin (PVL). A high proportion of MRSA-CC1 isolates were scn-negative (38.6%) and 52.9% of them were blaZ-negative. A multidrug resistance (MDR) phenotype was identified in 86% of MRSA isolates. The knowledge of other TetR-MRSA genetic lineages, in addition to CC398, is highly relevant, since most of them were MDR and some of them presented important virulence factors. Strains potentially associated with livestock (as the subpopulation CC1-t127-scn-negative) or with humans (as the CC45 lineage or the subpopulation CC1-scn-positive) have been found in this study. The use of tetracycline-resistance for detection, not only of CC398 but also of other LA-MRSA lineages should be tracked in the future. [ABSTRACT FROM AUTHOR]

Tetracyclines (TCs) are widely used for livestock, and the high prevalence of TC-resistant Escherichia coli in livestock has become a serious concern worldwide. In Japan, the National Action Plan on Antimicrobial Resistance in 2016 aimed to reduce the TC resistance rate in E. coli derived from livestock. Flavophospholipol (FPL), used as a feed additive, has an inhibitory effect on the spread of plasmid-mediated antimicrobial resistance. The number of TC-resistant E. coli was determined in pigs administered TCs and/or FPL to clarify the effect of FPL on reducing the number of TC-resistant E. coli in pigs. TC-resistant E. coli and their plasmids were then analyzed. The pigs were divided into four groups: control, doxycycline (DOXY; a TC), FPL, and a DOXY-FPL combination. Their feces were collected from the nursing period to the day before being transported to the slaughterhouse, followed by estimation of TC-resistant E. coli (colony-forming units [CFU]/g). The number of TC-resistant E. coli increased with the use of DOXY, suggesting that DOXY administration provides a selective pressure for TC-resistant E. coli. Supplementation with FPL as a feed additive significantly suppressed the increase in the number of TC-resistant E. coli, especially during the DOXY administration period. Transfer and growth inhibition analyses were performed for TC-resistant isolates. FPL inhibited the conjugational transfer and growth of a few TC-resistant E. coli isolates. These results suggest that FPL is effective against the spread of TC-resistant E. coli.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

Objectives: To determine the draft genome sequence and analyse the genetic features of a Staphylococcus pseudintermedius clinical isolate according to the main typing schemes available, with a special focus on antibiotic resistance. Methods: The strain was isolated from a case of otitis externa in a dog. Its identity and pattern of antibiotic susceptibility were determined using an automated system. The genome was sequenced using an Illumina platform. MLST, SCCmec typing, resistome, and mobile genetic elements were derived by comparative analysis using available specific databases. Results: S. pseudintermedius CAM1 isolate has a chromosome size of 2 652 610 bp. It showed a wide pattern of phenotypic resistance, comprising beta-lactams, macrolides and lincosamides, aminoglycosides, fluoroquinolones, tetracyclines, and trimethoprim-sulfamethoxazole. The genetic determinants of the underlying mechanisms were all found by in silico analysis of the genome. The mecA gene for methicillin resistance was harboured by the Vc type of the SCCmec. MLST of the strain was st551. Conclusion: By comparison with the MLST database of S. pseudintermedius and data from published molecular epidemiology studies, CAM1 is the first st551 strain recorded in Italy and, in the context of an already extremely wide antibiotic resistance pattern, it harbours also the tetK gene, the prevalence of which is rare in MDR S. pseudintermedius.

Background. Epidemiological studies of tetracycline (TE) resistance genes and integron gene cassettes, particularly in urine samples, are limited in Turkey. Objectives. To investigate antibiotic susceptibility profiles, extended-spectrum beta-lactamase (ESBL) positivity, tet gene types, class-I/-II integron gene cassettes, and clonal relationships among tet-resistant isolates of Escherichia coli from urine cultures of outpatients. Materials and methods. Isolates were identified using conventional methods and the automated Vitek® 2 Compact system. Antimicrobial susceptibility was performed for 19 antibiotics. The ESBL production was performed using the Kirby-Bauer disk diffusion test. The double disk synergy test was used for confirmatory testing. Polymerase chain reaction (PCR) was used to determine the presence of class-I/-II integron gene cassettes and tetA, tetB and tetD resistance genes. The pulsed-field gel electrophoresis typing was performed to identify clonal relations. Results. A total of 121 isolates were obtained and found to be resistant or sensitive to ampicillin and amikacin/imipenem. Resistance to ceftazidime, cefotaxime and ceftriaxone was determined to be 31.3%, 77.6% and 83.1%, respectively. Tetracycline resistance was detected in 82 isolates, mostly caused by the tetB gene. No tet gene was detected in the remaining 39 isolates. Although 64 out of 82 isolates carried a class-I integron, only 4 had a class-II integron (with sizes of 800-2900 base pairs). Furthermore, tet genes were identified with different size class-I integron gene cassettes. However, tet genes were not detected in any isolate identified with integron gene cassette II. Clonally, the isolates were found to be related in subgroups because they were community-acquired. Conclusions. This study showed that the tetB gene is most commonly found in E. coli isolates grown in urine samples from the Turkish population. [ABSTRACT FROM AUTHOR]

Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway. [ABSTRACT FROM AUTHOR]

ABSTRACTTigecycline is one of the last-resort antibiotics to treat severe infections. Recently, tigecycline resistance has sporadically emerged with an increasing trend, and Tet(X) family represents a new resistance mechanism of tigecycline. In this study, a novel chromosome-encoded tigecycline resistance gene, tet(X14), was identified in a tigecycline-resistant and colistin-resistant Empedobacter stercoris strain ES183 recovered from a pig fecal sample in China. Tet(X14) shows 67.14-96.39% sequence identity to the other variants [Tet(X) to Tet(X13)]. Overexpression of Tet(X14) in Escherichia coli confers 16-fold increase in tigecycline MIC (from 0.125 to 2 mg/L), which is lower than that of Tet(X3), Tet(X4) and Tet(X6). Structural modelling predicted that Tet(X14) shared a high homology with the other 12 variants with RMSD value from 0.003 to 0.055, and Tet(X14) can interact with tetracyclines by a similar pattern as the other Tet(X)s. tet(X14) and two copies of tet(X2) were identified on a genome island with abnormal GC content carried by the chromosome of ES183, and no mobile genetic elements were found surrounding, suggesting that tet(X14) might be heterologously obtained by ES183 via recombination. Blasting in Genbank revealed that Tet(X14) was exclusively detected on the chromosome of Riemerella anatipestifer, mainly encoded on antimicrobial resistance islands. E. stercoris and R. anatipestifer belong to the family Flavobacteriaceae, suggesting that the members of Flavobacteriaceae maybe the major reservoir of tet(X14). Our study reports a novel chromosome-encoded tigecycline resistance gene tet(X14). The expanded members of Tet(X) family warrants the potential large-scale dissemination and the necessity of continuous surveillance for tet(X)-mediated tigecycline resistance.

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the “One Health” approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene blaOXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments.

This study aimed to estimate the antimicrobial resistance (AMR) patterns and tetracycline-resistant gene profiles of Escherichia coli (E. coli) from broiler meat and livers sourced from live bird markets (LBMs) and supermarkets (SMs) in Chattogram, Bangladesh. In total, 405 samples were collected from SMs and LBMs, comprising muscle (n = 215) and liver (n = 190) samples. Disc diffusion tests were used to determine antimicrobial susceptibility profiles. PCR was used to identify E. coli and tetracycline-resistant genes. Over half (57%) of the chicken product samples were positive for E. coli. The AMR profiling of these isolates showed that the highest prevalence of resistance was against sulphamethoxazole–trimethoprim (89%), followed by tetracycline (87%), ampicillin (83%), and ciprofloxacin (61%). Among the antimicrobials listed by the World Health Organization as critically important, E. coli isolates were found to be resistant to cephalexin (37%), gentamicin (32%), and colistin sulfate (21%). A large proportion of E. coli demonstrated multi-drug resistance (MDR). Most (84%) of the tetracycline-resistant isolates encoded tetA. Of the remaining isolates, 0.5% encoded tetC, 6.0% encoded two genes, and 3.6% of isolates were tetD, which was newly identified by this study in Bangladesh. Broiler products in Bangladesh are frequently contaminated with multi-drug-resistant E. coli, with differential carriage of tetracycline genes. The prevalence of tetracycline resistance among E. coli indicates a concern for poultry health and welfare regarding the management of colibacillosis. It also indicates growing public health risks of AMR among broiler-associated pathogens, which can be transferred to humans via the food chain. Appropriate control measures should be developed and implemented, focused on the rational use of antimicrobials in poultry farming systems, to mitigate risk from this drug-resistant zoonotic pathogen from foods of animal origin and to protect public health.

ABSTRACT The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism’s virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Phototransformation is a key process affecting the fate of many antibiotics in the environment, but little is known about whether their photoproducts exert selective pressure on bacteria by inducing antibiotic resistance genes (ARGs). Here, we examined the expression of tetracycline resistance gene tet (M) of a fluorescent Escherichia coli whole-cell bioreporter influenced by the phototransformation of tetracycline. The presence of suspended smectite clay (montmorillonite or hectorite, 1.75 g/L) or dissolved humic substance (Pahokee Peat humic acid or Pahokee peat fulvic acid, 10 mg C/L) in aqueous solutions markedly facilitated the transformation of tetracycline (initially at 400 μg/L) with half-life shortened by 1.4-2.6 times. Despite the similar phototransformation ratios (80-90%) of the total loaded tetracycline after 60 min irradiation, the decreased ratios of cell fluorescence intensity (which was proportional to the expression amount of ARG tet (M)) were much higher with the two clays (94 and 93%) than with the two humic substances (44 and 69%) when compared to the respective dark controls. As illustrated by mass spectroscopic and chemical analyses, tetracycline was proposed to be mainly transformed to amide (ineffective in inducing ARGs) with the presence of clays by reaction with self-photosensitized singlet oxygen ( 1 O 2 ), while the humic substances might catalyze the production of another two demethylated and/or deaminated compounds (still effective in inducing ARGs) in addition to the amide compound via reaction with triplet excited state dissolved organic matter ( 3 DOM*). As clay minerals and humic substances are important soil constituents and ubiquitously present in surface environments, the observed clay and humic-dependent photooxidation pathways of tetracycline and the differing selective pressures of the associated products highlight the need for monitoring the transformation compounds of antibiotics and provide critical insight into the development of antibiotic treatment protocols.