Possibility of transfer and activation of 'silent' tetracycline resistance genes among Enterococcus faecalis under high-pressure processing.

In this study, the tetracycline resistance of Enterococcus faecalis strains isolated from food was determined and molecular analyses of the resistance background were performed by determining the frequency of selected tetracycline resistance genes. In addition, the effect of high-pressure stress (400 and 500 MPa) on the expression of selected genes encoding tetracycline resistance was determined, as well as changes in the frequency of transfer of these genes in isolates showing sensitivity to tetracyclines. In our study, we observed an increase in the expression of genes encoding tetracyclines, especially the tet(L) gene, mainly under 400 MPa pressure. The study confirmed the possibility of transferring genes encoding tetracyclines such as tet(M), tet(L), tet(K), tet(W) and tet(O) by horizontal gene transfer in both control strains and exposed to high-pressure. Exposure of the strains to 400 MPa pressure had a greater effect on the possibility of gene transfer and expression than the application of a higher-pressure. To our knowledge, this study for the first time determined the effect of high-pressure stress on the expression of selected genes encoding tetracycline resistance, as well as the possibility and changes in the frequency of transfer of these genes in Enterococcus faecalis isolates showing sensitivity to tetracyclines and possessing silent genes. Due to the observed possibility of increased expression of some of the genes encoding tetracycline resistance and the possibility of their spread by horizontal gene transfer to other microorganisms in the food environment, under the influence of high-pressure processing in strains phenotypically susceptible to this antibiotic, it becomes necessary to monitor this ability in isolates derived from foods.

GWAS and comparative genomics reveal candidate antibiotic resistance genes in the avian pathogen Gallibacterium anatis for six widespread antibiotics.

Gallibacterium anatis is a Gram-negative bacterium found in the respiratory and genital tracts of various animals, primarily poultry. Its association with septicemia and high mortality in poultry, along with the rise in multidrug-resistant strains, has amplified concerns. Recent research uncovered significant variability in antibiotic resistance profiles among G. anatis isolates from different Austrian flocks, and even between different organs within the same bird. In response, in the present study 60 of these isolates were sequenced and a combination of comparative genomics and genome-wide association study (GWAS) analysis was applied to understand the genetic variability of G. anatis across flocks and organs and to identify genes related to antibiotic resistance. The results showed that each flock harbored one or two strains of G. anatis with only a few strains shared between flocks, demonstrating a great variability among flocks. We identified genes associated with resistance to nalidixic acid, trimethoprim, cefoxitin, tetracycline, ampicillin and sulfamethoxazole. Our findings revealed that G. anatis may develop antibiotic resistance through two mechanisms: single-nucleotide mutations and the presence of specific genes that confer resistance. Unexpectedly, some tetracycline-resistant isolates lacked all known tetracycline-associated genes, suggesting the involvement of novel mechanisms of tetracycline resistance that require additional exploration. Furthermore, our functional annotation of resistance genes highlighted the citric acid cycle pathway as a potential key modulator of antibiotic resistance in G. anatis. In summary, this study describes the first application of GWAS analysis to G. anatis and provides new insights into the acquisition of multidrug resistance in this important avian pathogen.

Pentachlorophenol affects doxycycline and tetracycline resistance genes in soil by altering microbial structure.

Tetracycline antibiotics play a vital role in animal husbandry, primarily employed to uphold the health of livestock and poultry. Consequently, when manure is reintegrated into farmland, tetracycline antibiotics can persist in the soil. Simultaneously, to ensure optimal crop production, organochlorine pesticides (OCPs) are frequently applied to farmland. The coexistence of tetracycline antibiotics and OCPs in soil may lead to an increased risk of transmission of tetracycline resistance genes (TRGs). Nevertheless, the precise mechanism underlying the effects of OCPs on tetracycline antibiotics and TRGs remains elusive. In this study, we aimed to investigate the effects of OCPs on soil tetracycline antibiotics and TRGs using different concentrations of doxycycline (DOX) and pentachlorophenol (PCP). The findings indicate that PCP and DOX mutually impede their degradation in soil. Furthermore, our investigation identifies Sphingomonas and Bacillus as potential pivotal microorganisms influencing the reciprocal inhibition of PCP and DOX. Additionally, it is observed that the concurrent presence of PCP and DOX could impede each other's degradation by elevating soil conductivity. Furthermore, we observed that a high concentration of PCP (10.7 mg/kg) reduced the content of efflux pump tetA, ribosome protective protein tetM, tetQ, and passivating enzyme tetX. In contrast, a low PCP concentration (6.4 mg/kg) only reduced the content of ribosome protective protein tetQ. This suggests that PCP may reduce the relative abundance of TRGs by altering the soil microbial community structure and inhibiting the potential host bacteria of TRGs. These findings have significant implications in understanding the combined pollution of veterinary antibiotics and OCPs. By shedding light on the interactions between these compounds and their impact on microbial communities, this study provides a theoretical basis for developing strategies to manage and mitigate their environmental impact, and may give some information regarding the sustainable use of antibiotics and pesticides to ensure the long-term health and productivity of agricultural systems.

Tetracycline resistance in Listeria monocytogenes and L. innocua from wild black bears (Ursus americanus) in the United States is mediated by novel transposable elements.

IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria. Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears (Ursus americanus) from the United States, including 158 and 27 L. monocytogenes and L. innocua, respectively. Tetracycline resistance was the most prevalent resistance trait. In L. monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn916-like element Tn916.1039. In contrast, three distinct, novel tetracycline resistance elements (Tn5801.UAM, Tn5801.551, and Tn6000.205) were identified in L. innocua. Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria.

Prevalence and genetic basis of tetracycline resistance in Vibrioparahaemolyticus isolates recovered from food products in Shenzhen, China during 2013 to 2021.

Understanding tetracycline resistance in Vibrio parahaemolyticus from food products is crucial for effective control measures against this foodborne pathogen. This study aimed to investigate the prevalence, evolution routes, and mechanism of transmission of tetracycline resistance in Vibrio parahaemolyticus isolates collected from food products in Shenzhen, China. A total of 2342 non-duplicate Vibrio parahaemolyticus were isolated from 3509 food samples during the period 2013-2021. Among these 2342 Vibrio parahaemolyticus strains, 530 (21.37 %) were resistant to tetracycline. These tetracycline-resistant Vibrio parahaemolyticus strains were mainly isolated from shrimp samples, with the highest resistance rate (46.9 %) observed in 2019. Phylogenetic and genomic analyses of 387 isolates carrying the tet genes revealed that five different types of tet genes (tet(34), tet(A), tet(B), tet(M), and tet(E)) were present. The tet(A) gene was the most common (65 % of isolates), while tet(E) and tet(M) genes were only detected in specific years. Although tet(A) is the most commonly detected gene, it only encodes resistance in a low percentage of strains (47/129). On the other hand, the resistance rate is highest in isolates carrying tet(B) (41/55). Interestingly, V. parahaemolyticus carrying the tet genes were not necessarily tetracycline-resistant, and vice versa. A total of six different types of plasmids and two transposable units were found to carry the tet genes. V. parahaemolyticus strains that harbored these plasmids were often resistant to multiple antibiotics, indicating that horizontal transfer of antibiotic resistance genes is common among V. parahaemolyticus strains. Our findings suggest a high prevalence of tetracycline resistance in Vibrio parahaemolyticus strains recovered from food products in Shenzhen, China. These results provide valuable insight into the evolution and transmission of tetracycline resistance in foodborne Vibrio parahaemolyticus isolates and highlight the need for effective control measures to prevent the spread of antibiotic resistance.

Mechanism of antibiotic resistance development in an activated sludge system under tetracycline pressure.

The mechanism of antibiotic resistance (AR) development in an activated sludge system under tetracycline (TC) pressure was discussed and analyzed. According to the variation of macro-factors, including TC, COD, TN, TP, NH3-N, pH, heavy metals, and reactor settings, the tet genes respond accordingly. Consequently, the enrichment sites of tet genes form an invisible AR selection zone, where AR microorganisms thrive, gather, reproduce, and spread. The efflux pump genes tetA and tetB prefer anaerobic environment, while ribosome protective protein genes tetM, tetO, tetQ, tetT, and tetW were more concentrated in aerobic situations. As a corresponding micro-effect, different types of tet genes selected the corresponding dominant bacteria such as Thauera and Arthrobacter, suggesting the intrinsic relationship between tet genes and potential hosts. In summary, the macro-response and micro-effect of tet genes constitute an interactive mechanism with tet genes as the core, which is the crucial cause for the continuous development of AR. This study provides an executable strategy to control the development of AR in actual wastewater treatment plants from the perspective of macro-factors and micro-effects.

Exploring the third-generation tetracycline resistance of multidrug-resistant livestock-associated methicillin-resistant Staphylococcus aureus ST9 across healthcare settings in China.

BACKGROUND: The overuse of antibiotics in livestock is contributing to the burden of antimicrobial resistance in humans, representing a One Health challenge. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has recently become a growing concern, and ST9 is the major LA-MRSA lineage in China and has emerged in clinical settings. METHODS: Antimicrobial susceptibility testing was used to evaluate the tetracycline resistance of ST9 MRSA collections, and gene cloning experiments were performed to explore the resistance mechanisms. Whole-genome sequencing and comparative genomics were used to analyse the genetic features of clinical ST9 isolates. A phylogenetic tree was constructed to investigate the relationship of human- and livestock-derived ST9 isolates. RESULTS: Clinical ST9 isolates were found to possess several types of resistance genes and resistance-related mutations and were multidrug-resistant. Notably, all clinical ST9 isolates were resistant to third-generation tetracyclines. Cloning experiments showed that both the acquisition of the tetracycline resistance gene tet(L)/tet(63) and a mutation in the rpsJ gene contributed to third-generation tetracycline resistance. Phylogenetic analysis showed that the ST9 isolates collected in healthcare systems were probably transmitted from livestock. The ST9 lineage underwent multiple interspecies recombination events and gained many resistance elements. Furthermore, the resistance to third-generation tetracyclines may have evolved under tetracycline pressure in livestock. CONCLUSIONS: The evolution of ST9 MRSA in livestock and transmission of this clone between humans and livestock highlight the importance of establishing control strategies with the One Health approach to reduce the burden of antibiotic resistance.

Spatiotemporal distributions of sulfonamide and tetracycline resistance genes and microbial communities in the coastal areas of the Yangtze River Estuary.

In this paper, water and sediments were sampled at eight monitoring stations in the coastal areas of the Yangtze River Estuary in summer and autumn 2021. Two sulfonamide resistance genes (sul1 and sul2), six tetracycline resistance genes (tetM, tetC, tetX, tetA, tetO, and tetQ), one integrase gene (intI1), 16 S rRNA genes, and microbial communities were examined and analyzed. Most resistance genes showed relatively higher abundance in summer and lower abundance in autumn. One-way analysis of variance (ANOVA) showed significant seasonal variation of some ARGs (7 ARGs in water and 6 ARGs in sediment). River runoff and WWTPs are proven to be the major sources of resistance genes along the Yangtze River Estuary. Significant and positive correlations between intI1 and other ARGs were found in water samples (P < 0.05), implying that intI1 may influence the spread and propagation of resistance genes in aquatic environments. Proteobacteria was the dominant phylum along the Yangtze River Estuary, with an average proportion of 41.7%. Redundancy analysis indicated that the ARGs were greatly affected by temperature, dissolved oxygen, and pH in estuarine environments. Network analysis showed that Proteobacteria and Cyanobacteria were the potential host phyla for ARGs in the coastal areas of the Yangtze River Estuary.

Identification and Characterization of a Novel Major Facilitator Superfamily Efflux Pump, SA09310, Mediating Tetracycline Resistance in Staphylococcus aureus.

Drug efflux systems have recently been recognized as an important mechanism of multidrug resistance in bacteria. Here, we described the identification and characterization of a novel chromosomally encoded multidrug efflux pump (SA09310) in Staphylococcus aureus. SA09310 is a 43-kDa protein with 12 transmembrane helices. The conserved amino acid sequence motifs of the major facilitator superfamily (MFS) were identified in the protein SA09310, which indicated that SA09310 belonged to the MFS transporters. Expression of the sa09310 gene was induced by different types of antibiotics, including aminoglycoside, tetracycline, macrolides, and chloramphenicol. An sa09310 gene knockout mutant (Δsa09310) was constructed, and its susceptibility to 30 different antibiotics was evaluated. The Δsa09310 mutant exhibited increased sensitivity to tetracycline and doxycycline, with 64-fold- and 8-fold-decreased MICs, respectively. The mechanism of SA09310 mediation of tetracycline resistance was demonstrated by its ability to extrude intracellular tetracycline from within the cells into the environment. The efflux activity of SA09310 was further confirmed by ethidium bromide (EtBr) accumulation and efflux assays. In addition, the efflux activity of SA09310 was observed to be blocked by the known efflux pump inhibitor carbonyl cyanide chlorophenylhydrazone (CCCP), which provided direct evidence that suggested the H+-dependent activity of the SA09310 efflux pump. The conservation of SA09310 homologs in Staphylococcus indicated the universal function of these SA09310-like protein clusters. In conclusion, the function-unknown protein SA09310 has been identified and characterized as a tetracycline efflux pump mediating tetracycline resistance in S. aureus.

Tetracycline resistance potential of heterotrophic bacteria isolated from freshwater fin-fish aquaculture system.

AIMS: This study investigated the tetracycline resistance potential of heterotrophic bacteria isolated from twenty-four freshwater fin-fish culture ponds in Andhra Pradesh, India. METHODS AND RESULTS: A total of 261 tetracycline resistant bacteria (tetR) were recovered from pond water, pond sediment, fish gills, fish intestine, and fish feed. Bacteria with high tetracycline resistance (tetHR) (n = 30) that were resistant to tetracycline concentrations above 128  µg mL-1 were predominantly Lactococcus garvieae followed by Enterobacter spp., Lactococcus lactis, Enterobacter hormaechei, Staphylococcus arlettae, Streptococcus lutetiensis, Staphylococcus spp., Brevundimonas faecalis, Exiguobacterium profundum, Lysinibacillus spp., Stutzerimonas stutzeri, Enterobacter cloacae, and Lactococcus taiwanensis. Resistance to 1024 µg mL-1 of tetracycline was observed in L. garvieae, S. arlettae, Enterobacter spp., B. faecalis. Tet(A) (67%) was the predominant resistance gene in tetHR followed by tet(L), tet(S), tet(K), and tet(M). At similar concentrations of exposure, tetracycline procured at the farm level (69.5% potency) exhibited lower inhibition against tetHR bacteria compared to pure tetracycline (99% potency). The tetHR bacteria showed higher cross-resistance to furazolidone (100%) followed by co-trimoxazole (47.5%) and enrofloxacin (11%). CONCLUSIONS: The maximum threshold of tetracycline resistance at 1024 µg mL-1 was observed in S. arlettae, Enterobacter spp., B. faecalis, and L. garvieae and tet(A) was the major determinant found in this study.

Comparative metagenomics reveals poultry and swine farming are hotspots for multidrug and tetracycline resistance.

Antibiotic misuse in livestock is a major threat to human health, as bacteria are quickly developing resistance to them. We performed a comparative analysis of 25 faecal metagenomes from swine, poultry, cattle, and humans to investigate their resistance profiles. Our analysis revealed that all genes conferring resistance to antibiotic classes assessed except tetracyclines were more prevalent in poultry manure than in the remaining species. We detected clinically relevant antibiotic resistance genes, such as mcr-1 which confers resistance to polymyxins. Among them, extended-spectrum ß-lactamase blaCTX-M genes were particularly abundant in all species. Poultry manure was identified as a hotspot for multidrug resistance, which may compromise medical treatment options. Urgent actions in the livestock industry are imperative to hamper the emergence and spread of antibiotic resistance.

Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania.

BACKGROUND: Over one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing. METHODS: We assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP. RESULTS: Fifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes. CONCLUSION: The erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.

Tea polyphenols and catechins postpone evolution of antibiotic resistance genes and alter microbial community under stress of tetracycline.

Relying on the high mobility of water flow, the dissemination of antibiotic resistance genes (ARGs) in the water tends to be exacerbated and enlarged. It caused negative impacts on a wider scope of the environment. The ARGs dissemination monitoring and the methods efficiently reducing their concentration in water became the focus of interest. Green chemicals with antibacterial effects such as tea polyphenols (TPs) and catechins (CA) have been considered as auxiliary disinfectants for ARGs removal in the water environment. However, the antibacterial performance of TPs and CA under the stress of external antibiotics still lacks sufficient research. The results show that more operational taxonomic units can be observed in water samples with TPs and CA than in those without the ingredients under pressure of tetracycline. An unexpected increase along with the increase of ARGs concentrations and the diversity of microbial communities under the low-concentration TPs or CA (1 mg/L). Besides, under the stress of tetracycline, the inhibition of TPs was detected to be strengthened for increase of inti1 and tetC but weakened towards for the increase of tetA. Whilst CA substantially diminished abundances of tetC and tetA under tetracycline pressure. This research demonstrated that TPs and CA are able to assuage development of ARGs under the pressure of antibiotic in water system.

Disrupting the ArcA Regulatory Network Amplifies the Fitness Cost of Tetracycline Resistance in Escherichia coli.

There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (TetR) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of TetR E. coli during treatment with tetracycline. Targeted knockout of ArcA, identified by network analysis as a master regulator of this new compensatory physiological state, significantly compromised fitness of TetR E. coli during tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain, also resensitized TetR E. coli to tetracycline. We discovered that the potentiating effect of sertraline was eliminated upon knocking out arcA, demonstrating that the mechanism of potential synergy was through action of sertraline on the tetracycline-induced ArcA network in the TetR strain. Our findings demonstrate that therapies that target mechanistic drivers of compensatory physiological states could resensitize AMR pathogens to lost antibiotics. IMPORTANCE Antimicrobial resistance (AMR) is projected to be the cause of >10 million deaths annually by 2050. While efforts to find new potent antibiotics are effective, they are expensive and outpaced by the rate at which new resistant strains emerge. There is desperate need for a rational approach to accelerate the discovery of drugs and drug combinations that effectively clear AMR pathogens and even prevent the emergence of new resistant strains. Using tetracycline-resistant (TetR) Escherichia coli, we demonstrate that gaining resistance is accompanied by loss of fitness, which is restored by compensatory physiological changes. We demonstrate that transcriptional regulators of the compensatory physiologic state are promising drug targets because their disruption increases the susceptibility of TetR E. coli to tetracycline. Thus, we describe a generalizable systems biology approach to identify new vulnerabilities within AMR strains to rationally accelerate the discovery of therapeutics that extend the life span of existing antibiotics.

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Livestock wastewater is an important reservoir of antibiotic resistance genes (ARGs), with high environmental risks. We investigated the seasonal variations of distribution and removal of swine wastewater originated high-risk tetracycline resistance genes (TRGs) in horizontal subsurface flow constructed wetlands. The effects of exogenous addition of tetracycline (TC) and copper ion (Cu2+) on the abundance of TRGs in effluent with single and combined pollution of antibiotic and heavy metal were studied. The results showed that all the three high-risk TRGs (tetM, tetO and tetW) were detected in swine wastewater. Wetlands could effectively reduce the ARGs, with the absolute abundance of TRGs in effluent being decreased by 1.1-2.4 and 1.7-2.9 orders of magnitude in summer and winter compared with the influent, respectively. The abundance of TRGs in wetland soils showed the characte-ristics that the outflow side was lower than the inflow side, the non-rhizosphere area was lower than the rhizosphere area, and lower in winter than in summer. In summer and winter, single and combined pollution of TC and Cu2+ in swine wastewater would increase the abundance of TRGs in effluent compared with that in the control. The constructed wetland is suitable for controlling the environmental diffusion of ARGs in livestock wastewater.

The Dual Roles of Nano Zero-Valent Iron and Zinc Oxide in Antibiotics Resistance Genes (ARGs) Spread in Sediment.

Nanoparticles (NPs) are widely used and ubiquitous in the environment, but the consequences of their release into the environment on antibiotics resistance genes (ARGs), microbial abundance, and community, are largely unknown. Therefore, this study examined the effect of nano zero-valent iron (nZVI) and zinc oxide (nZnO) on tetracycline resistance genes (tet-ARGs) and class 1 integron (intI1) in sediment under laboratory incubation. The coexistence of NPs and tetracycline (TC) on tet-ARGs/intI1 was also investigated. It was found that nZVI and nZnO promoted tet-ARGs/intI1 abundance in sediment without TC but reduced the inducing effect of TC on tet-ARGs/intI1 in sediment overlaid with TC solution. Without TC, nZVI, intI1, and the bacterial community could directly promote tet-ARGs spread in nZVI sediment, while intI1 and bacterial abundance were the most directly important reasons for tet-ARGs spread in nZnO sediment. With TC, nZVI and bacterial community could reduce tet-ARGs abundance in nZVI sediment, while nZnO and bacterial community could directly promote tet-ARGs in nZnO sediment. Finally, these findings provided valuable information for understanding the role of NPs in promoting and reducing ARGs in the environment.

The enhanced degradation behavior of oxytetracycline by black soldier fly larvae with tetracycline resistance genes in the larval gut: Kinetic process and mechanism.

Black soldier fly larvae (larvae) can digest organic wastes and degrade contaminants such as oxytetracycline (OTC). However, compared to the kinetic processes and enhanced mechanisms used in the traditional microbial degradation of OTC, those employed by larvae are largely uncharacterized. To obtain further details, a combined analysis of larval development, larval nutritional values (crude protein, crude fat and the composition of fatty acids) and the expression of tetracycline resistance genes (TRGs) in the larval gut was performed for the degradation of OTC added to substrates and for oxytetracycline bacterial residue (OBR). When the larvae were exposed to the substrates, the degradation processes were enhanced significantly (P < 0.01), with a 4.74-7.86-fold decrease in the degradation half-life (day-1) and a 3.34-5.74-fold increase in the final degradation efficiencies. This result was attributed to the abundant TRGs (with a detection rate of 35.90%∼52.14%) in the larval gut. The TRGs presented the resistance mechanisms of cellular protection and efflux pumps, which ensured that the larvae could tolerate elevated OTC concentrations. Investigation of the TRGs indicated that enzymatic inactivation enhanced OTC degradation by larvae. These findings demonstrate that the larval degradation of antibiotic contaminants is an efficient method based on abundant TRGs in the larval gut, even though OTC degradation results in OBR. In addition, a more optimized system for higher reductions in antibiotic levels and the expansion of larval bioremediation to other fields is necessary.

Characterization of an MDR Lactobacillus salivarius isolate harbouring the phenicol-oxazolidinone-tetracycline resistance gene poxtA.

OBJECTIVES: To characterize the oxazolidinone resistance gene poxtA in a Lactobacillus salivarius isolate of pig origin. METHODS: L. salivarius isolate BNS11 was investigated for the presence of mobile oxazolidinone resistance genes by PCR. Antimicrobial susceptibility testing was performed by broth microdilution. Transfer experiments were conducted to assess horizontal transferability of the gene poxtA. WGS was carried out using a combination of Oxford Nanopore MinION/Illumina HiSeq platforms. The presence of translocatable units (TUs) carrying resistance genes was studied by PCR assays and subsequent sequence analysis. RESULTS: L. salivarius isolate BNS11 was positive for poxtA. WGS showed that it harboured two gene copies each of the poxtA and the fexB genes, which were located on the broad-host-range Inc18 plasmid pBNS11-37kb and in the chromosomal DNA, respectively. The plasmid-borne poxtA gene together with the genes fexB, vat(E) and erm(C) were located in an MDR region on plasmid pBNS11-37kb. Analysis of the genetic context showed that an approx. 11 kb poxtA-fexB fragment was integrated into the chromosomal DNA and two novel IS elements ISLasa1 and ISLasa2 were identified in this inserted fragment. PCR assays revealed that five different IS1216E-based TUs carrying the resistance genes poxtA, fexB, vat(E) or erm(C) were formed. CONCLUSIONS: To the best of our knowledge, this is the first report of the transferable oxazolidinone resistance gene poxtA in the genus Lactobacillus. In addition, the presence of IS1216E-based TUs will contribute to the persistence and accelerate the dissemination of resistance genes, including poxtA.

Functional and phylogenetic analysis of TetX variants to design a new classification system.

Recently, many TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. In this study, we identified essential residues including 329, 339, 340, 350, and 351 in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into TetX-A class, TetX-B class and TetX-C class. We further found that TetX-A class variants originated from Bacteroidetes, with some variants further evolving to TetX-C class and acquired by Enterobacteriaceae. On the other hand, our data showed that some variants genes belonging to TetX-A class evolved directly to TetX-B class, which was further transmitted to Acinetobacter spp. This new classification system may facilitate better clinical management of patients infected by TetX-producing strains.

Profiles of tetracycline resistance genes in paddy soils with three different organic fertilizer applications.

The rapid expansion of organic rice cultivation areas have been accompanied by increased application of organic fertilizers. The high prevalence of soil antibiotic resistance caused by organic fertilizer application poses a severe threat to the agricultural and soil ecosystems. To date, research efforts and understanding of the effects and mechanism of action of the various organic fertilizers on antibiotic resistance in paddy soils remain poorly investigated. Tetracycline resistance genes (TRGs, including tetB, tetC, tetL, tetZ, tetM, tetO, tetT, and tetX), class 1 integron-integrase gene (intI1) and bacterial communities were characterized using quantitative-PCR and Illumina MiSeq sequencing, in paddy soils exposed to inorganic fertilizer (NPK), animal-derived organic fertilizer (AOF, composted swine and/or chicken manure), plant-derived organic fertilizer (POF, rapeseed cake and/or astragalus) and commercial organic fertilizer (COF, composted of animal manure mix with crop residues) applications. Compared with NPK, AOF applications significantly increased the relative abundance of TRGs, which was predominantly expressed in the increase of the relative abundance of tetC, tetM, tetO, tetT, and tetX, while POF and COF had no significant effect on the relative abundance of TRGs. Principal coordinate analysis revealed that AOF and POF significantly altered bacterial communities in paddy soils relative to NPK, while COF had no significant change of bacterial communities. Variation partitioning analysis indicated that soil physicochemical properties were the decisive factors for the changes of TRGs in organic paddy fields. Furthermore, redundancy analysis and the Mantel test showed that TRG profiles in AOF applied paddy soils were strongly influenced by electrical conductivity (EC). Total nitrogen (TN) and organic matter (OM) affected the distribution of TRGs in COF and POF applied paddy soils through a different mechanism. This study provides insights into the impacts of different types of organic fertilizer on the profiles of TRGs in paddy soils.