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Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. However, the DRS of bacterial RNA is currently deficient in the yield of mRNA-mapping reads and has yet to be exploited for transcriptome-wide RNA modification mapping. Here, we showed that pre-processing of bacterial total RNA (size selection followed by ribosomal RNA depletion and polyadenylation) guaranteed high throughputs of sequencing data and considerably increased the amount of mRNA reads. This way, complex transcriptome architectures were reconstructed for Escherichia coli and Staphylococcus aureus and extended the boundaries of 225 known E. coli operons and 89 defined S. aureus operons. Utilizing unmodified in vitro-transcribed (IVT) RNA libraries as a negative control, several Nanopore-based computational tools globally detected putative modification sites in the E. coli and S. aureus transcriptomes. Combined with Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates were identified in the E. coli protein-coding transcripts, while none were detected in S. aureus. Altogether, we demonstrated the potential of Nanopore DRS in systematic and convenient transcriptome and epitranscriptome analysis.
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Escherichia coli , Secuenciación de Nanoporos , ARN Bacteriano , Análisis de Secuencia de ARN , Staphylococcus aureus , Transcriptoma , Escherichia coli/genética , Escherichia coli/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transcriptoma/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Análisis de Secuencia de ARN/métodos , Secuenciación de Nanoporos/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Operón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Nanoporos , Regulación Bacteriana de la Expresión Génica , Perfilación de la Expresión Génica/métodosRESUMEN
Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40â¯%). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.
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Microbioma Gastrointestinal , Infecciones por Klebsiella , Klebsiella pneumoniae , Rifampin , Microbioma Gastrointestinal/efectos de los fármacos , Animales , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/mortalidad , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Rifampin/uso terapéutico , Rifampin/farmacología , Masculino , Zidovudina/uso terapéutico , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Intestinos/microbiología , Intestinos/patología , Intestinos/efectos de los fármacos , Intestinos/inmunología , Ratones Endogámicos C57BL , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Sepsis/inmunología , Sepsis/mortalidad , Ratones , Inmunidad Innata/efectos de los fármacosRESUMEN
Acinetobacter baumannii (AB) infections have become a global public health concern due to the continued increase in the incidence of infection and the rate of resistance to carbapenems. This study aimed to investigate the genomic features of AB strains recovered from a tertiary hospital and assess the clinical implications of the findings. A total of 217 AB strains were collected between 2016 and 2018 at a tertiary hospital in Guangzhou, with 183 (84.33%) being carbapenem-resistant AB (CRAB), with the main mechanism being the carriage of the blaOXA-23 gene. The overall mortality rate of patients caused by such strains was 15.21% (n = 33). Artificial lung ventilation and the use of meropenem were mortality risk factors in AB-infected patients, while KL2 AB infection was negatively associated. Core genome multilocus sequence typing and clustering analysis were performed on the integrated AB genome collection from the NCBI database and this study to illustrate the population structure among China. The results revealed diverse core genome profiles (n = 17) among AB strains from China, and strains from this single hospital exhibited most of the core genome profiles (n = 13), suggesting genetic variability within the hospital and transmission across the country. These findings show that the high transmission potential of the CRAB strains and meropenem usage that confers a selective advantage of CRAB clinically are two major factors that pose significant challenges to the effective clinical management of AB infections. Understanding the genetic features and transmission patterns of clinical AB strains is crucial for the effective control of infections caused by this pathogen.
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This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.
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Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Microbiología de Alimentos , China/epidemiología , Farmacorresistencia Bacteriana Múltiple/genética , Antibacterianos/farmacología , Animales , Humanos , Pruebas de Sensibilidad Microbiana , Cólera/microbiología , Cólera/epidemiología , Vibrio cholerae/genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae no O1/genética , Vibrio cholerae no O1/efectos de los fármacos , Vibrio cholerae no O1/aislamiento & purificación , Plásmidos/genéticaRESUMEN
Nanopore sequencing technologies have enabled the direct detection of base modifications in DNA or RNA molecules. Despite these advancements, the tools for visualizing electrical current, essential for analyzing base modifications, are often lacking in clarity and compatibility with diverse nanopore pipelines. Here, we present Nanopore Current Events Magnifier (nanoCEM, https://github.com/lrslab/nanoCEM), a Python command-line tool designed to facilitate the identification of DNA/RNA modification sites through enhanced visualization and statistical analysis. Compatible with the four preprocessing methods including 'f5c resquiggle', 'f5c eventalign', 'Tombo' and 'move table', nanoCEM is applicable to RNA and DNA analysis across multiple flow cell types. By utilizing rescaling techniques and calculating various statistical features, nanoCEM provides more accurate and comparable visualization of current events, allowing researchers to effectively observe differences between samples and showcase the modified sites.
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The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum ß-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens.
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Secuenciación de Nanoporos , Plásmidos/genética , Genómica , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Bacterias/genética , Antibacterianos/farmacologíaRESUMEN
This study employs a comprehensive approach combining metagenomic analysis and bacterial isolation to elucidate the microbial composition, antibiotic resistance genes (ARGs), and virulence factors (VFGs) present in shrimps from market and supermarket. Metagenomic analysis of shrimps revealed a dominance of Proteobacteria and Bacteroidetes with Firmicutes notably enriched in some samples. On the other hand, the dominant bacteria isolated included Citrobacter portucalensis, Escherichia coli, Salmonella enterica, Vibrio species and Klebsiella pneumonaie. Metagenomic analysis unveiled a diverse spectrum of 23 main types and 380 subtypes of ARGs in shrimp samples including many clinical significant ARGs such as blaKPC, blaNDM, mcr, tet(X4) etc. Genomic analysis of isolated bacterial strains identified 14 ARG types with 109 subtype genes, which complemented the metagenomic data. Genomic analysis also allowed us to identify a rich amount of MDR plasmids, which provided further insights into the dissemination of resistance genes in different species of bacteria in the same samples. Examination of VFGs and mobile genetic elements (MGEs) in both metagenomic and bacterial genomes revealed a complex landscape of factors contributing to bacterial virulence and genetic mobility. Potential co-occurrence patterns of ARGs and VFGs within human pathogenic bacteria underlined the intricate interplay between antibiotic resistance and virulence. In conclusion, this integrated analysis for the first time provides a comprehensive view and sheds new light on the potential hazards associated with shrimp products in the markets. The findings underscore the necessity of ongoing surveillance and intervention strategies to mitigate risks posed by antibiotic-resistant bacteria in the food supply chain using the novel comprehensive approaches.
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Decápodos , Genes Bacterianos , Animales , Humanos , Antibacterianos/farmacología , Bacterias/genética , Plásmidos , Farmacorresistencia Microbiana/genética , CrustáceosRESUMEN
Antibiotic resistance is a global health issue, with Klebsiella pneumoniae (KP) posing a particular threat due to its ability to acquire resistance to multiple drug classes rapidly. OXA-232 is a carbapenemase that confers resistance to carbapenems, a class of antibiotics often used as a last resort for treating severe bacterial infections. The study reports the earliest known identification of six OXA-232-producing KP strains that were isolated in Zhejiang, China, in 2008 and 2009 within a hospital, two years prior to the first reported identification of OXA-232 in France. The four KP strains carry the OXA-232 gene and exhibit hypervirulent loci, suggesting a broader temporal and geographical spread and integration of this resistance and virulence than previously recognized with implications for public health. Global analysis of all OXA-232-bearing KP strains revealed that OXA-232-encoding plasmids are conservative, while the strains were very diverse suggesting the plasmid mediated transmission of this carbapenemase genes. Importantly, a large proportion of the OXA-232-bearing KP strains also carried virulence plasmids, in particular the recent emergence of ST15 type of KP that carried both OXA-232-encoding plasmids and hypervirulent (hv) plasmids in China since 2019, highlighting the importance of the emergence of this type of KP strains in clinical setting. The early detection and investigations of OXA-232 in these strains warrants the retrospective studies to uncover the true timeline of antibiotic resistance spread, which could provide valuable insights for shaping future strategies to tackle the global health crisis.
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Proteínas Bacterianas , Klebsiella pneumoniae , Klebsiella pneumoniae/genética , Estudios Retrospectivos , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/genética , Antibacterianos/farmacología , Plásmidos/genética , ChinaRESUMEN
The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with ß-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.
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Bacillus , Bacillus cereus/genética , Enterotoxinas/análisis , Microbiología de Alimentos , Prevalencia , Genómica , Contaminación de Alimentos/análisisRESUMEN
The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses grave threats to human health. These strains increased dramatically in clinical settings in China in the past few years but not in other parts of the world. Four isogenic K. pneumoniae strains, including classical K. pneumoniae, carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP) and CR-hvKP, were created and subjected to phenotypic characterization, competition assays, mouse sepsis model and rat colonization tests to investigate the mechanisms underlying the widespread nature of CR-hvKP in China. Acquisition of virulence plasmid led to reduced fitness and abolishment of colonization in the gastrointestinal tract, which may explain why hvKP is not clinically prevalent after its emergence for a long time. However, tigecycline treatment facilitated the colonization of hvKP and CR-hvKP and reduced the population of Lactobacillus spp. in animal gut microbiome. Feeding with Lactobacillus spp. could significantly reduce the colonization of hvKP and CR-hvKP in the animal gastrointestinal tract. Our data implied that the clinical use of tigecycline to treat carbapenem-resistant K. pneumoniae infections facilitated the high spread of CR-hvKP in clinical settings in China and demonstrated that Lactobacillus spp. was a potential candidate for anticolonization strategy against CR-hvKP.
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Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Klebsiella , Ratones , Humanos , Ratas , Animales , Tigeciclina/farmacología , Klebsiella pneumoniae/genética , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/epidemiología , Carbapenémicos/farmacología , Virulencia , Modelos Animales de Enfermedad , Antibacterianos/farmacologíaRESUMEN
Klebsiella pneumoniae is an important clinical bacterial pathogen that has hypervirulent and multidrug-resistant variants. Uniform Manifold Approximation and Projection (UMAP) was used to cluster genomes of 16 797 K. pneumoniae strains collected, based on core genome distance, in over 100 countries during the period 1937 to 2021. A total of 60 high-density genetic clusters of strains representing the major epidemic strains were identified among these strains. Using UMAP bedding, the relationship between genetic cluster, capsular polysaccharide (KL) types and sequence type (ST) of the strains was clearly demonstrated, with some important STs, such as ST11 and ST258, found to contain multiple clusters. Strains within the same cluster often exhibited significant diverse features, such as originating from different areas and being isolated in different years, as well as carriage of different resistance and virulence genes. These data enable the routes of evolution of the globally prevalent K. pneumoniae strains to be traced. Alarmingly, carbapenem-resistant K. pneumoniae strains accounted for 51.7% of the test strains and worldwide transmission was observed. Carbapenem-resistant and hypervirulent K. pneumoniae strains are mainly reported in China; however, these strains are increasingly reported in other parts of the world. Also identified in this study were several key genetic loci that facilitate development of a new K. pneumoniae typing method to differentiate between high- and low-risk strains. In particular, the acrR, ompK35 and hha genes were predicted to play a key role in expression of the resistance and virulence phenotypes.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Humanos , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Carbapenémicos , Virulencia/genética , Genómica , Antibacterianos/farmacología , beta-Lactamasas/genéticaRESUMEN
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.
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Resistencia a la Tetraciclina , Vibrio parahaemolyticus , Resistencia a la Tetraciclina/genética , Prevalencia , Filogenia , Antibacterianos/farmacología , Tetraciclina/farmacología , Vibrio parahaemolyticus/genética , China/epidemiologíaRESUMEN
In this work, we collected foodborne Salmonella strains in Shenzhen, China, during 2014-2017 and investigated the genetic profile of all cefotaxime-resistant isolates in the collection. The strains were subjected to antimicrobial susceptibility tests, whole-genome sequencing, bioinformatics analysis, and conjugation studies. A total of 79 cefotaxime-resistant Salmonella were identified and found to exhibit multidrug resistance. Resistance rate recorded during the study period increased from 1.9% to 9.1%. Salmonella Typhimurium was the predominant serovar, and CTX-M family genes were dominant among the ESBLs genes detected. Notably, CTX-M-bearing plasmids or transposons often contain other drug resistance genes. Furthermore, a combination of CTX-M-55 and CTX-M-65 genes was detected for the first time in foodborne Salmonella strains. Our findings reveal the prevalence and molecular characteristics of cefotaxime-resistant foodborne Salmonella strains in southern China. IMPORTANCE Cefotaxime-resistant Salmonella strains pose an increasing threat to human health by causing infections with limited treatment options. It is therefore necessary to undertake a surveillance on the prevalence of such strains and investigate the resistance and transmission mechanisms. In this work, various ESBL genes flanked by different IS located in different mobile genetic elements were detectable among cefotaxime-resistant Salmonella strains. These data show that the high prevalence and genotypic diversity of cefotaxime-resistant foodborne Salmonella strains in China are possibly attributed to the evolution and transmission of a wide range of multidrug resistance-encoding mobile genetic elements.
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Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major threat to public health due to its resistance to almost all antibiotics. It is associated with substantial morbidity and mortality and poses a significant challenge to healthcare systems around the globe. Based on our previous nationwide survey of carbapenem-resistant Enterobacteriaceae (CRE) in China, seven blaIMP-4-carrying CRKP isolates were identified, all exhibiting MDR and epidemiologically linked to four different regions in China. WGS analysis revealed that the seven blaIMP-4 genes were all located on plasmids, of which five blaIMP-4 genes were located on the IncHI5 plasmids and the other two belonged to the IncN and IncFIIK plasmids, respectively. Except for the IncHI5 plasmid, conjugation assays revealed that the IncN and IncFIIK plasmids could be transferred to the recipient strain Escherichia coli J53. This study revealed significant genetic variation and identified numerous resistance factors among blaIMP-4-carrying CRKP strains in China, suggesting that blaIMP-4-carrying CRKP strains evolved via multiple phylogenetic routes and highlighting a need for expanded surveillance and establishment of control measures to prevent dissemination of CRKP strains, and facilitate development of more effective antibiotic stewardship policies and infection control programs.
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Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Klebsiella , Humanos , Klebsiella pneumoniae/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Infecciones por Klebsiella/epidemiología , Filogenia , Antibacterianos/farmacología , China/epidemiología , Plásmidos/genética , Escherichia coli/genética , Genómica , Carbapenémicos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Plasmid-mediated quinolone resistance (PMQR) determinants, such as qnrVC genes, have been widely reported in Vibrio spp. while other types of PMQR genes were rarely reported in these bacteria. This study characterized the phenotypic and genotypic features of foodborne Vibrio spp. carrying qnrS, a key PMQR gene in Enterobacteriaceae. Among a total of 1,811 foodborne Vibrio isolates tested, 34 (1.88%) were found to harbor the qnrS gene. The allele qnrS2 was the most prevalent, but coexistence with other qnr alleles was common. Missense mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes were only found in 11 of the 34 qnrS-bearing isolates. Antimicrobial susceptibility tests showed that all 34 qnrS-bearing isolates were resistant to ampicillin and that a high percentage also exhibited resistance to cefotaxime, ceftriaxone, and trimethoprim-sulfamethoxazole. Genetic analysis showed that these phenotypes were attributed to a diverse range of resistance elements that the qnrS-bearing isolates harbored. The qnrS2 gene could be found in both the chromosome and plasmids; the plasmid-borne qnrS2 genes could be found on both conjugative and nonconjugative plasmids. pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of phenotypic resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would speed up the emergence of multidrug-resistant (MDR) pathogens that are resistant to the most important antibiotics used in treatment of Vibrio infections, suggesting that close monitoring of emergence and dissemination of MDR Vibrio spp. in both food samples and clinical settings is necessary. IMPORTANCE Vibrio spp. used to be very susceptible to antibiotics. However, resistance to clinically important antibiotics, such as cephalosporins and fluoroquinolones, among clinically isolated Vibrio strains is increasingly common. In this study, we found that plasmid-mediated quinolone resistance (PMQR) genes, such as qnrS, that have not been previously reported in Vibrio spp. can now be detected in food isolates. The qnrS2 gene alone could mediate expression of ciprofloxacin resistance in Vibrio spp.; importantly, this gene could be found in both the chromosome and plasmids. The plasmids that harbor the qnrS2 gene could be both conjugative and nonconjugative, among which the pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would accelerate the emergence of multidrug-resistant pathogens.
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Quinolonas , Vibrio , Ciprofloxacina/farmacología , Cefalosporinas , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Plásmidos/genética , Monobactamas , Vibrio/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Phenotypic resistance to fluoroquinolones due to mutational changes in the gyrA and parC genes is common among clinical Vibrio strains; the plasmid-mediated quinolone resistance (PMQR) qnrVC genes were also suggested to play a role in enhancing resistance development. This study investigated the prevalence of qnrVC genes in foodborne Vibrio strains collected in Shenzhen, China, during the period August 2015 and April 2017. A total of 1811 foodborne Vibrio strains were collected, mostly (73.8%) from shrimp samples and 20.2% of these strains were resistant to ciprofloxacin. Investigation of resistance mechanisms showed that mutations in the gyrA and parC genes were commonly associated with ciprofloxacin resistance. The presence of qnrVC genes was shown to enhance ciprofloxacin MIC in Vibrio strains and 69.7% of Vibrio strains that harbored target mutations also carried qnrVC genes, yet only 27.5% of the isolates not harboring such mutations carried the qnrVC genes. A total of 141 strains were found to carry the qnrVC alleles, with qnrVC5 and qnrVC1 being the most common types. Fourteen qnrVC variant genes that contained novel mutations were detectable, with 12 (85.7%) involving qnrVC5-like alleles. For the first time, we found a variant that was likely formed by the recombination of qnrVC1 and qnrVC5. The genetic context of the qnrVC genes found in this study was highly variable, with most being accompanied by mobile genetic elements and other resistance genes. The increasing prevalence of qnrVC genes in Vibrio and its contribution on mediating the development of ciprofloxacin resistance need to be further investigated.
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Farmacorresistencia Bacteriana , Vibrio , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana/genética , Prevalencia , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Vibrio/genética , Girasa de ADN/genética , MutaciónRESUMEN
Colistin resistance mediated by mcr-1-bearing plasmids poses a new challenge to treatment of Salmonella infections. To probe the scale of the problem that colistin resistance mediated by mcr-1 plasmids among Salmonella, the prevalence of mcr-1 in foodborne Salmonella recovered from 2014 to 2017 in Shenzhen, China and genetic profile of mcr-1 positive isolates were investigated. All mcr-1 positives Salmonella strains were collected from food products, characterized by PCR and MALDI-TOF, and subjected to antimicrobial susceptibility testing, whole-genome sequencing, bioinformatics analysis, and conjugation. Twenty-eight mcr-1-positive Salmonella strains were recovered from pork. The rate of recovery displayed an increasing trend and was often accompanied by multidrug resistance. Salmonella Typhimurium was the most prevalent serotypes. Comparative genomic analysis indicated that the mcr-1 gene was located on the transferable IncX4 plasmids, as well as the IncHI2 plasmids, in which the gene was associated with ISApl1. All two types of plasmids were often detected in zoonotic pathogen. Transferable 251K mcr-1-bearing IncHI2 type plasmids were frequently reported in human and food-producing animals, but this is first time to detect a certain number in food. These findings show that dissemination of these two types of plasmids is responsible for the increase in the prevalence of colistin resistance in Salmonella strains in recent years, leading to rapid emergence of MDR Salmonella upon acquisition of these two mcr-1-bearing plasmids. Transmission of IncX4 and IncHI2 plasmids in Salmonella would cause huge public health concerns in controlling foodborne infections caused by Salmonella.
Asunto(s)
Colistina , Proteínas de Escherichia coli , Animales , Antibacterianos/farmacología , China/epidemiología , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Proteínas de Escherichia coli/genética , Humanos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Salmonella typhimurium/genéticaRESUMEN
BACKGROUND: Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health. OBJECTIVES: To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio. METHODS: A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains. RESULTS: In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts. CONCLUSIONS: The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide.
Asunto(s)
Antibacterianos , Vibrio , Tipificación de Secuencias Multilocus , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , beta-Lactamasas/genética , Klebsiella pneumoniae/genética , Plásmidos , Escherichia coli/genética , Vibrio/genética , Genómica , China/epidemiologíaRESUMEN
We previously reported the recovery of five ST11 carbapenem resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains that harbored pLVPK-like virulence plasmids, yet molecular mechanisms underlying acquisition of virulence plasmid by ST11 K. pneumoniae have not been characterized. In this study, we showed that virulence plasmids in these CR-HvKP strains could be transferred to Escherichia coli strain EC600 via conjugation. Transmission of the virulence plasmids was found to involve formation of fusion plasmids with an Incl1 type conjugative plasmid and a small ColRNAI plasmid through homologous recombination and by insertion sequences IS26 and IS903B. The conjugative fusion event would transform different ST types of K. pneumoniae, in particular, the clinically prevalent ST11 or ST258 CRKP into CR-HvKP. Clinical factors that promote or suppress the occurrence of this fusion process should be further investigated to devise new approaches to halt such bacterial evolution trends.
RESUMEN
Metagenome assembly is a core yet methodologically challenging step for taxonomic classification and functional annotation of a microbiome. This study aims to generate the high-resolution human gut metagenome using both Illumina and Nanopore platforms. Assembly was achieved using four assemblers, including Flye (Nanopore), metaSPAdes (Illumina), hybridSPAdes (Illumina and Nanopore), and OPERA-MS (Illumina and Nanopore). Hybrid metagenome assembly was shown to generate contigs with almost same sizes comparable to those produced using Illumina reads alone, but was more contiguous, informative, and longer compared with those assembled with Illumina reads only. In addition, hybrid metagenome assembly enables us to obtain complete plasmid sequences and much more AMR gene-encoding contigs than the Illumina method. Most importantly, using our workflow, 58 novel high-quality metagenome bins were obtained from four assembly algorithms, particularly hybrid assembly (47/58), although metaSPAdes could provide 11 high-quality bins independently. Among them, 29 bins were currently uncultured bacterial metagenome-assembled genomes. These findings were highly consistent and supported by mock community data tested. In the analysis of biosynthetic gene clusters (BGCs), the number of BGCs in the contigs from hybridSPAdes (241) is higher than that of contigs from metaSPAdes (233). In conclusion, hybrid metagenome assembly could significantly enhance the efficiency of contig assembly, taxonomic binning, and genome construction compared with procedures using Illumina short-read data alone, indicating that nanopore long reads are highly useful in metagenomic applications. This technique could be used to create high-resolution references for future human metagenome studies.