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BackgroundMultidrug-resistant strains of the genus Aeromonas can produce various β-lactamases that confer resistance to a broad spectrum of β-lactams, which poses a significant public health threat due to their emergence and spread in clinical settings and natural environments. Therefore, a comprehensive investigation into the antibiotic resistance mechanisms of Aeromonas is scientifically significant.MethodsBetween 2018 and 2021, 78 clinical Aeromonas isolates were collected from human clinical specimens. The MicroScan WalkAway system and average nucleotide identity (ANI) analyses were used to classify the bacterial species. Antibiotic susceptibility was determined through the minimum inhibitory concentration (MIC) test via the agar dilution method. To determine the resistance mechanism and the structure of the resistance gene-related sequences, molecular cloning, whole-genome sequencing and bioinformatic analysis were performed.ResultsAmong the 78 Aeromonas isolates studied in this work, obtained from various specimens from different clinical departments, 77 were classified into seven known species by ANI analysis. Most of the isolates were A. caviae (34.6%, 27/78), followed by A. hydrophila (25.6%, 20/78). Multilocus sequence typing (MLST) revealed that they belonged to 72 sequence types (STs), including 52 new STs. A total of 334 resistance genes of 30 antibiotic resistance genotypes were identified from the genomes, more than half (55.99%, 187/334) of which were β-lactamase genes. The isolates showed much higher rates of resistance to penicillins (penicillin G, 98.7%) and first-generation cephalosporins (cefazolin, 96.2%), but lower resistance rates to fourth-generation cephalosporins (cefepime, 6.4%), monobactams (aztreonam, 5.1%), and carbapenems (imipenem, 1.3% and meropenem, 5.1%). Structural analyses of some β-lactamase genes (such as blaNDM-1 and blaPER-3) related sequences revealed that they were generally associated with mobile genetic elements.ConclusionThe investigation of the correlation between the distribution of β-lactamase genes and Aeromonas resistance phenotypes in this study suggested an urgent need for rigorous monitoring and control to counteract the escalating public health threat posed by the increase in Aeromonas strains harboring extended-spectrum β-lactamase and metallo-β-lactamase genes.

BackgroundAminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms.MethodsThe bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques.ResultsA novel aminoglycoside resistance gene, designated aph(3’)-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3’)-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3’)-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (Km, 4.22 ± 0.88 µM) and the highest catalytic efficiency [kcat/Km, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3’) proteins, APH(3’)-Ie contained all the conserved functional sites of the APH family. The aph(3’)-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance.ConclusionIn this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3’)-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.

BackgroundAeromonas species have been identified as agents responsible for various diseases in both humans and animals. Multidrug-resistant Aeromonas strains pose a significant public health threat due to their emergence and spread in clinical settings and the environment. The aim of this study was to determine a novel resistance mechanism against aminoglycoside antimicrobials in a clinical isolate.MethodsThe function of aac(6′)-Va was verified by gene cloning and antibiotic susceptibility tests. To explore the in vivo activity of the enzyme, recombinant proteins were expressed, and enzyme kinetics were tested. To determine the molecular background and mechanism of aac(6′)-Va, whole-genome sequencing and bioinformatic analysis were performed.ResultsThe novel aminoglycoside N-acetyltransferase gene aac(6′)-Va confers resistance to several aminoglycosides. Among the antimicrobials tested, ribostamycin showed the highest increase (128-fold) in the minimum inhibitory concentration (MIC) compared with the control strains. According to the MIC results of the cloned aac(6′)-Va, AAC(6′)-Va also showed the highest catalytic efficiency for ribostamycin [kcat/Km ratio = (3.35 ± 0.17) × 104 M−1 s−1]. Sharing the highest amino acid identity of 54.68% with AAC(6′)-VaIc, the novel aminoglycoside N-acetyltransferase constituted a new branch of the AAC(6′) family due to its different resistance profiles. The gene context of aac(6′)-Va and its close relatives was conserved in the genomes of species of the genus Aeromonas.ConclusionThe novel resistance gene aac(6′)-Va confers resistance to several aminoglycosides, especially ribostamycin. Our finding of a novel resistance gene in clinical A. hydrophila will help us develop more effective treatments for this pathogen’s infections.

BackgroundAchromobacter is a genus of gram-negative bacteria that can act as opportunistic pathogens. Recent studies have revealed that some species of Achromobacter show inherent resistance to β-lactams, but the resistance mechanisms of Achromobacter mucicolens have rarely been reported.MethodThe bacterium was isolated using standard laboratory procedures. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs). Genome sequencing was performed using the PacBio RS II and Illumina HiSeq 2500 platforms, and the Comprehensive Antibiotic Resistance Database (CARD) was used to annotate the drug resistance genes. The localization of the novel β-lactamase AMZ-1 was determined, and its characteristics were determined via molecular cloning and enzyme kinetic analysis. The phylogenetic relationship and comparative genomic analysis of the resistance gene-related sequences were also analyzed.ResultAchromobacter mucicolens Y3, isolated from a goose on a farm in Wenzhou, showed resistance to multiple antibiotics, including penicillins and cephalosporins. BlaAMZ–1 showed resistance to amoxicillin, penicillin G, ampicillin, cephalothin and cefoxitin, and the resistance activity could be inhibited by β-lactamase inhibitors. Enzyme kinetic analysis results showed that AMZ-1 has hydrolytic activity against a wide range of substrates, including cephalothin, amoxicillin, penicillin G, and cefoxitin but not ampicillin. The hydrolytic activity of AMZ-1 was greatly inhibited by avibactam but much more weakly inhibited by tazobactam. Mobile genetic elements could not be found around the blaAMZ–1-like genes, which are conserved on the chromosomes of bacteria of the genus Achromobacter.ConclusionIn this study, a novel AmpC gene, blaAMZ–1, from the animal-origin bacterium A. mucicolens Y3 was identified and characterized. It conferred resistance to some penicillins and first- and second-generation cephalosporins. The identification of this novel resistance gene will be beneficial for the selection of effective antimicrobials to treat associated infections.

BackgroundAminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention.MethodsThe bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3′)-Id gene and its phylogenetic relationship with other AMEs.ResultsA novel aminoglycoside 3′-O-phosphotransferase gene designated aph(3′)-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3′-O-phosphotransferase APH(3′)-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3′)-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3′)-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M−1/s−1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M−1/s−1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M−1/s−1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M−1/s−1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3′)-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3′)-Id gene.ConclusionIn this work, a novel aminoglycoside 3’-O-phosphotransferase gene designated aph(3′)-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.

BackgroundPantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance.MethodsA rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The β-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance gene-related sequences were analyzed by bioinformatic methods.ResultsThe isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for third-generation cephalosporins or aztreonam.ConclusionIn this study, a novel AmpC β-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance.

Background: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. Methods: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. Results: A novel aminoglycoside resistance gene, designated aph(3')-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (Km, 4.22 ± 0.88 µM) and the highest catalytic efficiency [kcat/Km, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3') proteins, APH(3')- Ie contained all the conserved functional sites of the APH family. The aph(3')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance. Conclusion: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3')-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes. [ABSTRACT FROM AUTHOR]

Background: Achromobacter is a genus of gram-negative bacteria that can act as opportunistic pathogens. Recent studies have revealed that some species of Achromobacter show inherent resistance to β-lactams, but the resistance mechanisms of Achromobacter mucicolens have rarely been reported. Method: The bacterium was isolated using standard laboratory procedures. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs). Genome sequencing was performed using the PacBio RS II and Illumina HiSeq 2500 platforms, and the Comprehensive Antibiotic Resistance Database (CARD) was used to annotate the drug resistance genes. The localization of the novel β-lactamase AMZ-1 was determined, and its characteristics were determined via molecular cloning and enzyme kinetic analysis. The phylogenetic relationship and comparative genomic analysis of the resistance gene-related sequences were also analyzed. Result: Achromobacter mucicolens Y3, isolated from a goose on a farm in Wenzhou, showed resistance to multiple antibiotics, including penicillins and cephalosporins. BlaAMZ-1 showed resistance to amoxicillin, penicillin G, ampicillin, cephalothin and cefoxitin, and the resistance activity could be inhibited by β-lactamase inhibitors. Enzyme kinetic analysis results showed that AMZ-1 has hydrolytic activity against a wide range of substrates, including cephalothin, amoxicillin, penicillin G, and cefoxitin but not ampicillin. The hydrolytic activity of AMZ-1 was greatly inhibited by avibactam but much more weakly inhibited by tazobactam. Mobile genetic elements could not be found around the blaAMZ-1-like genes, which are conserved on the chromosomes of bacteria of the genus Achromobacter. Conclusion: In this study, a novel AmpC gene, blaAMZ-1, from the animal-origin bacterium A. mucicolens Y3 was identified and characterized. It conferred resistance to some penicillins and first- and second- generation cephalosporins. The identification of this novel resistance gene will be beneficial for the selection of effective antimicrobials to treat associated infections. [ABSTRACT FROM AUTHOR]

Background: Pantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance. Methods: A rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The ß-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance generelated sequences were analyzed by bioinformatic methods. Results: The isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for thirdgeneration cephalosporins or aztreonam. Conclusion: In this study, a novel AmpC β-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance. [ABSTRACT FROM AUTHOR]