APH(3’)-Ie, an aminoglycoside-modifying enzyme discovered in a rabbit-derived Citrobacter gillenii isolate

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

BlaPSZ-1, a novel AmpC gene identified from a Pantoea isolate

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

Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm

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

cTFbase: a database for comparative genomics of transcription factors in cyanobacteria

BACKGROUND: Comprehensive identification and classification of the transcription factors (TFs) in a given genome is an important aspect in understanding transcriptional regulatory networks of a specific organism. Cyanobacteria are an ancient group of gram-negative bacteria with strong variation in genome size ranging from about 1.6 to 9.1 Mb and little is known about their TF repertoires. Therefore, we constructed the cTFbase database to classify and analyze all the putative TFs in cyanobacterial genomes, followed by genome-wide comparative analysis. DESCRIPTION: In the current release, cTFbase contains 1288 putative TFs identified from 21 fully sequenced cyanobacterial genomes. Through its user-friendly interactive interface, users can employ various criteria to retrieve all TF sequences and their detailed annotation information, including sequence features, domain architecture and sequence similarity against the linked databases. Furthermore, cTFbase provides phylogenetic trees of individual TF family, multiple sequence alignments of the DNA-binding domains and ortholog identification from any selected genomes. Comparative analysis revealed great variability of the TF sequences in cyanobacterial genomes. The high variance on the gene number and domain organization would be related to their diverse biological functions and their adaptation to various environmental conditions. CONCLUSION: cTFbase provides a centralized warehouse for comparative analysis of putative TFs in cyanobacterial genomes. The availability of such an extensive database would be of great interest for the community of researchers working on TFs or transcriptional regulatory networks in cyanobacteria. cTFbase can be freely accessible at and will be continuously updated when the newly sequenced cyanobacterial genomes are available

The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen

Salmonella enterica serovar Choleraesuis (S.Choleraesuis), a highly invasive serovar among non-typhoidal Salmonella, usually causes sepsis or extra-intestinal focal infections in humans. S.Choleraesuis infections have now become particularly difficult to treat because of the emergence of resistance to multiple antimicrobial agents. The 4.7 Mb genome sequence of a multidrug-resistant S.Choleraesuis strain SC-B67 was determined. Genome wide comparison of three sequenced Salmonella genomes revealed that more deletion events occurred in S.Choleraesuis SC-B67 and S.Typhi CT18 relative to S.Typhimurium LT2. S.Choleraesuis has 151 pseudogenes, which, among the three Salmonella genomes, include the highest percentage of pseudogenes arising from the genes involved in bacterial chemotaxis signal-transduction pathways. Mutations in these genes may increase smooth swimming of the bacteria, potentially allowing more effective interactions with and invasion of host cells to occur. A key regulatory gene of TetR/AcrR family, acrR, was inactivated through the introduction of an internal stop codon resulting in overexpression of AcrAB that appears to be associated with ciprofloxacin resistance. While lateral gene transfer providing basic functions to allow niche expansion in the host and environment is maintained during the evolution of different serovars of Salmonella, genes providing little overall selective benefit may be lost rapidly. Our findings suggest that the formation of pseudogenes may provide a simple evolutionary pathway that complements gene acquisition to enhance virulence and antimicrobial resistance in S.Choleraesuis

Identification and characterization of a novel β-lactamase gene, blaAMZ–1, from Achromobacter mucicolens

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

Molecular Characterization and Antimicrobial Susceptibility of Nasal Staphylococcus aureus Isolates from a Chinese Medical College Campus

Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935) S. aureus isolates, including 28 (3.0%, 28/935) MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%), ampicillin/sulbactam (83.3%) and trimethoprim/sulfamethoxazole (93.1%). 82%, (23/28) of the MRSA isolates and 66% (77/116) of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin—an agent commonly used for nasal decolonization. 16 different sequence types (STs), as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779) and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community

Sequencing and Genetic Variation of Multidrug Resistance Plasmids in Klebsiella pneumoniae

BACKGROUND: The development of multidrug resistance is a major problem in the treatment of pathogenic microorganisms by distinct antimicrobial agents. Characterizing the genetic variation among plasmids from different bacterial species or strains is a key step towards understanding the mechanism of virulence and their evolution. RESULTS: We applied a deep sequencing approach to 206 clinical strains of Klebsiella pneumoniae collected from 2002 to 2008 to understand the genetic variation of multidrug resistance plasmids, and to reveal the dynamic change of drug resistance over time. First, we sequenced three plasmids (70 Kb, 94 Kb, and 147 Kb) from a clonal strain of K. pneumoniae using Sanger sequencing. Using the Illumina sequencing technology, we obtained more than 17 million of short reads from two pooled plasmid samples. We mapped these short reads to the three reference plasmid sequences, and identified a large number of single nucleotide polymorphisms (SNPs) in these pooled plasmids. Many of these SNPs are present in drug-resistance genes. We also found that a significant fraction of short reads could not be mapped to the reference sequences, indicating a high degree of genetic variation among the collection of K. pneumoniae isolates. Moreover, we identified that plasmid conjugative transfer genes and antibiotic resistance genes are more likely to suffer from positive selection, as indicated by the elevated rates of nonsynonymous substitution. CONCLUSION: These data represent the first large-scale study of genetic variation in multidrug resistance plasmids and provide insight into the mechanisms of plasmid diversification and the genetic basis of antibiotic resistance