Antibiotic Resistance Gene Cluster Research Articles

Genome Sequence and Characterisation of Peribacillus sp. Strain AS_2, a Bacterial Endophyte Isolated from Alectra sessiliflora

Peribacillus sp. AS_2, a leaf endophytic bacterium isolated from the medicinal plant Alectra sessiliflora, was previously identified using the 16S rRNA gene sequence. The draft genome of AS_2 had a 5,482,853 bp draft circular chromosome, 43 contigs, N50 of 360,633 bp and an average G + C% content of 40.5%. Whole genome sequencing and phenotypic analysis showed that AS_2 was Gram-positive, endospore-forming, motile, and rod-shaped and it showed a high sequence similarity with P. frigoritolerans DSM 8801T. Genomic digital DNA–DNA hybridisation (dDDH) between strain AS_2 and Peribacillus frigoritolerans DSM 8801T and P. castrilensis N3T was 84.8% and 79.2%, respectively, and the average nucleotide identity (ANI) of strain AS_2 with P. frigoritolerans DSM 8801T and P. castrilensis N3T was 97.0% and 96.7%, respectively. The antiSMASH software predicted a total of eight secondary metabolite gene clusters comprising non-ribosomal peptide synthetase (NRPS) type koranimine, terpenes, and siderophore clusters. Strain AS_2 also displayed genes involved in endophytic lifestyle and antibiotic resistance gene clusters such as small multidrug resistance antibiotic efflux pumps (qacJs). Using the multilocus sequence analysis (MLSA), together with the phenotypic data and genomic analysis, we demonstrated that strain AS_2 is a subspecies of P. frigoritolerans DSM 8801T. Genome sequencing of Peribacillus sp. AS_2 from medicinal plants provides valuable genomic information and allows us to further explore its biotechnological applications.

Whole genome sequence and comparative genomics analysis of multidrug-resistant Staphylococcus xylosus NM36 isolated from a cow with mastitis in Basrah city

BackgroundStaphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed. ResultsThe genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature. ConclusionsThese findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.

Genomics of Aminoglycoside Resistance in Pseudomonas aeruginosa Bloodstream Infections at a United States Academic Hospital.

Pseudomonas aeruginosa frequently becomes resistant to aminoglycosides by the acquisition of aminoglycoside modifying enzyme (AME) genes and the occurrence of mutations in the mexZ, fusA1, parRS, and armZ genes. We examined resistance to aminoglycosides in a collection of 227 P. aeruginosa bloodstream isolates collected over 2 decades from a single United States academic medical institution. Resistance rates of tobramycin and amikacin were relatively stable over this time, while the resistance rates of gentamicin were somewhat more variable. For comparison, we examined resistance rates to piperacillin-tazobactam, cefepime, meropenem, ciprofloxacin, and colistin. Resistance rates to the first four antibiotics were also stable, although uniformly higher for ciprofloxacin. Colistin resistance rates were initially quite low, rose substantially, and then began to decrease at the end of the study. Clinically relevant AME genes were identified in 14% of isolates, and mutations predicted to cause resistance were relatively common in the mexZ and armZ genes. In a regression analysis, resistance to gentamicin was associated with the presence of at least one gentamicin-active AME gene and significant mutations in mexZ, parS, and fusA1. Resistance to tobramycin was associated with the presence of at least one tobramycin-active AME gene. An extensively drug-resistant strain, PS1871, was examined further and found to contain five AME genes, most of which were within clusters of antibiotic resistance genes embedded in transposable elements. These findings demonstrate the relative contributions of aminoglycoside resistance determinants to P. aeruginosa susceptibilities at a United States medical center. IMPORTANCE Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance. Mutations in the mexZ, fusA1, parR, pasS, and armZ genes were more common than acquisition of genes encoding aminoglycoside modifying enzymes. The whole-genome sequence of an extensively drug resistant isolate indicates that resistance mechanisms can accumulate in a single strain. Together, these results suggest that aminoglycoside resistance in P. aeruginosa remains problematic and confirm known resistance mechanisms that can be targeted for the development of novel therapeutics.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections.ObjectiveIn this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically.MethodsOne hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at −80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst’s latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk—v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student’s t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05.ResultsPhage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3–11) and temperature (45–65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome.Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1).ConclusionThe broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Antibiotic and metal resistance of Stenotrophomonas maltophilia isolates from Eboling permafrost of the Tibetan Plateau.

Whole-genome sequencing of pathogenic bacteria Stenotrophomonas maltophilia from a less polluted environment of permafrost can help understand the intrinsic resistome of both antibiotics and metals. This study aimed to examine the maximum minimum inhibitory concentration (MIC) of both antibiotics and metals, as well as antibiotic resistance genes and metal resistance genes annotated from whole-genome sequences. The permafrost S. maltophilia was sensitive to ciprofloxacin, tetracycline, streptomycin, and bacitracin, and resistant to chloramphenicol, trimethoprim-sulfamethoxazole, erythromycin, Zn2+, Ni2+, Cu2+, and Cr6+, with a lower maximum MIC, compared with clinical S. maltophilia. The former strain belonged to the lower antibiotic resistance gene (ARG) and metal resistance gene (MRG) clusters compared with the latter ones. The permafrost strain contained no or only one kind of ARG or MRG on a single genomic island, which explained the aforementioned lower maximum MIC and less diversity of ARGs or MRGs. The result indicated that the co-occurrence of antibiotic and metal resistance was due to a certain innate ability of S. maltophilia. The continuous human use of antibiotics or metals induced selective pressure, resulting in higher MIC and more diverse ARGs and MRGs in human-impacted environments.

Isolation and characterization of the novel bacteriophage vB_SmaS_BUCT626 against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia has been recognized as an emerging global opportunistic pathogen, and it is intrinsically resistant to most antibiotics, which makes the limited choice for treating S. maltophilia infections. Bacteriophage with the proper characterization is considered as a promising alternative treatment option to control S. maltophilia infections. In this study, we isolated a novel Siphoviridae bacteriophage vB_SmaS_BUCT626 with lytic activity against S. maltophilia. Phage vB_SmaS_BUCT626 can lysis 10 of 20 S. maltophilia and was relatively stable at a wide range of temperatures (4-70°C) and pH values (3.0-13.0) and exhibited good tolerance to chloroform. The genome of phage vB_SmaS_BUCT626 was a 61,662-bp linear double-stranded DNA molecule with a GC content of 56.2%, and contained 100 open-reading frames. It carried no antibiotic resistance, toxin, virulence-related genes, or lysogen-formation gene clusters. Together, these characteristics make phage vB_SmaS_BUCT626, a viable candidate as a biocontrol agent against S. maltophilia infection.

Complete Genome Analysis of Highly Pathogenic Non-O1/O139 Vibrio cholerae Isolated From Macrobrachium rosenbergii Reveals Pathogenicity and Antibiotic Resistance-Related Genes.

Non-O1/O139 Vibrio cholerae is a highly virulent pathogen that causes mass mortalities of various aquatic animals. In the present study, we sequenced the whole genome of non-O1/O139 V. cholerae GXFL1-4, isolated from Macrobrachium rosenbergii, to reveal the pathogenicity and antibiotic resistance. The result showed its genome contained two circular chromosomes and one plasmid with a total size of 4,282,243 bp, which harbored 3,869 coding genes. Among them, 3,047, 2,659, and 3,661 genes were annotated in the Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. In addition, 372 potential virulence genes were predicted based on the Virulence Factor Database (VFDB) database, such as type II, III, IV, and VI secretion systems related genes, flagella genes, and pilus formation or motility-related genes. Blast results in the Comprehensive Antibiotic Resistance Database (CARD) database showed that the strain contained 148 antibiotic resistance-related genes belonging to 27 categories, such as efflux pump complex antibiotic resistance genes and antibiotic resistance gene cluster genes. The Pathogen-Host Interaction (PHI) database annotated 320 genes related to pathogen-host interaction, such as T3SS, virulence regulatory factors, transcriptional regulators, and two-component response regulator related genes. The whole-genome analysis suggested that the pathogenic non-O1/O139 V. cholerae strain GXFL1-4 might have a complex molecular mechanism of pathogenicity and antibiotic resistance. This study provides a wealth of information about non-O1/O139 V. cholerae genes related to its pathogenicity and drug resistance and will facilitate the understanding of its pathogenesis as well as the development of prevention and treatment strategies for the pathogen.

Extensively resistant Acinetobacter baumannii isolate RCH52 carries several resistance genes derived from an IncC plasmid.

To identify the origins of resistance in a sporadic extensively resistant Acinetobacter baumannii isolate. The complete genome of RCH52 was determined by combining available Illumina short reads with MinION (Oxford Nanopore) long reads using Unicycler. Bioinformatic searches were used to identify features of interest. The complete genome of RCH52 revealed an unusual chromosomal region containing all of the antibiotic resistance genes, except tet39, which is in a plasmid. A 129 585 bp segment was bounded by inversely oriented copies of ISAba1 and included two groups of resistance genes separated by the large segment of the backbone of type 1 IncC plasmids that lies between the ARI-A and ARI-B resistance islands but does not include the replication region. The ISAba1-bounded segment was located in a novel integrative element that had integrated into the chromosomal thyA gene but provided a replacement thyA gene. Several resistance genes are derived from either the ARI-A or the ARI-B resistance islands found in IncC plasmids that have been brought together by an IS26-mediated deletion of the original plasmid. This non-replicating circular molecule (or translocatable unit) has been incorporated into a smaller ISAba1-bounded unit that includes oxa23 in Tn2008B via homologous recombination between sul2-CR2-floR segments found in both. The plasmids shared by most Gram-negative pathogens, including the broad host range IncC plasmids, have not been detected in Acinetobacter species. However, it seems likely that they can conjugate into members of this genus and contribute pre-existing clusters of antibiotic resistance genes.

Specific Inhibition of VanZ-Mediated Resistance to Lipoglycopeptide Antibiotics.

Teicoplanin is a natural lipoglycopeptide antibiotic with a similar activity spectrum as vancomycin; however, it has with the added benefit to the patient of low cytotoxicity. Both teicoplanin and vancomycin antibiotics are actively used in medical practice in the prophylaxis and treatment of severe life-threatening infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, Enterococcus faecium and Clostridium difficile. The expression of vancomycin Z (vanZ), encoded either in the vancomycin A (vanA) glycopeptide antibiotic resistance gene cluster or in the genomes of E. faecium, as well as Streptococcus pneumoniae and C. difficile, was shown to specifically compromise the antibiotic efficiency through the inhibition of teicoplanin binding to the bacterial surface. However, the exact mechanisms of this action and protein structure remain unknown. In this study, the three-dimensional structure of VanZ from E. faecium EnGen0191 was predicted by using the I-TASSER web server. Based on the VanZ structure, a benzimidazole based ligand was predicted to bind to the VanZ by molecular docking. Importantly, this new ligand, named G3K, was further confirmed to specifically inhibit VanZ-mediated resistance to teicoplanin in vivo.

Targeted Conservative Cointegrate Formation Mediated by IS26 Family Members Requires Sequence Identity at the Reacting End.

IS26 forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other's ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26 However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed.IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.

Pan-genome analysis of Riemerella anatipestifer reveals its genomic diversity and acquired antibiotic resistance associated with genomic islands.

Riemerella anatipestifer is a gram-negative bacterium that leads to severe contagious septicemia in ducks, turkeys, chickens, and wild waterfowl. Here, a pan-genome with 32 R. anatipestifer genomes is re-established, and the mathematical model is calculated to evaluate the expansion of R. anatipestifer genomes, which were determined to be open. Average nucleotide identity (ANI) and phylogenetic analysis preliminarily clarify intraspecies variation and distance. Comparative genomic analysis of R. anatipestifer found that horizontal gene transfer events, which provide an expressway for the recruitment of novel functionalities and facilitate genetic diversity in microbial genomes, play a key role in the process of acquiring and transmitting antibiotic-resistance genes in R. anatipestifer. Furthermore, a new antibiotic-resistance gene cluster was identified in the same loci in 14 genomes. The uneven distribution of virulence factors was also confirmed by our results. Our study suggests that the ability to acquire foreign genes (such as antibiotic-resistance genes) increases the adaptability of R. anatipestifer, and the virulence genes with little mobility are highly conserved in R. anatipestifer.

Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis.

Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacEΔ1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.

Long-term effects of manure and chemical fertilizers on soil antibiotic resistome

Applications of manure and chemical fertilizers can significantly modulate the bacterial communities in soil, but the effects on the soil antibiotic resistome and the underlying mechanisms remain largely unclear. In the present study, antibiotic resistance genes (ARGs) and bacterial communities were characterized using high-throughput quantitative PCR and Illumina HiSeq sequencing, respectively, in soils from a long-term (25 years) field experiment. Treatments included no fertilizers (control), chemical fertilizers (NPK), NPK with straw return (NPKS), pig manure (M), and M with chemical fertilizers (NPKM). Both NPK and NPKS decreased soil pH and induced a substantial shift in the bacterial communities, but had only moderate effect on the ARG diversity and abundance. The effect of straw return was hindered by the dominant influence of NPK. In comparison, additions of pig manure (M and NPKM) maintained the diversity of bacterial community and significantly influenced the ARG profiles by introducing extra ARGs from pig manure and boosting the soil indigenous ARG members. Manured soils harboured clusters of ARGs and transposase genes which were relatively independent of bacterial phylogenetic lineages, suggesting a strong co-occurrence of ARGs in soil bacteria which may result from horizontal gene transfers (HGT). Our results indicate the importance of HGT in the maintenance of ARG composition in agricultural soils, especially those receiving long-term manure applications.

Molecular analysis of antibiotic resistance gene clusters in Acinetobacter baumannii clinical isolates

This article was retracted by Editor in chief and authors after the paper has already been accepted.

Salmonella genomic island 1-J variants associated with change in the antibiotic resistance gene cluster in multidrug-resistant Salmonella enterica serovar Virchow isolated from humans, Taiwan, 2004–2006

Salmonella genomic island 1 (variant SGI1-J3) has been previously identified in multi-drug resistant (MDR) Salmonella enterica serovar Virchow isolated from humans in 1994. In this study, antimicrobial resistance, genotypes and genetic relationship were investigated in 96 S. Virchow isolates collected from humans in 2004–2006. XbaI-PFGE analysis separated 96 isolates into two main related clusters, I and II, which consisted of four major pulsotypes differing in prevalence by year. The majority of isolates were MDR to chloramphenicol, sulfonamide, trimethoprim and tetracyclines associated with antimicrobial resistance genes dfrA1, floR2, sulI and tet(G) of variant SGI1-J3. Among nine variants, we determined two novel variants, SGI1-J4 and -J5, which have undergone different homologous recombinational events resulting in partial deletions of the MDR region. The first one contained an empty integron structure and the second presented a deletion extending from the IS6100 element to the adjacent SGI1 backbone. SGI1-J3 is largely encountered in clonally related MDR S. Virchow isolates collected from humans, which spread vertically. The genomic island SGI1 appears to be largely responsible for the diversity of MDR phenotypes among S. Virchow isolates in Taiwan.

Bacterial mer operon-mediated detoxification of mercurial compounds: a short review

Mercury pollution has emerged as a major problem in industrialized zones and presents a serious threat to environment and health of local communities. Effectiveness and wide distribution of mer operon by horizontal and vertical gene transfer in its various forms among large community of microbe reflect importance and compatibility of this mechanism in nature. This review specifically describes mer operon and its generic molecular mechanism with reference to the central role played by merA gene and its related gene products. The combinatorial action of merA and merB together maintains broad spectrum mercury detoxification system for substantial detoxification of mercurial compounds. Feasibility of mer operon to coexist with antibiotic resistance gene (ampr, kanr, tetr) clusters enables extensive adaptation of bacterial species to adverse environment. Flexibility of the mer genes to exist as intricate part of chromosome, plasmids, transposons, and integrons enables high distribution of these genes in wider microbial gene pool. Unique ability of this system to manipulate oligodynamic property of mercurial compounds for volatilization of mercuric ions (Hg2+) makes it possible for a wide range of microbes to tolerate mercury-mediated toxicity.

A multiresistance megaplasmid pLG1 bearing a hylEfm genomic island in hospital Enterococcus faecium isolates

Enterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl Efm , encoding a putative hyaluronidase, is enriched among the hospital-associated polyclonal subpopulation of E. faecium.. The hyl Efm gene is described to be part of a genomic island and was recently identified to be plasmid-located. Here, we present a description of the structure, localization, and distribution of the putative pathogenicity factor hyl Efm and its putative island among 39 clinical isolates and elucidate the composition and host range of pLG1, a hyl Efm multiresistance plasmid of approximately 281.02 kb. The hyl Efm gene was located within a 17,824-bp element highly similar to the putative genomic island (GI) structure that had been previously described. This genomic region was conserved among 39 hyl Efm -positive strains with variation in a specific region downstream of hyl Efm in 18 strains. The putative hyl Efm was located on large plasmids (150–350 kb) in 37 strains. pLG1 could be horizontally transferred into four different E. faecium recipient strains ( n = 4) but not into E. faecalis ( n = 3). Sequencing of pLG1 resolved putative plasmid replication, conjugation, and maintenance determinants as well as a pilin gene cluster, carbon uptake and utilization genes, heavy metal and antibiotic resistance clusters. The hyl Efm transferable plasmid pLG1 bears additional putative pathogenicity factors and antibiotic resistance genes. These findings suggest horizontal gene transfer of virulence factors and antibiotic resistance gene clusters by a single genetic event (conjugative transfer) which might be triggered by heavy antibiotic use common in health care units where E. faecium is increasingly prevalent.

IS CR Elements Are Key Players in IncA/C Plasmid Evolution

In the recent paper by Call et al. (1), the authors show that sequence analysis of their IncA/C plasmids revealed that antimicrobial resistance genes were grouped in four main regions in addition to the region that harbors the blaCMY gene in an otherwise highly related plasmid backbone structure. The authors show that the floR resistance region is found in all three plasmids as well as in the pSN254 plasmid and is found in close proximity to what the authors (and also a previous paper [9]) call insA. The authors also note that another antibiotic resistance gene cluster includes the resistance gene aadA which is found in three of these plasmids and is always found adjacent to an insertion sequence termed insE (9). These two insertions, insE and insA, are typical of insertion sequences previously designated ISCR elements (6, 7). ISCR elements are closely related to an unusual family of insertion sequences called the IS91 family (6, 7). These elements are known to move by a process called rolling-circle replication, and a function of this process is the concomitant movement of additional sequences found upstream of their transposase genes (5). To be specific, insE is indeed ISCR2, and insF is ISCR16 (8). ISCR elements are now recognized as powerful antibiotic resistance gene capture and movement systems that are also capable of constructing extended clusters of antibiotic resistance genes on plasmids as well as on chromosomes (7). The IncA/C plasmid group is now known to be very widely distributed both geographically and in many species of bacteria because of the plasmids' promiscuous behavior (3, 4). The IncA/C reference plasmid, pRA1, isolated in 1971 from the fish pathogen Aeromonas hydrophila harbors ISCR2 (2), indicating that this element has been closely involved in recruiting antibiotic resistance genes with IncA/C plasmids for some time. ISCR2 is often found in close proximity to the sul2 gene and a truncated glmM gene as a conserved resistance array among IncA/C plasmids; ISCR2 is often called an ISVsa3-like IS element or simply orfA (2). Interestingly, a recent publication has indicated that the ICE element SXT shares a common ancestor with the IncA/C plasmids (2), and ISCR2 together with truncated copies of this element are always found as part as SXT, implicating ISCR2 in the construction of this element (6). The ISCR16 element appears to be a more recent addition to the IncA/C plasmids and has been found in only three plasmids so far and always adjacent to groEL or groES gene(s) (8). This also is very interesting as this element and the closely related elements ISCR3 and ISCR4 are also found adjacent to sections of groEL as well as resistance genes, which has allowed us to track the evolutionary history of this element (8). Therefore, it appears that ISCR elements are key players in the recent evolution of the IncA/C plasmid group. Furthermore, gene capture systems such as ISCR elements allied with the promiscuous nature of IncA/C plasmids provides a new and powerful combination for rapid dissemination of antibiotic resistance genes.

RSF1010-Like Plasmids in AustralianSalmonella entericaSerovar Typhimurium and Origin of Theirsul2-strA-strBAntibiotic Resistance Gene Cluster

Salmonella enterica serovar Typhimurium phage type 9 isolates resistant to streptomycin and sulfonamide have been recovered from both bovine and human sources in Australia. This study aimed to identify the resistance genes and their location. Polymerase chain reaction was used to screen for resistance genes and sul2 (sulphonamide resistance) and strA and strB (streptomycin resistance) were detected. A small streptomycin and sulfonamide resistance plasmid carrying the three resistance genes was recovered from these isolates by transformation and was shown to be essentially identical to the small IncQ plasmid RSF1010. The sequences of one plasmid, pSRC15, and RSF1010 differed at only a few positions that may be errors in the older sequence. RSF1010 has been recovered from many species and in many countries since its first isolation in the early 1970s. We conclude that this plasmid has persisted unchanged in the environment for over 30 years. The antibiotic resistance gene cluster containing strA, strB, and sul2 genes has clearly arisen from other known entities by a combination of transposition and homologous recombination using a short segment of homology. This resistance gene cluster is now widely distributed in plasmids and genomic islands in a number of contexts.

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1