Transposon Tn5 Research Articles

The inner membrane protein YhiM links copper and CpxAR envelope stress responses in uropathogenic E. coli.

Urinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 μg/ml) and to streptomycin (MIC > 100 μg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 μg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

Tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Construction of a Temperature-Sensitive Shuttle Vector between Lactic Acid Bacteria and Escherichia coli and Its Application to a Tn10-Based Random Mutagenesis Tool in Lactic Acid Bacteria.

In the present study, we have obtained a temperature-sensitive replication mutant in the Escherichia (E.) coli-lactic acid bacterium (LAB) shuttle vector pLES003-b carrying erythromycin-resistance gene by error-prone PCR technique. Among 858 clones obtained in the construction of the random mutation libraries of pLES003-b in the ori and repA regions, three clones could grow normally at 28 °C but not at 42 °C. One of the clones was designated as pLES003-b TS1. The sequencing analysis of pLES003-b TS1 revealed that the plasmid has four substitution mutations (376G > A, 435A > T, 914C > A, and 1996T > A) and one insertional mutation (1806_1807insA). Among those mutations, substitution mutation 914C > A, which leads to a CGC-to-AGC codon change at position 44 of the RepA protein (arginine-to-serine substitution mutation: R44S in RepA), was predicted to be a cause of temperature sensitivity. Therefore, the C-to-A substitution was introduced into the repA gene in pLES003-b using a site-directed mutagenesis method, and the resultant plasmid was electroporated into a Lactobacillus (L.) plantarum cell. The resultant transformant cannot grow at 42 °C in the presence of erythromycin, which is used as a selective marker, indicating that the R44S point mutation in the RepA protein may be crucial for temperature sensitivity. Furthermore, we have developed a new plasmid as an efficient genetic engineering tool for random insertional mutagenesis in LABs using a combination of transposon Tn10 and the temperature-sensitive replication system in pLES003-b. The resultant plasmid vector, which was designated pLES-Tn10-TS1, would be useful for genetic analysis of the functional molecule in lactic acid bacterial strains.

Worldwide Dissemination of blaKPC Gene by Novel Mobilization Platforms in Pseudomonas aeruginosa: A Systematic Review.

The dissemination of blaKPC-harboring Pseudomonas aeruginosa (KPC-Pa) is considered a serious public health problem. This study provides an overview of the epidemiology of these isolates to try to elucidate novel mobilization platforms that could contribute to their worldwide spread. A systematic review in PubMed and EMBASE was performed to find articles published up to June 2022. In addition, a search algorithm using NCBI databases was developed to identify sequences that contain possible mobilization platforms. After that, the sequences were filtered and pair-aligned to describe the blaKPC genetic environment. We found 691 KPC-Pa isolates belonging to 41 different sequence types and recovered from 14 countries. Although the blaKPC gene is still mobilized by the transposon Tn4401, the non-Tn4401 elements (NTEKPC) were the most frequent. Our analysis allowed us to identify 25 different NTEKPC, mainly belonging to the NTEKPC-I, and a new type (proposed as IVa) was also observed. This is the first systematic review that consolidates information about the behavior of the blaKPC acquisition in P. aeruginosa and the genetic platforms implied in its successful worldwide spread. Our results show high NTEKPC prevalence in P. aeruginosa and an accelerated dynamic of unrelated clones. All information collected in this review was used to build an interactive online map.

Loss of erm(B)-Mediated rRNA Dimethylation and Restoration of Erythromycin Susceptibility in Erythromycin-Resistant Enterococci Following Induced Linezolid Resistance.

Linezolid has been reported to restore erythromycin susceptibility in erythromycin-resistant Staphylococcus aureus. This phenomenon has not been reported in enterococci and the mechanisms involved therein are still unknown. The purpose of this study was to investigate the mechanisms involved and the effect of combining linezolid with erythromycin on erythromycin-resistant enterococci. Checkerboard techniques were used to determine drug interactions, and 12 of 14 isolates showed a synergistic effect between erythromycin and linezolid (fractional inhibitory concentration <0.5). We observed that the erm(B) gene, which encodes a dimethyltransferase responsible for erythromycin resistance, was expressed from transposon Tn1545 in the tested erythromycin-resistant enterococci. After exposure to linezolid, erm(B)-mediated rRNA dimethylation at A2071 could not be detected, and the erm(B) gene was lost following acquisition of erythromycin susceptibility. Thus, in conclusion, linezolid combined with erythromycin exerts a synergistic effect against erythromycin-resistant enterococci. Linezolid treatment suppressed erm(B)-mediated rRNA dimethylation at A2071, which could lead to loss of the erm(B) gene.

Screening and Characterization of Streptomyces spp. Isolated from Three Moroccan Ecosystems Producing a Potential Inhibitor of the Drug Efflux Pump AcrAB-TolC.

Traditional antimicrobial antibiotics are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. The antibiotic era is threatened by the ruthless rise of resistance in bacterial infections. A significant role in these resistance profiles is attributed to multidrug efflux pumps. Hence, much effort is being directed towards developing new compounds to overcome this problem. During our screening program of efflux pumps inhibitors (EPI) produced by bioactive Moroccan Actinobacteria, 210 isolates were screened for their antibacterial activities against Escherichia coli strains containing a system of efflux pump AcrAB-TolC, fully functional, and its mutant, inactivated due to the insertion of transposon Tn903 in AcrAB operon, using the method of agar disc diffusion. The results showed that 14 isolates were able to produce EPI as they were active against the wild type strain but not against the mutant in comparison with the synthetic inhibitor L-Phe-L-Arg-β-naphthylamide (PaβN). We focused on the highest EPI activity produced by four strains (Z332, Z35/G, Z385/b and 136). Taxonomic studies and the 16S rDNA sequence indicated that these strains belonged to the Streptomyces species. This work could contribute to the discovery of a new class of antibacterial agents that could expand the therapeutic arsenal.

Emergence of extensive multidrug-resistant Staphylococcus aureus carrying novel Sa-MRRlsa(E) in retail food

The aim of this study was to investigate the prevalence and genetic environment of the multidrug resistance gene lsa(E) in food-related Staphyloccocus aureus in China. 1463 S. aureus from retail food products in 39 Chinese cities were investigated to determine the prevalence of lsa(E). Furthermore, antimicrobial susceptibility testing, whole-genome sequencing (WGS), and complete genetic analysis were performed in lsa(E)-positive isolates. Thirty-five isolates (2.4%) were positive for the lsa(E) gene, which had an extensive multidrug-resistance phenotype. ST9-t899 and ST1-t4792 were the common sequence types in positive strains. The lsa(E) genes were located in two different types of novel multidrug resistance region (MRRlsa[E]) on the chromosome. The Sa-MRRlsa(E)-I were inserted into the lctP gene. The Sa-MRRlsa(E)-II were inserted into the crtP gene, and were comprised of seven antibiotic resistance genes (ARGs) interspersed with varieties of insertion sequences (ISs), transposons, and DNA invertase genes, showing a novel arrangement harboring lsa(E). Part of transposon Tn1546 was inserted downstream of lnu(B) in the novel Sa-MRRlsa(E)-II. Both types of Sa-MRRlsa(E) could be excised from the chromosome, indicating that Sa-MRRlsa(E) may be transferable. Our study is the first systematical investigation of lsa(E)-positive S. aureus in retail foods in China. It indicated that the origin of most food-related lsa(E)-positive S. aureus in China might be associated with livestock or poultry breeding farms, and these strains may be transmitted between animals and food. S. aureus carrying novel Sa-MRRlsa(E), especially, serve as reservoirs of antibiotic resistance traits, and warrant further attention.

Profiling of emerging pathogens, antibiotic resistance genes and mobile genetic elements in different biological wastewater treatment plants

The emergence and spread of antibiotic resistance through insufficiently treated effluents from wastewater treatment systems are detrimental to the receiving environment and human health. Metagenomic and transcriptomic approaches were employed to assess the diversity and the removal of bacterial pathogens, antibiotic-resistant genes (ARGs) and mobile genetic elements (MGEs) in three wastewater treatment plants (WWTPs) in Durban, South Africa. In total, 23 pathogenic bacterial genera, including enteric and emerging opportunistic pathogens, were found abundant in the samples. Aeromonas and Acinetobacter spp were the most dominant pathogens detected in the influent metagenomes, while the influent transcripts showed Escherichia and Acinetobacter spp as the most dominant pathogens. Shannon-Wiener indices showed that the bacterial diversity increased from influents to final effluents in two selected treatment plants. ARG types, including those conferring resistance to aminoglycosides, beta-lactamases, tetracycline and sulfonamide were abundant in both influent and effluent samples. Results further exposed that MGE-ARGs associations were the main drivers of ARG persistence into final effluents. This included 5 plasmids: R338-R151 (sulI), pRH-1238 (strB), pPM91 (aadA), pRH-1238 (aadA4–5), pRH-1238 (sulII); two class 1 integrons (aadA and arr) and 1 transposon Tn4351(tetX). In transcripts, MGE-ARG association showed two plasmids: pRH-1238 (aadA) and pPM91(aadA) and one hybrid plasmid R338-R151 (sulI). It was apparent across all the WWTPs that chlorination had little or no effect on MGE-ARG association . Combined, this study has highlighted the presence of bacterial pathogens, ARGs and MGEs in treated effluents, which can encourage the propagation of antibiotic resistance and potential sharing of genes in the downstream environments.

Generalised mutagenesis with transposon Tn5. A laboratory procedure for the identification of genes responsible for a bacterial phenotype and its regulation, illustrated with phenazine production in Pseudomonas chlororaphis

ABSTRACT Forward genetics involves the identification of an unknown DNA sequence (genotype) associated with the expression of that sequence (phenotype). This is often done by generating variants (mutants) of that feature. One of the most practical methods is transposon mutagenesis, which facilitates the genotype–phenotype association through a sequence tag. This six-session laboratory practical illustrates the concept of forward genetics in bacteria using the random DNA target selection of the Tn5 mini-transposon to generate a mutant library. The target bacterium is Pseudomonas chlororaphis subsp. aurantiaca strain SMMP3, whose broad-spectrum fungal antagonism is largely due to a diffusible, orange-pigmented antibiotic of the phenazine class. The library is screened on appropriate media to select clones with reduced colony pigmentation. Upon qualitative and quantitative phenotype confirmation, a subset of mutants is subjected to an arbitrary PCR to identify the interrupted genes through an in-silico comparative analysis of the sequencing products in public genomic databases. Students thus identify previously described or novel biosynthetic and regulatory genes involved in phenazine production. Along with the objective of illustrating a forward genetics approach, the exercise emulates a real scientific activity in which supervised students pursue a path to interpret the phenotypic consequences of gene knockouts.

RplI interacts with 5' UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Injectisome T3SS subunits as potential chaperones in the extracellular export of Pectobacterium carotovorum subsp. carotovorum bacteriocins Carocin S1 and Carocin S3 secreted via flagellar T3SS

Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22–6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8-16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Status quo of tet regulation in bacteria.

SummaryThe tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other natural or synthetic origins have been used for tetracycline‐dependent gene regulation (tet regulation) in at least 40 bacterial genera. Tet regulation serves several purposes such as conditional complementation, depletion of essential genes, modulation of artificial genetic networks, protein overexpression or the control of gene expression within cell culture or animal infection models. Adaptations of the promoters employed have increased tet regulation efficiency and have made this system accessible to taxonomically distant bacteria. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.

Antibiotic resistance and the presence of bla CfxA and bla CSP genes in β-lactamase-producing clinical Capnocytophaga isolates from a university hospital in Japan.

Introduction . Capnocytophaga species are common inhabitants of the oral cavity and can be responsible for systemic diseases in immunocompromised patients with granulocytopenia. Furthermore, it has been reported that some clinical isolates of Capnocytophaga species produce extended-spectrum β-lactamases (ESBLs).Gap statement. Information is lacking about the types of β-lactamase genes possessed by Capnocytophaga spp. and the antimicrobial susceptibility of Capnocytophaga spp. possessing each β-lactamase gene.Aim. The aim of this study was to investigate the presence of β-lactamase genes in clinical strains of β-lactamase-producing Capnocytophaga species isolated from clinical samples acquired at Shinshu University Hospital and examine the antimicrobial susceptibility of those strains.Methodology. The β-lactamase-producing Capnocytophaga species (n=49) were obtained from clinical specimens. PCR assays were used to detect bla CfxA, bla CSP, bla TEM, bla CepA/CblA and transposon Tn4555 genes. Southern hybridization assays were used to detect bla CfxA and bla CSP. The minimum inhibitory concentration of some β-lactams was determined using the E-test method.Results. PCR analysis indicated that the bla CfxA gene was present in 15 (30.6 %) and the bla CSP gene in 35 (69.3 %) of the 49 Capnocytophaga strains investigated, . Both bla CfxA and bla CSP genes were detected in a Capnocytophaga gingivalis strain. The PCR results were confirmed by Southern hybridization assays. Transposon Tn4555 was only detected in Capnocytophaga spp. harbouring the bla CfxA gene. All the β-lactamase-producing Capnocytophaga isolates were susceptible to ceftazidime-clavulanic acid, cefoxitin and imipenem. In contrast, most of the isolates were resistant to amoxicillin.Conclusions. The clinical isolates of Capnocytophaga spp. showed a high prevalence of the bla CSP gene in Japan. The presence of the bla CSP gene was distributed in Capnocytophaga sputigena as well as other Capnocytophaga spp. These results seem to suggest the dissemination of bla CfxA and bla CSP β-lactamase genes among Capnocytophaga species.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Spread of vancomycin-resistant Enterococcus faecium ST133 in the aquatic environment in Switzerland

The global dissemination of vancomycin-resistant enterococci (VRE) has become a serious public-health concern. Although outbreaks are typically caused by nosocomial transmission, contaminated food and water may contribute to the spread of VRE. The aim of this study was to assess the presence of VRE in flowing surface water bodies in Switzerland and to characterise the isolates. Surface water was sampled from rivers, streams and canals throughout Switzerland and was screened for the presence of VRE. Whole-genome sequencing was used to identify antimicrobial resistance genes and the phylogenetic similarity of the obtained isolates. VRE were detected in 6 (3.1%) of 191 water samples. The six VRE-containing samples were all collected near treated wastewater discharge sites. The six isolates were identified as Enterococcus faecium sequence type 133 (ST133) and harboured the vancomycin resistance-conferring vanA gene cluster on transposon Tn1546. They showed a close phylogenetic relationship to ST133 swine faecal isolates obtained during a previously reported screening in Switzerland. Our results suggest that surface water contributes to the environmental dissemination of VRE. Repeated identification of ST133 clones in geographically distinct water sampling sites and swine faecal samples collected in slaughterhouses may indicate a local dominance of this VRE lineage in Switzerland.

Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance.

This work aimed to determine how genes on transposon Tn1546 slow Bacillus subtilis spore germination and increase wet heat resistance, and to clarify the transposon's 3 gene spoVA operon's role in spore properties, since the seven wild-type SpoVA proteins form a channel transporting Ca2+ -dipicolinic acid (DPA) in spore formation and germination. Deletion of the wild-type spoVA operon from a strain with Tn1546 gave spores with slightly reduced wet heat resistance but some large decreases in germination rate. Spore water content and CaDPA analyses found no significant differences in contents of either component in spores with different Tn1546 components or lacking the wild-type spoVA operon. This work indicates that the SpoVA channel encoded by Tn1546 functions like the wild-type SpoVA channel in CaDPA uptake into developing spores, but not as well in germination. The essentially identical CaDPA and water contents of spores with and without Tn1546 indicate that low core water content does not cause elevated wet heat resistance of spores with Tn1546. Since wet heat resistance of spores of Bacillus species poses problems in the food industry, understanding mechanisms of spores' wet heat resistance is of significant applied interest.

F Plasmid, a Co-integrate of Two Major Replicons but their Functional States Controlled by the Transposon Tn1000 in E. coli K-12

F Plasmid, a Co-integrate of Two Major Replicons but their Functional States Controlled by the Transposon Tn1000 in E. coli K-12