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PubMed2005

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes.

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.

Amino Acid Sequence↗

PubMed2006

Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b.

Construction and integration of recombinant mini-Tn7 expression vectors into the chromosome of a surrogate, efflux-sensitized, and biosafe Pseudomonas aeruginosa host was validated as a generally applicable method for studies of uncharacterized bacterial efflux pumps. Using this method, the Burkholderia pseudomallei bpeEF-oprC operon was shown to encode a chloramphenicol and trimethoprim efflux pump.

Anti-Bacterial Agents↗

PubMed2002

Genes involved in bacitracin resistance in Streptococcus mutans.

Streptococcus mutans is resistant to bacitracin, which is a peptide antibiotic produced by certain species of Bacillus. The purpose of this study was to clarify the bacitracin resistance mechanism of S. mutans. We cloned and sequenced two S. mutans loci that are involved in bacitracin resistance. The rgp locus, which is located downstream from rmlD, contains six rgp genes (rgpA to rgpF) that are involved in rhamnose-glucose polysaccharide (RGP) synthesis in S. mutans. The inactivation of RGP synthesis in S. mutans resulted in an approximately fivefold-higher sensitivity to bacitracin relative to that observed for the wild-type strain Xc. The second bacitracin resistance locus comprised four mbr genes (mbrA, mbrB, mbrC, and mbrD) and was located immediately downstream from gtfC, which encodes the water-insoluble glucan-synthesizing enzyme. Although the bacitracin sensitivities of mutants that had defects in flanking genes were similar to that of the parental strain Xc, mutants that were defective in mbrA, mbrB, mbrC, or mbrD were about 100 to 120 times more sensitive to bacitracin than strain Xc. In addition, a mutant that was defective in all of the mbrABCD genes and rgpA was more sensitive to bacitracin than either the RGP or Mbr mutants. We conclude that RGP synthesis is related to bacitracin resistance in S. mutans and that the mbr genes modulate resistance to bacitracin via an unknown mechanism that is independent of RGP synthesis.

Anti-Bacterial Agents↗

PubMed2002

New carbenicillin-hydrolyzing beta-lactamase (CARB-7) from Vibrio cholerae non-O1, non-O139 strains encoded by the VCR region of the V. cholerae genome.

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a beta-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla(TEM) or primers for bla(CARB) gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the beta-lactamase was determined by bidirectional sequencing of a Sau3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this beta-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla(CARB-7) gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla(CARB-7) gene shared 93% identity with a locus situated inside V. cholerae's chromosome 2. These results strongly suggest the chromosomal location of the bla(CARB-7) gene, making this the first communication of a beta-lactamase gene located on the VCR island of the V. cholerae genome.

Amino Acid Sequence↗

PubMed2003

Genome-wide screening of Saccharomyces cerevisiae to identify genes required for antibiotic insusceptibility of eukaryotes.

The adverse reactions provoked by many antibiotics in humans are well documented but are generally poorly understood at the molecular level. To elucidate potential genetic defects that could give rise to susceptibility to prokaryote-specific antibiotics in eukaryotes, we undertook genome-wide screens using the yeast Saccharomyces cerevisiae as a model of eukaryotes; our previous work with a small number of yeast mutants revealed some specific gene functions required for oxytetracycline resistance. Here, the complete yeast deletion strain collection was tested for growth in the presence of a range of antibiotics. The sensitivities of mutants revealed by these screens were validated in independent tests. None of the approximately 4,800 defined deletion strains tested were found to be sensitive to amoxicillin, penicillin G, rifampin, or vancomycin. However, two of the yeast mutants were tetracycline sensitive and four were oxytetracycline sensitive; encompassed among the latter were mutants carrying deletions in the same genes that we had characterized previously. Seventeen deletion strains were found to exhibit growth defects in the presence of gentamicin, with MICs for the strains being as low as 32 micro g ml(-1) (the wild type exhibited no growth defects at any gentamicin concentration tested up to 512 micro g ml(-1)). Strikingly, 11 of the strains that were most sensitive to gentamicin carried deletions in genes whose products are all involved in various aspects of vacuolar and Golgi complex (or endoplasmic reticulum) function. Therefore, these and analogous organelles, which are also the principal sites of gentamicin localization in human cells, appear to be essential for normal resistance to gentamicin in eukaryotes. The approach and data described here offer a new route to gaining insight into the potential genetic bases of antibiotic insusceptibilities in eukaryotes.

Drug Resistance↗

PubMed2003

Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus.

Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that Staphylococcus aureus may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of S. aureus possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For S. aureus SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC(50)) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC(50) for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and trans(E)-flupentixol were found to reduce the proton motive force (PMF) of S. aureus by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential.

Drug Resistance, Multiple, Bacterial↗

PubMed2004

Genome-wide transcriptional profiling of the Escherichia coli response to a proline-rich antimicrobial peptide.

Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of Escherichia coli in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes in the expression levels of 70 bacterial genes from various functional categories were detected. Among these, 26 genes showed decreased expression, while 44 genes, including genes that are potentially involved in bacterial resistance to antimicrobials, showed increased expression. The generation of a transcriptional response under the experimental conditions used is consistent with the ability of Bac7(1-35) to interact with bacterial components and affect biological processes in this organism.

Anti-Bacterial Agents↗

PubMed2005

DNA sequence and comparative genomics of pAPEC-O2-R, an avian pathogenic Escherichia coli transmissible R plasmid.

In this study, a 101-kb IncF plasmid from an avian pathogenic Escherichia coli (APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the E. coli F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal E. coli strain from an apparently healthy bird, a Salmonella enterica serovar Typhimurium strain, and a uropathogenic E. coli strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.

Animals↗

PubMed2005

Antibiotic susceptibility testing of Mycoplasma genitalium by TaqMan 5' nuclease real-time PCR.

Mycoplasma genitalium is an important pathogen in male nongonococcal urethritis (NGU). Isolation of M. genitalium from clinical specimens by axenic culture is very difficult and time-consuming, and very few strains are available for antibiotic susceptibility testing. Primary isolation of M. genitalium by coculture with Vero cells improves the isolation rate significantly. However, some strains cannot be adapted to axenic culture. In this study, we determined the antibiotic susceptibility of M. genitalium strains grown in Vero cell culture with dilutions of antibiotics. Growth of M. genitalium was monitored by a quantitative PCR assay detecting a single-copy region of the mgpB adhesin gene. Growth inhibition in the presence of antibiotics was expressed as a percentage of the DNA load of controls grown in the absence of antibiotics. Eighteen strains were examined, including 6 new strains isolated from urethral swab specimens and 4 new strains isolated from urine specimens collected from Japanese men. Eight strains adapted to axenic culture were also tested by the conventional broth dilution method. The two methods had an acceptable correlation. Azithromycin was the most active drug against M. genitalium. Among the fluoroquinolones, moxifloxacin had the highest activity, with MICs ranging from 0.03 to 0.5 mg/liter, whereas ciprofloxacin and levofloxacin were considerably less active, with MICs ranging from 0.5 to 16 mg/liter and 0.25 to 4 mg/liter, respectively. MICs for tetracycline ranged from 0.125 to 4 mg/liter. This new method could increase the number of M. genitalium strains available for antibiotic susceptibility testing and significantly shorten the time from sampling to MIC results.

Animals↗

PubMed1997

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.

Bacteria↗

PubMed2005

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Bacterial Proteins↗

PubMed2006

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus.

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Alphaproteobacteria↗

PubMed1996

Survey of enterococcal susceptibility patterns in Belgium.

A national surveillance study was performed in order to identify the enterococci causing important infections, to determine their susceptibilities to first-choice agents for treatment, and to characterize the phenotypes and genotypes of the glycopeptide-resistant strains. A total of 472 isolates were collected between 15 January and 15 April 1993. The ability of the API rapid ID 32 STREP gallery to identify enterococci was evaluated. The majority of the Belgian enterococci were identified as E. faecalis (89.4%). E. faecium and other enterococci were present in small percentages only (9.1 and 1.5%, respectively). The API rapid ID 32 STREP system identified 88.6% of the strains with an excellent or very good identification score. For the majority of the strains with uncertain identification scores, the results of a single test only were aberrant. Only 2.3% of the strains remained unidentified. High-level aminoglycoside resistance was widespread in E. faecalis (streptomycin, 50.8%; gentamicin, 8.7%), and the emergence of ciprofloxacin resistance was found to be associated with aminoglycoside resistance. E. faecium is generally more resistant to a wide range of antibiotics, but glycopeptide-resistant strains (1.5%) have not yet become widespread.

Belgium↗

PubMed2005

Antifungal susceptibility and genotypical pattern of Microsporum canis strains.

Dermatophytes are a group of fungi that are capable of invading keratinized tissues of humans and other animals. Antifungal susceptibility analysis and genetic studies by random amplification of polymorphic DNA (RAPD), have been used to detect polymorphism as well as determining the possible resistance of dermatophytes to antifungals. The aim of this study was to evaluate the possible correlation between the antifungal susceptibility and genotypical pattern of Microsporum canis strains isolated in dogs and cats with dermatophytosis in Northeast Brazil. The antifungal susceptibility study was conducted using the broth microdilution test with griseofulvine, ketoconazole, itraconazole, and fluconazole. The genotypical analysis was performed using the RAPD method. The antifungal susceptibility analysis showed that all the strains of M. canis analyzed (n = 22) were sensitive to griseofulvine (0.25 microg/mL < or minimum inhibitory concentration (MIC) < or = 1 microg/mL), ketoconazole (0.25 microg/mL < or = MIC < or = 2 microg/mL), itraconazole (0.25 microg/mL < or = MIC < or = 1 microg/mL), and fluconazole (1 microg/mL < or = MIC < or = 16 microg/mL). The RAPD results showed that all analyzed strains are genetically similar. Thus, based on antifungal susceptibility analysis and RAPD data, a possible correlation can be shown between the antifungal susceptibility and the genotypical pattern of the strains of M. canis from Northeast Brazil.

Animals↗

PubMed2026

Whole genome analysis of a multidrug-resistant blaNDM-5-carrying Escherichia coli Sequence Type (ST) 167 strain isolated from seafood in Mumbai, India.

BACKGROUND: E. coli ST167 is an emerging extraintestinal pathogenic Escherichia coli (ExPEC) clone. This study reports the whole genome sequence analysis of a multidrug-resistant, blaNDM-5 harboring E. coli ST167 (EC121) isolated from seafood. The antibiotic susceptibility pattern was determined using the standard disc diffusion method. Genomic DNA was extracted, purified, and sequenced using the Illumina platform. The whole genome sequence was analyzed to determine the genome characteristics, including sequence type, serotype, phylogroup, antibiotic resistance genes, virulence attributes, and phylogenetic analysis. RESULTS: Phenotypically, this isolate was resistant to 26 of the 33 antibiotics tested, which correlated well with in-silico prediction. Multilocus sequence typing (MLST) analysis revealed that this strain belonged to sequence type 167, serotype O101:H9, and phylogroup A and harbored different virulence genes, suggesting it was a potential human pathogen. Many acquired antibiotic resistance genes were detected, including blaNDM-5, blaCMY-42, blaOXA-1, blaTEM-116, catA1, sul2, and tet(B). Point mutations in gyrA and parC responsible for quinolone resistance were also detected. CONCLUSION: The combinations of virulence and antibiotic resistance genes in this strain highlight the significant risk associated with emerging E. coli clonal types contaminating the seafood supply chain. Fecal contamination of seafood can contribute to the community dissemination of multidrug-resistant E. coli, necessitating effective monitoring measures.

Seafood↗

PubMed2005

Genome comparison analysis of molecular mechanisms of resistance to antibiotics in the Rickettsia genus.

In this study we describe molecular mechanisms of resistance to several classes of antibiotics within drug targets by in silico genome comparisons for bacteria of the genus Rickettsia. Apart from the mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species previously reported by our team, we found that typhus group (TG) rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein as compared to the spotted fever group rickettsiae (SFG), which could explain the natural resistance of SFG rickettsia to erythromycin. We found also that the genome of R. conorii contains an aminoglycoside 3'-phosphotransferase. Finally, either folA gene (encoding dihydrofolate reductase) and/or folP gene (encoding dihydropteroate synthase) was missing in the genome of rickettsial strains explaining the natural resistance to cotrimoxazole. Finally, multiple genes encoding for pump efflux were found especially in the genome of R. conorii that could be involved in resistance to antibiotics. Five specific ORFs related to antibiotic resistance have been identified in the genome of R. felis including a streptomycin resistance protein homologue, a class C beta-lactamase, a class D beta-lactamase, a penicillin acylase homologue, and an ABC-type multidrug transporter system. For the first time, using this approach, an experimental beta-lactamase activity has been shown for this bacterium. We believe that whole genome sequence analysis may help to predict several phenotypic characters, in particular resistance to antibiotics for obligate intracellular bacteria.

Amino Acid Sequence↗

PubMed2026

The chromosomal genome sequence of a staghorn coral, Acropora cf.  manni (Scleractinia: Acroporidae).

We present a genome assembly from a colony sample of Acropora cf. manni (staghorn coral; Cnidaria; Anthozoa; Scleractinia; Acroporidae). The assembly contains two haplotypes with total lengths of 459.26 megabases and 489.30 megabases. Most of haplotype 1 (94.39%) is scaffolded into 14 chromosomal pseudomolecules. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 18.48 kilobases. Metagenome binning recovered one bacterial bin from the phylum Chlamydiota.

Acropora cf. manni↗

PubMed2026

The genome sequence of a coronate scyphozoan jellyfish, Nausithoe racemosa (Komai, 1936) (Coronatae: Nausithoidae), and a metagenome-assembled genome of the associated cyanobacterium Moorena producens.

We present a genome assembly from a specimen of Nausithoe racemosa (coronate scyphozoan jellyfish; Cnidaria; Scyphozoa; Coronatae; Nausithoidae). The assembly contains two haplotypes with total lengths of 4 784.66 megabases and 4 868.20 megabases. Most of haplotype 1 (97.34%) is scaffolded into 20 chromosomal pseudomolecules. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 13.97 kilobases. From the metagenome data, we recovered one high-quality metagenome-assembled genome.

Coronatae↗