Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes

Colistin resistance has attracted substantial attention after colistin was considered as a last-resort drug for the treatment of infections caused by carbapenem-resistant and/or multidrug-resistant (MDR) Gram-negative bacteria in clinical settings. However, with the discovery of highly mobile colistin resistance (mcr) genes, colistin resistance has become an increasingly urgent issue worldwide. Despite many reviews, which summarized the prevalence, mechanisms, and structures of these genes in bacteria of human and animal origin, studies on the prevalence of mobile colistin resistance genes in aquaculture and their transmission between animals and humans remain scarce. Herein, we review recent reports on the prevalence of colistin resistance genes in animals, especially wildlife and aquaculture, and their possibility of transmission to humans via the food chain. This review also gives some insights into the routine surveillance, changing policy and replacement of polymyxins by polymyxin derivatives, molecular inhibitors, and traditional Chinese medicine to tackle colistin resistance.     

A novel Campylobacter jejuni sequence type from a culture-negative patient in the Gambia

The introduction of molecular diagnostic methods is crucial for improved understanding of the aetiology and epidemiology of bacterial infections in communities in resource poor settings. A blood sample from a 7 month old patient diagnosed with malaria in 2001 in a Gambian outpatient clinic was reported as culture negative after it was subjected to traditional bacterial culture protocols. We re-addressed the analysis of the blood sample from this case more recently (after 6.5 years in archival storage) in pilot work establishing 16S rRNA PCR in our molecular laboratory. Initial 16S rRNA PCR results confirmed the presence of bacterial DNA in the sample. 16S rRNA sequence analysis identified the organism as Campylobacter spp. In light of the molecular evidence we successfully grew the organism using appropriate culture conditions and subsequently biochemically confirmed that the isolate was Campylobacter jejuni. PCR and DNA sequencing of a set of seven C. jejuni housekeeping genes and in silico Multilocus Sequence Typing (MLST) analysis revealed that the isolate exhibits a novel sequence type (ST) of C. jejuni (ST 2928) and belongs to ST-443 clonal complex. This study demonstrates the potential for molecular tools to enhance the diagnosis of bacterial infections, which remain a major killer globally, not least in children in the developing world. Improvements in diagnostics are needed, and will be important not only for sick individuals but also for populations, where better measures of disease burden will contribute significantly to the improvement of public health policy.     

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez

Summary Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3 end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.     

Evolution of antibiotic resistance genes: the DNA sequence of a kanamycin resistance gene from Staphylococcus aureus

The kanamycin resistance gene from Staphylococcus aureus has been sequenced and its structure compared with similar genes isolated from Streptomyces fradiae and from two transposons, Tn5 and Tn903, originally isolated from Klebsiella pneumoniae and Salmonella typhimurium, respectively. The genes are all homologous but, since their common ancestor, have undergone extensive divergence, with more than 43% divergence between the closest pair. The phylogeny of the genes cannot be made congruent to the phylogeny of the taxa from which they were isolated without requiring rather improbable differences in rates. One is therefore led to conclude that there have been multiple occurrences of gene transfer between these species. Thus, although they are homologous, they are neither orthologous nor paralogous. It is suggested that homologous genes of this type be called xenologous.      

A novel large plasmid carrying multiple beta-lactam resistance genes isolated from a Klebsiella pneumoniae strain

Klebsiella pneumoniae clinical isolates were selected according to the results of antibiotic susceptibility tests. Most of them were resistant to multiple antibiotics, including ampicillin, ceftazidime, cefotaxime and aminoglycosides. Large plasmids were observed in these Kl. pneumoniae strains by pulsed-field gel electrophoresis with S1 nuclease digestion. The Kl. pneumoniae strains investigated produced one to two extrachromosomal bands with a mobility corresponding to 97 approximately 145 kbp linear DNA molecules. A 100 kbp plasmid, designated pK1, was observed in the multiply resistant strain K250. pK1 had sequences homologous to both the TEM-1 and the aphD probe which were associated with beta-lactam and aminoglycoside resistance. pK1 was transformed into Escherichia coli strain DH5alpha and was found to confer resistance to ampicillin, ceftazidime, cefotaxime and kanamycin. A 8 kbp BamHI DNA fragment of pK1 that carried the ampicillin resistance gene (minimum inhibitory concentration > 1000 microgram ml-1) was cloned into the BamHI site of pACYC184. Sequence determination showed that this cloned fragment carried a TEM-1 gene. These findings suggest that pK1 is novel in that it appears to carry genes for resistance to ampicillin, cefotaxime and ceftazidime, as well as kanamycin.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

Comparative genomics of closely related strains of Klebsiella pneumoniae reveals genes possibly involved in colistin resistance

Strains of colistin-resistant Klebsiella pneumoniae are emerging worldwide, due to the increased use of this molecule in antibiotic-resistant nosocomial infections. Comparative genomics was performed on three closely related K. pneumoniae strains isolated from three patients in a single hospital in Bologna, Italy. Two of these isolates are colistin-resistant, while the third is sensitive to this antibiotic. The designed bioinformatic approach detected, among the three analyzed genomes, single nucleotide polymorphisms, insertions and deletions, specific patterns of gene presence and absence, in a total of 270 genes. These genes were analyzed by automatic and manual methods, to identify those potentially involved in colistin resistance, based on the data available in the literature and on the mechanism of action of colistin, the alteration of the outer membrane. Three of the identified genes (waaL, rfbA, vacJ), all presenting non-synonymous substitutions in the colistin resistant strains, resulted to be of special interest, due to the specific function of their protein products, involved in the biosynthesis of the outer bacterial membrane.     

Comparison of type IV-pilin genes of Pseudomonas aeruginosa of various habitats has uncovered a novel unusual sequence

Abstract All known pilin sequences in Pseudomonas aeruginosa were amplified by a set of consensus primers located in the 5"-conserved region of pilA and the threonine-specific t-RNA following pilA . This also enabled the discovery of a novel pilin gene in a strain pair of clonal variants, which differs from known pilin genes in its increased GC-content. The mature protein has 173 amino acids making it the longest pilin known to date in P. aeruginosa . Different inserted sequences detected between the 3"-end of the pilin gene and the t-RNA in this strain and in strains with group I pilin genes seemed to be specific for each pilin group indicating a horizontal cotransfer of sequences.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

A novel type of phosphofructokinase from plants

A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.     

Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato.

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.     

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16-21 and ubiquitin 52-57 fragments, and second designed to mimic the ubiquitin 5-13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.     

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.     

Rapid construction of sequencing templates by random insertion of antibiotic resistance genes

We describe a novel and handy method for generating a population of templates for sequencing. The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I. The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid. DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background. We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants.     

Entire genomic sequence of novel canine papillomavirus type 13

Papillomaviruses are associated with benign and malignant neoplasias of the skin and mucous membranes. The sequence of a novel canine papillomavirus was determined from DNA detected in the oral cavity of a dog. The sequence of the novel virus canine papillomavirus type 13 (CPV13) shares the highest levels of similarity with the Tau papillomaviruses CPV2 and CPV7.     

The sequence of an antibiotic resistance gene from an antibiotic-producing bacterium: Homologies with transposon genes

The APH gene of a butirosin-producing Bacillus circulans has been cloned and sequenced; a comparison of the translated protein sequence with those from TN5 and TN903 indicates that they may have a common origin.     

A cell-penetrating peptide from a novel pVII-pIX phage-displayed random peptide library

A novel random peptide library was constructed using a phage-display format on the coat proteins pVII and pIX of filamentous bacteriophage. Panning against B-lymphocyte WI–L2 cells yielded one unique peptide-phage, denoted CHL8, that specifically bound to and penetrated the cells. Studies of each peptide derived from CHL8, denoted pep7 and pep9, established that only pep7 mediated the observed activity and only as a homodimer. Peptide libraries displayed on pVII–pIX should serve as a novel source of bioactive ligands for a variety of applications.