Michael T. Sweeney

ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: analysis of the regions that comprise 12 antimicrobial resistance genes

BACKGROUND In recent years, multiresistant Pasteurella multocida isolates from bovine respiratory tract infections have been identified. These isolates have exhibited resistance to most classes of antimicrobial agents commonly used in veterinary medicine, the genetic basis of which, however, is largely unknown. METHODS Genomic DNA of a representative P. multocida isolate was subjected to whole genome sequencing. Genes have been predicted by the YACOP program, compared with the SWISSProt/EMBL databases and manually curated using the annotation software ERGO. Susceptibility testing was performed by broth microdilution according to CLSI recommendations. RESULTS The analysis of one representative P. multocida isolate identified an 82 kb integrative and conjugative element (ICE) integrated into the chromosomal DNA. This ICE, designated ICEPmu1, harboured 11 resistance genes, which confer resistance to streptomycin/spectinomycin (aadA25), streptomycin (strA and strB), gentamicin (aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or tilmicosin/tulathromycin [msr(E)-mph(E)]. In addition, a complete bla(OXA-2) gene was detected, which, however, appeared to be functionally inactive in P. multocida. These resistance genes were organized in two regions of approximately 15.7 and 9.8 kb. Based on the sequences obtained, it is likely that plasmids, gene cassettes and insertion sequences have played a role in the development of the two resistance gene regions within this ICE. CONCLUSIONS The observation that 12 resistance genes, organized in two resistance gene regions, represent part of an ICE in P. multocida underlines the risk of simultaneous acquisition of multiple resistance genes via a single horizontal gene transfer event.

Discovery and Characterization of QPT-1, the Progenitor of a New Class of Bacterial Topoisomerase Inhibitors

ABSTRACT QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (−)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the β subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: structure and transfer

BACKGROUND Integrative and conjugative elements (ICEs) have not been detected in Pasteurella multocida. In this study the multiresistance ICEPmu1 from bovine P. multocida was analysed for its core genes and its ability to conjugatively transfer into strains of the same and different genera. METHODS ICEPmu1 was identified during whole genome sequencing. Coding sequences were predicted by bioinformatic tools and manually curated using the annotation software ERGO. Conjugation into P. multocida, Mannheimia haemolytica and Escherichia coli recipients was performed by mating assays. The presence of ICEPmu1 and its circular intermediate in the recipient strains was confirmed by PCR and sequence analysis. Integration sites were sequenced. Susceptibility testing of the ICEPmu1-carrying recipients was conducted by broth microdilution. RESULTS The 82 214 bp ICEPmu1 harbours 88 genes. The core genes of ICEPmu1, which are involved in excision/integration and conjugative transfer, resemble those found in a 66 641 bp ICE from Histophilus somni. ICEPmu1 integrates into a tRNA(Leu) and is flanked by 13 bp direct repeats. It is able to conjugatively transfer to P. multocida, M. haemolytica and E. coli, where it also uses a tRNA(Leu) for integration and produces closely related 13 bp direct repeats. PCR assays and susceptibility testing confirmed the presence and the functional activity of the ICEPmu1-associated resistance genes in the recipient strains. CONCLUSIONS The observation that the multiresistance ICEPmu1 is present in a bovine P. multocida and can easily spread across strain and genus boundaries underlines the risk of a rapid dissemination of multiple resistance genes, which will distinctly decrease the therapeutic options.

Molecular Basis of Macrolide, Triamilide, and Lincosamide Resistance in Pasteurella multocida from Bovine Respiratory Disease

ABSTRACT The mechanism of macrolide-triamilide resistance in Pasteurella multocida has been unknown. During whole-genome sequencing of a multiresistant bovine P. multocida isolate, three new resistance genes, the rRNA methylase gene erm(42), the macrolide transporter gene msr(E), and the macrolide phosphotransferase gene mph(E), were detected. The three genes were PCR amplified, cloned into suitable plasmid vectors, and shown to confer either macrolide-lincosamide resistance [erm(42)] or macrolide-triamilide resistance [msr(E)-mph(E)] in macrolide-susceptible Escherichia coli and P. multocida hosts.

Antimicrobial Resistance in Bovine Respiratory Disease Pathogens: Measures, Trends, and Impact on Efficacy

The introduction of newer antimicrobial agents over the past two decades has dramatically improved the treatment of bovine respiratory disease (BRD). In the same time period, the implementation of standardized susceptibility test methods and BRD-specific interpretive criteria has substantially improved the ability to detect clinical resistance in the BRD pathogens. Although overall levels of resistance to the newer antimicrobial agents are generally low, recent data have indicated the potential for emergence and dissemination of a resistant clone in cattle. These data indicate the need for long-term surveillance of antimicrobial resistance in the BRD pathogens and a better understanding of the epidemiology of antimicrobial resistance in these pathogens.

3-Arylpiperidines as potentiators of existing antibacterial agents

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Increased MICs of gamithromycin and tildipirosin in the presence of the genes erm(42) and msr(E)-mph(E) for bovine Pasteurella multocida and Mannheimia haemolytica.

Sir, Bovine respiratory disease (BRD) is one of the economically most important diseases in animal production, with global losses of the feedlot industry due to BRD being estimated to be over US

Analysis and comparative genomics of ICEMh1, a novel integrative and conjugative element (ICE) of Mannheimia haemolytica

3 billion per year. Antimicrobial agents, particularly macrolides, are commonly used to combat bacteria involved in BRD, such as Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. After the approval of the 16-membered macrolide tilmicosin (Micotil) in 1992 and the 15-membered triamilide tulathromycin (Draxxin) in 2005 for use in BRD, two new macrolides were approved during 2011 for the treatment of BRD. These are the 15-membered macrolide gamithromycin (Zactran) and the 16-membered macrolide tildipirosin (Zuprevo). Little is known about the mechanisms of resistance to these two new veterinary antimicrobial agents. Recently, three genes involved in macrolide resistance of P. multocida and M. haemolytica were identified: erm(42), coding for a novel rRNA methylase; msr(E), coding for an ABC transporter; and mph(E), coding for a macrolide phosphotransferase. The last two genes were present in an operon structure. To determine the role of erm(42) and msr(E)-mph(E) in macrolide and lincosamide resistance, these genes were cloned and expressed in P. multocida B130. A closer analysis of these clones for their MICs of selected macrolides and lincosamides revealed that erm(42) conferred resistance to erythromycin, tilmicosin and clindamycin, but not to the triamilide tulathromycin. Although erm(42)-carrying P. multocida B130 showed an 8-fold increase in the tulathromycin MIC to 16 mg/L (Table S1, available as Supplementary data at JAC Online), this MIC classified the P. multocida isolate as susceptible. In contrast, msr(E)-mph(E) conferred resistance to erythromycin, tilmicosin and tulathromycin, but not to the lincosamide clindamycin (Table S1). When the new macrolides became commercially available, we tested the aforementioned P. multocida B130 clones for their MICs of gamithromycin and tildipirosin by broth macrodilution according to CLSI recommendations. The recipient strain P. multocida B130 showed 8-fold lower MICs of 0.25 mg/L for both gamithromycin and tildipirosin, compared with tulathromycin (2 mg/L) (Table S1). In the presence of erm(42), the MIC of tildipirosin increased 128-fold to 32 mg/L, while that of gamithromycin increased only 16-fold to 4 mg/L. In the presence of msr(E)-mph(E), an opposite observation was made: the MIC of tildipirosin increased only 8-fold to 2 mg/L, while that of gamithromycin increased 256-fold to 64 mg/L. Based on these increases in the MICs, it appears that erm(42) has mainly an effect on the tildipirosin MIC, whereas msr(E)-mph(E) increases the gamithromycin MIC for P. multocida B130. To see whether naturally occurring P. multocida and M. haemolytica isolates from BRD cases that carry the genes erm(42) and/or msr(E)-mph(E) show similar MICs of gamithromycin and tildipirosin, a total of 40 P. multocida and 29 M. haemolytica isolates (Table 1), whose macrolide resistance gene status was determined by previously described PCR assays, were tested. These isolates were collected in the Pfizer Animal Health Susceptibility Surveillance Program for BRD between 1999 and 2007 from various states in the USA. The macrolide-susceptible P. multocida isolates (n1⁄48) showed low MICs of 0.25–0.5 mg/L for both gamithromycin and tildipirosin. Slightly higher MICs of 0.5–1 and 0.5–2 mg/L for gamithromycin and tildipirosin, respectively, were detected for the macrolidesusceptible M. haemolytica isolates (n1⁄47). These MICs are in agreement with the gamithromycin MIC50 and MIC90 values for 144 P. multocida and 142 M. haemolytica isolates obtained from animals enrolled in field studies in the USA. Moreover, the observed tildipirosin MICs are in the same range as determined for 105 P. multocida and 88 M. haemolytica isolates. Research letters

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition

OBJECTIVES The aim of this study was to identify and analyse the first integrative and conjugative element (ICE) from Mannheimia haemolytica, the major bacterial component of the bovine respiratory disease (BRD) complex. METHODS The novel ICEMh1 was discovered in the whole-genome sequence of M. haemolytica 42548 by sequence analysis and comparative genomics. Transfer of ICEMh1 was confirmed by conjugation into Pasteurella multocida recipient cells. RESULTS ICEMh1 has a size of 92,345 bp and harbours 107 genes. It integrates into a chromosomal tRNA(Leu) copy. Within two resistance gene regions of ∼ 7.4 and 3.3 kb, ICEMh1 harbours five genes, which confer resistance to streptomycin (strA and strB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)] and sulphonamides (sul2). ICEMh1 is related to the recently described ICEPmu1 and both ICEs seem to have evolved from a common ancestor. A region of ICEMh1 that is absent in ICEPmu1 was found in putative ICE regions of other M. haemolytica genomes, suggesting a recombination event between two ICEs. ICEMh1 transfers to P. multocida by conjugation, in which it also uses a tRNA(Leu) as the integration site. PCR assays and susceptibility testing confirmed the presence and activity of the ICEMh1-associated resistance genes in the P. multocida recipient. CONCLUSIONS These findings showed that ICEs, with structurally variable resistance gene regions, are present in BRD-associated Pasteurellaceae, can easily spread across genus borders and enable the acquisition of multidrug resistance via a single horizontal gene transfer event. This poses a threat to efficient antimicrobial chemotherapy of BRD-associated bacterial pathogens.

Complete Genome Sequence of Mannheimia haemolytica Strain 42548 from a Case of Bovine Respiratory Disease.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.