ESPECIFICACIONES AMBIENTALES GENERALES OBJETO
PERMISOS AMBIENTALES
ST5136 is the largest vertically expanding UK wide clone of Campylobacter jejuni.
Lopes Bruno1, Strachan Norval2, Ramjee Meenakshi1, Macrae Marion1, Forbes Ken1
1
School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom, AB25 2ZD
2
School of Biological Sciences, Cruickshank Building, University of Aberdeen, St. Machar Drive, Aberdeen, United Kingdom, AB24 3UU
Introduction: Campylobacter jejuni and Campylobacter coli are the most common causes of
bacterial gastroenteritis in the developed world. The aim of this study was to understand how antibiotics in the farm environment select for antibiotic resistant clones of Campylobacter. The diversity of tetracycline resistance determinants and quinolone resistances conferred by gyrA mutations in Campylobacter isolated form different host reservoirs was assessed.
Materials and methods: DNA from pure cultures of Campylobacter spp. from chicken, cattle,
sheep and humans in Grampian region of Scotland was extracted and whole genome sequenced using an Illumina HiSeq 2000 sequencer with 100 base paired-end sequencing and the FASTQ paired-end reads assembled using Velvet. The genomes were assessed for purity and submitted to the Bacterial Isolate Genome Sequence Database (BIGSdb) where alleles were tagged and the presence of the tetracycline resistance determinant CAMP1698 and the C257T gyrA mutation identified.
Results: Whole genome MLST analysis using 136 representative strains isolated from UK identified
that ST5136 is a UK wide clone, having emerged from ST464 through substantial genetic recombination. ST5136 was exclusively associated with chicken, turkey and humans and was the most prevalent strain harbouring the tet(O/32/O) 7-like determinant. The most common
tetracycline resistant alleles were tet(O/32/O)7, tet(O/32/O)8, 13. The tet(O/32/O) 7 variant was
chromosomally associated in ST5136. 99% (207/209) of CC464 strains were ciprofloxacin resistant and had the C257T gyrA mutation.
Conclusion: The tet(O) [Φ-m46.1] and tet(O)-like variants in Campylobacter were closely related.
The tet(O/32/O) from human gut bacteria and Campylobacter were also similar in sequence suggesting the potential for Campylobacter and other species to rapidly evolve through common DNA transfer systems. The increase in resistance to ciprofloxacin and tetracycline in clonally expanding populations of Campylobacter and the usage of these antibiotics in agriculture is discussed.
Emerging antibiotic resistance in ruminant Campylobacter in the United States
Tang Yizhi, Dai Lei, Wu Zuowei, Sahin Orhan, Zhang Qijing Department of Veterinary Microbiology and Preventive Medicine,
College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Ruminant Campylobacter contributes significantly to outbreaks and sporadic cases of foodborne illnesses in humans. Antibiotics such as fluoroquinolone (FQ) and florfenicol are frequently used in cattle for disease prevention and control in the U.S., but little is known about antibiotic resistance in bovine Campylobacter. To facilitate the control of antibiotic-resistant Campylobacter, we analyzed C. jejuni and C. coli isolates obtained from 35 feedlot cattle farms in multiple states. The results revealed high prevalence of FQ resistance: 35.4% in C. jejuni and 74.4% in C. coli. While most FQ-resistant Campylobacter isolates harbored resistance-conferring mutations in GyrA, some of the FQ-resistant isolates did not have any known mutations in GyrA, suggesting the presence of unknown mechanisms for FQ resistance. Molecular typing of FQ-resistant isolates further revealed that clonal expansion was involved in dissemination of FQ-resistant C. coli but not C. jejuni. Notably, florfenicol resistance, which was historically low in Campylobacter, also emerged in the bovine Campylobacter isolates. Whole genome sequencing analysis identified a novel cfr variant,
cfr(C), in the florfenicol-resistant isolates. The Cfr(C) ORF is divergent from Cfr and only shares
55.1% and 54.9% amino acid identity to Cfr and Cfr(B), respectively. Cloning of cfr (C) into C. jejuni NCTC11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to multiple classes of antibiotics including phenicols, lincosamides, pleuromutilins, and oxazolidinones. The cfr(C) gene was detected in 10% of the C. coli isolates, and molecular typing of the cfr(C)-positive C. coli isolates revealed its spread mainly via clonal expansion. These findings reveal the rising prevalence of FQ-resistant Campylobacter and the emergence of a novel multidrug resistant mechanism Cfr(C) in ruminant Campylobacter in the U.S. Acquisition of these resistance traits likely facilitates Campylobacter adaptation to the selection pressure from antibiotic usage in cattle.
Antimicrobial resistance in Helicobacter pylori – a 7 year perspective from Gastrointestinal Bacteria Reference Unit (GBRU), Public Health England
Kaindama Lukeki, Swift Craig, Godbole Gauri
Public Health England, Bacteriology Reference Department, National Infection Service,
London, UK
Objectives: Macrolide resistant Helicobacter pylori was recently been included in the WHO list of
"priority pathogens". National Guidelines for England and Wales recommend performing gastric biopsies for H. pylori culture and antimicrobial susceptibility testing for patients with dyspepsia who have failed first- and second-line treatment, have limited options due to drug hypersensitivity or live in an area with a high local resistance rate. We sought to determine antimicrobial resistance rates of H. pylori referrals to Gastrointestinal Bacteria Reference Unit (GBRU) over the last 7 years.
Methods: All gastric biopsies and H. pylori isolates referred to GBRU from January 2011 to
December 2016, were analysed. Culture for H. pylori was undertaken for all gastric biopsies. H.
pylori isolates underwent phenotypic susceptibility testing to using disc diffusion and E-tests. Data
were analysed using Excel 2010.
Results: 4430 clinical specimens (gastric biopsies and isolates) from 3767 patients were tested by
GBRU over a seven-year period (2011-2016), H. pylori was cultured in 1541 (34.7%). Phenotypic resistance (as % of culture positive specimens) was as follows: amoxicillin 4.0% (61), clarithromycin 72.7% (1121), levofloxacin 17.6% (271), metronidazole 86.4% (1331), rifampicin 13.7% (211), rifabutin 0.3% (5) and tetracycline 1.7% (26). Multidrug resistance (defined as resistance to ≥2 antimicrobial drugs) was found in 73.8% (1137) and mono-drug resistance in 18.4% (283). Resistance to ≥3 antimicrobial drugs was found in 24.8% (382). There were none identified with pan resistance (7 antibiotics) with eight resistant to 5 drugs and one resistant to 6 drugs (tetracycline, amoxicillin, clarithromycin, metronidazole, levofloxacin and rifampicin).
Conclusions: Our data suggest that secondary antimicrobial resistance rates of H. pylori in difficult-
to-treat dyspepsia are high in England and Wales. Urgent surveillance is needed to determine the incidence of primary resistance and risk factors for developing antimicrobial resistance in such patients to guide treatment and prevention strategies.
Targeting Campylobacter jejuni lytic transglycosylase for drug discovery
Jun Lin1, Focco van den Akker2, Ximin Zeng1, Jagamya Vijayaraghavan2, Ross Kaufhold2, Vijay Kumar2
1
Department of Animal Science, The University of Tennessee, Knoxville, TN, USA
2
Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA.
Production of β-lactamase is a major and threatening β-lactam resistance mechanism. To counteract β-lactam resistance, discovery of β-lactamase inhibitors has been the major emphasis in the past decades but has only led to partial success. Given the direct link between β-lactamase induction and cell wall metabolism in Gram-negative bacteria, inhibiting the induction of β- lactamase is a promising therapeutic strategy to counteract β-lactam resistance. Our recent studies have shown Campylobacter jejuni is a great model organism to test this hypothesis. In particular, the periplasmic lytic transglycosylase (LT) plays a critical role in regulating β-lactamase mediated β-lactam resistance in C. jejuni. In this study, we observed that the LT inhibitor bulgecin A inhibited the activity of the C. jejuni LT and significantly potentiated β-lactam antibiotic against resistant C. jejuni. To further develop LT inhibitor for combination therapy, we obtained the 2.16Å crystal structure of the C. jejuni LT; the fully refined structure revealed an interesting doughnut shape which is similar to the previously determined structures of E. coli SLT70 and P. aeruginosa MltE. It is remarkable that the doughnut-shaped feature is well maintained as C. jejuni LT is only 541 aa residues whereas Slt70 contains 618 residues. The complex structure of the C. jejuni LT with bulgecin A was also obtained, revealing active site residues important for LT activity. The active site of the C. jejuni LT situated in the catalytic domain is remarkably similar to that of E. coli Slt70 despite the only ~26% sequence identity. Together, this study strongly suggested that the soluble C. jejuni LT serves as an ideal representative target to perform LT-based drug discovery, and established a solid foundation for us to identify new lead compounds inhibiting LT using computational docking and high-throughput screening approaches in the future.