Title Participants "Beta-lactamase producing Haemophili and Neisseriae" "P. Piot" "Prevalence and distribution of beta-lactamase coding genes in third-generation cephalosporin-resistant Enterobacteriaceae from bloodstream infections in Cambodia" "Erika Vlieghe, T-D Huang, T Phe, P Bogaerts, C Berhin, Birgit De Smet, W.E. Peetermans, Jan Jacobs, Y. Glupczynski" "Resistance to third-generation cephalosporins in Gram-negative bacteria is emerging in Asia. We report the prevalence and distribution of extended-spectrum beta-lactamase (ESBL), AmpC beta-lactamase and carbapenemase-coding genes in cefotaxime-resistant Enterobacteriaceae isolates from bloodstream infections (BSI) in Cambodia. All Enterobacteriaceae isolated from BSI in adult patients at Sihanouk Hospital Centre of HOPE, Phnom Penh, Cambodia (2007-2010) were assessed. Antimicrobial susceptibility testing was carried out by disc diffusion and MicroScan according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Screening for ESBL, plasmidic AmpC and carbapenemase-coding genes was performed by multiplex polymerase chain reaction (PCR) sequencing assays. Identification of the ST131 clone was performed in all CTX-M-positive Escherichia coli, using PCR targeting the papB gene. Out of 183 Enterobacteriaceae, 91 (49.7 %) isolates (84 BSI episodes) were cefotaxime-resistant: E. coli (n = 68), Klebsiella pneumoniae (n = 17) and Enterobacter spp. (n = 6). Most episodes were community-acquired (66/84; 78.3 %). ESBLs were present in 89/91 (97.8 %) cefotaxime-resistant isolates: 86 (96.6 %) were CTX-M, mainly CTX-M-15 (n = 41) and CTX-M-14 (n = 21). CTX-M of group 1 were frequently associated with TEM and/or OXA-1/30 coding genes and with phenotypic combined resistance to ciprofloxacin, sulphamethoxazole-trimethoprim and gentamicin (39/50, 78.0 %). Plasmidic AmpC (CMY-2 and DHA-1 types) were found alone (n = 2) or in combination with ESBL (n = 4). Eighteen E. coli isolates were identified as B2-ST131-O25B: 11 (61.1 %) carried CTX-M-14. No carbapenemase-coding genes were detected. ESBL among Enterobacteriaceae from BSI in Cambodia is common, mainly associated with CTX-M-15 and CTX-M-14. These findings warrant urgent action for the containment of antibiotic resistance in Cambodia." "Quantifying antibiotic impact on within-patient dynamics of extended-spectrum beta-lactamase resistance" "Rene Niehus, Esther van Kleef, Yin Mo, Agata Turlej-Rogacka, Yehuda Carmeli, Herman Goossens, Evelina Tacconelli, Biljana Carevic, Liliana Preotescu, Surbhi Malhotra, Ben S. Cooper" "Antibiotic-induced perturbation of the human gut flora is expected to play an important role in mediating the relationship between antibiotic use and the population prevalence of antibiotic resistance in bacteria, but little is known about how antibiotics affect within-host resistance dynamics. Here we develop a data-driven model of the within-host dynamics of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. We use bla(CTX-M) (the most widespread ESBL gene family) and 16S rRNA (a proxy for bacterial load) abundance data from 833 rectal swabs from 133 ESBL-positive patients followed up in a prospective cohort study in three European hospitals. We find that cefuroxime and ceftriaxone are associated with increased bla(CTX-M) abundance during treatment (21% and 10% daily increase, respectively), while treatment with meropenem, piperacillin-tazobactam, and oral ciprofloxacin is associated with decreased bla(CTX-M) (8% daily decrease for all). The model predicts that typical antibiotic exposures can have substantial long-term effects on bla(CTX-M) carriage duration." "ACI-1 beta-lactamase is widespread across human gut microbiomes in Negativicutes due to transposons harboured by tailed prophages" "Harald Bruessow" "Antibiotic resistance is increasing among pathogens, and the human microbiome contains a reservoir of antibiotic resistance genes. Acidaminococcus intestini is the first Negativicute bacterium (Gram-negative Firmicute) shown to be resistant to beta-lactam antibiotics. Resistance is conferred by the aci1 gene, but its evolutionary history and prevalence remain obscure. We discovered that ACI-1 proteins are phylogenetically distinct from beta-lactamases of Gram-positive Firmicutes and that aci1 occurs in bacteria scattered across the Negativicute clade, suggesting lateral gene transfer. In the reference A. intestini RyC-MR95 genome, we found transposons residing within a tailed prophage context are likely vehicles for aci1's mobility. We found aci1 in 56 (4.4%) of 1,267 human gut metagenomes, mostly hosted within A. intestini, and, where could be determined, mostly within a consistent mobile element constellation. These samples are from Europe, China and the USA, showing that aci1 is distributed globally. We found that for most Negativicute assemblies with aci1, the prophage observed in A. instestini is absent, but in all cases aci1 is flanked by varying transposons. The chimeric mobile elements we identify here likely have a complex evolutionary history and potentially provide multiple complementary mechanisms for antibiotic resistance gene transfer both within and between cells." "Detection of beta-lactamase production in clinical Prevotella species by MALDI-TOF MS method" "Nurver Ulger Toprak, Oncu Akgul, József Sóki, Guner Soyletir, Elisabeth Nagy, Ingrid Wybo" "Penicillins, can be used in treatment of infections due to Prevotella species if they are susceptible to penicillin. Early and accurate preliminary detection of β-lactamase-producing isolates is crucial for treatment of infection. The aim of this study was to determine β-lactamase-producing Prevotella species by MALDI-TOF MS and screen them for the presence of cfxA gene, responsible for β-lactamase production. A total of 500 clinically relevant Prevotella isolates, collected from 13 countries for the previous European antibiotic resistance surveillance study, were tested. Susceptibility testing was performed against ampicillin and ampicillin/sulbactam by Etest methodology. EUCAST guidelines were used for susceptibility interpretations; the isolates with MIC value ≤ 0.5 for ampicillin were considered susceptible and >2 resistant. All Prevotella isolates, were tested for detection of β-lactamase activity by MALDI-TOF MS (Vitek® MS Research Use Only) system and the presence of the cfxA gene by PCR method. The susceptibility levels of the isolates to ampicillin/sulbactam and ampicillin were 99.6% and 43.4%, respectively. A total 59% of isolates presented β-lactamase activity and 60.8% were cfxA gene positive. Both these tests were positive for isolates in the resistant category. Additionally, >95% of the isolates (n = 65) which ampicillin MIC values ranged from >0.5 μg/mL to 2 μg/ml displayed β-lactamase activity. We also found that the MALDI-TOF MS-based β-lactamase assay delivers results in 2 h. We found a high concordance between the MALDI-TOF MS β-lactamase results in terms of cfxA β-lactamase gene presence. MALDI-TOF MS may serve as a simple and efficient alternative method of the existing phenotypic and PCR-based methods." "OXA-427, a new plasmid-borne carbapenem-hydrolysing class D beta-lactamase in Enterobacteriaceae" "Annette Schuermans" "OBJECTIVES: To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital. METHODS: OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53. RESULTS: Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%-29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid. CONCLUSIONS: OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period." "High abundance and diversity of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faeces and tonsils of pigs at slaughter" "Inge Van Damme, Cristina Garcia-Graells, Wauter Biasino, Tanuja Kumari Gullahally Manjegowda, Nadine Botteldoorn" "Identification of a metagenomic gene cluster containing a new class A beta-lactamase and toxin-antitoxin systems." "Ken Vercammen, Tamara Garcia-Armisen, Nathalie Goeders, Laurence Van Melderen, Josselin Bodilis, Pierre Cornelis" "Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different ?-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of ?-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to ?-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system." "Comparison of different phenotypic assays for teh detection of extended-sprectrum beta-lactamase production by inducible AmpC-producing Gram-negative bacilli" "Jan Verhaegen" "Routine detection of extended-spectrum β-lactamase (ESBL) production by AmpC-producing Enterobacteriaceae in microbiology laboratories is still a problem. The aim of this study was to compare the performance of four different phenotypic ESBL confirmation assays within this group of Enterobacteriaceae. A total of 83 AmpC-inducible Enterobacteriaceae were included in this study (58 clinical isolates with presumptive ESBL production and 25 molecularly characterized ESBL-producing isolates). Each isolate was tested for the presence of an ESBL enzyme by four phenotypic ESBL confirmation assays: ESBL Etests and combined double-disk synergy tests (CDDST), both on Mueller-Hinton (MH) agar with and without the use of cloxacillin, an AmpC inhibitor. Our study showed that performing a CDDST on MH agar with cefotaxime as the only indicator cephalosporin is not a reliable way to detect ESBL-encoding genes among chromosomal AmpC-producing Enterobacteriaceae due to its low sensitivity (52 %). The use of cloxacillin in this CDDST could only significantly increase the specificity of the CDDST when used with ceftazidime as the indicator [sensitivity (SN), 92 %; specificity (SP), 93 %]. Regarding ESBL Etest® strips, the sensitivity of the cefepime strip (80 %) was significantly higher compared to the cefotaxime and ceftazidime strips (16 % and 32 %, respectively). Adding cloxacillin to the MH agar improved the ESBL detection of each of these strips. We recommend the CDDST on MH agar supplemented with cloxacillin and ceftazidime or cefepime as the indicator cephalosporin as the most cost-efficient strategy to confirm ESBL production in inducible AmpC-producing Enterobacteriaceae." "Catheter-related phlebitis in calves associated with broad-spectrum beta-lactamase producting Escherichia coli" "Bart Pardon, Annemieke Smet, Peter De Schutter, Bonnie Valgaeren, Boudewijn Catry, Piet Deprez"