Increasing proportion of carbapenemase-producing Enterobacteriaceae and emergence of a MCR-1 producer through a multicentric study among hospital-based and private laboratories in Belgium from September to November 2015

Carbapenemase-producing Enterobacteriaceae (CPE) strains have been increasingly reported in Belgium. We aimed to determine the proportion of CPE among Enterobacteriaceae isolated from hospitalised patients and community outpatients in Belgium in 2015. For the hospitalised patients, the results were compared to a previous similar survey performed in the same hospitals in 2012. Twenty-four hospital-based and 10 private laboratories collected prospectively 200 non-duplicated Enterobacteriaceae isolates from clinical specimens. All isolates were screened locally by carbapenem disk diffusion using European Committee on Antimicrobial Susceptibility Testing methodology. Putative CPE strains with inhibition zone diameters below the screening breakpoints were referred centrally for confirmation of carbapenemase production. From September to November 2015, we found a proportion of clinical CPE of 0.55% (26/4,705) and of 0.60% (12/1,991) among hospitalised patients and among ambulatory outpatients respectively. Klebsiella pneumoniae (26/38) and OXA-48-like carbapenemase (28/38) were the predominant species and enzyme among CPE. One OXA-48-producing Escherichia coli isolated from a hospital was found carrying plasmid-mediated MCR-1 colistin resistance. Compared with the 2012 survey, we found a significant increased proportion of clinical CPE (0.55% in 2015 vs 0.25% in 2012; p = 0.02) and an increased proportion of hospitals (13/24 in 2015 vs 8/24 in 2012) with at least one CPE detected. The study results confirmed the concerning spread of CPE including a colistin-resistant MCR-1 producer in hospitals and the establishment of CPE in the community in Belgium

Potency of marbofloxacin for pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida: Comparison of growth media

Pharmacodynamic properties of marbofloxacin were established for six isolates each of the pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Three in vitro indices of potency were determined; Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Mutant Prevention Concentration (MPC). For MIC determination Clinical Laboratory Standards Institute guidelines were modified in three respects: (1) comparison was made between two growth media, an artificial broth and pig serum; (2) a high inoculum count was used to simulate heavy clinical bacteriological loads; and (3) five overlapping sets of two-fold dilutions were used to improve accuracy of determinations. Similar methods were used for MBC and MPC estimations. MIC and MPC serum:broth ratios for A. pleuropneumoniae were 0.79:1 and 0.99:1, respectively, and corresponding values for P. multocida were 1.12:1 and 1.32:1. Serum protein binding of marbofloxacin was 49%, so that fraction unbound (fu) serum MIC values were significantly lower than those predicted by correction for protein binding; fu serum:broth MIC ratios were 0.40:1 (A. pleuropneumoniae) and 0.50:1 (P. multocida). For broth, MPC:MIC ratios were 13.7:1 (A. pleuropneumoniae) and 14.2:1 (P. multocida). Corresponding ratios for serum were similar, 17.2:1 and 18.8:1, respectively. It is suggested that, for dose prediction purposes, serum data might be preferable to potency indices measured in broths

Phenotypic and genotypic characterisation of Neisseria gohorrhoeae isolates from New Zealand with reduced susceptibility to ceftriaxone : a thesis submitted to the College of Health in partial fulfilment of the requirements for the Master of Science in Microbiology at Massey University, New Zealand

Objectives Currently, ceftriaxone is the last remaining drug recommended for empirical treatment of gonorrhoea. Neisseria gonorrhoeae with reduced susceptibility to ceftriaxone have been isolated worldwide in countries such as Japan, France, Spain, Slovenia, Australia and Sweden. These have led to treatment failures and the emergence of ceftriaxone-resistant N. gonorrhoeae. Various mutations in penA (mosaic and nonmosaic), which encodes the penicillin-binding protein 2 (PBP2), have been reported to be the primary reason for reduced ceftriaxone susceptibility, but it can be reduced further by mutations in mtrR, porBIB and ponA. In this study, we aimed to determine the antimicrobial resistance patterns of New Zealand isolates of N. gonorrhoeae with reduced susceptibility to ceftriaxone and to characterise the penA, mtrR, porBIB and ponA in the isolates. Methods A total of 28 N. gonorrhoeae isolates with elevated ceftriaxone MIC (0.03 to 0.12 mg/L), collected from 2012 to 2015 and obtained from the Institute of Environmental Science and Research (ESR), were examined in this study. Samples came from laboratories in Auckland (26), Wellington (1) and Taranaki (1). The antimicrobial resistance of penicillin G, tetracycline, ciprofloxacin, azithromycin and ceftriaxone were determined through antimicrobial susceptibility test, using minimum inhibitory concentration (MIC) test strips. Polymerase chain reactions (PCRs) and sequencing to identify specific mutations in penA, mtrR, porBIB and ponA, that are associated with elevated minimum inhibitory concentrations (MICs) to ceftriaxone, were undertaken. The association between the phenotypic and genotypic results was investigated by comparing the presence of the number of mutated genes and the MIC level of ceftriaxone. Results Based on the AST results using MIC test strips and interpreted using The European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, 23 out of 28 isolates (82%) showed reduced susceptibility to ceftriaxone, with MICs of 0.03 to 0.06 mg/L. All of the isolates were resistant to ciprofloxacin, while 36%, 25% and 7% were resistant to penicillin G, tetracycline and azithromycin, respectively. Two azithromycin-resistant N. gonorrhoeae isolates were observed, and isolate 264 (azithromycin MIC: 4mg/L) also exhibited reduced susceptibility to ceftriaxone (MIC: 0.03 mg/L). A total of 21% (6/28) of the isolates produced ß- lactamase. The 23 isolates that conveyed reduced ceftriaxone susceptibility were found to harbour three or four mutated genes (penA, mtrR and/or porBIB and ponA). Reduced susceptibility to ceftriaxone among N. gonorrhoeae isolates in this study was associated with mosaic PBP2 (encoded by penA) with G545S/A501V mutations, with nonmosaic PBP2 with an A501V mutation, plus the presence of mutation in mtrR promoter with G120 and A121 alterations in PorBIB. A total of 65% (15/23) of the N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone harboured mosaic PBP2 XXXIV, a pattern found in N. gonorrhoeae associated with ceftriaxone treatment failures in Europe and Australia. The current study also revealed that the partial sequences of four mosaic PBP2 (M-2, M-3, M-4, M-5) were different from the common mosaic PBP2 sequences reported in various studies. Conclusion There is an association between the phenotypic and genotypic character of N. gonorrhoeae isolates expressing reduced susceptibility to ceftriaxone in this study population. Furthermore, the presence of important mosaic PBP2 that link to ceftriaxone treatment failure might be circulating among N. gonorrhoeae isolates in New Zealand . Keywords: Neisseria gonorrhoeae, ceftriaxone, reduced susceptibility, New Zealan

The continued value of disk diffusion for assessing antimicrobial susceptibility in clinical laboratories: Report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group

Expedited pathways to antimicrobial agent approval by the U.S. Food and Drug Administration (FDA) have led to increased delays between drug approval and the availability of FDA-cleared antimicrobial susceptibility testing (AST) devices.</jats:p

Direct susceptibility testing by disk diffusion on clinical samples : a rapid and accurate tool for antibiotic stewardship

We compared the accuracy of direct susceptibility testing (DST) with conventional antimicrobial susceptibility testing (AST), both using disk diffusion, on clinical samples. A total of 123 clinical samples (respiratory tract samples, urine, vaginal and abdominal abscess discharges, bile fluid and a haematoma punctate) were selected on various indications; direct inoculation on Mueller-Hinton agar and antibiotic paper disks were applied. In parallel, standard culture, identification and AST on the colonies grown overnight was executed. Both AST and DST were interpreted after identification of the isolates. The results from both AST and DST for 11 antibiotics tested on 97 samples with Gram-negative rods showed 93.4 % total agreement, 1.6 % minor discordances, 4.6 % major discordances and 0.4 % very major discordances. Analysing the discordant results, DST predominantly resulted in more resistant isolates than AST. This was mostly due to the presence of resistant mutants or an additional isolate. The remaining discordances were seen for isolates with inhibition zones close to the clinical breakpoint. For the 26 samples yielding staphylococci, a total agreement of 100 % was observed for the nine antibiotics tested. Overall, the highest percentage of discordant results occurred for the beta-lactam antibiotics amoxicillin-clavulanate (13.4 %) and cefuroxime (12.4 %). When used selectively and interpreted carefully, DST on clinical samples is potentially very useful in the management of critically ill patients, as the time to results is shortened by approximately 24 h. However, we recommend to communicate results with reservations and confirm by conventional AST

Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI screening parameters for the detection of extended-spectrum β-lactamase production in clinical Enterobacteriaceae isolates

Objectives To compare the performance of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints following their revision in 2010, for the detection of extended-spectrum β-lactamase (ESBL) production in Enterobacteriaceae. Methods 236 well-characterized clinical isolates (including 118 ESBL producers) were investigated by antibiotic disc testing with cefpodoxime, ceftriaxone, cefepime, cefotaxime EUCAST (5 μg/disc), ceftazidime EUCAST (10 μg/disc), cefotaxime CLSI (30 μg/disc) and ceftazidime CLSI (30 μg/disc) with the Kirby-Bauer method. Additionally, synergy phenomena were recorded between amoxicillin/clavulanic acid discs (20/10 μg/disc) and cefepime (30 μg/disc), EUCAST cefotaxime (5 μg/disc), EUCAST ceftazidime (10 μg/disc), CLSI cefotaxime (30 μg/disc) and CLSI ceftazidime [30 μg/disc; disc approximation method (DAM)]. Results Overall sensitivity of the cefotaxime EUCAST non-susceptible breakpoint equalled sensitivity of the cefotaxime CLSI ESBL screening breakpoint (99.2%). With the ceftazidime EUCAST non-susceptible breakpoint, 27/118 ESBL-producing isolates were not detected, whereas the ceftazidime CLSI ESBL screening breakpoint missed 41/118 ESBL-producing isolates. For cefpodoxime the resistant EUCAST breakpoint showed higher sensitivity for ESBL detection compared with the CLSI ESBL screening breakpoint/disc content (100% versus 98.3%, respectively). Sensitivities of ceftazidime and cefotaxime DAM with CLSI or EUCAST disc contents were comparable (sensitivities ranging from 84.7% to 89.8%). DAM with cefepime displayed the highest overall sensitivity (96.6%). In AmpC-producing isolates, synergy of amoxicillin/clavulanic acid with cefepime showed sensitivity and specificity for ESBL detection of 100% and 97.4%, respectively. Conclusions EUCAST non-susceptible breakpoints for ceftazidime and cefpodoxime detect more ESBL-producing Enterobacteriaceae isolates compared with corresponding CLSI ESBL screening breakpoints. Implementation of the cefepime DAM can facilitate ESBL screening, especially in strains producing an AmpC β-lactamase since the test shows high sensitivity and specificit

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP-flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP-flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC-828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador

Effect of β-lactamase inhibitors on in vitro activity of β-lactam antibiotics against Burkholderia cepacia complex species

Background: Bacteria belonging to the Burkholderia cepacia complex (Bcc) are an important cause of chronic respiratory tract infections in cystic fibrosis patients. Intrinsic resistance to a wide range of antimicrobial agents, including a variety of beta-lactam antibiotics, is frequently observed in Bcc strains. Resistance to beta-lactams is most commonly mediated by efflux pumps, alterations in penicillin-binding proteins or the expression of beta-lactamases. beta-lactamase inhibitors are able to restore the in vitro activity of beta-lactam molecules against a variety of Gram-negative species, but the effect of these inhibitors on the activity of beta-lactam treatment against Bcc species is still poorly investigated. Methods: In the present study, the susceptibility of a panel of Bcc strains was determined towards the beta-lactam antibiotics ceftazidime, meropenem, amoxicillin, cefoxitin, cefepime and aztreonam; alone or in combination with a beta-lactamase inhibitor (clavulanic acid, sulbactam, tazobactam and avibactam). Consequently, beta-lactamase activity was determined for active beta-lactam/beta-lactamase inhibitor combinations. Results: Clavulanic acid had no effect on minimum inhibitory concentrations, but addition of sulbactam, tazobactam or avibactam to ceftazidime, amoxicillin, cefoxitin, cefepime or aztreonam leads to increased susceptibility (at least 4-fold MIC-decrease) in some Bcc strains. The effect of beta-lactamase inhibitors on beta-lactamase activity is both strain-and/or antibiotic-dependent, and other mechanisms of beta-lactam resistance (besides production of beta-lactamases) appear to be important. Conclusions: Considerable differences in susceptibility of Bcc strains to beta-lactam antibiotics were observed. Results obtained in the present study suggest that resistance of Bcc strains against beta-lactam antibiotics is mediated by both beta-lactamases and non-beta-lactamase-mediated resistance mechanisms

Consequences of revised CLSI and EUCAST guidelines for antibiotic susceptibility patterns of ESBL- and AmpC β-lactamase-producing clinical Enterobacteriaceae isolates

Objectives This study aimed to: (i) analyse the antibiotic susceptibility testing (AST) profiles of extended spectrum β-lactamase (ESBL)- and AmpC β-lactamase-producing clinical Enterobacteriaceae isolates applying EUCAST 2013 AST guidelines; and (ii) evaluate discrepancies in AST profiles according to EUCAST 2010 guidelines, EUCAST 2013 guidelines, CLSI 2009 guidelines and CLSI 2013 guidelines. Methods The 195 ESBL- and/or AmpC β-lactamase-producing Enterobacteriaceae isolates used in this study were systematically characterized by disc diffusion AST interpreted according to the 2013 guidelines of EUCAST and CLSI, the EUCAST 2010 guidelines and the CLSI 2009 guidelines. Results Individual cephalosporin AST patterns according to EUCAST 2013 guidelines were described for individual ESBL and AmpC β-lactamase genotypes. Significant differences in the susceptibility rates of important cephalosporins such as cefepime, ceftazidime and cefotaxime applying EUCAST 2013 and CLSI 2013 AST guidelines were demonstrated for ESBL- and AmpC β-lactamase-producing isolates. Conclusions The confirmation of ESBL and/or AmpC β-lactamase production can support the selection of an adequate antibiotic drug therapy. Despite a harmonized CLSI and EUCAST ‘report as found' strategy for cephalosporins and ESBL-producing isolates, AST interpretation according to the CLSI 2013 and EUCAST 2013 guidelines shows significant differences in susceptibility rates for mainstay cephalosporins such as cefepime, ceftazidime and cefotaxime. Thus, further harmonization of clinical breakpoints is warrante

Evaluation of the Coverage of 3 Antibiotic Regimens for Neonatal Sepsis in the Hospital Setting Across Asian Countries.

Importance: High levels of antimicrobial resistance in neonatal bloodstream isolates are being reported globally, including in Asia. Local hospital antibiogram data may include too few isolates to meaningfully examine the expected coverage of antibiotic regimens. Objective: To assess the coverage offered by 3 antibiotic regimens for empirical treatment of neonatal sepsis in Asian countries. Design, Setting, and Participants: A decision analytical model was used to estimate coverage of 3 prespecified antibiotic regimens according to a weighted-incidence syndromic combination antibiogram. Relevant data to parameterize the models were identified from a systematic search of Ovid MEDLINE and Embase. Data from Asian countries published from 2014 onward were of interest. Only data on blood culture isolates from neonates with sepsis, bloodstream infection, or bacteremia reported from the relevant setting were included. Data analysis was performed from April 2019 to July 2019. Exposures: The prespecified regimens of interest were aminopenicillin-gentamicin, third-generation cephalosporins (cefotaxime or ceftriaxone), and meropenem. The relative incidence of different bacteria and their antimicrobial susceptibility to antibiotics relevant for determining expected concordance with these regimens were extracted. Main Outcomes and Measures: Coverage was calculated on the basis of a decision-tree model incorporating relative bacterial incidence and antimicrobial susceptibility of relevant isolates. Data on 7 bacteria most commonly reported in the included studies were used for estimating coverage, which was reported at the country level. Results: Data from 48 studies reporting on 10 countries and 8376 isolates were used. Individual countries reported 51 (Vietnam) to 6284 (India) isolates. Coverage varied considerably between countries. Meropenem was generally estimated to provide the highest coverage, ranging from 64.0% (95% credible interval [CrI], 62.6%-65.4%) in India to 90.6% (95% CrI, 86.2%-94.4%) in Cambodia, followed by aminopenicillin-gentamicin (from 35.9% [95% CrI, 27.7%-44.0%] in Indonesia to 81.0% [95% CrI, 71.1%-89.7%] in Laos) and cefotaxime or ceftriaxone (from 17.9% [95% CrI, 11.7%-24.7%] in Indonesia to 75.0% [95% CrI, 64.8%-84.1%] in Laos). Aminopenicillin-gentamicin coverage was lower than that of meropenem in all countries except Laos (81.0%; 95% CrI, 71.1%-89.7%) and Nepal (74.3%; 95% CrI, 70.3%-78.2%), where 95% CrIs for aminopenicillin-gentamicin and meropenem were overlapping. Third-generation cephalosporin coverage was lowest of the 3 regimens in all countries. The coverage difference between aminopenicillin-gentamicin and meropenem for countries with nonoverlapping 95% CrIs ranged from -15.9% in China to -52.9% in Indonesia. Conclusions and Relevance: This study's findings suggest that noncarbapenem antibiotic regimens may provide limited coverage for empirical treatment of neonatal sepsis in many Asian countries. Alternative regimens must be studied to limit carbapenem consumption