Susceptibility Testing Guidelines Research Articles

Evaluating the Role of Wastewaters as Reservoirs of Antibiotic-Resistant ESKAPEE Bacteria Using Phenotypic and Molecular Methods.

IntroductionWastewaters carrying thousands of human specimens from the community and representing the diversity of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) directly from the community mirror the extent of AR spread in the community and environment. This study aimed to investigate the occurrence and distribution of antibiotic-resistant ESKAPEE bacteria in the community versus clinical settings through monitoring nonclinical and clinical wastewaters.MethodologySeven wastewater samples were collected from different environmental sources. Isolates were obtained on general and selective media, biochemically characterized and antimicrobial-susceptibility tests performed by disk diffusion against 13 antibiotics according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines using MastDisc disk cartridges, and 16S rRNA metagenomic analysis was performed for two water samples.ResultsOf 43 isolates, all representatives of the ESKAPEE group were recovered from clinical wastewaters, but Gram-positive cocci were not obtained from nonclinical wastewaters. The most predominant isolate was Pseudomonas aeruginosa (n=15; 33%), followed by Escherichia coli (n=9; 20%). Complete (100%) resistance to eleven of the tested antibiotics was observed, with only a few isolates being susceptible to clarithromycin, amikacin, and gentamicin. The lowest (79%) resistance rate was observed for linezolid. The multiple antibiotic resistance (MAR) index was calculated, and the resistance phenotype was independent of the wastewater source, indicated by x2 (P=0.766). Metagenomic analysis replicated the results, as Pseudomonas spp., Acinetobacter spp., and Escherichia spp. were found to be predominant. The integrase gene (IntI1) was also amplified in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.ConclusionWastewaters are significant carriers of drug-resistant ESKAPEE bacteria and play an important role in their dissemination. This study endorses the periodic surveillance of water systems to evaluate the presence and burden of antibiotic-resistant pathogens.

Effects of Levofloxacin, Aztreonam, and Colistin on Enzyme Synthesis by P. aeruginosa Isolated from Cystic Fibrosis Patients

(1) Background: Cystic fibrosis (CF) is characterized by chronic pulmonary inflammation and persistent bacterial infections. P. aeruginosa is among the main opportunistic pathogens causing infections in CF. P. aeruginosa is able to form a biofilm, decreasing antibiotic permeability. LOX, a lipoxygenase enzyme, is a virulence factor produced by P. aeruginosa and promotes its persistence in lung tissues. The aim of this study is to evaluate if antibiotics currently used for aerosol therapy in CF are able to interfere with the production of lipoxygenase from open isolates of P. Aeruginosa from patients with CF. (2) Methods: Clinical isolates of P. aeruginosa from patients with CF were grown in Luria broth (LB). Minimum inhibitory concentration (MIC) was performed and interpreted for all isolated strains according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. We selected four antibiotics with different mechanisms of action: aztreonam, colistin, amikacin, and levofloxacin. We used human pulmonary epithelial NCI-H929 cells to evaluate LOX activity and its metabolites according to antibiotic action at increasing concentrations. (3) Results: there is a correlation between LOX secretion by clinical isolates of P. aeruginosa and biofilm production. Levofloxacin exhibits highly significant inhibitory activity compared to the control. Amikacin also exhibits significant inhibitory activity against LOX production. Aztreonam and colistin do not show inhibitory activity. These results are also confirmed for LOX metabolites. (4) Conclusions: among the evaluated antibiotics, levofloxacin and amikacin have an activity on LOX secretion.

Head‐to‐head comparison of CLSI, EUCAST, Etest and VITEK®2 results for Candida auris susceptibility testing

The susceptibility of 31 Candida auris clinical isolates was evaluated by four methods, namely the microdilution reference method according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as well as Etest and VITEK®2. Essential agreement between the two reference methods was 90%. Etest showed a better overall agreement with the reference methods (94% and 81% for CLSI and EUCAST, respectively) than VITEK®2 (70% and 72%, respectively). Discrepancies were found for fluconazole (FLC) and amphotericin B. Considering categorical agreement (CDC tentative breakpoints), the majority of isolates were considered FLC-resistant (93.6% and 80.6% by CLSI and EUCAST, respectively). Furthermore, all isolates were considered susceptible to echinocandins by all methods. Susceptibility results should be interpreted with care if the VITEK®2 system is used to guide therapeutic decisions for C. auris infections.

Detection of Colistin Resistance in Uropathogenic Carbapenem Resistant Klebsiella pneumoniae

Background: Colistin resistance among carbapenem-resistant Klebsiella pneumoniae (CRKp) strains at an accelerated rate poses a serious global threat which limits therapeutic options. Objective: In this study, 203 laboratory-archived uropathogenic K. pneumoniae isolates were screened for carbapenem and colistin resistance. Methods: Carbapenam resistance screening was performed using combined disc test. Colistin resistance was determined for CRKp isolates, according to clinical and laboratory standards institute- European Committee on Antimicrobial Susceptibility Testing guidelines. Polymerase Chain Reaction for carbapenamase genes (blaNDM-1, blaKPC-2 and blaOXA-1) and plasmid-mediated colistin resistance genes (mcr-1-2) were performed for the resistant isolates. Results: About 30% of the CRKp isolates were resistant to colistin; 57.1% and 35.7% of the isolates carried blaNDM-1 and blaOXA-1 gene, respectively. None of the isolates showed positive for blaKPC-2 and mcr (1-2) genes. Conclusion: Colistin resistance in the absence of a plasmid borne transfer mechanism was observed among our CRKp strains. Escalating colistin resistance is a crucial obstacle in treatment of K. pneumoniae infections and repurposing their usage is important to combat and control the resistance.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo–spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

First report of nosocomial outbreak of vancomycin-resistant Enterococcus faecium infection among COVID-19 patients hospitalized in a non-intensive care unit ward in Slovakia.

Enterococcus species account for most of the human enterococcal HAI and multidrug-resistant infections andhave become a major threat to modern public health. We examine the risein the number of vancomycin resistantE. faeciumblood stream and urinary tractinfections in a COVID-19 department during an epidemiologic outbreak investigation to detect and eliminate nosocomial clusters of the bacteria. Strain identification was performed by classical isolation andbiochemical andcultivation methods. Antibiotic testing results were interpreted according to European committee on antimicrobial susceptibility testing (EUCAST) guidelines. Six isolatedsamples underwent the whole genome sequencing (WGS) during the outbreak investigation. Isolate relatedness was determined using the core genome multi-locus sequence typing. WGS revealed two genotypically distinct VRE clusters, one of which had genetically closely related patients and environmental isolates. The cluster was terminated by enhanced infection control strategies. This study provides the first description of an outbreak caused by vanB-ST117 and vanA-ST17E. faeciumstrains among COVID-19 patients in Slovakia. This study can help to raise the awareness about the need for strict adherence to infection control measures and the implementation of rational antimicrobial stewardship as a routine part of COVID-19 management (Tab. 3, Fig. 3, Ref. 27). Text in PDF www.elis.sk Keywords: vancomycin-resistantEnterococcus faecium, antibiotic resistant, COVID-19, SARS-CoV-2, bacterial outbreak, healthcare-associated infection.

High level of intrinsic phenotypic antimicrobial resistance in enterobacteria from terrestrial wildlife in Gabonese national parks.

Data on the prevalence of antibiotic resistance in Enterobacteriaceae in African wildlife are still relatively limited. The aim of this study was to estimate the prevalence of phenotypic intrinsic and acquired antimicrobial resistance of enterobacteria from several species of terrestrial wild mammals in national parks of Gabon. Colony culture and isolation were done using MacConkey agar. Isolates were identified using the VITEK 2 and MALDI-TOF methods. Antibiotic susceptibility was analysed and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines. The preliminary test for ESBL-producing Enterobacteriaceae was performed by replicating enterobacterial colonies on MacConkey agar supplemented with 2 mg/L cefotaxime (MCA+CTX). Extended-spectrum beta-lactamase (ESBL) production was confirmed with the double-disc synergy test (DDST). The inhibition zone diameters were read with SirScan. Among the 130 bacterial colonies isolated from 125 fecal samples, 90 enterobacterial isolates were identified. Escherichia coli (61%) was the most prevalent, followed by Enterobacter cloacae (8%), Proteus mirabilis (8%), Klebsiella variicola (7%), Klebsiella aerogenes (7%), Klebsiella oxytoca (4%), Citrobacter freundii (3%), Klebsiella pneumoniae (1%) and Serratia marcescens (1%). Acquired resistance was carried by E. coli (11% of all E. coli isolates) and E. cloacae (3% of all E. cloacae) isolates, while intrinsic resistance was detected in all the other resistant isolates (n = 31); K. variicola, K. oxytoca, K. pneumoniae, E. cloacae, K. aerogenes, S. marcescens and P. mirabilis). Our data show that most strains isolated in protected areas in Gabon are wild type isolates and carry intrinsic resistance rather than acquired resistance.

In vitro activity of dalbavancin and other anti-staphylococcal agents against infecting isolates of methicillin-resistant coagulase-negative staphylococci.

Introduction. Dalbavancin was approved in Europe in 2015 for skin and soft tissue infections.Hypothesis/Gap Statement. Data on methicillin-resistant coagulase-negative staphylococci (MR-CNS) dalbavancin susceptibility are scarce.Aim. To assess the susceptibility of MR-CNS to dalbavancin and other anti-staphylococcal agents.Methodology. A total of 443 MR-CNS clinical isolates from patients hospitalized in a Greek university hospital during a 2.5-year period (January 2018 to June 2020) were included. The MICs for vancomycin, teicoplanin, linezolid and daptomycin were investigated by Etest and the MIC for dalbavancin was determined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in 196 isolates. The consumption of the aforementioned antimicrobials was calculated.Results. In total, 51 isolates were resistant to teicoplanin (11.5 %) and 211 (47.6 %) to linezolid; all were susceptible to vancomycin and daptomycin. Among 196 isolates tested, 32 (16.3 %) were resistant to dalbavancin. A significant increase of MIC during the study period was found for vancomycin, teicoplanin and daptomycin, while a decrease in linezolid's MIC was observed. Dalbavancin's MIC remained stable. No difference in consumption was observed among the studied anti-staphylococcal agents.Conclusion. An increase of vancomycin, teicoplanin and daptomycin MICs among MR-CNS was observed, whereas 47.6 % of isolates were non-susceptible to linezolid. Dalbavancin retains excellent potency against MR-CNS, even in the presence of non-susceptibility to other anti-staphylococcal antibiotics.

Antimicrobial Susceptibility among Gram-Negative Isolates in Pediatric Patients in Europe from 2013–2018 Compared to 2004–2012: Results from the ATLAS Surveillance Study

AimsData on antimicrobial resistance (AMR) in the paediatric patient population are scarce. This study assessed the AMR rates and phenotype distribution of Gram-negative isolates in paediatric patients in Europe from 2004–2012 and 2013–2018. MethodsIsolates that were collected were stratified by age groups (< 1, 1–5, 6–12, and 13–17 years) and regions (North-Western, Eastern and Southern Europe). Minimal inhibitory concentrations (broth microdilution) were interpreted according to European Committee on Antimicrobial Susceptibility Testing guidelines. Resistance rates and phenotype prevalence were identified for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Haemophilus influenzae. ResultsIn the overall paediatric patient population (0–17 years), extended-spectrum beta-lactamase (ESBL) production significantly decreased (from 20.7% to 15.4%, P < 0.0001) in Escherichia coli, whereas it increased for Klebsiella pneumoniae (from 35.0% to 39.2%, P = 0.015). Carbapenem resistance was highest for Acinetobacter baumannii (32.3%) compared with Klebsiella pneumoniae (4.7%) and Pseudomonas aeruginosa (12.4%) in 2013–2018, and rates were significantly increased relative to 2004–2012. There was no change in resistance to beta-lactam antimicrobials for Haemophilus influenzae. The lowest resistance rates for most organism groups were observed in North-Western Europe. ConclusionsThe results revealed a significant increase in Klebsiella pneumoniae isolates with an ESBL and carbapenem-resistance phenotype as well as in carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa from 2004–2012 to 2013–2018. Conversely, a decrease in ESBL E. coli was observed. Continued surveillance and awareness of resistance in these bacteria causing serious infections is crucial for improving treatment quality in paediatric patients.

Successfully controlling the incidence of multidrug-resistant Pseudomonas aeruginosa through antibiotic stewardship and infection control programmes at a Chinese university hospital.

The purpose of this study was to investigate the effect of imposing infection control programmes (ICPs) and antimicrobial stewardship (AMS) by monitoring the antimicrobial resistance of Pseudomonas aeruginosa. Antimicrobial susceptibility testing was performed in accordance with the Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. ICPs and AMS were initiated at the Fourth Hospital from 2013 to 2018. A total of 2,886 P.aeruginosa isolates were assessed. The antimicrobial resistance trends of the P.aeruginosa strains improved after the intervention measures. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) P.aeruginosa contributed to 18.5% and 3.5% of the total P.aeruginosa strains, respectively. Colistin was the most effective antibiotic against 97.6% of XDR-P.aeruginosa and 99.41% of MDR-P.aeruginosa. The consumption of alcohol-based hand gel (ABHG) increased from 0.6L to 10.8L per 1,000 patient-days (PD) (p=0.005). The yearly consumption of antibiotics decreased from 45 to 37.5 defined daily doses (DDD) per 1,000 PD(p=0.04). After 2013, the incidence rate of MDR-P.aeruginosa showed a significant decrease from 22% to 14.1% (p=0.04), and XDR-P.aeruginosa decreased from 5.8% to 0.9%. The use of ABHG was negatively related to MDR-P.aeruginosa morbidity (r=-0.86; p=0.021). The consumption of antibiotics was positively related to MDR-P.aeruginosa morbidity (r=0.86; p=0.021). Successful control of MDR-P.aeruginosa resistance was achieved by imposing comprehensive ICPs and AMS.

Evaluation of the European Committee on Antimicrobial Susceptibility Testing Guidelines for Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis-, Yersinia pestis- and Francisella tularensis-Positive Blood Cultures.

Rapid determination of bacterial antibiotic susceptibility is important for proper treatment of infections. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently published guidelines for rapid antimicrobial susceptibility testing (RAST) performed directly from positive blood culture vials. These guidelines, however, were only published for a limited number of common pathogenic bacteria. In this study, we evaluated the applicability of these guidelines to three Tier 1 bioterror agents (Bacillus anthracis, Yersinia pestis and Francisella tularensis) that require prompt antibiotic treatment to mitigate morbidity and mortality. We used spiked-in human blood incubated in a BACTEC™ FX40 system to determine the proper conditions for RAST using disc-diffusion and Etest assays. We found that reliable disc-diffusion inhibition diameters and Etest MIC values could be obtained in remarkably short times. Compared to the EUCAST-recommended disc-diffusion assays that will require adjusted clinical breakpoint tables, Etest-based RAST was advantageous, as the obtained MIC values were similar to the standard MIC values, enabling the use of established category breakpoint tables. Our results demonstrate the promising applicability of the EUCAST RAST for B. anthracis-, Y. pestis- or F. tularensis-positive blood cultures, which can lead to shorter diagnostics and prompt antibiotic treatment of these dangerous pathogens.

Rethinking Manure Application: Increase in Multidrug-Resistant Enterococcus spp. in Agricultural Soil Following Chicken Litter Application.

The current study investigated the impact of chicken litter application on the abundance of multidrug-resistant Enterococcus spp. in agricultural soil. Soil samples were collected from five different strategic places on a sugarcane farm before and after manure application for four months. Chicken litter samples were also collected. Enterococci were enumerated using the Enterolert®/Quanti-Tray 2000® system and confirm and differentiated into species using real-time PCR. The antibiotic susceptibility profile of the isolates was determined using the disk diffusion method following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The overall mean bacterial count was significantly higher (p < 0.05) in manure-amended soil (3.87 × 107 MPN/g) than unamended soil (2.89 × 107 MPN/g). Eight hundred and thirty-five enterococci (680 from soil and 155 from litter) were isolated, with E. casseliflavus being the most prevalent species (469; 56.2%) and E. gallinarum being the least (16; 1.2%). Approximately 56% of all the isolates were resistant to at least one antibiotic tested, with the highest resistance observed against tetracycline (33%) and the lowest against chloramphenicol (0.1%); 17% of E. faecium were resistant to quinupristin-dalfopristin. Additionally, 27.9% (130/466) of the isolates were multidrug-resistant, with litter-amended soil harbouring more multidrug-resistant (MDR) isolates (67.7%; 88/130) than unamended soil (10.0%; 13/130). All isolates were susceptible to tigecycline, linezolid and gentamicin. About 7% of the isolates had a multiple antimicrobial resistance index > 0.2, indicative of high antibiotic exposure. Although organic fertilizers are regarded as eco-friendly compared to chemical fertilizers for improving soil fertility, the application of untreated animal manure could promote the accumulation of antibiotics and their residues and antibiotic-resistant bacteria in the soil, creating an environmental reservoir of antimicrobial resistance, with potential human and environmental health risks.

Antimicrobial resistance profiles of Enterobacter cloacae and Klebsiella aerogenes a tertiary hospital in Turkey: A five-years study

Objective: Enterobacter cloacae and Klebsiella aerogenes species have multiple drug resistance and antibiotic resistance is a growing problem regarding to treating infections.&#x0D; Objective: The aim of this study was to determine and evaluate the antimicrobial resistance profiles of E. cloacae and K. aerogenes isolated from various clinical samples in our laboratory, retrospectively.&#x0D; Material and Methods: Totally 223 patients who applied to Karabuk University Training and Research Hospital microbiology laboratory between October2016-December2020 were included in this study. Conventional methods and automated systems were used for the identification and antibiotic susceptibilities of strains. Antibiotic susceptibility results were evaluated as per the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.&#x0D; Results: Total of 223 clinical samples (urine 68.6%, blood 12.6%, endotracheal aspirate 7.2%, wound 4.9%, sputum 3.6%, bronchoalveolar lavage fluid 2.7%, and ear fluid 0.4%) obtained from 223 patients; 119 (53%) females and 104 (47%) females, were analysed. The identified species were E. cloacae (132 strains, 59.2%) and K. aerogenes (91 strains, 40.8%). The Enterobacter cloacae and Klebsiella aerogenes positivity was detected as 30(13.4%) and 20(9.0%) in the samples. The highest resistance was found against cefixime at a rate of 60%; the lowest resistance was against amikacin, meropenem and imipenem ranged between 3% and 4% in both E. cloacae and K. aerogenes strains.&#x0D; Conclusions: Amikacin, imipenem and meropenem were the most effective antibiotics against E. cloacae and K. aerogenes. We may prefer TMP-SMX and ciprofloxacin, as oral antibiotic agents in the treatment of E. cloacae/K. aerogenes infections. Amikacin, gentamicin and carbapenems may be the first choice for parenteral antibiotics therapy

Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

Antibiotic Resistance in Pseudomonas aeruginosaIsolated from Hospital and Community-Acquired Infections: Current Status in Northern Cyprus

Objective: Pseudomonas aeruginosa is among the most common nosocomial opportunistic pathogens capable of acquiring multi-drug resistance. Recent data on antibiotic resistance in Northern Cyprus is limited. We aimed evaluate antibiotic susceptibility profiles of P. aeruginosa strains isolated from clinical samples and contribute to the update of antibiotic use policies in the country. Methods: Total of 504 P. aeruginosa strains sent to Microbiology Laboratory from various clinics from September 2016 to October 2019 were retrospectively analyzed. Clinical samples were cultured on %5 sheep blood agar and eosin-methylene blue agar. BD Phoenix™ 100 (Becton Dickinson Co., Sparks, Maryland, USA) automated bacterial identification system and conventional antimicrobial susceptibility testing was used. Antimicrobial susceptibility was determined in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The distribution of P. aeruginosa isolates according to hospital services, sample types, admission types, age group, and antibiotic resistance rates were investigated. Results: P. aeruginosa was mostly isolated from urine (37.3%), deep tracheal aspirate (17.9%) and abscess/wound material (17.9%) samples. Strains were most commonly obtained from the intensive care clinics (35.9%). Infections were observed mainly in &gt;65 age group (49.6%) and in-patients (70.8%) group. Antibiotic resistance rates were detected as 47.7% for aztreonam, 32% for cefepime, 26.3% for ceftazidime, 26.4% for imipenem, 25.6% for ciprofloxacin, 24.6% for meropenem, 19.7% for piperacillin-tazobactam, 13.5% for gentamicin, 12.2% for colistin and 5.3% for amikacin. Multi-drug resistant P. aeruginosa rate was 19.2%. When compared with 2010-2014, a substantial increase in rates of cefepime, ceftazidime, ciprofloxacin, piperacillin-tazobactam and carbapenem resistance was detected within the last three years. Conclusions: In this study, the highest resistance rates were detected against aztreonam and cefepime, and the highest susceptibility was observed for colistin and amikacin. These results may be used for empirical treatments, establishment of rational antibiotic use policies and prevention of emerging antibiotic resistance problem in Cyprus.

Detection of Extended spectrum beta lactamases in gram negative isolates causing urinary tract infection in Tertiary care centre

Introduction: Urinary Tract Infections (UTI’s) is the most commonly encountered infectious disease, affecting both the sexes in all age groups. This leads to consumption of large number of antibiotics and development of resistant strains leading to complications. Objective: The study was carried out to know the distribution pattern and to evaluate the sensitivity of ESBL producing urinary isolates from patients with UTI. Materials and Methods: A cross sectional study was conducted at Owaisi Hospital and Research Centre for 6months. A total of 392 samples were obtained from suspected UTI patients. Clean catch mid stream urine samples obtained were subjected to microscopy, culture and antibiotic sensitivity testing as per CLSI guidelines. Results: From the total of 392 samples, 200 samples i.e. 51% samples were culture positive. Among the culture positive cases, 69% were females, 35% were males. 38.8% of E.coli and 25% Klebsiella were predominant ESBL producers. Conclusion: This study revealed that majority of Gram negative urinary isolates are ESBL producers, and multidrug resistant. Thus strict policy with guidelines for antibiotic susceptibility testing and prescription should be followed to prevent further emergence of resistant strains and treatment failure. Keywords: Mid stream urine, Extended Spectrum Beta Lactamases, antibiotic susceptibility pattern.

Updated EUCAST Clinical Breakpoints against Aspergillus, Implications for the Clinical Microbiology Laboratory.

Azole resistance poses a problem for the management of patients with invasive aspergillosis. Former species are in fact groups of closely related species (or complexes); cryptic species frequently show high antifungal resistance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document (E.Def) 9.3.2 includes guidelines for antifungal susceptibility testing on Aspergillus spp. and clinical breakpoints for amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole against A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreus. New clinical breakpoints were released in February 2020 and one of the most relevant modifications was the definition of the new “susceptible, increased exposure” (formerly “intermediate”) category. Another relevant change was the adoption of the concept of area of technical uncertainty (ATU) that refers to problematic areas which involve uncertainty of susceptibility categorisation (e.g., when minimum inhibitory concentrations (MICs) for susceptible and resistant organisms overlap). To accommodate both the new “susceptible, increased exposure” category and the concept of ATU, MICs of azoles and amphotericin B that fall in the former “intermediate” category have been automatically categorized as either R (amphotericin B) or ATU (triazoles). Finally, EUCAST-AFST (Antifungal Susceptibility Testing) decided to adopt new breakpoints for less common species provided that the epidemiological cut-off value (ECOFF) is below or comparable to the breakpoint for the type species (A. fumigatus).

Antimicrobial Susceptibility Patterns and Wild-Type MIC Distributions of Anaerobic Bacteria at a German University Hospital: A Five-Year Retrospective Study (2015-2019).

Local antimicrobial susceptibility surveys are crucial for optimal empirical therapy guidelines and for aiding in antibiotic stewardship and treatment decisions. For many laboratories, a comprehensive overview of local antimicrobial susceptibility patterns of anaerobic bacteria is still lacking due to the long incubation time and effort involved. The present study investigates the antimicrobial susceptibility patterns and related clinical and demographic data of 2856 clinical isolates of anaerobic bacteria that were submitted for analysis to the Institute for Medical Microbiology and Hygiene of the Freiburg University Medical Center (a tertiary university medical center in Southern Germany) between 2015 and 2019. Antimicrobial susceptibility testing has been carried out according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guideline. Minimum inhibitory concentration (MIC)50 and MIC90 for penicillin, metronidazole, moxifloxacin, and clindamycin were established for Gram-positive anaerobes and for ampicillin-sulbactam, meropenem, metronidazole, moxifloxacin, and clindamycin for Gram-negative anaerobes. The distribution of MIC-values for various antibiotics against anaerobic bacteria was also established, especially for those having no specific breakpoints according to EUCAST guidelines. Most clinically relevant anaerobic bacteria originated from general surgery, neurological, and orthopedic wards. A high proportion of isolates were resistant to moxifloxacin and clindamycin indicating the importance of their susceptibility testing before administration. Based on our study metronidazole and other β-lactam/β-lactamase inhibitor combinations such as ampicillin-sulbactam remain suitable for empirical treatment of infections with anaerobic bacteria.

Implementation of rapid antimicrobial susceptibility testing combined with routine infectious disease bedside consultation in clinical practice (RAST-ID): a prospective single-centre study.

Recently, EUCAST released guidelines for rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles. The aim of our prospective single-centre clinical study was to assess the proportion of readable results and errors compared with routine antimicrobial susceptibility testing and the clinical consequences drawn by infectious disease (ID) physicians from RAST results during same-day bedside consultation. All positive blood cultures suitable for RAST from January to December 2019 were included and RAST results at 4 and 6 h compared with standard disc diffusion. The real-life impact of RAST on clinical decisions was assessed during same-day ID bedside consultation. The proportion of readable RAST results was significantly higher after 6 h of incubation compared with after 4 h (881/930 versus 642/847; P < 0.0001). Major and very major errors were rare (17/642 after 4 h and 12/881 after 6 h; P = 0.087). ID consultation was performed in 134 patients after the RAST result. Antimicrobial treatment was changed in 73 patients and 84 additional measures (i.e. imaging studies, surgery, additional resistance testing) were ordered in 62 patients. RAST according to EUCAST methods was easy to implement with a low number of major and very major errors after 6 h of incubation. ID physicians performing bedside consultations frequently used this information to change antimicrobial treatment and recommended additional measures.

Klebsiella pneumoniae Infections in the Intensive Care Unit: Risk Factors Related to Carbapenem Resistance and Patient Mortality

Klebsiella pneumoniae is the cause of complicated and difficult-to-treat nosocomial infections such as sepsis, urinary tract infection, catheter related infections, pneumonia and surgical site infections in intensive care units. The biggest problem in infections with K.pneumoniae is that treatment options are limited due to multiple antibiotic resistance and consequently the increased morbidity and mortality. The widespread and improper use of carbapenems can lead to epidemics that are difficult to control, especially in intensive care units because of the acquired resistance to this group of antibiotics. Outbreaks and sporadic cases caused by carbapenem resistant K.pneumoniae (CRKP) species have been reported all over the world in recent years with increased frequency. The aim of this study was to determine the risk factors related to carbepenem resistance and mortality caused by K.pneumoniae infections in a university hospital anesthesia intensive care unit. The study was conducted between January 1st, 2016, and December 31st, 2018. Retrospective data were obtained from the patient and laboratory-based surveillance records. Adult patients (≥ 18 years) with K.pneumoniae growth in the blood, urine, abscess and tracheal aspirate samples collected 48 hours after admission to the intensive care unit were considered as the relevant infection locus-related agent and treated with antibacterial therapy. Clinical samples collected from patients were inoculated onto 5% sheep blood and eosin-methylene-blue (EMB) agar except the blood samples. Blood samples were cultured in blood culture bottles and incubated in an automated system. Gram staining was performed for the samples showing growth signal within five days and then inoculated onto 5% sheep blood and EMB agar media and were incubated for 18-24 hours at 35.5-37°C. Identification of the isolates was performed using Bruker IVD MALDI Biotyper 2.3 (Bruker Daltonik GmbH, Bremen, Almanya) based on "matrix-assisted laser desorption/ionization time-of-mass spectrometry (MALDI-TOF MS)". K.pneumoniae isolates were identified by obtaining reliability scores of 2.0 and above in the study. Antibiotic susceptibility tests were performed with Phoenix 100 (BD, New Jersey, ABD) automated system. Interpretations were made according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Combination disk diffusion test and polymerase chain reaction based tests were used to show the presence of carbapenemase in CRKP isolates. A total of 88 patients with K.pneumoniae infection were included in the study. The mean age of the patients was 74 ± 15 (range= 21-93) years and 60.2% were female. CRKP was detected in 32 patients (36.4%) and carbapenem-sensitive K.pneumoniae (CSKP) was detected in 56 patients. The presence of OXA-48 was found to be 68.8% in the carbapenem screening test performed by combination disc method in patients with CRKP. Multivariate logistic regression analysis showed that previous use of colistin [Odds ratio (OR)= 19.108; 95% confidence interval (CI)= 2.027-180.133; p= 0.010] and aminoglycoside (OR= 12.189; 95% CI= 1.256-118.334; p= 0.031) was an independent risk factor in terms of CRCP among the patients with K.pneumoniae infection. The 28-day mortality rates were 71.9% in the CRKP group (23/32) and 37.5% in the CSKP group (21/56). Presence of CRKP in terms of 28-day mortality (OR= 5.146; 95% CI= 1.839-14.398; p= 0.002) was an independent risk factor. The data obtained in this study will guide for conducting effective and continuous surveillance studies and implementing rational antibiotic programs to prevent the increase in CRKP.