Emergence and persistent spread of carbapenemase-producing Klebsiella pneumoniae high-risk clones in Greek hospitals, 2013 to 2022.

BackgroundPreliminary unpublished results of the survey of carbapenem- and/or colistin-resistant Enterobacterales (CCRE survey) showed the expansion of carbapenemase-producing Klebsiella pneumoniae (CPKP) sequence type (ST) 39 in 12 of 15 participating Greek hospitals in 2019.AimWe conducted a rapid survey to determine the extent of spread of CPKP high-risk clones in Greek hospitals in 2022 and compare the distribution of circulating CPKP clones in these hospitals since 2013.MethodsWe analysed whole genome sequences and epidemiological data of 310 K. pneumoniae isolates that were carbapenem-resistant or 'susceptible, increased exposure' from Greek hospitals that participated in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE, 2013-2014), in the CCRE survey (2019) and in a national follow-up survey (2022) including, for the latter, an estimation of transmission events.ResultsFive K. pneumoniae STs including ST258/512 (n = 101 isolates), ST11 (n = 93), ST39 (n = 56), ST147 (n = 21) and ST323 (n = 13) accounted for more than 90% of CPKP isolates in the dataset. While ST11, ST147 and ST258/512 have been detected in participating hospitals since 2013 and 2014, KPC-2-producing ST39 and ST323 emerged in 2019 and 2022, respectively. Based on the defined genetic relatedness cut-off, 44 within-hospital transmission events were identified in the 2022 survey dataset, with 12 of 15 participating hospitals having at least one within-hospital transmission event.ConclusionThe recent emergence and rapid spread of new high-risk K. pneumoniae clones in the Greek healthcare system related to within-hospital transmission is of concern and highlights the need for molecular surveillance and enhanced infection prevention and control measures.

Analytical Performance and Potential Clinical Utility of EUCAST Rapid Antimicrobial Susceptibility Testing in Blood Cultures after Four Hours of Incubation.

EUCAST rapid antimicrobial susceptibility testing (RAST) provides antibiotic susceptibility results after 4 to 8 h of incubation. This study assessed the diagnostic performance and clinical usefulness of EUCAST RAST after 4 h. This was a retrospective clinical study performed on blood cultures with Escherichia coli and Klebsiella pneumoniae complex (K. pneumoniae and Klebsiella variicola) at Karolinska University Laboratory (Stockholm, Sweden). The rate of categorized RAST results and the categorical agreement (CA) of RAST with the standard EUCAST 16-to-20-h disk diffusion (DD) method for piperacillin-tazobactam, cefotaxime, ceftazidime, meropenem, and ciprofloxacin were analyzed, as well as the utility of RAST for adjusting the empirical antibiotic therapy (EAT) and the combination of RAST with a lateral flow assay (LFA) for extended-spectrum ß-lactamase (ESBL) detection. A total of 530 E. coli and 112 K. pneumoniae complex strains were analyzed, generating 2,641 and 558 readable RAST zones, respectively. RAST results categorized according to antimicrobial sensitivity/resistance (S/R) were obtained for 83.1% (2,194/2,641) and 87.5% (488/558) of E. coli and K. pneumoniae complex strains, respectively. The RAST result categorization to S/R for piperacillin-tazobactam was poor (37.2% for E. coli and 66.1% for K. pneumoniae complex). CA with the standard DD method was over 97% for all tested antibiotics. Using RAST, we detected 15/26 and 1/10 of the E. coli and K. pneumoniae complex strains that were resistant to the EAT. For patients treated with cefotaxime, RAST was used to detect 13/14 cefotaxime-resistant E. coli strains and 1/1 cefotaxime-resistant K. pneumoniae complex strain. ESBL positivity was reported the same day as blood culture positivity with RAST and LFA. EUCAST RAST provides accurate and clinically relevant susceptibility results after 4 h of incubation and can accelerate the assessment of resistance patterns. IMPORTANCE Early effective antimicrobial treatment has been shown to be crucial for improving the outcome of bloodstream infections (BSI) and sepsis. In combination with the rise of antibiotic resistance, this calls for accelerated methods for antibiotic susceptibility testing (AST) for effective treatment of BSI. This study assesses EUCAST RAST, an AST method that yields results in 4, 6, or 8 h after blood culture positivity. We analyzed a high number of clinical samples of Escherichia coli and Klebsiella pneumoniae complex strains and confirm that the method delivers reliable results after 4 h of incubation for the relevant antibiotics for treating E. coli and K. pneumoniae complex bacteremia. Furthermore, we conclude that it is an important tool for antibiotic treatment decision-making and early detection of ESBL-producing isolates.

[Precision diagnostics in clinical microbiology]. / Bred gensekvensering ger ökad säkerhet i infektionsdiagnostiken.

Genomic methods have had a major impact in clinical microbiology in the last decades. Microbial genomes are relatively small and therefore easier to characterise than human genomes. In both bacteriology and in virology, genomic methods have largely been used for molecular epidemiology, but also for molecular resistance testing of microorganisms. Targeted sequencing of predefined or isolated microorganisms was initially a dominant method but has gradually been supplemented with metagenomic diagnostics. Metagenomics aims at mapping all microorganisms - pathogenic as well as apathogenic - in a sample without determining in advance which agent(s) the analysis is targeting. Finally, there is also an increasing interest in mapping the significance of the microbiome, i.e. normal flora, both in health and disease.

Extended-Spectrum-ß-Lactamase- and Plasmid AmpC-Producing Escherichia coli Causing Community-Onset Bloodstream Infection: Association of Bacterial Clones and Virulence Genes with Septic Shock, Source of Infection, and Recurrence.

Invasive infections due to extended-spectrum-ß-lactamase- and pAmpC-producing Escherichia coli (ESBL/pAmpC-EC) are an important cause of morbidity, often caused by the high-risk clone sequence type (ST131) and isolates classified as extraintestinal pathogenic E. coli (ExPEC). The relative influence of host immunocompetence versus microbiological virulence factors in the acquisition and outcome of bloodstream infections (BSI) is poorly understood. Herein, we used whole-genome sequencing on 278 blood culture isolates of ESBL/pAmpC-EC from 260 patients with community-onset BSI collected from 2012 to 2015 in Stockholm to study the association of virulence genes, sequence types, and antimicrobial resistance with severity of disease, infection source, ESBL/pAmpC-EC BSI low-risk patients, and patients with repeated episodes. ST131 subclade C2 comprised 29% of all patients. Factors associated with septic shock in multivariable analysis were patient host factors (hematologic cancer or transplantation and reduced daily living activity), presence of the E. coli virulence factor iss (increased serum survival), absence of phenotypic multidrug resistance, and absence of the genes pap and hsp Adhesins, particularly pap, were associated with urinary tract infection (UTI) source, while isolates from post-prostate biopsy sepsis had a low overall number of virulence operons, including adhesins, and commonly belonged to ST131 clades A, B, and subclade C1, ST1193, and ST648. ST131 was associated with recurrent episodes. In conclusion, the most interesting finding is the association of iss with septic shock. Adhesins are important for UTI pathogenesis, while otherwise low-pathogenic isolates from the microbiota can cause post-prostate biopsy sepsis.

Prediction of bloodstream infection caused by extended-spectrum ß-lactamase-producing Enterobacterales in patients with suspected community-onset sepsis.

OBJECTIVES: In severe infections, time to appropriate therapy is decisive for survival. Patients with bloodstream infection caused by extended-spectrum ß-lactamase-producing Enterobacterales (EPE-BSI) often receive inadequate empirical treatment. This study aimed to identify risk factors, to evaluate a previously suggested risk score and to suggest a new score for facilitating empirical treatment choice. METHODS: Predictors for EPE-BSI were assessed through a retrospective case-control design. The diagnostic performance of the two scores was evaluated. Included patients had blood cultures sampled at four EDs in Stockholm (2012-2015), were admitted, and received antibiotics with activity against Gram-negative bacilli. RESULTS: A total of 277 EPE-BSI cases and 400 controls were included. The strongest predictor of EPE-BSI was prior EPE-positive culture (cases 33% vs. controls 3%; multivariate (MV) OR = 19.1). Recent EPE-positivity within ≤3 months had a univariate OR of 32.8. Other major predictors were recent prostate biopsy (14% vs. 1%; MV OR = 22.2) and healthcare abroad (6% vs. 2%; MV OR = 3.9). Several previously suggested risk factors were not associated with EPE-BSI. The previously developed Utrecht score had a sensitivity of 54% and a specificity of 77%. The Stockholm score suggested herein (prior EPE-positive culture/prostate biopsy/healthcare abroad) showed comparable sensitivity (50%) but better specificity (96%). Prediction in patients lacking major predictors was difficult and caused high false-positive rates, which would cause unnecessary overtreatment. CONCLUSIONS: Prior EPE-positive culture, especially recently sampled, prostate biopsy and healthcare abroad were the strongest risk factors for community-onset EPE-BSI in Stockholm. Local data are needed when evaluating risk-scoring models before implementation.

USA300 methicillin-resistant Staphylococcus aureus in Stockholm, Sweden, from 2008 to 2016.

Methicillin-resistant Staphylococcus aureus (MRSA) USA300 isolates have been recognized globally, not only in community but also in healthcare settings. USA300 isolates were initially resistant only to methicillin, but resistance to non-ß-lactams has emerged with time. To evaluate the prevalence and antimicrobial susceptibility of USA300 isolates in Stockholm, we conducted a nine-year retrospective study. Of 5359 consecutive MRSA cases in Stockholm, isolates from 285 cases were USA300 strains according to the pulsed-field gel electrophoresis pattern. Of these cases, repeated isolates with altered antibiotic resistance patterns were observed in six individuals. Therefore, antimicrobial susceptibility testing was performed on totally 291 isolates. To study the phylogenetic relatedness of isolates in transmission events and genomic resistance traits, 35 isolates were further studied by whole genome sequencing (WGS). The incidence of MRSA was increased from 17.6 per 100,000 inhabitants in 2008 to 37.3 per 100,000 inhabitants in 2016, while the proportion of USA300 cases declined from 6.6% in 2008 to 2.6% in 2016. Among the USA300 isolates, 73.5% were community-associated, 21.3% healthcare-associated, and 5.2% had unknown acquisition. The highest resistance rate among non-ß-lactams was found in erythromycin (86%), followed by fluoroquinolones (68-69%). 57% of the isolates were resistant to both erythromycin and fluoroquinolone. Simultaneous resistance to four non-ß-lactam antibiotic classes was found in six isolates. Four isolates were susceptible to all non-ß-lactam antibiotics. Ceftaroline, daptomycin, linezolid, mupirocin, rifampicin, teicoplanin, telavancin, trimethoprim-sulfamethoxazole and vancomycin retained full activity in the study. WGS analysis indicated that isolates from an outbreak were phylogenetically closely related. In conclusion, USA300 MRSA isolates in Stockholm have neither been limited to the community setting, nor remained susceptible to non-ß-lactam agents. WGS is becoming a useful tool in tracing transmission events. The results herein provide the most up-to-date and comprehensive information regarding status of USA300 strains in this geographic area.

Rapid EUCAST disc diffusion testing of MDR Escherichia coli and Klebsiella pneumoniae: inhibition zones for extended-spectrum cephalosporins can be reliably read after 6†h of incubation.

Objectives: The need for rapid antibiotic susceptibility testing increases with escalating levels of antimicrobial resistance in Enterobacteriaceae. Our objective was to evaluate the accuracy of reading EUCAST disc diffusion, ROSCO ESBL and carbapenemase detection kits and the Mast Carbapenemase Activity Test (CAT-ID) disc, after 6 h of incubation. Methods: We used a collection of 128 isolates of Escherichia coli and Klebsiella pneumoniae with a wide variety of resistance mechanisms. Inhibition zones read from digital photo images with the BD Kiestra™ Total Lab Automation System after 6 h of incubation were compared with standard reading, after 18 h, of the same Mueller-Hinton agar plates. Results: For WT isolates, zones were generally smaller at 6 h than at 18 h. Cefotaxime had excellent categorical agreement of 99%, despite the high number of challenge isolates. However, for some other antimicrobials, hetero-resistant subpopulations were commonly invisible at 6 h, which resulted in an unacceptable number of errors when using standard EUCAST breakpoints. Accurate ESBL detection was possible at 6 h for isolates lacking other ß-lactamases. Carbapenemase detection was not reliable after 6 h. Conclusions: Inhibition zone reading at 6 h is an accurate method for susceptibility testing of extended-spectrum cephalosporins for Enterobacteriaceae. For other antimicrobials, 6 h reading can be used for preliminary reports of clearly resistant or susceptible isolates, preferably with application of adjusted breakpoints including an area of uncertainty between susceptible and resistant values.

Methicillin-resistant Staphylococcus aureus in Stockholm, Sweden: Molecular epidemiology and antimicrobial susceptibilities to ceftaroline, linezolid, mupirocin and vancomycin in 2014.

Methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. The aim of the present study was to investigate the molecular epidemiology and antimicrobial susceptibilities of MRSA strains in Stockholm, Sweden in 2014. Pulsed-field gel electrophoresis (PFGE) was used to characterise the strains. Antimicrobial susceptibilities to ceftaroline, linezolid and mupirocin were determined by the disc diffusion method. Etest was used to determine vancomycin susceptibility and to confirm resistance to ceftaroline, mupirocin and linezolid in non-susceptible strains. High-level ceftaroline-resistant strains [minimum inhibitory concentration (MIC)≥4mg/L] were confirmed by the broth microdilution method. spa typing was carried out on strains that were non-susceptible to the antibiotics tested. In total, 743 consecutive non-duplicate MRSA strains recovered in Stockholm in 2014 were investigated. PFGE analysis of the isolates revealed a population with 271 different PFGE patterns and three non-typeable strains. No PFGE type accounted for >10% of all strains. The most common PFGE types were MRSA-00-02 (6.9%) and MRSA-05-02 (4.6%). MRSA-05-02 is a USA300-like strain. The antimicrobial susceptibilities of the strains were as follows: ceftaroline, 98.5%; linezolid, 100%; mupirocin, 99.3%; and vancomycin, 100%. Two strains with spa t001 displayed ceftaroline MICs of 4mg/L. Three strains with spa types t002, t064 and t437 showed high-level mupirocin resistance (MIC>1024mg/L). In conclusion, there was a diverse genetic population among the MRSA isolates and no predominant genotype was found. This study identified a few strains with high-level ceftaroline resistance, high-level mupirocin resistance and high-risk genotypes.

[Vancomycin resistant Staphylococcus epidermidis caused prosthesis infection. Linezolid and rifampicin healed the complicated infection]. / Vankomycin-resistent Staphylococcus epidermidis gav protesinfektion - Linezolid och rifampicin läkte den komplicerade infektionen.

Coagulase-negative Staphylococci, including Staphylococcus epidermidis, are common pathogens in orthopedic prosthesis infections. Operation and prolonged treatment with rifampicin in combination with another antibiotic is often required. Coagulase-negative Staph-ylococci are frequently multi-resistant, but resistance to vancomycin is rare in Sweden. Linezolid is an alternative, however it is only recommended for up to 4 weeks treatment due to risk of hematological side effects. We have successfully used prolonged treatment with linezolid and rifampicin in a patient suffering from a complicated prosthetic joint infection caused by a vancomycin resistant Staphyloccous epidermidis strain.

Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom.

Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of bla(NDM-1).

Pneumonia and bacteremia due to Kytococcus schroeteri.

Kytococcus schroeteri, a saprophyte of the human skin, may cause serious infections in the immunocompromised host. Here, we describe a case of pneumonia and bacteremia due to Kytococcus schroeteri in an immunocompromised patient, successfully treated with linezolid and trimethoprim-sulfamethoxazole.