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Resumen Introduccion El objetivo de este estudio es la caracterizacion de cepas de Streptococcus pyogenes con fenotipo mucoide y su comparacion con las cepas no mucoides aisladas entre abril y agosto de 2016. Material y metodos Se llevo a cabo la caracterizacion y el estudio de sensibilidad antimicrobiana de todos los aislados. Se determino el tipo emm y se analizaron los genes de exotoxinas speA, speB, speC, speF, speG, speH, speJ, speZ y ssa. Se recogieron datos clinicos y demograficos. Resultados De 96 aislados analizados, el 47% presentaron un fenotipo mucoide, y de estos ultimos, el 95,5% presentaron los genes speA-speB-speF-speG-ssa y genotipo emm3. La principal manifestacion clinica entre todos los pacientes fue faringoamigdalitis (77,1%) que evoluciono a escarlatina en el 67,5% de los casos. Conclusion Se describe la circulacion de una cepa de aspecto mucoide con perfil de toxinas speA-speB-speF-speG-ssa y genotipo emm3.1 considerado de los mas frecuentes y mas virulentos de SGA.

Increased relative bacterial load of KPC-producing Klebsiella pneumoniae (KPC-KP) within the intestinal microbiota has been associated with KPC-KP bacteremia. Prospective observational study of KPC-KP adult carriers with a hospital admission at recruitment or within the three prior months (January 2018 to February 2019). A qPCR-based assay was developed to measure the relative load of KPC-KP in rectal swabs (RLKPC, proportion of blaKPC relative to 16S rRNA gene copy number). We generated Fine-Gray competing risk and Cox regression models for survival analysis of all-site KPC-KP infection and all-cause mortality, respectively, at 90 and 30 days. The median RLKPC at baseline among 80 KPC-KP adult carriers was 0.28% (range 0.001% to 2.70%). Giannella Risk Score (GRS) was independently associated with 90-day and 30-day all-site infection (adjusted subdistribution hazard ratio [aHR] 1.23, 95% CI = 1.15 to 1.32, P 7, aHR 2.96, 95% CI = 0.97 to 9.07, P = 0.057). KPC-KP relative intestinal load was independently associated with all-cause mortality in our clinical setting, after adjusting for age and severe KPC-KP infection. Our study confirms the utility of GRS to predict infection risk in patients colonized by KPC-KP., This work was supported by: (i) Plan Estatal de I+D+I 2013-2016, cofinanced by the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación and the Fondo Europeo de Desarrollo Regional-FEDER (FIS PI16/01631-KLEBCOM granted to E.P.N.); (ii) Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, and cofinanced by the European Development Regional Fund “A Way to Achieve Europe” Operative Program Smart Growth 2014–2020, Spanish Network for Research in Infectious Diseases (REIPI) (RD16/0016/0008); and (iii) Consejería de Salud y Familias, Junta de Andalucía (RH-0065-2020 granted to E.P.N.).

Objectives To detect a potential hidden dissemination of the blaOXA-48 gene among Proteus mirabilis isolates obtained from a single centre. Methods P. mirabilis from diverse clinical samples presenting an ESBL phenotype or obtained from blood cultured from 2017 to 2019 were evaluated. Bacterial identification was performed using MALDI-TOF MS. MICs were determined using International Organization for Standardization (ISO) standard microdilution and interpreted following EUCAST guidelines. WGS was performed using both short- and long-read technologies and assemblies were done using Unicycler. Resistomes were assessed using the ResFinder database. SNPs were detected using the PATRIC bioinformatics platform. Cloning experiments were performed using the pCRII-TOPO cloning kit. Results Thirty-one out of 108 (28.7%) isolates were positive for blaOXA-48 and blaCTX-M-15. Twenty-nine out of 31 of the isolates were susceptible to temocillin, piperacillin/tazobactam, ertapenem and meropenem, whereas only 2/31 showed a resistance phenotype against these antibiotics. Both blaOXA-48 and blaCTX-M-15 genes were detected within the same chromosomally integrated new transposon in all isolates. The resistant isolates displayed a single mutation located in the putative promoter upstream of blaOXA-48. Cloning experiments confirmed that the mutation was responsible for the resistance phenotype. Conclusions The presence of a chromosomal copy of blaOXA-48 did not confer resistance to carbapenems, but a single mutation in the promoter could lead to an increase in resistance. This study shows a hidden circulation of OXA-48-positive, but carbapenem- and piperacillin/tazobactam-susceptible, P. mirabilis isolates that can become resistant to β-lactams after a single mutation.

We gratefully acknowledge the collaboration of the Unit of Microbiology and the Maimonides Biomedical Research Institute of Cordoba (IMIBIC)., Multidrug-resistant Gram-negative bacteria (MDR-GNB) are microorganisms that have acquired resistance to extended-spectrum antibacterials and constitute an emerging threat to public health. Although carriers are an important source of transmission in healthcare settings, data about risk factors for MDR-GNB carriage are limited. Therefore, we aimed to identify risk factors for MDR-GNB carriage upon intensive care unit (ICU) admission and to optimise screening strategies. We conducted a case–control study. Admissions of adult patients to the ICU of a 1000-bed hospital during a year were included. We collected sociodemographic, clinical and microbiological data and performed a multivariate logistic regression model. A total of 1342 patients resulted in 1476 episodes of ICU admission, 91 (6.2%) of whom harboured MDR-GNB (38.5% women; median age 63.9 years). The most frequently isolated pathogens were Escherichia coli (57%) and Klebsiella pneumoniae (16%). The most frequent resistance mechanism was production of extended-spectrum beta lactamases. MDR-GNB carriage was associated to liver cirrhosis (OR 6.54, 95% CI 2.17–19.17), previous MDRGNB carriage (OR 5.34, 1.55–16.60), digestive surgery (OR 2.83, 1.29–5.89) and length of hospital stay (OR 1.01 per day, 1.00–1.03). Several risk factors for MDR-GNB carriage upon admission to a high-risk setting were identified; the main comorbidity was liver cirrhosis.

[Objectives] To monitor quantitatively the extent of intestinal colonisation by KPC-producing Klebsiella pneumoniae (KPC-Kp) in colonised patients who receive selective digestive decontamination (SDD) with oral gentamicin., [Methods] We developed a real-time quantitative PCR (qPCR) method for determination of the relative load of blaKPC (RLKPC) within the gut microbiota. Clinical validation was performed using a culture method as the gold standard and receiver operating curve (ROC) analysis. Fifteen patients were observationally and prospectively followed for one year. Clinical, microbiological variables and rectal swab samples were collected at 0 (baseline), 14 and 30 days and monthly thereafter., [Results] Clinical validation performed on 111 rectal swab samples demonstrated that the PCR method detected 17% more positives than the culture method. ROC curve analysis documented excellent agreement between both methods (area under the curve, 0.96; 95% confidence interval 0.93–0.99). The RLKPC decreased in 6/15 (40%) and 7/12 (58.3%) patients on days 14 and 30, respectively. Persistent eradication was observed in 2/12 (16.7%), 3/9 (33.3%), 4/8 (50%) and 7/8 (87.5%) patients at 1, 3, 6 and 12 months, respectively, with a median time of 150 days (range 30–270) to persistent eradication. Gentamicin-resistant KPC-Kp isolates were identified in 4/15 (26.7%) patients. The rates of infections (57.1% vs. 12.5%, P = 0.119) and deaths (71.4% vs. 0%, P = 0.007) were higher among patients with high baseline RLKPC., [Conclusion] Following SDD, a rapid reduction on intestinal load is observed when the colonising KPC-Kp isolate is susceptible to gentamicin; however, persistent eradication at the end of SDD is low. Intestinal carriage of KPC-Kp persists after three months in about one third of patients., This work was supported by research funds ‘FIS PI16/01631’ granted to EPN from Plan Estatal de I+D+I 2013-2016, co-financed by the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación and the Fondo Europeo de Desarrollo Regional (FEDER); and Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0008) ‐ co‐financed by European Development Regional Fund ‘A way to achieve Europe’, operative program Intelligent Growth 2014–2020. EPN holds a research contract from Consejería de Salud y Familias, Junta de Andalucía (RH-0065-2020I).

This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser deso...

IntroductionInfections caused by carbapenemase-producing Enterobacterales are frequent and associated with high rates of mortality. Intestinal carriers are at increased risk of infection by these microorganisms. Decolonisation strategies with antibiotics have not obtained conclusive results. Faecal microbiota transplantation (FMT) could be an effective and safe strategy to decolonise intestinal carriers of KPC-producing Klebsiella pneumoniae (KPC-Kp) but this hypothesis needs evaluation in appropriate clinical trials.Methods and analysisThe KAPEDIS trial is a single-centre, randomised, double-blind, placebo-controlled, phase 2, superiority clinical trial of FMT for eradication of intestinal colonisation by KPC-Kp. One hundred and twenty patients with rectal colonisation by KPC-Kp will be randomised 1:1 to receive encapsulated lyophilised FMT or placebo. The primary outcome is KPC-Kp eradication at 30 days. Secondary outcomes are: (1) frequency of adverse events; (2) changes in KPC-Kp relative load within the intestinal microbiota at 7, 30 and 90 days, estimated by real-time quantitative PCR analysis of rectal swab samples and (3) rates of persistent eradication, KPC-Kp infection and crude mortality at 90 days. Participants will be monitored for adverse effects throughout the intervention.Ethics and disseminationEthical approval was obtained from Reina Sofía University Hospital Institutional Review Board (approval reference number: 2019-003808-13). Trial results will be published in peer-reviewed journals and disseminated at national and international conferences.Trial registration numberNCT04760665.

There is very limited information about OXA-48-producing Klebsiella oxytoca. The aim of this study was to describe the phenotypic and molecular characterization of OXA-48-producing K. oxytoca isolates that caused an outbreak in a hospital in Tunisia. Nineteen OXA-48-producing K. oxytoca were isolated from 2013 to 2016 in the University Hospital Farhat Hached, Sousse, Tunisia. Antibiotic susceptibility testing was performed by broth microdilution. Carbapenemase activity was investigated using the modified carbapenem inactivation method (mCIM). Phenotypic tests were also carried out to detect extended-spectrum β-lactamases. PCR was used to test for the presence of carbapenemase genes (blaIMP, blaVIM, blaNDM, blaSPM, blaAIM, blaDIM, blaGIM, blaSIM, blaKPC, blaBIC and blaOXA-48). Genetic relatedness among isolates was investigated using rep-PCR. Whole genome sequencing (WGS) was performed in three representative isolates. mCIM was positive in all isolates. None of the isolates presented an ESBL phenotype. All strains were susceptible to cefoxitin, ceftazidime, cefepime, aztreonam, imipenem, meropenem, fluoroquinolones, aminoglycosides and colistin, and resistant to piperacillin-tazobactam, ertapenem, ticarcillin and ampicillin-sulbactam. All isolates presented the blaOXA-48 gene located in a ca. 63 kb IncL plasmid, which carried no additional resistance genes. They belonged to the new ST220. Isolates from this study did not co-express an ESBL, which could complicate their detection in clinical laboratories. As OXA-48 has been mostly reported in K. pneumoniae there is a risk that the production of this enzyme is not suspected in the less common species K. oxytoca. These difficulties could play an important role in the hidden spread of this enzyme. This research was funded in part by grants PI14/01911 (to L.M.-M.), REIPI RD16/0016/0008 and REIPI RD16CIII/0004/0002, all from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, and co-financed by the European Development Regional Fund “A Way to Achieve Europe” ERDF. This study did not receive any specific grant from commercial or not-for-profit sectors. Sí

The objective of this study is to characterize Streptococcus pyogenes isolates with a mucoid phenotype and to compare them with non-mucoid isolates obtained between April and August 2016.Identification and antimicrobial susceptibility were performed in all isolates. The emm type and exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analyzed. Clinical and demographic data were collected.From 96 isolates analyzed, 47% had a mucoid phenotype and 95.5% of them presented speA-speB-speF-speG-ssa genes and emm3 genotype. The main clinical manifestation was pharyngotonsillitis (77.1%) evolving to scarlet fever in 67.5% of the cases.This study describes the circulation of a mucoid phenotype strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype considered one of the most frequent and virulent of SGA.

Background The management and indication of empiric treatment in Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp)-colonized patients should be improved. Methods A prospective cohort of 94 patients colonized by KPC-Kp was followed for 90 days to validate (i) the Giannella risk score (GRS) to predict the development of any type of KPC-Kp infection and (ii) the INCREMENT-CPE score (ICS) to predict 30-day mortality in patients with infection. Both scores were combined to recommend appropriate empiric treatment. The predictive ability of the scores was measured by calculating the area under the receiver operating characteristic (AUROC) curve. Results The GRS showed an AUROC curve for infection due to KPC-Kp of 0.92 (95% confidence interval [CI], .87-.98). The optimal cutoff point was fixed at

Background The best available treatment against carbapenemase-producing Enterobacteriaceae (CPE) is unknown. The objective of this study was to investigate the effect of appropriate therapy and of appropriate combination therapy on mortality of patients with bloodstream infections (BSIs) due to CPE. Methods In this retrospective cohort study, we included patients with clinically significant monomicrobial BSIs due to CPE from the INCREMENT cohort, recruited from 26 tertiary hospitals in ten countries. Exclusion criteria were missing key data, death sooner than 24 h after the index date, therapy with an active antibiotic for at least 2 days when blood cultures were taken, and subsequent episodes in the same patient. We compared 30 day all-cause mortality between patients receiving appropriate (including an active drug against the blood isolate and started in the first 5 days after infection) or inappropriate therapy, and for patients receiving appropriate therapy, between those receiving active monotherapy (only one active drug) or combination therapy (more than one). We used a propensity score for receiving combination therapy and a validated mortality score (INCREMENT-CPE mortality score) to control for confounders in Cox regression analyses. We stratified analyses of combination therapy according to INCREMENT-CPE mortality score (0–7 [low mortality score] vs 8–15 [high mortality score]). INCREMENT is registered with ClinicalTrials.gov, number NCT01764490. Findings Between Jan 1, 2004, and Dec 31, 2013, 480 patients with BSIs due to CPE were enrolled in the INCREMENT cohort, of whom we included 437 (91%) in this study. 343 (78%) patients received appropriate therapy compared with 94 (22%) who received inappropriate therapy. The most frequent organism was Klebsiella pneumoniae (375 [86%] of 437; 291 [85%] of 343 patients receiving appropriate therapy vs 84 [89%] of 94 receiving inappropriate therapy) and the most frequent carbapenemase was K pneumoniae carbapenemase (329 [75%]; 253 [74%] vs 76 [81%]). Appropriate therapy was associated with lower mortality than was inappropriate therapy (132 [38·5%] of 343 patients died vs 57 [60·6%] of 94; absolute difference 22·1% [95% CI 11·0–33·3]; adjusted hazard ratio [HR] 0·45 [95% CI 0·33–0·62]; p

Objectives: Our aim was to characterize the putative MBL of the environmental strain Zhongshania aliphaticivorans isolated from a marine environment.Methods: The putative MBL was identified in silico using the NCBI database. The β-lactamase gene was cloned into different Escherichia coli backgrounds. Kinetic parameters were determined using the purified enzyme.Results: The enzyme named ZHO-1 shared 51% amino acid identity with the acquired class B carbapenemases IMP-1, KHM-1 and DIM-1. Expression of the blaZHO-1 gene in a susceptible E. coli resulted in a carbapenemase phenotype. Kinetic parameters determined from purified ZHO-1 enzyme showed that it had significant hydrolytic activity against most β-lactams including penicillins, cephalosporins and carbapenems, with the exception of aztreonam and cefepime.Conclusions: This study adds to the knowledge regarding environmental species as a reservoir of possible clinically relevant MBLs.

External validation of the INCREMENT-CPE risk score (ICS) for 30-day all-cause mortality is needed. There is also scarce information about whether colistin resistance influences the prognosis of carbapenem-resistant Klebsiella pneumoniae (CRKp) bacteraemia. In this study, the ability of ICS to predict all-cause mortality in the KAPECOR cohort was calculated using the area under the receiver operating characteristic (AUROC) curve. The association of colistin resistance with mortality was studied. The ICS showed an AUROC curve of 0.77 (95% CI 0.68–0.86). A cut-off of 8 points showed 96.8% sensitivity and 50.7% specificity. Mortality of low-risk patients was not different in those treated with monotherapy versus combination therapy. However, mortality of high-risk patients treated with combination therapy (37.8%) was significantly lower than in those treated with monotherapy (68.4%) (P = 0.008). To study the prognostic significance of colistin resistance, 83 selected cases of bacteraemia due to colistin-susceptible CRKp were obtained from the INCREMENT cohort for comparison. Colistin resistance could not be shown to be associated with higher mortality in either the high-risk ICS group [adjusted odds ratio (aOR) = 1.56, 95% CI 0.69–3.33; P = 0.29] or in 37 ICS-matched pairs (aOR = 1.38, 95% CI 0.55–3.42; P = 0.49), or in a sensitivity analysis including only KPC isolates (aOR = 1.81, 95% CI 0.73–4.57; P = 0.20), but the precision of estimates was low. These results validate ICS for all-cause mortality and to optimise targeted therapy for CRKp bacteraemia. Colistin resistance was not clearly associated with increased mortality., This study was supported by Plan Nacional de I+D+i 2013–2016, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/0001; RD16/0016/0008], co-financed by the European Regional Development Fund ‘A way to achieve Europe’, Operative Program Intelligent Growth 2014–2020.

Objectives To describe the case of a patient with infection due to a KPC-producing Klebsiella pneumoniae (K. pneumoniae) isolate developing ceftazidime-avibactam resistance with restored carbapenem susceptibility during ceftazidime-avibactam therapy. To review the clinical/microbiological cure and survival rates using carbapenems in other similar case reports and case series. Patients and methods A patient with an intra-abdominal infection due to K. pneumoniae producing the KPC-48 variant (L169P-A172T) (resistant to ceftazidime/avibactam and susceptible to carbapenems) who was treated with imipenem-cilastatin in combination with tigecycline and gentamicin. The literature was reviewed in order to summarise the in vivo (clinical/microbiological cure and survival rate) use of carbapenems in this emerging scenario. Results The patient was successfully treated with the indicated regimen. In other reported cases (mostly with pneumonia) all-cause mortality was 50% and clinical cure was 62.5%. Meropenem-vaborbactam has been successful used in an additional case. Conclusions A carbapenem-based regimen of combination therapy seems to be an option for treating patients infected with K. pneumoniae resistant to ceftazidime/avibactam and susceptible to carbapenems, at least when the risk of mortality is low.

This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive urine samples detected with the luminometry screening Coral UTI Screen

Background Infections caused by KPC-producing Klebsiella pneumoniae (KPC-Kp) are not well represented in pivotal trials with ceftazidime-avibactam (CAZ-AVI). This study aimed to investigate its efficacy in a single-centre cohort of patients infected with KPC-Kp. Methods A retrospective observational study was conducted of consecutive patients treated for > 72 hours with CAZ-AVI for KPC-Kp infections. Fourteen-day clinical response was considered when none of these criteria were present: i) the patient died before day 14, ii) treatment with CAZ-AVI at day 14 for persistence of symptoms or signs of infection, iii) recurrence. A multivariate logistic regression model was used to identify factors predictive of 14-day clinical failure. A propensity score to receive targeted initial treatment with CAZ-AVI was used as a covariate of the analysis. Results Forty-seven patients were included. The median age was 70 years and the median Charlson index was 4. The most frequent sources of infection were intraabdominal (n = 18; 38.3%) followed by pneumonia (n = 14; 29.8%). Twenty-five patients (53.2%) had septic shock. CAZ-AVI was used as monotherapy in 34 (72.3%) of the cases. CAZ-AVI resistance was detected after CAZ-AVI therapy in six of 47 (12.7%) patients. Thirty-day crude mortality was 23.4% (n = 11). The 14-day clinical response rate was 59.6% (n = 28). Pneumonia (OR 7.57; 95% CI 1.45–39.43; P = 0.01), and INCREMENT-CPE score > 7 points (OR 6.73; 95% CI 1.39–34.94; P = 0.02) were associated with 14-day clinical failure. Conclusions CAZ-AVI offers an advance for the treatment of KPC-Kp infections. In patients with pneumonia or an INCREMENT-CPE score > 7 points it may be reasonable to use CAZ-AVI in combination.

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification. Sysmex España S.L. financially supported the study, providingreagents and equipment. Additionally, Yuliya Zboromyrska receivedtravel grants from Sysmex España S.L. The funder had no role in data analysis and interpretation, decision to publish, or preparation of the manuscript.

Combination therapy including colistin and a carbapenem has been found to be associated with lower mortality in the treatment of bloodstream infections (BSI) due to KPC-producing Klebsiella pneumoniae when the isolates show a meropenem or imipenem MIC of, This study was funded by Planes Nacionales de I+D+i 2008-2011 and 2013-2016, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0010 and REIPI RD16/0016/0001; RD16/0016/0001; RD16/00167008) and cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020.