Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13). CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

Real-life use of cefiderocol for salvage therapy of severe infections due to carbapenem-resistant Gram-negative bacteria.

Treatment of infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) is challenging and new active antibiotics are needed urgently. This study describes the efficacy and safety of cefiderocol in a retrospective series of 13 patients with severe CR-GNB infection and limited treatment options. Pseudomonas aeruginosa was the predominant CR-GNB (n=8), followed by Burkholderia cepacia (n=3), Sthenotrophomona maltophilia (n=1) and KPC-producing Klebsiella pneumoniae (n=1). The source of infection was nosocomial pneumonia in 92.3% of cases (12/13), of which 11 cases were ventilator-associated pneumonia. Five patients were lung transplant recipients (38.5%). The median duration of treatment was 10 days (range 6-21 days). No severe adverse effects required reducing the dose or interrupting the treatment. Clinical and microbiological cure were assessed 7 days after the end of treatment, and achieved in 84.6% (11/13) of patients. Crude mortality at day 28 was observed in 23.1% (3/13) of cases. Cefiderocol is a valid alternative for the treatment of susceptible CR-GNB infections in patients with limited therapeutic options.

Hidden dissemination of carbapenem-susceptible OXA-48-producing Proteus mirabilis.

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 ß-lactams after a single mutation.

Caracterización de estreptococos beta-hemolíticos del grupo A con fenotipo mucoide aislados en un hospital de tercer nivel / Characterization of group A beta-haemolytic streptococcus with mucoid phenotype isolated in a tertiary hospital

Introducción: El objetivo de este estudio es la caracterización de cepas de Streptococcus pyogenes con fenotipo mucoide y su comparación con las cepas no mucoides aisladas entre abril y agosto de 2016. Material y métodos: Se llevó a cabo la caracterización y el estudio de sensibilidad antimicrobiana de todos los aislados. Se determinó el tipo emm y se analizaron los genes de exotoxinas speA, speB, speC, speF, speG, speH, speJ, speZ y ssa. Se recogieron datos clínicos y demográficos. Resultados: De 96 aislados analizados, el 47% presentaron un fenotipo mucoide, y de estos últimos, el 95,5% presentaron los genes speA-speB-speF-speG-ssa y genotipo emm3. La principal manifestación clínica entre todos los pacientes fue faringoamigdalitis (77,1%) que evolucionó a escarlatina en el 67,5% de los casos. Conclusión: Se describe la circulación de una cepa de aspecto mucoide con perfil de toxinas speA-speB-speF-speG-ssa y genotipo emm3.1 considerado de los más frecuentes y más virulentos de SGA.(AU)

Introduction: 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. Material and methods: 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. Results: 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. Conclusion: 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.(AU)

Community-acquired bacteraemia by Klebsiella pneumoniae producing KPC-3 and resistant to ceftazidime/avibactam.

OBJECTIVES: To describe the clinical and microbiological features of a case of community-acquired infection by KPC-producing K. pneumoniae (KPCKP) resistant to ceftazidime/avibactam (CAZ-AVI). METHODS: Identification of microorganisms was performed with MALDI Biotyper CA System (BrukerDaltonics, Madrid, Spain). Antimicrobial susceptibility testing was performed using Sensitre EURGNCOL panels (Thermo Fisher Scientific, Madrid, Spain) and gradient strips (Etest, bioMérieux, Madrid, Spain) in the case of CAZ-AVI, using EUCAST breakpoints for interpretation. Whole genome sequencing of blood culture and rectal swab isolates was performed using the Illumina NovaSeq 6000 sequencing system, with 2 × 150-bp paired-end reads (Illumina, Inc.). RESULTS: Blood culture and rectal swab KPCKP isolates were resistant to carbapenems and to CAZ-AVI. The blood culture isolate showed susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX), but the rectal swab culture isolate was resistant to this antibiotic. Both isolates belonged to clonal lineage ST512, harboured a single copy of blaKPC-3 gene, and showed 16 single nucleotide polymorphisms (SNP) between them and 38 SNPs with regard to the first KPC-3 producing K. pneumoniae isolated in our hospital in an initial outbreak in 2012. Genome-wide resistome analysis revealed the presence of a IncFIB(K) plasmid harbouring sul1 and dfrA12 genes only in the rectal swab culture isolate, which may explain its resistance to TMP-SMX. CONCLUSIONS: Resistance to ceftazidime-avibactam is an emerging nosocomial problem. This case shows that CAZ-AVI-resistant KPCKP strains may disseminate into the community and cause serious infections.

Prognostic Significance of the Relative Load of KPC-Producing Klebsiella pneumoniae within the Intestinal Microbiota in a Prospective Cohort of Colonized Patients.

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 < 0.001). RLKPC (adjusted hazard ratio [aHR] 1.04, 95% CI = 1.01 to 1.07, P = 0.008) and age (aHR 1.05, 95% CI = 1.01 to 1.10, P = 0.008) were independent predictors of 90-day all-cause mortality in a Cox model stratified by length of hospital stay (LOHS) ≥20 days. An adjusted Cox model for 30-day all-cause mortality, stratified by LOHS ≥14 days, included RLKPC (aHR 1.03, 95% CI = 1.00 to 1.06, P = 0.027), age (aHR 1.10, 95% CI = 1.03 to 1.18, P = 0.004), and severe KPC-KP infection (INCREMENT-CPE score >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. IMPORTANCE The rapid dissemination of carbapenemase-producing Enterobacterales represents a global public health threat. Increased relative load of KPC-producing Klebsiella pneumoniae (KPC-KP) within the intestinal microbiota has been associated with an increased risk of bloodstream infection by KPC-KP. We developed a qPCR assay for quantification of the relative KPC-KP intestinal load (RLKPC) in 80 colonized patients and examined its association with subsequent all-site KPC-KP infection and all-cause mortality within 90 days. Giannella Risk Score, which predicts infection risk in colonized patients, was independently associated with the development of all-site KPC-KP infection. RLKPC was not associated with all-site KPC-KP infection, possibly reflecting the large heterogeneity in patient clinical conditions and infection types. RLKPC was an independent predictor of all-cause mortality within 90 and 30 days in our clinical setting. We hypothesize that KPC-KP load may behave as a surrogate marker for the severity of the patient's clinical condition.

Characterization of group A beta-haemolytic streptococcus with mucoid phenotype isolated in a tertiary hospital.

INTRODUCTION: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Proof­of­concept study to quantify changes in intestinal loads of KPC-producing Klebsiella pneumoniae in colonised patients following selective digestive decontamination with oral gentamicin.

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.

Randomised, double-blind, placebo-controlled, phase 2, superiority trial to demonstrate the effectiveness of faecal microbiota transplantation for selective intestinal decolonisation of patients colonised by carbapenemase-producing Klebsiella pneumoniae (KAPEDIS).

INTRODUCTION: Infections 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 ANALYSIS: The 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 DISSEMINATION: Ethical 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 NUMBER: NCT04760665.

Risk Factors for Multidrug-Resistant Gram-Negative Bacteria Carriage upon Admission to the Intensive Care Unit.

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 MDR-GNB 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.

Characterization of OXA-48-producing Klebsiella oxytoca isolates from a hospital outbreak in Tunisia.

OBJECTIVE: 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. METHODS: 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 ß-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. RESULTS: 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. CONCLUSION: 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.

Characterization of group A beta-haemolytic streptococcus with mucoid phenotype isolated in a tertiary hospital. / Caracterización de estreptococos beta-hemolíticos del grupo A con fenotipo mucoide aislados en un hospital de tercer nivel.

INTRODUCTION: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Ceftazidime-avibactam in the treatment of infections caused by KPC-producing Klebsiella pneumoniae: factors associated with clinical efficacy in a single-center cohort.

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.

Use of carbapenems in the combined treatment of emerging ceftazidime/avibactam-resistant and carbapenem-susceptible KPC-producing Klebsiella pneumoniae infections: Report of a case and review of the literature.

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.

External validation of the INCREMENT-CPE mortality score in a carbapenem-resistant Klebsiella pneumoniae bacteraemia cohort: the prognostic significance of colistin resistance.

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.

Combination of Coral UTI ScreenTM system, gram-stain and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnosis of urinary tract infections directly from urine samples.

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 ScreenTM system underwent Gram staining and identification of the causative organism was performed by MALDI-TOF Microflex LT mass spectrometer (Bruker Daltonics, Germany). Subsequently, the results were compared with those of conventional culture identification using WIDER MIC/id system (Francisco Soria Melguizo SA, Spain). Considering the conventional approach as the gold standard, the proposed algorithm presented both a high specificity (98.1%) and a positive likelihood ratio of 37.42. The implementation of this algorithm would allow diagnosis of urinary tract infection in less than an hour in 92.4% of positive samples. This combination of techniques would be useful particularly for patients with severe UTI, pyelonephritis or urinary sepsis.

ZHO-1, an intrinsic MBL from the environmental Gram-negative species Zhongshania aliphaticivorans.

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 ß-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 ß-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.

A multicentre study investigating parameters which influence direct bacterial identification from urine.

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.

Risks of Infection and Mortality Among Patients Colonized With Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: Validation of Scores and Proposal for Management.

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 <7 and ≥7 (92.9% sensitivity, 84.8% specificity); infection developed in 6.3% patients in the 0-6 GRS group and in 84.8% patient in the ≥7 GRS group. According to the ICS, the severity of the infection was also significantly higher in the ≥7 GRS group. The ICS showed an AUROC of 0.78 (95% CI, .65-.91) for 30-day all-cause mortality among patients with infection. A classification and regression tree analysis confirmed the GRS cutoff point at 7, and selected ≥12 points to predict a KPC-Kp infection with a high ICS. Conclusions: Our results validate the GRS and ICS for indicating empiric therapy in KPC-Kp-colonized patients.