Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Colistin versus polymyxin B: A pragmatic assessment of renal and neurological adverse effects and effectiveness in multidrug-resistant Gram-negative bacterial infections.

OBJECTIVES: Our study aimed to evaluate the real-world data on renal and neurological adverse effects and effectiveness of colistimethate sodium (CMS) and polymyxin B (PMB). MATERIALS AND METHODS: An observational prospective study was performed on inpatients receiving CMS and PMB for multidrug-resistant Gram-negative bacterial infections. CMS dose was titrated to renal function, and serum creatinine was assessed daily. The incidence of nephrotoxicity, the primary outcome, was evaluated based on an increase in serum creatinine from baseline as well as by the Risk, Injury, Failure, Loss of kidney function, and End-stage renal disease criteria. Neurological adverse effects were assessed based on clinical signs and symptoms, and the causality and severity were assessed by the Naranjo scale and modified Hartwig-Siegel scale, respectively. The effectiveness of polymyxin therapy was ascertained by a composite of microbiological eradication of causative bacteria and achievement of clinical cure. Thirty-day all-cause mortality was also determined. RESULTS: Between CMS and PMB, the incidence of nephrotoxicity (59.3% vs. 55.6%, P = 0.653) or neurotoxicity (8.3% vs. 5.6%, P = 0.525) did not significantly differ. However, reversal of nephrotoxicity was significantly more with patients receiving CMS than PMB (48.4% vs. 23.3%, P = 0.021). Favorable clinical outcomes (67.6% vs. 37%, P < 0.001) and microbiological eradication of causative bacteria (73.1% vs. 46.3%, P = 0.001) were significantly more with CMS than PMB. Patients treated with CMS had lower all-cause mortality than those with PMB treatment (19.4% vs. 42.6%, P = 0.002). CONCLUSION: There is no significant difference in the incidence of renal and neurotoxic adverse effects between CMS and PMB when CMS is administered following renal dose modification. CMS shows better effectiveness and lower mortality compared to PMB.

Extended infusion of piperacillin-tazobactam versus intermittent infusion in critically ill egyptian patients: a cost-effectiveness study.

Extended infusion of piperacillin/tazobactam over 4 h has been proposed as an alternate mode of administration to the 30-min intermittent infusion to optimize treatment effects in patients with gram-negative bacterial infections. The study aimed to evaluate the extended infusion regimen of piperacillin/tazobactam in standings of efficacy, safety, and cost to the intermittent one in the treatment of gram-negative bacterial infections. A prospective randomized comparative study was performed on 53 patients, 27 in the intermittent infusion group and 26 in the extended infusion group. The primary outcome was the mean number of days to clinical success and the percentage of patients who were clinically cured after treatment. The secondary outcomes included mortality, readmission within 30-days, and cost-effectiveness analysis based on the mean number of days to clinical success. The clinical success rate was comparable in the two groups. Days on extended infusion were significantly lower than intermittent infusion (5.7 vs 8.9 days, respectively, p = 0.0001) as well as days to clinical success (4.6 vs 8.5 days, respectively, p = 0.026). The extended infusion was superior to the intermittent infusion regarding cost-effectiveness ratio ($1835.41 and $1914.09/expected success, respectively). The more cost-effective regimen was the extended infusion. Both regimens had comparable clinical and microbiological outcomes.

Neonatal multidrug-resistant gram-negative infection: epidemiology, mechanisms of resistance, and management.

Infants admitted to the neonatal intensive care unit, particularly those born preterm, are at high risk for infection due to the combination of an immature immune system, prolonged hospitalization, and frequent use of invasive devices. Emerging evidence suggests that multidrug-resistant gram-negative (MDR-GN) infections are increasing in neonatal settings, which directly threatens recent and ongoing advances in contemporary neonatal care. A rising prevalence of antibiotic resistance among common neonatal pathogens compounds the challenge of optimal management of suspected and confirmed neonatal infection. We review the epidemiology of MDR-GN infections in neonates in the United States and internationally, with a focus on extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales and carbapenem-resistant Enterobacterales (CRE). We include published single-center studies, neonatal collaborative reports, and national surveillance data. Risk factors for and mechanisms of resistance are discussed. In addition, we discuss current recommendations for empiric antibiotic therapy for suspected infections, as well as definitive treatment options for key MDR organisms. Finally, we review best practices for prevention and identify current knowledge gaps and areas for future research. IMPACT: Surveillance and prevention of MDR-GN infections is a pediatric research priority. A rising prevalence of MDR-GN neonatal infections, specifically ESBL-producing Enterobacterales and CRE, compounds the challenge of optimal management of suspected and confirmed neonatal infection. Future studies are needed to understand the impacts of MDR-GN infection on neonatal morbidity and mortality, and studies of current and novel antibiotic therapies should include a focus on the pharmacokinetics of such agents among neonates.

Evaluation of the Neonatal Sequential Organ Failure Assessment and Mortality Risk in Preterm Infants With Late-Onset Infection.

Importance: Infection in neonates remains a substantial problem. Advances for this population are hindered by the absence of a consensus definition for sepsis. In adults, the Sequential Organ Failure Assessment (SOFA) operationalizes mortality risk with infection and defines sepsis. The generalizability of the neonatal SOFA (nSOFA) for neonatal late-onset infection-related mortality remains unknown. Objective: To determine the generalizability of the nSOFA for neonatal late-onset infection-related mortality across multiple sites. Design, Setting, and Participants: A multicenter retrospective cohort study was conducted at 7 academic neonatal intensive care units between January 1, 2010, and December 31, 2019. Participants included 653 preterm (<33 weeks) very low-birth-weight infants. Exposures: Late-onset (>72 hours of life) infection including bacteremia, fungemia, or surgical peritonitis. Main Outcomes and Measures: The primary outcome was late-onset infection episode mortality. The nSOFA scores from survivors and nonsurvivors with confirmed late-onset infection were compared at 9 time points (T) preceding and following event onset. Results: In the 653 infants who met inclusion criteria, median gestational age was 25.5 weeks (interquartile range, 24-27 weeks) and median birth weight was 780 g (interquartile range, 638-960 g). A total of 366 infants (56%) were male. Late-onset infection episode mortality occurred in 97 infants (15%). Area under the receiver operating characteristic curves for mortality in the total cohort ranged across study centers from 0.71 to 0.95 (T0 hours), 0.77 to 0.96 (T6 hours), and 0.78 to 0.96 (T12 hours), with utility noted at all centers and in aggregate. Using the maximum nSOFA score at T0 or T6, the area under the receiver operating characteristic curve for mortality was 0.88 (95% CI, 0.84-0.91). Analyses stratified by sex or Gram-stain identification of pathogen class or restricted to infants born at less than 25 weeks' completed gestation did not reduce the association of the nSOFA score with infection-related mortality. Conclusions and Relevance: The nSOFA score was associated with late-onset infection mortality in preterm infants at the time of evaluation both in aggregate and in each center. These findings suggest that the nSOFA may serve as the foundation for a consensus definition of sepsis in this population.

Análise do consumo de antimicrobianos e infecções relacionadas à assistência à saúde após implantação de um programa de gestão de antimicrobianos em unidade de tratamento intensivo neonatal do Rio de Janeiro / Analysis of antimicrobial consumption and healthcare-associated infections after an antimicrobial stewardship program implantation in neonatal intensive care of Rio de Janeiro / Análisis del consumo de antimicrobianos y las infecciones asociadas a la atención de la salud después de la implantación de un programa de administración de antimicrobianos en cuidados intensivos neonatales de Río de Janeiro

Justificativa e objetivos. Programas de gestão de antimicrobianos (PGA) podem contribuir para otimizar o uso de antimicrobianos em unidades de tratamento intensivo neonatais (UTINEO). O objetivo deste estudo foi mensurar o consumo de antimicrobianos, dentre eles os carbapenêmicos e infecções relacionadas à assistência à saúde (IRAS), especificamente as causadas por bactérias Gram-negativas resistentes a carbapenêmicos (BGN-CR) em neonatos após a implantação de um PGA. Métodos: Estudo prospectivo descritivo do consumo de antimicrobianos, dentre eles os carbapenêmicos; e das taxas de IRAS em uma UTINEO, durante 1 ano de seguimento. O consumo fo i medido em dias de terapia/1000 pacientes-dia (DOT/1000PD). Resultados: Em setembro de 2017 o PGA foi implementado com os seguintes elementos-chave: auditoria de antibióticos/feedback, restrição de antimicrobianos-alvo, medida do consumo de antimicrobianos e maior rapidez na liberação de resultados de culturas. Entre setembro de 2017 e setembro de 2018 admitimos 308 pacientes, totalizando 2223 pacientes-dia. A mediana de consumo total de antimicrobianos foi de 1580 DOT/1000PD (variação de 1180,7 a 2336,6/mês) sem tendência de aumento e a de carbapenêmicos 12 DOT/1000PD (variação de 0 a 163,2/mês). O consumo de carbapenêmicos foi reduzido entre abril a setembro de 2018 (valor de p =0,07) quando comparado com os primeiros seis meses. Oito IRAS foram registradas, correspondendo a uma densidade de incidência de 3,6/1000 pacientes-dia. Não foram reportadas BGN-CR causando IRAS. Conclusões: O consumo total de antimicrobianos não apresentou aumento ao longo do ano após implantação do PGA. No entanto, houve redução significativa do consumo de carbapenêmicos. Não foram verificadas IRAS por BGN-CR no período do estudo.(AU)

Background and objectives: Antimicrobial stewardship programs (ASPs) could contribute to optimize antimicrobial use within neonatal intensive care units (NICUs). The aim of this study was to measure the antimicrobial consumption, including carbapenems and healthcare-associated infections (HAI), specifically infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) in neonates, after implementation of an ASP. Methods: A prospective descriptive study of antimicrobial and carbapenem consumption; and healthcare-associated rate in a NICU, during a one-year follow-up. The consumption was measured in days of therapy /1000 patients-days(DOT/1000PD). Results: In September 2017, the ASP was implemented, with the following core components: antibiotic audit and feedback, restriction of target antimicrobials, measure of antimicrobial consumption and improvement of results from microbiologic laboratory. Between September 2017 and September 2018, we admitted 308 patients, totalizing 2223 patient-days. The median of total antimicrobial consumption was 1580 DOT/1000PD (range from 1180.7 to 2336.6/month and of carbapenems 12 DOT/1000PD (range from 0 to 162.3/month). The carbapenem consumption was reduced between April and September of 2018 (p value=0.07) when we compared the first six months of the study. Eight HAI were detected, corresponding to density of incidence of 3.6/1000 patient-days. No HAI due to CR-GNB was reported. Conclusion: The total antimicrobial consumption did not increase during all the year after the ASP implantation. Although there was a significant reduction of carbapenem consumption. Carbapenem-resistant bacteria was not found in NICU causing HAI.(AU)

Justificación y objetivos: Los programas de optimizatión de uso de antimicrobianos (POA) podrían contribuir a optimizar el uso de antimicrobianos dentro de las unidades de cuidados intensivos neonatales (UCIN). El objetivo de este estudio fue medir el consumo de antimicrobianos incluidos los carbapenems y las infecciones asociadas a la atención de la salud (IAAs), especificamente las infecciones causadas por bacterias Gram negativas resistentes a carbapenems (CR-GNB) en neonatos, después de la implementación de un POA. Métodos: Un estudio descriptivo prospectivo del consumo de antimicrobianos y carbapenems; y la tasa de IAAs en una UCIN durante un año de seguimiento. El consumo se midió en días de terapia (DOT) / 1000 pacientes-días. Resultados: En septiembre de 2017, se implementó el POA con los siguientes componentes principales: auditoría y retroalimentación de antibióticos, restricción de antimicrobianos objetivo, medición del consumo de antimicrobianos y mejora de los resultados del laboratorio microbiológico. Entre septiembre de 2017 y septiembre de 2018, admitimos 308 pacientes, totalizando 2223 días-paciente. La mediana del consumo total de antimicrobianos fue de 1580 DOT / 1000PD (rango de 1180.7 a 2336.6 / mes y de carbapenems 12 (rango de 0 a 162.3 / mes). El consumo de carbapenem se redujo entre abril y septiembre de 2018 (valor p = 0.07) cuando comparamos los primeros seis meses del estudio, se detectaron ocho IAAs, lo que corresponde a la densidad de incidencia de 3.6 / 1000 días-paciente No se informó ningún IAA debido a CR-GNB. Conclusiones: El consumo total de antimicrobianos no aumentó durante todo el año posterior a la implantación de POA. Aunque hubo una reducción significativa del consumo de carbapenem. No se encontraron bacterias resistentes a carbapenem en la UCIN que causa IAA.(AU)

Attributable burden in patients with carbapenem-nonsusceptible gram-negative respiratory infections.

OBJECTIVE: We aimed to describe the clinical and economic burden attributable to carbapenem-nonsusceptible (C-NS) respiratory infections. METHODS: This retrospective matched cohort study assessed clinical and economic outcomes of adult patients (aged ≥18 years) who were admitted to one of 78 acute care hospitals in the United States with nonduplicate C-NS and carbapenem-susceptible (C-S) isolates from a respiratory source. A subset analysis of patients with principal diagnosis codes denoting bacterial pneumonia or other diagnoses was also conducted. Isolates were classified as community- or hospital-onset based on collection time. A generalized linear mixed model method was used to estimate the attributable burden for mortality, 30-day readmission, length of stay (LOS), cost, and net gain/loss (payment minus cost) using propensity score-matched C-NS versus C-S cohorts. RESULTS: For C-NS cases, mortality (25.7%), LOS (29.4 days), and costs ($81,574) were highest in the other principal diagnosis, hospital-onset subgroup; readmissions (19.4%) and net loss (-$9522) were greatest in the bacterial pneumonia, hospital-onset subgroup. Mortality and readmissions were not significantly higher for C-NS cases in any propensity score-matched subgroup. Significant C-NS-attributable burden was found for both other principal diagnosis subgroups for LOS (hospital-onset: 3.7 days, P = 0.006; community-onset: 1.5 days, P<0.001) and cost (hospital-onset: $12,777, P<0.01; community-onset: $2681, P<0.001). CONCLUSIONS: Increased LOS and cost burden were observed in propensity score-matched patients with C-NS compared with C-S respiratory infections; the C-NS-attributable burden was significant only for patients with other principal diagnoses.

Evaluation of quick sequential organ failure assessment and systemic inflammatory response syndrome in patients with gram negative bloodstream infection.

BACKGROUND: The quick sequential organ failure assessment (qSOFA) score predicts mortality in patients with suspected infection. We sought to understand how well qSOFA and the Systemic Inflammatory Response Syndrome (SIRS) criteria predict gram negative bacteraemia. METHODS: We prospectively evaluated 99 patients with gram negative bloodstream infection from a single tertiary centre. We assessed the utility of SIRS and qSOFA for their rate of positivity and association with early delivery of antibiotics (<3 h). RESULTS: The SIRS criteria had the highest positivity rate amongst patients with gram negative bacteraemia (85%) compared to the qSOFA criteria (25%) on the day of first positive culture. Positive SIRS criteria was the only score associated with delivery of antibiotics within 3 h (Relative risk 3.5, 95% Confidence interval 1.3 to 12.5, p = < 0.02). CONCLUSION: In patients with gram negative bloodstream infection SIRS criteria was the most common positive risk score and had a higher association with early delivery of antibiotics when compared to qSOFA.

Assessment and Management of Infection in Alcoholic Hepatitis.

Severe alcoholic hepatitis (SAH) is a condition characterized by jaundice and liver failure that develops after heavy and prolonged alcohol consumption. Infection frequently complicates the natural history of the disease and is independently associated with mortality. Objective recognition and recording of infection are therefore essential in the evaluation of therapeutic interventions and for antibiotic stewardship. This review will evaluate infections that complicate SAH at admission and beyond. Factors that associate with the development of infection will be identified and clinical and laboratory techniques available to identify infection will be discussed. Common pathogens and frequently used antibiotics will be reviewed and recommendations will be made for the management of infection for SAH patients. New techniques to assess infection earlier and more precisely may improve diagnosis and treatment of this important driver of mortality in SAH.

Cefepime/sulbactam as an enhanced antimicrobial combination therapy for the treatment of MDR Gram-negative infections.

BACKGROUND: ß-Lactam (BL)/ß-lactamase inhibitor (BLI) combinations are widely used for the treatment of Gram-negative infections. Cefepime has not been widely studied in combination with BLIs. Sulbactam, with dual BL/BLI activity, has been partnered with very few BLs. We investigated the potential of cefepime/sulbactam as an unorthodox BL/BLI combination against MDR Gram-negative bacteria. METHODS: In vitro activity of cefepime/sulbactam (1:1, 1:2 and 2:1) was assessed against 157 strains. Monte Carlo simulation was used to predict the PTA with a number of simulated cefepime combination regimens, modelled across putative cefepime/sulbactam breakpoints (≤16/≤0.25 mg/L). RESULTS: Cefepime/sulbactam was more active (MIC50/MIC90 8/8-64/128 mg/L) compared with either drug alone (MIC50/MIC90 128 to >256 mg/L). Activity was enhanced when sulbactam was added at 1:1 or 1:2 (P<0.05). Reduction in MIC was most notable against Acinetobacter baumannii and Enterobacterales (MIC 8/8-32/64 mg/L). Pharmacokinetic/pharmacodynamic modelling highlighted that up to 48% of all isolates and 73% of carbapenem-resistant A. baumannii with a cefepime/sulbactam MIC of ≤16/≤8 mg/L may be treatable with a high-dose, fixed-ratio (1:1 or 1:2) combination of cefepime/sulbactam. CONCLUSIONS: Cefepime/sulbactam (1:1 or 1:2) displays enhanced in vitro activity versus MDR Gram-negative pathogens. It could be a potential alternative to existing BL/BLI combinations for isolates with a cefepime/sulbactam MIC of 16/8 mg/L either as a definitive treatment or as a carbapenem-sparing option.

Agrobacterium spp. nosocomial outbreak assessment using rapid MALDI-TOF MS based typing, confirmed by whole genome sequencing.

Background: A number of episodes of nosocomial Agrobacterium spp. bacteremia (two cases per year) were observed at Bern University Hospital, Switzerland, from 2015 to 2017. This triggered an outbreak investigation. Methods: Cases of Agrobacterium spp. bacteremias that occurred between August 2011 and February 2017 were investigated employing line lists, environmental sampling, rapid protein- (MALDI-TOF MS), and genome-based typing (pulsed field gel electrophoresis and whole genome sequencing) of the clinical isolates. Results: We describe a total of eight bacteremia episodes due to A. radiobacter (n = 2), Agrobacterium genomovar G3 (n = 5) and A. pusense (n = 1). Two tight clusters were observed by WGS typing, representing the two A. radiobacter isolates (cluster I, isolated in 2015) and four of the Agrobacterium genomovar G3 isolates (cluster II, isolated in 2016 and 2017), suggesting two different point sources. The epidemiological investigations revealed two computer tomography (CT) rooms as common patient locations, which correlated with the two outbreak clusters. MALDI-TOF MS permitted faster evaluation of strain relatedness than DNA-based methods. High resolution WGS-based typing confirmed the MALDI-TOF MS clustering. Conclusions: We report clinical and epidemiological characteristics of two outbreak clusters with Agrobacterium. spp. bacteremia likely acquired during CT contrast medium injection and highlight the use of MALDI-TOF MS as a rapid tool to assess relatedness of rare gram-negative pathogens in an outbreak investigation.

Fight Against Antimicrobial Resistance: We Always Need New Antibacterials but for Right Bacteria.

Antimicrobial resistance in bacteria is frightening, especially resistance in Gram-negative Bacteria (GNB). In 2017, the World Health Organization (WHO) published a list of 12 bacteria that represent a threat to human health, and among these, a majority of GNB. Antibiotic resistance is a complex and relatively old phenomenon that is the consequence of several factors. The first factor is the vertiginous drop in research and development of new antibacterials. In fact, many companies simply stop this R&D activity. The finding is simple: there are enough antibiotics to treat the different types of infection that clinicians face. The second factor is the appearance and spread of resistant or even multidrug-resistant bacteria. For a long time, this situation remained rather confidential, almost anecdotal. It was not until the end of the 1980s that awareness emerged. It was the time of Vancomycin-Resistance Enterococci (VRE), and the threat of Vancomycin-Resistant MRSA (Methicillin-Resistant Staphylococcus aureus). After this, there has been renewed interest but only in anti-Gram positive antibacterials. Today, the threat is GNB, and we have no new molecules with innovative mechanism of action to fight effectively against these bugs. However, the war against antimicrobial resistance is not lost. We must continue the fight, which requires a better knowledge of the mechanisms of action of anti-infectious agents and concomitantly the mechanisms of resistance of infectious agents.

Assessment of Gill Pathological Responses in Yellowfin Sea Bream (Acanthopagrus Latus) Under Aeromonas Hydrophila Exposure.

Bacterial diseases in cultured fish are considered the main problem with aquaculture system in Iran. The gills are multifunctional organs responsible for respiration, osmoregulation, nitrogenous waste excretion, and acid-base balance. Moreover, they are very sensitive to water contamination. Aeromonas hydrophila (A. hydrophila) is an opportunist pathogen responsible for a wide range of diseases in different species of fish. The gill histological alterations were used to assess the effects of A. hydrophila exposure on yellowfin sea bream, Acanthopagrus latus (A. latus). In this regard, 90 A. latus were exposed to the concentrations of A. hydrophila (103 and 106 CFU/ml) for three weeks. The most histopathological alterations in the gill of the exposed fish included hypertrophy and hyperplasia of the epithelial cells, lamellar fusion, club shaping of gill lamellae, lifting of the epithelium and edema of lamellae with large sub-epithelial space, blood congestion, and hypertrophy and hyperplasia of the mucosal cells. The histopathological alterations were observed in the gill of fish exposed to higher levels of A. hydrophila (106 CFU/ml) consisted of aneurysm and hemorrhage with blood congestion. According to the obtained results of this study, A. hydrophila could cause severe histopathological changes in the gill of A. latus and decrease gas change capability in yellowfin sea bream. Furthermore, the findings of the present study suggested that histopathological changes of the gill provide helpful information about the environmental conditions and as particular biomarkers may help the evaluation of fish general health.

Effective implementation of the Accelerate Pheno™ system for positive blood cultures.

Using conventional methods, organism identification (ID) and antibiotic susceptibility testing (AST) results are available ∼1.5-3 days after positive blood culture. New technologies can reduce this time to 8-12 h, allowing therapy to be optimized substantially sooner. To make full use of fast ID and AST results requires overcoming various hurdles to effective implementation, including restructuring laboratory workflows to optimize timeliness of results and modifying clinical pathways to respond more quickly when results are available. Efficient laboratory procedures and clinical interventions coupled with fast and accurate identification and AST results have the potential to substantially reduce overall costs and provide more-sophisticated and effective patient management.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Attributable clinical and economic burden of carbapenem-non-susceptible Gram-negative infections in patients hospitalized with complicated urinary tract infections.

BACKGROUND: Gram-negative complicated urinary tract infections (cUTIs) can have serious consequences for patients and hospitals. AIM: To examine the clinical and economic burden attributable to Gram-negative carbapenem-non-susceptible (C-NS; resistant/intermediate) infections compared with carbapenem-susceptible (C-S) infections in 78 US hospitals. METHODS: All non-duplicate C-NS and C-S urine source isolates were analysed. A subset had principal diagnosis ICD-9-CM codes denoting cUTI. Collection time (<3 vs ≥3 days after admission) determined isolate classification as community or hospital onset. Mortality, 30-day re-admissions, length of stay (LOS), hospital cost and net gain/loss in US dollars were determined for C-NS and C-S cases, with the C-NS-attributable burden estimated through propensity score matching. Three subgroups with adequate patient numbers were analysed: cUTI principal diagnosis, community onset; other principal diagnosis, community onset; and other principal diagnosis, hospital onset. FINDINGS: The C-NS-attributable mortality risk was significantly higher (58%) for the other principal diagnosis, hospital-onset subgroup alone (odds ratio 1.58, 95% confidence interval 1.14-2.20; P < 0.01). The C-NS-attributable risk for 30-day re-admission ranged from 29% to 55% (all P < 0.05). The average attributable economic impact of C-NS was 1.1-3.9 additional days LOS (all P < 0.05), US$1512-10,403 additional total cost (all P < 0.001) and US$1582-11,848 net loss (all P < 0.01); overall burden and C-NS-attributable burden were greatest in the other principal diagnosis, hospital-onset subgroup. CONCLUSION: Greater clinical and economic burden was observed in propensity-score-matched patients with C-NS infections compared with C-S infections, regardless of whether cUTI was the principal diagnosis, and this burden was most severe in hospital-onset infections.

Validation of a clinical decision tree to predict if a patient has a bacteraemia due to a ß-lactamase producing organism.

BACKGROUND: In recent years, several scores and algorithms have been developed in order to guide empirical antibiotic treatment in patients with gram-negative bacilli (GNB) bacteraemia according to the risk of extended-spectrum ß-lactamase (BL) producing. Some of these algorithms do not have easy applicability or present some limitations in their validation. The aim of our study was to validate a recently designed decision tree in our prospective cohort of bacteraemia due to gram-negative bacilli. METHODS: We prospectively identified and analyzed all bacteraemia due to gram-negative bacilli in adult patients in our centre between January 2015 and December 2016. Previously developed clinical decision tree was used to classify patients in each of the terminal nodes. Patients were classified as BL group according to whether they were producers of any type of BL. The statistical power of the tree was analyzed by receiver operating characteristics (ROC) curve and by calculation of C-statistics. RESULTS: A total of 448 episodes of bacteraemia were included; 132 (29.5%) were BL group; 68 (15.1%) ESBL producing, 43 (9.6%) due to AmpC and 21 (4.7%) isolates of Pseudomonas aeruginosa. The original clinical decision tree was modified according to the results of our multivariate analysis. The modified tree has a sensitivity of 71%, specificity of 92%, predictive positive value (PPV) of 79% and predictive negative value (NPV) of 88% generating an ROC curve with a C-statistic of 0.76. CONCLUSIONS: An easy-to-apply clinical decision tree could be used at the exact moment of diagnosis and adjust the empirical antibiotic treatment in patients with gram-negative bacilli bacteraemia.

Assessment and comparison of bacterial load levels determined by quantitative amplifications in blood culture-positive and negative neonatal sepsis.

Bacterial sepsis remains a major cause of mortality and blood cultures are the gold standard of laboratory diagnosis even though they lack sensitivity in neonates. Culturenegative sepsis, also known as clinical sepsis, has long been considered a diagnosis in neonatal intensive care units because, as well as culture-positive infants, culture-negative neonates have worse prognosis in comparison with non-infected ones. Quantitative amplifications are used to detect bacterial infections in neonates but results are considered only in a qualitative way (positive or negative). The aim of the present study was to determine and compare bacterial load levels in blood culture-positive and culture-negative neonatal sepsis. Seventy neonates with clinical and laboratory evidence of infection admitted at three neonatal intensive care units were classified as blood culture-positive or culture-negative. Blood samples obtained at the same time of blood cultures had bacterial load levels assessed through a 16S rDNA qPCR. Blood cultures were positive in 29 cases (41.4%) and qPCR in 64 (91.4%). In the 29 culture-positive cases, 100% were also positive by qPCR, while in the 41 culture-negative cases, 35 (85.4%) were positive by qPCR. Bacterial load levels were in general < 50 CFU/mL, but were significantly higher in culture-positive cases (Mann-Whitney, p = 0.013), although clinical and laboratory findings were similar, excepting for deaths. In conclusion, the present study has shown that blood culture-negative neonates have lower bacteria load levels in their bloodstream when compared to blood culture-positive infants.