Fecal carriage of extended-spectrum beta-lactamase-producing Enterobacterales in healthy Spanish schoolchildren.

Background: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are a serious threat among emerging antibiotic resistant bacteria. Particularly, the number of cases of ESBL-E infections reported in children has been increasing in recent years, and approved antibiotic treatments for this age group are limited. However, information regarding the prevalence of colonization in European children, risk factors associated with colonization, and the characteristics of the colonizing strains is scarce. The aims of this study were to determine the prevalence of ESBL-E colonization in fecal samples of apparently healthy schoolchildren, to identify lifestyle routines associated with colonization, and to characterize clonal relationships and mechanisms of resistance in ESBL-E isolates. Methods: A cohort of 887 healthy children (3-13 years old) from seven primary and secondary schools in the Madrid metropolitan area was recruited between April-June 2018, and sociodemographic information and daily habits were collected. Fecal samples were screened for ESBL-E carriage in selective medium. ESBL-E isolates were further characterized by assessing molecular epidemiology (PFGE and MLST), ESBL gene carriage, and antibiotic resistance profile. This information was analyzed in conjunction with the metadata of the participants in order to identify external factors associated with ESBL-E carriage. Results: Twenty four ESBL-E, all but one Escherichia coli, were detected in 23 children (prevalence: 2.6%; 95% CI: 1.6-3.6%). Of these, seven contained the blaCTX-M-14 allele, five the blaCTX-M-15, five the blaSHV-12, three the blaCTX-M-27, three the blaCTX-M-32, and one the blaCTX-M-9. Significant clonal diversity was observed among the isolates that grouped into 22 distinct clusters (at <85% similarity of PFGE profile). ESBL-producing E. coli isolates belonged to 12 different STs, with ST10 (25%) and ST131 (17%) being the most frequent. Apart from ß-lactams, resistance to trimethoprim/sulfamethoxazole (46%), ciprofloxacin (33%), levofloxacin (33%), tobramycin (21%), and gentamicin (8%) were the most frequently detected. Conclusion: The prevalence of ESBL-E in the studied cohort of children was lower than the average colonization rate previously detected in Europe for both children and adults. E. coli was the main ESBL-producing species detected and CTX-M were the most frequently identified ESBLs. High ST diversity suggests polyclonal dissemination. Compared to other STs, ST131 isolates were associated with resistance to various antimicrobials.

The classification of Staphylococcus aureus strains by biofilm production differs depending on the method used.

INTRODUCTION: Strains can be classified in terms of biofilm production from quantitative absorbance values collectively by dividing strains into tertile ranks or individually by calculating the optical density for the negative control. However, these methods have not been compared in a large sample of Staphylococcus aureus strains. Therefore, our objective was to analyze the agreement between both methods in terms of biomass production and metabolic activity of their biofilm. METHODS: We classified 233 S. aureus strains by biomass production and metabolic activity using the crystal violet and XTT assays, respectively. Strains were classified as low, moderate, or high biofilm producers according to tertile or optical density. RESULTS: We found no agreement between both methods (p<0.001 and p=0.028, respectively). CONCLUSIONS: We consider strains' biofilm classification by optical density to be a more reliable method, as it depends on the individual absorbance of each strain.

The classification of Staphylococcus aureus strains by biofilm production differs depending on the method used / La clasificación de cepas de Staphylococcus aureus según la producción de biopelícula varía dependiendo del método utilizado

IntroductionStrains can be classified in terms of biofilm production from quantitative absorbance values collectively by dividing strains into tertile ranks or individually by calculating the optical density for the negative control. However, these methods have not been compared in a large sample of Staphylococcus aureus strains. Therefore, our objective was to analyze the agreement between both methods in terms of biomass production and metabolic activity of their biofilm.MethodsWe classified 233 S. aureus strains by biomass production and metabolic activity using the crystal violet and XTT assays, respectively. Strains were classified as low, moderate, or high biofilm producers according to tertile or optical density.ResultsWe found no agreement between both methods (p<0.001 and p=0.028, respectively).ConclusionsWe consider strains’ biofilm classification by optical density to be a more reliable method, as it depends on the individual absorbance of each strain.

IntroducciónLas cepas se pueden clasificar en términos de producción de biopelícula a partir de valores cuantitativos de absorbancia dividiendo de forma colectiva las cepas en rangos por terciles o individualmente calculando la densidad óptica para el control negativo. Sin embargo, estos métodos no se han comparado en una gran muestra de cepas de Staphylococcus aureus. Por lo tanto, nuestro objetivo fue analizar la concordancia entre ambos métodos en términos de producción de biomasa y actividad metabólica de la biopelícula.MétodosSe clasificaron 233 cepas de S. aureus por producción de biomasa y actividad metabólica utilizando los ensayos de cristal violeta y de XTT, respectivamente. Las cepas se clasificaron como altamente, moderadamente o bajamente productoras de biopelícula según terciles o densidad óptica.ResultadosNo encontramos concordancias entre ambos métodos (p<0,001 y p=0,028, respectivamente).ConclusionesConsideramos que la clasificación del biofilm de cepas por densidad óptica es un método más fiable, ya que depende de la absorción individual de cada cepa.

Inhibition of LpxC Increases the Activity of Iron Chelators and Gallium Nitrate in Multidrug-Resistant Acinetobacter baumannii.

Infections caused by multidrug-resistant Acinetobacter baumannii would benefit from the development of novel treatment approaches. Compounds that interfere with bacterial iron metabolism, such as iron chelators and gallium nitrate, have previously been shown to have antimicrobial activity against A. baumannii. In this study, we characterize the effect of LpxC inhibitors on the antimicrobial activity of previously characterized iron chelators, 2,2'-bipyridyl (BIP) and deferiprone (DFP), and gallium nitrate (Ga(NO3)3) against A. baumannii reference strains and multidrug-resistant clinical isolates. The LpxC inhibitor LpxC-2 was synergistic with BIP for 30% of strains tested (FICI values: 0.38-1.02), whereas inhibition with LpxC-4 was synergistic with BIP for 60% of strains tested (FICI values: 0.09-0.75). In time-kill assays, combinations of BIP with both LpxC inhibitors demonstrated synergistic activity, with a more than 3 log10 reduction in bacterial counts compared to BIP alone. LpxC-2 was synergistic with Ga(NO3)3 for 50% of strains tested (FICI values: 0.27-1.0), whereas LpxC-4 was synergistic with Ga(NO3)3 for all strains tested (FICI values: 0.08-≤0.50). In time-kill assays, combinations of Ga(NO3)3 with LpxC-2 and LpxC-4 decreased the growth of both strains compared to each compound separately; however, only the combination with LpxC-4 met the defined criteria for synergy. These results identify a novel synergy between two antimicrobial classes against A. baumannii strains.

How should microbiology laboratories interpret cultures of the sonicate of closed needleless connectors? / ¿Cómo deben los laboratorios de microbiología interpretar los cultivos del «sonicado» de los conectores cerrados sin aguja?

Introduction: Our objective was to determine whether there is a cut-off in the needleless connectors’ (NCs) cultures that when combined with skin cultures it was as efficient as conventional superficial cultures to rule-out catheter colonization (CC) and catheter-related bloodstream infection (CRBSI). Methods: During 10 months, we collected samples and then we analyzed the validity values of skin+NCs cultures for CC and CRBSI considering the best cut-off showing at least >90% of specificity to have a high negative predictive value using a ROC curve. Results: We collected a total of 167 catheters. The optimal cut-off of NCs culture was 1000cfu/NC. The validity values for CC and CRBSI combining skin cultures and NCs cultures using the selected cut-off were, respectively: S, 42.9%/16.7%; SP, 83.6%/75.8%; PPV, 27.3%/2.5%; and NPV, 91.0%/96.0%. Conclusions: The combination of skin cultures and quantitative NCs cultures could be used for ruling-out CC and CRBSI.(AU)

Introducción: Nuestro objetivo fue determinar si existe un punto de corte en los cultivos de conectores sin aguja (NC) que, cuando se combina con cultivos de piel, sea tan eficiente como los cultivos superficiales convencionales para descartar colonización de catéter (CC) y bacteriemia relacionada con el catéter (BRC). Métodos: Durante 10 meses se coleccionaron muestras, y después se analizaron los valores de validez de los cultivos de piel + NC para CC y BRC considerando el mejor punto de corte aquel que mostrara al menos >90% de especificidad para tener un alto valor predictivo negativo usando una curva ROC. Resultados: Se estudiaron un total de 167 catéteres. El punto de corte óptimo del cultivo de NC fue de 1.000ufc/NC. Los valores de validez para CC y BRC combinando cultivos de piel y cultivos de NC utilizando el punto de corte seleccionado fueron, respectivamente: S: 42,9/16,7%; ES: 83,6/75,8%; VPP: 27,3/2,5% y VPN: 91,0/96,0%. Conclusiones: La combinación de cultivos de piel y cultivos cuantitativos de NC podría usarse para descartar CC y BRC.(AU)

How should microbiology laboratories interpret cultures of the sonicate of closed needleless connectors?

INTRODUCTION: Our objective was to determine whether there is a cut-off in the needleless connectors' (NCs) cultures that when combined with skin cultures it was as efficient as conventional superficial cultures to rule-out catheter colonization (CC) and catheter-related bloodstream infection (CRBSI). METHODS: During 10 months, we collected samples and then we analyzed the validity values of skin+NCs cultures for CC and CRBSI considering the best cut-off showing at least >90% of specificity to have a high negative predictive value using a ROC curve. RESULTS: We collected a total of 167 catheters. The optimal cut-off of NCs culture was 1000cfu/NC. The validity values for CC and CRBSI combining skin cultures and NCs cultures using the selected cut-off were, respectively: S, 42.9%/16.7%; SP, 83.6%/75.8%; PPV, 27.3%/2.5%; and NPV, 91.0%/96.0%. CONCLUSIONS: The combination of skin cultures and quantitative NCs cultures could be used for ruling-out CC and CRBSI.

The classification of Staphylococcus aureus strains by biofilm production differs depending on the method used.

INTRODUCTION: Strains can be classified in terms of biofilm production from quantitative absorbance values collectively by dividing strains into tertile ranks or individually by calculating the optical density for the negative control. However, these methods have not been compared in a large sample of Staphylococcus aureus strains. Therefore, our objective was to analyze the agreement between both methods in terms of biomass production and metabolic activity of their biofilm. METHODS: We classified 233 S. aureus strains by biomass production and metabolic activity using the crystal violet and XTT assays, respectively. Strains were classified as low, moderate, or high biofilm producers according to tertile or optical density. RESULTS: We found no agreement between both methods (p<0.001 and p=0.028, respectively). CONCLUSIONS: We consider strains' biofilm classification by optical density to be a more reliable method, as it depends on the individual absorbance of each strain.

Randomized clinical trial analyzing maintenance of peripheral venous catheters in an internal medicine unit: Heparin vs. saline.

BACKGROUND: Peripheral venous catheters (PVCs) require adequate maintenance based on heparin or saline locks in order to prevent complications. Heparin has proven effective in central venous catheters, although its use in PVCs remains controversial. Our hypothesis was that saline locks are as effective as heparin locks in preventing problems with PVCs. The objective of the present study was to compare phlebitis and catheter tip colonization rates between PVCs locked with saline and those locked with heparin in patients admitted to an internal medicine department (IMD). METHODS: We performed a 19-month prospective, controlled, open-label, randomized clinical study of patients with at least 1 PVC admitted to the IMD of our hospital. The patients were randomized to receive saline solution (PosiFlush®, group A) or heparin (Fibrilin®, group B) for daily maintenance of the PVC. Clinical and microbiological data were monitored to investigate the frequency of phlebitis, catheter tip colonization, and catheter-related bloodstream infection (C-RBSI), as well as crude mortality, days of hospital stay, and days of antimicrobial treatment. RESULTS: We assessed 339 PVCs (241 patients), of which 192 (56.6%) were locked with saline (group A) and 147 (43.4%) with heparin (group B). The main demographic characteristics of the patients were distributed equally between the 2 study groups. The median (IQR) catheter days was 5 (3-8) for both groups (p = 0.64). The frequency of phlebitis was 17.7% for group A and 13.3% for group B (p = 0.30). The frequency of colonization of PVC tips was 14.6% and 12.2% in groups A and B, respectively (p = 0.63). Only 2 episodes of C-RBSI were detected (1 patient in group A). Saline lock was not an independent factor for phlebitis or catheter colonization. CONCLUSIONS: Our study revealed no statistically significant differences in the frequency of phlebitis and catheter tip colonization between PVCs locked with saline and PVCs locked with heparin. We suggest that PVC can be maintained with saline solution, as it is safer and cheaper than heparin.

Evaluation of the Alfred™ turbidity monitoring system (Alifax®) following sonication in the diagnosis of central venous catheter colonization.

The conventional diagnostic techniques for catheter colonization (CC) take at least 48 h to yield results. Therefore, new diagnostic procedures that speed up the time necessary for results are needed. Our main objective was to assess the efficacy of the combination of sonication, turbidity monitoring, and MALDI-TOF to detect CC and catheter-related bloodstream infection (C-RBSI). For 1 year, we assessed central venous catheter (CVC) tips that arrived at the microbiology laboratory from adult patients admitted to our institution. CVC tips were cut, inoculated into 2.5 ml of BHI, and sonicated for 1 min. The suspension was then processed using Gram stain, quantitative culture (gold standard), and preincubation on the Alfred™ system. We analyzed the validity values of our new diagnostic approach for prediction of CC and C-RBSI and compared them with those of the gold standard. We collected a total of 167 catheters, 33 (19.8%) of which were colonized. We confirmed 21 episodes of C-RBSI. The distribution of microorganisms in colonized CVCs was as follows: Gram-positive, 68.4%; Gram-negative, 5.3%; and yeasts, 26.3%. The validity values for CC and C-RBSI using the new procedure were as follows: S, 39.4%/61.9%; Sp, 100%/100%; PPV, 100%/100%; and NPV, 87.0%/94.8%. The combination of sonication with a pre-incubation period based on turbidity monitoring using the Alfred™ system followed by MALDI-TOF proved to be a useful tool that was faster than conventional culture for ruling out C-RBSI. Future studies are needed to assess the clinical and economic impact of this diagnostic approach.

Can dalbavancin be used as a catheter lock solution?

Purpose. The new lipoglycopeptide dalbavancin has only been approved for acute bacterial skin and skin structure infections. However, its alternative use as a catheter lock solution could facilitate the conservative management of catheter-related bloodstream infection. Our objective was to assess the stability and activity of dalbavancin alone and in combination with heparin against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) biofilms. We also compared the results with those obtained with vancomycin alone and in combination with heparin.Methodology. We used a 96-well plate in vitro model based on 24 h biofilms of MRSA and MRSE (ATCC 43300, ATCC 35984 and one clinical strain of each). The biofilms were exposed to dalbavancin (0.128 mg ml-1) and vancomycin (5 mg ml-1) alone and in combination with heparin (60 IU). The median percentage reductions in metabolic activity, biomass, bacterial load, and cell viability for each solution were compared.Results. Dalbavancin combined with heparin significantly reduced the median [interquartile range (IQR)] percentage of metabolic activity in MRSA biofilms compared with vancomycin [90.0 % (70.4-92.9 %) versus 35.0 % (14.8-59.6 %), P=0.006]. For the remaining variables studied, the combination was not inferior to vancomycin for MRSA and MRSE.Conclusions. Dalbavancin proved to be active against MRSA and MRSE biofilms. The combination of dalbavancin with heparin is a promising catheter lock solution that has the advantage of locking the catheter at home for 7 days.

Activity of maltodextrin and vancomycin against staphylococcus aureus biofilm.

We aimed to assess the anti-biofilm activity of vancomycin, maltodextrin, and their combination against vancomycin resistant Staphylococcus aureus (VRSA) and vancomycin-susceptible S. aureus (VSSA) strains based on an in vitro static model. Biofilms of 4 VSSA and 2 VRSA strains were grown in a 96-well static model. Vancomycin 2 mM, maltodextrin 10 mM, and both in combination were tested using tetrazolium salt (XTT), resazurin, and cfu/well counts. The efficacy of the antimicrobial solutions was expressed as the percentage reduction in metabolic activity with each method. Overall percentage reduction in the metabolic activity of VSSA was 79.3%, 34%, and 75.7% for vancomycin, maltodextrin, and their combination (p<0.001). Overall percentage reduction in metabolic activity of VRSA was 46.7%, 27.8%, and 34.6% for vancomycin, maltodextrin, and their combination (p>0.05). Maltodextrin did not improve the anti-biofilm efficacy of vancomycin in VSSA or in VRSA biofilms. XTT cannot replace cfu counts as a means of quantifying cell viability. Futures studies are needed to assess the synergistic effects of other non-antimicrobial molecules combined with vancomycin.

The NeutraClear® needleless connector is equally effective against catheter colonization compared to MicroClave®.

INTRODUCTION: Neutral-valve closed-system connectors can reduce the frequency of catheter colonization. Commercially available closed system connectors need to be tested and compared with each other to assess how they protect against contamination. We aimed to compare, in vitro, the efficacy of connectors NeutraClear® and MicroClave® against contamination under conditions of daily clinical practice. METHODS: The model consisted of a set of 200 blood culture bottles (BCBs) with a cannula inserted (100 closed with NeutraClear® and 100 closed with MicroClave®) that were assessed in two experiments while instilling 1 mL of saline: manipulation based on the standard of care and manipulation using gloves impregnated with a 0.05 McFarland Staphylococcus aureus solution. The BCBs were incubated in a BACTEC System at 37°C under continuous shaking for up to 7 days. When a bottle turned positive, 100 µL of the fluid was cultured. The positivity rate and time to positivity of the BCB in each experiment was compared. RESULTS: In the aseptic model in the NeutraClear® and MicroClave® groups, only 1 BCB and 2 BCBs were positive, respectively, (p = 0.55). In the contaminated model, all BCBs were positive in both groups at the end of the incubation time. We did not find differences for the MTP between NeutraClear® and MicroClave® (36.04 vs. 20.13 hours, p = 0.09). CONCLUSIONS: The NeutraClear® needleless connector proved to be as efficient as the MicroClave® connector in the prevention of catheter colonization and migration of S. aureus from the surface to the inside of the hub in an in vitro model.

Comparison of the XTT and resazurin assays for quantification of the metabolic activity of Staphylococcus aureus biofilm.

We assessed whether resazurin was as efficient as XTT in the measurement of the metabolic activity of 209 clinical Staphylococcus aureus biofilm using an vitro model comparing the percentage of formazan and resorufin. The overall categorical agreement was 61.2% (r=0.024), which means that resazurin can not substitute XTT.

Assessment of biofilm production in Candida isolates according to species and origin of infection / Determinación de la producción de biopelícula en aislados de Candida de acuerdo con las especies de Candida y el origen de la infección

The biofilm production (BP) of 200 clinical strains of Candida isolated during 2010-2013 were assessed using an in vitro model and a comparison of the results was made between species and between origins of the infections. The BP was assessed using the crystal violet assay, and the strains were classified as low, moderate, or high biofilm producers. Candida tropicalis had the highest values for BP, which varied depending on the origin of the infection. Assessment of BP is a key diagnostic tool that enables us to better understand Candida infections

Desde 2010 a 2013 evaluamos la producción de biopelícula (PB) en 200 cepas clínicas de Candida y comparamos los resultados de las especies de Candida entre los orígenes de la infección mediante un modelo in vitro. La PB se determinó con el ensayo de cristal violeta y las cepas se clasificaron como baja, moderada o altamente productoras de biopelícula. C. tropicalis tuvo los valores más altos de PB, y la PB en Candida varió dependiendo del origen de la infección. La determinación de la PB es una herramienta diagnóstica importante para entender mejor las infecciones por Candida

Assessment of biofilm production in Candida isolates according to species and origin of infection.

The biofilm production (BP) of 200 clinical strains of Candida isolated during 2010-2013 were assessed using an in vitro model and a comparison of the results was made between species and between origins of the infections. The BP was assessed using the crystal violet assay, and the strains were classified as low, moderate, or high biofilm producers. Candida tropicalis had the highest values for BP, which varied depending on the origin of the infection. Assessment of BP is a key diagnostic tool that enables us to better understand Candida infections.

Does biomass production correlate with metabolic activity in Staphylococcus aureus?

We assessed agreement between the crystal violet binding assay and the XTT assay in the classification of biofilm production in 492 Staphylococcus aureus strains from bacteremic patients. We found that the overall correlation between the procedures was 46.5%. Biomass production and metabolic activity must be assessed simultaneously.

Corrigendum to "sonicating multi-lumen sliced catheter tips after the roll-plate technique improves the detection of catheter colonization in adults" [J. Microbiol. Methods 122 (2016) 20-22].

The Maki technique is the standard method for detecting catheter tip (CT) colonization. However, some "multi-lumen" catheters finish in a vaulted fornix and end at different distances from the CT. Therefore, we compared the traditional Maki technique with the sonication method using several cross-cut fragments of the CT. Our objective was to assess the yield of the Maki technique followed by sonication in the detection of adult CT colonization and catheter-related bloodstream infection (C-RBSI). For 3months, we prospectively performed CT cultures of polyurethane catheters from adult patients admitted to our institution. First, we performed CT culture using the Maki technique on blood agar plates and then sonicated small fragments of CTs in 5ml of BHI followed by culture of 100µl of the sonicate. We included a total of 252 CVCs, with overall colonization and C-RBSI rates of 14.3% (36/252) and 5.9% (15/252). Of the 36 colonized CVCs, 21 (58.3%) were detected both by Maki and sonication, 6 (16.7%) were detected only by Maki technique, and 9 (25.0%) only by sonication method. Among 15 episodes with concomitant bacteremia, both techniques were positive and concordant in 11 cases (73.3%) and in 4 cases (26.7%) sonication was the only positive technique. Our study shows that both techniques are complementary. We recommend sonicating fragments of the CT from patients with bacteremia of unknown origin and a negative CT culture by the Maki technique.

Use of MALDI-TOF to detect colonized vascular catheter tips after 6 and 12h of incubation.

We analyzed by MALDI-TOF MS 80 catheter tips after 6h and 12h of incubation and the sensitivity of each incubation period for the identification of colonization and C-RBSI was, respectively, 9.5%-NA and 42.9%-28.6%. Despite MALDI-TOF MS cannot be used to predict catheter colonization, it may rule out C-RBSI.