Clinical Outcome of Patients with Escherichia coli Isolated from Catheter Lumens and/or Peripheral Blood Cultures: A Retrospective Analysis.

BACKGROUND: Escherichia coli commonly causes catheter-related bloodstream infection (C-RBSI) in specific populations. The differential time to positivity (DTTP) technique is the recommended conservative procedure for diagnosing C-RBSIs. METHODS: We conducted a retrospective study of episodes in which E. coli was isolated from catheter lumens obtained using the DTTP technique. Microbiological and clinical data were obtained based on the DTTP technique as either catheter colonization, C-RBSI, or non-C-RBSI. RESULTS: A total of 89 catheter blood cultures were included, classified as follows: catheter colonization, 33.7%; C-RBSI, 9.0%; and non-C-RBSI, 57.3%. Only 15.7% of the catheters were withdrawn, with no positive catheter-tip cultures. We found no statistically significant differences in catheter type, antibiotic treatment, or clinical outcome among the groups, except for the frequency of catheter lock therapy or in the frequency of successful treatment. Mortality was associated with C-RBSI in only one patient. CONCLUSION: E. coli bacteremia diagnosed by the DTTP technique was classified as non-catheter-related in most patients. As the majority of the catheters were retained, E. coli bacteremia could not be microbiologically confirmed as catheter-related by the catheter-tip culture. Future studies are needed to assess the profitability of the DTTP technique for diagnosing E. coli C-RBSIs.

Addressing catheter lock therapy: Does heparin reduce the bioactivity of dalbavancin when together in solution during freezing?

INTRODUCTION: The possible use of dalbavancin as a catheter lock solution was previously demonstrated by our study group. However, it was needed to assess whether heparin could affect dalbavancin bioactivity during freezing storage. METHODS: We tested the bioactivity of a dalbavancin+heparin (DH) vs. dalbavancin (D) against Staphylococcal biofilms comparing DH median value of cfu counts and metabolic activity with that obtained for D before and during storage under freezing up to 6 months. RESULTS: Despite there was a slight decrease in the median percentage reduction of metabolic activity at month 3 in Staphylococcus epidermidis between DH and D (97.6 vs. 100, p=0.037), considering the clinical criteria, no significant reduction in any of the variables tested was observed at the end of the experiment between D and DH solutions. CONCLUSION: The addition of heparin to a dalbavancin lock solution did not affect its bioactivity against staphylococcal biofilms irrespective of its preservation time under freezing.

Validation of the antibacterial effect of topically applied tranexamic acid using in vitro and in vivo models.

Background: Several studies have shown that tranexamic acid (TXA), an antifibrinolytic, reduces postoperative infection rates. Recent in vitro research showed that TXA alone and in combination with vancomycin and gentamicin had a synergistic effect against some staphylococcal strains. In the present study, this synergistic effect was validated in samples from patients with staphylococcal periprosthetic infection (PPI) and in an in vivo model. Methods: We tested 19 clinical strains (5 Staphylococcus aureus and 14 coagulase-negative staphylococci [CoNS]) against 10 mg/ml TXA alone and in combination with serial dilutions of vancomycin and gentamicin. The standardized microtiter plate method was used. The minimal inhibitory concentration (MIC) were calculated using standard visualization of well turbidity. We also used an S. aureus (ATCC29213) murine subcranial PPI model to compare the synergistic effect of TXA and gentamicin with that of TXA or gentamicin alone after 4 days of monitoring. The mice were euthanized, and disks were removed for analysis of cfu/ml counts and cell viability rate. Biofilm structure of both in vitro and in vivo samples was also analyzed using scanning electron microscopy (SEM). Results: When TXA was combined with vancomycin or gentamicin, the MIC decreased in 30% of the strains studied. According to species, the MIC50 for vancomycin and gentamicin alone and in combination with TXA against S. aureus strains was the same. This was also the case for CoNS with vancomycin and its corresponding combination, whereas with gentamicin and TXA, a reduction in MIC50 was observed (2 dilutions). In addition, in the in vivo model, the mean (SD) log cfu/ml and cell viability rate obtained from the implant was lower in the group of mice treated with TXA and gentamicin than in those treated only with TXA or gentamicin. SEM images also corroborated our findings in strains in which the MIC was reduced, as well as the in the mice implants, with the area occupied by biofilm being greater in samples treated only with gentamicin or TXA than in those treated with TXA+gentamicin. Conclusion: We confirm that combining TXA with vancomycin or gentamicin exerts a synergistic effect. However, this only occurs in selected strains.

Corrigendum: Tranexamic acid in combination with vancomycin or gentamicin has a synergistic effect against staphylococci.

[This corrects the article DOI: 10.3389/fmicb.2022.935646.].

Understanding the diagnosis of catheter-related bloodstream infection: real-time monitoring of biofilm growth dynamics using time-lapse optical microscopy.

Background: The differential time to positivity (DTTP) technique is recommended for the conservative diagnosis of catheter-related bloodstream infection (C-RBSI). The technique is based on a 120-minute difference between microbial growth in blood drawn through the catheter and blood drawn through a peripheral vein. However, this cut-off has failed to confirm C-RBSI caused by Candida spp. and Staphylococcus aureus. Objective: We hypothesized that the biofilm of both microorganisms disperses faster than that of other microorganisms and that microbial load is rapidly equalized between catheter and peripheral blood. Therefore, our aim was to compare the biofilm dynamics of various microorganisms. Methods: Biofilm of ATCC strains of methicillin-resistant Staphylococcus epidermidis, methicillin-susceptible S. aureus, Enterococcus faecalis, Escherichia coli and Candida albicans was grown on silicon disks and analyzed using time-lapse optical microscopy. The time-lapse images of biofilms were processed using ImageJ2 software. Cell dispersal time and biofilm thickness were calculated. Results: The mean (standard deviation) dispersal time in C. albicans and S. aureus biofilms was at least nearly 3 hours lower than in biofilm of S. epidermidis, and at least 15 minutes than in E. faecalis and E. coli biofilms. Conclusion: Our findings could explain why early dissemination of cells in C. albicans and S. aureus prevents us from confirming or ruling out the catheter as the source of the bloodstream infection using the cut-off of 120 minutes in the DTTP technique. In addition, DTTP may not be sufficiently reliable for E. coli since their dispersion time is less than the cut-off of 120 minutes.

An In Vitro Study to Assess the Best Strategy for the Chemical Debridement of Periprosthetic Joint Infection.

Irrigation and debridement using an irrigation solution is a fundamental step during the surgical treatment of both acute and chronic periprosthetic joint infection (PJI). However, there is no consensus on the optimal solution, nor is there sufficient evidence on the optimal irrigation time and combination of solutions. Therefore, it is necessary to determine which solution or combination of solutions is most efficacious against biofilm, as well as the optimal irrigation time. We conducted an experimental in vitro model by inoculating stainless steel discs with ATCC strains of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and a clinical strain of Staphylococcus epidermidis. The discs were all irrigated with commonly used antiseptic solutions (10% and 3% povidone iodine, hydrogen peroxide, 3% acetic acid, and Bactisure™) for 1 min, 3 min, and 5 min and their combinations for 9 min (3 min each) vs. sterile saline as a positive control. We evaluated the reduction in biofilm based on colony-forming unit (cfu) counts and in combination assays, also based on cell viability and scanning electron microscopy. All antiseptics alone reduced more than 90% of cfu counts after 1 min of irrigation; the worst results were for hydrogen peroxide and 3% acetic acid. When solutions were sequentially combined, the best results were observed for all those starting with acetic acid, in terms of both reduction of log cfu/mL counts and viable cells. We consider that a combination of antiseptic solutions, particularly that comprising the sequence acetic acid + povidone iodine + hydrogen peroxide, would be the best option for chemical debridement during PJI surgery.

In Vitro Efficacy of Dalbavancin as a Long-Acting Anti-Biofilm Agent Loaded in Bone Cement.

Based on previous studies by our group in which we demonstrated that dalbavancin loaded in bone cement had good elution capacity for the treatment of biofilm-related periprosthetic infections, we now assess the anti-biofilm activity of dalbavancin and compare it with that of vancomycin over a 3-month period. We designed an in vitro model in which we calculated the percentage reduction in log cfu/mL counts of sonicated steel discs contaminated with staphylococci and further exposed to bone cement discs loaded with 2.5% or 5% vancomycin and dalbavancin at various timepoints (24 h, 48 h, 1 week, 2 weeks, 6 weeks, and 3 months). In addition, we tested the anti-biofilm activity of eluted vancomycin and dalbavancin at each timepoint based on a 96-well plate model in which we assessed the percentage reduction in metabolic activity. We observed a significant decrease in the dalbavancin concentration from 2 weeks of incubation, with sustained anti-biofilm activity up to 3 months. In the case of vancomycin, we observed a significant decrease at 1 week. The concentration gradually increased, leading to significantly lower anti-biofilm activity. The percentage reduction in cfu/mL counts was higher for dalbavancin than for vancomycin at both the 2.5% and the 5% concentrations. The reduction in log cfu/mL counts was higher for S. epidermidis than for S. aureus and was particularly more notable for 5% dalbavancin at 3 months. In addition, the percentage reduction in metabolic activity also decreased at 3 months in 5% dalbavancin and 5% vancomycin, with more notable values recorded for the latter.

In vitro study to assess modulation of Candida biofilm by Escherichia coli from vaginal strains.

Background: Vulvovaginal candidiasis (VVC) is caused by biofilm formation and epithelial invasion. In addition, Escherichia coli (EC) can establish a vaginal intracellular reservoir modulating Candida spp. biofilm production. We aimed to analyze the behavior of Candida albicans (CA) and EC biofilm both in single cultures and in co-cultures. Methods: We prospectively collected CA and EC isolates from vaginal swabs over 6 months. We selected positive cultures with both CA and EC (cases) and a comparator group with either CA or EC (controls). We analyzed overall biomass production and metabolic activity in single cultures and in co-cultures based on staining assays, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) to assess biofilm occupation. We also analyzed clinical manifestations. Results: We cultured 455 samples, 16 (3.5%) of which had CA and EC (cases); only CA or EC (controls) was detected, respectively, in 72 (15.8%) and 98 (21.5%). Biomass production and metabolic activity were significantly more pronounced in co-cultures in both groups. CLSM and SEM, on the other hand, showed the biofilm of each species to be significantly reduced when they were cultured together, with higher values in CA (percentage biofilm reduction: CA, 95.8% vs. EC, 36.2%, p < 0.001). There were no clinically significant differences between co-infected patients and patients infected only by C. albicans. Conclusion: Ours is the first study assessing co-cultures of CA and EC in a large collection of samples. We observed that coinfection of CA and EC was unusual (3.5%) and promoted high biomass, whereas microscopy enabled us to detect a reduction in biofilm production when microorganisms were co-cultured. No differences in symptoms were observed.

Determination of the Elution Capacity of Dalbavancin in Bone Cements: New Alternative for the Treatment of Biofilm-Related Peri-Prosthetic Joint Infections Based on an In Vitro Study.

Antibiotic-loaded bone cement is the most widely used approach for the treatment of biofilm-induced septic sequelae in orthopedic surgery. Dalbavancin is a lipoglycopeptide that acts against Gram-positive bacteria and has a long half-life, so we aimed to assess whether it could be a new alternative drug in antibiotic-loaded bone cement for the treatment of periprosthetic joint infections. We assessed the elution capacity of dalbavancin and compared it with that of vancomycin in bone cement. Palacos®R (Heraeus Medical GmbH, Wehrheim, Germany) bone cement was manually mixed with each of the antibiotics studied at 2.5% and 5%. Three cylinders were obtained from each of the mixtures; these were weighed and incubated in 5 mL phosphate-buffered saline at 37°C under shaking for 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 168 h, and 336 h. PBS was replenished at each time point. The samples were analyzed using high-performance liquid chromatography (vancomycin) and mass cytometry (dalbavancin). Elution was higher than the minimum inhibitory concentration (MIC)90 for both antibiotics after 14 days of study. The release of vancomycin at 14 days was higher than of dalbavancin at each concentration tested (p = 0.05, both). However, the cumulative release of 5% dalbavancin was similar to that of 2.5% vancomycin (p = 0.513). The elution capacity of dalbavancin reached a cumulative concentration similar to that of vancomycin. Moreover, considering that the MIC90 of dalbavancin is one third that of vancomycin (0.06 mg/L and 2 mg/L, respectively) and given the long half-life of dalbavancin, it may be a new alternative for the treatment of biofilm-related periprosthetic infections when loaded in bone cement.

Tranexamic Acid in Combination With Vancomycin or Gentamicin Has a Synergistic Effect Against Staphylococci.

Background: Tranexamic acid (TXA) is an antifibrinolytic agent applied in orthopedic surgery and has been proven to reduce post-surgery infection rates. We previously showed that TXA also had an additional direct antimicrobial effect against planktonic bacteria. Therefore, we aimed to evaluate whether it has a synergistic effect if in combination with antibiotics. Materials and Methods: Three ATCC and seven clinical strains of staphylococci were tested against serial dilutions of vancomycin and gentamicin alone and in combination with TXA at 10 and 50 mg/ml. The standardized microtiter plate method was used. Minimal inhibitory concentrations (MICs) were calculated by standard visualization of well turbidity (the lowest concentration at which complete absence of well bacterial growth was observed by the researcher) and using the automated method (the lowest concentration at which ≥80% reduction in well bacterial growth was measured using a spectrophotometer). Results: Tranexamic acid-10 mg/ml reduced the MIC of vancomycin and gentamicin with both the standard method (V: 1-fold dilution, G: 4-fold dilutions) and the automated turbidity method (vancomycin: 8-fold dilutions, gentamicin: 8-fold dilutions). TXA-50 mg/ml reduced the MIC of gentamicin with both the standard turbidity method (6-fold dilutions) and the automated turbidity method (1-fold dilutions). In contrast, for vancomycin, the MIC remained the same using the standard method, and only a 1-fold dilution was reduced using the automated method. Conclusion: Ours was a proof-of-concept study in which we suggest that TXA may have a synergistic effect when combined with both vancomycin and gentamicin, especially at 10 mg/ml, which is the concentration generally used in clinical practice.

Efficacy of Povidone Iodine Against Microbial Biofilms in Breast Implants With Different Textures: Results From an in vitro Study.

Background: In the practice of breast augmentation and reconstruction, implant irrigation with various solutions has been widely used to prevent infection and capsular contracture, but to date, there is no consensus on the optimal protocol to use. Recently, application of povidone iodine (PI) for 30 min has shown in vitro to be the most effective irrigating formula in reducing contamination in smooth breast implants. However, as 30 min is not feasible intraoperatively, it is necessary to determine whether shorter times could be equally effective as well as to test it in both smooth and textured implants. Methods: We tested the efficacy of 10% PI at 1', 3', and 5' against biofilms of 8 strains (2 ATCC and 6 clinical) of Staphylococcus spp. on silicone disks obtained from Mentor® and Polytech® implants of different textures. We analyzed the percentage reduction of cfu counts, cell viability and bacterial density between treatment (PI) and control (sterile saline, SS) groups for each time of application. We consider clinical significance when > 25% reduction was observed in cell viability or bacterial density. Results: All textured implants treated with PI at any of the 3 exposure times reduced 100% bacterial load by culture. However, none of the implants reached enough clinical significance in percentage reduction of living cells. Regarding bacterial density, only 25-50 µm Polytxt® Polytech® implants showed significant reduction at the three PI exposure times. Conclusion: PI is able to inhibit bacterial growth applied on the surface of breast implants regardless of the exposure time. However, no significant reduction on living cells or bacterial density was observed. This lack of correlation may be caused by differences in texture that directly affect PI absorption.

Effect of Tranexamic Acid against Staphylococcus spp. and Cutibacterium acnes Associated with Peri-Implant Infection: Results from an In Vitro Study.

Tranexamic acid (TXA) is extensively used in orthopedic surgery and traumatology as an antifibrinolytic agent to control intra- and postoperative bleeding and, therefore, indirectly, to reduce postsurgery infection rates. The hypothesis of an additional antibiotic effect against microorganisms associated with periprosthetic joint infection needs to be further evaluated. We aimed to assess whether TXA could reduce bacterial growth using an in vitro model. ATCC and clinical strains of staphylococci and Cutibacterium acnes were tested against TXA in both planktonic and sessile forms. We recorded the percent reduction in the following variables: log CFU/mL by microbiological culture, percentage of live cells by confocal laser scanning microscopy, and, additionally in sessile cells, metabolic activity by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay. Variables were compared between groups using the Kruskal-Wallis test, and the results were reported as median (interquartile range [IQR]). Statistical significance was set at a P value of <0.05. Clinical significance was defined as a reduction of ≥25%. TXA at 50 mg/mL led to a slight reduction in CFU counts (4.5%). However, it was at 10 mg/mL that the reduction reached 27.2% and 33.0% for log CFU/mL counts and percentage of live cells, respectively. TXA was not efficacious for reducing preformed 24-h mature staphylococci and 48-h mature C. acnes biofilms, regardless of its concentration. TXA did not exert an antimicrobial effect against bacterial biofilms. However, when bacteria were in the planktonic form, it led to a clinically and statistically significant reduction in bacterial growth at 10 mg/mL. IMPORTANCE The possible use of TXA as an antibiotic agent in addition to its antifibrinolytic effect may play an important role in the prevention of prosthetic joint infection.

A Dalbavancin Lock Solution Can Reduce Enterococcal Biofilms After Freezing.

INTRODUCTION: We previously demonstrated the efficacy of a frozen dalbavancin-heparin (DH) lock solution against biofilms of staphylococci. However, as enterococci also commonly cause catheter-related bloodstream infections (C-RBSI), we assessed the bioactivity of frozen dalbavancin (D) and DH against enterococci. METHODS: Over 6 months, we compared the bioactivity of a solution of DH (1 mg/ml) with that of D in terms of cfu counts and metabolic activity against biofilms of Enterococcus faecalis and Enterococcus faecium (four strains each). For each solution, we individually compared results obtained at each time point (months 3 and 6) with baseline (month 0). We also compared the median DH value of each variable at baseline and at months 3 and 6 of freezing with the values obtained for D alone. We used both statistical and clinical criteria when results were within 25% of the reference value. RESULTS: At the end of the experiment (month 6), neither a statistically nor a clinically significant reduction in the bioactivity of D solution was observed in terms of cfu count and metabolic activity against enterococcal biofilms. Regarding the DH solution, we found both statistical and clinical significance in the median percentage reduction in metabolic activity between months 0 and 6 in E. faecalis strains (51.8% vs. 77.8%, P = 0.007). Moreover, after freezing, the DH solution lost significant bioactivity compared with the D solution, especially in E. faecalis. CONCLUSION: A dalbavancin lock solution can be frozen for up to 6 months with no negative effect on its bioactivity against enterococcal biofilms. However, when combined with heparin, its efficacy was reduced. Therefore, we recommend that if lock therapy with frozen dalbavancin is used in the management of enterococcal C-RBSI, heparin should be added simultaneously at the time of catheter lock.

Determination of the ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify high-biofilm-producing strains.

Background: The traditional method for assessing the capacity of a microorganism to produce biofilm is generally a static in vitro model in a multi-well plate using the crystal violet (CV) binding assay, which takes 96 h. Furthermore, while the method is simple to perform, its reproducibility is poor. Objective: We evaluated whether matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) could make it possible to differentiate between high-and low-biofilm-producing microorganisms on 24-h cultures of Staphylococcus aureus and Candida albicans. Methods: We included 157 strains of S. aureus and 91 strains of C. albicans obtained from the blood cultures of patients with bacteremia/candidemia. We tested biofilm production using the CV binding assay as the gold standard to classify strains as low or high biofilm producers. We then applied MALDI-TOF MS to create a machine learning-based predictive model using 40 strains of S. aureus and C. albicans, each with extreme absorbance values, and validated this approach with the remaining 117 and 51 strains using the random forest algorithm and the support vector machine algorithm, respectively. Results: Overall, 81.2% of the S. aureus strains (95/117) and 74.5% of the C. albicans strains (38/51) used for validation were correctly categorized, respectively, as low and high-biofilm-producing. Conclusion: Classification based on MALDI-TOF MS protein spectra enables us to predict acceptable information about the capacity of 24-h cultures of S. aureus and C. albicans to form biofilm.

Antiparasitic Effects of Potentially Toxic Beetles (Tenebrionidae and Meloidae) from Steppe Zones.

Arthropods and specifically beetles can synthesize and/or sequester metabolites from dietary sources. In beetle families such as Tenebrionidae and Meloidae, a few studies have reported species with toxic defensive substances and antiparasitic properties that are consumed by birds. Here we have studied the antiparasitic activity of extracts from beetle species present in the habitat of the Great Bustard (Otis tarda) against four pathogen models (Aspergillus niger, Meloidogyne javanica, Hyalomma lusitanicum, and Trichomonas gallinae). The insect species extracted were Tentyria peiroleri, Scaurus uncinus, Blaps lethifera (Tenebrionidae), and Mylabris quadripunctata (Meloidae). M. quadripunctata exhibited potent activity against M. javanica and T. gallinae, while T. peiroleri exhibited moderate antiprotozoal activity. The chemical composition of the insect extracts was studied by gas chromatography coupled with mass spectrometry (GC-MS) analysis. The most abundant compounds in the four beetle extracts were hydrocarbons and fatty acids such as palmitic acid, myristic acid and methyl linoleate, which are characteristic of insect cuticles. The presence of cantharidin (CTD) in the M. quadripunctata meloid and ethyl oleate (EO) in T. peiroleri accounted for the bioactivity of their extracts.