Antibiofilm Substances Research Articles

Sellado antibiótico de reservorios subcutáneos: estabilidad de soluciones con vancomicina

Subcutaneous reservoirs are a type of central venous catheter. When using central venous catheters, healthcare workers need to avoid two major risks: clot formation and bacterial infections. To prevent and avoid catheter contamination in both hospitalized patients and outpatients, several strategies have been carried out, such as the so-called ” antibiotic-based catheter lock solution”. Therefore, it has been suggested to implement the use of solutions with antimicrobial agents, to which anticoagulant and/or antibiofilm substances are often added. However, the stability of such solutions needs to be tested by techniques such as high performance liquid chromatography (HPLC), in addition to antimicrobial efficacy testing, in order to establish patient safety. In consequence, this literature review aims to include the most clinically representative research towards these aspects, to demonstrate the behaviour of antibiotic-based catheter lock solutions under different conditions of storage and use. In particular, this review focuses on solutions containing vancomycin. According to the studies consulted, vancomycin solutions with sodium citrate (chelating agent) present the best stability characteristics in terms of physicochemical properties and efficacy.. Keywords: Vancomycin; heparin; sodium citrate; stability; subcutaneous implantable central venous device

A novel antibacterial and anti-biofilm material: Biosynthetic silver nanoparticles mediated by extract from Oplopanax elatus adventitious roots

The search for plant resources involved in the biosynthesis of silver nanoparticles (AgNPs) is crucial for the diversity of AgNP materials. Oplopanax elatus is a valuable medicinal shrub and its adventitious root (AR) culture is a modern method for producing the raw material. To explore an efficient biosynthetic AgNPs, this study synthesized AgNPs using O. elatus AR extract (OE-AgNPs) and the characterization of OE-AgNPs was performed. Finally, the antibacterial and anti-biofilm effects of OE-AgNPs were investigated. The result showed that OE-AgNPs had an obvious absorption peak of nanosilver with a spherical shape and a silver-like face-centered cubic structure, which the OE-AgNPs were capped by compounds with aliphatic amine, amide, carbonyl, and hydroxyl groups of O. elatus AR extract, indicating a successful synthesis of OE-AgNPs. The 10–40 nm particles were the most abundant in the OE-AgNPs, accounting for 70%. OE-AgNPs had the strongest antibacterial activity against Escherichia coli and Pseudomonas aeruginosa among six bacterial species tested, with a minimum inhibitory concentration (MIC) of 0.75 μg/mL, and the cell contents of E. coli and P. aeruginosa leaked out by OE-AgNP treatment, and bacterial respiration was inhibited. After OE-AgNP treatment, biofilms of E. coli and P. aeruginosa were efficiently scavenged with a scavenging rate of more than 60%; the extracellular polysaccharide concentrations in biofilms were also ruduced. The findings of this study suggested that OE-AgNPs could be used as a potential antibacterial and antibiofilm substance to apply in the product production and indicated the feasibility of expanding the application of O. elatus ARs.

Antimicrobial and biofilm inhibition effects of p-anisaldehyde against Vibrio parahaemolyticus

Vibrio parahaemolyticus is a food-borne bacterial pathogen that seriously threatens the safety of seafood and human health. Biofilm formation is an important cause of pathogenicity and effective natural antibiofilm substances are urgently needed. Herein, we investigated the antimicrobial and antibiofilm effects of p-anisaldehyde (PAA) from Pimpinella anisum L. against V. parahaemolyticus, along with its application on salmon. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PAA against V. parahaemolyticus was 2 μL/mL. PAA inhibited the growth, motility and adhesion of V. parahaemolyticus in a dose-dependent manner. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) confirmed that cell membrane integrity was disrupted and the ultrastructure of cells was damaged. Transcriptome analysis showed that treatment with PAA resulted in significant differences in the expression of 2415 genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that differentially expressed genes related to biofilm formation, flagella mobility and quorum sensing (QS) were significantly altered in V. parahaemolyticus. Notably, PAA could also inhibit V. parahaemolyticus in salmon via fumigation, suggesting its potential application in food preservation. This study identified PAA as a potential natural antimicrobial agent to inhibit biofilm formation of V. parahaemolyticus and thereby protect salmon against infection.

Exploration of the Main Antibiofilm Substance of Lactobacillus plantarum ATCC 14917 and Its Effect against Streptococcus mutans.

Dental plaque, a complex biofilm system established by cariogenic bacteria such as Streptococcus mutans (S. mutans), is the initiator of dental caries. Studies have found that the cell-free supernatant (CFS) of Lactobacilli could inhibit S. mutans biofilm formation. However, the main antibiofilm substance of the Lactobacilli CFS that acts against S. mutans is unclear. The present study found that the CFS of Lactobacillus plantarum (L. plantarum) ATCC 14917 had the strongest antibiofilm effect among the five tested oral Lactobacilli. Further bioassay-guided isolation was performed to identify the main antibiofilm substance. The antibiofilm effect of the end product, named 1-1-4-3, was observed and the structure of it was elucidated by using Q-TOF MS, 2D NMR and HPLC. The results showed that several components in the CFS had an antibiofilm effect; however, the effect of 1-1-4-3 was the strongest, as it could reduce the generation of exopolysaccharides and make the biofilm looser and thinner. After structure elucidation and validation, 1-1-4-3 was identified as a mixture of lactic acid (LA) and valine. Additionally, LA was shown to be the main antibiofilm substance in 1-1-4-3. In summary, this study found that the antibiofilm effect of the L. plantarum CFS against S. mutans was attributable to the comprehensive effect of multiple components, among which LA played a dominant role.

The effect of fluoroquinolones and antioxidans on biofilm formation by Proteus mirabilis strains

BackgroundFluoroquinolones are a group of antibiotics used in urinary tract infections. Unfortunately, resistance to this group of drugs is currently growing. The combined action of fluoroquinolones and other antibacterial and anti-biofilm substances may extend the use of this therapeutic option by clinicians. The aim of the study was to determine the effect of selected fluoroquinolones and therapeutic concentrations of ascorbic acid and rutoside on biofilm formation by Proteus mirabilis.Materials and methodsThe study included 15 strains of P. mirabilis isolated from urinary tract infections in patients of the University Hospital No. 1 dr A. Jurasz in Bydgoszcz (Poland). The metabolic activity of the biofilm treated with 0.4 mg/ml ascorbic acid, 0.02 µg/ml rutoside and chemotherapeutic agents (ciprofloxacin, norfloxacin) in the concentration range of 0.125–4.0 MIC (minimum inhibitory concentration) was assessed spectrophotometrically.ResultsBoth ciprofloxacin and norfloxacin inhibited biofilm formation by the tested strains. The biofilm reduction rate was correlated with the increasing concentration of antibiotic used. No synergism in fluoroquinolones with ascorbic acid, rutoside or both was found. The ascorbic acid and rutoside combination, however, significantly decreased biofilm production.ConclusionsOur research proves a beneficial impact of ascorbic acid with rutoside supplementation on biofilm of P. mirabilis strains causing urinary tract infections.

The impact of titanium oxide nanoparticles and low direct electric current on biofilm dispersal of $Bacillus~cereus$ and $Pseudomonas~aeruginosa$: A comparative study

Bacteria growing in biofilms cause a wide range of environmental, industrial and public health risks. Because biofilm bacteria are very resistant to antibiotics, there is an urgent need in medicine and industry to develop new approaches to eliminating bacterial biofilms. One strategy for controlling these biofilms is to generate an antibiofilm substance locally at the attachment surface. Direct electric current (DC) and nanoparticles (NPs) of metal oxides have outstanding antimicrobial properties. In this study we evaluated the effect of titanium oxide nanoparticle (TiO$_2$-NP) concentrations from 5 to 160 $\mu$g/mL on Bacillus cereus and Pseudomonas aeruginosa biofilms, and compared this with the effect of a 9 V, 6 mA DC electric field for 5, 10 and 15 min. TiO$_2$-NPs were characterized using transmission and scanning electron microscopes, X-ray diffraction and FTIR. They exhibited an average size of 22-34 nm. The TiO$_2$-NP concentrations that attained LD50 were $104 \pm 4$ $\mu$g/mL and $63 \pm 3$ $\mu$g/mL for B. cereus and P. aeruginosa, respectively. The eradication percentages obtained by DC at 5, 10, and 15 min exposure were 21%, 29%, and 33% respectively for B. cereus and 30%, 39%, and 44% respectively for P. aeruginosa. Biofilm disintegration was verified by exopolysaccharide, protein content and cell surface hydrophobicity assessment, as well as scanning electron microscopy. These data were correlated with the reactive oxygen species produced. The results indicated that both DC and TiO$_2$-NPs have a lethal effect on these bacterial biofilms, and that the DC conditions used affect the biofilms in a similar way to TiO$_2$-NPs at concentrations of 20-40 $\mu$g/mL.

1259. Activity of eravacycline against staphylococci isolated from periprosthetic joint infections

BackgroundPerirosthetic joint infections (PJIs) are costly and difficult to treat. The most common causes of PJIs are Staphylococcus aureus and Staphylococcus epidermidis. Eravacycline is a newer tetracycline with promising activity against Gram-positive and negative bacteria which is approved for treatment of complicated intraabdominal infections. Here, the in vitro activity of eravacycline was assessed against bacteria associated with PJI.Methods185 staphylococcal isolates, including 38 methicillin-resistant S. aureus (MRSA), 64 methicillin-susceptible S. aureus (MSSA), 62 methicillin-resistant S. epidermidis (MRSE) and 21 methicillin-susceptible S. epidermidis (MSSE) strains were studied. Minimum inhibitory concentrations (MICs) were determined according to Clinical and Laboratory Standards Institute guidelines (range of 0.06-64 µg/ml tested). Results were analyzed using susceptible breakpoints from EUCAST (≤0.25 µg/ml) and the FDA (≤0.06 µg/ml). Minimum biofilm bactericidal concentrations (MBBCs) were determined using a modification of the Calgary biofilm method. Briefly, biofilms were formed on pegged lids in trypticase soy broth, after which the pegged lids were rinsed in phosphate buffered saline (PBS), transferred to a plate containing dilutions of eravacycline in cation-adjusted Mueller Hinton broth (CAMHB) and incubated for 20-24h. Finally, the pegged lids were again rinsed in PBS and transferred to a plate containing CAMHB and incubated for 24h. The MBBC was the lowest concentration with no visible growth.ResultsMIC50/90 (range) in µg/ml for MRSA, MSSA, MRSE, and MSSE were 0.125/0.125 (≤0.06-0.25), ≤0.06/0.125 (≤0.06-0.25). 0.125/1 (≤0.06-2), and 0.25/1 (≤0.06-1), respectively. Using the EUCAST susceptible breakpoint, 100% of isolates would be considered susceptible, whereas only 54% would be considered susceptible using the FDA breakpoint. MBBC50/90 (range) in µg/ml for MRSA and MSSA were both 8/16 (4-16); for MRSE and MSSE, the values were 4/16 (2-32) and 8/16 (2-32), respectively.ConclusionOur data suggest that the FDA susceptible breakpoint may need re-evaluation. Eravacycline has low anti-staphylococcal biofilm activity.DisclosuresRobin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)

1193. Human Transcriptomic Analysis of Periprosthetic Joint Infection

BackgroundPeriprosthetic joint infection (PJI), a devastating complication of total joint replacement, is of incompletely understood pathogenesis and may sometimes be challenging to clinically distinguish from other causes of arthroplasty failure.MethodsWe characterized human gene expression in 93 specimens derived from surfaces of resected arthroplasties, comparing transcriptomes of subjects with infection- versus non-infection-associated arthroplasty failure.ResultsDifferential gene expression analysis confirmed the association of 28 previously investigated biomarkers with PJI- bactericidal/permeability increasing protein (BPI), cathelicidin antimicrobial peptide (CAMP), chemokines CCL3, CCL4, and CXCL2, colony stimulating factor 2 receptor (CSF2RB), colony stimulating factor 3 (CSF3), alpha-defensin (DEFA4), receptor CD64B, intercellular adhesion molecule 1 (ICAM1), IFNG, IL13RA2, IL17D, IL1A, IL1B, IL1RN, IL2RA, IL2RG, IL5RA, IL6, IL8, lipopolysaccharide binding protein (LBP), lipocalin (LCN2), lactate dehydrogenase C (LDHC), lactotransferrin (LTF), matrix metallopeptidase 3 (MMP3), peptidase inhibitor 3 (PI3), and vascular endothelial growth factor A (VEGFA), as well as identified three novel molecules with diagnostic potential for detection of PJI- chemokine CCL20, coagulation factor VII (F7), B cell receptor FCRL4. Comparative analysis of infections caused by staphylococcal versus non-staphylococcal and Staphylococcus aureus versus Staphylococcus epidermidis showed significant elevated expression of IL13, IL17D, and metalloprotease protein MMP3 in staphylocococcal infections, and increased expression of IL1B, IL8, and platelet factor PF4V1 in S. aureus infections. Pathway analysis of over-presented genes suggested activation of host immune response and cellular maintenance and repair functions in response to invasion of infectious agents.ConclusionOur study provides new potential targets for diagnosis of PJI and targets for differentiation of PJI-associated infectious agents.DisclosuresMatthew P. Abdel, MD, Dr. Abdel receives royalties from Stryker on certain hip and knee products, and is a paid consultant for Stryker. (Consultant) Robin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)

875. Pseudo-outbreak of Clostridioides (Clostridium) difficile amongst post-operative Oncology Patients

Background Clostridioides difficile infection rates are subject to infection prevention surveillance as a quality measure within the hospital setting. A large spike in Clostridioides difficile infections in post-operative patients, the majority of whom were gastrointestinal surgery (GIS) patients, was noted within a six month period (June through November 2019) at our comprehensive cancer center. These patients had been housed in one of two inpatient units and there was appropriate concern that this represented a C. difficile outbreak possibly related some type of infection control breach.MethodsIn an effort to query case relatedness, whole genome sequencing was performed using Illumina MiSeq instrumentation and chemistry with Illumina Nextera XT library chemistry. Assembly and core genome multilocus sequence typing analysis were performed with Ridom SeqSphere+ software. Cases were classified as community or hospital acquired based on the National Healthcare Safety Network (NHSN) definitions.ResultsThere were 23 samples submitted for possible whole genome sequencing (WGS). 5 samples were unable to be grown therefore WGS was not completed; 16 were found to be unrelated (51 or more allelic differences); 2 of the 18 isolates were found to be possibly related (7 to 50 allelic differences). There were no isolates found to be definitively related (zero to 6 allelic differences).ConclusionGiven the overwhelming unrelatedness of the isolates via whole genome sequencing, this increase of C. difficile cases, identified by routine surveillance within two inpatient units, was determined to be representative of a pseudo-outbreak rather than an outbreak. This study has implications on public health reporting. National Healthcare Safety Network definitions are used to identify healthcare facility-onset C. difficile infections (CDI). The majority of cases in this study met the definition of healthcare facility-onset, and thus were reported as such, despite being genetically unrelated. This raises the concern that a significant percentage of C. difficile infections may be currently misclassified as hospital-associated and this may have negative, unfair consequences for hospitals, such as implications on reimbursement.DisclosuresRobin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)

Antimicrobial and antibiofilm activities of marine sponge-associated bacteria against multidrug-resistant Staphylococcus spp. isolated from canine skin

Dogs play important roles in our society, thus the concern for their health becomes imperative. Staphylococcus spp. are commensal bacterium frequently isolated from canine skin and recognized as zoonotic agents. These bacteria have been becoming increasingly resistant to antimicrobials used to treat infections and to produce biofilm, which further increases their virulence capability and resistance. In this context, sponges-associated bacteria are known as prolific sources of substances with antimicrobial activities, representing a potential to integrate the arsenal of drugs for clinical use. In this study, 121 strains of Staphylococcus isolated from healthy or infected dogs were characterized according to their resistance to antimicrobials, as well as to their biofilm production ability. From the total of strains, 82 were resistant to at least one antimicrobial and 40 were multidrug-resistant (MDR). Furthermore, 117 out of 121 were capable to produce biofilm, and within those 36 were classified as strong biofilm producers. A set of fifteen bacterial strains previously isolated from marine sponges were also evaluated for antimicrobial and antibiofilm activities. Among the marine bacteria with antimicrobial activity, eight inhibited the growth of more than 50% of the MDR Staphylococcus. In addition, the cell-free supernatant obtained from five sponge-associated bacteria cultures was able to disaggregate more than 50% of the mature biofilm staphylococcal cells. The organic extracts (256 μg/mL) from two potential strains, Pseudomonas fluorescens H40 and H41, dissociated the biofilm of a strain classified as MDR and strong biofilm producer in 88.5% and 91.3%, respectively. These marine Pseudomonas strains also exhibited a strong activity of antimicrobial and antibiofilm substances. The results suggest that the sponge-associated bacteria analyzed could be potential sources of antimicrobial and antibiofilm substances against MDR and biofilm producers Staphylococcus isolated from canine skin.

1834. Incremental Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice

BackgroundPolymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic.MethodsThis is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture.ResultsWe reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4).ConclusionOverall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

691. Activity of TNP-2092 Against Biofilms Formed by Prosthetic Joint Infection-Associated Staphylococci

BackgroundInfection occurs in ~1–2% of prosthetic joint replacement surgeries, with staphylococci being the most common cause. TNP-2092 is an investigational drug composed of rifamycin and quinolizinone pharmacophores conjugated via a stable linker. Here, we determined TNP-2092’s in vitro activity against biofilms formed by staphylococci associated with prosthetic joint infection and compared activity to that of ciprofloxacin and rifampin alone and in combination, as well as to daptomycin and vancomycin.MethodsA total of 80 staphylococcal isolates (40 Staphylococcus aureus and 40 Staphylococcus epidermidis) were studied. Planktonic state minimum inhibitory concentrations (MICs) of TNP-2092, ciprofloxacin, rifampin, ciprofloxacin + fixed concentration (1 mg/mL) rifampin, daptomycin and vancomycin were determined following CLSI guidelines. Tween-80（0.002%）was added to TNP-2092 to prevent drug binding to plastic plates. Minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm bactericidal concentration (MBBCs) were determined as follows. Bacteria were grown in TSB to logarithmic phase and adjusted to a turbidity of 0.5 McFarland; 150 µL aliquots were transferred to individual wells of 96-well flat-bottom plates and the plates covered with 96-pegged lids. Plates were incubated on a shaker for 5 hours at 37℃. Pegged lids were rinsed using 200 µL PBS/well and placed into a microtiter plate containing serial 2-fold drug dilutions in CAMHB Plates were incubated for 20–24 hours at 37°C and MBICs read by visual turbidity. Pegged lids were rinsed with PBS and placed into plates filled with 200 µL CAMHB/well and incubated for 20–24 hours at 37°C after which MBBCs were determined by assessing visual turbidity.ResultsResults shown in the table.ConclusionTNP-2092 has promising in vitro activity against prosthetic joint infection-associated staphylococcal biofilms. DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid

BackgroundWe recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/ciz266). The method we described included a DNA extraction/purification step, which can be time consuming and costly. Here, we evaluated an extraction-free approach to 16S rRNA gene PCR/sequencing.Methods28 SF samples derived from explanted clinically-infected CIEDs were tested. Cases were categorized as “culture-positive” (C-P) if ≥20 cfu/10 mL were recovered, and as “culture-negative” (C-N), if <20 cfu/10 mL were detected in SF culture. The extraction-free method consisted of a single step of lysis at 1,000 rpm, 95°C for 5 minutes using an Eppendorf ThermoMixer®. DNA extraction (the comparator method) was performed using the ZymoBIOMICSTM Kit with modifications. Samples were processed using both methods, followed by amplification of the 16S rRNA gene and bidirectional Sanger sequencing. Crossing points (CPs) generated by the two approaches were compared. Organisms detected by the two PCR methods were compared with those detected with culture.ResultsOf the 28 samples tested, 13 were C-N and 15 C-P. The extraction-free method generated an amplicon in 13/15 C-P cases, with CPs ranging from 26 to 36 cycles vs. 100% (15/15) detected with the DNA extraction method and Cps of 19 to 32. Usable sequence length for the extraction-free method was of 359 (interquartile range, 307–390) vs. 390 (interquartile range, 308–396) base pairs with DNA extraction. Genus-level concordance between bacteria detected by culture in C-P samples and those found using the extraction-free and extraction methods was 92% (12/13) and 93% (14/15), respectively. Bacteria were detected by the extraction method in 2/13 C-N specimens, with none detected with an extraction-free method.ConclusionThe described extraction-free method may be suitable for testing SF derived from CIEDs using 16S rRNA gene PCR/sequencing, saving time and cost. More studies are needed to establish clear cutoffs for interpretation of results and to assess for PCR inhibitors in the studied specimen-type.DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

1517. Multidrug-Resistant Escherichia coli ST131 Late-Onset Neonatal Sepsis in Premature Twins Linked to Contaminated Maternal Frozen Breast Milk

BackgroundSequence type 131 (EC-ST131) is a prevalent cause of extraintestinal Escherichia coli infection, including in neonates, and accounts for a majority of multidrug-resistant strains. Rare reports of neonatal unit outbreaks have emerged, with one linking the source to freshly expressed breast milk (BM) sharing. We report on premature twin girls whose infection was linked to their mother’s contaminated frozen BM.MethodsBlood culture isolates were from twin girls born at 24–1/7 weeks’ gestation who developed severe sepsis caused by ampicillin- and gentamicin-resistant E. coli on days 11 (Baby A; died) and 8 (Baby B; survived) of life; both neonates had sterile blood cultures at birth, and received orogastric feeds using frozen BM provided by their mother. Five remaining frozen BM samples predating onset of sepsis were thawed and cultured; E. coli resistant to ampicillin and gentamicin was recovered from 1 collected on day 5 of life. DNA was extracted from cultured isolates using the Zymo Research Quick-DNA™ Fungal/Bacterial Miniprep kit, sequencing libraries prepared (Nextera® XT PE), and sequencing (Illumina MiSeq® with V2 2 X 250 bp chemistry) completed for the 2 blood and 1 BM isolates. Multilocus Sequence Typing (MLST) and core genome MLST (cgMLST) analyses were performed using SeqSphere+, version 5.1.0 (Ridom, Munster, DE) software, with 2513 alleles analyzed for cgMLST.ResultsThe 2 blood and 1 BM isolates were typed as ST131 by MLST and were indistinguishable by cgMLST. Of the 2513 alleles queried, only 263 (10.5%) differed from the ST131 strain SCB34 (figure). Thus, our EC-ST131 cluster isolates belong to the same clonal complex as, but are genetically divergent from, SCB34. In addition to the E. coli described above isolated from a BM sample, all 5 frozen BM samples grew multiple morphotypes of coagulase-negative staphylococci. The mother had acute chorioamnionitis and was treated with intravenous ampicillin, gentamicin, and metronidazole for 48 hours immediately after delivery.ConclusionDespite frozen BM’s persistent antibacterial activity, it is a potential source of multidrug-resistant bacteria for neonates, as evidenced by this report of EC-ST131 neonatal sepsis in premature twins. DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Biofilms: Architecture, Resistance, Quorum Sensing and Control Mechanisms.

Biofilm is a mode of living employed by many pathogenic and environmental microbes to proliferate as multicellular aggregates on inert inanimate or biological substrates. Several microbial diseases are associated with biofilms that pose challenges in treatment with antibiotics targeting individual cells. Bacteria in biofilms secrete exopolymeric substances that contribute to architectural stability and provide a secure niche to inhabiting cells. Quorum sensing (QS)plays essential roles in biofilm development. Pathogenic bacteria in biofilms utilize QS mechanisms to activate virulence and develop antibiotic resistance. This review is a brief overview of biofilm research and provides updates on recent understandings on biofilm development, antibiotic resistance and transmission, and importance of QS mechanisms. Strategies to combat biofilm associated diseases including anti-biofilm substances, quorum quenching molecules, bio-surfactants and competitive inhibitors are briefly discussed. The review concludes with updates on recent approaches utilized for biofilm inhibition and provides perspectives for further research in the field.

Potential anti-biofilm producing marine actinomycetes isolated from sea sediments in Thailand

Marine actinomycetes are well known as novel resources of bioactive compounds including anti-biofilm agents with various therapeutic applications. In this study, one hundred and one marine actinomycetes were isolated from sea sediments collected from Andaman sea and the Gulf of Thailand. Morphological study and partial 16S rDNA sequence analysis revealed that most of isolates belong to genus Streptomyces (n = 90, 89.11%) and the rest are rare actinomycetes (n = 11, 10.89%) including Actinopolymorpha, Actinomycetospora, Dietzia Nocardiopsis, Micromonospora and Mycobacterium. Among them, ten and thirteen strains significantly reduced more than 60% of biofilm formation of Escherichia coli and Staphylococcus aureus, respectively. But no such activity was found against Pseudomonas aeruginosa. Further analysis of their supernatants indicated that most of marine actinomycetes secreted non-toxic anti-biofilm agents and some accompanied with protease activity. These are preferred characteristics of anti-biofilm substances that potentially restricted biofilm forming of E. coli and S. aureus and simultaneously prevented bacteria to develop resistance.

Antimicrobial and Antibiofilm Activity of Unionid Mussels from the North of Portugal

The populations of freshwater mussels belonging to the family Unionidae have been facing drastic changes in terms of diversity and numbers caused by constant aggressions on their natural habitat. Nevertheless, bivalves are capable of developing strategies of defense to overcome potential aggressors. The present work aimed to assess the potential antibacterial capacity of different species of unionid mussels from the north of Portugal. For this purpose, circulating cells (hemocytes), fluids, and mucus were obtained by nonlethal methods from the species Anodonta anatina (Linnaeus), Anodonta cygnea (Linnaeus), Potomida littoralis (Cuvier), and Unio delphinus (Spengler), and tested against bacterial reference strains and multidrug-resistant isolates. The cellular fraction of A. anatina, A. cygnea, and P. littoralis showed antibacterial activity, detected by the agar disc diffusion method, against Bacillus subtilis ATCC 6683, Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606. Circulating cells from P. littoralis and A. anatina also inhibited Listeria monocytogenes ATCC 19111 and A. cygnea has also inhibited a multidrug-resistant isolate of Pseudomonas putida. The plasma of all mentioned freshwater mussels, used directly or diluted, showed great ability to hamper or inhibit the biofilm formation of Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. Anodonta cygnea hampered the biofilm formation by Enterococcus faecalis ATCC 29212 as well. Overall, these results showed that not only cells play a relevant role in the immune system of these species but also the plasma, which likely contains antibiofilm substances. Anodonta cygnea stood out by presenting the best antibacterial inhibition potential.

Plant extract activities as antioxidant and antibiofilm against chicken gut bacteria

The occurrence of microbial resistance against antibiotic due to the subtherapeutic dosage of antibiotic growth promoter (AGP) in poultry can be prevented by the antibiofilm substance. Plant secondary compounds have some activities like antioxidant, antimicrobial, and antibiofilm. This research was conducted to obtain the plant with the highest activity of antibiofilm and also antioxidant by analyzing several plant secondary compounds as antioxidant and antibiofilm against chicken’s gut bacteria. The tested plants were clove leaves, leaffruit plants, mangosteen peel, cashew nut shell, guava leaves, and bay leaves. These plants were extracted with methanol or n-hexane using sonication method. The antioxidant activity as the IC50 value of the plant methanol extracts were determined using α,α-diphenyl-β-picrylhydrazyl (DPPH) assay. The biofilm inhibition activity was tested against Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus ATCC® 29213TM using methanol and n-hexane extracts. All of the samples had antioxidant activity. The clove leaves and leaffruit plants had the highest antioxidant activity, while mangosteen peel extract in methanol had the highest antibiofilm activity against all tested bacteria. The species of bacteria also affected the antibiofilm activity. E. coli and S. enteritidis were more resistant to antibiofilm then S. aureus. Mangosteen peel extract which showed high antioxidant and antibiofilm activity is potential to be used as a feed additive to control the pathogenic bacteria.

Assessing Phagolysosomal Escape of Staphylococcus epidermidis RP62A in Intracellularly Infected Human Fibroblasts

S. epidermidis causes chronic prosthetic joint infection; identifying how it can evade immune killing and persist to cause chronic infection will inform strategies to prevent and treat S. epidermidis PJI. Previously, we showed that S. epidermidis clinical PJI isolates can persist intracellularly in fibroblasts and osteoblasts, and that they reside primarily within phagolysosomes. Others have shown that some S. Aureus strains are able to escape the phagolysosome and enter the cytoplasm. Here, we used intracellular pH monitoring to assess whether S. epidermidis RP62A can similarly escape the phagolysosome. S. Aureus strain 6850—known to escape the phagolysosome—and S. epidermidis RP62A were grown overnight in TSB at 37°C and labeled with FITC. Human lung fibroblast MRC5 cells were infected at a multiplicity of infection of 15. Cells were infected for either 2 or 6 hours. Cells were then incubated in the presence or absence of chloroquine and analyzed by flow cytometry. The invasion rate with chloroquine (total internalized bacteria), expressed as arbitrary fluorescence units (AFU), was determined and normalized to the mean fluorescence intensity of each corresponding bacterial preparation. Experiments were done in triplicate and the average AFU were used. Because the quenching of FITC fluorescence is proportional to acidic pH, the difference in AFU (ΔAFU) in the presence or absence of chloroquine, normalized to the invasion rate, was used as readout for pH. Results are shown in the figure. At 2 hours post infection, both S. Aureus 6850 and S. epidermidis RP62A were in acidic compartments. At 6 hours post infection, S. Aureus 6850 appeared to be in the cytoplasm, as indicated by a pH close to neutral. In contrast, the environment of S. epidermidis RP62A had become more acidic suggesting lack of phagolysosomal escape. Differences of ΔAFU suggest escape of S. Aureus 6850 but not S. epidermidis RP62A from a phagolysosomal to a cytoplasmic environment 6 hours following infection of MRC5 cells. As not all S. Aureus strains can escape the phagolysosome, with some requiring longer times before escaping, more isolates and longer time points are needed to definitely conclude that S. epidermidis cannot escape the phagolysosome. R. Patel, ASM: Board Member, None. CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Grant recipient. Curetis: Consultant, Monies paid to my employer. A patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Patents, Patents, any money is paid to my employer. Actelion: DSMB, Money paid to my employer ASM and IDSA: Editor’s stipends, Editor’s stipends. NBME, Up-to-Date and the Infectious Diseases Board Review Course: NBME, Up-to-Date and the Infectious Diseases Board Review Course, Honoraria. Roche, ASM, and IDSA: Travel reimbursement, Travel reimbursement

Lichen biodiversity: A source of secondary metabolites active against Candida biofilms

The frequency of infections involving fungal biofilms (in particular associated with Candida yeasts) as well as their resistance to current treatments, justified the need to discover new anti-biofilm substances [1]. Lichens correspond to a symbiotic association between an alga and/or a cyanobacteria and a fungus and are a reservoir for novel compounds with therapeutic potential. For these reasons, this research program was focused on the access to original compounds from lichens from the French Limousin region [2]. A screening of forty lichen extracts was performed against planktonic Candida albicans by evaluating MICs. Moreover, the activity of these extracts was studied against the development (up to 48h) of C. albicans biofilms on polystyrene substrates, using the XTT method. These studies showed that none of extracts active against planktonic C. albicans (MIC> 0.1 mg/mL). However, eleven extracts demonstrated a significant anti-biofilm activity (p < 0.05) for concentrations less than 50 µg/mL. Among them, the Cladonia parasitica extract (43 µg/mL at 24h) was selected for further investigation. HPLC analysis showed poor chemical diversity of the extract which contained 72% thamnolic acid. This depside was active against Candida biofilms at 14.5 and 33 µg/mL at 24 and 48h respectively. Among the most active extracts, Evernia prunastri, Ramalina fraxinea, R. fastigiata, Xanthoparmelia conspersa and Peltigera hymenina displayed significant anti-biofilm activity (p < 0.05) for concentrations less than 10 µg/mL; in particular for E. prunastri and R. fastigiata whose activity was observed as soon as 24h and persisted at 48h. This work demonstrated the anti-biofilm activity of lichens from local biodiversity and thamnolic acid against C. albicans and highlighted the potential of lichens to fight Candida biofilms.