Cellular variability of RpoS expression underlies subpopulation activation of an integrative and conjugative element.

Conjugative transfer of the integrative and conjugative element ICEclc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICEclc transfer rates and activation of two key ICEclc promoters (P(int) and P(inR)) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on P(inR), whereas one of the gene products from the P(inR)-controlled operon (InrR) transmits activation to P(int) and other ICEclc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the P(int) and P(inR) promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated P(int) and P(inR), whereas a double-copy rpoS-mcherry-containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate P(inR) and thus ICEclc transfer. Double promoter-reporter fusions confirmed that expression of P(inR) is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from P(int) is dominated by intrinsic noise, indicating it is specific to the ICEclc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

Wet chemical silver treatment of endotracheal tubes to produce antibacterial surfaces.

Mechanically ventilated patients in hospitals are subjected to an increased risk of acquiring nosocomial pneumonia that sometimes has a lethal outcome. One way to minimize the risk could be to make the surfaces on endotracheal tubes antibacterial. In this study, bacterial growth was inhibited or completely prevented by silver ions wet chemically and deposited onto the tube surface. Through the wet chemical treatment developed here, a surface precipitate was formed containing silver chloride and a silver stearate salt. The identity and morphology of the surface precipitate was studied using x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray powder diffraction. Leaching of silver ions into solution was examined, and bacterial growth on the treated surfaces was assayed using Pseudomonas aeruginosa wild type (PAO1) bacteria. Furthermore, the minimum inhibitory concentration of silver ions was determined in liquid- and solid-rich growth medium as 23 and 18 microM, respectively, for P. aeruginosa.

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

Saccharomyces cerevisiae, a tool to study the β-oxidation through the synthesis of polyhydroxyalkanoates

Summary Polyhydroxyalkanoates (PHAs) represent a family of polyesters naturally synthesized by a wide variety of bacteria. Through their thermoplastic and elastomeric qualities, together with their biodegradable and renewable properties, they are predicted to be a good alternative to the petroleum- derived plastics. Nevertheless, as PHA production costs using bacteria fermentation are still too high, PHA synthesis within eukaryotic systems, such as plants, has been elaborated. Although the costs were then efficiently lowered, the yield of PHAs produced remained low. In this study, Saccharomyces cerevisae has been used as another eukaryotic model in order to reveal the steps which limit PHA production. These cells express the PHA synthase of Pseudomonas aeruginosa and the PHAs obtained were analyzed to understand the flux of fatty acids towards and through the peroxisomal β-oxidation core cycle, generating the main substrate of the PHA synthase. When S. cerevisiae wild-type cells are grown in a media containing glucose as carbon source as well as fatty acids, the PHA monomer composition is largely influenced by the nature of the external fatty acid used. Thus, even-chain PHA monomers are generated from oleic acid (18:1Δ9cis) and odd- chain PHA monomers are generated from heptadecenoic acid (17:1Δ. 10 cis). Moreover, PHA synthesis is dependent on the first two enzymes of the 0-oxidation core cycle, the acyl-CoA oxidase and the multifunctional enzyme enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA dehydrogenase. S. cerevisiae mutant cells growing on oleic or heptadecenoic acid and deficient in either the R-3- hydroxyacyl-CoA dehydrogenase or in the 3-ketothiolase activity, the last β-oxidation cycle steps, surprisingly contained PHAs of predominantly even-chain monomers. This is also noticed in wild- type and mutants grown on glucose or raffinose, indicating that the substrate used for PHA synthesis is generated from the degradation of intracellular short- and medium-chain fatty acids by the 3- oxidation cycle. Inhibition of fatty acid biosynthesis by cerulenin blocks the synthesis of PHAs from intracellular fatty acids but still enables the use of extracellular fatty acids for polymer production. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed towards the peroxisomal β-oxidation pathway. In this thesis, no increase of the yield of PHA produced could be obtained. But the PHA synthesis confirmed the carbon flux into and through the β-oxidation core cycle and unveiled the existence of novel mechanisms. It is thus a good tool to study in vivo the flux of carbons in S. cerevisiae cells. Résumé Les polyhydroxyalkanoates (PHAs) sont une famille de polyesters naturellement synthétisés par un grand nombre de bactéries. Ayant des propriétés de thermoplastiques, d'élastomères et étant des ressources biodégradables et renouvelables, les PHAs représentent une bonne alternative aux plastiques dérivés du pétrole. Pour pallier aux coûts considérables de la production de PHAs par fermentation bactérienne, la synthèse de PHAs par des systèmes eucaryotes telles les plantes a été élaborée. Les coûts ont ainsi efficacement été diminués, mais le rendement de PHAs produits reste faible. Dans cette étude, Saccharomyces cerevisiae a été utilisé comme autre modèle eucaryote pour révéler les étapes limitantes de la production de PHAs. Les PHAs obtenus dans les cellules exprimant la F'HA synthase de Pseudomonas aeruginosa ont été analysés afin de comprendre le flux d'acides gras vers et à travers le cycle péroxisomal de la β-oxidation, principal producteur du substrat de la PHA synthase. Lorsque la souche S. cerevisiae de type sauvage se développe dans un milieu contenant du glucose et des acides gras, la composition des monomères de PHAs est influencée par la nature des acides gras extracellulaires. Ainsi, les monomères pairs sont générés par l'acide oléique (18:1Δ9cis), tandis que les impairs le sont par l'acide heptadécénoïque (17:1Δ10cis). La synthèse de PHAs est dépendante des deux premières enzymes de la β-oxidation; l'acyl-CoA oxidase et l'enzyme multifonctionnelle enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA déshydrogénase. Les souches mutantes ne possédant pas les activités de la R-3-hydroxyacyl-CoA déshydrogénase ou de la 3- ketothiolase contiennent, en présence d'acide oléique ou heptadécénoïque, des PHAs composés essentiellement de monomères pairs. Cela a également été observé en présence de glucose ou de raffinose uniquement. Le substrat utilisé pour la synthèse de PHAs a ainsi été généré par la dégradation d'acides gras intracellulaires à chaîne courte et moyenne via le cycle de la β-oxidation. L'inhibition de la synthèse d'acides gras par la cérulénine a bloqué la synthèse de PHAs par les acides gras internes. Ces résultats ont révélés l'existence d'un cycle futile par lequel des intermédiaires à chaîne courte et moyenne de la synthèse cytoplasmique d'acides gras sont dirigés vers le cycle péroxisomal de la β-oxidation. Dans cette étude, le rendement de PHAs produits reste inchangé, mais l'analyse des PHAs permet de confirmer le flux de carbones vers et à travers le cycle péroxisomal de la β-oxidation et l'existence de nouveaux méchanismes a été dévoilée. Cette synthèse s'avère être un bon outil pour étudier in vivo le flux de carbones dans les cellules de S. cerevisiae.

Characterization of a plant-derived peptide displaying water clarifying and antimicrobial activities

SUMMARY Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment- friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components was unknown. Here, we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Pseudomona, Streptococcus and Legionella species. Structural modeling of the peptide coupled to the functional analysis of synthetic peptide derivatives delineated distinct structural determinants for the flocculation and antibacterial activities. Our results suggest that a glutamine-rich portion of the polypeptide is involved in the sedimentation process; alternatively, the antibacterial activity depends on a amphiphilic loop. Assembly of multiple copies of this loop into a branched peptide derivative strongly enhances antibacterial activity without displaying hemolytic effect. In conclusion, this polypeptide displays the unprecedented feature of combining efficient water purification and disinfectant properties indicating different molecular mechanisms involved in each case. This work not only identified the features responsible for these activities but also provides useful information that has implications for the further development of this cationic polypeptide as a potent antibacterial agent. RESUME L'eau potable est actuellement une ressource limitée dans le monde. La production d'eau propre à la consommation exige des traitements complexes, incluant la clarification des particules en suspension ainsi que sa désinfection par des additifs chimiques. Les extraits de la graine d'un arbre tropical, Moringa oleifera, sont utilisés traditionnellement en Afrique afin de clarifier l'eau. Quoique la nature exacte des composants actifs était inconnue, on a pu mettre en évidence un polypeptide cationique contenu dans ces graines, capable de sédimenter de manière efficace des particules minérales en suspension ainsi que des bactéries. Ce travail a aussi mis en évidence que ce polypeptide a une activité bactéricide, permettant une désinfection d'eau fortement contaminée. De plus, nous avons démontré que ce polypeptide est efficace contre de nombreuses souches bactériennes pathogènes, également celles résistantes aux antibiotiques comme Pseudomonas, Streptococcus et Legionella. L'analyse de la structure moléculaire de ce polypeptide, couplée à son analyse fonctionnelle a mis en évidence deux domaines structuraux distinct, un pour l'activité de floculation et l'autre pour l'activité antibactérienne. Nos résultats suggèrent que le domaine riche en glutamine est impliqué dans le processus de sédimentation et que l'activité antimicrobienne dépend d'un domaine formant une boucle amphiphilique. En ramifiant plusieurs copies de cette boucle on a pu augmenter de manière significative l'activité antibactérienne. En conclusion, nous avons pu démontrer que ce polypeptide à la capacité unique de combiner des propriétés de purification et de désinfection de l'eau, ce qui implique des mécanismes moléculaires distincts pour ces deux activités. Ce travail a permis d'identifier les domaines du polypeptide qui sont responsables de ses activités et offre une perspective pour le développement d'un nouvel agent antimicrobien.

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment.

Indicazioni e risultati della laparostomia retroperitoneale nel trattamento della pancreatite acuta necrotizzante infetta [Indications and results of retroperitoneal laparostomy in the treatment of infected acute necrotizing pancreatitis]

The aim of this study is to describe personal experience with retroperitoneal laparostomy in the management of infected acute necrotizing pancreatitis. The presence of an infected phlegmon requires surgical debridement and drainage. The surgical approach can be either an anterior laparotomy with irrigation and drainage (which can be either an open or closed laparotomy) or a posterior laparostomy. Three patients (2 men and 1 woman) presented with an unfavourable course of their acute necrotizing pancreatitis despite the administration of broad spectrum antibiotics. A posterior laparostomy with necrosectomy and drainage was performed. The postoperative course was slowly favorable in all 3 cases. Abdominal CT is the best modality for the detection and follow-up of pancreatic necrosis. CT-guided fine needle aspiration can detect superinfection of areas of necrosis. Posterior laparostomy presents several advantages compared to an anterior approach. There is no contamination of the peritoneal cavity; the integrity of the abdominal wall is respected. The necrosectomy is equally complete and the drainage is better as it is direct and posterior.

The role of potassium in inflammasome activation by bacteria.

Many Gram-negative bacteria possess a type III secretion system (TTSS( paragraph sign)) that can activate the NLRC4 inflammasome, process caspase-1 and lead to secretion of mature IL-1beta. This is dependent on the presence of intracellular flagellin. Previous reports have suggested that this activation is independent of extracellular K(+) and not accompanied by leakage of K(+) from the cell, in contrast to activation of the NLRP3 inflammasome. However, non-flagellated strains of Pseudomonas aeruginosa are able to activate NLRC4, suggesting that formation of a pore in the cell membrane by the TTSS apparatus may be sufficient for inflammasome activation. Thus, we set out to determine if extracellular K(+) influenced P. aeruginosa inflammasome activation. We found that raising extracellular K(+) prevented TTSS NLRC4 activation by the non-flagellated P. aeruginosa strain PA103DeltaUDeltaT at concentrations above 90 mm, higher than those reported to inhibit NLRP3 activation. Infection was accompanied by efflux of K(+) from a minority of cells as determined using the K(+)-sensitive fluorophore PBFI, but no formation of a leaky pore. We obtained exactly the same results following infection with Salmonella typhimurium, previously described as independent of extracellular K(+). The inhibitory effect of raised extracellular K(+) on NLRC4 activation thus reflects a requirement for a decrease in intracellular K(+) for this inflammasome component as well as that described for NLRP3.

Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves.

Physical damage and disease are known to lead to changes in the oxylipin signature of plants. We searched for oxylipins produced in response to both wounding and pathogenesis in Arabidopsis leaves. Linoleic acid 9- and 13-ketodienes (KODEs) were found to accumulate in wounded leaves as well as in leaves infected with the pathogen Pseudomonas syringae pv. tomato (Pst). Quantification of the compounds showed that they accumulated to higher levels during the hypersensitive response to Pst avrRpm1 than during infection with a Pst strain lacking an avirulence gene. KODEs are Michael addition acceptors, containing a chemically reactive alpha,beta-unsaturated carbonyl group. When infiltrated into leaves, KODEs were found to induce expression of the GST1 gene, but vital staining indicated that these compounds also damaged plant cells. Several molecules typical of lipid oxidation, including malonaldehyde, also contain the alpha,beta-unsaturated carbonyl reactivity feature, and, when delivered in a volatile form, powerfully induced the expression of GST1. The results draw attention to the potential physiological importance of naturally occurring Michael addition acceptors in plants. In particular, these compounds could act directly, or indirectly via cell damage, as powerful gene activators and might also contribute to host cell death.

Tigecycline in combination with other antimicrobials: a review of in vitro, animal and case report studies.

Tigecycline has been investigated in combination with other antibacterials against a wide range of susceptible and multiresistant Gram-positive and Gram-negative bacteria. Combinations have been analysed in vitro, in animal models and in human case reports. In vitro, tigecycline combined with other antimicrobials produces primarily an indifferent response (neither synergy nor antagonism). Nevertheless, synergy occurred when tigecycline was combined with rifampicin against 64-100% of Enterococcus spp., Streptococcus pneumoniae, Enterobacter spp. and Brucella melitensis isolates. Combinations of tigecycline with amikacin also showed synergy for 40-100% of Enterobacter spp., Klebsiella pneumoniae, Proteus spp. and Stenotrophomonas maltophilia isolates. Moreover, bactericidal synergisms occurred with tigecycline plus amikacin against problematic Acinetobacter baumannii and Proteus vulgaris, and with colistin against K. pneumoniae. Data from animal experiments and case reports, although limited, displayed consistent beneficial activity of tigecycline in combination with other antibacterials against multiresistant organisms, including vancomycin against penicillin-resistant S. pneumoniae in experimental meningitis, gentamicin against Pseudomonas aeruginosa in experimental pneumonia, daptomycin against Enterococcus faecium endocarditis, and colistin against K. pneumoniae bacteraemia and P. aeruginosa osteomyelitis. Antagonism was extremely rare in vitro and was not reported in vivo. Thus, tigecycline may be combined with a second antimicrobial as part of a combination regimen.

Determination of bacterial quantity by sonication in vacum-assisted closure (VAC) foams used for treatment of chronic wounds

Background: Negative pressure wound treatment is increasingly used through a Vacuum-Assisted Closure (VAC) device in complex wound situations. For this purpose, sterile polyurethane (PU) and polyvinyl alcohol (PVA) foam dressings are fitted to the wound size and covered with an adhesive drape to create an airtight seal. Little information exists about the type and quantity of microorganisms within the foams. Therefore, we investigated VAC foams after removal from the wound using a validated method (sonication) to detect the bacterial bioburden in the foam consisting as microbial biofilms.Methods: We prospectively included VAC foams (PU and PVA, KCI, Rümlamg, Switzerland) without antibacterial additions (e.g. silver), which were removed from wounds in patients with chronic ulcers from January 2007 through December 2008. Excluded were patients with acute wound infection, necrotizing fasciitis, underlying osteomyelitis or implant. Removed foams from regular changes of dressing were aseptically placed in a container with 100 ml sterile Ringer's solution. Within 4 hours after removal, foams were sonicated for 5 min at 40 kHz (as described in NEJM 2007;357:654). The resulting sonication fluid was cultured at 37°C on aerobic blood agar plates for 5 days. Microbes were quantified as No. of colony-forming units (CFU)/ml sonication fluid and identified to the species level.Results: A total of 68 foams (38 PU and 30 PVA) from 55 patients were included in the study (median age 71 years; range 33-88 years, 57% were man). Foams were removed from the following anatomic sites: sacrum (n=29), ischium (n=18), heel (n=13), calves (n=6) and ankle (n=2). The median duration of being in place was 3 days (range, 1-8 days). In all 68 foams, bacteria were found in large quantities (median 105 CFU/ml, range 102-7 CFU/ml sonication fluid. No differences were found between PU and PVA foams. One type of organisms was found in 11 (16%), two in 17 (24%) and 3 or more in 40 (60%) foams. Gram-negative rods (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) were isolated in 70%, followed by Staphylococcus aureus (20%), koagulase-negative staphylococci, streptococci (8%), and enterococci (2%).Conclusion: With sonication, a high density of bacteria present in VAC foams was demonstrated after a median of 3 days. Future studies are needed to investigate whether antimicrobial-impregnated foams can reduce the bacterial load in foams and potentially improve wound healing.