High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Inhibition of COX-2 signaling favors E. coli during urinary tract infection.

BACKGROUND: To avoid the overuse of antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), acting via cyclooxygenase (COX) inhibition, have been used to reduce pain and as an alternative treatment for uncomplicated urinary tract infections (UTIs). However, clinical studies evaluating NSAIDs versus antibiotics have reported an increased risk of acute pyelonephritis. Therefore, we hypothesized that COX inhibition could compromise the innate immune response and contribute to complications in patients with uncomplicated UTI. RESULTS: We here demonstrate that in particular COX-2 inhibition led to decreased expression of the antimicrobial peptides psoriasin and human ß-defensin-2 in human uroepithelial cells. Psoriasin expression was altered in neutrophils and macrophages. COX-2 inhibition also had impact on the inflammasome mediated IL-1ß expression in response to uroepithelial E. coli infection. Further, COX-2 inhibition downregulated free radicals and the epithelial barrier protein claudin 1, favoring infectivity. In addition, conditioned media from COX-2 inhibited uroepithelial cells infected with E. coli failed to activate macrophages. CONCLUSIONS: Taken together, our data suggests an adverse innate immune effect of COX-2 inhibition on uroepithelial cells during UTI.

MV140 mucosal bacterial vaccine improves uropathogenic E. coli clearance in an experimental model of urinary tract infection.

MV140 is a mucosal vaccine of inactivated whole bacteria (E. coli, K. pneumoniae, E. faecalis, P. vulgaris) with clinical efficacy against recurrent urinary tract infections (UTIs). Here, MV140 was evaluated in a murine model of acute uropathogenic E. coli (UPEC)-induced UTI using the UTI89 strain. MV140 vaccination resulted in UPEC clearance, concomitant with increased influx of myeloid cells in urine, CD4+ T cells in the bladder, and a systemic adaptive immune response to both MV140-containing E. coli and UTI89.

A Synthetic Cyclized Antimicrobial Peptide with Potent Effects against Drug-Resistant Skin Pathogens.

Dermal infections requiring treatment are usually treated with conventional antibiotics, but the rise of bacterial resistance to first-line antibiotics warrants alternative therapeutics. Here, we report that a backbone-cyclized antimicrobial peptide, CD4-PP, designed from the human host defense peptide LL-37, has strong direct antibacterial effects on antibiotic sensitive as well as resistant-type strains and clinical isolates of common skin pathogens in the low (<2) µM range. In addition, it influences innate immunity in keratinocytes, and treatment with CD4-PP is able to clear bacterial infections in infected keratinocytes. Additionally, CD4-PP treatment significantly reduces the wound area in a lawn of keratinocytes infected with MRSA. In conclusion, CD4-PP has the potential to serve as a future drug treating wounds infected with antibiotic-resistant bacteria.

Antibiotic knowledge, attitudes and reported practice during pregnancy and six months after birth: a follow- up study in Lao PDR.

BACKGROUND: Antibiotics are important medicines to prevent maternal and child morbidity and mortality. Women's knowledge and attitudes towards antibiotic use influence their practice. When they become mothers, this may be mirrored in the use of antibiotics for their newborn children. The current study aimed to assess knowledge, attitudes, and reported practice of pregnant women regarding antibiotic use and antibiotic resistance as well as their approach towards antibiotic use for their newborn babies. METHODS: This was a follow-up study with data collected via structured interviews between September 2019 and August 2020 in Feuang (rural) and Vangvieng (urban) districts in Vientiane province, Lao PDR. We identified and invited all women attending antenatal care in their third trimester of pregnancy in the selected areas. Using a structured questionnaire at third trimester of pregnancy we captured data on knowledge regarding antibiotic use and resistance. We collected information on attitudes and reported practice at two time points: (i) at third trimester of pregnancy and (ii) 6 months after birth. Univariate analysis and frequency distributions were used to study pattern of responses. Chi-square and Mann-Whitney tests were used to compare categorical and continuous variables respectively. P value < 0.05 was considered statistically significant. RESULTS: We surveyed 539 women with a mean age of 25 years. Two oral antibiotics, i) ampicillin and ii) amoxicillin were correctly identified by 68 and 47% of participants respectively. Only 24% of women (19% in Feuang and 29% in Vangvieng) answered correctly that antibiotics are effective against bacterial infections. The most prevalent response was "I don't know" suggesting the questions were challenging. Significantly less women would use antibiotics from a previous illness for their child than for themselves (16% vs 29%), however they would be more willing to use antibiotics for their baby even in case of mild symptoms (29% vs 17% while pregnant). The majority of antibiotics were prescribed by healthcare providers and 46% of children with the common cold received antibiotics. CONCLUSIONS: Women's knowledge was sub-optimal, still, they manifested appropriate attitudes towards antibiotic use during pregnancy and for their child. Nearly half of children received antibiotics for the common cold. There is a need for context adapted programs aiming at improving women's knowledge, as well as healthcare providers, emphasising rational antibiotic prescribing during pregnancy and for children.

Diabetes downregulates the antimicrobial peptide psoriasin and increases E. coli burden in the urinary bladder.

Diabetes is known to increase susceptibility to infections, partly due to impaired granulocyte function and changes in the innate immunity. Here, we investigate the effect of diabetes, and high glucose on the expression of the antimicrobial peptide, psoriasin and the putative consequences for E. coli urinary tract infection. Blood, urine, and urine exfoliated cells from patients are studied. The influence of glucose and insulin is examined during hyperglycemic clamps in individuals with prediabetes and in euglycemic hyperinsulinemic clamped patients with type 1 diabetes. Important findings are confirmed in vivo in type 2 diabetic mice and verified in human uroepithelial cell lines. High glucose concentrations induce lower psoriasin levels and impair epithelial barrier function together with altering cell membrane proteins and cytoskeletal elements, resulting in increasing bacterial burden. Estradiol treatment restores the cellular function with increasing psoriasin and bacterial killing in uroepithelial cells, confirming its importance during urinary tract infection in hyperglycemia. In conclusion, our findings present the effects and underlying mechanisms of high glucose compromising innate immunity.

Perceptions and reported practices of pregnant women and mothers of children under two years of age regarding antibiotic use and resistance in Vientiane province, Lao PDR: a qualitative study.

BACKGROUND: Understanding pregnant women and mothers' perceptions towards antibiotic use and resistance is essential for appropriate antibiotic use and limiting antibiotic resistance. This study aimed to explore perceptions and reported practices of pregnant women and mothers with children under two years of age regarding correct antibiotic use and antibiotic resistance in Vientiane Province, Lao PDR. METHODS: The study employed an exploratory qualitative research design using focus groups discussions (FGDs). Participants were purposively selected based on: being pregnant at third trimester and attending antenatal care and mothers with children under two years of age, attending the health facility for postpartum visit /vaccinations. Six focus group discussions were conducted in September 2019 with a total of 55 women. The FGDs were transcribed verbatim, data were analyzed first by coding then categorizing the data as we looked for patterns and themes by using the qualitative content analysis. RESULTS: Most participants had some understanding of antibiotics but wrongly believed antibiotics can be used to treat viral disease. Over half of the participants had heard the term "antibiotic resistance", but often believed it was their bodies, not the bacteria that developed antibiotic resistance. During pregnancy and for their infants, women preferred to use antibiotics only when prescribed by a doctor. Outside of pregnancy however, consuming antibiotics without a prescription was commonly reported. Participants wanted more information about the indications for antibiotic use and antibiotic resistance. CONCLUSIONS: More effort is required to increase the level of understanding, and practice of mothers to promote optimal antibiotic use. Mothers' desire to learn more, and their fundamental concern for their children, can be used to promote appropriate antibiotic use. Awareness raising should be complemented by efforts to address other determinants of inappropriate antibiotic use, including educating healthcare workers, and pharmacists and addressing health service determinants that contribute to inappropriate antibiotic use.

A stable cyclized antimicrobial peptide derived from LL-37 with host immunomodulatory effects and activity against uropathogens.

The increasing antibiotic resistance among uropathogenic bacteria warrants alternative therapeutic strategies. We demonstrate the potential of the synthetic peptide CD4-PP, designed by dimerization and backbone cyclization of the shortest antimicrobial region of human cathelicidin, LL-37. CD4-PP is active against clinical and type strains of common uropathogens Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa at concentrations substantially below cellular cytotoxic levels and induced membrane deformation and leakage in E. coli and P. aeruginosa. Furthermore, CD4-PP treatment prevented the formation of new biofilm and dissolved mature biofilm created by E. coli and P. aeruginosa and targeted curli amyloid in E. coli biofilms. In addition, CD4-PP also induced production of LL-37 by uroepithelial cells and increased the expression of tight junction proteins claudin-14 and occludin. During uroepithelial cell infection, CD4-PP significantly reduced uropathogen survival when treatment was given at the start of infection. Low micromolar of CD4-PP treatment initiated after 2 h was successful with all tested species, except P. aeruginosa where CD4-PP was unable to reduce survival, which could be attributed by early biofilm formation. Finally, we demonstrated that urinary catheter pieces coated with saline fluid supplemented with CD4-PP reduced the attachment of E. coli, giving it a potential clinical application.

Effect of anticoagulant and platelet inhibition on the risk of bacteremia among patients with acute pyelonephritis: a retrospective cohort study.

BACKGROUND: An increasing number of patients are being prescribed anticoagulants and platelet inhibitors (antithrombotic treatment). Basic research has suggested an association between antithrombotic treatment and bacteremia during kidney infection. Here, we investigated the association between antithrombotic treatment, bacteremia and acute kidney injury in patients with acute pyelonephritis. METHODS: A retrospective cohort study was conducted in a large university hospital in Sweden. Data were retrieved from electronic medical records for adult patients with acute pyelonephritis in 2016. The main outcome was bacteremia and secondary outcome acute kidney injury. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated through multiple logistic regression. Treatment with different groups of antithrombotic agents were compared to no antithrombotic treatment. RESULTS: 1814 patients with acute pyelonephritis were included, in whom bacteremia developed in 336 (18.5%). Low-molecular-weight heparin (LMWH) at prophylactic doses was associated with a lower risk of bacteremia, compared to no antithrombotic treatment (OR 0.5; 95% CI 0.3-0.7). Other antithrombotic treatments were not associated with a risk of bacteremia. Additionally, patients with prophylactic doses of LMWH had a lower risk of acute kidney injury (OR 0.5; 95% CI 0.3-0.8). CONCLUSIONS: We found no association between antithrombotic treatment and an increased risk of bacteremia during acute pyelonephritis. Conversely, patients with prophylactic doses of LMWH had a slightly reduced risk of bacteremia. LMWH at prophylactic doses was also associated with a lower risk of acute kidney injury. Our results suggest that it is safe to continue antithrombotic treatment during acute pyelonephritis, in regards to bacteremia and acute kidney injury risk.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

Antibiotic Prescribing in Connection to Childbirth: An Observational Study in Two Districts in Lao PDR.

Overuse and misuse of antibiotics has frequently been reported for obstetric conditions and procedures, which may impact both the mother and the unborn baby and increase antibiotic resistance. This study aimed to investigate the antibiotic prescribing pattern in connection to childbirth in two districts in Lao PDR. It is a cross-sectional observational study. Antibiotic prescription data related to childbirth was collected via reviews of medical records in two district hospitals and five health centers in Lao PDR from September 2019 to November 2020. In total, antibiotic prescription data for 1777 women were extracted from their medical records. It was found that all women received antibiotics during in-patient care irrespective of delivery mode. When in hospital, 85.5% of the women who underwent a caesarean section got antibiotic treatment for 5 days and women who had a vaginal delivery usually had antibiotic treatment for one day or less. All the women got oral antibiotics for an additional 4-5 days upon discharge. Antibiotic prescription rate in connection to childbirth was very high in comparison with the WHO guidelines, and antibiotics were used extensively in the participating health facilities. Interventions to guide appropriate prescribing behavior in relation to childbirth are urgently needed in Lao PDR.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.

Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1ß, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed. KEY MESSAGES: • Mohanty et al. "HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients." • Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes. • Our key observations are: 1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37. 2. HIF-1 activation increased IL-1ß and IL-8 in human uroepithelial cells treated with high glucose concentration. 3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes. • We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Knowledge, Attitudes, Perception and Reported Practices of Healthcare Providers on Antibiotic Use and Resistance in Pregnancy, Childbirth and Children under Two in Lao PDR: A Mixed Methods Study.

BACKGROUND: Overuse and misuse of antibiotics contribute unnecessarily to antibiotic resistance (ABR), and are thereby global health threats. Inappropriate prescriptions of antibiotics during pregnancy, delivery and early childhood are widespread across the world. This study aimed to assess knowledge, attitudes, and reported practices of healthcare providers (HCPs) and to explore their perceptions regarding antibiotic use and ABR related to pregnancy, childbirth, and children under two in Lao PDR. METHODS: This is a mixed methods study with data collection in 2019 via structured interviews among 217 HCPs (medical doctors/assistant doctors, midwives/nurses, pharmacists/assistant pharmacists and drug sellers), who prescribed/dispensed antibiotics in one rural and one urban district in Vientiane province and individual qualitative interviews with 30 HCPs and stakeholders. RESULTS: Of the HCPs, 36% had below average knowledge regarding antibiotic use and ABR, and 67% reported prescribing antibiotics for uncomplicated vaginal delivery. Half of the HCPs did not believe that their prescribing contributed to ABR, and only 9% had participated in antibiotic education. CONCLUSION: A substantial number of HCPs had suboptimal knowledge and prescribed antibiotics unnecessarily, thereby contributing to ABR. Continuous education and regular supervision of HCPs is recommended to improve the use of antibiotics related to pregnancy, childbirth, and young children.

Dendritic Hydrogels Induce Immune Modulation in Human Keratinocytes and Effectively Eradicate Bacterial Pathogens.

Infections caused by antibiotic-resistant bacteria are globally a major threat, leading to high mortality rates and increased economic burden. Novel treatment strategies are therefore urgently needed by healthcare providers to protect people. Biomaterials that have inherent antibacterial properties and do not require the use of antibiotics present an attractive and feasible avenue to achieve this goal. Herein, we demonstrate the effect of a new class of cationic hydrogels based on amino-functional hyperbranched dendritic-linear-dendritic copolymers (HBDLDs) exhibiting excellent antimicrobial activity toward a wide range of clinical Gram-positive and Gram-negative bacteria, including drug-resistant strains isolated from wounds. Intriguingly, the hydrogels can induce the expression of the antimicrobial peptides RNase 7 and psoriasin, promoting host-mediated bacterial killing in human keratinocytes (HaCaT). Moreover, treatment with the hydrogels decreased the proinflammatory cytokine IL-1ß, reactive nitrogen species (NO), and mitochondrial reactive oxygen species (ROS) in S. aureus-infected HaCaT cells, conjunctively resulting in reduced inflammation.

Metformin strengthens uroepithelial immunity against E. coli infection.

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

Ag@ZnO Nanoparticles Induce Antimicrobial Peptides and Promote Migration and Antibacterial Activity of Keratinocytes.

Antibacterial activity of silver nanoparticles is often associated with toxicity to the host. We here report that noncytotoxic doses of silver nanoparticles coated with zinc oxide, Ag@ZnO, can stimulate proliferation and migration of human keratinocytes, HaCaT, with increased expression of Ki67 and vinculin at the leading edge of wounds. Interestingly, Ag@ZnO stimulates keratinocytes to produce the antimicrobial peptides hBD2 and RNase7, promoting antibacterial activity against both extracellular and intracellular Staphylococcus aureus isolated from wounds. Overall, these results suggest that Ag@ZnO has the potential to significantly improve treatment outcomes in clearing wound infection.

Activation of NLRP3 by uropathogenic Escherichia coli is associated with IL-1ß release and regulation of antimicrobial properties in human neutrophils.

The NLRP3 inflammasome and IL-1ß have recently been linked to the severity of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection (UTI). However, not much is known about the contribution of NLRP3 to the antimicrobial properties of neutrophils and the release of IL-1ß during UPEC infection. The purpose of this study was to elucidate the mechanisms behind UPEC-induced IL-1ß release from human neutrophils, and to investigate the contribution of the NLRP3 inflammasome in neutrophil-mediated inhibition of UPEC growth. We found that the UPEC strain CFT073 increased the expression of NLRP3 and increased caspase-1 activation and IL-1ß release from human neutrophils. The IL-1ß release was mediated by the NLRP3 inflammasome and by serine proteases in an NF-κB-and cathepsin B-dependent manner. The UPEC virulence factors α-hemolysin, type-1 fimbriae and p-fimbriae were all shown to contribute to UPEC mediated IL-1ß release from neutrophils. Furthermore, inhibition of caspase-1 and NLRP3 activation increased neutrophil ROS-production, phagocytosis and the ability of neutrophils to suppress UPEC growth. In conclusion, this study demonstrates that UPEC can induce NLRP3 and serine protease-dependent release of IL-1ß from human neutrophils and that NLRP3 and caspase-1 can regulate the antimicrobial activity of human neutrophils against UPEC.

Anti-biofilm activity of chlorhexidine digluconate against Candida albicans vaginal isolates.

OBJECTIVES: Recurrent vulvovaginal candidiasis (RVVC) causes significant morbidity. Candida albicans is the main pathogen associated with both sporadic and recurrent candidiasis. Due to unsatisfactory treatment effect, the impact of chlorhexidine digluconate and fluconazole alone or in combination on C. albicans and biofilm was investigated. METHODS: Vaginal C. albicans isolates from 18 patients with recurrent candidiasis and commensals from 19 asymptomatic women were isolated by culture. Crystal violet, XTT and colony forming unit assay were used to analyze the effect of chlorhexidine digluconate and fluconazole on growth of C. albicans, formation of new and already established, mature, biofilm. RESULTS: Fluconazole reduced the growth of planktonic C. albicans. However, in established biofilm, fluconazole had no effect on the candida cells and was not able to disperse and reduce the biofilm. By contrast, chlorhexidine digluconate had a direct killing effect on C. albicans grown both planktonically and in biofilm. Chlorhexidine digluconate also dispersed mature biofilm and inhibited formation of new biofilm. No major differences were observed between commensal isolates and candida causing recurrent vulvovaginitis with respect to biofilm or growth after chlorhexidine digluconate treatment. CONCLUSION: Biofilm is a problem in patients with recurrent vulvovaginal candidiasis reducing the effect of antifungal treatment. Development of new treatment strategies are urgently needed to decrease the recurrences. In already established biofilm, chlorhexidine digluconate dispersed the biofilm and was more effective in eradicating candida compared to fluconazole. Future treatment strategy may thus be a combination of chlorhexidine digluconate and fluconazole and prophylactic use of chlorhexidine digluconate to prevent biofilm formation and restrict infections.