Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria

Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae–Escherichia coli, E. coli–Enterococcus faecalis, K. pneumoniae–E. faecalis, and K. pneumoniae–Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.     

Interactions of microorganisms within a urinary catheter polymicrobial biofilm model

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2′-O-methyl-RNA fluorescence in situ hybridization (LNA/2′OMe-FISH), we studied the interactions of four species commonly associated with catheter-associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24-well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (S_r) values (0.1–0.54) in all combinations tested, whereas C. albicans had higher repeatability S_r values (0.36–1.18). The study showed the complexity of early-stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.     

Role of nutrient limitation in the competition between uropathogenic strains of Klebsiella pneumoniae and Escherichia coli in mixed biofilms

Klebsiella pneumoniae and Escherichia coli form mixed species biofilms in catheter-associated urinary tract infections. Recently, a detrimental effect of K. pneumoniae over E. coli was observed in mixed species biofilms grown in an artificial urine medium. The mechanism behind this competitive interaction was studied. K. pneumoniae partially outcompeted E. coli in early-stage batch-fed biofilms, whereas both microorganisms co-exist at longer times (K. pneumoniae:E. coli ratio, 55:1), as shown by cell counts and confocal microscopy. E. coli cells were scattered along the K. pneumoniae biofilm. Biofilm supernatants did not appear to contain either antimicrobial or anti-biofilm activities against E. coli. Biofilms grown under continuous flow prevented interspecies competition. K. pneumoniae showed both increased siderophore production and better growth in iron-limited media compared to E. coli. In summary, these results indicate the importance of nutrient (particularly iron) competition in the modulation of the bacterial composition of mixed species biofilms formed by uropathogenic K. pneumoniae and E. coli.     

Photodynamic and peptide-based strategy to inhibit Gram-positive bacterial biofilm formation

Abstract

The self-produced extracellular polymeric matrix of biofilms renders them difficult to eliminate once they are established. This makes the inhibition of biofilm formation key to successful treatment of biofilm infection. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides offer a new approach as antibiofilm strategies. In this study sub-lethal doses of aPDT (with chlorin-e6 (Ce6-PDT) or methylene blue (MB-PDT)) and the peptides AU (aurein 1.2 monomer) or (AU)₂K (aurein 1.2?C-terminal dimer) were combined to evaluate their ability to prevent biofilm development by Enterococcus faecalis. Biofilm formation was assessed by resazurin reduction, confocal microscopy, and infrared spectroscopy. All treatments successfully prevented biofilm development. The (AU)₂K dimer had a stronger effect, both alone and combined with aPDT, while the monomer AU had significant activity when combined with Ce6-PDT. Additionally, it is shown that the peptides bind to the lipoteichoic acid of the E. faecalis cell wall, pointing to a possible key mechanism of biofilm inhibition.     

Biohybrid Polymer-Antimicrobial Peptide Medium against Enterococcus faecalis

Antimicrobial peptides (AMPs) are conserved evolutionary components of the innate immune system that are being tested as alternatives to antibiotics. Slow release of AMPs using biodegradable polymers can be advantageous in maintaining high peptide levels for topical treatment, especially in the oral environment in which dosage retention is challenged by drug dilution with saliva flow and by drug inactivation by salivary enzymatic activity. Enterococcus faecalis is a multidrug resistant nosocomial pathogen and a persistent pathogen in root canal infections. In this study, four ultra-short lipopeptides (C16-KGGK, C16-KLLK, C16-KAAK and C16-KKK) and an amphipathic α-helical antimicrobial peptide (Amp-1D) were tested against E. faecalis. The antibacterial effect was determined against planktonic bacteria and bacteria grown in biofilm. Of the five tested AMPs, C16-KGGK was the most effective. Next C16-KGGK was formulated with one of two polymers poly (lactic acid co castor oil) (DLLA) or ricinoleic acid-based poly (ester-anhydride) P(SA-RA). Peptide-synthetic polymer conjugates, also referred to as biohybrid mediums were tested for antibacterial activity against E. faecalis grown in suspension and in biofilms. The new formulations exhibited strong and improved anti- E. faecalis activity.     

Real-time PCR method to detect Enterococcus faecalis in water

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day^?1. Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

猪源乳酸菌的分离、鉴定及其生物学特性

【目的】将分离自猪肠道粘膜、食糜和粪便的乳酸菌,通过产乳酸能力、生长性能、耐酸和耐胆盐性能及抑菌能力评价,筛选适应养猪生产的潜在益生特性的菌株。【方法】共分离获得155株乳酸菌纯菌株,从中筛选出4株产酸能力较强的乳酸菌,结合生理生化试验及细菌16S rRNA测序鉴定其种属,评价候选乳酸菌的生长情况、耐酸、耐胆盐及抑菌特性。【结果】综合变色时间(8 h)、pH值(3.9)和乳酸含量(100 mmol/L),筛选出4株(L45、L47、L63和L79)候选菌株,经鉴定依次为罗伊氏乳杆菌、植物乳杆菌、约氏乳杆菌和粪肠球菌。该4株乳酸菌均可在体外快速生长;L47和L79能够耐受pH 2.5的酸性环境,L47能够耐受0.5%胆盐环境;各乳酸菌上清液与指示菌共培养,发现对E coli K88和沙门氏菌均产生了抑制作用,其中L47上清液对指示菌的抑制作用较强。【结论】L47具有较好的产酸性能与生长性能、可耐受猪胃酸和肠道胆盐环境,对E.coli K88和沙门氏菌具有较好的抑制作用,说明该乳酸菌具有潜在的益生特性。     

Polyamine‐independent growth and biofilm formation,and functional spermidine/spermine N‐acetyltransferases in Staphylococcus aureus and Enterococcus faecalis

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram‐positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine‐free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N‐acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine‐independent growth and biofilm formation, and presence of functional polyamine N‐acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.     

Non-competitive product inhibition in lactic acid fermentation from glucose

Summary A kinetic study regarding product inhibition in lactic acid fermentation by Streptococcus faecalis, which produces l-lactic acid, was performed in a chemostat at various feed concentrations of glucose (10, 20, and 30 g/l) at pH 7.0. Steady-state kinetic constants for the specific consumption rate of glucose and the specific production rate of lactic acid were determined at a residual glucose concentration below 2 g/l, which was accomplished in a chemostat. All the parameters, the specific growth rate, the specific consumption rate of glucose, and the specific production rate of lactic acid, were definitely related to non-competitive inhibition with regard to the concentration of the product, lactic acid.Offprint requests to: K. Hiyama     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Genetic Recombination in Klebsiella pneumoniae An Approach to Genetic Linkage Mapping

Genetic recombination was observed between two different strains of Klebsiella pneumoniae, which is a non-motile and encapsulated bacterium belonging to the family Enterobacteriaceae and has about 55% of its DNA content as GC. The mode of recombination seemed to be similar to that of the F-factor mediated conjugation in Escherichia coli. One strain acted as the donor and the other as the recipient, and a relatively large fragment of the donor's chromosome was transferred unilaterally and unidirectionally by cell to cell contact. No genetic factor which is associated with the recombination has been identified. The genetic linkage map of K. pneumoniae was analyzed various mutants derived from the two strains. It was found that the 28 markers so far investigated were arranged linearly in a single linkage group, and that the genetic linkage map of K. pneumoniae, like that of E. coli, could be considered circular. The proposed genetic linkage map of K. pneumoniae was quite similar to that of E. coli or Salmonella typhimurium. The close similarities in this map among the three species suggest a possibility that K. pneumoniae may have differentiated from an ancestor common all three species.     

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3–1C^V and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 μg ml^?1, respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.     

Molecular mechanisms of Streptococcus pneumoniae‐targeted autophagy via pneumolysin,Golgi‐resident Rab41, and Nedd4‐1‐mediated K63‐linked ubiquitination

Streptococcus pneumoniae is the most common causative agent of community‐acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin‐ and ubiquitin‐p62‐LC3 cargo‐dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae‐containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41‐positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48‐ and K63‐linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4‐1 was recruited to PcAV and played a pivotal role in K63‐linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4‐1‐mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell‐invaded pneumococci.     

Surface properties of Enterococcus faecalis cells isolated from chicken hearts determine their low ability to form biofilms

Enterococcus faecalis is one of the most significant bacterial pathogens associated with the first-week mortality of chickens. Here, the surface properties of bacterial cells and the selected virulence factors of E. faecalis strains isolated from the hearts of clinically healthy broiler chickens were studied. Investigations were carried out on live and autoclaved cells. E. faecalis (ATCC 29212) was used as a reference strain. The bacterial cells revealed different haemolytic activities. Their surface free energy was dominated by the hydrophobic component. The cell walls of the bird isolates showed slightly weaker acidic characteristics than those of E. faecalis (ATCC 29212). Moreover, the bacterial cells from the chicken hearts showed higher electrophoretic mobility and surface electrical charge than the reference strain, and consequently demonstrated a low ability to form biofilms.     

Antimicrobial activity of enterocins from Enterococcus faecalis SL-5 against Propionibacterium acnes, the causative agent in acne vulgaris, and its therapeutic effect

A lactic acid bacterial strain was isolated from human fecal specimen and identified as Enterococcus faecalis SL-5. The isolated strain showed antimicrobial activity against Gram-positive pathogens assayed, especially the highest activity against Propionibacterium acnes. The antimicrobial substance was purified and verified as a bacteriocin (named ESL5) of E. faecalis SL-5 by activity-staining using P. acnes as an indicator. N-terminal sequence of ESL5 was determined (MGAIAKLVAK) and sequence analysis revealed that it is almost identical to the some of enterocins including L50A/B of E. faecium L50 and MR10A/B of E. faecalis MRR 10-3. From the sequencing data of L50A/B structural genes, the nucleotide sequence showed 100% identity with that of the MR10A/B structural genes, implying that ESL5 is an equivalent of enterocin MR10. Meanwhile, we also tested the therapeutic effect of anti-P. acnes activity in patients with mild to moderate acne because of its pathogenic role to acne vulgaris. For this purpose, a concentrated powder of CBT SL-5 was prepared using cell-free culture supernatant (CFCS) of E. faecalis SL-5 and included in a lotion for application in the patients. The study showed that CBT SL-5 lotion significantly reduced the inflammatory lesions like pustules compared to the placebo lotion. Therefore our results indicate that the anti-P. acnes activity produced by E. faecalis SL-5 has potential role to the treatment of acne as an alternative to topical antibiotics. These authors contributed equally to this work.     

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84–7.25 CFU cm^?2 at 192?h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.     

Enterococcus faecalis pCF10‐encoded surface proteins PrgA,PrgB (aggregation substance) and PrgC contribute to plasmid transfer,biofilm formation and virulence

Enterococcus faecalis pCF10 transfers at high frequencies upon pheromone induction of the prgQ transfer operon. This operon codes for three cell wall‐anchored proteins – PrgA, PrgB (aggregation substance) and PrgC – and a type IV secretion system through which the plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to plasmid transfer, biofilm formation and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 transfer at wild‐type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone‐responsive, conjugative plasmids of E. faecalis have retained Prg‐like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation and widespread lateral gene transfer.     

Characterization of Candida albicans and Staphylococcus aureus polymicrobial biofilm on different surfaces

BackgroundStaphylococcus aureus and Candida albicans have been co-isolated from biofilm-associated diseases such as denture stomatitis, periodontitis, and burn wound infections, as well as from medical devices. However, the polymicrobial biofilm of both microorganisms has not been fully characterized.AimsTo characterize the polymicrobial biofilm of C. albicans and S. aureus in terms of microbial density, synergy, composition, structure, and stability against antimicrobials and chemical agents.MethodsCrystal violet assay was used to measure the biofilm formation. Scanning electron microscopy and confocal microscopy were used to analyze the structure and chemical composition of the biofilms, respectively.ResultsSupplemented media with fetal bovine serum (FBS) decreased the biofilm formation of S. aureus and the polymicrobial biofilm. For C. albicans, depending on the culture media, the addition of glucose or FBS had a positive effect in biofilm formation. FBS decreased the adhesion to polystyrene wells for both microorganisms. Supplementing the media with glucose and FBS enhanced the growth of C. albicans and S. aureus, respectively. It seems that C. albicans contributes the most to the adhesion process and to the general structure of the biofilms on all the surfaces tested, including a catheter model. Interestingly, S. aureus showed a great adhesion capacity to the surface of C. albicans in the biofilms. Proteins and β-1,6-linked polysaccharides seem to be the most important molecules in the polymicrobial biofilm.ConclusionsThe polymicrobial biofilm had a complex structure, with C. albicans serving as a scaffold where S. aureus adheres, preferentially to the hyphal form of the fungus. Detection of polymicrobial infections and characterization of biofilms will be necessary in the future to provide a better treatment.