Amino acid starvation and iron limitation facilitate the biofilm formation of Klebsiella pneumoniae within urine.

Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG(0)) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. We present the first systematic genome-wide investigation of the role of 3',5'-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG(0)) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.

Biofilm formation by uropathogenic Escherichia coli: a complicating factor for treatment and recurrence of urinary tract infections

IntroductionBacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core.Materials and MethodsRabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECAPG) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECAPG is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECALPS) free of ECAPG. The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies.ResultsThe results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECALPS, suggesting that it induced the anti-ECA antibody responses. Only the presence of ECAPG could be demonstrated in the Rc mutant strain R4/O28.ConclusionsThese results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECALPS. Since ECAPG is not immunogenic unless combined with some proteins, it is likely that ECAPG-protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.

As an important zoonotic pathogen, Staphylococcus aureus has led to serious mastitis and endometritis in infected dairy cows. In this study, a total of 164 strains of S. aureus were isolated from dairy cows in Xinjiang Province, China, and subjected to assays to determine drug susceptibility and biofilm (BF) formation ability. Enterotoxin-related genes were detected, and the transcription levels of genes related to BF formation were determined by using reverse transcription-quantitative polymerase chain reaction. Moreover, the pathogenicity of isolates with different BF formation abilities was determined by measuring their hemolysis activity, half lethal dose (LD50) and organ bacterial load. The results showed that 86.0% of S. aureus isolates could form BF. Among them, 42.1% of the strains had weak BF formation ability, and most strains with a strong BF formation ability were ica gene carriers. The S. aureus isolates displayed multidrug resistance and their drug resistance was positively correlated with their BF formation ability. Moreover, 96.3% of the S. aureus isolates carried enterotoxin genes. Among them, the detection rates of the novel enterotoxin genes were higher than those of conventional enterotoxin genes. Furthermore, isolates with a strong BF formation ability had higher LD50 but lower hemolysis ability and organ bacterial load than those of the isolates with weak or no BF ability. However, isolates without BF ability produced more severe pathological changes than those of isolates with strong BF formation ability. These findings suggest that higher BF ability and presence of novel enterotoxin genes are important characteristics of S. aureus isolates from dairy cows in Xinjiang Province, China, and such isolates may pose potential threats to food safety.

ABSTRACTPseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections.

Enterobacterial common antigen (ECA) has attracted considerable interest since the original publication by Kunin in 1962. In the present study we demonstrated this antigen directly in the urine from patients with urinary tract infections (UTI) elicited by enterobacteria. Sheep erythrocytes were incubated with UTI urine; this resulted in their coating with ECA, which was studied by means of hemagglutination by anti-ECA serum. Test tube hemagglutination and the more simple slide hemagglutination were employed and with both procedures similar results were obtained. Positive results were observed in 94-99% of urine specimens from enterobacterial UTI collected in The Buffalo Children's Hospital in the 1960s. ECA in urine could also be demonstrated by hemagglutination inhibition. In this test, antibodies in anti-ECA serum were neutralized as a result of incubation of this serum with urine, and agglutination by the antiserum of sheep erythrocytes coated with a standard ECA preparation was prevented or reduced. By means of this latter test, ECA could be demonstrated in 67-88% of urine specimens from enterobacterial UTI. The possible diagnostic application of these tests has been discussed.

Iron Salts Perturb Biofilm Formation and Disrupt Existing Biofilms of Pseudomonas aeruginosa

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

Background: Biofilms have been reported as important virulent markers associated with drug resistance in urinary tract infections (UTIs) in humans and dogs. However, in veterinary medicine, researches involving biofilm formation, treatments and preventions have been limited; yet, it is still possible to find few studies demonstrating biofilm-forming bacteria associated with different comorbidities such as otitis, wound infections, UTIs, and endometritis. These studies generally select dogs with chronic and recurrent infections, which could be an important factor in antibiotic resistance. We aimed to evaluate biofilms in sporadic cystitis regarding prevalence and drug resistance.Materials, Methods & Results: Urine samples were collected by cystocentesis from 36 client-owned dogs under clinical and laboratory suspicion of non-recurrent urinary bladder infection (cystitis). Urine was aseptically plated onto blood agar, MacConkey, and CLED, followed by incubation for 24 to 48 h. Definitive identification of a potential pathogen was made by subculture collected from an isolated colony to obtain a pure culture. The gram staining method and specific biochemical tests (phenol red fermentation, lysine, phenylalanine, citrate, sulfide-indole-motility, and urease) were used to distinguish and classify the bacteria. After identification, the bacteria were tested for antimicrobial susceptibility by a standard disk diffusion method, using the following antimicrobials: amoxicillin with clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, clindamycin, cefazolin, cephalothin, erythromycin, gentamicin, norfloxacin, and sulfamethoxazole-trimethoprim. The biofilm-forming ability was determined based on a culture in Congo red agar (CRA), where biofilm producer strains formed black colonies with a dry crystalline surface, while non-biofilm producer strains formed red colonies with a smooth surface. A crystal violet dye assay was used to confirm the CRA results. Of the 36 urine samples collected from dogs with suspected cystitis, a total of 37 isolates were obtained, from mixed or pure cultures. The most prevalent bacteria were Escherichia coli (11/37), followed by Staphylococcus spp. (8/37), Proteus spp. (7/37), and Enterococcus spp. (5/37). Other less prevalent bacteria were Klebsiella spp., Streptococcus spp., and Enterobacter spp. As for biofilm-forming ability, 67.6% (25/37) of the 37 bacterial isolates had biofilm formation in CRA and 54.05% (20/37) on the microplates containing crystal violet dye. There was no statistical difference in antimicrobial susceptibility between biofilm producer and non-biofilm producer bacteria.Discussion: We found a high proportion (> 54%) of in vitro biofilm-forming ability by different bacteria, which may indicate that biofilms may also be formed in vivo, in simple cystitis. Antimicrobial resistance was not noticed in bacteria capable of forming a biofilm; however, in a future study it is important to evaluate bacterial resistance in vivo, considering the possibility of having a different response than in vitro. In addition, the problem of the presence of a biofilm in vivo is that it can nullify the antimicrobial efficacy of therapeutic agents even with in vitro susceptibility. Besides the possibility of slow or incomplete diffusion of antibiotics through the extracellular matrix of the biofilm, aspects like hydration level, pCO2, pO2, pH, pyrimidine, and divalent cation concentration that negatively influence antimicrobial activity in vitro can also cause undesirable effects at the profound layers of the biofilm. In conclusion, all of the genera of bacteria isolated from dog’s sporadic cystitis in this study were able to form a biofilm in vitro. The pathogenicity and antibiotic resistance of bacteria appears unrelated to biofilm formation in vitro.Keywords: sessile bacteria, urine, simple cystitis, antibiotic resistance.

Background: The probiotics’ auto-aggregation and biofilm formation abilities have a significant role in the development of biotechnological processes.Objective: The aim of this study was to evaluate the biofilm formation and auto-aggregation abilities of novel, targeted aqua-probiotics isolated from aquatic organisms.Methods: The biofilm formation abilities of Lactobacillus delbrueckii str. UZ-1, Lactiplantibacillus plantarum str. R3, Lactococcus str. UZ-2, Enterococcus faecium str. R2, Pediococcus acidilactici str. N from the culture collection of the Microbiology of the Academy of Sciences of the Republic of Uzbekistan, Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R and from the culture collection of the Southern Federal University of Russa and Lacticaseibacillus rhamnosus str. 1A and Enterococcus str. 9-3 from the culture collection of the Armenian National Agrarian University were assessed.Results: According to the investigations, the biofilm formation abilities of Lactobacillus delbrueckii str. UZ-1, Lactiplantibacillus plantarum str. R3, Lactococcus str. UZ-2, Enterococcus faecium str. R2, Pediococcus acidilactici str. N, Bacillus subtilis str. 1R, Bacillus amyloliquefaciens str. 4R, Bacillus amyloliquefaciens str. 5R, Lacticaseibacillus rhamnosus str. 1A and Enterococcus str. 9-3 were 0.119 ± 0.05D, 0.113 ± 0.065D, 0.196 ± 0.04D, 0.116 ± 0.01D, 0.152 ± 0.05D, 0.74 ± 0.15D, 2.621± 0.55D, 1.831 ± 0.45D, and 0.227 ± 0.04D and 0.483 ± 0.15D respectively. The highest rate of auto-aggregation was shown by Bacillus amyloliquefaciens str. 5R, and Bacillus amyloliquefaciens str. 4R was the strain with the highest ability to form biofilm. These two Bacillus strains are also distinguished by the highest DNA protective properties and relatively low antioxidant activity. Despite the fact that Bacillus amyloliquefaciens str. 5R showed the highest rate of auto-aggregation after 2 hours, this strain showed the lowest level of auto-aggregation among the studied strains after 24 hours. The Enterococcus str. 9-3 strain with the highest antioxidant activity showed 0.483 ± 0.15D biofilm formation ability.Conclusion: The novel targeted aquaprobiotics have distinct biofilm formation and aggregation properties, which are important to consider when planning appropriate biotechnological processes, requiring specific membrane properties of probiotics.Graphical Abstract: Membrane properties of novel targeted aquaprobiotics.Keywords: Lactobacilli, aqua-probiotic, antioxidant activity, biofilm formation, aggregation, Enterococcus str. 9-3

SspA positively controls exopolysaccharides production and biofilm formation by up-regulating the algU expression in Pseudoalteromonas sp. R3

Presence of virulence factor genes (gelE and esp) and biofilm formation in clinical Enterococcus faecalis and Enterococcus faecium isolated from urinary tract infection in Isfahan, Iran

In response to adverse conditions, many bacterial species can switch from a planktonic growth to a surface associated growth mode and form biofilm communities. A key factor triggering the formation of biofilms in a multitude of bacterial species is the second messenger bis-(3’-5’)cyclic dimeric guanosine (c-di-GMP). The biosynthesis of c-di-GMP by condensation of two GTP molecules is performed by diguanylate cyclases (DGCs). DGCs consist of catalytic GGDEF domains in combination with N-terminal, environment sensing regulatory domains. A significant fraction of DGCs are linked to N-terminal sequences of unknown function indicating that c-di-GMP signaling is linked to numerous undiscovered environmental and cellular signals. In this study structural and biochemical analysis on the DGC YdeH from E. coli was undertaken, to elucidate its regulatory mechanism. Three-dimensional structures of YdeH were determined, which reveal in the regulation of YdeH. The N-terminal sensory domain of YdeH shows a new fold, a four helical bundle, which harbors a zinc-binding site compromised of three histidines and one cysteine. It could be shown that the DGC activity of YdeH is inhibited by zinc binding to the N- terminal sensory domain with an inhibition constant in the femtomolar range. A model for the inhibition of YdeH by zinc is proposed, in which upon zinc binding the linker between the regulatory domain and the enzymatic domain is fixed in a conformation, which prevents the productive encounter of the two GGDEF domains. In the structures of YdeH, substrate and product binding to the active site could be shown, however the dimeric arrangement of the two DGC domains, each harboring only one half of the active site, are not in a competent constellation. With the help of the determined structures of YdeH a model of a competent dimer was generated, which provides insights into the regulation of YdeH. Product binding to the inhibitory site of YdeH was shown in the crystal structures and inhibition by c-di-GMP was demonstrated in enzymatic experiments. YdeH represents the first example of a biological zinc-sensor that exerts its downstream effects post-transcriptionally and the first example of a metal sensory c-di-GMP signaling protein. A protocol for the enzymatic large-scale synthesis of c-di-GMP by using the DGC YdeH from E. coli was developed and optimized. In contrast to the chemical synthesis of c-di- GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within one day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes. In biofilms cells are entrapped within a extracellular polymeric matrix. One component of this matrix is the poly-β-1,6-N-Acetyl-glucosamine (poly-1,6-GlcNAc), which is synthesized and exported by the four proteins of the pgaABCD operon. PgaC and PgaD are responsible for the synthesis of poly-1,6-GlcNAc and are allosterically regulated by c-di-GMP. The deacetylase PgaB and the outer membrane protein PgaA are involved in the modification and export of the poly-1,6-GlcNAc chain. For PgaA and PgaB an expression and purification protocol was established and resulted in stable and homogenous proteins. The predicted deacetylase activity of PgaB was demonstrated in vitro with an activity assay, which is suitable for rapid screening of different reaction conditions and for the search of inhibitors for PgaB and PgaC, which are of specific pharmaceutical interest.

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.

Exopolysaccharides (EPS) are of high significance in bacterial biofilm formation. However, the effects of EPS cluster(s) on biofilm formation in Paenibacillus species are little known. In this study, we have shown that Paenibacillus polymyxa WLY78, a N2-fixing bacterium, can form biofilm. EPS is the major component of the extracellular matrix. The genome of P. polymyxa WLY78 contains two putative gene clusters (designated pep-1 cluster and pep-2 cluster). The pep-1 cluster is composed of 12 putative genes (pepO-lytR) co-located in a 13 kb region. The pep-2 cluster contains 17 putative genes (pepA-pepN) organized as an operon in a 20 kb region. Mutation analysis reveals that the pep-2 cluster is involved in EPS biosynthesis and biofilm formation. Disruption of the pep-2 cluster also leads to the enhancement of motility and change of the colony morphology. In contrast, disruption of the pep-1 cluster does not affect EPS synthesis or biofilm formation. More importantly, the biofilm allowed P. polymyxa WLY78 to fix nitrogen in aerobic conditions, suggesting that biofilm may provide a microaerobic environment for nitrogenase synthesis and activity.