Proteus mirabilis outcompetes Klebsiella pneumoniae in artificial urine medium through secretion of ammonia and other volatile compounds

Klebsiella pneumoniae and Proteus mirabilis form mixed biofilms in catheter-associated urinary tract infections. However, co-inoculation of P. mirabilis with K. pneumoniae in artificial urine medium (AUM) resulted in a drastic reduction of K. pneumoniae cells in both biofilm and planktonic growth. Here, the mechanism behind this competitive interaction was studied. Both pH and aqueous ammonia (NH3aq) increased in mixed cultures (to 9.3 and 150 mM, respectively), while K. pneumoniae viable cells dramatically diminished over time (>6-log reduction, p < 0.05). Mixed cultures developed in either 2-(N-morpholino) ethanesulfonic acid (MES)-buffered AUM (pH 6.5) or AUM without urea did not show bacterial competition, evidencing that the increase in pH and/or NH3aq concentration play a role in the competitive interaction. Viability of K. pneumoniae single-species cultures decreased 1.5-log in alkaline AUM containing 150 mM NH3aq after 24 h inoculation, suggesting that ammonia is involved in this inter-species competition. Besides NH3aq, additional antimicrobials should be present to get the whole competitive effect. Supernatants from P. mirabilis-containing cultures significantly diminished K. pneumoniae viability in planktonic cultures and affected biofilm biomass (p < 0.05). When subjected to evaporation, these supernatants lost their antimicrobial activity suggesting the volatile nature of the antimicrobial compounds. Exposure of K. pneumoniae to volatile compounds released by P. mirabilis significantly decreased cell viability in both planktonic and biofilm cultures (p < 0.05). The current investigation also evidenced a similar bactericidal effect of P. mirabilis volatiles over Escherichia coli and Morganella morganii. Altogether, these results evidence the secretion of ammonia and other volatile compounds by P. mirabilis, with antimicrobial activity against gram-negative uropathogens including K. pneumoniae. This investigation provides novel insight into competitive inter-species interactions that are mediated by production of volatile molecules.Fil: Juarez, Guillermo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Centro de Estudios Biomédicos, Básicos, Aplicados y Desarrollo. Departamento de Ciencias Biológicas y Biomédicas; ArgentinaFil: Mateyca, Andrea Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Galvan, Estela Maria. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

Escherichia coli is the most frequent agent of urinary tract infections in humans. The emergence of uropathogenic multidrug-resistant (MDR) E. coli strains that produce extended spectrum β-lactamases (ESBL) has created additional problems in providing adequate treatment of urinary tract infections. We have previously reported the antimicrobial activity of 1,8-cineole, one of the main components of Rosmarinus officinalis volatile oil, against Gram negative bacteria during planktonic growth. Here, we evaluated the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of MDR ESBL-producing uropathogenic E. coli clinical strains by carrying out different technical approaches such as counting of viable cells, determination of biofilm biomass by crystal violet staining, and live/dead stain for confocal microscopy and flow cytometric analyses. The plant compound showed a concentration- and time-dependent antibiofilm activity over pre-formed biofilms. After a 1 h treatment with 1% (v/v) 1,8-cineole, a significant decrease in viable biofilm cell numbers (3-log reduction) was observed. Biofilms of antibiotic-sensitive and MDR ESBL-producing E. coli isolates were sensitive to 1,8-cineole exposure. The phytochemical treatment diminished the biofilm biomass by 48-65% for all four E. coli strain tested. Noteworthy, a significant cell death in the remaining biofilm was confirmed by confocal laser scanning microscopy after live/dead staining. In addition, the majority of the biofilm-detached cells after 1,8-cineole treatment were dead, as shown by flow cytometric assessment of live/dead-stained bacteria. Moreover, phytochemical-treated biofilms did not fully recover growth after 24 h in fresh medium. Altogether, our results support the efficacy of 1,8-cineole as a potential antimicrobial agent for the treatment of E. coli biofilm-associated infections.Fil: Vázquez, Nicolás Martín. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mariani, Florencia. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Pablo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Moreno, Silvia. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galvan, Estela Maria. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Tn5AraOut mutagenesis for the identification of Yersinia pestis genes involved in resistance towards cationic antimicrobial peptides

Bacterial pathogens display a variety of protection mechanisms against the inhibitory and lethal effects of host cationic antimicrobial peptides (CAMPs). To identify Yersinia pestis genes involved in CAMP resistance, libraries of DSY101 (KIM6 caf1 pla psa) minitransposon Tn5AraOut mutants were selected at 37°C for resistance to the model CAMPs polymyxin B or protamine. This approach targeted genes that needed to be repressed (null mutations) or induced (upstream P(BAD) insertions) for the detection of CAMP resistance, and predictably for improved pathogen fitness in mammalian hosts. Ten mutants demonstrated increased resistance to polymyxin B or protamine, with the mapped mutations pointing towards genes suspected to participate in modifying membrane components, genes encoding transport proteins or enzymes, or the regulator of a ferrous iron uptake system (feoC). Not all the mutants were resistant to both CAMPs used for selection. None of the polymyxin B- and only some protamine-resistant mutants, including the feoC mutant, showed increased resistance to rat bronchoalveolar lavage fluid (rBALF) known to contain cathelicidin and β-defensin 1. Thus, findings on bacterial resistance to polymyxin B or protamine don't always apply to CAMPs of the mammalian innate immune system, such as the ones in rBALF.Fil: Guo, Jitao. Peking University Health Science Center. Department of Microbiology; ChinaFil: Nair, Manoj K. M.. University of Pennsylvania. School of Veterinary Medicine; Estados UnidosFil: Galvan, Estela Maria. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. University of Pennsylvania; Estados UnidosFil: Liu, Shu Lin. Peking University Health Science Center. Department of Microbiology; ChinaFil: Schifferli, Dieter M.. University of Pennsylvania. School of Veterinary Medicine; Estados Unido

Exposure of multidrug-resistant Klebsiella pneumoniae biofilms to 1,8-cineole leads to bacterial cell death and biomass disruption

Klebsiella pneumoniae is a common cause of health-care associated infections. The rise of antibiotic resistance and the ability to form biofilm among K. pneumoniae strains are two key factors associated with antibiotic treatment failure. The present study investigates the antibiofilm activity of 1,8-cineole against preformed biofilms of multidrug-resistant extended-spectrum β-lactamase-producing K. pneumoniae clinical isolates. To evaluate the antibiofilm activity, cellular viability was analyzed by colony-forming units counting and live/dead staining. In addition, biofilm biomass was evaluated by crystal violet and the biofilm matrix was stained with calcofluor white and observed by confocal laser scanning microscopy. A time- and concentration-dependent effect of the phytochemical over biofilm cell viability was observed revealing that 1% (v/v) 1,8-cineole during 1 h was the optimal treatment condition displaying a significant reduction of cell viability in the preformed biofilms (2.5?5.3 log cfu/cm2). Furthermore, confocal laser scanning microscopy after SYTO-9 and propidium iodide staining showed that 1,8-cineole was capable of killing bacteria throughout all layers of the biofilm. The compound also caused a biofilm disruption (30?62% biomass reduction determined by crystal violet staining) and a significant decrease in biofilm matrix density. Altogether, our results demonstrate that 1,8-cineole is a promising candidate as a novel antibiofilm agent against multidrug-resistant K. pneumoniae strains producing extended-spectrum β-lactamases, given its capability to disrupt the structure and to kill cells within the biofilm.Fil: Vázquez, Nicolás Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Moreno, Silvia. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galvan, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentin

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.Fil: Juarez, Guillermo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Galvan, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentin

1,8-cineole as an antimicrobial and antibiofilm agent against multidrug resistant Klebsiella pneumoniae

Klebsiella pneumoniae is a common cause of antimicrobial-resistant opportunistic infections in hospitalized patients, including urinary tract infections. The emergence of multidrug-resistant (MDR) strains producing extended-spectrum β-lactamases (ESBL) and/or carbapenemases, in combination with the capacity to produce biofilm has created additional problems in providing adequate antibiotic treatment of urinary tract infections. Biofilms are complex bacterial communities adhered to biotic or abiotic surfaces that are surrounded by an extracellular matrix composed of exopolysaccharides, proteins and nucleic acids that give them differential phenotypic properties associated with greater resistance to antibiotics. 1,8-cineole, one of the main components of Rosmarinus officinalis volatile oil, has shown antimicrobial activity against non-MDR Gram negative bacteria (including K. pneumoniae) during planktonic growth. Here, we evaluated the antimicrobial and antibiofilm activity of 1,8-cineole against planktonic and pre-formed mature biofilms of non-MDR and MDR ESBL-producing K. pneumoniae clinical strains isolated from urinary tract infections. Killing curves were performed in planktonic cultures by adding 1% (v/v) 1,8-cineole for 5-180 min and counting viable cells (cfu/ml). Results showed variable decrease of K. pneumoniae viability (ranging from 0.5 to 4 log reduction) after phytochemical treatment, not related to their antibiotic resistance profile. Regarding biofilms, all tested strains formed robust biomass after 48 h, as determined by crystal violet staining (Abs 595nm > 1). One-hour treatment with 1% (v/v) 1,8-cineole partially disrupted biofilm biomasses (34 to 62% reduction in crystal violet staining). Additionally, a variable decrease in cell viability (between 0,5 and 4 log reduction of ufc/cm2 ) was observed by viable cell counting, regardless if they were or not MDR. Two MDR ESBL-producing K. pneumoniae strains, presenting different susceptibility to 1,8-cineole, were chosen to study their extracellular matrix in biofilms by confocal laser scanning microscopy after calcofluor white staining. Noteworthy, differences in the extracellular matrix structure were observed between strains, that could account for differences in 1,8-cineole susceptibility. Altogether, our results show that some antibiotic-sensitive and MDR ESBL-producing K. pneumoniae isolates were sensitive to 1,8-cineole exposure and support the efficacy of 1,8-cineole as a potential antimicrobial agent for the treatment of planktonic and biofilm-associated infections caused by MDR K. pneumoniae.Fil: Vázquez, Nicolás Martín. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Laboratorio de Biotecnologia Bacteriana.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moreno, Silvia. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galvan, Estela Maria. Laboratorio de Biotecnologia Bacteriana.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB) y XVI Annual Meeting of the Argentinean Society for General Microbiology (SAMIGE)ArgentinaSociedad Argentina de Bioquímica e Investigación en Biología MolecularSociedad Argentina de Microbiología Genera

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.Fil: Galvan, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mateyca, Andrea Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Xanthan chain length is modulated by increasing the availability of the polysaccharide copolymerase protein GumC and the outer membrane polysaccharide export protein GumB

Xanthan is a polysaccharide secreted by Xanthomonas campestris that contains pentameric repeat units. The biosynthesis of xanthan involves an operon composed of 12 genes (gumB to gumM). In this study, we analyzed the proteins encoded by gumB and gumC. Membrane fractionation showed that GumB was mainly associated with the outer membrane, whereas GumC was an inner membrane protein. By in silico analysis and specific globomycin inhibition, GumB was characterized as a lipoprotein. By reporter enzyme assays, GumC was shown to contain two transmembrane segments flanking a large periplasmic domain. We confirmed that gumB and gumC mutant strains uncoupled the synthesis of the lipid-linked repeat unit from the polymerization process. We studied the effects of gumB and gumC gene amplification on the production, composition and viscosity of xanthan. Overexpression of GumB, GumC or GumB and GumC simultaneously did not affect the total amount or the chemical composition of the polymer. GumB overexpression did not affect xanthan viscosity; however, a moderate increase in xanthan viscosity was achieved when GumC protein levels were increased 5-fold. Partial degradation of GumC was observed when only that protein was overexpressed; but co-expression of GumB and GumC diminished GumC degradation and resulted in higher xanthan viscosity than individual GumB or GumC overexpression. Compared with xanthan from the wild-type strain, longer polymer chains from the strain that simultaneously overexpressed GumB and GumC were observed by atomic force microscopy. Our results suggest that GumB-GumC protein levels modulate xanthan chain length, which results in altered polymer viscosity.Fil: Galvan, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ielmini, María V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Patel, Yamini N.. Fermentation Sciences; Estados UnidosFil: Bianco, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Franceschini, Esteban Andres. Comision Nacional de Energía Atómica; ArgentinaFil: Schneider, Jane C.. Fermentation Sciences; Estados UnidosFil: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Adhesive properties of YapV and paralogous autotransporter proteins of Yersinia pestis

Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs.Fil: Nair, Manoj Kumar Mohan. University Of Pennsylvania; Estados UnidosFil: De Masi, Leon. University Of Pennsylvania; Estados UnidosFil: Yue, Ming. University Of Pennsylvania; Estados UnidosFil: Galvan, Estela Maria. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. State University Of Pennsylvania; Estados UnidosFil: Chen, Huaiqing. University Of Pennsylvania; Estados UnidosFil: Wang, Fang. University Of Pennsylvania; Estados UnidosFil: Schifferli, Dieter M.. University Of Pennsylvania; Estados Unido