Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry.

BACKGROUND: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. RESULTS: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim-sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. CONCLUSIONS: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens.

Synthesis and biological activities of some new isonicotinic acid 2-(2-hydroxy-8-substituted-tricyclo[7.3.1.0(2.7)]tridec-13-ylidene)-hydrazides.

A series of several new isoniazid derivatives, isonicotinic acid 2-(2-hydroxy-8-substituted-tricyclo[7.3.1.0(2.7)]tridec-13-ylidene)-hydrazides, were synthesized and fully characterized. These new isoniazid derivatives were studied regarding their antibacterial activity and cytotoxicity, as well as their influences on some metabolizing enzymes. The best anti-mycobacterial activity was observed in the case of compounds containing alkyl side chains in the 8 position of tricyclo[7.3.1.0(2.7)]tridec-13-ylidene group. On contrary, the antimicrobial activity of these new compounds against various non-tuberculosis strains showed the best activity to be with the phenyl side chain of compound 6. It proved also to be the most toxic, inducing apoptosis and blocking the cell cycle in G0/G1 phase. The cell cycle was blocked in G0/G1 phase also by compound 3, but this compound did not show any toxicity. All compounds induced the expression of NAT1 and NAT2 genes in HT-29 cell line, and the expression of CYP1A1 in HT-29 and HCT-8 cell lines. The expression level of CYP3A4 was increased by compounds 1, 6 and 7 in HCT-8 cells. These results indicated that the activation of other metabolizing pathways, apart from those of isoniazid, take place. It might also point out the possibility of an increased isoniazid acetylation ratio by co-administration with new compounds in slow acetylators.

Molecular screening of carbapenemase-producing Gram-negative strains in Romanian intensive care units during a one year survey.

This is the first study, to our knowledge, performed on a significant number of strains (79 carbapenem-resistant Enterobacteriaceae and 84 carbapenem-resistant non-fermenting Gram-negative rods, GNRs) isolated from tissue samples taken from patients in the intensive care units of two large hospitals in Bucharest, Romania, between 2011 and 2012. The results revealed a high prevalence and great diversity of carbapenemase genes (CRG), in both fermenting and non-fermenting Gram-negative carbapenem-resistant strains. The molecular screening of carbapenem-resistant GNRs revealed the presence of worldwide-distributed CRGs (i.e. blaOXA-48 and blaNDM-1 in Enterobacteriaceae and blaOXA-23, blaVIM-4, blaOXA-10-like, blaOXA-60-like, blaSPM-like and blaGES-like in non-fermenting GNRs), reflecting the rapid evolution and spread of carbapenemase producers, particularly in hospitals. Rapid identification of the colonized or infected patients is required, as are epidemiological investigations to establish the local or imported origin of the respective strains.

Interaction of bacteria isolated from clinical biofilms with cardiovascular prosthetic devices and eukaryotic cells.

PURPOSE: To identify the relationships between some infectious agents implicated in cardiovascular diseases with the cellular substrate and prosthetic devices in the presence of antibiotics. SPECIFIC OBJECTIVES: Strains isolation and identification, comparative study of antibiotic resistance of planktonic (disk diffusion, E-test, automatic systems) and sessile (using original experimental models for in vitro development of monospecific biofilms) bacterial cells, virulence assays (adherence and invasion of HeLa cells, slime test, soluble virulence factors expression), dynamic study of biofilm development on inert substrata, under the influence of antibiotics, the influence of cellular and soluble bacterial fractions on HeLa cells (by flow cytometry and real-time PCR). RESULTS: The identified strains were isolated from different sources, the etiology being dominated by Gram-negative non-fermentative bacilli, Gram-positive cocci and yeasts, harboring invasion enzymes responsible for development of systemic infections. The isolated strains exhibited a high level of antibiotic resistance to beta-lactams, aminoglycosides and quinolones, and an evident tendency of colonizing the cellular and inert substrate, the degree of colonization depending on the physico-chemical nature of the substrate. By comparison with planktonic ones, the sessile bacterial strains expressed a changed profile of antibiotic resistance, this aspect being very important for the readjustment of the treatment and prevention of infections associated with prosthetic devices. In vitro experiments suggested that different fractions of S. aureus cultures could trigger the release of proinflammatory (TNF-α, IL-1b, IL-6) and anti-inflammatory (IL-8) cytokines and induced apoptosis in HeLa cells.

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing.

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.

Screening of molecular virulence markers in Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from clinical infections.

Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections.

Quantitative real-time PCR study of the influence of probiotic culture soluble fraction on the expression of Pseudomonas aeruginosa quorum sensing genes.

The opportunistic pathogen Pseudomonas (Ps.) aeruginosa causes severe infections, particularly in immunocompromised individuals and patients with cystic fibrosis (CF). A serious side effect of antibiotic therapy in Ps. aeruginosa infections is the development of resistance to antibiotics. During the infection process Ps. aeruginosa forms biofilms, rendering bacterial cells more resistant to disinfectants, antibiotics and the action of host immune defense effectors. Pseudomonas aeruginosa employs the intercellular communication system, known as quorum sensing (QS) to coordinate the expression of tissue-damaging factors. Since the QS systems controls the production of different virulence factors, it is possible that the inhibition of its regulatory activity to severely compromise the ability of Ps. aeruginosa to cause infections in humans. Many studies have shown that some probiotic strains exhibit inhibitory activity on different virulence properties of pathogenic bacteria (adherence to cellular or inert substrate, soluble virulence factors expression). The aim of the present study was to investigate by real-time RT-qPCR the influence of probiotic culture soluble factors on the QS genes expression in 30 Ps. aeruginosa strains isolated from patients hospitalized in the National Institute for Cardiovascular Infections, Prof. C.C. Iliescu Fundeni Hospital, Bucharest. The results of the real time RT-qPCR have shown that in all Ps. aeruginosa strains grown in the presence of probiotic culture sterile filtrates, the level of QS genes expression was reduced comparatively with those from control cultures. In conclusion, these results proved that the inhibition of virulence factors regulation mechanisms by soluble molecules secreted by probiotics could represent an interesting way pathogenicity and virulence attenuation in Ps. aeruginosa nosocomial strains.

Subinhibitory concentrations of phenyl lactic acid interfere with the expression of virulence factors in Staphylococcus aureus and Pseudomonas aeruginosa clinical strains.

The discovery of intra- and intercellular communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria, without interfering with their growth. In this study, we investigated the ability of subinhibitory concentrations (sIC) of phenyl lactic acid (PLA), known to be produced by Lactobacillus probiotic strains, to attenuate the virulence and pathogenicity of Pseudomonas aeruginosa (as experimental model of intercellular bacterial communication in Gram-negative bacteria) and Staphylococcus aureus (as experimental model of intercellular bacterial communication in Gram-positive bacteria) by interfering with the coordinated expression of different virulence factors implicated in the pathogenicity of these opportunistic strains. Our results showed that sIC of PLA decreased the ability of the tested strains to adhere both to the cellular and inert substrata and induced changes in the adherence patterns as well as in the cell morphology. The sIC of PLA induced a significant decrease of sheep red blood cells haemolysins, lecithinase and caseinase and stimulated lipase and gelatinase production by Pseudomonas strains. The sIC of PLA induced an important and constant increase of the Pseudomonas growth inhibition zones diameters for all tested antibiotics, demonstrating the potential use of PLA in the design of new synergic antimicrobial associations active on multiresistant and biofilm-growing P, aeruginosa strains. The present study has proved the role of sIC of PLA released by Lactobacillus probiotic strains in the attenuation of P. aeruginosa and S. aureus virulence and pathogenicity, by interfering with different processes depending on cell density and regulated by quorum sensing (i.e. growth rate, expression of adhesion molecules and secretion of soluble, enzymatic factors) and altering the success of these pathogens in the colonization of a sensitive host and the development of an infectious process. Our results demonstrate that this probiotic soluble products could be used as a new, ecological anti-infective strategy with great therapeutic and preventive value in the biomedical field (especially in the treatment of chronic infections produced by multiresistant and biofilm forming microorganisms), but also in the management of the environmental quality, agriculture and industrial field by reducing the chemical burden delivered in the external medium and by preventing the surfaces colonization with microorganisms and the development of natural biofilms.

In vivo experimental model for the study of the influence of subinhibitory concentrations of phenyllactic acid on Staphylococcus aureus pathogenicity.

The discovery of communication systems regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with their growth. In this paper the authors describe the effect of subinhibitory concentrations of phenyl-lactic acid (PLA) on the pathogenicity of Staphylococcus aureus in holoxenic mice. The animals were inoculated by oral (p.o.), intranasal (i.n.), intravenous (i.v.) and intraperitoneal (i.p.) routes with S. aureus wild and PLA-treated cultures. The mice were followed up during 16 days after infection and the body weight, mortality and morbidity rate were measured every day. The microbial charge was studied by viable cell counts in lungs, spleen, intestinal mucosa and blood. The present study has proved that PLA, besides its microbicidal activity, could attenuate in subinhibitory concentrations the virulence and pathogenicity of S. aureus strains, as demonstrated by in vivo holoxenic mouse infection experimental model. Our results are accounting for the hypothesis that subinhibitory concentrations of PLA, which are not affecting the bacterial cell viability, are probably interfering with the intracellular communication and the sequential and coordinated expression of different virulence factors, altering the success of this pathogen in the colonization of sensitive hosts and the development of an infectious process.