Agnieszka E. Laudy

Synthesis and antimicrobial and nitric oxide synthase inhibitory activities of novel isothiourea derivatives

The reaction of substituted benzylhalides, or of halomethyl derivatives of thiophene or furane, with thiourea or its derivatives yielded the respective isothioureas as hydrohalide salts. The products (a total of 17, including 16 novel compounds) were tested for activity against five Gram-positive and nine Gram-negative bacterial strains, six yeast species and two protozoan species. The most active against Gram-positive bacteria were S-(2,4-dinitrobenzyl)isothiourea hydrochloride (MIC range for four out of five strains tested: 12.5–25 μg/mL) and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide (MIC range: 12.5–50 μg/mL). The lowest MICs of novel isothioureas for yeast and Gram-negative bacteria ranged between 50 and 100 μg/mL. Nine novel isothioureas showed appreciable genotoxicity in the Bacillus subtilis ‘rec-assay’ test, the most potent being S-2-(5-nitrofuran-2-ylmethyl)isothiourea and S-(2-nitrobenzyl) isothiourea. At 10 μM concentration, S-(3,4-dichlorobenzyl)isothiourea hydrochloride and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide inhibited Ca2+/calmodulin-dependent (non-inducible) nitric oxide synthase activity in normal rat brain homogenates stronger (p < 0.05) than the reference drug 7-nitroindazole (by 78, 76 and 60%, respectively); ten other new isothiourea derivatives significantly inhibited the activity to a lower extent (by 28–60%). These results extend the list of promising isothioureas with substantial activity in vitro and suggest that an in-depth study of toxicity, antimicrobial properties in vivo and nitric oxide synthase isoform selectivity of selected novel compounds is warranted.

German Cockroaches (Blattella Germanica L.) as a Potential Source of Pathogens Causing Nosocomial Infections

German cockroaches collected in two hospitals were examined bacteriologically. Twenty strains of different taxons were isolated, all considered to be potentially pathogenic to humans. Some strains were resistant to antibacterial drugs widely used for treatment of patients: Gram-negative rods were resistant to amoxicillin/clavulanic acid, Pseudomonas spp. additionally resistant to co-trimoxasole. Strains of Staphylococcus equorum, S. hominis were methicillin-resistant, a strain of S. epidermidis showed MLSb mechanism of resistance. Determination of the minimum inhibitory concentration (MIC) for the disinfectant sodium dichloroisocyanurate showed it was in the range 500- 2000 mg. L−1. The MIC for glucoprotamine was from 1.453 to 500 mg. L−1. A strain of S. epidermidis was less sensitive (MIC = 125 mg. L−1) to glucoprotamine than other staphylococci, Serratia marcescens was resistant to that agent (MIC = 500 mg. L−1). Product containing glucoprotamine was ineffective against strains of S. epidermidis MLSb and Pseudomonas putida. Our data suggest that cockroaches might serve as a source of infection, by acting as carriers and reservoirs of bacterial strains in nosocomial outbreaks.

Modification of the Susceptibility of Gram- Negative Rods Producing ESβLS to β-Lactams by the Efflux Phenomenon

The production of β-lactamases is the most important mechanism of Gram-negative rod resistance to β-lactams. Resistance to ceftazidime and cefepime in clinical isolates of Enterobacteriaceae (especially ESβL-positive E. coli and K. pneumoniae) and P. aeruginosa is life-threatening. However, all strains of the above mentioned species possess chromosomally encoded RND efflux pump systems in addition to β-lactamase production. The main goal of this study was to assess the role of efflux pump systems in cefepime and/or ceftazidime resistant phenotypes of ESβL-positive clinical strains of Enterobacteriaceae and P. aeruginosa. The influence of the efflux pump inhibitor PAβN on the minimum inhibitory concentration (MIC) values of tested cephalosporins was species-dependent. Generally, a significant reduction (at least four-fold) of β-lactam MICs was observed in the presence of PAβN only in the case of P. aeruginosa clinical isolates as well as the ESβL-producing transformant PAO1161 ΔampC. The usage of this agent resulted in the restoration of susceptibility to cefepime and/or ceftazidime in the majority of the P. aeruginosa ESβL-positive strains with low and moderate resistance to the above cephalosporins. Moreover, an outer membrane permeabilizing effect in the presence of PAβN was identified. Strain-dependent β-lactamase leakage upon PAβN or β-lactam treatment was demonstrated. The most important observation was the restoration of susceptibility of P. aeruginosa WUM226 to cefepime (MIC decrease from 32 to 4 mg/L) and ceftazidime (MIC decrease from 128 to 4 mg/L) in the presence of PAβN, which occurred despite an almost complete lack of β-lactamase leakage from bacterial cells. In conclusion, these data indicate that RND efflux pumps can modify the susceptibility to β-lactams in Gram-negative rods producing ESβLs. However, this phenomenon occurs only in P. aeruginosa strains and was not observed among E. coli and K. pneumoniae strains, representing the Enterobacteriaceae family.

The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods.

Background Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. Methodology The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAβN (Phe-Arg-β-naphtylamide) were measured. Results The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAβN, were significantly (≥4-fold) reduced, decreasing to 25–1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAβN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains’ susceptibility to antibiotics. Conclusions The effects of PAβN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains.

Anti-Pentatrichomonas hominis activity of newly synthesized benzimidazole derivatives — in vitro studies

Pentatrichomonas hominis, a parasitic protozoan often detected in human diarrheic stools, is the cause of severe morbidity in newborns and children, particularly in tropical zones. The flagellate is resistant to many disinfectants and anti-protozoan drugs. Therefore in this study we have synthesized three novel 4,5,6,7-tetrabromobenzimidazole (TBBI) derivatives carrying a polyfluoroalkyl substituent in position 2 of the benzimidazole scaffold, namely 2-trifluoromethyl-TBBI (CF3-TBBI), 2-nonafluorobutyl-TBBI (C4F9-TBBI), and 2-nonadecafluorononyl-TBBI (C9F19-TBBI), that next we tested for their in vitro activity against P. hominis.Widely applied anti-protozoal drug, metronidazole as a reference was used. All the investigated agents were added to 24 h P. hominis cultures; each of them was administered at three different concentrations. Number of the moving trichomonads was determined and compared with the control cultures. Different anti-trichomonal activity occurred depending on a kind of compound and its concentration. C4F9-TBBI was the most effective TBBI derivative tested: the agent, at the highest concentration 24.2 μg/ml, after 72 h reduced the number of viable trichomonads to 44.3%; C9F19-TBBI, at the concentration 24 μg/ml reduced the number of the flagellates to 58.5%. Paradoxically, metronidazole after the same time given at the highest concentration increased trophozoite counts by 464.6% in comparison with the control cultures (100%).

The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria

The potential role of non-antibiotic medicinal products in the treatment of multidrug-resistant Gram-negative bacteria has recently been investigated. It is highly likely that the presence of efflux pumps may be one of the reasons for the weak activity of non-antibiotics, as in the case of some non-steroidal anti-inflammatory drugs (NSAIDs), against Gram-negative rods. The activity of eight drugs of potential non-antibiotic activity, active substance standards, and relevant medicinal products were analysed with and without of efflux pump inhibitors against 180 strains of five Gram-negative rod species by minimum inhibitory concentration (MIC) value determination in the presence of 1 mM MgSO4. Furthermore, the influence of non-antibiotics on the susceptibility of clinical strains to quinolones with or without PAβN (Phe-Arg-β-naphthylamide) was investigated. The impacts of PAβN on the susceptibility of bacteria to non-antibiotics suggests that amitriptyline, alendronate, nicergoline, and ticlopidine are substrates of efflux pumps in Gram-negative rods. Amitriptyline/Amitriptylinum showed the highest direct antibacterial activity, with MICs ranging 100–800 mg/L against all studied species. Significant decreases in the MIC values of other active substances (acyclovir, atorvastatin, and famotidine) tested with pump inhibitors were not observed. The investigated non-antibiotic medicinal products did not alter the MICs of quinolones in the absence and in the presence of PAβN to the studied clinical strains of five groups of species.

Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods

Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-β-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9), GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15), OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544) and OXA-10 (5 isolates with OXA-74 and one with OXA-142). The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide.

Strains of Enterobacter cloacae Isolated in a Hospital Environment – Some Phenotypic and Genotypic Properties

The aim of this study was to characterise the 34 Enterobacter cloacae strains isolated from different places in a hospital, either from patients who were infected or colonised and from the body surface of German cockroaches (Blatella germanica) caught in hospitals. A number of factors were determined: the ability of E. cloacae strains to adhere to the HEp-2 cell line, their susceptibility to drugs, the activity of disinfectant agents to planktonic cells and effectiveness of those disinfectants to bacteria that are sessile on catheters as well as the Enterobacterial Repetitive Intergenic Consensus – Polymerase Chain Reaction (ERIC-PCR) profile of the strains. On the basis of a statistical analysis of all the phenotypic results we were able to distinguish a few clusters. The majority of them consisted of strains isolated from colonisation and infection of patients. Some clusters consisted of E. cloacae from all the test sites. Some strains showed similarities of their phenotypic properties as well as the ERIC-PCR profile. Epidemiological studies are continuing.