Staphylococcal Resistance Patterns, blaZ and SCCmec Cassette Genes in the Nasopharyngeal Microbiota of Pregnant Women.

Antimicrobial resistance in Staphylococcus spp. colonising the nasopharynx can create risk factors of therapeutic treatment failure or prophylaxis in pregnant women. Resistance is mostly encoded on plasmids (e.g., blaZ gene for penicillinase synthesis) or chromosomes (e.g., mecA and mecC for methicillin resistance). The mecA gene is part of the chromosomal mec gene cassette (SCCmec), which is also located on the plasmid. The disc diffusion method for the selected drugs (beta-lactams, fluoroquinolones, streptogramins, aminoglicosides, macrolides, oxasolidinones, tetracyclines and other groups) was used. PCR for blaZ, mecA and mecC genes and SCCmec cassette detection and typing were performed. S. aureus (54.4%) and S. epidermidis (27.9%) were the most prevalent and showed the highest diversity of resistance profiles. The blaZ, mecA and mecC genes were reported in 95.6%, 20.6% and 1.5% of isolates, respectively. The highest resistance was found to beta-lactams, commonly used during pregnancy. Resistance to a variety of antimicrobials, including benzylpenicillin resistance in blaZ-positive isolates, and the existence of a very high diversity of SCCmec cassette structures in all staphylococci selected from the nasopharyngeal microbiota of pregnant women were observed for the first time. Knowledge of the prevalence of antimicrobial-resistant staphylococci in the nasopharynx of pregnant women may be important for the appropriate treatment or prophylaxis of this group of patients.

Synthesis and Antimicrobial Activity of New Mannich Bases with Piperazine Moiety.

A series of novel Mannich bases were designed, synthesized, and screened for their antimicrobial activity. The target compounds were synthesized from 4-(3-chlorophenyl)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione and different piperazine derivatives. The structures of the products were confirmed by 1H and 13C NMR and elemental analysis. The activity of piperazine derivatives against bacteria (Gram-positive: Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus luteus, Bacillus cereus, and Bacillus subtilis; Gram-negative: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Proteus mirabilis) and yeasts (Candida glabrata, Candida krusei, and Candida parapsilosis) was determined by the minimum inhibitory concentration and minimum bactericidal concentration values. Significant activity was observed against Gram-positive bacteria, mainly staphylococci (PG7-PG8) and bacteria of the genes of Micrococcus and Bacillus (PG1-3), as well as selected strains of Gram-negative bacteria, including bacteria of the Enterobacteriaceae family (PG7), while all tested compounds showed high fungistatic activity against Candida spp. yeasts, especially C. parapsilosis, with MICs ranging from 0.49 µg/mL (PG7) to 0.98 µg/mL (PG8) and 62.5 µg/mL (PG1-3). In conclusion, the results obtained confirm the multidirectional antimicrobial activity of the newly synthesized piperazine derivatives. Furthermore, in silico studies suggest that the tested compounds are likely to have good oral bioavailability. The results obtained will provide valuable data for further research into this interesting group of compounds. The library of compounds obtained is still the subject of pharmacological research aimed at finding new interesting biologically active compounds.

Design, Synthesis, Antibacterial Evaluations and In Silico Studies of Novel Thiosemicarbazides and 1,3,4-Thiadiazoles.

The emergence of drug-resistant bacterial strains continues to be one of the major challenges of medicine. For this reason, the importance of searching for novel structures of antibacterial drugs chemically different from the currently known antibiotics is still of great importance. In this study, we synthesized the thiosemicarbazide and 1,3,4-thiadiazole derivatives and tested them for antibacterial activity. In in vitro tests, we examined the activity of the synthesized substances against Gram-positive and Gram-negative bacteria strains. While all 1,3,4-thiadiazoles tested lacked significant activity, the antimicrobial response of the thiosemicarbazides was moderate and it was also dependent on the type and position of the substituent on the phenyl ring. The highest activity towards all Gram-positive bacteria strains was shown by all three linear compounds containing the trifluoromethylphenyl group in the structure. The MIC (minimum inhibitory concentration) values were in the range of 3.9-250 µg/mL. Additionally, we try to explain the mechanism of the antibacterial activity of the tested compounds using the molecular docking to DNA gyrase and topoisomerase IV, following previous reports on the molecular basis of the activity of thiosemicarbazides. Docking simulations allow the purposing dual mechanism of the antibacterial activity of the synthesized compounds through inhibition of topoisomerase IV DNA gyrase with the moderate prevalence of the topoisomerase pathway.

Influence of Thiazolidine-2,4-Dione Derivatives with Azolidine or Thiosemicarbazone Moieties on Haemophilus spp. Planktonic or Biofilm-Forming Cells.

Biofilm, naturally formed by microorganisms as integrated surface-bound communities, is one of the reasons for the development of antimicrobial resistance. Haemophilus spp. are common and representative opportunistic Gram-negative rods forming from the upper respiratory tract microbiota. The aim of this paper was to evaluate the influence of thiazolidine-2,4-dionebased azolidine and chlorophenylthiosemicarbazone hybrids against both planktonic and biofilm-forming Haemophilus spp. cells. The in vitro activity against planktonic and biofilm-forming cells of the tested compounds were evaluated by using the broth microdilution method. These activities were detected against reference and clinical strains of Haemophilus spp. on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The target compounds showed potential activity against planktonic cells with MIC = 62.5⁻500 mg/L and biofilm-forming cells with MBIC = 62.5⁻1000 mg/L. The observed anti-adhesive properties of the tested compounds were reversible during long-term incubation in a lower concentration of compounds.

Synthesis, Structural Studies and Molecular Modelling of a Novel Imidazoline Derivative with Antifungal Activity.

Six novel imidazoline derivatives were synthesized and tested in antifungal assays. One of the compounds, N-cyclohexyl-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide showed moderate activity against several clinical strains of Candida albicans. Its structure was solved by X-ray crystallography and its mode of action was deduced using molecular modelling. It was found to be similar to that of fluconazole. The potential for further optimization including SAR of the compound is briefly discussed.

Prevalence of Culturable Bacteria and Yeasts in the Nasopharynx Microbiota during the Physiological Course of Pregnancy.

The aim of the study was to compare the prevalence of the nasopharyngeal carriage of culturable microorganisms in the microbiota of asymptomatic women with a physiological pregnancy (PW) and nonpregnant women (NPW). Nasopharyngeal swabs were collected from 53 PW and 30 NPW to detect bacterial and fungal colonization. Isolates were identified using the culture method and the MALDI-TOF MS technique. The nasopharyngeal microbiota (NPM) partially differed between PW and NPW. These differences in the frequency of nasopharyngeal colonization between the PW and NPW groups were not statistically significant (p > 0.05); all cases were colonized by bacteria and only two cases in the PW group were colonized by yeasts, namely, Rhodotorula spp. High levels of staphylococcal colonization, including predominantly coagulase-negative staphylococci and S. aureus in the nasopharyngeal sample, were present in both groups. The reduced number of Gram-negative rods colonized in the cases studied was seen in samples from the NPW group, particularly with Enterobacterales, and anaerobic Cutibacterium spp. were isolated only in the PW group (p < 0.05). Moreover, a higher carriage rate of Enterobacter aerogenes colonization was statistically significant (p < 0.05) and correlated with the NPW group. Pregnancy may disturb the composition of the NPM represented by commensals and opportunistic bacteria and promote yeast colonization as compared to nonpregnant women.

Antioxidant and antimicrobial properties of an extract rich in proteins obtained from Trametes versicolor.

Introduction: Bioactive proteins and peptides generated from fruit, vegetables, meat or fish have great potential as functional food or substitutes for antibiotics. In recent years it has also been demonstrated that the fungus kingdom could be a source of these compounds. The study investigated the bioactivity of an extract of the lignicolous fungus Trametes versicolor and its hydrolysate. Material and Methods: The fungus was collected in a mixed forest in October, extracted and hydrolysed. To inspect the protein and peptide profiles before and after hydrolysis, matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometric analysis was performed. To evaluate the antioxidant properties of the preparations, 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) radical scavenging assays were used. The activity of the fungus extract and hydrolysate against Aeromonas veronii, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis was determined by the minimum inhibitory concentration and minimum bactericidal concentration values. Results: The extract and its hydrolysate showed almost 100% ABTS•+ and DPPH• radical scavenging with a low half maximal inhibitory concentration. The water extract and hydrolysate of T. versicolor exhibited antimicrobial activity against two S. aureus strains, E. coli, P. aeruginosa and Salmonella Typhimurium. Conclusion: These results provide compelling evidence that the analysed fungus extract and its hydrolysate hold promise with their antibacterial and antioxidant properties.

Plasmid-Mediated Fluoroquinolone Resistance Genes in Quinolone-Susceptible Aeromonas spp. Phenotypes Isolated From Recreational Surface Freshwater Reservoir.

Aeromonas spp. are recognized as opportunistic pathogens causing diseases. Infections in humans can result mainly in gastrointestinal and wound diseases with or without progression to septicemia. Although Aeromonas spp. are not known uropathogens and they rarely cause urinary tract infection, we hypothesize that the presence of these bacteria in the water and the contact during, e.g., recreational and bathing activity can create the conditions for the colonization of the human body and may result to diseases in various locations, including the urinary tract. Our study presents the occurrence of aeromonad fluoroquinolone-susceptible phenotypes with the presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes in a natural freshwater reservoir occasionally used for recreational activities. Sixty-nine isolates collected during the bathing period were identified by mass spectrometry and screened for the presence of fluoroquinolone-resistant phenotypes and genotypes. Fluoroquinolone susceptibility was determined as minimal inhibitory concentration values. PMQR qnr genes were detected by PCR. Isolates comprising eight species, namely, mainly Aeromonas veronii (50.7% isolates) and Aeromonas media (24.6% isolates) and rarely Aeromonas eucrenophila, Aeromonas caviae, Aeromonas bestiarum, Aeromonas ichthiosmia, and Aeromonas hydrophila, were selected. All isolates were phenotypically susceptible either to ciprofloxacin or levofloxacin. Unexpectedly, at least one to three of the PMQR genes were detected in 42.0% of the fluoroquinolone-susceptible Aeromonas spp. phenotypes. Mainly the qnrS (34.8% isolates) and qnrA (14.5% isolates) determinants were detected. In conclusion, the freshwater reservoir occasionally used for bathing was tainted with aeromonads, with a high occurrence of opportunistic pathogens such as A. veronii and A. media. MALDI-TOF MS is a powerful technique for aeromonad identification. Our data reveals the mismatch phenomenon between fluoroquinolone-susceptible aeromonad phenotypes and the presence of plasmid-mediated qnr resistance genes. It suggests that phenotypically susceptible bacteria might be a potential source for the storage and transmission of these genes. The exposure during, e.g., a recreational activity may create the potential risk for causing infections, both diagnostically and therapeutically difficult, after expressing the resistance genes and quinolone-resistant strain selection.

Prevalence of susceptibility patterns of opportunistic bacteria in line with CLSI or EUCAST among Haemophilus parainfluenzae isolated from respiratory microbiota.

The application of CLSI and EUCAST guidelines led to many discrepancies. Various doubts have already appeared in preliminary stages of microbiological diagnostics of Haemophilus spp. A total of 87 H. parainfluenzae isolates were obtained from throat or nasopharyngeal swabs from adults 18 to 70 years old, both healthy volunteers and patients with chronic diseases between 2013 to 2015 in eastern Poland. Haemophilus spp. were identified by colony morphology, Gram-staining, API NH and MALDI-TOF MS technique. Both susceptibility to various antimicrobials and phenotypes of Haemophilus spp. resistance to beta-lactams were determined. Statistically significant association between applied guidelines and drug resistance patterns were observed to as follows: ampicillin, cefuroxime, cefotaxime, amoxicillin-clavulanate, azithromycin, tetracycline and trimethoprim-sulfamethoxazole. Resistance phenotypes according to CLSI vs. EUCAST were as follows: 3.4% vs. 8.0% for BLNAR and 6.9% vs. 19.5% for BLPACR isolates. In conclusion, this is the first study that reports comparative analysis of drug susceptibility interpretation using CLSI and EUCAST of haemophili rods from human respiratory microbiota in Poland. In case of susceptible, increased exposure (formerly intermediate) category of susceptibility within H. parainfluenzae isolates we have observed EUCAST as more restrictive than CLSI. Moreover, BLNAI and BLPAI phenotype isolates have been observed, as well as BLPBR using only CLSI or EUCAST guidelines, respectively.

Prevalence of Resistance to ß-Lactam Antibiotics and bla Genes Among Commensal Haemophilus parainfluenzae Isolates from Respiratory Microbiota in Poland.

(1) Background: Beta-lactams are the most frequently used antimicrobials, and are the first-line drugs in many infectious diseases, e.g., pneumonia, otitis media. Due to this fact, various bacteria have developed resistance to this group of drugs. (2) Methods: Eighty-seven Haemophilus parainfluenzae isolates were obtained from adults 18-70 years old in eastern Poland. The presence of 10 bla genes and 2 substitutions in ftsI reported as the most frequent in H. parainfluenzae were analyzed. (3) Results: Among 57 beta-lactam-resistant isolates, 63.2% encoded bla genes; blaTEM-1 predominated (54.4%), followed by blaOXA (19.3%), blaDHA (12.3%), blaSHV (10.5%), blaGES (7.0%), blaCMY (5.3%), blaVEB (1.8%) and blaROB-1 (1.8%). Lys-526 was the most common substitution in ftsI gene. The resistance genotypes were as follows: gBLNAS (17.5%), low-gBLNAR I (1.8%), low-gBLNAR II (1.8%), gBLNAR II (15.8%), gBLPAS (15.8%), gBLPAR (19.3%), gBLPBS I (8.8%) and gBLPBS II (1.8%); (4) Conclusions: This has been the first study to report on the high diversity of bla genes in H. parainfluenzae isolates in Poland. High sensitivity and specificity of benzylpenicillin test, as well as PCR of bla genes were shown, indicating that these methods may be useful as tools for the rapid screening of beta-lactamase prevalence and resistance to beta-lactams among H. parainfluenzae isolated from respiratory microbiota.

Antibiotic Resistance and Genotypes of Helicobacter pylori Strains in Patients with Gastroduodenal Disease in Southeast Poland.

The aim of this study was to investigate genetic diversity of Helicobacter pylori virulence markers to predict clinical outcome as well as to determine an antibiotic susceptibility of H. pylori strains in Poland. Gastric biopsies from 132 patients with gastrointestinal disorders were tested for presence of H. pylori with the use of rapid urease test, microbial culture, and polymerase chain reaction (PCR) detection. The genetic diversity of 62 H. pylori positive samples was evaluated by detection of cagA and PCR-typing of vacA and iceA virulence-associated genes. Most common H. pylori genotypes were cagA(+)vacAs1m2 (27.4%) and cagA(-)vacAs2m2 (24.2%). In logistic regression analysis, we recognized the subsequent significant associations: gastritis with ureC, i.e., H. pylori infection (p = 0.006), BMI index (p = 0.032); and negatively with iceA1 (p = 0.049) and peptic ulcer with cagA (p = 0.018). Thirty-five H. pylori strains were cultured and tested by E-test method showing that 49% of strains were resistant to at least one of the tested antibiotics. This is the first study that reports the high incidence and diversity of allelic combination of virulence genes in gastroduodenitis patients in Poland. Genotyping of H. pylori strains confirmed the involvement of cagA gene and vacAs1m1 genotype in development and severity of gastric disorder.

Inhibitory effect of 1,2,4-triazole-ciprofloxacin hybrids on Haemophilus parainfluenzae and Haemophilus influenzae biofilm formation in vitro under stationary conditions.

Haemophilus parainfluenzae and Haemophilus influenzae, upper respiratory tract microbiota representatives, are able to colonize natural and artificial surfaces as biofilm. The aim of the present study was to assay the effect of ten 1,2,4-triazole-ciprofloxacin hybrids on planktonic or biofilm-forming haemophili cells in vitro under stationary conditions on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The reference strains of H. parainfluenzae and H. influenzae were included. The broth microdilution microtiter plate (MTP) method with twofold dilution of the compounds, or ciprofloxacin (reference agent) in 96-well polystyrene microplates, was used. The optical density (OD) reading was made spectrophotometrically at a wavelength of 570 nm (OD570) both to measure bacterial growth and to detect biofilm-forming cells under the same conditions with 0.1% crystal violet. The following values of parameters were estimated for 1,2,4-triazole-ciprofloxacin hybrids - MIC = 0.03-15.63 mg/L, MBIC = 0.03-15.63 mg/L, MBIC/MIC = 0.125-8, depending on the compound, and for ciprofloxacin - MIC = 0.03-0.06 mg/L, MBIC = 0.03-0.12 mg/L, MBIC/MIC = 1-2. The observed strong anti-adhesive properties (95-100% inhibition) of the tested compounds were reversible during long-term incubation at subinhibitory concentrations. Thus, 1,2,4-triazole-ciprofloxacin hybrids may be considered as starting compounds for designing improved agents not only against planktonic but also against biofilm-forming Haemophilus spp. cells.