Overuse and misuse of broad-spectrum antibiotics increases the risks for the disruption of normal bacterial flora and the development of antimicrobial resistance. Oral broad-spectrum antimicrobials, such as quinolones, cephalosporins, and macrolides, were more frequently prescribed in Japan than in Europe or the United States. In particular, the use of oral quinolones was increased from 2009 to 2013 in Japan. Therefore, the National Action Plan on antimicrobial resistance was formulated in April 2016; it aimed to reduce oral broad-spectrum antimicrobial use by 50% by 2020. We opted to promote appropriate quinolone use through lectures for all medical staff including physicians. This study aimed to evaluate the trend of the oral quinolone use and the susceptibility of Escherichia coli and Klebsiella pneumoniae from 2013 to 2020 in our hospital. To investigate oral antibiotic use, the monthly median days of therapy per 100 patient days were investigated among inpatients and outpatients, excluding those with chronic diseases that required long term quinolone prescriptions. Additionally, the data on susceptibility of E. coli and K. pneumoniae to levofloxacin were analyzed. The use of oral quinolones for outpatients had significantly decreased by approximately 50% from 2013 to 2020, but no significant differences were observed in the use of oral quinolones for inpatients and the susceptibility of the bacteria to levofloxacin. Further rigorous antimicrobial stewardship interventions are necessary for optimization of oral quinolone use and minimizing the prevalence of resistant pathogens.

Pseudomonas aeruginosa is highly versatile gram-negative pathogen and accounts for high morbidity and mortality rates in cystic fibrosis (CF) patients and other immunocompromised individuals. The infamous resistance of this pathogen to multiple currently available antibacterial agents and the weak pipeline for effective antibiotic agents has accelerated the development of multidrug-resistant (MDR) P. aeruginosa strains leading to rejuvenated clinical interest in the “last resort” cationic antimicrobial agent- polymyxins. However, there are increasing reports of resistance to polymyxins in P. aeruginosa. This is a serious therapeutic challenge for treating devastating infections caused by this pathogen. The resistance to polymyxins is demonstrated to be multifactorial and factors conferring resistance to polymyxins at physiologically relevant conditions remains to be poorly understood. Here we show that the presence of physiological levels of calcium (Ca²⁺) in CF lungs enhances resistance in P. aeruginosa to polymyxin B (Pol B) in both sessile and planktonic environments. Several two-component systems earlier identified to control resistance to Pol B in P. aeruginosa including PhoPQ, PmrAB and ParRS, shows no significant involvement in this resistance indicating the involvement of other novel Ca²⁺-dependent mechanisms. In support of this hypothesis, the transcription of two component systems and several other genes involved in Pol B resistance mechanisms described thus far, are downregulated in the presence of increased Ca²⁺ levels. Through random chemical mutagenesis, we identified three genes contributing to Ca²⁺-induced resistance: PA2803, PA3237, and PA5317. Genome-wide RNA-Seq analyses revealed that the transcription of PA2803 and PA3237 is induced by elevated Ca²⁺. Further, both the outer membrane and inner membrane permeability increases during growth at elevated Ca²⁺. Proteomic and transcriptomic profiling uncovered several Ca²⁺-dependent differences in the membrane structure, including Wzz, PhoP/Q, and OprE. Currently, we aim to elucidate the Ca²⁺-dependent membrane lipid modifications that confer resistance. This will improve our understanding of the molecular mechanisms responsible for Ca²⁺-induced PolB resistance in P. aeruginosa.

Fatty acids are well-known molecules from diverse sources that are toxic to microorganisms. As infectious diseases become a severe threat to human health and economy due to the dramatic emergence of antibiotic resistance, a quest for novel antibacterial agents from natural sources, including the marine ecosystem, has increased last few decades. Seaweeds, mainly marine algae, are being studied as a potential source of bioactive molecules to combat the new trend of acquired resistance in microbes. Algae contain plenty of different fatty acids including ω3 (i.e., 16:4 ω3 and 18:4 ω3), eicosapentaenoic (20:5 ω3), α-linolenic (18:3 ω-3), octadecatetraenoic (18:4 ω-3), and arachidonic (20:4 ω-6) acid. These fatty acids have a wide range of direct and indirect inhibitory effects majorly by destabilizing bacterial cell membranes. Therefore, the present study summarised and discussed the available pieces of evidence related to fatty acid activities in bacterial growth inhibition. Following the PRISMA guidelines, the literature mining was conducted using PubMed, Scopus, Web of Science, and Google Scholar databases published within the year 2011 to 2020. The search was limited to in vitro research regarding the antibacterial role of fatty acids isolated from marine algae. The literature search revealed 570 potentially relevant records, of which 28 were relevant in vitro studies that investigated the role of fatty acids for antibacterial effects, were included finally. Fatty acids were reported to be isolated following the bioassay-guided fractionation of algal extracts, followed by chromatographic techniques, and identified by nuclear magnetic resonance (NMR). The included studies showed various algal species as a potential source of fatty acids that were significant in inhibiting different bacterial strains, limited to gram-positive: Bacillus subtilis, Enterococcus faecalis, and Staphylococcus aureus, as well as gram-negative: Escherichia coli, Pseudomonas aeruginosa, for example. Present in vitro evidence-based review highlighted the cellular and molecular actions of fatty acids that positively affect bacterial growth inhibition. The evidence thus recommends that the algal fatty acids might act as potential antibacterial agents, thereby can be used as pharmaceutical products for infectious diseases.

Pseudomonas aeruginosa is an opportunistic pathogen which is considered as one of the important causes of hospital-acquired infections such as pneumonia, wound infections, urogenital and intra-abdominal sepsis. Phages are hopeful alternatives for antibiotics and they can eliminate the biofilms and pathogens effectively. The objectives of this work are assessing the characterization and anti-biofilm activity of a naturally isolated phage on P. aeruginosa. Morphological analysis by transmission electron microscopy revealed the isolated phage is an icosahedral particle with ~50 nm diameter The isolated phage has a species-specific host range for P. aeruginosa. The analysis of the phage proteins resulted in a pattern with about 10 proteins and a major band of ~35 kDa. The phage exhibited 86.82 and 86.86% biofilm inhibition at PFU of 10 and 100, respectively. Furthermore, the phage is effective on some antibiotic resistant clinically-isolated P. aeruginosa. The results of this study shows that the natural phages are effective agents against biofilms. Due to increasing resistant pathogens to antibiotics, alternative agents such as phages are promising tools for treating bacterial infections.

The ageing of the population – especially in developed countries – has brought on many societal challenges and has significantly contributed to the burden on healthcare infrastructures worldwide. Urinary tract infections (UTIs) are the third most common infections in humans, representing an important factor of morbidity, both among outpatients and hospitalized patients. The elderly are at a higher risk of developing UTIs, both due to anatomical and physiological changes, lifestyle factors, immobility and the frequent use of urinary catherers. A retrospective observational study was performed at a 1820-bed primary- and tertiary-care teaching hospital (University of Szeged) regarding the epidemiology and resistance of UTIs in patients aged ≥65 years. Identification of the isolates was carried out using VITEK 2 ID/AST and MALDI-TOF MS. Antibiotic resistance in these isolates was assessed based on the WHO AWaRe (Access, Watch, Reserve) classification of antimicrobials. During the 10-year study period, n=4214 (421.4±118.7/year) and n=4952 (495.2±274.6) laboratory-confirmed UTIs were recorded in inpatients and outpatients, respectively. The patients presented with the following demographic characteristics: a. outpatients: median age: 75 years (65-96), 39.7% male; b. inpatients: median age: 76 years (65-98), 71.3% male. The causative agents showed differentiation among inpatients and outpatients: Escherichia coli (25.7% vs. 48.1%; p=0.001), Enterococcus spp. (21.5% vs. 20.2%; p>0.05), Klebsiella spp. (16.3% vs. 16.2%; p>0.05), Pseudomonas spp. (13.4% vs. 4.4%; p=0.001); Proteus-Providencia-Morganella group (11.1% vs. 4.6%; p=0.001); Candida spp. (5.9% vs. 0.5%; p=0.001); Citrobacter-Enterobacter-Serratia group (2.9% vs. 1.8%; p<0.05). Significant differences were observed in the resistance rates among inpatient and outpatient isolates for many Access and Watch antibiotics; in addition, resistance rates were higher in these UTI pathogens, compared to the previously recorded rates in the region. Continuous surveillance of resistance rates in bacteria affecting vulnerable patient populations is needed for antimicrobial stewardship and to the ensure the selection of appropriate therapy in these patients.

Staphylococcus aureus (S. aureus) is an important causative agent in human infections. Biofilm-production is an important virulence factor of many pathogens, often leading to chronic infections. There has been significant interest in assessing the possible relationship between the multidrug-resistant (MDR) status and the biofilm-producer phenotype. In the present study, the biofilm-production rates in clinical methicillin-susceptible [MSSA] and resistant [MRSA] S. aureus isolates were characterized; in addition, the correlation between their antibiotic resistance and biofilm-forming capacity was also assessed. A total of three hundred (n=300) S. aureus isolates (MSSA and MRSA isolates in equal measure) were included in this study. Identification of the isolates was carried out using the VITEK 2 ID/AST automated system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests. Biofilm-production was assessed using phenotypic methods, including the crystal violet (CV) tube-adherence method and the Congo red agar (CRA) plate method. There were significant differences among MSSA and MRSA isolates regarding susceptibility-levels to commonly-used antibiotics (erythromycin, clindamycin and ciprofloxacin: p<0.001, gentamicin: p=0.023, sulfamethoxazole/trimethoprim: p=0.027, rifampin: p=0.037). In the CV tube adherence-assay, 37% (n=56) of MSSA and 39% (n=58) of MRSA isolates were positive for biofilm-production, while during the use of CRA plates, 41% (n=61) of MSSA and 44% (n=66) of MRSA were positive. Biofilm-positive isolates were most common from catheter-associated infections. Overall, no associations were found between methicillin-resistance and biofilm-production; however, resistance to erythromycin, clindamycin and rifampin was associated with biofilm-positivity (p=0.004, p<0.001 and p<0.001, respectively). The relationship between the MDR phenotype and biofilm-positivity in S. aureus has been studied extensively, but the results available in the literature are still inconclusive.

We use conjugation to investigate the linkage and potential for horizontal transfer of a limited number of clusters of antibiotic resistance genes that dominate the Extended-Spectrum Beta-Lactamase antibiotic resistance gene landscape in samples of Extraintestinal E. coli recovered from two West Coast hospitals. These include: a) an integron with two aminoglycoside phosphotransferases b) two functionally-overlapping groups genes, encompassing diverse β-lactamases and aminoglycoside acetyltransferases in combinations of up to 4 genes that we name AG1 and AG2 for “association group 1 and 2”. We characterize 139 sequenced, multidrug resistant clinical strains from two different hospitals for the ability to transfer carbenicillin resistance by conjugation. We made the following observations: a) β-lactamase-bearing conjugative plasmids largely (95%) belonged to the IncF plasmid incompatibility group and 97% of these have combinations of IncF replicons; b) AG1 and AG2 gene combinations can spread by conjugation but this is not consistently the case; higher-order combinations tend to appear in non-conjugating strains. The observed patterns of conjugation transfer of CG1 and CG2 genes suggest that conjugation plays an important role in the early stages of evolution of multidrug resistance, making genes available for the generation of adaptive solutions, and that these adaptive solutions are highly heterogeneous, driven by selection rather than by co-mobilization. In fact, our results suggest that the formation of more complex combinations occurs at the expense of their ability to be mobilized.

A novel complex of antimicrobial peptides with antifungal and cytotoxic activity was derived from the alkalophilic fungus Emericellopsis alkalina VKPM F-1428 isolated from soda soil. Novel peptides were assigned “Emericellipsins”, so-called because of their source fungus. The complete primary structure and detailed biological activity were determined for the five of them.

The dominant peptaibol - emericellipsin A (EmiA) showed strong antifungal, cytotoxic properties. The inhibitory activity of the major compound, EmiA against azole-resistant pathogenic Aspergillus spp., Candida spp. and Cryptococcus spp. was similar to amphotericin B. In addition, EmiA demonstrated low cytotoxic activity to the normal cell line (human postnatal fibroblasts, HPF) but possessed cancer selectivity to human myelogenous leukaemia (K-562) and human colon cancer (НСТ-116) cell lines. Emericellipsin A revealed negligible hemolytic activity at concentrations of 0–20 μM, making it a low-toxicity compound regarding normal human cells, but with a potentially high therapeutic index. The inhibited effect of EmiA on biofilm formation of clinical pathogens Staphylococcus aureus and Candida albicans was determined. The results obtained suggest EmiA as a natural compound with a prospect for using it to develop a promising antifungal agent for invasive mycoses therapy, especially for treatment multi-drug resistant aspergillosis and cryptococcosis

Appropriate professional competencies and attitudes are of critical importance for healthcare-personnel to effectively prevent, diagnose and treat infectious diseases, and to curb the spread of antimicrobial resistance. The aim of our study was to evaluate the knowledge and attitude of junior doctors on infectious diseases and antimicrobial resistance in Hungary. A self-administered, 47-item questionnaire was developed by an expert panel, including questions on demographic characteristics, source of knowledge on antimicrobials, knowledge-level (30 items, three different subject areas) and attitudes (10 items). Following instrument development, pilot testing was performed among junior doctors at the Faculty of Medicine, University of Szeged, between January-December 2018. Descriptive statistics and nonparametric tests were performed by IBM SPSS Statistics 22.0. Internal consistency measures (Cronbach’s α, Kuder–Richardson KR-20) and the test-retest analysis both showed acceptable reliability. Based on the responses to the questionnaire, an attitude score (0-7) was determined. Among the n=146 respondents, 57.5% (n=84) has polled female, with an average age of 29.1±3.2 years. 41.8% (n=61) and 42.5% (n=62) of respondents cited the Internet and scientific papers as relevant sources of information, respectively. 71.2% (n=104) were satisfied with their academic achievements during their graduate studies. The number of correct answers among the respondents were 15.5±3.8 overall (range: 2-22), 36.3% did not reach an acceptable (≥15) score. Number of correct answers from the respective subject areas were: medical microbiology 6.0±1.8, epidemiology/infection control 5.4±1.7, and infectology 4.3±1.6. Good academic achievements corresponded to better results in the knowledge-based questions (13.7±3.7 vs. 16.3±3.6; p<0.001). Spearman-correlations were significant among the number of correct answers within the individual subject areas and with the overall number of correct responses. 95.1% of residents presented with an appropriate attitude (score≥5), while no correlation was shown between attitude scores and knowledge levels. Our instrument may be an effective tool for the identification of knowledge gaps related to infectious diseases among young prescribers in the early years of their professional career.

Antibiotics are medicines of critical importance, but the emergence of antibiotic-resistant bacteria (ABR) is serious threat to healthcare institutions worldwide. One of the most important hallmarks in the development of resistance is their imprudent use, including their non-prescription procurement or their use in self-medication, without the supervision of a healthcare-professional. Many international campaigns have aimed to promote and enhance the understanding of the general population regarding the dangers of ABR, including the European Antibiotic Awareness Week (EAAW) and the World Antibiotic Awareness Week (WAAW); nevertheless, several studies have highlighted that the Internet has become one of principal sources of health-related information for many people. The aim of this infodemiological study was to assess the changes in antibiotic-related Internet queries worldwide and to identify the possible association between the information seeking behaviour for antibiotics and various infectious diseases. Qualitative and quantitative data, and spatio-temporal distribution of queries on the topic of antibiotics were extracted from the Google Trends analysis tool for the time-period between 2010.01.01-2020.12.31, corresponding to all searches worldwide. In addition, search intensity data were also collected for keywords “flu”, “common cold”, “UTI”, “sore throat”, “cough”, and “sinus infection”. Search intensity was expressed as relative search volume (RSV), a normalized score ranging between 0-100. Independent sample t-test, ANOVA and Pearson-correlation were performed by IBM SPSS Statistics 22.0. Search intensity for antibiotic-related information (based on the keyword “antibiotic”) has increased by 54.4% (55.5±5.6 vs. 85.7±8.4) between 2010 and 2020, based on RSV values. Countries with the highest search intensity were Romania (RSV: 100), the Philippines (RSV: 58), Jamaica (RSV: 50) and the USA (RSV: 43). The most common related queries were “what is an antibiotic” (RSV range: 54-81), “antibiotic resistance” (RSV range: 60-69), “antibiotic for UTI” (RSV range: 18-40), “antibiotic cream” (RSV range: 12-36) and “antibiotic side effects” (RSV range: 15-27). EAAD and WAAW-related searches corresponded to RSVs <1 throughout the study. Antibiotic-related educational campaigns (EAAD and WAAW) in November did not have significant effects on RSV values (66.6±15.5 vs. 67.9±14.1; p>0.05). Strong positive correlations were found between antibiotic-related online queries and searches for flu (R=0.561), UTIs (R=0.884), sore throat (R=0.734), cough (R=0.780), sinus infection (R=0.553) and the common cold (R=0.535); in every case, p values <0.001 were recorded. Analysis of antibiotic-related Internet queries may be a valuable source of information of collective health utilization trends. The results suggest that antibiotic-related educational campaigns did not influence the temporal distribution of Internet searches on this topic. Noteworthy associations were seen between information-seeking behaviour on commonly occurring infectious ailments (where self-medication with antibiotic commonly occurs) and antibiotic-related queries.