Antibiotic resistance profiles in cultivable microbiota isolated from some romanian natural fishery lakes included in Natura 2000 network.

BACKGROUND: The present study aims the characterization of antibiotic resistance phenotypes and encoding genes in bacterial strains isolated from some Romanian aquatic fishery lowland salted lakes. MATERIAL/METHODS: This study was conducted on 44 bacterial strains, mainly belonging to species used as microbiological indicators of fecal pollution isolated from four natural fishery lakes. All strains were tested for their antibiotic susceptibility by disk diffusion method. Simplex and multiplex PCR were performed to identify the ß-lactams antibiotic resistance genes (blaNMD, blaOXA-48, blaVIM, blaIMP, blaCTX-M, blaTEM), sulfonamides (Sul1, Sul2), tetracyclines (TetA, TetB, TetC, TetD, TetM), aminoglycosides (aac3Ia), vancomycin (VanA, VanB, VanC), macrolides (ermA, ermB, ermC) as well as the plasmid-mediated quinolone resistance (PMQR) markers (QnrA, QnrB, QnrS), and class 1 integrons (Int1, drfA1-aadA1). RESULTS: The Enterococcus spp. isolates exhibited phenotypic resistance to vancomycin (35 %) and macrolides (erythromycin) (75 %); from the vancomycin - resistant strains, 5 % harboured VanA (E. faecalis), while the erythromycin resistant isolates were positive for the ermA gene (E. faecalis - 10 %, E. faecium - 5 %). The Gram- negative rods (GNR) exhibited a high level of resistance to ß-lactams: cefuroxime (63 %), cefazolin (42 %), ceftriaxone (8 %), ceftazidime and aztreonam (4 % each). The genetic determinants for beta-lactam resistance were represented by blaCTX-M-like (33 %), blaNDM-like and blaIMP-like (8.33 %) genes. The resistance to non-ß-lactam antibiotics was ascertained to the following genes: quinolones (QnrS - 4.16 %); sulfonamides (Sul1-75 %, Sul2-4.16 %); aminoglycosides (aac3Ia - 4.16 %); tetracyclines (tetA - 25 %, tetC - 15 %). The integrase gene was found in more than 50 % of the studied strains (58.33 %). CONCLUSIONS: The cultivable aquatic microbiota from fishery lakes is dominated by enterococci and Enterobacterales strains. The GNR strains exhibited high levels of ß-lactam resistance mediated by extended spectrum beta-lactamases and metallo-ß-lactamases. The Enterococcus sp. isolates were highly resistant to macrolides and vancomycin. The high level and diversity of resistance markers, correlated with a high frequency of integrons is suggesting that this environment could act as an important reservoir of antibiotic resistance genes with a great probability to be horizontally transmitted to other associated species from the aquatic sediments microbiota, raising the potential zoonotic risk for fish consumers.

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.

Antimicrobial Susceptibility Testing for Corynebacterium Species Isolated from Clinical Samples in Romania.

Antimicrobial resistance is one of the most important public health issues. Besides classical multidrug resistance species associated with medical care involved in superficial or invasive infections, there are strains less commonly associated with hospital or outpatient setting's infections. Non-diphtheria Corynebacterium spp. could produce infections in patients with or without immune-compromised status. The aim of our study was to determine the susceptibility to antimicrobial agents to Corynebacterium spp. from clinical samples collected from Romanian hospitalized individuals and outpatients. Twenty Corynebacterium strains were isolated and identified as Corynebacterium striatum (n = 7), Corynebacterium amycolatum (n = 7), C. urealyticum (n = 3), Corynebacterium afermentans (n = 2), and Corynebacterium pseudodiphtheriticum (n = 1). All isolates have been tested for antibiotic susceptibility by standardized disc diffusion method and minimal inhibitory concentration (MIC) tests. Seventeen isolates demonstrated multidrug resistance phenotypes. The molecular support responsible for high resistance to quinolones for ten of these strains was determined by the detection of point mutation in the gene sequence gyrA.

Snapshot of Phylogenetic Groups, Virulence, and Resistance Markers in Escherichia coli Uropathogenic Strains Isolated from Outpatients with Urinary Tract Infections in Bucharest, Romania.

BACKGROUND: Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections worldwide, including Romania. To the best of our knowledge, this is the first study performed on a significant number of community-acquired (CA) UPEC strains isolated from Romanian outpatients, aiming to evaluate and establish potential correlations among the phylogenetic groups (PG), resistance profiles, and the virulence factors (VF) genes of the CA-UPEC isolates. MATERIALS/METHODS: The present study was performed on a total of 787 UPEC nonrepetitive isolates consecutively isolated during one month from outpatients with CA-UTIs, visiting one of the biggest laboratories in Bucharest, Romania, receiving patients from all over the country. The strains identification was performed by MALDI TOF and the susceptibility patterns were tested using Microscan according to CLSI guidelines. PCR assays were performed to detect the presence of different VFs (fimH gene encoding for type 1 fimbriae, afaBC for A fimbriae, sfaDE for S fimbriae, KpsMTII for capsule, hlyA for haemolysin A, hlyD for haemolysin D, and cnf-1 for tumor necrosis factor), the phylogenetic groups (PG) A, B1, B2, and D, and the extended spectrum beta-lactamases (ESBLs) genes. RESULTS: The 787 CA-UPEC strains were isolated predominantly from female patients (90.95%) of >30 years (~74%). The resistance rates were 47.52% for ampicillin, 41.16% for tetracycline, 24.39% for cotrimoxazole, 19.18% for amoxicillin-clavulanic acid, 15.50% for cefazolin, 14.99% for ciprofloxacin, and 14.86% for levofloxacin; 35.19% of the investigated strains were MDR and 9.03% ESBL producers (from which 42.25% were positive for blaCTX-M, 38.02% for blaTEM, and 19.71% for blaSHV). FimH was the most frequent virulence gene (93.90%) followed by hlyD (44.34%); afaBC (38.24%); KpsMTII (32.65%); sfaDE (23.88%); hlyA (12.45%); and cnf-1 (7.75%). The distribution of the analyzed UPEC strains in phylogenetic groups was different for non-MDR and MDR strains. Overall, 35% of the strains belonged to the phylogenetic group B2 (harboring the yjaA gene); 27% to group B1 (confirmed by the presence of the TspE4C2 fragment); 16% to group D; and 22% to group A. The CA-UPEC strains included in PG B1 and PG B2 proved to be the most virulent ones, the number of strains carrying multiple VFs (>3) being significantly larger as compared to strains belonging to PG A and PG D) (p<0,0001). The presence of one or two ESBL genes was significantly associated (p =0.0024) with PGs A and D. CONCLUSIONS: Our findings showed that the community UPEC strains circulating in Bucharest, Romania, belong predominantly to group B2 and >90% harbored the fimH gene. High MDR resistance rates were observed, as well as extended VF profiles, highlighting the importance of this type of studies for improving the epidemiological surveillance and the therapeutic or prophylactic management of the respective infections, in the context of antibiotic resistance emergence.

Invasive Fungal Infection in Romania: Changing Incidence and Epidemiology During Six Years of Surveillance in a Tertiary Hospital.

The present study aimed to evaluate the incidence of candidemia, the trend of species distribution and the antifungal susceptibility patterns of all invasive strains of Candida spp. isolated over a 6-year period in an Eastern European University hospital. A total number of 156 isolates were reidentified by MALDI-TOF and tested for susceptibility by SensititreTM YeastOne™. Isolates were assigned as non-susceptible or belonging to the wild type according to the new CLSI (2017, 2018) break points and epidemiological cut-off values. C. parapsilosis (37.82%) was the most frequently isolated yeast, followed by C. albicans (26.28%). The general tendency of the species distribution during the surveyed period shifted towards an increase in C. parapsilosis and C. lusitaniae isolates. Fluconazole resistance was present in 23.52% of C. glabrata, 5.08% of C. parapsilosis and in none of the C. albicans, C. tropicalis and C. lusitaniae isolates. Echinocandin resistance was present only in 1(5.88%) C. glabrata isolate. The study emphasises the importance of monitoring local epidemiologic data and antifungal susceptibility trends due to the diversity of affected patient groups in our hospital.

In vitro activity of echinocandins against 562 clinical yeast isolates from a Romanian multicentre study.

The study presents the echinocandin susceptibility profile of a multi-centre collection of pathogenic yeast isolates from Romanian tertiary hospitals. The 562 isolates were identified using ID32C strips, MALDI-TOF MS and DNA sequencing. Minimal inhibitory concentrations (MICs) of caspofungin (CAS), micafungin (MCA), and anidulafungin (ANI) were assessed and interpreted according to EUCAST guidelines. Minimal fungicidal concentrations (MFC) were determined by plating content from the clear MIC wells. The activity was considered fungicidal at MFC/MIC ≤ 4. The three echinocandins had strongly correlated MICs and high percentages of MIC essential agreement. Most often, MCA had the lowest MICs, followed by CAS and ANI. Against C. parapsilosis and C. kefyr, CAS had the lowest MIC values. The MIC50 values were between 0.03 and 0.25 mg/l, except C. parapsilosis. The MIC90 values were usually one dilution higher. MFCs and MICs were weakly correlated. ANI and MCA had the lowest MFC values. The MFC50 values were between 0.06 and 0.5 mg/l, except C. parapsilosis, C. guilliermondii, and C. dubliniensis. The MFC90 values were usually two dilutions higher. Based on EUCAST breakpoints, 47 isolates (8.4%) were resistant to at least one echinocandin, most often ANI. Most resistant isolates were of C. albicans, C. glabrata, and C. krusei. There were 17 isolates (3%) resistant to echinocandins and fluconazole and most belonged to the same three species. MCA and ANI had the highest rates of fungicidal activity. The high rates of echinocandin resistance and significant multidrug resistance make prophylaxis and empiric therapy difficult.

Bacillus cereus strain isolated from Demodex folliculorum in patients with topical steroid-induced rosaceiform facial dermatitis.

The aim of the study was to identify Bacillus species from the Demodex folliculorum of patients with topical steroidinduced facial rosaceiform dermatitis. Of the 75 patients examined, 20% had clinical spinulosis, while 18.66% had dermoscopic features of Demodex: follicular plugs and tails. Of the 17.33% positive patients identified upon microscopy for Demodex, samples for bacterial culture were plated on trypticase soy Colombia agar. Identification was performed by microorganisms grown method mass spectrometry. We identified a strain of Bacillus cereus.

Bacillus cereus strain isolated from Demodex folliculorum in patients with topical steroid-induced rosaceiform facial dermatitis

Abstract: The aim of the study was to identify Bacillus species from the Demodex folliculorum of patients with topical steroidinduced facial rosaceiform dermatitis. Of the 75 patients examined, 20% had clinical spinulosis, while 18.66% had dermoscopic features of Demodex: follicular plugs and tails. Of the 17.33% positive patients identified upon microscopy for Demodex, samples for bacterial culture were plated on trypticase soy Colombia agar. Identification was performed by microorganisms grown method mass spectrometry. We identified a strain of Bacillus cereus.

Antagonistic activities of some Bifidobacterium sp. strains isolated from resident infant gastrointestinal microbiota on Gram-negative enteric pathogens.

The gastrointestinal microbiota contributes to the consolidation of the anti-infectious barrier against enteric pathogens. The purpose of this study was to investigate the influence of Bifidobacterium sp. strains, recently isolated from infant gastrointestinal microbiota on the in vitro growth and virulence features expression of enteropathogenic bacterial strains. The antibacterial activity of twelve Bifidobacterium sp. strains isolated from human feces was examined in vitro against a wide range of Gram negative pathogenic strains isolated from 30 infant patients (3 days to 5 years old) with diarrhea. Both potential probiotic strains (Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium catenulatum, Bifidobacterium breve, Bifidobacterium ruminantium) and enteropathogenic strains (EPEC, EIEC, Klebsiella pneumoniae, Salmonella sp., Yersinia enterocolitica, Pseudomonas aeruginosa) were identified by MALDI-TOF and confirmed serologically when needed. The bactericidal activity, growth curve, adherence to the cellular HEp-2 substratum and production of soluble virulence factors have been assessed in the presence of different Bifidobacterium sp. cultures and fractions (whole culture and free-cell supernatants). Among the twelve Bifidobacterium sp. strains, the largest spectrum of antimicrobial activity against 9 of the 18 enteropathogenic strains was revealed for a B. breve strain recently isolated from infant intestinal feces. The whole culture and free-cell supernatant of B. breve culture decreased the multiplication rate, shortened the log phase and the total duration of the growth curve, with an earlier entrance in the decline phase and inhibited the adherence capacity to a cellular substratum and the swimming/swarming motility too. These results indicate the significant probiotic potential of the B. breve strain.

MECHANISMS OF RESISTANCE TO CIPROFLOXACIN AND GENETIC DIVERSITY OF ESCHERICHIA COLI STRAINS ORIGINATING FROM URINE CULTURES PERFORMED FOR ROMANIAN ADULTS.

Urinary tract infections (UTI) with Escherichia coli are among the most common infections presenting in general practice. Fluoroquinolones (FQs) are relied on for their empirical therapy but recent reports indicate a concerning increase in the percentage of FQ-resistant E. coli isolates in many countries, including Romania. Sixty E. coli strains with ciprofloxacin resistance and cephalosporin susceptibility isolated from urine specimens of non-hospitalized patients during a five-month period (October 2014 - February 2015) were further analyzed to determine the molecular basis of FQ resistance (i.e. mutations in chromosomal gyrA, gyrB, parC genes and presence of plasmid-borne qnrA, qnrB, qnrS, and aac(6'-Ib-cr genes), the phylogenetic background (i.e. phylogenetic groups A, B1, B2, C, D, E, F or clade I), O25b/ST131 status, and genetic relatedness inferred from the XbaI pulsed-field gel electrophoresis (PFGE) profiles as a measure of isolate-specific genetic composition. The PCR-based phylotyping showed that most strains were assigned to non-B2 phylogenetic groups (i.e. group A/21 strains, group B1/14 strains, group B2/10 strains, group C/8 strains, group D/3 strains, group F/4 strains). Already described chromosomal mutations associated to FQ resistance were found, the strains being double gyrA mutants (i.e. Ser83Leu, Asp87Asn) with one or two parC mutations (e.g. Ala56Thr, Ser80Ile, Glu84Gly). Seven percent of the strains harboured plasmid-borne genes qnrS1 (2 strains) and aac(6'-Ib-cr (2 strains). Based on the PCR results, 15% of the strains were members of the O25b/ST131 clone and possessed the gyrA/parC allele combination which is considered as hallmark of H30 subclone. PFGE genotyping revealed a genetically diverse population of FQ-resistant E. coli. ST131 strains displayed more homogeneous PFGE profiles than non-ST131. The ST131 cluster extended to 77.74% similarity versus 60% overall. These findings underscore the need for ongoing surveillance to capture the complexity of the emerging population of FQ-resistant strains disseminated across our community.

Screening for group B streptococcus: a private laboratory experience.

We examined group B streptococcus (GBS) isolates colonizing women at the 35-37 weeks of pregnancy. A total of 257 group B streptococcus (GBS) isolates for serotyped using direct agglutination with a set of commercially available antisera (Ia, Ib, II, III, IV, and V) and tested for susceptibility to antimicrobials (penicillin, macrolides, lincosamides, fluoroquinolones and tetracyclines). Fourteen isolates could not be serotyped with the antisera set used in the study. Serotype III was the predominant serotype (33%), followed by serotypes V (23%), and Ia (20%). Whereas all isolates were susceptible to penicillin, the rates of susceptibility to the other antimicrobials tested were the following: 91% for ofloxacin, 80% for clindamycin, 77% for erythromycin, and 4% for tetracycline. More than half (67%) of the macrolide resistant isolates belonged to serotypes V and III. A systematic surveillance of the autochthonous GBS serotypes, performed at the level of laboratories processing a high number of human specimens, is mandatory for strengthening the national epidemiological GBS surveillance. While penicillin remains the drug of choice for intrapartum prophylaxis, the resistance of autochthonous GBS isolates to other antibiotics should be actively monitored.