Examining the Prevalence and Antibiotic Susceptibility of S. aureus Strains in Hospitals: An Analysis of the pvl Gene and Its Co-Occurrence with Other Virulence Factors.

S. aureus is a pathogenic bacterium that causesinfections. Its virulence is due to surface components, proteins, virulence genes, SCCmec, pvl, agr, and SEs, which are low molecular weight superantigens. SEs are usually encoded by mobile genetic elements, and horizontal gene transfer accounts for their widespread presence in S. aureus. This study analyzed the prevalence of MRSA and MSSA strains of S. aureus in two hospitals in Greece between 2020-2022 and their susceptibility to antibiotics. Specimens collected were tested using the VITEK 2 system and the PCR technique to detect SCCmec types, agr types, pvl genes, and sem and seg genes. Antibiotics from various classes were also tested. This study examined the prevalence and resistance of S. aureus strains in hospitals. It found a high prevalence of MRSA and that the MRSA strains were more resistant to antibiotics. The study also identified the genotypes of the S. aureus isolates and the associated antibiotic resistances. This highlights the need for continued surveillance and effective strategies to combat the spread of MRSA in hospitals. This study examined the prevalence of the pvl gene and its co-occurrence with other genes in S. aureus strains, as well as their antibiotic susceptibility. The results showed that 19.15% of the isolates were pvl-positive and 80.85% were pvl-negative. The pvl gene co-existed with other genes, such as the agr and enterotoxin genes. The results could inform treatment strategies for S. aureus infections.

A Tale of Two Pandemics: Antimicrobial Resistance Patterns of Enterococcus spp. in COVID-19 Era.

Although the COVID-19 pandemic has held the spotlight over the past years, the antimicrobial resistance (AMR) phenomenon continues to develop in an alarming manner. The lack of strict antibiotic regulation added to the overuse of antimicrobials fueled the AMR pandemic. This paper aims to analyze and identify the impact of the COVID-19 pandemic on antibiotic resistance patterns of Enterococcus spp. The study was designed as a retrospective observational study. Enterococcus spp. infections data were collected from one academic hospital in Cluj-Napoca, Romania over 18 months. A statistical analysis was performed to compare antibiotic resistance phenotypes identified. We recorded an increase in the isolation rates of Enterococcus spp. strains, from 26 isolates (26.53%) during Period A (November 2020-April 2021) to 42 strains (42.85%) during Period C (November 2021-April 2022). The number of strains with resistance to vancomycin increased from 8 during Period A to 17 during Period C. Of the total 36 strains with resistance to vancomycin, 25 were identified as E. faecium. SARS-CoV-2 patients (n = 29) proved to be at risk to develop an E. faecium co-infection (n = 18). We observed that strains with resistance to ampicillin (n = 20) and vancomycin (n = 15) are more often isolated from these patients. All changes identified in our study are to be considered in the light of COVID-19 pandemic, highlighting the threatening AMR phenomenon in Romania. Further studies should be performed to quantify the worldwide effects of these pandemics.

Easy and Affordable: A New Method for the Studying of Bacterial Biofilm Formation.

BACKGROUND: Bacterial biofilm formation (BBF) proves itself to be in the spotlight of microbiology research due to the wide variety of infections that it can be associated with, the involvement in food spoilage, industrial biofouling and perhaps sewage treatment. However, BBF remains difficult to study due to the lack of standardization of the existing methods and the expensive equipment needed. We aim to describe a new inexpensive and easy to reproduce protocol for a 3D-printed microfluidic device that can be used to study BBF in a dynamic manner. METHODS: We used the SolidWorks 3D CAD Software (EducationEdition 2019-2020, Dassault Systèmes, Vélizy-Villacoublay, France) to design the device and the Creality3D Ender 5 printer (Shenzhen Creality 3D Technology Co., Ltd., Shenzhen, China) for its manufacture. We cultivated strains of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. For the biofilm evaluation we used optical coherence tomography (OCT), scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy and crystal violet staining technique. RESULTS: Based on the analysis, Enterococcus faecalis seems to produce more biofilm in the first hours while Pseudomonas aeruginosa started to take the lead on biofilm production after 24 h. CONCLUSIONS: With an estimated cost around €0.1285 for one microfluidic device, a relatively inexpensive and easy alternative for the study of BBF was developed.

Descriptive Analysis of Circulating Antimicrobial Resistance Genes in Vancomycin-Resistant Enterococcus (VRE) during the COVID-19 Pandemic.

COVID-19 offers ideal premises for bacteria to develop antimicrobial resistance. In this study, we evaluated the presence of several antimicrobial resistance genes (ARG) in vancomycin-resistant Enterococcus (VRE) isolated from rectal swabs from patients at a hospital in Cluj-Napoca, Romania. Rectal swabs were cultivated on CHROMID® VRE (bioMérieux, Marcy-l' Étoile, France) and positive isolates were identified using MALDI-TOF Mass Spectrometry (Bruker Daltonics, Bremen, Germany) and further analyzed using the PCR technique for the presence of the following ARGs: van A, van B, tet(M), tet(L), ermB, msrA, mefA, aac(6')-Im, aph(2)-Ib, ant(4')-Ia, sul1, sul2, sul3, and NDM1. We isolated and identified 68 isolates of Enterococcus faecium and 11 isolates of Enterococcus faecalis. The molecular analysis showed 66 isolates positive for the vanA gene and eight positive for vanB. The most frequent association of ARG in VRE was vanA-tet(M)-ermB. There was no statistically significant difference between Enterococcus faecium and Enterococcus faecalis regarding ARGs. Our work proves that during the COVID-19 pandemic, highly resistant isolates of Enterococcus were present in patients in the intensive care unit; thus, better healthcare policies should be implemented for the management and control of these highly resistant isolates in the future.

Enterococcus raffinosus, Enterococcus durans and Enterococcus avium Isolated from a Tertiary Care Hospital in Romania-Retrospective Study and Brief Review.

(1) Background: This paper aims to provide a description of non-faecalis non-faecium enterococci isolated from a tertiary care hospital in Romania and to briefly review the existing literature regarding the involvement of Enterococcus raffinosus, Enterococcus durans and Enterococcus avium in human infections and their antimicrobial resistance patterns; (2) Methods: We retrospectively analyzed all Enteroccocus species isolated from the "Prof. Dr. O. Fodor" Regional Institute of Gastroenterology and Hepatology from Cluj-Napoca during one year focusing on non-faecalis non-faecium Enterococci. A brief review of the literature was performed using case reports involving Enterococcus raffinosus, Enterococcus durans and Enterococcus avium; (3) Results: Only 58 out of 658 Enteroccocus isolates were non-faecalis non-faecium and met the inclusion criteria. These species were isolated more often (p < 0.05) from the surgical ward from mixed etiology infections with E. coli. In our review, we included 39 case reports involving E. raffinosus, E. durans and E. avium; (4) Conclusions: Isolation of non-faecalis non-faecium enterococci displays an emerging trend with crucial healthcare consequences. Based on the analysis of the case reports, E. avium seems to be involved more often in neurological infections, E. durans in endocarditis, while E. raffinosus displays a more heterogenous distribution.

Curcumin and Vitamin C Attenuate Gentamicin-Induced Nephrotoxicity by Modulating Distinctive Reactive Species.

Gentamicin remains widely used in all age groups despite its well-documented nephrotoxicity; however, no adjuvant therapies have been established to counteract this side effect. Our study aimed to experimentally determine whether curcumin and vitamin C have nephroprotective effects and whether certain reactive species could be used as markers of early gentamicin nephrotoxicity. Wistar adult male rats were evenly distributed into four groups: control, gentamicin, curcumin and gentamicin, vitamin C and gentamicin (gentamicin: 60 mg/kg/day, intraperitoneally, 7 days). We determined renal function (urea, creatinine), oxidative stress (malondialdehyde, nitric oxide, 3-nitrotyrosine, total oxidative stress), and antioxidant and anti-inflammatory status (thiols, total antioxidant capacity, interleukin-10). Nephrotoxicity was successfully induced, as shown by the elevated creatinine levels in the gentamicin group. In contrast, supplementation with curcumin and vitamin C prevented an increase in urea levels while decreasing total oxidative stress levels compared to the gentamicin group. Moreover, vitamin C and curcumin distinctively modulate the levels of nitric oxide and malondialdehyde. Histological analysis showed more discrete lesions in rats that received vitamin C compared to the curcumin group.

Evidence of SARS-CoV-2 Virus in the Middle Ear of Deceased COVID-19 Patients.

The presence of SARS-CoV-2 in the middle ear reveals the etiopathogenesis of otitis media in COVID-19, as well as an epidemiological risk during otologic examination and surgical procedures in COVID-19 patients. The study included 8 deceased patients with COVID-19. Tissue samples from the middle ear were subjected to virology, histopathology, scanning (SEM) and transmission (TEM) electron microscopy investigation. Ethmoidal mucosa samples were processed for virology analyses. qPCR resulted positive for 75% of nasal mucosa samples and 50% of middle ear samples. Ct values showed lower viral loads in middle ear samples. A proportion of 66.6% patients with positive results in the nasal mucosa showed positive results in the middle ear, and the subtype analysis of the complete genome sequences indicated B.1.1.7 lineage for all samples. In histopathological and SEM samples, no pathological aspects were identified. TEM revealed on the background of death critical alteration of cellular morphology, suggestive structures resembling SARS-CoV-2, goblet cells and immune cells. SARS-CoV-2 can be present in the middle ear of COVID-19 patients even if there is not clinical evidence of acute otitis media. Otolaryngologists could be particularly exposed to COVID-19 infection.

Respiratory Nasal Mucosa in Chronic Rhinosinusitis with Nasal Polyps versus COVID-19: Histopathology, Electron Microscopy Analysis and Assessing of Tissue Interleukin-33.

(1) Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most studied rhinological disorders. Modifications of the respiratory nasal mucosa in COVID-19 patients are so far unknown. This paper presents a comparative morphological characterization of the respiratory nasal mucosa in CRSwNP versus COVID-19 and tissue interleukin (IL)-33 concentration. (2) Methods: We analyzed CRSwNP and COVID-19 samples through histopathology, scanning and transmission electron microscopy and performed proteomic determination of IL-33. (3) Results: Histopathologically, stromal edema (p < 0.0001) and basal membrane thickening (p = 0.0768) were found more frequently in CRSwNP than in COVID-19. Inflammatory infiltrate was mainly eosinophil-dominant in CRSwNP and lymphocyte-dominant in COVID-19 (p = 0.3666). A viral cytopathic effect was identified in COVID-19. Scanning electron microscopy detected biofilms only in CRSwNP, while most COVID-19 samples showed microbial aggregates (p = 0.0148) and immune cells (p = 0.1452). Transmission electron microscopy of CRSwNP samples identified biofilms, mucous cell hyperplasia (p = 0.0011), eosinophils, fibrocytes, mastocytes, and collagen fibers. Extracellular suggestive structures for SARS-CoV-2 and multiple Golgi apparatus in epithelial cells were detected in COVID-19 samples. The tissue IL-33 concentration in CRSwNP (210.0 pg/7 µg total protein) was higher than in COVID-19 (52.77 pg/7 µg total protein) (p < 0.0001), also suggesting a different inflammatory pattern. (4) Conclusions: The inflammatory pattern is different in each of these disorders. Results suggested the presence of nasal dysbiosis in both conditions, which could be a determining factor in CRSwNP and a secondary factor in COVID-19.

Accelerated lipid peroxidation in a rat model of gentamicin nephrotoxicity.

Kidney disease represents a burden for the health care system worldwide. As the prevalence continues to rise, discovering new biomarkers of early kidney damage has become crucial. Oxidative stress (OS) represents one of the main factors involved in the early stages of many syndromes leading to kidney damage. Therefore, it must be studied in detail. To date, many studies have focused on OS in advanced stages of acute kidney injury (AKI), with great success. The aim of the present study was to ascertain whether even mild renal function impairment can be linked to specific systemic markers of OS and systemic antioxidants in order to pinpoint certain biomarkers for early kidney damage. We used male rats (Rattus norvegicus) in which we induced kidney damage by injecting gentamicin for 7 days. Blood was collected 24 h after the last dose of gentamicin. Urea, creatinine, 3-nitrotyrosine (3-NT), nitric oxide (NO), malondialdehyde (MDA), thiols (TS), total oxidative stress (TOS), and interferon-γ (IFN-γ) were determined. In addition, for the antioxidant status we measured total antioxidant capacity (TAC) and interleukin-10 (IL-10). Our results demonstrated that the rats had mild renal impairment consistent with a pre-AKI stage due to the nephrotoxic effect of gentamicin. However, TOS, MDA and NO were significantly higher in the gentamicin group compared to the control group. In addition, TAC was higher in the control group. Hence, OS markers reach higher levels and may potentially be used as markers of kidney damage even in cases of mild renal function impairment.

COVID-19 and Pneumocystis jirovecii Pulmonary Coinfection-The First Case Confirmed through Autopsy.

Background: Establishing the diagnosis of COVID-19 and Pneumocystisjirovecii pulmonary coinfection is difficult due to clinical and radiological similarities that exist between the two disorders. For the moment, fungal coinfections are underestimated in COVID-19 patients. Case presentation: We report the case of a 52-year-old male patient, who presented to the emergency department for severe dyspnea and died 17 h later. The RT-PCR test performed at his admission was negative for SARS-CoV-2. Retesting of lung fragments collected during autopsy revealed a positive result for SARS-CoV-2. Histopathological examination showed preexisting lesions, due to comorbidities, as well as recent lesions: massive lung thromboses, alveolar exudate rich in foam cells, suprapleural and intra-alveolar Pneumocystisjirovecii cystic forms, and bilateral adrenal hemorrhage. Conclusion: COVID-19 and P.jirovecii coinfection should be considered, particularly in critically ill patients, and we recommend the systematic search for P. jirovecii in respiratory samples.

Chronic Rhinosinusitis: MALDI-TOF Mass Spectrometry Microbiological Diagnosis and Electron Microscopy Analysis; Experience of the 2nd Otorhinolaryngology Clinic of Cluj-Napoca, Romania.

(1) Background: Chronic rhinosinusitis (CRS) represents a wide range of infectious-inflammatory processes affecting, simultaneously, the nose and paranasal sinuses mucosa. The paper presents outcomes of the investigation of CRS microbiological characteristics in a group of 32 patients. (2) Methods: The purulent samples were collected during functional endoscopic sinus surgery. Agar plates were incubated and examined. All types of colonies were identified using Matrix-Assisted Laser Desorption - Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS). For scanning electron microscopy, samples were fixed and sputter-coated with 10 nm gold and analyzed using a scanning electron microscope. For transmission electron microscopy, samples were fixed, postfixed, and dehydrated. After polymerization, ultrathin sections were collected on carbon coated copper grids and analyzed with Jeol JEM1010 TEM. (3) Results: Positive microbiological diagnosis was obtained in 62.5% of cases. The most frequent species found are Staphylococcus aureus and Streptococcus constellatus subsp. pharyngis. Corynebacterium aurimucosum and Eggerthia catenaformis were unreported species in CRS until the present. Biofilm was evidenced in 43.7% of sinus mucosa samples. Ciliary disorientation, atrophy, and no ciliated cells were also identified. (4) Conclusion: The microbial factor-pathogen or opportunistic-is one of the most important pathological links in chronic rhinosinusitis. MALDI-TOF MS allows easily and quickly identification of germs.

Infectious Esophagitis in Romanian Children: From Etiology and Risk Factors to Clinical Characteristics and Endoscopic Features.

OBJECTIVES: The aim of this study is to provide information about prevalence, etiology, risk factors, clinical characteristics and endoscopic features of various types of infectious esophagitis in children. METHODS: We performed a total of 520 upper gastrointestinal tract endoscopies in Pediatric Clinic II, Emergency Hospital for Children, Cluj-Napoca. Indications for endoscopy in our cohort were gastrointestinal tract symptoms such as dysphagia, heartburn, or appetite loss. RESULTS: The prevalence of infectious esophagitis in the study population was 2.11% (11 patients). Candida albicans (C. albicans) was the most frequent cause. Our data illustrates that herpes simplex virus (HSV)-induced esophagitis is common in immunocompromised patients and should be systematically suspected in cases of severe dysphagia, heartburn, or hematemesis. In the present study, all cytomegalovirus (CMV) esophagitis patients were immunocompromised. Immunodeficiency (81.8%) and prolonged antibiotic therapy with broad-spectrum antibiotics were by far the most important risk factors involved in the pathogenicity of the disease. Dysphagia, appetite loss, heartburn, epigastralgia, and hematemesis were the main clinical manifestations. Infectious esophagitis was associated with significant mortality. In four patients, endoscopy during life showed signs of infectious esophagitis; however, the precise etiology was only established post-mortem, in the pathological anatomy laboratory department. A risk factor involved in pathogenesis of post-mortem diagnosed infectious esophagitis is the DiGeorge syndrome for CMV and HSV patients. CONCLUSIONS: The study illustrates that infectious esophagitis should be considered in immunocompromised infants with prolonged antibiotic therapy with broad-spectrum antibiotics.

Correlated quantification using microbiological and electrochemical assays for roxithromycin determination in biological and pharmaceutical samples.

Microbiological and electrochemical assays, applying the cylinder-plate and differential pulse voltammetry as techniques, are reported for the quantitative determination of roxithromycin in serum and solid pharmaceutical form. The microbiological assay is based upon the inhibitory effect of this drug on the strain Bacillus subtilis ATCC 9372 used as the test microorganism. Linearity of the calibration curve was observed over the concentration range of 8.37-83.70 µg mL-1, with relative standard deviation values less than 5.0%. The electrochemical behavior of roxithromycin was studied at a graphite screen-printed electrode modified with graphene by using cyclic voltammetry and differential pulse voltammetry. The current value of the oxidative peak obtained for roxithromycin at 0.65 V vs. Ag/AgCl in 0.03 mol L-1 phosphate buffer solution (pH 7.0) with a scan rate of 0.1 V-1 is a linear function of the concentration in a range of 4.19-83.70 µg mL-1 (5-100 µmol L-1). A comparative study was carried out and both methods were applied for the determination of roxithromycin in solid dosage forms and spiked serum. The bioassay results of human serum samples were in accordance with the electrochemical ones (R2 = 0.988, P < 0.001), and the Bland-Altman method also showed good agreement between the values obtained by both procedures. Moreover, the statistical comparison indicated that there was no significant difference between the proposed techniques regarding both accuracy and precision.

Toxoplasmosis in Transplant Recipients, Europe, 2010-2014.

Transplantation activity is increasing, leading to a growing number of patients at risk for toxoplasmosis. We reviewed toxoplasmosis prevention practices, prevalence, and outcomes for hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT; heart, kidney, or liver) patients in Europe. We collected electronic data on the transplant population and prevention guidelines/regulations and clinical data on toxoplasmosis cases diagnosed during 2010-2014. Serologic pretransplant screening of allo-hematopoietic stem cell donors was performed in 80% of countries, screening of organ donors in 100%. SOT recipients were systematically screened in 6 countries. Targeted anti-Toxoplasma chemoprophylaxis was heterogeneous. A total of 87 toxoplasmosis cases were recorded (58 allo-HSCTs, 29 SOTs). The 6-month survival rate was lower among Toxoplasma-seropositive recipients and among allo-hematopoietic stem cell and liver recipients. Chemoprophylaxis improved outcomes for SOT recipients. Toxoplasmosis remains associated with high mortality rates among transplant recipients. Guidelines are urgently needed to standardize prophylactic regimens and optimize patient management.

Molecular epidemiology of the community-associated methicillin-resistant staphylococcus aureus clones: a synthetic review.

The article presents a synthetic molecular characterization of the methicillin-resistant Staphylococcus aureus and describes the most important community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clones that circulate nowadays in the world: the main molecular and epidemiological characteristics, as well as notions related to the clinic of infections produced by these clones and their antibiotic resistance spectrum. The predominant clone of CA-MRSA in North America is USA300 - ST8-IV in North America, in Australia - Queensland (Qld) MRSA (ST93-IV), in Europe - ST80-IV, in Asia there is a high heterogeneity of clones population, in Africa the distribution of CA-MRSA clones is unclear, and in South America - USA 1100 and USA300-Latin American variant are predominant. The molecular diagnosis is performed by highly specialized institutions. The knowledge of clones allows the study of antibiotic resistance spectrum for each one, a fact of great importance for medical practice. Molecular epidemiology of the CA-MRSA shows that lowly restricted sales of antibiotics in shops and pharmacies, as well as medical prescribing practices without a laboratory investigation, especially in Eastern Europe and Asia, contribute to the development of new MRSA clones with increased resistance to antibiotics.

Cytomegalovirus infection in immunosuppressed patients after kidney transplantation.

The first kidney transplantation was performed in 1951 and ever since then living donor transplantation became a more and more important solution for patients with end-stage renal disease (ESRD). Renal transplantation is a life-saving procedure. Morbidity and mortality on waiting-lists are strongly correlated with the time of dialysis and end-stage renal disease is one of the most important causes of death; this is the reason why transplantation has to be performed as soon as possible in order to reduce the time of dialysis. Once the transplantation is performed, a number of complications may occur in post-transplant evolution, the most important of which is rejection. The rejection may appear through several mechanisms, but one of the most frequent causes of rejection is cytomegalovirus (CMV) infection. It is very important to have a precocious and fast diagnosis of CMV infection in order to maintain the functionality and survival of the graft. PP65 CMV antigenemia has proven its effectiveness in detecting and monitoring the CMV infection in transplanted patients. In the laboratory of the Clinical Institute of Urology and Renal Transplantation (ICUTR) of Cluj Napoca the CMV infection is evidenced by two methods: PP65antigenemia and IgM antibody identification by chemiluminiscence.

Antibacterial activity of different natural honeys from Transylvania, Romania.

Honey is used in food industry and medicine due to its nutritive, therapeutic and dietetic qualities. The microbiological characteristics of 10 unpasteurized honey samples of known origin, collected from Transylvania beekeepers (Romania) were determined. The antibacterial activity of these types of honey against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella enteritidis, Salmonella anatum, Salmonella choleraesuis, Bacillus cereus, Bacillus subtilis subsp. spizizenii and Listeria monocytogenes strains was studied. The most sensitive to the antibacterial activity were the two staphylococus strains (the largest diameter of inhibition zone was 18 mm) and B. subtilis strains (13.5 mm). The strains of B. cereus, E. coli, L. monocytogenes and Salmonella spp. were found to present resistance to some of the honey samples. Manna, sunflower and polyfloral honeys presented high antibacterial activity while acacia and linden honeys had a lower activity in terms of the number of sensible strains. Statistical analysis shows that the type of strains and the type of honey have influence on the diameter of inhibition.