Acta Microbiol. Hell., Volume 69, Issue 4 (December 2024) – 10 articles

Viruses transmitted by arthropods (arboviruses) pose a global public health threat. Sporadic cases or outbreaks caused by West Nile virus, Crimean–Congo haemorrhagic fever virus, tick-borne encephalitis virus, and phleboviruses are often reported in Europe. Recently, they expanded their distribution in geographic areas where they had never been observed before, while tropical viruses, like Dengue, Chikungunya, and Zika, started to cause autochthonous cases and outbreaks following the return of viraemic travellers from endemic countries. The primary or secondary vectors of these viruses are established in Europe, and the incidence of arboviral diseases is expected to increase due to several anthropogenic and/or environmental factors (mainly climate change, which affects the survival and amplification of the arthropod vectors). This is an update on the emerging arboviruses in Europe, associated challenges, and future perspectives. Full article

Community-acquired urinary tract infections (UTIs) represent a significant public health issue, primarily due to the increasing antibiotic resistance among uropathogens. This study assesses the resistance status of uropathogenic community Enterobacterales to various antibiotics, particularly aminoglycosides, and determines the prevalence of aminoglycoside-modifying enzyme (AME) genes, while investigating the coexistence of 16S rRNA methylating enzymes. We analyzed 628 clinical isolates of Enterobacterales obtained from 4282 cytobacteriological urine examinations at the Pasteur Institute Casablanca, Morocco, collected from October 2018 to December 2021. Identification and antibiotic susceptibility testing were conducted using the VITEK 2^® COMPACT system, following CA-SFM guidelines. DNA extraction utilized the heat shock method, and subsequent PCR was performed. Gram-negative bacteria accounted for 85% of isolates, with Enterobacterales representing 91% of this group. E. coli (73%) and Klebsiella pneumoniae (20%) were the most common species among Enterobacterales. Resistance was particularly high for ampicillin (76.7%) and amoxicillin-clavulanate (58%). Among aminoglycosides, gentamicin and tobramycin resistance rates were 33.5% and 35%, respectively, while amikacin resistance was observed in 21.3% of isolates. High frequencies of AME genes were detected, with AAC(3′)-IIa (27.7%) and AAC(6′)-Ib (25.9%) being the most prevalent. Notably, no 16S rRNA methylation genes (rmtA, rmtB, rmtC, rmtD) were found. All tested strains exhibited biofilm-forming capacity, with K. pneumoniae demonstrating intense biofilm production. The study highlights a concerning trend of antibiotic resistance among uropathogenic Enterobacterales in the community setting, correlating genotype with resistance phenotype and emphasizing the need for enhanced surveillance and targeted treatment strategies. Full article

Ebola virus (EBOV) causes Ebola virus disease (EVD), a severe and often fatal hemorrhagic fever. Although much research has focused on host miRNA expression during EBOV infection, it has been discovered that EBOV itself also produces miRNAs. However, further studies are needed to fully comprehend the role of these EBOV-encoded miRNAs in infection and disease development. This study aimed to identify known and novel EBOV-encoded miRNAs and their potential functions in the pathogenic mechanisms of EBOV. We reanalyzed previously available small RNASeq data to identify the miRNAs and predict their cellular targets and functions. We identified four EBOV-encoded miRNAs—EBOV-mir-M1 (4390–4414), EBOV-mir-M4, EBOV-mir-M2 (8288–8309), and EBOV-mir-M3 (9885–9906)—expressed specifically in Ebola-infected human adult retinal pigment epithelial (ARPE) cells. EBOV-mir-M1 (4390–4414) was expressed up to 19 times more than the other three miRNAs. The identified miRNAs were predicted to target genes associated with pathways such as calcium signaling, MAPK signaling, type I interferon signaling, and cytokine-mediated signaling, which play critical roles in Ebola infection and pathogenesis. This study contributes to our understanding of the role of EBOV-encoded miRNAs in infection and pathogenesis by demonstrating the expression of these miRNAs in human ARPE cells, providing insights into the mechanisms underlying EBOV pathogenesis. Full article

In early 2020, Greece was affected by the SARS-CoV-2 epidemic, and since then, the continuous emergence of fast-spreading variants has caused surges of new SARS-CoV-2 infections. In this study, we performed genomic, phylogenetic, and epidemiological analyses to investigate the SARS-CoV-2 epidemic in northwestern Greece. From March 2020 to February 2022, nasopharyngeal samples obtained from patients suspected to have COVID-19 were tested for SARS-CoV-2 detection. Complete SARS-CoV-2 genomic sequences were generated from selected positive samples. Northwestern Greece experienced four distinct waves of the epidemic following the first wave, which was mainly observed in Attica and other parts of Greece. The positivity index was rising throughout the pandemic waves in several geographical units, with the highest levels recorded in prominent tourist destinations characterized by high agricultural density. The phylogenetic analyses revealed 34 different lineages, with B.1, B.1.1, B.1.1.305, B.1.1.318, B.1.177, B.1.1.7, B.1.617.2, AY.43, and BA.1 being the most prevalent lineages in the region. Although multiple lineages were co-circulating, each pandemic wave was dominated by a different lineage. The SARS-CoV-2 epidemic in northwestern Greece was characterized by the successive introduction of new lineages, resulting in surges of new SARS-CoV-2 infections. Full article

The cycle threshold (Ct) value of PCR, used to detect SARS-CoV-2 viral load, has been associated with disease severity in adults, but few studies have examined these relationships in pediatric patients. The aim of this study was to evaluate the relationship between the Ct value at hospital admission and the duration of fever, length of hospital stay, and the need for medical interventions in pediatric patients with COVID-19. We conducted a retrospective study of 124 pediatric patients with a positive nasopharyngeal swab for SARS-CoV-2 between April 2020 and March 2022. A negative correlation was observed between the Ct value and the duration of fever (p = 0.012), with a similar trend for length of hospital stay (p = 0.25). A low Ct value was also associated with the development of MIS-C (p = 0.026) and the need for respiratory support and steroid therapy (p = 0.036). The Ct value may be useful in stratifying pediatric patients with COVID-19, helping to predict the duration of fever and hospital stay, as well as the need for medical interventions. Full article

Biofilms are bacterial communities on surfaces within an extracellular matrix. Targeting biofilm-specific bacteria is crucial, and natural compounds with reported antibiofilm activity have garnered significant interest. The study evaluated the antibacterial and antibiofilm activity of Erythrina senegalensis leaf extract against multidrug-resistant (MDR) Gram-negative bacteria, including Salmonella Typhimurium, S. Typhi, S. Enteritidis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The leaf extract was prepared using aqueous and ethanol solvents, and qualitative phytochemical screening revealed the presence of various bioactive compounds such as tannins, saponins, cardiac glycosides, flavonoids, terpenoids, alkaloids, anthraquinone, reducing sugar, and ketones. A Kirby–Bauer disc diffusion assay was performed to test the susceptibility of antibiotics, and the antibacterial efficacy of the aqueous and ethanol extracts of E. senegalensis was determined using the cup-plate method, while the antibiofilm activities were determined using the crystal violet titer-plate method. The aqueous and ethanol extracts of E. senegalensis revealed the presence of tannins, saponins, cardiac glycosides, flavonoids, terpenoids, alkaloids, anthraquinone, reducing sugar, and ketones. The study found that the Gram-negative bacteria isolates that were MDR were S. Typhimurium, S. Enteritidis, and P. aeruginosa, while K. pneumoniae was resistant to beta-lactam and fluoroquinolones, and S. Typhi was susceptible to all antibiotics tested. Statistically, susceptibility to antibiotics had an inverse, weak, and significant relationship with biofilm production (r = −0.453, −0.106, −0.124, −0.106, −0.018, n = 10, p < 0.05). The aqueous extract showed good biofilm inhibition against K. pneumoniae and P. aeruginosa, and poor biofilm inhibition against S. Enteritidis, while S. Typhimurium and S. Typhi exhibited no biofilm inhibition. The ethanol extract did not demonstrate any antibiofilm activity against the tested Gram-negative pathogens. The study suggests that the Gram-negative bacteria’s capacity to form biofilms is negatively associated with their antibiotic resistance phenotypes, and the aqueous extract of E. senegalensis exhibited moderate antibiofilm activity against K. pneumoniae, P. aeruginosa, and S. Enteritidis. Full article

Tuberculosis has had a long-standing prevalence in Greece over the last century, going hand in hand with the country’s overall socioeconomic situation. The current study explores aspects of tuberculosis in Greece since the interwar period and up to the present. Apparently, ignorance of the nature of the disease coupled with false medical perceptions sealed the fate of patients while unwittingly influencing public opinion and making societies hostile toward patients. Such paradigms are of great and timeless value for the cohesion and functioning of societies, not excluding modern ones. Although the present-day situation in Greece is utterly different from that in the previous century, considering the limited number of cases and minimal impact of the disease, new challenges have emerged, such as the under-reporting of cases, the emergence of multidrug-resistant tuberculosis, latent tuberculosis or the management of large refugee flows in the Eastern Mediterranean involving citizens from countries with a high prevalence of the disease. On the other hand, the study of tuberculosis is nowadays more urgent than ever, given the global concerns regarding the future of the disease in the midst of the COVID-19 pandemic, which has put enormous pressure on national health systems. Full article

The United States Centers for Disease Control and Prevention (CDC) has categorized Clostridioides difficile infection (CDI) as a significant concern in extended-care facilities, hospitals, and outpatient clinics. However, little is known about CDI in low- and middle-income countries. This study determined CDI prevalence and impact in outpatient adults presenting with diarrhea in Nigeria. Toxigenic culture and PCR were used to detect and validate C. difficile. Prior antibiotic use, medical history, and demographic data were also obtained. Descriptive and inferential statistics were used for data analysis. The patient demographics were 35.48% (22/62) for the 18–24 years age group and 32.26% (20/62) for both the 25–30 years age group and the 31+ years group, with an average age of 29.7 years. Forty-eight percent of the patients (30/62) tested positive for CDI, and the prevalence increased with age. Most patients (86.67%, 52/60) reported moderate/severe cases of diarrhea and 67.7% had no knowledge of antibiotics. The results showed that 62.30% of the cases were hospitalized with the duration of diarrhea being significantly associated (r = 0.98, p ˂ 0.001) with the length of hospitalization. These results suggest that C. difficile is common among diarrhea patients in this population and that Nigerian hospitals’ infection prevention and control measures must include this pathogen. Full article

Neutrophil gelatinase-associated lipocalin (NGAL) is a protein secreted by activated neutrophils and certain tissues. The aim of this study was to investigate the prognostic role of urinary neutrophil gelatinase-associated lipocalin (uNGAL) in hospitalized COVID-19 patients with regard to mortality. The prospective observational cohort study involved 86 hospitalized adult COVID-19 patients. Patients’ urine samples were collected upon admission and 48–72 h after admission. General anamnesis and clinical status information were obtained from medical records. Within 24 h of sample collection, urine aliquots were centrifuged, the supernatant was separated, and samples were frozen at −80 °C until uNGAL concentration measurement, which was performed within two years of sample collection. The patients were categorized into groups based on disease outcome (survived/deceased). Data analysis was performed to identify predictive factors and diagnostic indicators for the unfavorable outcome group using logistic regression and ROC curve methods. Logistic regression associated age, cardiomyopathy, invasive mechanical ventilation, and uNGAL concentration (follow-up sampling) with mortality. ROC analysis identified uNGAL concentration (follow-up sampling) as an indicator of mortality, with a cut-off value of >23.8 ng/mL. This study concludes that there is an association between disease outcome and uNGAL concentration in COVID-19 patients. However, understanding the dynamics of uNGAL concentration during COVID-19 remains limited, which is crucial for its effective clinical application. Full article

Newcastle disease, which affects poultry and is endemic in many nations across the world, is caused by Avian Paramyxovirus-1 (APMV-1). This experimental study was conducted from January to June 2021 at the National Veterinary Institute (NVI) to evaluate the virus viability and antibody titer of Newcastle disease vaccines (Hichner’s B1, Lasota, and ThermostableI2) stored at different temperature storage conditions. Chickens (12 treatment groups and 1 control group) were vaccinated and challenged with the virulent ND virus (0.5 × 10^6.5 embryonic lethal dose fifty (ELD₅₀)). The immune responses (antibody titers) of chickens were evaluated using hemagglutination (HA) and hemagglutination inhibition (HI) assays. The Newcastle disease vaccines (Hachiner’s B1 (ND-HB1), ND-Lasota, and ND-Thermostable I2) stored at +4 °C HI-induced antibody titers of 151 (±103.3), 136 (±53.4), and 145 (±91) on day 14, respectively, whereas on day 21, they increased to 160 (±82) for ND-HB1 and 144 (±74.5) for ND-Lasota. ND-Thermostable I2 showed a decrement to 133 (±44.8). All three vaccines stored at different temperature storage conditions (+4, +23, and +30 °C) used in this experiment induced antibody titers greater than 128 on day 28 post-vaccination, except the Newcastle disease vaccine Thermostable I2 stored at +30 °C. The vaccines collected from private veterinary drugstores (customer vaccines Hachiner’s B1 and ND-Thermostable I2) used in this experiment induced very low antibody titers, less than 128 antibody titers, from days 14 to 21. Statistically significant induced mean antibody titers were observed for chickens that received vaccines stored at different temperature storage conditions for 72 h (p < 0.05), except for the ND-HB1 mean HI-induced antibody titer at days 7 and 28. Further, vaccine protection was confirmed by inoculation of both the vaccinated (treatment groups) and control groups by the virulent ND virus, where the control group started dying three days post-challenge but all chicks that received the vaccines survived. Overall, this study showed the impact of temperature storage conditions on the antibody titer and their effect on the titer of the viable virus in the vaccine, and thereby its protective capacity, warranting appropriate cold chain management of the vaccines along the value chain. Full article