Lipooligosaccharide architecture in Acinetobacter modestus CM11G: a non-pathogenic strain.

This study explored the application of ddPCR and qPCR methods in identifying Naegleria fowleri from both clinical and environmental water samples. Strong agreement between ddPCR and qPCR methods over clinical DNA samples was observed. Naegleria fowleri was present in surface water samples from Lake Pontchartrain during our study period. The ability of N. fowleri to survive in brackish water is therefore a potential risk factor for people who engage in water-related recreational activities. The ddPCR performance demonstrated in this study on clinical and environmental samples lead to greater confidence of ddPCR technology on field application.

BackgroundHepatitis A virus (HAV) strains detected in environmental and clinical samples were analysed to characterize the genotypes of HAV circulating in Greece. Fifty (50) sewage samples were collected from Patras (South-Western Greece) and Alexandroupolis (North-Eastern Greece) from 2007 until 2009, accordingly. The clinical samples derived from an HAV outbreak involved populations from three neighbouring prefectures of North-Eastern Greece (Xanthi, Rodopi, and Evros). HAV particles were detected by nested RT-PCR, using a previously validated set of primers to amplify a 290-bp fragment encompassing the 5'-NTR. Positive HAV samples were confirmed by sequencing of the PCR product. To determine the relatedness between the different isolated sequences, a phylogenetic tree was constructed.ResultsResults showed a 100% prevalence of genotype I, and particularly subgenotype IA. The analyzed HAV strains were closely related between them with the percentage of nucleotide identity ranging between 96% and 100%.ConclusionsThe study revealed the major prevalence of circulating strains of IA genotype in Greece and underlined the usefulness of molecular methods for the detection and typing of viruses in both environmental and clinical samples. The present study is, to our knowledge, the first in Greece to depict the simultaneous molecular characterization of HAV strains isolated from both clinical and environmental samples.

BackgroundVibrio parahaemolyticus has emerged as the leading cause of seafood-associated infections worldwide. Previous studies have shown that V. parahaemolyticus can be detected in both environmental and clinical samples. However, the molecular characteristics of V. parahaemolyticus isolated from these sources remain unknown.ResultsThis study examined 128 strains of V. parahaemolyticus isolated from clinical and environmental samples collected between 2020 and 2023 in Huzhou, China. We identified 73 serotypes; O10:K4, O3:K6, and O4:KUT were the dominant serotypes among clinical isolates. We examined the proliferation and motility of major epidemic strains from environmental and clinical samples. Genetic diversity and evolution were assessed by average nucleotide identity (ANI), phylogenetic tree construction, and multilocus sequence typing (MLST). Furthermore, we identified 13 novel sequence types (STs) among the environmental isolates, indicating that V. parahaemolyticus strains are widely distributed and evolve rapidly in the environment in Huzhou, China. We found 206 virulence genes among these isolates, indicating that environmental isolates possess numerous virulence genes. Additionally, we detected 4 strains carrying the tdh or trh gene, which may increase their pathogenicity. The prediction results of antibiotic resistance genes shown that environmental isolates may carry up to 104 resistance genes, compared to 30 in clinical isolates.ConclusionsWe observed that the environmental serotypes of V. parahaemolyticus exhibit greater diversity compared to clinical isolates, which are predominantly concentrated in three major serotypes. Furthermore, a considerable genetic distance was found between most clinical and environmental isolates. Notably, some clinical isolates show a closer genetic proximity to environmental isolates. Additionally, the distribution of virulence genes, specifically T3SS and tdh, significantly differs among isolates from these two distinct sources. The prediction results for antibiotic resistance genes suggest that environmental isolates may harbor a broader spectrum of resistance genes. The findings of this study provide new insights into the phylogenetic relationships between V. parahaemolyticus strains from clinical and environmental sources, and they enhance the MLST database.

Background: The increasing prevalence of multidrug-resistant bacterial infections, particularly within healthcare-associated settings, poses a global public health threat. Among these pathogens, Acinetobacter species, notably Acinetobacter baumannii, have gained recognition as opportunistic nosocomial pathogens with a concerning propensity for antimicrobial resistance. Understanding their clinical and antimicrobial profile is crucial for addressing the challenges posed by these infections.Objectives: To assess the prevalence of Acinetobacter species in distinct clinical samples.To analyze Acinetobacter species' clinical profiles and patterns of antibiotic sensitivity using a range of clinical samples.Materials and Methods: A cross-sectional study was conducted in a tertiary care hospital in South India, approved by the Institutional Ethics Committee. Patients with confirmed Acinetobacter infections were included, and clinical samples were collected from the critical care unit. Demographics, comorbidities, clinical presentations, and outcomes were recorded. Laboratory processing included standard protocols for sample analysis, with confirmation through advanced methods such as PCR.Results: Out of 82 participants, 48.78% showed positive cultures for Acinetobacter species. Prevalence varied by sample type, with urine samples exhibiting the highest positivity (50%). Clinical analysis of 40 infected patients revealed a mean age of 43.72 ± 17.34 years. Musculoskeletal symptoms (17.5%) and obstetric complications (17.5%) were prominent clinical presentations. Antibiotic sensitivity analysis demonstrated that 51% of isolates were multidrug-resistant, with high resistance to key antibiotics. Commonly resistant antibiotics included Cefepime, Piperacillin, Levofloxacin, Imipenem, Meropenem, and Amikacin.Conclusion: This study adds to the growing understanding of Acinetobacter infections, emphasizing the need for awareness, infection control, and judicious antibiotic use. The multidisciplinary clinical presentations and high resistance rates underscore the complexity of managing Acinetobacter infections, requiring a holistic approach in healthcare settings. The findings inform strategies for infection prevention, surveillance, and therapeutic approaches in the global effort to combat antimicrobial resistance

Epidemic cholera caused by toxigenic Vibrio cholerae O1 is a major health problem in several developing countries. Traditional methods for identifying V. cholerae involve cultural, biochemical and immunological assays which are cumbersome and often take several days to complete. In the present study, a direct cell multiplex PCR was developed targeting the ompW, ctxB and rfbO1 genes for confirmation of V. cholerae , its toxigenicity and serogroup O1, respectively from clinical and environmental samples. The detection sensitivity of the multiplex PCR was 1.9 x 10 3 V. cholerae per PCR reaction. A total of 31 environmental samples and 45 clinical V. cholerae isolates from different outbreaks were examined by the PCR. The assay was simple and specific, as there was no requirement for DNA extraction and no amplification was observed with other homologous bacteria used. The assay can be very useful for rapid surveillance of toxigenic V. cholerae O1 in environmental water samples, as well as for confirmation of clinical isolates.

Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples. Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL. To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%. It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

ABSTRACTBackgroundTyping Aspergillus species is crucial for understanding the sources of infection in hospital environments.ObjectivesThis study analysed clinical and air samples as well as their relationship with the clinical forms of aspergillosis. Additionally, we examined the usefulness of the Short Tandem Repeats (STR) technique with two highly discriminatory markers for analysing the Aspergillus fumigatus (A. fumigatus) profile.Patients/MethodsSeventy‐five air samples (September 2013–July 2014) and 116 clinical samples (2009–2014) were collected in a university hospital. Seventy‐two samples were typed by STR with two markers, MC3 and MC5.ResultsOf the 75 air samples collected, 10 were positive in the Bone Marrow Transplant unit, a ventilated unit with HEPA filters as were 18 in the Haematology ward, a naturally ventilated unit. Of the 116 clinical samples of Aspergillus spp., 95 were identified as A. fumigatus. High diversity was found, with 42 genotypes in 67 clinical samples and four in five environmental samples. Most isolates were collected during the demolition and renovation of the Emergency unit in the Hospital from 2013 to 2014. Genotype 1 was found in several units during different years. Despite the heterogeneity, identical genotypes were observed three times at short intervals in the same or different wards. Some of these identical genotypes were confirmed as possible clones by genome sequencing while others' genotyping matches failed to be confirmed.ConclusionDespite the diversity of clinical and environmental samples, useful correlations can be established in invasive aspergillosis surveillance programs by using this simple STR method as a preliminary step.

Molecular markers in the epidemiology and diagnosis of coccidioidomycosis

Pseudomonas species are common pathogenic Gram negative bacteria frequently found in both environmental and clinical samples. They are opportunistic pathogen and could be multidrug resistant (MDR); their presence in the environment signifies threat to the public health. Water from Awba dam is used as municipal water supply for drinking, fishing and agricultural purposes in the University of Ibadan which gives reason for proper monitoring. The study was designed to isolate and characterize Pseudomonas species present in Awba dam reservoir and also to compare its antimicrobial resistance pattern with clinical isolates. Pseudomonas was isolated using Centrimide agar (Oxoid) and identification was done to species level using classical method. Disk diffusion method was applied to test antibiotic sensitivity according to clinical and laboratory standard guidelines. A total of 200 bacteria isolates were obtained from water and sediment, of which 30 were found positive for Pseudomonas giving a prevalence of 15.0% (30/200). Eighteen clinical samples were collected from University College Hospital with about 50% from urine, 27.8% (Ear), 11.1% (wound) and 5.6% each from pleural effusion and nasopharyngeal tube. Two species of Pseudomonas; aeruginosa and fluorescens were identified. Out of the 10 antibiotics used, environmental isolates showed no resistance to gentamicin, amikacin, ciprofloxacin, tetracycline and imipinem while clinical isolates showed 83.3, 16.7, 83.3, 100 and 11.1% resistance to the antibiotics respectively. Amikacin and Imipinem was the most effective drug for both environmental and clinical Pseudomonas isolates. High rates of MDR Pseudomonas (94.4%) from clinical strains and 13.3% of the environmental strains were observed. In conclusion, the result shows that Pseudomonas isolates from the clinical specimens are more exposed to antibiotics, and MDR isolates in the water system can be transferred to humans and animals.

Whole-genome sequencing (WGS) of influenza A virus (IAV) is crucial for identifying diverse subtypes and newly evolved variants and for selecting vaccine strains. In developing countries, where facilities are often inadequate, WGS is challenging to perform using conventional next-generation sequencers. In this study, we established a culture-independent, high-throughput native barcode amplicon sequencing workflow that can sequence all influenza subtypes directly from a clinical specimen. All segments of IAV in 19 clinical specimens, irrespective of their subtypes, were amplified simultaneously using a two-step reverse transcriptase PCR (RT-PCR) system. First, the library was prepared using the ligation sequencing kit, barcoded individually using the native barcodes, and sequenced on the MinION MK 1C platform with real-time base-calling. Then, subsequent data analyses were performed with the appropriate tools. WGS of 19 IAV-positive clinical samples was carried out successfully with 100% coverage and 3,975-fold mean coverage for all segments. This easy-to-install and low-cost capacity-building protocol took only 24 h complete from extracting RNA to obtaining finished sequences. Overall, we developed a high-throughput portable sequencing workflow ideal for resource-limited clinical settings, aiding in real-time surveillance, outbreak investigation, and the detection of emerging viruses and genetic reassortment events. However, further evaluation is required to compare its accuracy with other high-throughput sequencing technologies to validate the widespread application of these findings, including WGS from environmental samples. IMPORTANCE The Nanopore MinION-based influenza sequencing approach we are proposing makes it possible to sequence the influenza A virus, irrespective of its diverse serotypes, directly from clinical and environmental swab samples, so that we are not limited to virus culture. This third-generation, portable, multiplexing, and real-time sequencing strategy is highly convenient for local sequencing, particularly in low- and middle-income countries like Bangladesh. Furthermore, the cost-efficient sequencing method could provide new opportunities to respond to the early phase of an influenza pandemic and enable the timely detection of the emerging subtypes in clinical samples. Here, we meticulously described the entire process that might help the researcher who will follow this methodology in the future. Our findings suggest that this proposed method is ideal for clinical and academic settings and will aid in real-time surveillance and in the detection of potential outbreak agents and newly evolved viruses.

Antimicrobial resistance in Escherichia coli presents a global challenge associated with nosocomial infections and increased mortality rates. Understanding resistance profiles is crucial for guiding treatment strategies and ensuring effective antibiotic use. This study aimed to investigate the prevalence and in vitro resistance of E. coli to community-acquired and nosocomial infections. Various clinical samples from 700 patients were cultured on MacConkey’s medium and blood agar. The disk diffusion method was used to determine the antibiotic susceptibility profile of the E. coli isolates following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Urine, pus, seminal fluid, vaginal swabs, and other body fluids were among the clinical samples analyzed. Of the 112 E. coli isolates, 48.2% were from inpatients and 51.8% were from outpatients, with the majority (66%) isolated from urine samples. Higher resistance levels were observed in the urinary isolates than that in the previously recorded data from the same institutions. Notably, isolates exhibited high resistance to penicillin (98.2%), ampicillin (97.3%), first-generation cephalosporins (90.2%), erythromycin (72.2%), and roxithromycin (95.4%), whereas lower resistance was noted against piperacillin-tazobactam (25.0%), nitrofurantoin (12.5%), and imipenem (9.8%). The overall multidrug resistance rate was 62.5%, with higher rates observed in nosocomial infections (70%) compared to community-acquired isolates (55.6%). However, this difference was not statistically significant (p>0.05). This study underscores the prevalence of E. coli isolates (27.0%) and highlights the concerning level of resistance, particularly to older antibiotics. These findings emphasize the importance of judicious antibiotic use and ongoing surveillance.

Aim: Extended-spectrum beta-lactamases (ESBL) play an important role in the development of resistance to beta-lactam antibiotics in Gram-negative bacteria. ESBL-producing bacteria can cause outbreaks, especially in hospital infections, which can lead to inadequate treatment, prolonged hospital stay, and increased mortality rates. This study aimed to investigate the antibiotic resistance profiles and ESBL production rates in Escherichia coli and Klebsiella spp. species isolated from various clinical samples in a tertiary care hospital. Materials and Methods: A total of 315 bacterial isolates were included in the study between September 2022-January 2023. 213 of them were E.coli (67.6%) and 102 were Klebsiella spp. (32.4%). Antibiotic susceptibilities of the isolates; ampicillin, amoxicillin-clavulanate, piperacillin/tazobactam, cefuroxime, cefixime, cefotaxime, gentamicin, amikacin, ciprofloxacin, trimethoprim-sulfamethoxazole, imipenem and meropenem were tested. ESBL production was evaluated with a double disk synergy test. The results were interpreted according to EUCAST criteria. Results: ESBL positivity rate was determined as 35.2% in E.coli isolates and 52.9% in Klebsiella spp. isolates. This rate was found as 22% in pediatric patients and 42% in adult patients. When evaluated according to gender, ESBL positivity was found to be significantly higher in male patients. According to clinical sample types, ESBL positivity was seen most in endotracheal aspirate and wound samples. According to the disk diffusion test, the highest resistance was detected against beta-lactam group antibiotics and the lowest resistance was detected against carbapenems. Conclusion: ESBL production continues to be a significant resistance problem in hospital infections. This study draws attention to the importance of detecting ESBL-positive isolates with the double disk synergy test. Incorrect and unnecessary antibiotic use is one of the main risk factors for increasing ESBL positivity rates. Therefore, early detection of ESBL-positive bacteria, isolation precautions and reconsideration of antibiotic policies are of great importance.

Sappinia is a free-living amoeba, a single-celled living organism found in the environment. It has recently been attributed to being an opportunistic human pathogen. There are two known species of Sappinia: S. diploidea and S. pedata. The current study detected Sappinia species using morphological and genetic approaches in various environmental and clinical samples in the Thi-Qar province of southern Iraq. The study was conducted from February - September 2020; one hundred and two samples for this study were obtained from various environmental and clinical sources. PCR was carried out with a positive culture after the samples were cultivated on an NN-agar medium. Overall, the study showed that 53(51.96%) samples were positive on morphological characters as well as PCR analysis showed that only 47 (46.07%) ) of Sappinia morphologically positive samples were positive by using a specific primer. Sappinia spp. were observed in all types of samples of environmental and clinical sources except CSF, and clinical ear samples were negative. The current study showed two species of Sappinia that have similar morphology but differ in the diameter of trophozoite and cyst, number of nuclei, and cyst morphology. The current study will pave the way for additional epidemiological research by considering the presence of potential Sappinia species in various samples, whether from environmental or clinical sources, to better understand the function of Sappinia as a potential health danger to people. Keyword: Sappinia spp. Free-living amoeba, opportunistic amoeba, Thi-Qar, Iraq

As an infectious disease that poses a significant threat to the rapidly growing pig breeding industry, the detection of Haemophilus parasuis (HPS) is often compromised by various interfering substances present in the test sample during quantitative real-time PCR (qPCR). The rapid detection of HPS is important for the isolation of infectious pigs and their treatment. We designed and optimized a rapid qPCR test to detect the INFB gene of HPS in clinical and environmental samples on pig farms. The method was evaluated for its specificity, sensitivity, repeatability, anti-interference capability, and its ability to detect HPS in clinical samples. The results indicated that the method was specific for the detection of HPS when evaluated against pathogens and intestinal probiotics found in pig farms. By using a seven-fold dilution series of the recombinant plasmid DNA in triplicate, it was determined that the lowest limit of detection (LOD) for this method was less than 10 copies/µL. The results of inter-batch and intra-batch repeatability tests showed that the coefficient of variation (CV) was consistently below 1%. Furthermore, the impact of 14 endogenous and exogenous interfering substances on the Ct values detected by the HPS qPCR was found to be less than 5% when compared to the Ct values obtained in the absence of interfering substances. A total of 248 clinical samples were analyzed using the HPS qPCR, commercial kits, and corresponding national standards, yielding positive rates of 9.27%, 6.05%, and 9.27%, respectively. Notably, the positive and negative percent agreement between the detection method developed in this research and the national standard was 100%. These findings demonstrate that the established detection method is suitable for epidemiological research on HPS and for diagnosing clinical samples containing interfering substances, thereby providing essential technical support for the prevention and control of HPS.

L–Arabinose-based coordination polymers were synthesized by the oxidation of L–arabinose using warm HNO3 to form arabinaric acid and allowing the resulting arabinaric acid to react with the different metal ions such as zinc(II), lead(II), copper(II), iron(III), and chromium(III). Only zinc(II) and lead(II) ions produced crystalline coordination polymers suitable for analysis while copper(II) produced a gummy solid. Preliminary characterization of the coordination polymers was based on their solubility, melting points, UV–Visible spectra, and infrared spectra. The coordination polymers were insoluble in common organic solvents and in acidic aqueous solutions but soluble in moderately basic aqueous solutions (pH 12). The zinc(II) and lead(II) coordination polymers have melting points above 300°C while the copper(II) polymer melts from 218–225°C. UV–Visible spectra showed that these polymers have maximum absorption in the ultraviolet region. Zinc(II), lead(II), and copper(II) arabinarate coordination polymers had maximum absorbance at λmax = 217 nm, λmax = 240 nm, and λmax = 220 nm, respectively. The infrared spectra of the coordination polymers indicated the association of the arabinarate ligand with the metal ions. Introduction and incorporation of molecular species such as azobenzene and molecular iodine to the coordination polymers were also investigated. Infrared spectra obtained from the azobenzene inclusion experiments showed additional infrared absorption bands below 1000 cm –1 implying absorption of azobenzene within the polymers. Iodometric titrations also confirmed the inclusion of iodine in the coordination polymers. The potential of the coordination polymers to incorporate lead(II) ions in aqueous solutions was also investigated using column elution of different sample concentrations of lead(II). Atomic Absorption Spectrometric analysis illustrated significant lead reduction of up to 80.70 ppm of lead(II) on the eluted samples. Introduction Rapid industrialization in the modern world today causes severe pollution in the environment. This may be due to toxic, inorganic, or non-biodegradable materials released to the environment as waste products during industrial processes. Heavy metals, like lead, are among the most harmful of the environmental elemental pollutants. Lead contamination from excessive exposure has been found to cause several health hazards and long term detrimental effects. Prominent conventional processes for the removal of lead from aqueous mixtures, such as sludge separation, chemical precipitation, and electrochemical treatments are relatively expensive and can cause secondary pollutants (Manahan, 1993 as cited in Acabal et al., 2000). Due to the problems of pollution, “green” industrial productions of compounds and their derivatives based on renewable starting materials and biodegradable products are developed (Kiely, 2001). Carbohydrates are biodegradable, widely available, and generally economical. Hence, their study for chemical and industrial applications would be very sensible. Because of their ease in functionalization forming sugar derivatives, they could serve as valuable building blocks for potentially useful polymers (Abrahams et al., 2003). Heavy metal sequestering properties of certain types of these polymers provide an alternative and potentially economical treatment process for heavy metal removal and recovery.