Swab Samples Research Articles

Development and Validation of Duplex PCR Assay for Simultaneous Detection and Differentiation of Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry

Background: Mycoplasmosis caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically significant disease in both commercial and backyard poultry. The present work aimed at developing a duplex PCR assay for quick, accurate detection and differentiation of MG and MS and validating it for screening purpose. Methods: Duplex PCR assay was standardized with primers designed specific to the mgc2 gene for MG and hemagglutinin vlhA gene for MS with amplicon size of 150 bp and 787 bp, respectively. Validation of molecular detection of MG and MS with duplex PCR assay was performed with a total of 179 pooled oropharyngeal swab samples from backyard poultry flocks from different parts of India. Result: The duplex PCR assay showed, 133/179 (74.3%), 39/179 (21.78%) and 35/179 (19.55%) positivity for MG, MS and both MG and MS, respectively. Duplex PCR assay showed sensitivity of detection limit of 10-6 of 1 mg/ml of template DNA. Comparison of results of duplex PCR assay with single PCR of field samples showed perfect agreement between two assays (k= 0.90). The duplex PCR validated in the study would be useful in rapid screening/ surveillance, diagnosis and differentiation of MG and MS in field samples in cost-effective manner and to devise effective control strategies.

A Survey for Atypical Brucella Species in Wild-Caught Marine Toads (Rhinella marina) Using Qualitative PCR: A Pilot Study

Abstract The genus Brucella is a group of intracellular bacterial pathogens documented to cause brucellosis, a zoonotic disease of worldwide concern. In the past decade, reported cases of atypical Brucella species in amphibians have increased. These species are considered atypical based on their tropism, phenotypic characteristics, and phylogeny differences. Atypical Brucella species can be misdiagnosed due to their similarity to classical Brucella species and closely related genera. Recent literature has provided evidence for the zoonotic potential of atypical Brucella linked to amphibians. Therefore, understanding the prevalence of these atypical Brucella species in captive amphibian populations is significant for amphibian health, conservation, and public health. This pilot study evaluated the prevalence of atypical Brucella species in a population of marine toads (Rhinella marina) using a validated pan-Brucella qualitative PCR. Samples were analyzed from 56 asymptomatic wild-caught marine toads from Miami, FL, USA, using skin swab samples. Brucella DNA was not detected in any of the skin swab samples analyzed. This pilot study represents a viable non-invasive sampling technique and PCR assay for rapidly screening populations of amphibians for the presence of atypical Brucella species.

Integration of surface swab with optical microscopy for detection and quantification of bacterial cells from stainless-steel surfaces.

Swab sampling is a common method for recovering microbes on various environmental surfaces. Its successful application for a specific target depends on the proper swab method and the following detection assay. Herein, we evaluated critical factors influencing surface swab sampling, aiming to achieve the optimal detection and quantification performance of optical detection for bacterial cells on stainless-steel surfaces. Our results showed the recovery rate of Salmonella enterica (SE1045) cells from the 10×10cm2 stainless-steel surface reached up to 92.71±2.19% when using ammonia bicarbonate-moistened polyurethane foam swabs for gentle collection, followed by ultrasound-assisted release in NH4HCO3 solution. Among the six different foam swabs, the Puritan™ Sterile Large Foam Swab contributed the lowest background noise and highest recovery efficiency when integrated with the optical detection assay. Notably, our method exhibited a strong linear relationship (r2=0.9983) between the detected cell numbers and the theoretical number of SE1045 cells seeded on surfaces in the range of 104-107 Colony Forming Units (CFU), with a limit of detection of 7.2×104 CFU 100cm-2. This integration was completed within 2 h, exhibiting the applicable potential in various settings.

Advancing SARS‐CoV‐2 Variant Detection with High Affinity Monoclonal Antibodies and Plasmonic Nanostructure

AbstractA nanoplasmonic biosensor for the precise detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by leveraging advances in nanostructured sensing materials and highly selective monoclonal antibodies is presented. The sensor integrates plasmonic Au nanostructures optimized for surface‐enhanced Raman scattering (SERS), enhancing sensitivity through unique light interactions at the nanoscale. Coupled with exclusive antibodies that specifically target SARS‐CoV‐2 and its evolving variants, this sensor demonstrates remarkable selectivity and versatility. Validated with 270 clinical samples, it demonstrates a sensitivity of 98.9% and specificity of 100%. More importantly, the virus in nasopharyngeal swab samples collected over 3 years, marking the long‐term, large‐scale clinical validation of the nanoplasmonic biosensor for SARS‐CoV‐2 is been successfully detected. Furthermore, the integration of this sensor with face masks enables the detection of airborne SARS‐CoV‐2, highlighting its potential for routine management of coronavirus disease 2019 (COVID‐19).

Incidence and Antibiotic Resistance Patterns of Staphylococcus aureus and Pseudomonas aeruginosa Isolated from Fomites in Critical Units of General Hospital Katsina

Study’s Excerpt Investigation of the role of fomites in the transmission of nosocomial pathogens, specifically Staphylococcus aureus and Pseudomonas aeruginosa, within various units of General Hospital Katsina was carried out. The results reveal high contamination of fomites in the male ward, with significant bacterial colony counts, and the differing susceptibility profiles of S. aureus and P. aeruginosa to commonly used antibiotics. There is need for improved infection control and antibiotic stewardship in the region. Full Abstract Nosocomial infections, predominantly caused by Staphylococcus aureus and Pseudomonas aeruginosa, continue to be a major public health challenge, especially in developing countries, due to their associated morbidity and mortality. This study aimed to isolate and identify bacterial pathogens from fomites in the Accident and Emergency (A&E) unit, male ward, and female ward of General Hospital Katsina and to evaluate their antimicrobial susceptibility patterns. A total of 90 swab samples were collected from frequently touched items such as beddings, door handles, floor surfaces, scissors, and forceps across the three locations. Standard microbiological techniques were utilized for the isolation and identification of S. aureus and P. aeruginosa, followed by antibacterial susceptibility testing using the disc diffusion method. Bacterial growth was observed in 58 (64.4%) samples, with S. aureus accounting for 32 (55.2%) and P. aeruginosa for 26 (44.8%) of the isolates. The male ward exhibited the highest contamination levels, with a mean colony count of 725.25 CFU/plate, significantly higher than the female ward (487.67 CFU/plate) and the A&E unit (48 CFU/plate), as confirmed by a one-way ANOVA (p < 0.05). Chi-square analysis revealed no statistically significant association between bacterial species and hospital location (p = 0.823). The susceptibility testing showed that S. aureus isolates were generally susceptible to Gentamycin, Ciprofloxacin, and Pefloxacin but resistant to Amoxicillin. Conversely, P. aeruginosa displayed high resistance to Gentamycin and Septrin. Conclusively, these findings revealed the critical role of fomites in the transmission of pathogens and highlighted the need for enhanced infection control measures and targeted antibiotic stewardship programs to mitigate the risks associated with these infections.

PSVII-25 Contributions to bacterial diversity of the piglet pioneer microbiome

Abstract The establishment of the gastrointestinal tract microbiome of the neonate, or pioneer microbiome, can predetermine the health and performance of the newborn. Previous work has established that both maternal and environmental sources are capable of shifting the pioneer microbial composition. Despite this, proportional quantifications of the maternal and environmental factors that influence the bacterial diversity of the piglet pioneer microbiome have yet to be evaluated. Therefore, the objective of this study was to determine the contributions from both maternal and environmental origins on piglet pioneer bacterial diversity. Sterile swab samples were collected from farrowing crates prior to (EMPTYCRATE) and after gilts were housed (FULLCRATE; n = 5; Landrace x Yorkshire x Duroc), gilt vaginas at farrowing (BIRTHCANAL), and piglet rectums on d 0 (pre-suckling), 3, and 10 post-farrowing and at weaning (21.6 ± 1.0 d post-farrowing). Colostrum (COLOSTRUM) from a representative number of teats was also collected from each gilt at farrowing into sterile collection cups. All samples were stored at -80°C until subsequent analyses. Gene sequencing for microbiome analysis targeted the V4 hypervariable region of the 16S rRNA bacterial gene. Sequence quality was assessed utilizing DADA2, and taxon analysis and diversity metrics were processed in the qiime2 pipeline. Alpha diversity (within sample) metrics were analyzed with Kruskal-Wallis, and beta diversity (between sample) metrics were assessed with PERMANOVA. Alpha diversity analyses included Pielou’s evenness index (i.e., within sample distribution), Shannon’s diversity index (i.e., within sample richness and evenness), observed OTUs (i.e., within sample richness), and Faith’s diversity index (i.e. within sample richness and phylogeny). Pielou’s evenness index (P = 0.027) and Shannon’s diversity index (P = 0.086) indicated differences between EMPTYCRATE and FULLCRATE. However, no differences were detected between the environmental sources for observed OTUs or Faith’s diversity index. Piglet samples by day were different for observed OTUs (P = 0.002), Faith’s diversity index (P = 0.0002), and Shannon’s diversity index (P = 0.073), which all consider bacterial richness within the samples. Specifically, the piglet d 3 samples were the least for these three metrics; however, there were no differences in Pielou’s evenness index for all piglet by day samples. There were no differences for all alpha diversity metrics between BIRTHCANAL and COLOSTRUM. Beta diversity metrics included unweighted UniFrac (i.e., between sample bacterial presence or absence) and weighted UniFrac (i.e., between sample bacterial presence or absence and abundance). Evaluation between the maternal contributions BIRTHCANAL and COLOSTRUM indicated differences for both unweighted UniFrac (P = 0.011) and weighted UniFrac (P = 0.007). Bacterial diversity between the environmental origins EMPTYCRATE and FULLCRATE were also different for unweighted UniFrac (P = 0.012) and weighted UniFrac (P = 0.038). There were no differences in beta diversity between all piglet by day samples. These results indicate that both maternal and environmental sources contribute to changes in piglet pioneer microbial diversity during early neonatal life.

PSV-16 Evaluation of upper respiratory and fecal microbiomes in neonatal beef calves

Abstract There is a critical lack of information on the timing of colonization of bovine respiratory disease-related bacteria in the upper respiratory tract, potentially affecting calf health and disease onset. The objective of this study was to evaluate the upper respiratory tract and fecal microbiomes of neonatal beef calves during the first 24 h of life compared with their dams at the time of birth. Late-gestation commercial beef cows (n = 28) were assigned to individual, soil-surfaced pens (6.25 x 2.29 m) fitted with canvas tarps on shared fences to limit contact between unrelated pairs. Left nasal (LN), right nasal (RN), fecal, and vaginal swab samples were collected from cows at a single time point promptly after parturition. Swabs of the LN, RN, and rectum were collected from calves at birth (0 h), 6, 12, and 24 h post-parturition. Samples were flash-frozen and stored at -80°C until processing. DNA was extracted from swabs using Power Soil Pro kit. Extracted DNA was used to prepare DNA libraries for full-length 16S rRNA gene sequencing on a MinION Mk1C device. Sequenced reads were classified using Centrifuge and used in microbiome analyses conducted in R to estimate the changes in the microbiome according to animal type (cow versus calf), time of collection, and sample type (nasal, fecal, or vaginal). Upper respiratory microbiomes significantly differed (P < 0.005) between cows and their offspring at birth. In nasal microbiomes, animal type explained 12.6% (P = 0.002) and 18.5% (P = 0.001) of variance in beta diversity in left and right cavities, respectively. Animal type explained 42.1% of the beta diversity variance in fecal microbiomes (P = 0.001). Nasal microbiomes at birth were significantly more dispersed in dams compared with their calves in both the LN (P = 0.03) and RN (P = 0.03). Similarly, fecal microbiomes of dams were significantly more dispersed (P < 0.0001) than those of their offspring. There was a clear transition of the microbial communities associated with calf age in the upper respiratory and fecal niches. Calf age accounted for 2% (P = 0.02), 3.5% (P = 0.001), and 28% (P = 0.001) of the variance in the beta diversity of the LN, RN, and fecal microbiomes, respectively. After adjusting for calf age, the laterality of nasal microbiomes was not significantly (P = 0.682) associated with the composition of microbial communities. In conclusion, calf microbial communities were dissimilar to that of their dam at birth. Additionally, microbial community transition was evident with each subsequent sampling time point, potentially influenced by the presence of the dam.

PSIX-2 Skin microbial compositions differ among dog body sites and are affected by dietary probiotics

Abstract The study aimed to characterize the skin microbiota across four distinct body sites and investigate the impact of dietary probiotics on the skin microbiota of healthy adult dogs. Healthy adult dogs [n = 60; mean age = 6.2 ± 2.8 yr; mean body weight (BW) = 13.1 ± 4.0 kg] were randomly assigned into three treatment groups and fed for 60 d: a control kibble diet (CON; n = 20; 29.0% crude protein, 10.1% crude fat, 9.9% crude fiber, 7.1% ash, as-fed), CON supplemented with Bifidobacterium longum 548 (CON-B; n = 20), or CON supplemented with Bifidobacterium longum 548 and Lactobacillus rhamnosus 451 (CON-B-L; n = 20). All dogs were individually housed in a temperature- and humidity-controlled research facility. Protocols were approved by the facility’s Institutional Animal Care and Use Committee before the study. On d 60, skin swab samples were collected from the dorsal lumbar area, right axilla, right groin, and right ear concave pinnae of each dog using sterile swabs. These samples were placed into bead tubes and stored at -20°C until analysis. DNA extraction from skin swab samples was followed by sequencing the V4 region of 16S rRNA using Illumina sequencing on a MiSiq. Sequence data were processed through QIIME 2.0, and statistical analysis was performed using R 4.3.1. to test differences between body sites and diets, with a significance threshold set at P < 0.05. The predominant bacterial phyla observed across all body sites were Actinobacteria (40 ± 21.7%), Firmicutes (31.2 ± 17.9 %), Proteobacteria (17.9 ± 13.3%), and Bacteroidetes (6.6 ± 6.1%). Additionally, Acidobacteria, Gemmatimonadetes, and Verrucomicrobia were exclusive to the groin region. Considering data from all body sites, diets affected relative abundances of phyla and genera in dogs. At phylum level, dogs fed the CON-B diet displayed greater (P < 0.05) relative abundance of Actinobacteria compared with those on the CON diet. The CON-B diet also resulted in a greater (P < 0.05) relative abundance of Firmicutes than dogs on either the CON or CON-B-L diets. At genus level, dogs on the CON-B diet showed significantly greater (P < 0.05) relative abundance of Clostridium, Clostridiaceae unclassified, and Lachnospiraceae unclassified compared with the other diet groups. Dogs on the CON diet displayed a greater (P < 0.05) relative abundance of Dietzia compared with those on probiotic-supplemented diets. These findings underscore the variation in skin microbial composition across distinct body sites, contributing valuable data to limited information available on the skin microbiota of dogs. Moreover, consumption of probiotic influenced the skin microbial compositions in dogs, which warrant further studies to elucidate the relationship between probiotics, skin microbiota, and skin health in dogs.

Optical detection and enumeration of Escherichia coli and Salmonella enterica using a low-magnification light microscope

A rapid and cost-effective method for detecting bacterial cells from surfaces is critical to food safety, clinical hygiene, and pharmacy quality. Herein, we established an optical detection method based on a gold chip coating with 3-mercaptophenylboronic acid (3-MPBA) to capture bacterial cells, which allows for the detection and quantification of bacterial cells with a standard light microscope under low-magnification (10×) objective lens. Then, integrate the developed optical detection method with swab sampling to detect bacterial cells loading on stainless-steel surfaces. Using Salmonella enterica (SE1045) and Escherichia coli (E. coli OP50) as model bacterial cells, we achieved a capture efficiency of up to 76.0 ± 2.0 % for SE1045 cells and 81.1 ± 3.3 % for E. coli OP50 cells at 103 CFU/mL upon the optimized conditions, which slightly decreased with the increasing bacterial concentrations. Our assay showed good linear relationships between the concentrations of bacterial cells with the cell counting in images in the range of 103 -107 CFU/mL for SE1045, and 103 -108 CFU/mL for E. coli OP50 cells. The limit of detection (LOD) was 103 CFU/mL for both SE1045 and E. coli OP50 cells. A further increase in sensitivity in detecting E. coli OP50 cells was achieved through a heat treatment, enabling the LOD to be reduced as low as 102 CFU/mL. Furthermore, a preliminary application succeeded in assessing bacterial contamination on stainless-steel surfaces following integration with the approximately 40 % recovery rate, suggesting prospects for evaluating the bacteria from surfaces. The entire process was completed within around 2 h, costing merely a few dollars per sample. Considering the low cost of standard light microscopes, our method holds significant potential for practical industrial applications in bacterial contamination control on surfaces, especially in low-resource settings.

HomeRNA self-blood collection enables high-frequency temporal profiling of presymptomatic host immune kinetics to respiratory viral infection: a prospective cohort study.

Early host immunity to acute respiratory infections (ARIs) is heterogenous, dynamic, and critical to an individual's infection outcome. Due to limitations in sampling frequency/timepoints, kinetics of early immune dynamics in natural human infections remain poorly understood. In this nationwide prospective cohort study, we leveraged a Tasso-SST based self-blood collection and stabilization tool (homeRNA) to profile detailed kinetics of the presymptomatic to convalescence host immunity to contemporaneous respiratory pathogens. We enrolled non-symptomatic adults with recent exposure to ARIs who subsequently tested negative (exposed-uninfected) or positive for respiratory pathogens. Participants self-collected blood and nasal swabs daily for seven consecutive days followed by weekly blood collection for up to seven additional weeks. Symptom burden was assessed during each collection. Nasal swabs were tested for SARS-CoV-2 and common respiratory pathogens. 92 longitudinal blood samples spanning the presymptomatic to convalescence phase of eight SARS-CoV-2-infected participants and 40 interval-matched samples from four exposed-uninfected participants were subjected to high-frequency longitudinal profiling of 785 immune genes. Generalized additive mixed models (GAMM) were used to identify temporally dynamic genes from the longitudinal samples and linear mixed models (LMM) were used to identify baseline differences between exposed-infected (n = 8), exposed-uninfected (n = 4), and uninfected (n = 13) participant groups. Between June 2021 - April 2022, 68 participants across 26 U.S. states completed the study and self-collected a total of 691 and 466 longitudinal blood and nasal swab samples along with 688 symptom surveys. SARS-CoV-2 was detected in 17 out of 22 individuals with study-confirmed respiratory infection, of which five were still presymptomatic or pre-shedding, enabling us to profile detailed expression kinetics of the earliest blood transcriptional response to contemporaneous variants of concern. 51% of the genes assessed were found to be temporally dynamic during COVID-19 infection. During the pre-shedding phase, a robust but transient response consisting of genes involved in cell migration, stress response, and T cell activation were observed. This is followed by a rapid induction of many interferon-stimulated genes (ISGs), concurrent to onset of viral shedding and increase in nasal viral load and symptom burden. Finally, elevated baseline expression of antimicrobial peptides were observed in exposed-uninfected individuals. We demonstrated that unsupervised self-collection and stabilization of capillary blood can be applied to natural infection studies to characterize detailed early host immune kinetics at a temporal resolution comparable to that of human challenge studies. The remote (decentralized) study framework enables conduct of large-scale population-wide longitudinal mechanistic studies. This study was funded by R35GM128648 to ABT for in-lab developments of homeRNA and data analysis, a Packard Fellowship for Science and Engineering from the David and Lucile Packard Foundation to ABT, and R01AI153087 to AW.

Virulence Genes Prevalence and Enterobacterial Repetitive Intergenic Consensus‐PCR Profiles of Goose‐Derived Campylobacter jejuni Isolates

ABSTRACTCampylobacter jejuni is a causative agent of gastroenteritis in humans worldwide, and wild and domestic poultry carry of this bacterium in their gastrointestinal tract. Molecular studies to determine the pathogenicity, origin, and epidemiological relationships among C. jejuni isolates from poultry such as chicken, turkey, and goose consumed as human food are important for public health and infection control. This study aimed to investigate the prevalence of virulence genes and Enterobacterial Repetitive Intergenic Consensus (ERIC‐PCR) based genotyping of C. jejuni isolates obtained from goose cloacal swab samples. For this purpose, PCR analysis of flaA, racR, dnaJ, pldA, cadF, cdtC, ciaB, cdtB, cdtA, virB11, and wlaN virulence genes and ERIC‐PCR analysis of 50 C. jejuni isolates were performed. The emerged genetic profiles and antimicrobial resistance genes regarding the isolates were interpreted with the existing multi‐drug resistance (MDR) findings. Virulence gene positivity was detected as 88%, 84%, 82%, 82%, 80%, 80%, 72%, 30%, and 18% for flaA, racR, dnaJ, pldA, cadF, cdtC, ciaB, cdtB, and cdtA, respectively. VirB11 and wlaN genes were not detected among the C. jejuni isolates. Virulence genes‐based genotyping revealed that the C. jejuni isolates exhibited 22 profiles (A–V). As a result of ERIC‐PCR analysis, the C. jejuni isolates showed heterogeneous distribution, exhibiting 14 different ERIC‐PCR profiles (Cluster I [Cl‐I]–Cluster XIV [Cl‐XIV]). The MDR positivity was detected in 7 (14%) of the C. jejuni isolates. Tetracycline and ciprofloxacin were the antibiotics most frequently included in the MDR profiles. There was no clear correlation between ERIC‐PCR profiles, virulence gene profiles, and MDR profiles. However, isolates with triple‐MDR resistant to ampicillin, tetracycline, and ciprofloxacin showed significant heterogeneity in both ERIC‐PCR profile and virulence gene‐based genetic profile, all of which were positive for ciaB and flaA genes. These results indicate that carriage of the C. jejuni isolates with high gene prevalence and MDR profiles by geese may pose a risk for Campylobacter infections in humans.

A one-pot, one-step CRISPR platform (CRISPR-one) for nucleic acid detection: Application for the detection of Mycoplasma pneumoniae

The conventional CRISPR-mediated detection technologies typically involve two separate steps: target pre-amplification and CRISPR-mediated signal detection. This multi-step procedure is not only cumbersome but also prone to cross-contamination. Here, we propose a new CRISPR-mediated nucleic acid detection methodology, termed CRISPR-one (CRISPR-mediated testing in one-tube, one-pot). At a constant temperature, CRISPR-one carries out both multiple cross displacement amplification (MCDA) for target pre-amplification and CRISPR/AapCas12b-based cleavage for signal detection in a single reaction pot. The novel CRISPR-one technique is employed for detecting Mycoplasma pneumoniae (M. pneumoniae), a causative agent for community-acquired pneumonia in children. M. pneumoniae CRISPR-one targets the CARDS (community-acquired respiratory distress syndrome toxin) gene and performs at 60 ºC for 50 min with only basic instrumentation required. The results can be easily visualized through a real-time fluorescence instrument, with the entire process taking less than an hour. The lowest detection limit of M. pneumoniae CRISPR-one was 50 fg (∼55 copies) of genomic DNA per reaction, and no cross-reactivity with non-M. pneumoniae templates was observed. In clinical applications, the CRISPR-one assay demonstrated a high level of agreement with both the microfluidic chip method (Kappa value: 0.95) and real-time PCR (Kappa value: 0.93) when analyzing 135 oropharynx swab samples. There is no statistical difference in detection efficiency between our assay and other methods. Together, the CRISPR-one developed here offers a rapid, convenient, highly sensitive, and specific approach for pathogen detection (e.g., M. pneumoniae), serving as an invaluable tool for clinical diagnosis and epidemiologic surveillance, particularly in resource-limited laboratories.

Inducible clindamycin-resistant and biofilm formation in the Staphylococcus aureus isolated from healthcare worker’s anterior nasal carriage

ObjectiveThe purpose of this study is a new update on the resistance profile, Macrolide–Lincosamide–Streptogramin B resistance mechanisms and biofilm formation in the Staphylococcus aureus isolated from health care workers (HCWs) nasal carriage at a children’s teaching hospital in Babol (Northern Iran).ResultsA total of 143 non-repetitive nasal swab samples were collected from volunteers, where 53.8% (n; 77/143) were HCWs, 33.6% (n; 48/143) medical students, and 12.6% (n; 18/143) resident students. The prevalence of nasal carriers of S. aureus was 22.4% (n; 32/143), among them, 40.6% (n; 13/32) were identified as methicillin-resistant Staphylococcus aureus (MRSA( carriers. Antimicrobial susceptibility testing showed that erythromycin (68.8%, n; 22/32) and ciprofloxacin (15.6%, n; 5/32) had the highest and lowest resistance rate, respectively. The frequency of resistance genes in the strains was as follows; ermC (n; 17/32, 53.1%), ermA (n; 11/32, 34.4%), ermB (n; 6/32, 18.7%), ereA (n; 3/32, 9.4%). Moreover, 50.0% (n; 16/32), 28.1% (n; 9/32) and 21.8% (n; 7/32) of isolates were strongly, weakly and moderately biofilm producer, respectively. Macrolides-lincosamides-streptogramins B (MLSB) antibiotic resistance among S. aureus isolates from HCWs nasal carriage have found significant prevalence rates throughout the globe. It is crucial to remember that the development of biofilms and MLS B antibiotic resistance are both dynamic processes.

Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection.

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

The Occurrence of Chlamydia felis in Cats and Dogs in Hungary.

The World Health Organization (WHO) estimates that many human infections are zoonoses, creating a worldwide public health challenge. Among Chlamydia species, Chlamydia felis is the leading cause of conjunctivitis in cats and is a prominent zoonotic species. This study aimed to determine the occurrence and risk of chlamydiosis in cats and dogs in Szeged, Hungary, and surrounding areas. The total nucleic acids from conjunctival swab samples of symptomatic and asymptomatic animals were extracted using an automated nucleic acid extraction system. After that, DNA was amplified by pan-chlamydia PCR. Bacterial and fungal cultures were also performed to detect other microorganisms. Of the 93 animals, 32 (34.4%) were positive for pan-chlamydia PCR. The positivity rates were 33.3% (26/78) in cats and 40.0% (6/15) in dogs. Furthermore, the positivity rates were 37.2% (16/43) in the cat shelter, 42.4% (14/33) in the veterinary clinic, and 11.7% (2/17) in household pets. In total, 103 species were identified through culture-based examinations, including 97 (94.2%) bacterial and 6 fungal (5.8%) species. From both human and animal health perspectives, it is essential to have a detailed understanding of the circumstances of chlamydiosis, given the global impact of zoonotic diseases.

Molecular Detection of Bacterial vaginosis Isolated from Preterm Labor Patients in Wasit City, Iraq

The aim of this study was to evaluate the role of bacterial vaginosis in preterm labor. There is growing evidence that infections, particularly those that spread from the lower genital tract, might trigger preterm labor. One of the main causes of newborn morbidity and mortality is preterm birth. This is a cross-sectional study which was carried out in the labor facilities in local hospital in Wasit. A total 90 swab samples were collected by gynecologist from patients in Preterm and Full-term labor Bacteriological diagnosis was done using Molecular quantification methods have been reported recently, but the specific risk factors they might identify remain unclear. We carried our study on 90 pregnant women, divided into two groups. First group delivered preterm and other group at full term. There are 38 out of 45 women who had no bacterial infection in the group of full-term pregnancy compared to 19 out of 45 in the preterm group , so the incidence of BV was significantly more in preterm group. The commonest isolated pathogen was G. vaginalis, followed by Megasphaera and Atopobium vaginalis. More than one-third (37.8%) of patients with preterm delivery have Gardnerella infection when compared to only 6.7% of those who delivered at term , Around one-quarter (26.7%) of patients who delivered preterm were diagnosed with Megasphaera. Only 4.4% of full-term patients had positive tests for this infection and 22.2% of patients with preterm labour compared to 4.4% of full-term pregnancies diagnosed with Atopobium infection.

Distribution of Respiratory Infection Viruses in 2019-2020 Season and Determination of Oseltamivir Resistance of Influenza Viruses

Objective: This study aimed to determine the seasonal distribution of the factor data of viral agents determined by the multiplex PCR method in routine practice in patients admitted to the hospital with upper respiratory tract infection symptoms and to detect oseltamivir resistance in viruses that are positive for Influenza A (H1N1). Methods: Nasopharyngeal swab and bronchoalveolar lavage samples of 354 patients between the ages of 0-94 who were admitted to the Sakarya University Sakarya Training and Research Hospital with symptoms of upper respiratory tract infection between 12 September 2019 and 19 February 2020 were studied with the multiplex PCR method. Oseltamivir resistance was investigated in 11 samples selected from Influenza A (H1N1) positive samples by Sanger sequence analysis method. Results: One or more respiratory viruses were identified in 233 (66%) of 354 respiratory samples evaluated. Of these, 64 (27.5%) are Influenza A/H1N1, 4 (1.7%) are Influenza A/H3N2, 24 (10.3%) are Influenza B, 64 (27.5) are Respiratory. Syncytial Virus A/B (RSV A/B), 58 (24.9%) Rhinovirus/Enterovirus, 18 (7.7%) Adenovirus, 12 (5.2%) Human Metapneumo Virus (hMPV) ), 10 (4.3%) Bocavirus, 4 (1.7%) Parainfluenza 1, 7 (3%) Parainfluenza 3, 3 (1.3%) Parainfluenza 4, 5 (2.1%) were found to be Coronavirus HKU1 and 13 (5.6%) were found to be Coronavirus NL63. More than one factor was detected simultaneously in 45 of 233 positive samples (19.3%). Oseltamivir resistance was not found in any of the 11 influenza A/H1N1 positive samples. Conclusion: No oseltamivir resistance was observed in any of the Influenza A/H1N1 positive samples evaluated in this study. Periodic analysis of influenza A/H1N1 strains for oseltamivir resistance is necessary to guide empirical treatment.

Toward Analysis at the Point of Need: A Digital Microfluidic Approach to Processing Multi-Source Sexual Assault Samples.

Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short-tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non-sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12h post-coitus (PC), the new method identifies single-source (male) STR profiles. When applied to samples collected 24-72h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre-processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.

Nasopharyngeal carriage of Streptococcus pneumoniae among children aged 30 days to <60 months in Beijing and Shenzhen, China (2018–2021) during pneumococcal conjugate vaccine introduction and the coronavirus disease (COVID-19) pandemic

ObjectivesTo describe the carriage rate, serotype distribution, and antimicrobial susceptibility patterns of Streptococcus pneumoniae (S. pneumoniae) nasopharyngeal (NP) isolates among healthy children aged 30 days to <60 months in the cities of Beijing and Shenzhen during 2018–2021.MethodsA NP swab sample was collected among four annual cohorts of healthy children at routine well-child visits. S. pneumoniae was identified by culture, optochin sensitivity and bile solubility, serotypes determined by latex agglutination and Quellung, and antimicrobial susceptibility testing performed using E-test strips.ResultsS. pneumoniae NP carriage was 13.1% (645/4,911), with the highest S. pneumoniae carriage prevalence (15.3%) observed in 25 to <60 months. The carriage prevalence was 15.1% in children 13–24 months, 13.2% in children 7–12 months, and 8.2% in children 30 days to 6 months (P < 0.01). Living with siblings [20.0% vs. 9.4%: OR: 2.42 (95% CI: 2.05–2.87)] or attending day-care [31.8% vs. 11.3%: OR: 3.67 (95% CI: 2.94–4.57)] increased the risk (P < 0.01). During the period (January 2020–April 2021) of strict non-pharmaceutical interventions to prevent and control the COVID-19 pandemic, the proportion of children with S. pneumoniae colonization declined from 16.0% (94/587) to 5.8% (108/1,848) in Beijing while increasing from 14.5% (64/443) to 18.6% (379/2,033) in Shenzhen. Among S. pneumoniae isolates, 36.7% (237/645) belonged to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes, 64.3% (408/645) were non-PCV13 serotypes, including 20.8% (134/645) non-serotypeable S. pneumoniae (NST). A total of 158/644 isolates (24.5%) were MDR. For the PCV13 isolates, MDR was detected in 36.3% (86/237) of isolates; in comparison, 17.6% (72/407) of non-PCV13 serotypes, including NST, were MDR (P < 0.01). S. pneumoniae NP carriage was detected in 10.7% of children with previous pneumococcal vaccination (PCV7 or PCV13 only) compared with 14.9% in children without previous pneumococcal vaccination.ConclusionsThe highest S. pneumoniae carriage prevalence were found in the oldest age group (25 to <60 months) and in children living with siblings or attending day-care. Vaccination with PCV7 or PCV13 was associated with lower PCV13-serotype colonization. In Beijing, S. pneumoniae carriage significantly declined during the COVID-19 pandemic.

Microbiome resilience of three-toed box turtles (Terrapene carolina triunguis) in response to rising temperatures.

The gastrointestinal (GI) microbiome of chelonians (testudines) plays an important role in their metabolism, nutrition, and overall health but the GI microbiome of three-toed box turtles (Terrapene carolina triunguis) has yet to be characterized. How the GI microbiome responds to rapidly rising environmental temperatures has also not been studied extensively in ectotherms, specifically chelonians. In this study, twenty (20) T.c.triunguis were split into control and experimental groups. The experimental group experienced 4.5°C increases every two weeks while the control group stayed at a constant ambient temperature (24°C) through the entirety of the experiment. Before each temperature increase, all turtles had cloacal swab samples taken. These samples underwent DNA extraction followed by 16S rRNA gene sequencing and microbial community analyses. Differences in diversity at the community level in the controls compared to the experimental groups were not statistically significant, indicating microbiome resilience to rapid temperature changes in T.c.triunguis, although some differentially abundant lineages were identified. Interestingly, an amplicon sequence variant belonging to the Erysipelothrix spp. was exclusively enriched in the highest temperature group relative to controls. Overall, our work suggests that there may be an innate robustness to rapid temperature swings in the microbiome of T.c.triunguis which are native to temperate North America. Despite this resilience, Erysipelothrix spp. was enriched at the highest temperature. Phylogenetic analysis of this amplicon variant showed that it is a close relative of Erysipelothrix rhusiopathiae, a pathogen of zoonotic importance associated with both wildlife and livestock.