Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Inferential properties with a novel two parameter Poisson generalized Lindley distribution with regression and application to INAR(1) process.

Based on the well-known Poisson (P) distribution and the new generalized Lindley distribution (NGLD) developed by using gamma (α,θ) and gamma (α-1,θ) distributions, a new compound two-parameter Poisson generalized Lindley (TPPGL) distribution is proposed in this paper and thereon systematically explores the mathematical properties. Closed form expressions are assembled for such properties including the probability generating function, moments, skewness, kurtosis, etc. The likelihood-based method is used for estimating the parameters followed by a broad Monte Carlo simulation study. To further motivate the proposed model, a count regression model and a first order integer valued autoregressive process are constructed based on the novel TPPGL distribution. The empirical importance of the proposed models is confirmed through application to four real datasets.

Application of MALDI-TOF mass spectrometry, and DNA sequencing-based SLST and MLST analysis for the identification of Cronobacter spp. isolated from environmental surveillance samples.

Cronobacter spp. are emerging infectious foodborne bacteria that can cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. Although, little is known about its reservoirs or transmission routes, it has been linked to powdered infant formula worldwide. Three Cronobacter spp. (C. sakazakii, C. malonaticus, and C. turicensis) have been described as more virulent, and isolated frequently from infant meningitis cases. The estimated mortality rates are as high as 80% in infants. Thus, surveillance and typing of Cronobacter spp. isolated from food and environmental samples is essential to prevent contamination and spread of this pathogen. In this study, we have characterized 83 Cronobacter isolates recovered from various environmental samples by conventional microbiologic protocols. Species identification was accomplished by VITEK 2 system and real-time PCR analysis. Subsequently, these isolates were analyzed using VITEK MS system. Single locus sequence typing (SLST) was achieved by characterizing the regions of 16S rRNA and rpoB genes. Multilocus sequence typing (MLST) was performed by sequence characterization of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. VITEK MS system identified, the majority of isolates as Cronobacter sakazakii with a high confidence value (99.9%). MLST analysis ascertained 12 distinct clonal complexes (CC1, CC4, CC8, CC13, CC17, CC21, CC31, CC40, CC52, CC64, CC73, and CC83) for the recovered C. sakazakii isolates. The results suggest that the MALDI-TOF MS is a reliable diagnostic tool for rapid species identification whereas 7-loci MLST is a powerful technique to discriminate and differentiate Cronobacter spp. isolates.

Infant wheezing and prenatal antibiotic exposure and mode of delivery: a prospective birth cohort study.

Introduction: Assessments on whether prenatal antibiotic exposure and mode of delivery increase the risk of wheezing in infants and toddlers are inconsistent. Our goal is to evaluate the association between prenatal antibiotic use and Cesarean section with three subtypes of wheezing in infancy.Methods: An ongoing prospective three generations cohort study provides data on prenatal antibiotic use and mode of delivery. Respective questionnaire data was used to distinguish three subtypes of wheezing: any wheezing, infectious wheezing, and noninfectious wheezing. Repeated measurements of wheezing at 3, 6, and 12 months were analyzed using generalized estimation equations. Latent transition analysis assessed patterns of infant wheezing development in the first year of life.Results: The prevalence of any wheezing was highest at 12 months (40.1%). The prevalence of infectious wheezing was higher (3 months 23.8%, 6 months 33.5%, 12 months 38.5%) than of noninfectious wheezing (3 months 13.0%, 6 months 14.0%, 12 months 11.1%). About 11-13% of children had both infectious and noninfectious wheezing at 3, 6, and 12 months (3 months 10.7%, 6 months 13.9%, 12 months 13.1%). Children born via Cesarean section have approximately a 70-80% increase in the risk of any wheezing (RR = 1.83, 95% CI 1.29-2.60) and of infectious wheezing (RR = 1.72, 95% CI 1.18-2.50).Conclusions: Analyses of infectious and noninfectious wheezing subtypes suggests that children born by Cesarean sections may be more susceptible to infectious wheezing, warranting investigations into microbial factors of infectious wheezing. No significant associations were found between prenatal antibiotic exposure and wheezing types.

Passive Source Localization Using Compressive Sensing.

This paper presents an underwater passive source localization method by forming an underdetermined linear inversion problem. The signal strength on a specified grid is evaluated using sparse reconstruction algorithms by exploiting the spatial sparsity of the source signals. Our strategy leads to a high ratio of measurements to sparsity (RMS), an increase in the peak sharpness with a low side lobe level, and minimization of the dimensionality of the problem due to the formulation of the system equation of the multiple snapshots based on the data correlation matrix. Furthermore, to reduce the computational burden, pre-locating with Bartlett is presented. Our proposed technique can perform close to Bartlet and white noise gain constraint processes in the single-source scenario, but it can give slightly better results while localizing multiple sources. It exhibits the respective characteristics of traditionally used Bartlett and white noise gain constraint methods, such as robustness to environmental/system mismatch and high resolution. Both the simulated and experimental data are processed to demonstrate the effectiveness of the method for underwater source localization.

A Comparative Evaluation Study of Growth Conditions for Culturing the Isolates of Campylobacter spp.

Campylobacter is one of the leading causes of foodborne travelers' diarrhea worldwide. Although a large number cases of campylobacteriosis go undiagnosed or unreported, it is considered as the second most common foodborne illness in the USA affecting over 1.3 million individuals every year. Of various Campylobacter species, C. jejuni, C. coli, and C. lari have been accounted for causing more than 99% of human infections. Thus, there is a need to have efficient isolation method to protect public health on food safety and monitoring the burden of campylobacteriosis. Nevertheless, it is a challenging task as the exposure of environmental stress during isolation process makes Campylobacter species less culturable. Sixteen Campylobacter spp. were used to evaluate the current protocols used in Campylobacter isolation. For optimal recovery, a range of growth media (Bolton, Columbia, Muller Hinton, CVA Campy and mCCDA), incubation temperatures, and additional supplements (including antibiotics) were tested. Blood agars without antibiotics were sufficient for the initial recovery. Afterward, the isolates could grow on agars without any supplements, and in some cases growth was observed in the presence of antibiotics. Incubation at 37 °C was found to be the optimal temperature for the recovery and the growth of most species. Additionally, a food adulteration study was also carried out by artificially contaminating three food matrices that included egg, milk, and infant cereal, with two isolates of C. jejuni and C. coli. Results of this study should provide the insight for culturing and isolation of Campylobacter from food and other sources.

Evaluation of Cronobacter Growth and Phenotypic Variation Under Modified Culture Conditions.

Cronobacter sakazakii is an opportunistic pathogen known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. It has been isolated from a wide range of food and environmental samples, and has been linked to outbreaks associated with powdered infant formula. This study was carried out to assess variations in growth conditions (temperature, pH, and sugar supplement) and to establish how these changes impact phenotypic characteristics for successful recovery and identification of Cronobacter, particularly for routine surveillance purposes. A total of six Cronobacter isolates were tested to evaluate the above growth conditions, including three ATCC Cronobacter reference and three environmental isolates obtained from regulatory sample screening. Although only slight changes in colony-forming units were observed across the pH range and the sugars tested, the morphology was significantly impacted by changes in these growth factors. Incubation between 30 and 50 °C resulted in growth after 24 h, and the growth was slower at ambient temperature and colony formation was most robust at 30 °C. Results of this study suggest that 30 °C may be suitable for recovery of some Cronobacter strains, and minor variations in growth conditions can alter colony morphology and appearance. Expression of unique biological characteristics based on phenotypic observations may be beneficial for differentiating various Cronobacter strains.

Genetic Characterization of Cronobacter sakazakii Recovered from the Environmental Surveillance Samples During a Sporadic Case Investigation of Foodborne Illness.

Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness.

Threshold levels of 25-hydroxyvitamin D and parathyroid hormone for impaired bone health in children with congenital ichthyosis and type IV and V skin.

BACKGROUND: Patients with congenital ichthyosis, especially those with darker skin types, are at increased risk of developing vitamin D deficiency and rickets. The relationships between 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH) and bone health have not been studied previously, in ichthyosis. OBJECTIVES: To determine the threshold levels of 25(OH)D and PTH for impaired bone health in children with congenital ichthyosis. METHODS: In this cross-sectional study, 119 children with ichthyosis and 168 controls were recruited. Serum 25(OH)D, PTH, calcium, phosphate and alkaline phosphatase (ALP) were measured. Radiological screening for rickets was carried out only in children with ichthyosis. RESULTS: Forty-seven children with ichthyosis had either clinical or radiological evidence of rickets. The correlation between serum 25(OH)D and PTH showed that a serum level of 25(OH)D 8 ng mL(-1) was associated with a significant increase in PTH. The correlation between PTH and ALP showed that a serum PTH level of 75 pg mL(-1) was associated with a significant increase in ALP levels. Of the different clinical phenotypes of ichthyosis, both autosomal recessive congenital ichthyosis (ARCI) and epidermolytic ichthyosis (EI) were found to have significantly increased PTH, ALP and radiological rickets scores compared with common ichthyosis. CONCLUSIONS: Serum levels of 25(OH)D ≤ 8 ng mL(-1) and PTH ≥ 75 pg mL(-1) significantly increases the risk for development of rickets [odds ratio (OR) 2·8; 95% confidence interval (CI) 1·05-7·40; P = 0·04] in ichthyosis. Among the different types, patients with ARCI (OR 4·83; 95% CI 1·74-13·45; P < 0·01) and EI (OR 5·71; 95% CI 1·74-18·79; P < 0·01) are at an increased risk of developing rickets.

RVOT pseudoaneurysm post biventricular repair for tetralogy of Fallot with single pulmonary artery.

Pseudoaneurysm of the right ventricular outflow tract (RVOT), post repair for tetralogy of Fallot (TOF), is a rare occurrence with few cases reported in literature. TOF with single pulmonary artery is in itself a rare occurrence. RVOT pseudoaneurysm in a case of TOF with single pulmonary artery has not been reported to the best of our knowledge. RVOT pseudoaneurysm is a catastrophic complication which has very few symptoms and has to be picked up early to avoid dire consequences. We have reported such a rare occurrence to highlight the importance of looking out for such complications in rare presentations where anatomy is altered. Supplementary information: The online version contains supplementary material available at 10.1007/s12055-023-01558-9.

Cytokine and matrix metalloproteinase expression in fibroblasts from peri-implantitis lesions in response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. METHODS: Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. RESULTS: Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1ß, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1ß, MCP-1, and MMP-1 compared to periodontitis fibroblasts. CONCLUSIONS: Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.

Indoor Bacterial and Fungal Burden in "Moldy" versus "Non-Moldy" Homes: A Case Study Employing Advanced Sequencing Techniques in a US Metropolitan Area.

The presence of fungi in the indoor environment is associated with allergies and other respiratory symptoms. The aim of this study was to use sequencing and molecular methods, including next-generation sequencing (NGS) approaches, to explore the bacterial and fungal communities and their abundance in the indoor environment of houses (n = 20) with visible "moldy" (HVM) and nonvisible "non-moldy" (HNM) in Memphis, TN, USA. Dust samples were collected from air vents and ground surfaces, and the total DNA was analyzed for bacteria and fungi by amplifying 16S rRNA and ITS genes on the Illumina Miseq. Results indicated that Leptosphaerulina was the most abundant fungal genus present in the air vent and ground samples from HNM and HVM. At the same time, the most abundant bacterial genera in the air vent and ground samples were Propionibacterium and Streptococcus. The fungi community diversity was significantly different in the air vent samples. The abundance of fungal species known to be associated with respiratory diseases in indoor dust samples was similar, regardless of the visibility of fungi in the houses. The existence of fungi associated with respiratory symptoms was compared with several parameters like dust particulate matter (PM), CO2 level, temperature, and humidity. Most of these parameters are either positively or negatively correlated with the existence of fungi associated with respiratory diseases; however, none of these correlations were significant at p = 0.05. Our results indicate that implementing molecular methods for detecting indoor fungi may strengthen common exposure and risk assessment practices.

A Single-Laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

BACKGROUND: Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains has caused a major health care burden worldwide. Cronobacter is a group of Gram-negative bacteria that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp., and Vibrio spp. is crucial for the source tracking of contaminated food, as well as to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. OBJECTIVE: This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like isolates of public health importance. METHOD: A total of 226 isolates recovered from various food, environmental surveillance samples, and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates, whole genome sequencing (WGS) and DNA sequencing of 16S rRNA partial region were also performed for species identification. RESULTS: The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system were comparable to data obtained by the VITEK MS system. CONCLUSIONS: The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates and bacterial isolates from additional sources, in most cases. HIGHLIGHTS: The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

Synthesis and evaluation of 2-phenylamino-1,4-naphthoquinones derivatives as potential hypoglycaemic agents.

Due to the severe side effects revealed by most of the currently used antidiabetic medicines, search for finding new and safe drugs to manage diabetes is continued. Naphthoquinones possessing strong antioxidant properties have been employed as candidates for diabetes therapy. Present study is aimed at finding the antioxidant and hypoglycaemic potential of some novel derivatives of 2-phenylamino-1,4-naphthoquinones (PAN) including chloro, nitro, methyl and bromo (5a-d) derivatives synthesized by single pot experiment. Product crystals were purified by TLC and characterized by FT-IR. The antioxidant potential of the compounds was assayed through DPPH radical scavenging and reducing power activities noted as UV-vis. absorbance. The DPPH assay has showed the powerful antioxidant activity of nitro and bromo derivatives, while the nitro derivative showed the significant reduction potential towards FRAP assay. Hypoglycaemic potential of the compounds was studied in rat animal model. All synthesized compounds revealed better hypoglycaemic activity; however, the chloro-derivative exhibited the more potent hypoglycaemic activity showing about 43% reduction in the mean blood glucose levels of the treated animals. As the bioreduction of naphthoquinones may be influenced by changing its redox properties, it has been noticed that the e-donating resonance effect (+R) of 'chloro' group has shown the significant effects on biological activity through stabalization of its imine form which limits the potential of generation of free radicals during bioreduction of quinones and thus has been proposed as the reason of its hypoglycaemic activity. Future studies employing the properties of e-donating groups of PAN may optimize the drug-receptor interaction for better drug designing and drug development strategies against diabetes and also for the clinical trials.

MALDI-TOF Mass Spectrometry and 16S rRNA Gene Sequence Analysis for the Identification of Foodborne Clostridium Spp.

BACKGROUND: Clostridium is a genus of Gram-positive, spore-forming, anaerobic bacteria comprising approximately 100 species. Some Clostridium spp. (C. botulinum, C. perfringens, C. tetani, and C. difficile) have been recognized to cause acute food poisoning, botulism, tetanus, and diarrheal illness in humans. Thus, rapid identification of Clostridium spp. is critical for source-tracking of contaminated food and to understand the transmission dynamics of these foodborne pathogens. OBJECTIVE: This study was carried out to rapidly identify Clostridium-like isolates by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and rRNA sequencing methods. METHOD: Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using the VITEK MS system. Sequence characterization of the 16S rRNA region was done on an ABI 3500xL Genetic Analyzer. RESULTS: The VITEK MS system identified 28 of the 33 Clostridium-like isolates with a high confidence value (99.9%); no identification was observed for the remaining five isolates. Nucleotide sequencing of the 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, 11 distinct Clostridium spp. (Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, and Clostridium subterminale) were recognized among the 33 Clostridium-like isolates. One of the Clostridium-like isolates was identified as Citrobacter amalonaticus by both diagnostic methods. The generated 16S rRNA sequences matched completely (100%) with sequences available in GenBank for Clostridium and Citrobacter species. Species identification attained using the VITEK MS for the Clostridium-like isolates was comparable to that from the 16S rRNA sequencing-based data. CONCLUSIONS: The VITEK MS and 16S rRNA sequence analysis can be implemented in the species identification of Clostridium spp. isolates of public health importance. HIGHLIGHTS: MALDI-TOF MS and 16S rRNA sequencing can be used in the species identification of Clostridium species.

HCV-genotypes: a review on their origin, global status, assay system, pathogenecity and response to treatment.

The present report is a review article on various aspects of Hepatitis C virus (HCV) genotypes and their subtypes. HCV has six genotypes and several subtypes showing important epidemiological and clinical implications. The information based on previous studies and presented through this article highlight the origin, classification and causes of genetic diversity, global status, detection assays, pathogenicity and response to treatment of HCV-genotypes. The six genotypes differ in 30-35% of nucleotide sites over the complete genome. The difference in genomic composition of sub-types of genotype is usually found to be 20-25%. The variability remains more frequent in structural genes as compared to non-structural or untranslated genes. Both genotypes and their sub-types show a varied prevalence globally and raise several issues related to their transmission and treatment of HCV-infection. All this information has a great significance while planning future strategies for eradication and therapeutic management of HCV. In addition, these reports produce a further scope for more studies to unravel the mystery behind HCV-genotypes and formulate guidelines to resolve this public health problem noted worldwide.

Application of Ribosomal Internal Transcribed Spacer 1, Internal Transcribed Spacer 2, and Large-Subunit D1-D2 Regions as the Genetic Markers to Identify Fungi Isolated from Different Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

BACKGROUND: In September 2012, a multistate fungal meningitis outbreak started across 20 states in the United States. It affected 753 individuals and caused 64 deaths who received contaminated spinal injections. In a previous study, we analyzed 26 environmental samples collected from the manufacturing premises of a compounding company to determine the possible cause of an outbreak and identified 14 distinct fungal species. OBJECTIVES: In this follow-up study, we have analyzed 198 environmental samples collected from three additional compounding company premises located in the United States for the presence of pathogenic fungi. METHODS: Environmental swab samples were initially examined by standard microbiological methods. Subsequently, DNA sequencing was performed on all of the 25 recovered fungal isolates at the D1-D2 domain of the large subunit (LSU) ribosomal RNA (rRNA) and the internal transcribed spacer (ITS) regions. RESULTS: Sequence analysis of the ITS1, ITS2, and LSU rRNA regions confirmed the presence of the following fungal species in the environmental samples analyzed: (i) Pestalotiopsis cocculi from the region Ia; (ii) Epicoccum nigrum and Trichaptum biforme from the region Ib; (iii) Nigrospora sphaerica and Fusarium sp. from the region II; and (iv) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Species identification of 25 recovered fungal isolates matched, in most cases, at 3 sequenced loci (ITS1, ITS2, and LSU). HIGHLIGHTS: DNA sequencing of ITS1, ITS2, and LSU D1-D2 regions can be used to perform fungal typing and in implementing effective environmental monitoring programs of public health importance.

Species Identification of Campylobacter jejuni and Campylobacter coli Isolates from Raw Poultry Products by MALDI-TOF MS and rRNA Sequence Analysis.

BACKGROUND: Campylobacter is a genus of Gram-negative bacteria. It is considered one of the leading cause of diarrheal illness worldwide. Incidence of human campylobacteriosis has increased significantly during recent decades; the increase has been directly linked with the advancement made in Campylobacter detection methods. C. jejuni and C. coli are the two major human-pathogenic species that can colonize poultry and cause foodborne illness and outbreak. OBJECTIVE: This study was carried out to rapidly identify Campylobacter-like isolates recovered from raw poultry products by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS and DNA sequencing of 16S and 23S ribosomal RNA (rRNA) regions. METHODS: Twenty-seven isolates of Campylobacter-like organisms were isolated from raw poultry products and cultured on blood agars for MALDI-TOF MS analysis and DNA isolation. For each isolate, one to two colonies were directly spotted on the VITEK MS analysis. Genomic DNA was extracted from overnight bacterial culture. Afterward, two-directional Sanger sequencing of rRNA gene was performed to confirm species identification on an ABI 3500xL Genetic Analyzer. RESULTS: The VITEK MS could provide species-level identification for all 27 Campylobacter-like bacterial isolates (including 13 isolates as C. jejuni and 14 isolates as C. coli). Species identification attained by the VITEK MS matched completely with the rRNA sequence characterization data. CONCLUSIONS: Based on the restricted number of target strains and agar plates, MALDI-TOF MS and rRNA sequence analysis can be used for rapid identification of C. jejuni and C. coli isolates. HIGHLIGHTS: MALDI-TOF MS and rRNA sequencing can provide species identification of C. jejuni and C. coli isolates.

Association of Virulence and Antibiotic Resistance in Salmonella-Statistical and Computational Insights into a Selected Set of Clinical Isolates.

The acquisition of antibiotic resistance (AR) by foodborne pathogens, such as Salmonella enterica, has emerged as a serious public health concern. The relationship between the two key survival mechanisms (i.e., antibiotic resistance and virulence) of bacterial pathogens is complex. However, it is unclear if the presence of certain virulence determinants (i.e., virulence genes) and AR have any association in Salmonella. In this study, we report the prevalence of selected virulence genes and their association with AR in a set of phenotypically tested antibiotic-resistant (n = 117) and antibiotic-susceptible (n = 94) clinical isolates of Salmonella collected from Tennessee, USA. Profiling of virulence genes (i.e., virulotyping) in Salmonella isolates (n = 211) was conducted by targeting 13 known virulence genes and a gene for class 1 integron. The association of the presence/absence of virulence genes in an isolate with their AR phenotypes was determined by the machine learning algorithm Random Forest. The analysis revealed that Salmonella virulotypes with gene clusters consisting of avrA, gipA, sodC1, and sopE1 were strongly associated with any resistant phenotypes. To conclude, the results of this exploratory study shed light on the association of specific virulence genes with drug-resistant phenotypes of Salmonella. The presence of certain virulence genes clusters in resistant isolates may become useful for the risk assessment and management of salmonellosis caused by drug-resistant Salmonella in humans.

Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates.

BACKGROUND: The antifungal drug resistance has become an emerging problem in the management of candida infections worldwide. The objective of this study was to examine the efficacy of epigallocatechin 3-O-gallate (EGCG) alone and in combination with fluconazole/ketoconazole drugs against oral Candida isolates. METHODS: Minimum inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) of EGCG against 60 oral Candida isolates and 4 ATCC strains were determined. Synergism of EGCG with azole drugs was evaluated by checkerboard micro-dilution method and calculated fractional inhibitory concentration index (FICI). Candida cells' ultrastructure was studied by electron microscopy. RESULTS: MIC and MFC values of EGCG were in the range of 3.91-15.63 and 15.63-31.25µg/mL, respectively. Minimum biofilm inhibitory concentration (MBIC) range of EGCG (62.5-125µg/mL), was less than the ketoconazole (64-256µg/mL) and fluconazole (128-512µg/mL). The combination of EGCG with fluconazole/ketoconazole exhibited synergistic effects (ΣFICI≤0.50). EGCG with azole drugs showed high sensitivity against the tested isolates in growth curve assays. Against the biofilm, the susceptibility of fluconazole/ketoconazole significantly increased (3 to 5 fold), after combination with EGCG (MBIC/4) (P≤0.001). Electron microscopy of EGCG treated cells showed deformation of cell structure, ruptured cell wall and release of intracellular content. In molecular docking experiments, a strong interaction was observed between EGCG and fungal cell membrane molecule ergosterol. CONCLUSION: We conclude that EGCG synergistically enhanced the antifungal potential of azole drugs. The synergistic potential of EGCG might be helpful in preventing the development of drug resistance, in lowering the drug dosage, and thus minimizing adverse effects.