The effect of COVID-19 on cancer incidences in the U.S.

Fundamental data analysis assists in the evaluation of critical questions to discern essential facts and elicit formerly invisible evidence. In this article, we provide clarity into a subtle phenomenon observed in cancer incidences throughout the time of the COVID-19 pandemic. We analyzed the cancer incidence data from the American Cancer Society [1]. We partitioned the data into three groups: the pre-COVID-19 years (2017, 2018), during the COVID-19 years (2019, 2020, 2021), and the post-COVID-19 years (2022, 2023). In a novel manner, we applied principal components analysis (PCA), computed the angles between the cancer incidence vectors, and then added lognormal probability concepts in our analysis. Our analytic results revealed that the cancer incidences shifted within each era (pre, during, and post), with a meaningful change in the cancer incidences occurring in 2020, the peak of the COVID-19 era. We defined, computed, and interpreted the exceedance probability for a cancer type to have 1000 incidences in a future year among the breast, cervical, colorectal, uterine corpus, leukemia, lung & bronchus, melanoma, Hodgkin's lymphoma, prostate, and urinary cancers. We also defined, estimated, and illustrated indices for other cancer diagnoses from the vantage point of breast cancer in pre, during, and post-COVID-19 eras. The angle vectors post the COVID-19 were 72% less than pre-pandemic and 28% less than during the pandemic. The movement of cancer vectors was dynamic between these eras, and movement greatly differed by type of cancer. A trend chart of cervical cancer showed statistical anomalies in the years 2019 and 2021. Based on our findings, a few future research directions are pointed out.

Examining the Effect of Contactless Intergenerational Befriending Intervention on Social Isolation Among Older Adults and Students' Attitude Toward Companionship: Content Analysis.

BACKGROUND: Intergenerational friendship, a mechanism of social support, is an effective intervention to reduce the increasing risk of social isolation (SI) and develop companionship in the older adult population. The COVID-19 pandemic provided a unique opportunity to examine the psychosocial intervention of befriending via technology use as a primary form of contactless socialization. OBJECTIVE: The study aims to explore the effectiveness of the befriending intervention through a contactless, intergenerational service-learning project on older adult emotions, especially boredom and loneliness as the key attributes of SI, and on students' attitude toward companionship. METHODS: During the months of January to April 2022 , undergraduate students enrolled in a health administration course with a special focus on culture were asked to be involved in a contactless, intergenerational service-learning project (n=46). In this study, contactless intervention meant communication using the telephone and apps such as FaceTime and Zoom. Students were paired with older adults to have at least a 30-minute weekly conversation, for 8 weeks, via telephone or an internet-based app such as FaceTime. Students were asked to write a half-page diary after each interaction and a 1-page reflection at the end of the fourth week and at the end of the service-learning project. At the completion of the project, the researchers also surveyed the older adults to assess the impact of the project using a 5-item open-ended questionnaire. Following a heuristic approach and content analysis, student artifacts (110,970 words; 118-page, single-spaced Microsoft Word document) and the older adult surveys were analyzed using MAXQDA, (VERBI GmbH). Qualitative data were extracted to assess the impact of service learning on SI by measuring the attributes of boredom and loneliness among 46 older adults. Students' attitudes toward companionship were also assessed using data from their diaries and reflections. RESULTS: Overall, three major constructs were identified: (1) meaningful engagement, defined as feeling safe, having increased confidence, and having reduced boredom; (2) internal motivation to participate in the weekly interaction, defined as discussion about daily life experience, level of happiness, and ability to exert personal control over the situation; and (3) intergenerational befriending, defined as perceived benefits from the friendly nature of the interaction, ability to comfortably connect with students, and positive feeling and attitude toward the student. CONCLUSIONS: The contactless, intergenerational befriending intervention reduced boredom and loneliness among older adults and enhanced positive attitude and confidence among university students. Students helped older adults to develop digital skills for the use of apps and social media. Older adults showed interest in the intervention and shared their daily life experiences with the students, which helped to reduce the gap between generations. Findings indicate the effectiveness of an intergenerational service-learning intervention on SI reduction and increased positive attitude among college students.

Mechanism of fungal remediation of wetland water: Stropharia rugosoannulata as promising fungal species for the development of biofilters to remove clinically important pathogenic and antibiotic resistant bacteria in contaminated water.

Mycoremediation uses mushroom forming fungi for remediation of sites contaminated with biotic and abiotic contaminants. The root-like hyphae of many fungi, the mycelia, have been used to remediate soil and water. In this study mushroom mycelia biofilters were evaluated for remediation efficacy of wetland water polluted with crow feces containing antibiotic resistant (AMR) bacteria. Three strains of fungi, Pleurotus ostreatus, Stropharia rugosoannulata, and Pleurotus pulmonarius, were allowed to develop dense mycelia for 3-5 weeks on wood chips within cylindrical jars. Biofilter jars were incubated with wetland water (WW) obtained from a crow roost area that was additionally spiked with AMR bacteria isolated from previous crow fecal collections. E. coli, Staphylococcus aureus, Enterococcus faecium, Campylobacter jejuni, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enteritidis were added at concentrations of 1,500-3,500 CFU/100 ml. Remediation was calculated from bacterial counts or gene copy numbers (GCN), before and after passage of water through jars. Stropharia and P. pulmonarius biofilters remediated all bacteria, but Klebsiella, in the range of 43-78%, after 1 h. Incubation of water for 24 h showed Stropharia remediation to be superior relative to other tested fungi. Percent remediation varied as follows: S. aureus-100%, E. faecium-97%, C. jejuni-59%, P. aeruginosa-54%, E. coli-65% and S. enteritidis-27%. The mechanism of remediation was tested by removing the mycelium from the biofilter column after passage of water, followed by extraction of DNA. Association of bacterial DNA with the mycelia was demonstrated by qPCR for all bacteria, except S. aureus and Salmonella. Depending on the bacteria, the GCN ranged from 3,500 to 54,000/250 mg of mycelia. Thus, some of the ways in which mycelia biofilters decrease bacteria from water are through bio-filtration and bio-absorption. Active fungal growth and close contact with bacteria appear necessary for removal. Overall these results suggest that mushroom mycelia biofilters have the potential to effectively remediate water contaminated with pathogenic and AMR bacteria.

An Exploratory Analysis of the Association Between Hospital Labor Costs and the Quality of Care.

Introduction: The competent healing touch of a caregiver is a critical component to the care patients receive. The more skilled the provider, the higher the likelihood outcomes will be delivered in a safe and effective manner. Unfortunately, in recent years, hospitals in the United States have faced immense financial pressures that are threatening their economic sustainability and patients' access to care in the future. Through the recent COVID-19 pandemic, the cost of delivering healthcare has continued to escalate, while the demand for patient care has exceeded many hospitals' capacity. Most troubling is the impact that the pandemic has had on the healthcare workforce, which has resulted in many hospitals struggling to fill vacancies at ever-increasing cost - all while under immense pressure to deliver quality patient care. What remains uncertain is whether the increase in labor costs has been matched with a commensurate rise in the quality of care or if quality has deteriorated as the labor force mix has changed to include more contract and temporary staff. Thus, in the enclosed study, we sought to determine what association, if any, exists between hospitals' cost of labor and the quality of care delivered. Methods and Models: Drawing from a representative national sample of nearly 3214 short-term acute care hospitals' common quality measures from the year 2021, we examined the labor cost-quality relationship via multivariate linear and logistic regression and found there is a persistent negative association across all quality outcome variables studied. Discussion: These findings lead us to believe simply paying more for hospital labor does not, by itself, ensure a positive patient outcome. We contend cautious use of temporary staff, measured adoption of short-term financial incentives, and robust staff development all should be considered as features of future workforce planning.

Leveraging mHealth and Virtual Reality to Improve Cognition for Alzheimer's Patients: A Systematic Review.

BACKGROUND: Alzheimer's Disease (AD) is a global problem affecting 58 million people, expected to reach a prevalence of 88 million people by 2050. The disease affects the brain, memory, cognition, language, and motor movement. Many interventions have sought to improve memory and cognition. mHealth and virtual reality (VR) are two such interventions. OBJECTIVES: To analyze studies from the last 10 years with older adults with AD to ascertain the effectiveness of telehealth techniques such as mHealth and VR for memory care. METHODS: In accordance with the Kruse Protocol and reported in accordance with PRISMA 2020, five reviewers searched four research databases (PubMed, CINAHL, Web of Science, and ScienceDirect) on 3 August 2022 for studies with strong methodologies that fit the objective statement. RESULTS: Twenty-two studies from 13 countries were analyzed for trends. Four interventions (mHealth/eHealth, VR, mHealth + VR, game console, and telephone) used RCT, quasi-experimental, pre-post, observational, and mixed methods. These interventions improved cognition, memory, brain activity, language, depression, attention, vitality, quality of life, cortical atrophy, cerebral blood flow, neuro plasticity, and mental health. Only three interventions reported either no improvements or no statistically significant improvements. Cost, time, training, and low reimbursement were barriers to the adoption of these interventions. CONCLUSION: mHealth and VR offer interventions with positive effectiveness for memory care for AD. The long-term effect of this improvement is unclear. Additional research is needed in this area to establish clinical practice guidelines.

Assessing the Effectiveness of Intergenerational Virtual Service-Learning Intervention on Loneliness and Ageism: A Pre-Post Study.

BACKGROUND: Service-learning is an effective intervention to solve social issues. The purpose of this study is to assess the effectiveness of intergenerational virtual service-learning on loneliness and ageism. METHOD: This study used a pre-post design. A group of undergraduate students were randomly assigned to a "service-learning" project (n = 18). They were paired with seniors (n = 22) to have at least a 30-min weekly virtual interaction for six weeks. The following scales were used: the Aging Semantic Differential (ASD) Scale, the UCLA Loneliness Scale, a one-item researcher generated Likert-rating of loneliness, and two-item researcher generated Likert-rating of student competence. RESULTS: Among college students, the service-learning group showed lower ASD and ageism scores at the post-test compared to the non-service-learning group, t (1, 40) = -2.027, p = 0.049; t (1, 40) = -2.102, p = 0.042, respectively. Among seniors, loneliness scores on the UCLA Scale and the one-item scale of loneliness dropped significantly from pre- to post-interactions with students, t (1, 19) = 2.301, p = 0.033, and t (1, 22) = 2.412, p = 0.009, respectively. CONCLUSION: Virtual service-learning is an effective way to solve social issues such as loneliness and ageism.

Mechanisms of Social Interaction and Virtual Connections as Strong Predictors of Wellbeing of Older Adults.

Socially engaged older adults are less likely to decline in health and happiness and have a higher quality of life. Building upon this premise, examination was conducted on the domains of social determinants of health, specifically the social and community context per Healthy People 2030 objectives. These mechanisms of social interaction, in the form of group activities, community engagement, and virtual interactions via email or text message, were assessed using hierarchical regression analysis to find out their association with wellbeing, depression symptoms, and cognition of older adults. The data included a total of 4623 sample of older adults from the National Health and Aging Trend Study (NHATS) Round 8. The results showed that social support explained a 40.3% unique variance on wellbeing. The use of text message and email had a moderating effect on community engagement and self-reported depression level in older adults. Findings suggest that community programs, shared group activities, or technology training workshops can improve social interaction and support cognition and reduce depression in older adults. Directions for future research include examining human behaviors and perceptions and increasing technology training sessions to promote independence of older adults and increase their social connections. In addition, participant involvement in interventions would enhance the possibility of success of such endeavors.

The use of digital technology for social wellbeing reduces social isolation in older adults: A systematic review.

This systematic review emphasizes the need for technology use in older adults to reduce social isolation. With the advancement of technology over the years, the effectiveness of interventions based on its use can be examined to see how these can address the problem of social isolation and enhance social wellbeing. We focus on identifying how older adults can most benefit from affordable and accessible technology use and how the training and implementation of such interventions can be tailored to maximize their beneficial effect. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to select relevant studies. We analyzed 25 articles, performed a narrative analysis to identify themes, and quality of life indicators connected to technology use and wellbeing. Engagement of older adults at the community-level, following best practices from the Community-Based Participatory Research can facilitate effective practices to deliver technology based social isolation interventions and increase digital use self-efficacy in older adults. Mobile technology-based applications not only help families to stay connected, but also link older adults to resources in healthcare and encourage physical and mental well-being. Use of technology devices address cognitive, visual, and hearing needs, and increase digital use self-efficacy in older adults, particularly helpful during necessary social distancing or self-quarantine during the COVID-19 pandemic.

Genotypic analyses and antimicrobial resistance profiles of Campylobacter jejuni from crows (Corvidae) of United States and India reflect their respective local antibiotic burdens.

AIM: The study examined the hypothesis that crow-borne Campylobacter can function as environmental reservoirs and indicators of antibiotic resistance (AR) determinants circulating in a human population. METHODS AND RESULTS: Two species of crows from Washington (WA), United States, and Kolkata, India, respectively, were examined for their ability to carry antibiotic resistant Campylobacter. Campylobacter jejuni was the only species isolated by selective agar plating from crow faecal samples. Disk diffusion method used to compare the AR profile of the isolates showed tetracycline (TET) resistance to be the most prevalent (27%) among WA isolates, followed by ciprofloxacin (CIP; 24%). Among Kolkata isolates, nalidixic acid resistance was most common (36%), followed by CIP (27%). The AR profile demonstrated by crow isolates of WA reflects those reported by the US National Antimicrobial Resistance Monitoring System for human isolates (2007-2011), where resistance to TET was most prevalent (≈45%), followed by quinolones (≈24%). The Kolkata crow isolates reflected the AR profile of human clinical isolates from India, where 97% resistance was shown to quinolones, followed by TET (18%). Multilocus sequence typing of 37 isolates, including 11 water isolates from the crow roost area, showed 24 different sequence types (STs). Seventeen of these were previously found in wild birds, 2 in human diarrhoea, 4 in poultry and 8 in environmental water. One isolate was found in both water and faeces, though from different sites within WA. CONCLUSIONS: The results indicate that crows most likely acquire the AR from anthropogenic sources. Although they are colonized by specific STs, rarely isolated from humans, they can facilitate the spread of AR. SIGNIFICANCE AND IMPACT OF THE STUDY: By studying two areas in different continents, this research demonstrates that Campylobacter borne by crows can function as environmental reservoirs and indicators of AR determinants that circulate in a human population. This information will be of importance to scientists from the medical and poultry industries.

American Crows as Carriers of Extra Intestinal Pathogenic E. coli and Avian Pathogenic-Like E. coli and Their Potential Impact on a Constructed Wetland.

The study examines whether crows are carriers of extraintestinal pathogenic E. coli (ExPEC) and avian pathogenic E. coli (APEC)-like strains, and if wetland roost areas contribute to their spread. A total of 10 crow feces (n = 71) and 15 water E. coli isolates (n = 134) from a wetland area could be characterized as potentially ExPEC based on the presence of ≥2 of the five cardinal genes iutA, kpsMT2, papEF, pap A/C, papG, sfa/foc, and afa/dra, while six fecal and 14 water isolates could be characterized as potentially APEC-like based on the presence of plasmid associated genes: iutA, episomal iss, ompT, hlyF and iroN. A total of 32 fecal and 27 water isolates tested carried plasmids based on incompatibility typing. Plasmids from 34 of 38 isolates tested could be transferred to another E. coli strain by conjugation with the antibiotic resistance (AR) profile being transferred, indicating their potential to be transferred to indigenous and non-pathogenic strains in the wetland. APEC-like plasmids could be transferred in six of eight isolates tested. Pathogenic E. coli of importance to the medical community and poultry industry may be detected in high levels in surface water due to corvid activity. Regardless of their role in health or disease, water in wetlands and streams can serve as a media for the dissemination of AR and virulence traits of bacteria, with corvids acting as potential vectors for farther dissemination.

Emergence of Genetic Diversity and Multi-Drug Resistant Campylobacter jejuni From Wild Birds in Beijing, China.

Campylobacter jejuni (C. jejuni) is considered as an opportunistic zoonotic pathogen that may cause gastroenteritis in humans and other animals. Wild birds may be as potential vectors of C. jejuni around urban and suburban areas. Here, 520 samples were collected from 33 wild bird species in urban and suburban areas, Beijing. In total 57 C. jejuni were isolated from seven species. It was found that Nineteen (33.33%, 19/57) isolates were resistant to at least one of 11 antibiotics, especially streptomycin (36.84%) and four isolates resistant to all. Nineteen (33.33%, 19/57) isolates were multi-drug resistance. Multilocus sequence typing (MLST) analysis of the isolates showed that 36 different sequence types (STs) belonged to four Clonal complexes and unassigned. Twenty STs (55.56%) and six alleles among them were first detected. Virulence genes including flaA, cadF, and the cytolethal distending toxin (CDT) gene cluster, were detected in all isolates, but truncated cdt gene clusters only detected in the isolates from the crow, daurian jackdaw and silver pheasant. In conclusion, it was the first detection of C. jejuni involved truncated cdt gene clusters from the silver pheasant. These wild birds around urban and suburban areas may pose potential public health problems as reservoir vectors of C. jejuni.

Antibiotic Resistance of E. coli Isolated From a Constructed Wetland Dominated by a Crow Roost, With Emphasis on ESBL and AmpC Containing E. coli.

Information on the dissemination of antibiotic resistance mechanisms in the environment as well as wild life is needed in North America. A constructed wetland (where ∼15,000 American crows roost) was sampled on the University of Washington Bothell Campus for the presence of antibiotic resistant E. coli (ARE). Crow droppings from individual birds and grab samples of water were collected in 2014-2015. E. coli were isolated by selective agar plating. The most frequent antibiotic resistance (AR) of the fecal isolates was to ampicillin (AMP) (53%), followed by amoxicillin-clavulanic acid (AMC) (45%), streptomycin (S) (40%), and nalidixic acid (NA) (33%). Water isolates had similar AR pattern and ∼40% were multidrug resistant. Isolates from water samples collected during storm events showed higher resistance than isolates from no rain days to tetracycline, AMP, AMC, NA, and gentamycin. Extended spectrum beta lactamase (ESBL) containing E. coli with the bla ctx-M was found in three water and nine fecal isolates while bla cmy-2 in 19 water and 16 fecal isolates. Multilocus Sequence Typing analysis (MLST) yielded 13 and 12 different sequence types (STs) amongst fecal and water isolates, many of which could be correlated to livestock, bird, and humans. MLST identified ESBL E. coli belonging to the clinically relevant ST131 clone in six fecal and one water isolate. Three STs found in feces could be found in water on the same dates of collection but not subsequently. Thus, the strains do not appear to survive for long in the wetland. Phylogenetic analysis revealed similar distribution of the water and fecal isolates among the different phylo-groups, with the majority belonging to the commensal B1 phylo-group, followed by the pathogenic B2 phylo-group. This study demonstrates that corvids can be reservoirs and vectors of ARE and pathogenic E. coli, posing a significant environmental threat.

Campylobacter jejuni Colonization in the Crow Gut Involves Many Deletions within the Cytolethal Distending Toxin Gene Cluster.

Campylobacter spp. are major causes of gastroenteritis worldwide. The virulence potential of Campylobacter shed in crow feces obtained from a roost area in Bothell, Washington, was studied and compared with that from isolates from other parts of Washington and from a different crow species 7,000 miles away in Kolkata, India. Campylobacter organisms were isolated from 61% and 69% of the fecal samples obtained from Washington and Kolkata, respectively, and were confirmed to be C. jejuni The cytolethal distending toxin (CDT) gene cluster from these isolates revealed a truncated sequence of approximately 1,350 bp. Sequencing of the gene cluster revealed two types of mutations: a 668-bp deletion across cdtA and cdtB and a 51-bp deletion within cdtB Some strains had additional 20-bp deletions in cdtB In either case, a functional toxin is not expected; a functional toxin is produced by the expression of three tandem genes, cdtA, cdtB, and cdtC Reverse transcriptase PCR with total RNA extracted from the isolates showed no expression of cdtB A toxin assay performed with these isolates on HeLa cells failed to show cytotoxic effects on the cells. However, the isolates were able to colonize the chicken ceca for a period of at least 4 weeks, similar to that of a clinical isolate. Other virulence gene markers, flagellin A and CadF, were present in 100% of the isolates. Our study suggests that crows carry the bacterium C. jejuni but with a dysfunctional toxin protein that is expected to drastically reduce its potential to cause diarrhea.IMPORTANCE Campylobacters are a major cause of gastroenteritis in humans. Since outbreaks have most often been correlated with poultry or unpasteurized dairy products, contact with farm animals, or contaminated water, historically, the majority of the studies have been with campylobacter isolates from poultry, domestic animals, and human patients. However, the bacterium has a broad host range that includes birds. These reservoirs need to be investigated, because the identification of the source and a determination of the transmission routes for a pathogen are important for the development of evidence-based disease control programs. In this study, two species of the human-commensal crow, from two different geographical regions separated by 7,000 miles of land and water, have been examined for their ability to cause disease by shedding campylobacters. Our results show that the crow may not play a significant role in campylobacteriosis, because the campylobacter organisms they shed produce a nonfunctional toxin.

Ubiquity, diversity and physiological characteristics of Geodermatophilaceae in Shapotou National Desert Ecological Reserve.

The goal of this study was to gain insight into the diversity of culturable actinobacteria in desert soil crusts and to determine the physiological characteristics of the predominant actinobacterial group in these crusts. Culture-dependent method was employed to obtain actinobacterial strains from desert soil samples collected from Shapotou National Desert Ecological Reserve (NDER) located in Tengger Desert, China. A total of 376 actinobacterial strains were isolated and 16S rRNA gene sequences analysis indicated that these isolates belonged to 29 genera within 18 families, among which the members of the family Geodermatophilaceae were predominant. The combination of 16S rRNA gene information and the phenotypic data allowed these newly-isolated Geodermatophilaceae members to be classified into 33 "species clusters," 11 of which represented hitherto unrecognized species. Fermentation broths from 19.7% of the isolated strains showed activity in at least one of the six screens for antibiotic activity. These isolates exhibited bio-diversity in enzymatic characteristics and carbon utilization profiles. The physiological characteristics of the isolates from different types of crusts or bare sand samples were specific to their respective micro-ecological environments. Our study revealed that members of the family Geodermatophilaceae were ubiquitous, abundant, and diverse in Shapotou NDER, and these strains may represent a new major group of potential functional actinobacteria in desert soil.

Development of a sensitive detection method for stressed E. coli O157:H7 in source and finished drinking water by culture-qPCR.

A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

Effects of prolonged chlorine exposures upon PCR detection of Helicobacter pylori DNA.

The effect of low doses of free chlorine on the detection of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. Detection of sequences targeted to the ureA gene from preparations containing 107 cells/ml decreased about 2-4 logs by days 9 and 14, respectively. When duplicate suspensions of the 107 cells/ml were exposed to higher levels of chlorine, 0.2-2.2 mg/l, by day 9 and 14 there were 5 and 6 log decreases, respectively, in the detection of ureA gene. H. pylori target sequences (within suspended, intact cells at densities of 102-103 cells /ml) were rendered undetectable by qPCR analysis after 17 h of continuous exposure to low chlorine levels common to treated drinking water distribution systems. The persistence of DNA sequences within treated distribution systems detectable by qPCR may be as brief as 17 h especially for bacteria such as H. pylori which are known to occur in very low numbers within treated distribution systems. This study suggests that degradation of H. pylori DNA target sequences by chlorine levels commonly found within treated water distribution systems occurs within the average water retention times (2-3 days) commonly found in these systems.

Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water.

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

Importance of flagella and enterotoxins for Aeromonas virulence in a mouse model.

A genetic characterization of eight virulence factor genes, elastase, lipase, polar flagella (flaA/flaB, flaG), lateral flagella (lafA), and the enterotoxins alt, act, and ast, was performed using polymerase chain reaction with 55 drinking water and nine clinical isolates. When 16 Aeromonas hydrophila strains, seven Aeromonas veronii strains, and seven Aeromonas caviae strains exhibiting different combinations of virulence factor genes were tested in immunocompromised mice by intraperitoneal injection, only those strains that had one or more of the enterotoxins flaA, flaB, and either flaG or lafA showed signs of being virulent. The correlation was seen in 97% (29/30) of the strains, which included strains from drinking water. Thus, Aeromonas water isolates have the potential to be pathogenic in immunocompromised hosts.

Development of a rapid identification method for Aeromonas species by multiplex-PCR.

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.