Pathogens, Volume 4, Issue 2 (June 2015) – 15 articles , Pages 157-421

IL-17-producing Th17 cells are of critical importance in host defense against oropharyngeal candidiasis (OPC). Speculation about defective Th17 responses to oral C. albicans infection in the context of HIV infection prompted an investigation of innate and adaptive immune responses to Candida albicans in transgenic mice expressing the genome of HIV-1 in immune cells and displaying an AIDS-like disease. Defective IL-17 and IL-22-dependent mucosal responses to C. albicans were found to determine susceptibility to OPC in these transgenic mice. Innate phagocytes were quantitatively and functionally intact, and individually dispensable for control of OPC and to prevent systemic dissemination of Candida to deep organs. CD8+ T-cells recruited to the oral mucosa of the transgenic mice limited the proliferation of C. albicans in these conditions of CD4+ T-cell deficiency. Therefore, the immunopathogenesis of OPC in the context of HIV infection involves defective T-cell-mediated immunity, failure of crosstalk with innate mucosal immune effector mechanisms, and compensatory cell responses, which limit Candida infection to the oral mucosa and prevent systemic dissemination. Full article

Major waterborne (enteric) pathogens are relatively well understood and treatment controls are effective when well managed. However, water-based, saprozoic pathogens that grow within engineered water systems (primarily within biofilms/sediments) cannot be controlled by water treatment alone prior to entry into water distribution and other engineered water systems. Growth within biofilms or as in the case of Legionella pneumophila, primarily within free-living protozoa feeding on biofilms, results from competitive advantage. Meaning, to understand how to manage water-based pathogen diseases (a sub-set of saprozoses) we need to understand the microbial ecology of biofilms; with key factors including biofilm bacterial diversity that influence amoebae hosts and members antagonistic to water-based pathogens, along with impacts from biofilm substratum, water temperature, flow conditions and disinfectant residual—all control variables. Major saprozoic pathogens covering viruses, bacteria, fungi and free-living protozoa are listed, yet today most of the recognized health burden from drinking waters is driven by legionellae, non-tuberculous mycobacteria (NTM) and, to a lesser extent, Pseudomonas aeruginosa. In developing best management practices for engineered water systems based on hazard analysis critical control point (HACCP) or water safety plan (WSP) approaches, multi-factor control strategies, based on quantitative microbial risk assessments need to be developed, to reduce disease from largely opportunistic, water-based pathogens. Full article

Opportunistic premise plumbing pathogens are responsible for a significant number of infections whose origin has been traced to drinking water. These opportunistic pathogens represent an emerging water borne disease problem with a major economic cost of at least $1 billion annually. The common features of this group of waterborne pathogens include: disinfectant-resistance, pipe surface adherence and biofilm formation, growth in amoebae, growth on low organic concentrations, and growth at low oxygen levels. Their emergence is due to the fact that conditions resulting from drinking water treatment select for them. As such, there is a need for novel approaches to reduce exposure to these pathogens. In addition to much-needed research, controls to reduce numbers and human exposure can be instituted independently by utilities and homeowners and hospital- and building-operators. Full article

Controlling organic carbon levels in municipal water has been hypothesized to limit downstream growth of bacteria and opportunistic pathogens in premise plumbing (OPPPs). Here, the relationships between influent organic carbon (0–15,000 µg ozonated fulvic acid /L) and the number of total bacteria [16S rRNA genes and heterotrophic plate counts (HPCs)] and a wide range of OPPPs (gene copy numbers of Acanthamoeba polyphaga, Vermamoeba vermiformis, Legionella pneumophila, and Mycobacterium avium) were examined in the bulk water of 120-mL simulated glass water heaters (SGWHs). The SGWHs were operated at 32–37 °C, which is representative of conditions encountered at the bottom of electric water heaters, with water changes of 80% three times per week to simulate low use. This design presented advantages of controlled and replicated (triplicate) conditions and avoided other potential limitations to OPPP growth in order to isolate the variable of organic carbon. Over seventeen months, strong correlations were observed between total organic carbon (TOC) and both 16S rRNA gene copy numbers and HPC counts (avg. R² > 0.89). Although M. avium gene copies were occasionally correlated with TOC (avg. R² = 0.82 to 0.97, for 2 out of 4 time points) and over a limited TOC range (0–1000 µg/L), no other correlations were identified between other OPPPs and added TOC. These results suggest that reducing organic carbon in distributed water is not adequate as a sole strategy for controlling OPPPs, although it may have promise in conjunction with other approaches. Full article

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. Full article

Waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. These diseases are directly related to environmental deterioration and pollution. Despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. Proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. Powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. Quantitative microbial risk assessment (QMRA) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. This review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. This review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of QMRA approach to protect public health. Full article

Arenaviruses include multiple human pathogens ranging from the low-risk lymphocytic choriomeningitis virus (LCMV) to highly virulent hemorrhagic fever (HF) causing viruses such as Lassa (LASV), Junin (JUNV), Machupo (MACV), Lujo (LUJV), Sabia (SABV), Guanarito (GTOV), and Chapare (CHPV), for which there are limited preventative and therapeutic measures. Why some arenaviruses can cause virulent human infections while others cannot, even though they are isolated from the same rodent hosts, is an enigma. Recent studies have revealed several potential pathogenic mechanisms of arenaviruses, including factors that increase viral replication capacity and suppress host innate immunity, which leads to high viremia and generalized immune suppression as the hallmarks of severe and lethal arenaviral HF diseases. This review summarizes current knowledge of the roles of each of the four viral proteins and some known cellular factors in the pathogenesis of arenaviral HF as well as of some human primary cell-culture and animal models that lend themselves to studying arenavirus-induced HF disease pathogenesis. Knowledge gained from these studies can be applied towards the development of novel therapeutics and vaccines against these deadly human pathogens. Full article

To examine the impact of environmental factors on Legionella in drinking water distribution systems, the growth and survival of Legionella under various conditions was studied. When incubated in tap water at 4 °C, 25 °C, and 32 °C, L. pneumophila survival trends varied amongst the temperatures, with the stable populations maintained for months at 25 °C and 32 °C demonstrating that survival is possible at these temperatures for extended periods in oligotrophic conditions. After inoculating coupons of PVC, copper, brass, and cast iron, L. pneumophila colonized biofilms formed on each within days to a similar extent, with the exception of cast iron, which contained 1-log less Legionella after 90 days. L. pneumophila spiked in a model drinking water distribution system colonized the system within days. Chlorination of the system had a greater effect on biofilm-associated Legionella concentrations, with populations returning to pre-chlorination levels within six weeks. Biofilms sampled from drinking water meters collected from two areas within central Arizona were analyzed via PCR for the presence of Legionella. Occurrence in only one area indicates that environmental differences in water distribution systems may have an impact on the survival of Legionella. These results document the impact of different environmental conditions on the survival of Legionella in water. Full article

Aichi virus 1 (AiV-1), belonging to the genus Kobuvirus in the family Picornaviridae, has been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. AiV-1 is globally distributed and has been detected in various types of environmental samples, such as sewage, river water, groundwater, and shellfish. Recent environmental studies revealed that this virus could be detected in higher frequency and greater abundance than other human enteric viruses. These findings suggest that AiV-1 could potentially be an appropriate indicator of viral contamination in the environment because of its high prevalence in water environments as well as structural and genetic similarity with some of the other important enteric viruses. Further studies on the occurrence and fate of AiV-1 in environments, even in combination with clinical studies of many regions, are needed for a better understanding of their epidemiology, temporal and geographical distribution, environmental stability, and potential health risks to humans. Full article

Group A rotaviruses (RV) are the major cause of acute gastroenteritis in infants and young children globally. Waterborne transmission of RV and the presence of RV in water sources are of major public health importance. In this paper, we present the Global Waterborne Pathogen model for RV (GloWPa-Rota model) to estimate the global distribution of RV emissions to surface water. To our knowledge, this is the first model to do so. We review the literature to estimate three RV specific variables for the model: incidence, excretion rate and removal during wastewater treatment. We estimate total global RV emissions to be 2 × 10¹⁸ viral particles/grid/year, of which 87% is produced by the urban population. Hotspot regions with high RV emissions are urban areas in densely populated parts of the world, such as Bangladesh and Nigeria, while low emissions are found in rural areas in North Russia and the Australian desert. Even for industrialized regions with high population density and without tertiary treatment, such as the UK, substantial emissions are estimated. Modeling exercises like the one presented in this paper provide unique opportunities to further study these emissions to surface water, their sources and scenarios for improved management. Full article

Serratia marcescens is a Gram negative bacterium (Enterobacteriaceae) often associated with infection of insects. In order to find pathogenic bacteria with the potential to control scarab larvae, several bacterial strains were isolated from the hemocoel of diseased Phyllophaga spp (Coleoptera:Scarabaeidae) larvae collected from cornfields in Mexico. Five isolates were identified as Serratia marcescens by 16S rRNA gene sequencing and biochemical tests. Oral and injection bioassays using healthy Phyllophaga blanchardi larvae fed with the S. marcescens isolates showed different degrees of antifeeding effect and mortality. No insecticidal activity was observed for Spodoptera frugiperda larvae (Lepidoptera: Noctuidae) by oral inoculation. S. marcescens (Sm81) cell-free culture supernatant caused significant antifeeding effect and mortality to P. blanchardi larvae by oral bioassay and also mortality by injection bioassay. Heat treated culture broths lost the ability to cause disease symptoms, suggesting the involvement of proteins in the toxic activity. A protein of 50.2 kDa was purified from the cell-free broth and showed insecticidal activity by injection bioassay towards P. blanchardi. Analysis of the insecticidal protein by tandem- mass spectrometry (LC-MS/MS) showed similarity to a Serralysin-like protein from S. marcescens spp. This insecticidal protein could have applications in agricultural biotechnology. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 10³ CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 10⁵. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens. Full article

Staphylococcus aureus is one of the top five pathogens contributing to acquired foodborne illnesses causing an estimated quarter million cases every year in the US. The objectives of this study were to determine the prevalence of Methicillin Susceptible S. aureus (MSSA) and Methicillin Resistant S. aureus (MRSA) in retail beef livers, beef, and pork meats sold in Tulsa, Oklahoma and to characterize the recovered strains for their virulence and antimicrobial resistance. Ninety six chilled retail beef (50 beef livers and 46 beef other cuts), and 99 pork meat samples were collected. The prevalence in beef livers was 40/50 (80%) followed by other beef cuts 23/46 (50%) then pork 43/99 (43.3%). No isolates were positive for MRSA since none harbored the mecA or mecC gene. A total of 334 recovered S. aureus isolates (143 beef livers, 76 beef, and 115 pork isolates) were screened for their antimicrobial susceptibility against 16 different antimicrobials and their possession of 18 different toxin genes. Multidrug resistance was more prevalent in the pork isolates followed by beef then beef livers. The prevalence of enterotoxin genes such as seg, seh, and sei and the toxic shock syndrome gene tst was higher in the pork isolates than in the beef ones. The hemolysin genes, particularly hlb, were more prevalent in isolates from beef livers. Molecular typing of a subset of the recovered isolates showed that they are highly diverse where spa typing was more discriminatory than PFGE. The alarmingly high incidence of S. aureus in retail beef livers in this study should raise awareness about the food safety of such meat products. Full article

The continuously improving sophistication of molecular engineering techniques gives access to novel classes of bio-therapeutics and new challenges for their production in full respect of the strengthening regulations. Among these biologic agents are DNA based vaccines or gene therapy products and to a lesser extent genetically engineered live vaccines or delivery vehicles. The use of antibiotic-based selection, frequently associated with genetic manipulation of microorganism is currently undergoing a profound metamorphosis with the implementation and diversification of alternative selection means. This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the bio-therapeutic world. Full article