Biological control of Phlebotomus papatasi larvae by using entomopathogenic nematodes and its symbiotic bacterial toxins.

The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) - Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 - were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.

Biological control of Phlebotomus papatasi larvae by using entomopathogenic nematodes and its symbiotic bacterial toxins

@# The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) – Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 – were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.

A quantitative method to assess the role of indoor air decontamination to simultaneously reduce contamination of environmental surfaces: testing with vegetative and spore-forming bacteria.

Indoor air can spread pathogens, which can be removed/inactivated by a variety of means in healthcare and other settings. We quantitatively assessed if air decontamination could also simultaneously reduce environmental surface contamination in the same setting. Two types of vegetative bacteria (Staphylococcus aureus and Acinetobacter baumannii), and a bacterial spore-former (Geobacillus stearothermophilus) were tested as representative airborne bacteria. They were separately aerosolized with a Collison nebulizer into a 24-m3 aerobiology chamber and air samples collected with a programmable slit-to-agar sampler. Settling airborne particles were collected on culture plates placed at, and collected from, five different locations on the floor of the chamber with a custom-built remote plate-placement and -retriever system. Experimentally contaminated air in the chamber was decontaminated for 45 min with a device based on HEPA filtration and UV light. The plates were incubated and CFU counted. The device reduced the viability levels of all tested bacteria in the air by >3 log10 (>99·9%) in 45 min. Based on two separate tests, the average reductions in surface contamination for S. aureus, A. baumannii and G. stearothermophilus were respectively, 97, 87 and 97%. We thus showed that air decontamination could substantially and simultaneously reduce the levels of surface contamination in the same setting irrespective of the type of pathogen present. SIGNIFICANCE AND IMPACT OF THE STUDY: The innovative and generic test protocol described can quantitatively assess the reduction in environmental surface contamination from microbial decontamination of indoor air in the same setting. This added advantage from air decontamination has implications for infection prevention and control in healthcare and other settings without the need for additional expense or effort. Continuous operation of an air decontamination device, such as the one tested here, can lead to ongoing reductions in pathogens in air and on environmental surfaces.

Assessing the activity of microbicides against bacterial spores: knowledge and pitfalls.

Bacterial endospores (spores) have a higher intrinsic resistance to microbicides as compared to other microbial forms, most likely due to their impermeable outer layers and low water content. Though structural differences between the spores of various bacterial species may account for observed variations in their resistance to microbicides, flaws in methods for testing the sporicidal activity of microbicides often exaggerate the differences. This has major implications when considering the selection of one or more surrogates to assess microbicides against clinically relevant spore-formers such as Clostridium difficile. The mounting significance of Cl. difficile as a pathogen is leading to a corresponding increase in the number of commercially available microbicidal formulations claiming activity against its spores without proper differentiation between the product's sporistatic and sporicidal actions. In this review we critically assess the situation and the implications of product claims on the field use of microbicidal products.

Disinfectant wipes are appropriate to control microbial bioburden from surfaces: use of a new ASTM standard test protocol to demonstrate efficacy.

BACKGROUND: The use of disinfectant pre-soaked wipes (DPW) to decontaminate high-touch environmental surfaces (HTES) by wiping is becoming increasingly widespread in the healthcare environment. However, DPW are rarely tested using conditions simulating their field use, and the label claims of environmental surface disinfectants seldom include wiping action. AIM: To evaluate the new E2967-15 standard test specific to wipes, particularly their ability to decontaminate surfaces and to transfer acquired contamination to clean surfaces. METHODS: ASTM Standard E2967-15 was used by three independent laboratories to test the efficacy of five types of commercially available wipe products. All data generated were pulled together, and reproducibility and repeatability of the standard were measured. FINDINGS: All the commercial DPW tested achieved a >4log10 (>99.99%) reduction in colony-forming units (CFU) of Staphylococcus aureus and Acinetobacter baumanii with 10s of wiping, but only one DPW containing 0.5% accelerated H2O2 prevented the transfer of bacteria to another surface. CONCLUSION: This newly introduced standard method represents a significant advance in assessing DPW for microbial decontamination of HTES, and should greatly assist research and development, and in making more relevant and reliable claims on marketed DPW.

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms.

AIM: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. METHODS AND RESULTS: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip-fed reactor; the biofilms were treated with either 0.5 mg l(-1) or 1.0 mg l(-1) of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine-treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic-resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. CONCLUSIONS: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.

Survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus.

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

Allen Denver Russell Memorial Lecture, 2006. The use of microbicides in infection control: a critical look at safety, testing and applications.

Microbial pathogens continue as major threats to health. Indeed, many ongoing societal changes are enhancing our vulnerability and exposure to several frank and opportunistic pathogens. This, together with rampant antimicrobial resistance and reduced prospects for newer drugs and vaccines, is forcing a higher reliance on microbiocides in infection prevention and control. That this reliance may not be well-founded becomes apparent from a closer look at current ways of testing and registering microbiocides, their label claims as well as human and environmental safety of certain widely used microbicidal chemicals. Many methods to test microbiocides for registration are flawed and/or entail test conditions irrelevant to field use. Pathogens listed on product labels may not be among those amenable to interruption through microbiocide use. The wide variations and discrepancies in existing national/regional regulations for registering microbiocides for sale stifle innovation. This is a critical look at the above-mentioned issues with emphasis on chemicals meant for use on environmental surfaces and medical devices. It highlights better ways to test microbiocides and to attain global harmonization of testing and product registration. It also details the known and potential dangers of microbiocide use and what to consider in choosing such formulations for optimal safety and effectiveness. End users are advised to be more critical and prudent in the selection and application of microbicidal chemicals, manufacturers are encouraged to explore infection control products and technologies that are safer in the workplace and for the environment, and regulators are urged to review and update the requirements and procedures for premarket review of microbiocide efficacy data and label claims. Independent investigations are also urgently needed to document the proportion of nosocomial infections that would be amenable to prevention through chemical disinfection of environmental surfaces.

Impact of concentration, temperature, and pH on inactivation of Salmonella spp. by volatile fatty acids in anaerobic digestion.

It is known that the presence of volatile fatty acids may play a role in the inactivation of pathogens for systems that employ an acid phase reactor. This study was conducted to investigate the influence of volatile fatty acids on the inactivation of Salmonella spp. over a range of digestion temperatures. In this study, digesters that were treating municipal wastewater treatment plant sludges were operated at temperatures that ranged from 35 to 49 degrees C and had a solids residence time of 15 days. Samples collected from the effluent of the digesters were dosed with solutions containing acetic, propionic, and butyric acids alone and in mixtures, and the dosed effluents were analyzed for Salmonella spp. over time. In the first round of testing, the digester effluents were dosed with individual organic acids and also a mixture containing all three volatile fatty acids over a range of concentrations from 750 to 6000 mg/L, and the pH of the samples was fixed at a value of 5.5. In the second round of testing, the sample sludges were spiked with a fixed amount of organic acid mixture, and the pH was varied from 4.5 to 7.5. The reduction of Salmonella spp. in digester effluents, when dosed with volatile organic acids, was found to depend on pH, temperature, the chain length of the acids, and the concentration and composition of the acids present. Increases in temperature appeared to increase the inhibitory effects of the volatile organic acids. At mesophilic temperatures, acidic pHs resulted in a greater inhibition of Salmonella spp.; whereas at higher temperatures neutral pHs were found to be more inhibitory. The results suggest that acid phase digesters that operate at elevated temperatures and low pH can achieve substantial reduction of Salmonella spp.

Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.

A new formula with reduced ethanol content (55%) in combination with 10% propan-1-ol, 5.9% propan-1.2-diol, 5.7% butan-1.3-diol and 0.7% phosphoric acid exhibited a broad spectrum of virucidal activity. In quantitative suspension tests, with and without protein load, this formulation reduced the infectivity titre of seven enveloped (influenza A and B, herpes simplex 1 and 2, bovine corona, respiratory syncytial, vaccinia, hepatitis B, bovine viral diarrhoea) and four non-enveloped (hepatitis A, polio, rota, feline calici) viruses >10(3)-fold within 30s. In comparative testing, only 95% ethanol showed similar levels of activity. In fingerpad tests, the formulation produced a log10 reduction factor of the titre of poliovirus type 1 (Sabin) of 3.04 in 30s compared with 1.32 by 60% propan-2-ol. Testing against feline calicivirus produced a log10 reduction factor of 2.38 by the test formulation; in contrast, the log10 reduction factors with 70% ethanol and 70% propan-1-ol were 0.68 and 0.70, respectively.

Predicting serum gastrin levels among men during Ramadan fasting.

Increased gastric acidity is common when fasting during Ramadan. Our study aimed to develop a regression equation to predict fasting serum gastrin levels using parameters commonly analysed in clinical laboratories. Fasting blood samples from six men were taken on days 1, 10, 19, 26 and 28 of Ramadan. Serum gastrin, total cholesterol, urea and uric acid were analysed. All 5 samples from each man were included in multiple regression analysis and the prediction equation obtained was: serum gastrin, pg/mL = 198.27-0.199 total cholesterol (mg/dL) + 2.525 urea (mg/dL)--103.238 uric acid (mg/dL) + 10.923 uric acid (mg/dL)2 + 3.683 body mass index, r2 = 0.75, P < 0.001. This equation might be used to estimate gastrin levels and plan dietary and medicinal measures to avoid high gastric acidity during Ramadan.

Predicting serum gastrin levels among men during Ramadan fasting

Increased gastric acidity is common when fasting during Ramadan. Our study aimed to develop a regression equation to predict fasting serum gastrin levels using parameters commonly analysed in clinical laboratories. Fasting blood samples from six men were taken on days 1, 10, 19, 26 and 28 of Ramadan. Serum gastrin, total cholesterol, urea and uric acid were analysed. All 5 samples from each man were included in multiple regression analysis and the prediction equation obtained was: serum gastrin, pg/mL = 198.27-0.199 total cholesterol [mg/dL] + 2.525 urea [mg/dL]--103.238 uric acid [mg/dL] + 10.923 uric acid [mg/dL]2 + 3.683 body mass index, r2 = 0.75, P < 0.001. This equation might be used to estimate gastrin levels and plan dietary and medicinal measures to avoid high gastric acidity during Ramadan

Investigation of opportunistic pathogens in municipal drinking water under different supply and treatment regimes.

Changing regulations to lower disinfectant byproducts in drinking water is forcing utilities to switch disinfection from chlorine to monochloramine. It is generally unknown whether this will impact positively or negatively on the microbiological quality of drinking water. A utility in Florida, using water with relatively high organic carbon levels from deep wells in several wellfields, made the decision to change its disinfection regime from chlorine to chloramine in order to meet the new regulations. To assess the impacts of such a change on the microbiology of its water supplies, it undertook a number of studies before and after the change. In particular, the presence of the opportunistic pathogens Legionella and Mycobacterium, and also the composition of drinking-water biofilms, were examined. A preliminary synthesis and summary of these results are presented here. Legionella species were widely distributed in source waters and in the distribution system when chlorine was the disinfectant. In some samples they seemed to be among the dominant biofilm bacteria. Following the change to monochloramine, legionellae were not detected in the distribution system during several months of survey; however, they remained detectable at point of use, although with less species diversity. A variety of mycobacteria (21 types) were widely distributed in the distribution system when chlorine was the disinfectant, but these seemed to increase in dominance after chloramination was instituted. At point of use, only four species of mycobacteria were detected. Other changes occurring with chloramination included (a) an altered biofilm composition, (b) increased numbers of total coliforms and heterotrophs and (c) nitrification of water storage tanks. The results suggested that consideration should be given to the microbiological effects of changing disinfection regimes in drinking-water and distribution system biofilms.

Microbicides and the environmental control of nosocomial viral infections.

Viruses are important causes of acute and chronic diseases in humans. Newer viruses are still being discovered and those that are already known are being incriminated in the aetiology of clinical conditions with hitherto unknown causes. Apart from frequently causing infections in the general community, many types of viruses are also significant nosocomial pathogens. While it is generally agreed that we underestimate the proportion of nosocomial infections that are viral, due to a lack of routine monitoring, viruses easily account for more than 30% of the cases of hospital-acquired infections in many paediatric settings. Indeed, the relative importance of viruses in this respect is increasing due to a number of societal and demographic changes as well as alterations in healthcare practices. Safe vaccines against many common nosocomial viral agents are currently unavailable while there is also a virtual lack of effective and affordable chemotherapy against them. There is, therefore, renewed emphasis on preventive strategies by better understanding of the relative importance of various vehicles in the nosocomial spread of viruses and by infection control using microbicides. This, in turn, has stimulated considerable interest in the development of formulations that are not only safer but which also have demonstrated activity against major types of nosocomial viral pathogens. Further, much work is now underway to design better methods to assess the virucidal activity of microbicides used to decontaminate hands, reusable medical devices and environmental surfaces in critical areas of healthcare settings. It is anticipated that these approaches will result in reducing the health and economic impact of nosocomial infections due to viruses.

Norovirus cross-contamination during food handling and interruption of virus transfer by hand antisepsis: experiments with feline calicivirus as a surrogate.

While there is good epidemiological evidence for foods as vehicles for norovirus transmission, the precise means of spread and its control remain unknown. The feline calicivirus was used as a surrogate for noroviruses to study infectious virus transfer between hands and selected types of foods and environmental surfaces. Assessment of the potential of selected topicals in interrupting such virus transfer was also made. Ten microliters of inoculum of feline calicivirus deposited onto each fingerpad of adult subjects was allowed to air dry and the contaminated area on individual fingerpads was pressed (10 s at a pressure of 0.2 to 0.4 kg/cm2) onto 1-cm-diameter disks of ham, lettuce, or brushed stainless steel. The virus remaining on the donor and that transferred to the recipient surfaces was eluted and plaque assayed. Virus transfer to clean hands from experimentally contaminated disks of ham, lettuce, and stainless steel was also tested. Nearly 46 +/- 20.3, 18 +/- 5.7, and 13 +/- 3.6% of infectious virus was transferred from contaminated fingerpads to ham, lettuce, and metal disks, respectively. In contrast, approximately 6 +/- 1.8, 14 +/- 3.5, and 7 +/- 1.9% virus transfer occurred, respectively, from ham, lettuce, and metal disks to hands. One-way analysis of variance test showed that pretreatment (washing) of the fingerpads either with water or with both topical agent and water significantly (P < 0.05) reduced virus transfer to < or = 0.9%, as compared with < or = 2.3 and < or = 3.4% transfer following treatments with either 75% (vol/vol) ethanol or a commercial hand gel containing 62% ethanol, respectively. Despite wide variations in virus transfer among the targeted items used, intervention agents tested reduced virus transfer significantly (P < 0.05) when compared with that without such treatments (71 +/- 8.9%). These findings should help in a better assessment of the potential for cross-contamination of foods during handling and also assist in developing more effective approaches to foodborne spread of norovirus infections.

A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus.

Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).

A quantitative study of the survival of two species of Candida on porous and non-porous environmental surfaces and hands.

AIMS: In spite of the importance of many species of Candida as human pathogens, little is known about their ability to survive on animate and inanimate surfaces. Such information is essential in understanding the vehicles and modes of their spread, and in designing proper infection control strategies against them. The aim of this study was to generate comparative quantitative data in this regard. METHODS AND RESULTS: The survival of one clinical isolate each of Candida albicans and C. parapsilosis on two types of hard inanimate surfaces (glass and stainless steel) and two types of fabrics (100% cotton and a blend of 50% cotton and 50% polyester) was evaluated under ambient conditions (air temperature 22 +/- 2 degrees C; relative humidity 45-62%) using quantitative test protocols. The survival of C. albicans was also assessed on human skin, using the fingerpads of adult volunteers as carriers. Each carrier surface received 10 microl of the test suspension containing a soil load to simulate body fluids. When dried on glass and stainless steel carriers, C. albicans and C. parapsilosis remained viable for at least three and 14 days, respectively. Both species could survive for at least 14 days on both types of fabric. On the skin, 20% of the viable C. albicans remained detectable one hour post-inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: This quantitative and comparative study demonstrated the potential for, and differences in the ability of clinically significant species of Candida to remain viable on porous and non-porous inanimate surfaces as well as on human hands. These results should help in understanding the epidemiology of nosocomial infections due to Candida, and in designing better prevention and control strategies against them.

Testing chemical germicides against Candida species using quantitative carrier and fingerpad methods.

Six disinfectants were tested against Candida albicans, C. parapsilosis and C. tropicalis using quantitative carrier tests based on glass (QCT-1) and metal (QCT-2) surfaces. C. albicans was also used to test four topical agents by a fingerpad method. Hard water (200 ppm as CaCO(3)) was the product diluent. In preliminary tests with QCT-1 and QCT-2, the testing was with or without a soil load; subsequent tests and fingerpad tests included soil. In QCT-1 and QCT-2, each carrier received 10 microL (5.0 x 10(6) - 1.0 x 10(7)colony forming units) of Candida, and was air dried for 1 h, then exposed to 1 mL or 50 microL of test product at 22 +/- 2 degrees C for up to 10 min. Controls received an equivalent volume of saline. For fingerpad tests, each digit received 10 microL of inoculum, which was allowed to dry and exposed to 1 mL of test product for 20 s. Inoculated plates of Sabouraud's dextrose agar were held for 48 h at 30 degrees C and colonies counted to determine reductions in colony forming units. In tests on both hard surfaces and fingerpads, ethanol and products based on ethanol reliably and rapidly inactivated all the Candida species tested. Products with sufficient potency to have tuberculocidal claims produced substantial reductions in the titre of C. albicans, although some showed a lesser reduction in titre of C. tropicalis and C. parapsilosis. This may reflect differences in cell hydrophobicity between Candida species, and highlights the need for care in selecting a suitable surrogate for disinfectant tests. The quantitative carrier and fingerpad protocols are suitable for assessing the activity of disinfectants and topical antiseptics against candida.

Comparison of static and dynamic disinfection models for bacteria and viruses in water of varying quality.

Disinfection studies rarely use natural waters due to demands exerted on the applied disinfectants and lack of consistent disinfectant residuals. This study compared the degree of disinfection achieved in natural waters between conventional batch (static) models and a system of similar volume where disinfectant residuals were maintained at constant levels (dynamic). In the latter, disinfectant was delivered through a hollow fibre cartridge from a slipstream of a full-scale (chloramine) or pilot (chlorine) water treatment plant. The test organisms (hepatitis A virus, poliovirus, MS-2, Mycobacterium terrae and Enterococcus durans) were selected with different resistance to the disinfectants. In general, for water of "good" quality, the differences between the two systems were often small or not apparent for monochloramine. However, for low chlorine residuals, or when additional demand was placed on the disinfectant, differences between the two systems became more apparent. Little difference was seen between disinfection of the test organisms singly or in mixtures, but injury of vegetative bacteria with monochloramine was very apparent. This system could be useful for understanding the fluctuations in disinfection efficacy that may occur in source water of varying quality, or in distribution systems, as disinfectant residuals decline.