Biomedical subjects

I L Pepper

Publications and source records attributed to I L Pepper.

At least 37 records · Page 2Linked to original sources

PubMed1997

Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner.

This two year study evaluated the prevalence of indicator bacteria and specific pathogens in 10 'normal' kitchens in the United States. In Phase I, none of the kitchens was cleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens were monitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms and faecal coliforms. Almost all locations at all households exhibited contamination, with the sink and sponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations in sink and sponge samples were very high, with 63 and 67% of all samples being positive, respectively. Escherichia coli was detected in 16.7% of all sink surfaces and 33.3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobial disinfectant cleaner which families were encouraged to use but not forced to do so; in some cases, the product was used infrequently or not at all. This regimen did not demonstrate any consistent reduction in the incidence of bacterial contamination. By contrast, in the final phase of the study where disinfectant use was targeted for surfaces soon after contamination with foods or hands, the incidence of contamination decreased dramatically. These data show that normal kitchens can easily be contaminated with a variety of bacterial contaminants including faecal coliforms, E. coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobial agents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likely to reduce the incidence of bacterial contaminants.

Bacteria↗

PubMed1997

Detection of viable Giardia cysts by amplification of heat shock-induced mRNA.

Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.

Animals↗

PubMed1997

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.

Campylobacter jejuni↗

PubMed1996

Detection of infectious enteroviruses by an integrated cell culture-PCR procedure.

Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with > or = 3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.

Animals↗

PubMed1996

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1995

Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction.

This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 x 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 microliters (0.2 PFU/l tap water).

Humans↗

PubMed1995

Comparison of PCR and cell culture for detection of enteroviruses in sludge-amended field soils and determination of their transport.

PCR and cell culture assays for enteroviruses were conducted on soil samples collected from an experimental farm that had received mesophilic anaerobically digested sludge for the past 7 years. Of 24 samples assayed, 21 samples were positive by PCR, implying that at least some viral nucleic acid sequences remained intact. However, these viral particles were unable to infect the Buffalo Green Monkey cell line used in subsequent cell culture assays. It is significant that positive PCR detection of nucleic acid sequences occurred even though the most recent sludge application was 3 months prior to soil sampling. Viral nucleic acid sequences were detected by PCR at points vertically and laterally displaced from sludge injections, illustrating significant transport of viruses. Rainfall and irrigation events may have contributed to viral transport.

Animals↗

PubMed1994

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1994

Cell culture and PCR determination of poliovirus inactivation by disinfectants.

Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 10(2) PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.

Cells, Cultured↗

PubMed1994

A method to detect enteroviruses in sewage sludge-amended soil using the PCR.

PCR detection of seeded poliovirus type 1 in sludge-amended soil was made possible by utilizing Sephadex G-50 and Chelex-100 resins to remove compounds present in sludge-amended soil that may inhibit PCR. With this method, enteroviruses indigenous to an anerobically digested sludge were detected by PCR in 10 different soils amended with this sludge.

Enterovirus↗

PubMed1994

Detection of naturally occurring enteroviruses and hepatitis A virus in undigested and anaerobically digested sludge using the polymerase chain reaction.

Four undigested and four anaerobically digested sewage sludge samples were analyzed for enteroviruses and hepatitis A virus using seminested and double polymerase chain reaction (PCR), respectively. For enteroviruses, all eight samples were positive when detection was by seminested PCR. Using cell culture all samples except two digested sludge samples were positive. For hepatitis A virus, seven out of eight samples were positive by PCR detection. In all samples, PCR inhibitory substances were removed by passage through Sephadex G-50 and Chelex 100 columns. Overall the PCR methodology was highly successful in identifying the presence of both viruses; however, with this methodology, there was no indication as to whether enteroviruses or hepatitis A viruses not confirmed in cell culture were infectious.

Anaerobiosis↗

PubMed1993

Hazards from pathogenic microorganisms in land-disposed sewage sludge.

Sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are precipitated from wastewater and sewage during primary, secondary, and tertiary sewage treatment. Present in these sludges are significant numbers of microorganisms that include viral, bacterial, protozoan, fungal, and helminth pathogens. The treatment of sludge to reduce biochemical oxygen demand, solids content, and odor is not always effective in reducing numbers of pathogens. This becomes a public health concern because the infectious dose for some of these pathogens may be as low as 1 particle (virus) to 50 organisms (Giardia). When sludge is applied to land for agricultural use and landfill compost, these pathogens can survive from days (bacteria) to months (viruses) to years (helminth eggs), depending on environmental conditions. Shallow aquifers can become contaminated with pathogens from sludge and, depending on groundwater flow, these organisms may travel significant distances from the disposal site. Communities that rely on groundwater for domestic use can become exposed to these pathogens, leading to a potential disease outbreak. Currently, methods to determine the risk of disease from pathogens in land-disposed sludge are inadequate because the sensitivity of pathogen detection is poor. The application of recombinant DNA technology (gene probes and polymerase chain reaction) to environmental samples may provide increased sensitivity for detecting specific pathogens in land-disposed sludge and greatly improved risk assessment models for our exposure to these sources of pathogens.

Hazardous Waste↗

PubMed1993

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.

Bacteria↗

PubMed1993

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1993

Detection of enteroviruses in groundwater with the polymerase chain reaction.

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.

Base Sequence↗

PubMed1993

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.

Animals↗

PubMed1992

Persistence of viruses in desert soils amended with anaerobically digested sewage sludge.

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log(10) reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 x 10 Pa for both soils) and temperatures of 15, 27, and 40 degrees C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40 degrees C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40 degrees C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15 degrees C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log(10) reduction per day in clay loam and sandy loam soils, respectively. At 27 degrees C, this rate increased to 0.629 log(10) reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15 degrees C (k = 0.064 log(10) reduction per day, clay loam; k = 0.095 log(10) reduction per day, sandy loam) and 27 degrees C (k = 0.133 log(10) reduction per day, clay loam; k = 0.154 log(10) reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40 degrees C. No reduction of PRD-1 was observed after 28 days at 15 degrees C and after 16 days at 27 degrees C. At 40 degrees C, the inactivation rates were 0.208 log(10) reduction per day in amended clay loam soil and 0.282 log(10) reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15 degrees C, within 3 days at 27 degrees C, and within 2 days at 40 degrees C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.

Journal Article↗

PubMed1992

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

2,4-Dichlorophenoxyacetic Acid↗