Gerard N. Stelma

The isolation and identification of Mycobacterium avium complex (MAC) recovered from Los Angeles potable water, a possible source of infection in AIDS patients

Los Angeles water was investigated as a possible source of Mycobacterium avium complex (MAC) infection in patients with AIDS. MAC consists of M. avium (MA), M. intracellulare (MI) and Mycobacterium X (MX) (positive for MAC by DNA probe but not MA or MI). The study included 13 reservoirs supplying 90% of the water to the City of Los Angeles, ten major hospitals and 58 dwellings. Water samples were filtered through 0.45 μm Millipore filters and decontaminated with either sodium hydroxide and/or oxalic acid and cetylpyridinium chloride. The decontaminated filters were placed on selective Middlebrook 7H10 medium and observed for growth at three and eight weeks. Coccobacillary acid‐fast bacilli were subcultured; Tween and nitrate negative isolates were identified by DNA hybridization probes. Eight morphotypes were observed and nontubercu‐lous mycobacteria (NTM) were recovered from 12 (92%) reservoirs, 54 (95%) homes, and ten (100%) hospitals. Twenty‐six (34%) of the NTM were probe positive for MAC. MA was isol...

Persistence of Nontuberculous Mycobacteria in a Drinking Water System after Addition of Filtration Treatment

ABSTRACT There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.

Isolation and Identification of Nontuberculous Mycobacteria from Foods as Possible Exposure Sources

A variety of foods collected from local supermarkets and produce stands were examined as possible sources of nontuberculous mycobacterial exposure. Food samples were combined with sterile ultrapure water and manually shaken. To remove large particles, the suspensions were filtered through a sterile strainer, centrifuged, and the supernatants were discarded. The food pellets were stored at -75 degrees C. The pellets were treated with either oxalic acid or sodium hydroxide-sodium citrate solutions to reduce contamination by nonmycobacterial organisms. Decontaminated pellets were cultured on both Middlebrook 7H10C agar and Middlebrook 7H10C agar with supplemental malachite green. Plates were observed for growth at 2 and 8 weeks. Isolates demonstrating acid-fastness were identified to species using polymerase chain reaction and restriction enzyme analysis. Nontuberculous mycobacteria (NTM) were recovered from 25 of 121 foods. Six different species of NTM were isolated, the most predominant being Mycobacterium avium.

Molecular Comparison of Mycobacterium avium Isolates from Clinical and Environmental Sources

ABSTRACT We collected Mycobacterium avium isolates from clinical and drinking-water sources and compared isolates among themselves and to each other using molecular methods. Four clinical isolates were related to water isolates. Groups of indistinguishable clinical isolates were identified. The groups of identical clinical isolates suggest a common source of exposure.

Nonpoint pollution from animal sources and shellfish sanitation

Many of the microorganisms pathogenic to both animals and man are transmitted via the fecal-oral route. Most of these pathogens could conceivably be transmitted through a shellfish vector. Bacteria potentially transmitted from animal to man via shellfish include most of the salmonellae, Yersinia enterocolitica , Yersinia pseudotuberculosis , Escherichia coli O157:H7, Campylobacter jejuni , and Listeria monocytogenes . The protozoa most likely to be transmitted this way are Giardia lamblia and Cryptosporidium spp. Because the enteric viruses are highly species-specific, they are not likely to be transmitted from animals to humans. There are environmental data showing that bacterial pathogens shed by both domestic and wild animals have been isolated from shellfish. However, there is little epidemiological evidence that illness outbreaks have been caused by shellfish harvested from waters polluted by animals. Unfortunately, epidemiological observations are of limited value because most illnesses are probably not recorded. In addition, more than half of the recorded outbreaks are of unknown etiology, and more than half of the shellfish implicated in illness outbreaks cannot be traced to their points of origin. More lenient bacteriological standards should not be established for waters affected only by animal pollution until health effects studies have been performed, and an indicator that differentiates between human and nonhuman fecal pollution is available. Most of the pollution that originates from domestic animals could be eliminated by simple and inexpensive measures.

Evaluation of media for recovery of aerosolized bacteria

Disease transmission by airborne bacteria is well known. Bacterial burden in indoor air is estimated by sampling the air and estimating Colony Forming Units (CFU) using a variety of media. In this study, the recovery of bacteria, after aerosolization in an aerosol chamber, and employing a variety of media, was compared to that achieved using Tryptic Soy Agar medium. The total number of cells present was determined by direct microscopy. All trials were conducted at approximately the same relative humidity (RH) and temperature using the same collection device. Twelve species of bacteria were tested and a total of 120 media or media combinations were evaluated. Recovery on 64 media formulations was significantly lower for all strains examined, and therefore, excluded from further consideration for the purposes of this study. Data for 56 of the media are presented. Three species (Bacillus subtilis, Staphylococcus aureus andSerratia marcescens) were selected as representative for reporting and testing recovery success. It is concluded that, for the media included in the study, there are large differences in recovery and successful recovery is related both to the effect of aerosolization and the type of medium employed for recovery. Brain Heart Infusion Agar (with horse serum), Tryptic Soy Agar and Mueller Hinton Agar yielded the best recoveries of aerosolized cultures. The most important finding was that only a small fraction of the airborne bacterial populations, enumerated by direct microscopy, could be recovered on any of the media tested, suggesting that culturable bacterial count is not a satisfactory means of estimating air microbial pollution.

Characterization of Aeromonas Virulence Using an Immunocompromised Mouse Model

An immunocompromised mouse model was used to characterize Aeromonas strains for their ability to cause opportunistic, extraintestinal infections. A total of 34 isolates of Aeromonas (A. hydrophila [n = 12]), A. veronii biotype sobria [n = 7], A. caviae [n = 4], A. enchelia [n = 4], A. allosaccharophila [n = 2], A. salmonicida (n = 4), and A. bestiarum [n = 1]) were introduced by intraperitoneal injection into immunocompetent or chemically compromised (using cyclophosphamide) mice. The ability of each isolate to persist in the liver and spleen tissue was monitored at 24 hours after exposure. A majority of A. hydrophila and A veronii v. sobria strains, but none of the isolates of other Aeromonas species, were capable of persistent colonization (<300 cells/mg spleen and liver tissue at 24 hours). The presence or absence of several putative virulence factors (cytotoxicity to HEp-2, lipase activity, elastase activity, and hemolysis) were determined for each isolate using in vitro tests. There were no correlations between the presence or absence of biochemical test results for putative virulence factors and persistence of the isolate in spleen and liver tissue at 24 hours.

Production of Enterotoxin by Vibrio vulnificus Isolates

Weakly virulent isolates of Vibrio vulnificus that were lethal only to simultaneously iron-overloaded and immunosuppressed mice were tested for ability to cause fluid accumulation in the permanently ligated rabbit ileal loop. Unlike the highly virulent isolates, which caused septicemia and death in rabbits, these isolates caused significant fluid accumulation in the rabbit loops. Fluid accumulation was also observed when culture filtrates were tested, indicating the existence of an enterotoxin. Enterotoxin activity did not correlate with the hemolysin or protease activities. Only one of three enterotoxigenic isolates caused diarrhea when administered to temporarily ligated rabbit ileal loops, suggesting involvement of some other pathogenic determinant(s) such as colonization.

Identification of Microcystis aeruginosa Peptides Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and Immunogenic Peptides

Background The cyanobacterium species Microcystis aeruginosa produces microcystin and an array of diverse metabolites believed responsible for their toxicity and/or immunogenicity. Previously, chronic rhinitis patients were demonstrated to elicit a specific IgE response to nontoxic strains of M. aeruginosa by skin-prick testing, indicating that cyanobacteria allergenicity resides in a non-toxin–producing component of the organism. Objectives We sought to identify and characterize M. aeruginosa peptide(s) responsible for allergic sensitization in susceptible individuals, and we investigated the functional interactions between cyanobacterial toxins and their coexpressed immunogenic peptides. Methods Sera from patients and extracts from M. aeruginosa toxic [MC(+)] and nontoxic [MC(–)] strains were used to test IgE-specific reactivity by direct and indirect ELISAs; 2D gel electrophoresis, followed by immunoblots and mass spectrometry (MS), was performed to identify the relevant sensitizing peptides. Cytotoxicity and mediator release assays were performed using the MC(+) and MC(–) lysates. Results We found specific IgE to be increased more in response to the MC(–) strain than the MC(+) strain. This response was inhibited by preincubation of MC(–) lysate with increasing concentrations of microcystin. MS revealed that phycocyanin and the core-membrane linker peptide are the responsible allergens, and MC(–) extracts containing these proteins induced β-hexosaminidase release in rat basophil leukemia cells. Conclusions Phycobiliprotein complexes in M. aeruginosa have been identified as the relevant sensitizing proteins. Our finding that allergenicity is inhibited in a dose-dependent manner by microcystin toxin suggests that further investigation is warranted to understand the interplay between immunogenicity and toxicity of cyanobacteria under diverse environmental conditions. Citation Geh EN, Ghosh D, McKell M, de la Cruz AA, Stelma G, Bernstein JA. 2015. Identification of Microcystis aeruginosa peptides responsible for allergic sensitization and characterization of functional interactions between cyanobacterial toxins and immunogenic peptides. Environ Health Perspect 123:1159–1166; http://dx.doi.org/10.1289/ehp.1409065

Research considerations for more effective groundwater monitoring

Since numerous pathogens occur in feces, water is monitored for fecal contamination using indicator organisms rather than individual pathogens. Although this approach is supported by health effects data in recreational waters, it is questionable when used for drinking water. Most outbreaks in groundwater occur in systems that have not violated the US EPAs maximum contaminant limit (MCL) for total coliforms within 12 months before the outbreak. Additionally, environmentally stable viruses and parasites are often detected in drinking water samples with no detectable indicators. Recent detections of Escherichia coli O157:H7 and Campylobacter jejuni in groundwaters in the apparent absence of indicators also cast some doubt on the worth of indicators for fecal bacterial pathogens. Individual pathogen monitoring is now technically achievable but currently unreasonable due to the number of possible pathogens and the costs involved. Several alternatives to pathogen monitoring could significantly reduce the frequency at which pathogens occur in waters testing negative for indicators: (i) increasing sample volumes for indicators, (ii) increasing monitoring frequency, (iii) using a suite of indicators, (iv) using a more conservative polymerase chain reaction (PCR) method, (v) sampling when fecal contamination is most likely present or (vi) any combination of these options.