Howard Kator

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

Phylogenetic relationships among plant and animal parasites, and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.

Induction of Skin Ulcers in Atlantic Menhaden by Injection and Aqueous Exposure to the Zoospores of Aphanomyces invadans

Abstract The infectivity and role of Aphanomyces invadans in the etiology of skin ulcers in Atlantic menhaden Brevoortia tyrannus were investigated with two laboratory challenges. In the first experiment, Atlantic menhaden received subcutaneous injections with secondary zoospores from one of three cultures of Aphanomyces: WIC (an endemic isolate of A. invadans in Atlantic menhaden from the Wicomico River, Maryland), PA7 (an isolate of A. invadans from striped snakehead Channa striata (also known as chevron snakehead), infected with epizootic ulcerative syndrome from Thailand), and ATCC-62427 (an isolate from Atlantic menhaden from North Carolina). Fish were injected with 1.9 × 102 (WIC-low), 1.9 × 103 (WIC-high), 5.2 × 102 (PA7), or 6.0 × 102 (ATCC-62427) zoospores and held in static water at 23.5°C (6‰ salinity) for 21 d. Both low and high doses of WIC caused incipient, granulomatous lesions after 5 d. Fish injected with the high-dose WIC died within 7 d. All fish injected with the low-dose WIC were dead...

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro.

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.

High salinity relay as a postharvest processing strategy to reduce vibrio vulnificus levels in Chesapeake Bay oysters (Crassostrea virginica).

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to <30 most probable number (MPN) per g and the impact on oyster mortality were assessed in the lower Chesapeake Bay. Two relay experiments were performed during the summer and fall of 2010. Oysters collected from three grow-out sites, a low salinity site (14 to 15 practical salinity units [psu]) and two moderate salinity sites (22 to 25 psu), were relayed directly to a high salinity site (≥30 psu) on Virginias Eastern Shore. Oysters were assayed for V. vulnificus and Vibrio parahaemolyticus (another Vibrio species of concern) densities at time 0 prior to relay and after 7 and 14 days of relay, using the FDA MPN enrichment method combined with detection by real-time PCR. After 14 days, both V. vulnificus and V. parahaemolyticus densities were ≤0.8 MPN/g, and decreases of 2 to 3 log in V. vulnificus densities were observed. Oyster mortalities were low (≤4%) even for oysters from the low salinity harvest site, which experienced a salinity increase of approximately 15 psu. Results, although preliminary and requiring formal validation and economic analysis, suggest that high salinity relay could be an effective PHP method.

Seasonal occurrence of mesophilic Aeromonas spp. as a function of biotype and water quality in temperate freshwater lakes

Abstract Densities of mesophilic aeromonads were compared in Virginia freshwater lakes with trophic classifications ranging from mesotrophic to hypereutrophic. Aeromonad concentrations were independent of trophic status and did not correlate statistically with water quality parameters used to measure trophic state (total phosphorus, chlorophyll a and Secchi depth). Bacterial indicators (fecal coliforms and Escherichia coli ) used to assess the public health safety of recreational waters correlated with mesophilic aeromonads only when sewage pollution occurred. Overall ( n = 101–107) there were small negative correlations of aeromonad densities with dissolved oxygen ( r = −0.30, P = r = −0.30, P = 0.05). During frequent sampling from April through October 1991, mean mesophilic aeromonad concentrations ranged from 10 3–4 cells 100 ml −1 with the lowest values measured during mid-summer. Over the course of twelve months, 273 aeromonad isolates were identified as belonging to the following biotypes: A. sobria (54%), A. hydrophila (29%), A. caviae (10%), and undetermined (7%). Seasonally, the frequency of isolation of A. sobria , considered the most pathogenic biotype, increased from 17 to 69% when water temperatures exceeded 20°C in a hypereutrophic lake. These temperatures typically occur during periods of peak recreational usage. Phenotypic characteristics associated with virulence were positive in 70% (hemolysis) and 42% (autoagglutination) of glycerol-stored strains ( n = 85), and in 15% (autoagglutination) of strains immediately processed ( n = 73). S-layer proteins associated with invasiveness were not found in 14 strains which autoagglutinated. To evaluate the public health significance of aeromonads in nutrient enriched lakes, our results demonstrate a need to couple investigations of mesophilic aeromonad ecology with virulence and biotype assessments.

Effects of sunlight and autochthonous microbiota onEscherichia coli survival in an estuarine environment

The effects of sunlight and the indigenous microbiota onEscherichia coli survival were examined with membrane diffusion chambers deployed in Chesapeake Bay shellfish growing waters. Chambers, fitted with an “upper” UV and visible light-transmitting copolymer film and “lower” semipermeable polycarbonate membrane, were deployed parallel to the water surface to maximize light exposure. Maximum values of a coefficient, kdens, describing changes in culturable cell densities after exposure to sunlight, were 1.7 h−1 and 0.7 h−1 in preliminary tank and in situ experiments, respectively. Mortality and sublethal stress, the latter measured with an electrochemical detection technique, were largest during the first 4 h of exposure. Owing to the light-attenuating properties of Chesapeake Bay water, light-induced cell mortality was significantly reduced at 0.25 m compared with surface exposed cells, and was undetected at 0.5–1.0 m except during seasons of maximal light penetration. Combined exposure to both sunlight and the autochthonous microbiota yielded significantly greater mortality than for either factor alone.

Use of Salmonella typhimurium WG49 to enumerate male-specific coliphages in an estuary and watershed subject to nonpoint pollution

The occurrence of male-specific RNA (FRNA) coliphages, proposed as indicators of enteric viruses, was determined in an estuary subject to nonpoint pollution that included fecal inputs from livestock. A host originally developed for detecting FRNA phages in sewage was applied to water and sediment samples. Phages were enumerated using the host Salmonella typhimurium WG49 containing an Escherichia coli plasmid coding for sex pili, and the female parent strain WG45. FRNA phages and fecal coliforms were enumerated in samples collected seasonally from an estuary and associated feeder streams and densities related to selected environmental parameters. Mean phage densities enumerated on WG49 ranged from < 1 to 50 100 ml−1 water and < 13 to 7200 100 g−1 dry sediment. Examination of 300 phages from estuarine and freshwater samples showed that ⩾ 99% were RNase-resistant, ⩾ 94% were lytic to the female parent salmonella strain (WG45), ⩽ 9% were lytic to male E. coli C3000, and none were lytic to female E. coli C. RNase resistant phages lytic to both salmonella strains were noncontractile flexible tailed phages and those lytic to male salmonella or E. coli hosts were filamentous phages. Electron micrographs of the only RNase-sensitive phage recovered that plaqued only male hosts showed cubic phage particles adsorbed to sex pili. Parallel enumerations of environmental samples on WG45 and WG49 yielded equal or greater phage densities on the former host. Purified phages from these samples were lytic to certain salmonella serovars recovered from the environment but did not cross react with fecal coliform or heterotrophic bacteria isolated from the environment. Although the WG49 host was inappropriate to estuarine and freshwater samples examined because of interference by somatic phages, WG45 and WG49 should be examined as hosts for enumerating salmonella phages. Similarly, the public health significance of somatic phages detected by these hosts should be determined. FRNA phages, with a single exception (1187 samples), were not detected in a condemned shellfish growing area subject to nonpoint pollution. This observation questions the application of FRNA phages as indicators of fecal contamination in waters impacted by diffuse fecal inputs.

Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay

ABSTRACT Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis.

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.