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.

Quantitative PCR assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and application to environmental samples and fishes from the Chesapeake Bay.

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.

Method for measuring microbial degradation and mineralization of(14)C-labeled chitin obtained from the blue crab,Callinectes sapidus.

A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described.(14)C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D-[1-(14)C]-glucosamine and allowing for its incorporation into the exoskeleton. The cuticle had a total organic carbon content of 0.48 mg C mg(-1) with a specific radioactivity of 6,356 CPM mg(-1). Glucosamine, i.e., chitin content as determined colorimetrically, was 22% (w/w). Microbial degradation and mineralization rates were assessed in batch culture using(14)C-chitin as substrate and York River water as inoculum. Replicate flasks were sampled daily for enumeration of chitinoclastic bacteria and the radiolabel recovered as particulate(14)C-chitin or(14)CO2. The amount of(14)CO2 generated was directly proportional to the loss of particulate(14)C-chitin, with 96% of the added label recovered as the sum of both phases. The maximum rate of mineralization was 207 mg day(-1) g(-1) seeded(14)C-chitin at 20°C. Highest chitinoclastic bacterial counts corresponded to the period of maximum rate of chitinolysis. It is suggested that the rate of chitin mineralization is limited by exoenzymatic depolymerization and not by chitin concentration.

In situ development of sublethal stress in Escherichia coli: effects on enumeration.

Development of sublethal stress in Escherichia coli exposed in situ to estuarine waters was examined during various seasons. An electrochemical detection technique was utilized to derive a stress index based upon the difference between a predicted electrochemical response time in Trypticase soy broth or EC medium at 44.5 degrees C estimated from a standard curve for unstressed cells and an observed response time for cells exposed to seawater. This stress index was related to recovery efficiencies of seawater-exposed cells, using a variety of standard and resuscitative enumeration procedures. Stress was further studied by determination of the adenylate energy charge. Sublethal stress as measured by the electrochemical detection method was an inverse function of water temperature, with maximum stress occurring after exposure to temperatures below 10 degrees C. Total adenylates and ATP decreased dramatically at low temperatures, although energy charge remained relatively constant under various environmental conditions. Decreases in E. coli ATP suggest that ATP may not be an adequate measure of biomass for in situ stressed cells. Discrepancies in enumeration efficiency were most pronounced at temperatures below 10 degrees C. Resuscitative procedures for solid-media techniques increased the recovery of stressed cells under cold water conditions but were not as effective as the standard most-probable-number procedure.

Seasonal variation in survival of Escherichia coli exposed in situ in membrane diffusion chambers containing filtered and nonfiltered estuarine water.

Human fecal Escherichia coli isolates were exposed over a seasonal cycle to estuarine water in diffusion chambers filled with double-filtered (0.45 and 0.2 microns) and nonfiltered water. Laboratory manipulations of E. coli cultures before estuarine exposure were reduced to minimize sublethal stress, and nonselective or resuscitative enumeration techniques were employed to maximize recovery of stressed cells. E. coli was capable of extended survival during in situ exposure to estuarine water, provided eucaryotes were excluded from diffusion chambers. Survival was directly related to temperature in absence of the eucaryote component of the natural microbiota. Although it was not possible to prevent eventual bacterial contamination in double-filtered water, there was no direct evidence that such contamination affected E. coli survival. Conversely, E. coli disappearance was most pronounced at warmer temperatures in the presence of the natural microbiota, and decline coincided with increasing eucaryote densities. In contrast, the decline of E. coli during winter was similar in both filtered and nonfiltered seawater.