Method for determining the specific growth rate of distinct microbial populations in a non-homogeneous system

Abstract

The present invention pertains to a molecular biology-based method and kit for measuring the specific growth rate (or cell doubling time) of distinct microbial populations. The method and kit can be used to analyze mixed culture samples that have been exposed to chloramphenicol or other protein synthesis inhibitors for defined times. In a preferred embodiment, the method of the invention (also referred to herein as FISH-RiboSyn) is an in situ method that utilizes fluorescence in situ hybridization (FISH) with probes that target: (1) the 5′ or 3′ end of precursor 16S rRNA; or (2) the interior region of both precursor 16S rRNA and mature 16S rRNA. Images can be captured for a defined exposure time and the average fluorescent intensity for individual cells can be determined. The rate of increase of the whole cell fluorescent intensity is used to determine the specific growth rate. The method of the invention can be attractive for rapidly measuring the specific growth rate (or cell doubling time) of distinct microbial populations within a mixed culture in industries such as environmental systems (water and wastewater treatment systems), bioremediation (optimization of conditions for microbial growth), public health (identification of rapidly growing infectious microbes), and homeland security (identification of rapidly growing bioterrorism agents).

1 . A method for determining the specific growth rate of a microbial population, comprising:
 contacting a non-homogeneous system with at least one protein synthesis inhibitor that inhibits the secondary processing of precursor 16S rRNA but does not inhibit the production of precursor 16S rRNA, wherein the non-homogeneous system potentially comprises a microbial population;   obtaining samples from the non-homogeneous system over time following said contacting; and   determining the rate of pre16S rRNA accumulation of the microbial population in the samples, if present, wherein the rate of pre16S rRNA accumulation relative to the 16S rRNA is indicative of the specific growth rate.   
     
     
         2 . The method of  claim 1 , wherein the at least one protein synthesis inhibitor is selected from the group consisting of chloramphenicol, lincomycin, and erythromycin. 
     
     
         3 . The method of  claim 1 , wherein said determining comprises contacting the samples with a labeled hybridization probe targeting the precursor 16S rRNA of the microbial population, and detecting a signal from the probe, wherein the signal is indicative of the number of ribosomes present in each sample. 
     
     
         4 . The method of  claim 3 , wherein the probe targets the 5′ end or 3′ end of precursor 16S rRNA. 
     
     
         5 . The method of  claim 3 , wherein the probe targets the interior region of both precursor 16S rRNA and mature 16S rRNA. 
     
     
         6 . The method of  claim 1 , wherein said determining comprising carrying out fluorescence in situ hybridization (FISH) with an oligonucleotide probe targeting the precursor 16S rRNA of the microbial population. 
     
     
         7 . The method of  claim 6 , wherein the probe targets the 5′ or 3′ end of precursor 16S rRNA. 
     
     
         8 . The method of  claim 6 , wherein the probe targets the interior region of both precursor 16S rRNA and mature 16S rRNA. 
     
     
         9 . The method of  claim 1 , wherein said determining comprises contacting the samples with primers targeting the precursor 16S rRNA of the microbial population, wherein an amplification product is indicative of the number of ribosomes present in each sample. 
     
     
         10 . The method of  claim 1 , wherein the non-homogeneous system is a mixed cell culture comprising a plurality of distinct microbial populations. 
     
     
         11 . The method of  claim 1 , wherein the non-homogeneous system is water. 
     
     
         12 . The method of  claim 1 , wherein the non-homogeneous system is a biological sample. 
     
     
         13 . The method of  claim 1 , wherein the non-homogeneous system is a bodily fluid. 
     
     
         14 . The method of  claim 1 , wherein the microbial population is bacteria. 
     
     
         15 . The method of  claim 1 , wherein the microbial population is a phosphorus accumulating microorganism. 
     
     
         16 . The method of  claim 1 , wherein the microbial population is nitrifying bacteria. 
     
     
         17 . The method of  claim 1 , wherein the microbial population is a methanogen. 
     
     
         18 . The method of  claim 1 , wherein the microbial population is selected from the group consisting of  Nitrospira  spp.,  Nitrosospira  spp.,  Nitrobacter  spp.,  Nitrosomonas  spp.,  Clostridium  spp.,  Bacillus  spp., methogenic archaea, coliform,  Salmonella  spp., and  Bacteroides  spp. 
     
     
         19 . The method of  claim 1 , further comprising inputting the rate of pre16S rRNA accumulation of the microbial population into a computer algorithm that calculates the specific rate of ribosome synthesis. 
     
     
         20 . The method of  claim 1 , further comprising recording the specific growth rate or specific rate of ribosome synthesis of the microbial population in physical or electronic media. 
     
     
         21 . The method of  claim 1 , further comprising comparing the specific growth rate of the microbial population within the non-homogeneous system with that of a known reference microbial population. 
     
     
         22 . A kit for determining the specific growth rate of a microbial population, comprising packaging; and a compartment containing one or more oligonucleotide probes or primers that target sequence within the precursor 16S rRNA and/or mature 16S rRNA. 
     
     
         23 . The kit of  claim 22 , wherein the probe or primers target the 5′ or 3′ end of precursor 16S rRNA, or the interior region of both precursor 16S rRNA and mature 16S rRNA. 
     
     
         24 . The kit of  claim 22 , further comprising at least one component selected from the group consisting of a protein synthesis inhibitor, a reagent to conduct an amplification reaction, means for obtaining a biological or environmental sample, and a set of instructions relating information regarding components of the kit and/or how to measure specific growth rate of a microbe.