SUMMARY 


Mycolic-acid containing Actinomycetes (mycolata) belong to the 
suborder Corynebacterineae within the order Actinomycetales. This group 
contains filamentous bacteria that produces thick foam or scum in activated 
wastewater treatment plants. This foam retains up to 40% of the suspended 
solids affecting the process. Some species of this group are opportunistic 
human pathogens and there is a need to detect them quickly to avoid that 
they could become a human health problem. 

Several identification systems such as the miniaturized system API 
ZYM, API ID32 C and MicroLog have been used to characterize mycolata 
species. Sometimes, biochemistry profiles obtained by API systems cannot 
be able to distinguish among all the species. However, the MicroLog system 
is the only one to discriminate at species level. In order to characterize these 
species this system detects mycolic acids which are only present in mycolata 
microorganisms. 

Nocardia strains have been characterized using primers previously 
described by other authors with the use of the polimerase chain reaction 
(PCR). Moreover, Gordonia (which is the genus most frequently associated 
with foam problems) and Rhodococcus strains isolated from activated sludge 
have been characterized with primers designed in this work. Besides, the 
opportunistic pathogen species R. equi have been detected using specific 
primers. 

The output in the isolation of mycolata species has been improved by 
three decontamination treatments applied to foam samples reducing the fast 
growth of the microbiota. Three culture media have been selected to isolate 
the most important genera of mycolata found in foam. 
Fluorescent in situ hybridization technique (FISH) has been used to 
directly detect the main foam-producing species avoiding the tedious and 
long-time consuming traditional culture methods. Two cell permeabilization 
methods have been optimized to allow the access of different probes into the 
target. Initially, the general Bacteria specific probe EUB 338 was used to 
compare the efficiency of both methods. Although with both methods no 
significant differences have been observed, the method proposed by De los 
Reyes et al. (1998) is considered more suitable than the one proposed by 
Carr et al. (2003) due to the absent of disturbance of the cell morphology. 

Furthermore, specific probes (Myb-736 and Myc-657) were used to 
detect the mycolata group. Gordonia amarae have been also detected by the 
use of a species-specific probe G.am-205. With this probe, G. amarae strains 
have been detected in both pure culture and in environmental samples. It 
was also observed that enrichment of foam samples increases the 
hybridization signals.