Detection of Antibiotic and Heavy Metal Resistance Genes

There have been relatively few studies focusing on detection of antibiotic or heavy metal resistance genes in situ, in environments such as soil. Most research has concentrated on detection of resistance genes in clinical and environmental isolates. Resistance genes have been detected in soil using conventional DNA extraction and PCR methods. Chee-Sanford et al. (2001) screened for eight tet genes in groundwater associated with swine production facilities. Tetracycline resistance genes were found as far as 250 m

Table 11.1. Cloning and sequencing of aromatic PKS gene clusters from actinomycetes (reproduced from Hopwood 1997)

Host

Polyketide

PKS genes

Cloning strategy

S. coelicolor

actinorhodin

act

Complementation

S. rimosus

oxytetracycline

otc

Resistance/

complementation

S. glaucescens

tetracenomycin

tcm

Complementation

S. violaceoruber

granaticin

gra

act probe

S. coelicolor

spore pigment

whiE

Complementation/act probe

S. peucetius

daunorubicin

dps

act/tcm probes

S. cinnamonensis

unknown

mon

act probe

S. halstedii

spore pigment

sch

act probe

S. curacoi

spore pigment

cur

act probe

Sac. hirsuta

unknown

hir

act probe

S. roseofulvus

frenolicin

fren

act probe

S. griseus

griseusin

gris

act probe

S. venezuelae

jadomycin

jad

act probe

S.spp. C5

daunorubicin

dau

act probe

Kib. aridum

unknown

ard

act probe

S. fradiae

urdamycin

urd

tcm/act probes

S. nogalater

nogalamycin

sno

act probe

S. argillaceus

mithramycin

mtm

act probe

downstream from waste lagoons, highlighting the danger posed by use of antibiotics in agriculture and the risk of contamination of drinking water with antibiotic resistant bacteria. In a different study a detection limit of 102-103 copies of the tet(M) gene per gram was achieved using a nested PCR method with TC-DNA (Agerso et al. 2004). The gene was detected in farmland soil previously amended with pig slurry containing resistant bacteria; the number of positive samples from farmland soils 1 year after manure treatment was significantly higher than in samples of garden soil not treated with manure.

Exogenous plasmid isolation has been used to detect resistance genes in soil bacteria. This method allows capture of plasmids from the total bacterial fraction of an environmental sample without the necessity to culture the host organism. Smit et al. (1998) investigated mercury resistance plasmids in soil populations using exogenous isolation, and identified plasmids of 10-50 kb carrying resistance to copper, streptomycin and chlorampheni-col. These authors amended soil with mercuric chloride and found this to subsequently increase the recovery of resistance plasmids. Plasmids have also been captured from polluted soils and slurrys (Top et al. 1994; Smalla et al. 2000). The former authors identified multiple antibiotic resistance genes from isolated plasmids.

In addition to multi-resistance plasmids antibiotic resistance genes are situated on transposable elements that can associate with other elements such as chromosomes and plasmids. These transposable elements include transposons and integrons, which can be transferred horizontally. Integrons are thought to play an important role in the evolution of antibiotic resistance as they contain mobile gene cassettes which bear a recombination site known as a 59-base element (59-be) that is recognised by the integron-encoded integrase IntI (Hall et al. 1991). Most cassette genes described are antibiotic or quaternary ammonium compound resistance genes. However, recent studies have revealed that the cassette gene pool is far more diverse than previously thought. Stokes et al. (2001) designed PCR primers to conserved regions within the 59-be of gene cassettes, allowing detection of a large number of novel genes. Using these primers, 123 cassette types were recovered from Antarctic and Australian soils and sediments, with very few represented in clone libraries more than once, indicating the large size of the cassette gene pool. Most open reading frames (ORFs) did not match known sequences, again illustrating the diversity of these gene sequences. Further studies revealed a further 41 environmental gene cassettes, giving a total of 164 directly sampled from natural environments by PCR (Holmes et al. 2003). There are several classes of integrons, the most commonly studied being class 1 integrons that are commonly associated with antibiotic-resistant bacteria. Recent studies have detected novel integron classes in soils: Nield et al. (2001) classified three new integron classes and Nemergut et al. (2004) an additional 14 classes, demonstrating the immense variety of these elements present in the environment.

Antibiotic resistance genes have proved useful as molecular markers for studying the antibiotic-producing streptomycetes in soil. Self-resistance genes in antibiotic-producing bacteria are usually clustered with the biosynthesis genes and may indicate potential for antagonism in addition to resistance. The presence of indigenous antibiotic resistance and production genes in soil TC-DNA was investigated and allowed detection of specific genesbyPCRamplification. Thegenesinvolvedinstreptomycinproduction were detected in diverse samples of natural and agricultural Brazilian soils (Huddleston et al. 1997). This indicated the presence of a population of actinomycetes with the potential for antibiotic production, probably in excess of 103 colony-forming units (cfu) g-1, which is about the detection limit for DNA from soil samples using PCR. Using a similar approach the distribution of the gene sttR involved in self-resistance to streptothricin was studied in a wide range of soils from Europe and sub-tropical regions. DNA sequencing of the PCR products from TC-DNA from two Cuban soils, sugar cane and maize soil, were the only samples to provide PCR products. Since the first round of PCR yielded a small amount of DNA that was not sufficient for DNA sequencing, the PCR products were reamplified with the same primers before being sequentially purified and sequenced (Watyam 2003; Anukool et al. 2004). The Blast results confirmed that these sequences had homology to streptothricin resistance genes from known streptothricin producers in the databases.

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