Copper-resistance in Enterobacteriaceae and other Proteobacteria from children’s intestine Sánchez-Valenzuela A, Morales E, Rodríguez M, López-Espinosa MJ, Coque MT, Sunyer J, Baquero F. Department of Microbiology, Ramón y Cajal University Hospital, IRYCIS, Madrid, and Spanish Project for Environment and Childhood Research (INMA) Sabadell, Spain. INTRODUCTION MATERIALS AND METHODS
The interest of the study of copper (Cu) resistance in the gut flora is based on: A total of 167 intestinal samples from healthy children (≤ 8 years old) from different Spanish a) The need of detecting Cu-R potentially pathogenic bacteria, as Cu is increasingly towns were plated on MacConkey Petri square plates, containing a copper sulfate gradient used as disinfectant in farms, but also in intensive care unit in hospitals. ranging from 0 to 512 µg/ml (0 – 2.05mM). Due to the absence (in academia and probably in b) The possible cross resistance between Cu-R and antibiotic resistance. nature) of well-defined cut-offs defining copper susceptibility or resistance, and the expected c) Cu-R organisms as markers of the exposure of humans to this heavy metal. gradient distribution of copper concentrations in nature, the evaluation of the inhibitory effect of copper was done by dividing the surface of the plate in three identical sectors, corresponding to We studied the prevalence of Cu-R in fecal samples of children belonging to the cohort copper concentrations of < 170 µg/ml, 170-340 µg/ml, and >340 µg/ml. Colonies growing beyond of Spanish Project for Environment and Childhood Research (INMA, a threshold of 340 µg/ml (1.36mM) were considered presumptively as copper-resistant, and http://www.proyectoinma.org/). identified by MALDI-TOF technology.
RESULTS Table 1. Percentage of positive samples per number of resistant species. Resistant species per sample Fig.1. Percentage of positive samples containing high (blue) or intermediate (red) copper-resistant colonies. 1 sp 2 sp 3 sp 4 sp MIC 340-512 µg/ml 61,46% 27,52% 9,17% 1,83% MIC 170-340 µg/ml 90,11% 9,89% - - % Table 2. Frequency of Cu-R colonies (170-340, 340-512 µg/ml) among resistant (>170 µg/ml) strains of bacterial genera. Genera Escherichia Enterobacter Citrobacter Klebsiella Raoultella Pseudomonas MIC 340-512 µg/ml 11,19 25 32,68 31,24 4/6 5/5 MIC 170-340 µg/ml 52,03 69,31 75,28 59,6 2/6 - Genera Acinetobacter Kluyvera Hafnia Comamonas Non-identified MIC 340-512 µg/ml 2/2 1/1 1/1 1/1 6/9 MIC 170-340 µg/ml - - - - 6/9
Considering all 69 isolates of predominantly environmental species, where high copper resistance reaches 30.72%, the proportion of Cu-R among all 207 E. coli (predominantly enteric) is significantly lower, 11.19% (p<0.001). At MIC 170-340 µg/ml, where Cu-R reaches 70.57% on environmental species, the proportion of Cu-R among all E. coli (predominantly enteric) is also significantly lower, 52.03% (p<0.01).
CONCLUSIONS REFERENCES
-The Cu-R is significantly higher in predominantly environmental microorganisms, - Bertazzo A et al (1996). Trace Elem Res, 52: 37-53. suggesting that environmental pollution with copper could be the source of copper - Heijerick DG et al (2006). Environ Toxicol Chem, 25: 858-864. resistance in bacteria from children’s intestine. - Callan AC et al (2012). Arch Environ Contam Toxicol, 62: 714-722. - Glorennec P et al (2012). Environ Int, 45: 129-134. - Oyoo-Okoth E et al (2013). Int J Hyg Environ Health, 216: 8-16.