In vitro study of the effect of cationic biocides on bacterial population dynamics and susceptibility

Abstract

Cationic biocides (CBs) are widely used in domestic and public hygiene and to control biofouling and microbial contamination in industry. The increased use of biocides has led to concern regarding possible reductions in biocide effectiveness. Domestic drain microcosms were stabilized for 5 months and then exposed to polyhexamethylene biguanide (PHMB) at 0.1, 0.2, and 0.4g liter(-1) over 6 months and characterized throughout by differential culture, together with eubacterial-specific PCR-denaturing gradient gel electrophoresis. Additionally, MICs and minimal bactericidal concentrations (MBCs) for bacteria previously isolated from a domestic drain (n = 18) and the human skin (n = 13) were determined before, during, and after escalating, sublethal exposure (14 passages) to two quaternary ammonium compounds (QAC1 and QAC2), the bisbiguanide chlorhexidine (CHX), and PHMB. Exposure of the drain microcosm to PHMB did not decrease the total viable count although significant (P < 0.01) decreases in recovery were observed for the gram-positive cocci with associated clonal expansion of pseudomonads (from ca. 0.1% of the population to ca. 10%). This clonal expansion was also manifested as elevations in bacteria that could grow in the presence of PHMB, CHX, and QAC1. Decreases in susceptibility (greater than twofold) occurred for 10/31 of the test bacteria for QAC1, 14/31 for QAC2, 10/31 for CHX, and 7/31 for PHMB. Exposure of microcosms to PHMB targeted gram-positive species and caused the clonal expansion of pseudomonads. In terms of prolonged-sublethal passage on CBs, exposure to all the biocides tested resulted in susceptibility decreases for a proportion of test bacteria, but refractory clones were not generated.

FIG. 1.

Total and viable counts of domestic drain microcosm biofilms before and throughout exposure to increasing amounts of PHMB, as indicated by the dotted lines (mg ml⁻1): a, 0.1; b, 0.2; and c, 0.4. Data are means ± standard deviations from two separate sample pans analyzed in triplicate. Filled circles, total cell count; open triangles, vital cell count; filled squares, dead cell count (BacLight); open circles, viable cell count (plate counts).

FIG. 2.

Negative image of a parallel DGGE gel showing eubacterial community fingerprints for microcosm samples before PHMB addition and after a 2-month exposure to PHMB at 0.1, 0.2, and 0.4 g liter⁻¹ and control untreated fermentor. Excised band identities (a to o) are given in Table 9.