J Clin Pathol: first published as 10.1136/jcp.33.3.288 on 1 March 1980. Downloaded from J Clin Patho/ 1980; 33: 288-296 Antiseptic and antibiotic resistance in Gram-negative bacteria causing urinary tract infection DJ STICKLER AND B THOMAS From the Department of Applied Biology, University of Wales Institute of Science and Technology. King Edward V1I A venue, Cardiff UK SUMMARY A collection of 802 isolates of Gram-negative bacteria causing urinary tract infections was made from general practice, antenatal clinics, and local hospitals. The organisms were tested for their sensitivity to chlorhexidine, cetrimide, glutaraldehyde, phenyl mercuric nitrate, a phenolic formulation, and a proprietary antiseptic containing a mixture of picloxydine, octyl phenoxy polyethoxyethanol, and benzalkonium chloride. Escherichia coli, the major species isolated, proved to be uniformly sensitive to these agents. Approximately 10°,! of the total number of isolates, however, exhibited a degree of resistance to the cationic agents. These resistant organisms were members of the genera Proteus, Providencia, and Pseudomonas; they were also generally resistant to five, six, or seven antibiotics. It is proposed therefore that an antiseptic policy which involves the intensive use of cationic antiseptics might lead to the selection of a flora of notoriously drug- resistant species. copyright. During the course of studies on the development of minimum inhibitory concentrations (MICs) of up urinary tract infection in patients undergoing to 800 ,sg/ml chlorhexidine,'4 well above the level of intermittent bladder catheterisation in the early 10-50 jug/ml orginally reported to inhibit the growth stages of paraplegia, observations were made on the of Gram-negative species.5 effect of the repeated application of the antiseptic These chlorhexidine-resistant strains were isolated http://jcp.bmj.com/ chlorhexidine on the bacterial flora of the urethral from an unusual clinical situation in which repeated meatus.' 2 The bladder management of these patients exposure to the antiseptic over periods of up to involved the washing of the penis with aqueous 3-4 weeks takes place. It is perhaps understandable solutions of chlorhexidine (600 jtg/ml) before the that resistant strains should be isolated from a insertion of the catheter at approximately 8-hour situation where such a strong selective pressure is intervals. The flora was examined daily before and operating. However, as resistance to agents like after application of the antiseptic, and the general chlorhexidine is not generally screened for in clinical on September 24, 2021 by guest. Protected pattern that emerged from this work was that for laboratories it is not possible to say whether resis- the first few days after trauma the meatus carried a tance to antiseptics is limited to such special cir- Gram-positive flora which the chlorhexidine usually cumstances or is a more general phenomenon. The eliminated. A Gram-negative population developed objective of this study was to observe the sensitivity subsequently, however, and proved to be more of Gram-negative organisms causing urinary tract refractory to chlorhexidine. In particular, Proteus infections, in a variety of types of patients, to a mirabilis, Pseudomonas aeruginosa, Providencia number of antiseptics and disinfectants. A col- stuartii, and Klebsiella frequently survived the lection of organisms was therefore made from application of the antiseptic, and in laboratory general practice, antenatal clinics, and inpatients at tests many of the strains demonstrated an ability to five local hospitals. The sensitivity of these isolates grow in media containing 200 ,ug/ml of the agent. to chlorhexidine, cetrimide, glutaraldehyde, phenyl Subsequently, some of the Pr. mirabilis and P. mercuric nitrate, a phenolic formulation, and a stuartii strains from this source were shown to have proprietary antiseptic containing a mixture of picloxydine, octylphenoxy polyethoxyethanol, and benzalkonium chloride was determined. In addition, Received for publication 5 September 1979 the antibiotic resistance patterns were recorded to 288 J Clin Pathol: first published as 10.1136/jcp.33.3.288 on 1 March 1980. Downloaded from Antiseptic and antibiotic resistance in Gram-negative bacteria causing urinary tract infections 289 explore any possible relationship between disinfect- colony formation after overnight incubation at 370C ant and drug resistance. was taken as the MIC for that strain. The tests were routinely performed in duplicate and, with each Methods and material batch of strains tested, E. coli 10418 and Pr. mirabilis 61 were included as control reference organisms with BACTERIAL STRAINS known MICs. The clinical isolates used in this study all originated from cases ofsignificant bacilluria ( > 105 viable cells/ ANTIBIOTIC SENSITIVITY ml urine). They were obtained over a 12-month The antibiotic sensitivity patterns of the isolates were period from a hospital laboratory which served determined by seeding plates of DST agar (Oxoid several hospitals and local health centres in the Ltd) with young log phase broth cultures (0-1 ml) Cardiff area. To avoid duplication, repeated and applying U3 Multodiscs (Oxoid Ltd), which are isolates of the same species from individual patients impregnated with the following antibiotics: gen- were excluded from the survey. The organisms were tamicin (10 )ug), colistin (10 ,pg), nitrofurantoin identified using the methods of Cowan and Steel6 (200 pug), sulphafurazole (500 tig), kanamycin (30 and in some cases with the API 20E Microtube ,ug), ampicillin (25 jug), sulphamethoxazole/tri- System (API Products Ltd). Escherichia coli NCTC methoprim (25 ,tg), and tetracycline (50 ,ug). E. 10418 and Pr. mirabilis 613 were used as control coli NCTC 10418 was used as sensitive control with organisms in the sensitivity testing. each batch of strains tests. ANTIBACTERIAL AGENTS Results The chlorhexidine digluconate and the cetrimide were obtained from ICI Ltd. Glutaraldehyde was In a 12-month period, over 1000 isolates of Gram- purchased from Sigma Ltd and phenyl mercuric negative bacteria were obtained from urinary nitrate from BDH Ltd. The phenolic preparation infections. E. coli proved to be by far the most contained 16% v/v of a mixture of chloroxylenol, prevalent species, and after six months when some copyright. chlorocresol, chlorophene, sodium 0-phenylphenate, 369 E. coli isolates had been investigated, the sen- and sodium pentachlorophenate in a basis of sitivity testing of this species was discontinued. The anionic emulsifier and alcohol (Hycolin, Pearson species distribution of the isolates examined in the Ltd. Clough Road, Hull). The sixth antibacterial survey is shown in Table 1. The results of the sen- agent was the proprietary antiseptic Resiguard sitivities of these organisms to the six antibacterials (Nicholas Laboratories Ltd) which contains pic- are summarised in Figures 1 to 6. The histograms loxydine digluconate (1 % w/v), octyl phenoxy represent the percentage of isolates having each MIC, http://jcp.bmj.com/ polyethoxyethanol (11-0% w/v), and benzalkonium grouped on the basis of the multiplicity of their chloride (12% w/v). antibiotic resistance. It can be seen that E. coli was uniformly sensitive to the antibacterial, having MIC DETERMINATIONS MICs of the six agents equivalent to that of the As the inoculum history of bacterial cultures used in control E. coli strain 10418 and well below the antimicrobial sensitivity tests may affect the results recommended use concentrations of these agents. obtained7 a standard procedure was adopted for The situation is different with Proteus, P. stuartii, on September 24, 2021 by guest. Protected testing the clinical isolates. A single colony from a isolated urinary tract pure culture of each strain growing on MacConkey Table 1 Bacterial species from infections over a 12-month period agar after overnight incubation at 370C was inocu- lated into nutrient broth (Oxoid Nutrient Broth No. Species Number of isolates 2). After 18 hours' incubation at 370C, volumes (5 of a 10-4 dilution of the broth culture, each E. coli 369* Atl) Pr. mirabilis 139 containing approximately 103 viable cells, were Pr. morganii 31 dropped on to the surface of nutrient agar plates Pr. vulgaris 10 Pr. rettgeri 1 containing various concentrations of the anti- Klebsiella 167 bacterials. A standard method was adopted for the P. stuartii 24 The were added to Ps. aeruginosa 35 preparation of the plates. agents Citrobacter 12 molten agar that had been allowed to cool to 500C, Enterobacter 9 and the plates were poured immediately. When set, Acinetobacter 3 then Serratia marcesens 2 the plates were dried for 20 minutes at 370C and Total 802 used directly. The lowest concentration of the agent preventing *Number of isolates obtained in first six months of the study. J Clin Pathol: first published as 10.1136/jcp.33.3.288 on 1 March 1980. Downloaded from 290 Stickler and Thomas No of isolates in each group MIC of Multiplicity E col' 10418 of antibiotic <.1fA/m resistance 100 u U 0 0) 52 ,u 0 0C 29 copyright. E col/i Proteus Klebsiel /a Pseudomonas http://jcp.bmj.com/ Providen cia I Other species 5 a 10 50 200 800 1600 ;10 50 200 800 1600 MIC Chlorhexidine (Jug/ml) on September 24, 2021 by guest. Protected Fig. 1 Sensitivity of the isolates to chlorhexidine. and Pseudomonas, where MICs substantially higher antiseptics and disinfectants are concerned. For the than those of the control strain were recorded for the purpose of this study it was decided that if the MIC cationic antiseptics. value for an organism was greater than the con- In deciding whether strains are resistant or centration of the agent normally recommended for sensitive to antiseptics or disinfectants, it is difficult routine use, then that isolate was designated as to know what criterion to apply. With antibiotics it resistant. When this criterion is applied (Table 2) it is usual to designate organisms as resistant if they appears that all the isolates were sensitive to are able to multiply in the maximum concentration glutaraldehyde, the phenolic mixture, and phenyl- of the drug attainable at the site of the infection.