Survival of Staphylococcus pseudintermedius in

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fluid resulted in false positive diagnosis of Gram-negative ..... Staphylococcus pseudintermedius fue capaz de sobrevivir en soluciones de tinción Quick.
Vet Dermatol 2017; 28: 333–e71

DOI: 10.1111/vde.12435

Survival of Staphylococcus pseudintermedius in modified Romanowsky staining solutions Angus Misan, Wei Yee Chan

, Darren Trott and Peter B. Hill

School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy, South Australia 5371, Australia Correspondence: Peter B. Hill, Companion Animal Health Centre, School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy SA 5371, Australia. E-mail [email protected]

Background – Stains that are used regularly for patient-side diagnosis to rapidly identify bacterial and fungal infections could become contaminated by common pathogens, such as Staphylococcus pseudintermedius, during slide immersion. Hypothesis/Objectives – To determine whether the inoculation of S. pseudintermedius into modified Romanowsky type stains (Quick Dipâ) results in viable bacterial contamination and whether this is influenced by the addition of organic debris (canine hair and skin). Methods – A clinical isolate of S. pseudintermedius was inoculated into clean and organically contaminated Quick Dipâ solutions (methanol fixative, eosin, methylene blue), and positive (broth) and negative (bleach) controls. Each solution was tested for the presence of viable bacteria by counting the number of colony forming units (CFU/mL) at various time points. Solutions also were examined under high power microscopy to count the number of visible bacteria at each time point. Results – Staphylococcus pseudintermedius was able to survive in the clean and contaminated Quick Dipâ stains for at least one hour, but by 24 h no viable bacteria remained. Survival of the bacteria was not supported in the fixative at any time point. Staphylococcus pseudintermedius remained visible under high power microscopy for up to 2 weeks in all organically contaminated solutions of the Quick Dipâ set. Conclusions and clinical importance – Staphylococcus pseudintermedius only remains viable in eosin and methylene blue for short periods of time, but the prolonged visibility of dead organisms could theoretically lead to the misdiagnosis of cytology samples.

Introduction One of the most commonly used staining procedures in clinical cytology is the sequential application of methanol fixative, eosin and methylene blue (a modification of the Romanowsky–Giemsa stain, commonly referred to as DiffQuikâ or Quick Dipâ). These stains are used to rapidly identify bacterial and fungal infections for patient-side diagnosis. The recommended staining procedure for these stains involves dipping glass slides into the staining solutions, rather than adding stains to the slides. Therefore, during staining of specimens for cutaneous cytological examination it is theoretically possible that bacteria or fungi on slides could be transferred to the staining solutions, leading to contamination. This would become increasingly likely if staining solutions were used frequently and not often replaced. If the stain contaminants were able to survive for a prolonged period, samples subsequently stained may be misdiagnosed. Therefore, it is advisable to determine whether or not common bacteria are able to survive in such stains as viable contaminants.

Accepted 20 January 2017 Sources of Funding: This project was funded by the School of Animal and Veterinary Sciences, University of Adelaide. Conflict of Interest: No conflicts of interest have been declared. © 2017 ESVD and ACVD, Veterinary Dermatology, 28, 333–e71.

Some stains are known to have bactericidal properties whereas others have no disinfectant action and allow growth of bacteria.1,2 Previous studies have shown that Pseudomonas aeruginosa can survive for variable periods of time in eosin and methylene blue stains of the Quick Dipâ set,1 and that Merck’s methylene blue has minimal disinfectant action.2 However, both gentian and crystal violet stains have significant disinfectant action against Gram-positive bacteria, including the Staphylococcus genus.2 In several circumstances, contamination of stains has led to false positive results. For example, fungal contamination of Grocott light-green counterstain solution led to the misdiagnosis of yeast infections in two separate cases.3 Gram-stained clinical samples of cerebrospinal fluid resulted in false positive diagnosis of Gram-negative bacilli due to bacterial contamination of the piped deionized water that was used to make the Gram stain solutions.4 Previous studies also have highlighted misdiagnosis and unnecessary therapy following contamination of other media, including Gram-negative bacilli in Amies transport medium used for the transport of swabs,5 and fungal contaminants in Hanks’ solution.6 Staphylococcus pseudintermedius is the most common cutaneous pathogen in the dog, regularly isolated from cases of pyoderma and otitis externa.7,8 This pathogen is commonly stained using Romanowsky-type stains. There have been no previously published data on whether or not this common pathogen is able to survive in and 333

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contaminate staining solutions. Staphylococcus pseudintermedius is a common contaminant of the environment, contaminating clothing in veterinary hospitals,9 and household areas commonly used by pets.10 Although it is unknown if stains are bactericidal to S. pseudintermedius, previous research indicates that bleach is bactericidal, even in low concentrations,8 and that oils derived from natural products can inhibit growth.7 The aim of this study was to determine whether the inoculation of S. pseudintermedius into eosin and methylene blue stains could result in viable bacterial contamination and whether this is influenced by the addition of organic debris (canine hair and skin).

Materials and methods Bacteria and inoculum size A methicillin-sensitive isolate of S. pseudintermedius originating from a dog with otitis externa was used for this study (isolate number V13/2/347). The isolate was stored in brain heart infusion broth (Becton Dickinson Pty Ltd; Sydney, NSW, Australia) with 20% glycerol (Ajax Finechem; Cheltenham, Victoria, Australia) at 80°C. Fresh cultures were grown on 5% Columbia sheep blood agar (Thermo Fisher Scientific; Melbourne, Victoria, Australia) and incubated at 37°C for 18 h. Fresh pure colonies were suspended in 7 mL of phosphate buffered saline (PBS) pH 7.3 (Thermo Fisher Scientific) to generate a 0.5 McFarland standard with a corresponding optical density at 600 nm of 0.1 (0.08–0.13) as determined by a spectrophotometer (BioPhotometer plus, Eppendorf; Macquarie Park, Australia). Preliminary studies found that this suspension contained approximately 1.5 9 108 colony forming units (CFU)/mL. An inoculation volume was calculated by determining the concentration of solution required to be able to visualize and count bacteria under 10009 oil-immersion magnification. It was concluded that a solution with 7 9 106 CFU/mL allowed sufficient visualization of bacteria after 1 h of incubation at room temperature in Mueller–Hinton broth. This concentration was achieved for each experiment by inoculating 2.86 mL of solution (controls, fixative, eosin, methylene blue) with a 140 lL aliquot of a 0.5 McFarland standard suspension to make up a 3 mL solution.

Staining and control solutions The positive control for this experiment was cation-adjusted Mueller– Hinton broth (Becton Dickinson Pty Ltd). The negative control was sodium hypochlorite (White King bleach, 42 g/L, Pental Products; Shepparton, Victoria, Australia). The staining system used was Quick Dipâ (Point of Care Diagnostics Scientific; Artarmon, New South Wales, Australia). The staining set comprised methanol (>99% w/v) as a fixative, Quick Dipâ 1 stain (eosin Y