Mohammad Ahangarzadeh Rezaee MSc*, Qorban Behzadiyan-Nejad PhD*, Shahin Najjar Pirayeh MSc*, Parviz Owlia PhD**^●

Pseudomonas aeruginosa is nearly ubiquitous in nature and is quite innocu-ous in most environments. However, P. aeruginosa can cause severe and life-threatening infections in immunosuppressed hosts such as patients with burns; patients suffering from respiratory diseases, chemotherapy cancer patients, and children and young adults with cystic fibrosis.^1–4 This opportunistic pathogen produces a number of unique virulence factors. Extracellular toxins, proteases, hemolysins, and exopolysacchar-ides are some of the types of virulence factors that have been implicated in the pathogenicity of P. aeruginosa.^1,5–7

The extracellular glycocalyx (alginate) is produced in copious amounts by mucoid strains of P. aeruginosa. Alginate is a linear copolymer composed of D-mannuronic acid and L-gluronic acid.^2,8 There is also evidence that alginate provides a barrier against penetration by aminoglycosides.² The aim of this study was to compare the levels of resistance of mucoid and non-mucoid strains of P. aeruginosa to gentamicin, amikacin and tobramycin.

Table. Comparative in vitro resistance of mucoid and non-mucoid isolates of Pseudomonas aeruginosa to antibiotics.

M = mucoid; NM = non-mucoid.

Bacterial suspensions of 10⁴ CFU/mL were spread onto the surface of Müller-Hinton agar plates by swab. Fifteen minutes after inoculating the plates with bacteria, three antimicrobial disks were applied using sterile forceps. The plates were inverted and incubated overnight at 35°C for 18 hours, after which the diameter of each zone of inhibition was measured.⁹

Mucoid strains were identified using the Muir method,¹⁰ in which a thin film of suspension was prepared and air-dried, the film was covered with a piece of filter paper the size of the smear, and the slide was flooded with Ziehl-Neelsen carbol fuchsin and heated to steaming for 30 seconds by a low Bunsen flame.

The slide was gently rinsed with 95% ethanol and then with distilled water. Mordant solution was added for 20 seconds and then washed with distilled water. Decolorization was performed using ethanol. For counterstaining, 0.3% methylene blue was used for 30 seconds prior to examination of the preparations under the oil immersion lens. The cells were stained red, and the capsules blue.¹⁰

The susceptibility of mucoid and non-mucoid strains of P. aeruginosa to the antibiotics were determined using SAS, version 6. A p value of less than 0.05 was considered significant.

From the 133 clinically isolated P. aeruginosa strains, 43 were identified to be mucoid by the Muir staining method.

The in vitro activities of antibiotics against mucoid and non-mucoid strains of P. aeruginosa are shown in the Table. Overall, there was more to gentamicin (n = 103) and tobramycin (n = 101) than to amikacin (n = 80) (Figure).

Statistical analysis showed that mucoid strains were significantly more resistant than non-mucoid strains to gentamicin, amikacin and tobramycin (p = 0.001, 0.0002 and 0.009, respectively).

It appears that alginate, an extracellular glycocalyx, probably acts as a barrier against aminoglycosides.² In earlier studies, we showed that subinhibitory concentrations of gentamicin reduced alginate production in mucoid strains of P. aeruginosa.^11,12 Govan and Fyfe reported that the mucoid forms of P. aeruginosa are more resistant to carbencillin, flucloxacillin and tobramycin than the non-mucoid isolates.¹³ In contrast, Demko and Thomassen observed increased sensitivity of alginate-producing strains to carbencillin, tobra-mycin and ticarcillin.⁵ Nevertheless, there is evidence that alginate provides an ionic barrier against penetration of aminoglycoside antibiotics.² Slack and Nichols used antibiotic diffusion through agar as a criterion for direct measurement of the permeability of the alginate layer to antibiotics.⁷ They found that, with the exception of β-lactams, alginate did in fact impede the penetration of antibiotics such as aminoglycosides. However, Gordon et al observed that the alginate-to-anti-biotic ratio could greatly influence the perceived permeability barrier. When this ratio is high, aminoglycosides (but not β-lactams) are retained in the alginate layer. However, low alginate-to-antibiotic ratios quickly result in disruption of the gel structure and faster penetration of amino-glycosides.⁸ They suggested that high levels of antibiotic saturate the negative charge of alginate and result in a breakdown in the permeability layer. In a study by Rastegar Lari of 2,122 patients admitted to Tohid Burn Center in Tehran from 1995–1997, 3,365 bacterial strains including P. aeroginosa (73.39%), Staphylococcus aureus (9.1%) and other organisms (17%) were isolated.¹⁴ The frequency of P. aeruginosa resistance to gentamicin, carbencillin, cotrimoxazole, ceftizox-ime and tetracycline was over 95% in their study.

Figure. In vitro activities of gentamicin, amikacin and tobramycin against 133 clinical isolates of P. aeruginosa.

Ciofu et al characterized 42 paired mucoid and non-mucoid P. aeruginosa isolates collected from Danish cystic fibrosis patients in 1997 by RiboPrinting, antibiotic susceptibility (cipro-floxacin, tobramycin, meropenem, colistin, ceftazidime, piperacillin, azteronam, meropenem) and b -lactamase activity.¹⁵ They determined that the non-mucoid/mucoid minimum inhibitory concentration (MIC) ratio for tobramycin was 1.3, and that mucoid isolates were generally more susceptible to antibiotics. This result is in direct contrast to our findings and may be due to the differences in the methods used: they determined susceptibility by MIC, whereas we used the disk diffusion method.

In the current investigation, mucoid strains of P. aeroginosa were more resistant to gentamicin, amikacin and tobramycin as compared to non-mucoid strains. Overall, there was more resistance to gentamicin, followed by tobramycin and amikacin.

The results of this study suggest that the capsule may act as a barrier against aminoglycosides. Results indicate that familiarity with the type of P. aeruginosa (mucoid or non-mucoid) can aid in the selection of appropriate antibiotic therapy. Nevertheless, antibiograms must be performed before antibiotic therapy is begun.