Comparison of bacterial culture results obtained from three different sampling locations in dogs and cats with chronic nasal disease

ABSTRACT

Aims

To assess agreement of bacterial culture results from samples taken from nasal discharge, the nasal cavity and nasal biopsy from dogs and cats with nasal disease.

Methods

Nineteen dogs and 21 cats with different nasal diseases (chronic rhinitis, n = 30; neoplasia, n = 7; sinonasal aspergillosis, n = 3) were prospectively enrolled in the study. Nasal swabs were taken bilaterally from nasal discharge at the nares, the nasal cavity, and one nasal mucosal biopsy per side. All samples were subjected to aerobic bacterial culture. Kappa statistics were used to evaluate agreement for the most prevalent bacterial species between sampling sites.

Results

A positive culture result for at least one bacterial species was detected in 80% of samples from nasal discharge/nares, 92% of nasal cavity samples, and 75% of biopsy samples. The mean agreement between the three sampling sites for positive vs. negative culture results was never greater than moderate and the precision of the estimates of agreement varied widely.

The most frequently isolated bacterial species in dogs were Staphylococcus pseudintermedius, Staphylococcus spp. and Streptococcus spp. In cats, Pasteurella spp. and Staphylococcus felis were the bacterial species cultured most frequently.

For the most prevalent cultured species, Staphylococcus spp., mean agreement between sites was never greater than fair and the precision again varied widely.

Conclusion

This study indicates that bacterial culture results in feline and canine nasal disease are site-specific and there was no evidence from this study for consistency between sites within a patient for many bacterial species. Consequently, if bacterial culture results from nasal swabs are used to guide therapeutic antimicrobial choice, different treatments may be selected depending on the site of culture. As a consequence, there is no evidence from this study that nasal bacterial cultures should be recommended as a routine diagnostic measure.

KEYWORDS:

• Bacterial infection
• canine
• feline
• chronic rhinitis
• nasal neoplasia

Introduction

The bodies of animals and humans are populated with a multitude of different bacterial species (Li et al. 2019). Most interactions between host and microorganism do not result in disease, because the two entities have a symbiotic relationship (Dethlefsen et al. 2007). As the commensal bacterial flora is well developed in the upper airways, interpretation of bacterial culture results from that area is difficult, and the rationale for antibiotic treatment is questionable in many cases. Primary bacterial rhinitis is considered to be an uncommon cause of chronic nasal disease in dogs and cats. However, mucosal impairment and secondary bacterial infection are common and can be induced by different causes, including inflammatory chronic rhinitis; fungal, viral or parasitic infection; neoplasia; foreign bodies; or dental-related nasal disease (Cohn 2020; Reed 2020). Antibiotic treatment frequently results in temporary improvement of clinical signs (Cohn 2020). An aetiological role for bacteria has been discussed in dogs and cats with chronic rhinitis, as most of these patients show considerable improvement in clinical signs after receiving antibiotics (Lobetti 2009).

Surveys of the bacterial microflora of the nasal cavities, tonsils and pharynx of clinically healthy dogs and cats have found many types of aerobic and facultative anaerobic bacteria. Greater numbers of organisms can normally be cultured from the rostral rather than from the caudal nasal cavity. Although there are marked individual variations in nasal and pharyngeal bacteria, a specific range of bacterial species can be found in both locations (Smith 1961; Clapper and Meade 1963; Lee-Fowler and Reinero 2012).

Some authors have postulated that cultivating nasal discharge might be of limited benefit, because commensal bacteria of the oropharynx will likely be detected (Schulz et al. 2006) and as primary bacterial rhinitis is considered a rare condition, bacterial cultures from nasal samples are thought to be of little use or even misleading (Cohn 2020). Others recommend deep tissue or swab samples from the nasal cavity for bacterial culture instead (Cohn 2020). However, it has been shown that the sampling method can influence nasal culture results in cats (Johnson and Kass 2009) and it remains unclear which bacterial sampling method and location should be used in dogs and cats with chronic nasal disease or whether cultivating nasal discharge at the nares, within the nasal cavity or by nasal biopsy is still appropriate at all. Dorn et al. (2017) suggested that the focus of diagnostic efforts should rather be on evaluation of bacterial colonisation as a whole and the factors promoting bacterial overgrowth and dysbiosis in certain areas. Nevertheless, many veterinarians still focus on culture and sensitivity testing in dogs and cats with nasal disease, potentially missing the primary disease process. The significance of nasal bacterial cultures is thus controversial.

The aim of the present study was to assess the most common bacterial species cultured from dogs and cats with nasal disease from nasal discharge, the nasal cavity and nasal biopsies and to evaluate the level of agreement for positive cultures and for the most prevalent species. Our hypothesis was that bacterial cultures obtained from three different locations within the nose would yield a low level of agreement.

Materials and methods

Study design

The study was approved by the ethical committee of the Department of Veterinary Sciences of the Ludwig Maximilian University of Munich (LMU Munich, Germany) (63-12-04-2016) and was conducted from 2016 to 2019.

Dogs and cats that were presented for a diagnostic work-up of nasal disease at the Clinic of Small Animal Medicine (LMU Munich) were prospectively enrolled in the study. The diagnostic work-up included CT, rhinoscopy and collection of nasal swab and biopsy samples. Pre-treatment with antibiotics was not defined as an exclusion criterion.

Sample collection

All samples were collected while patients were under general anaesthesia for rhinoscopic work-up. Three samples per nasal side were taken from each patient: one swab (swab with Amies transport medium; Sarstedt, Nuembrecht, Germany) from nasal discharge at the nostrils or, if absent, from the nostril itself. Second, after cleaning and disinfection of the skin surrounding the nostril, the nasal cavity was sampled by advancing a swab into the nasal cavity up to the medial canthus of the eye and rotating the swab. Finally, biopsies of the nasal mucosa were taken, either guided by endoscopy during rhinoscopy or blind, using sterilised biopsy forceps after flushing the nasal cavity with sterile saline. All tissue samples were taken at the level of the medial canthus of the eye. The biopsies were transferred onto a cotton swab. All samples were submitted for culture on the day of sampling.

Bacteriological examination

Samples were submitted for bacterial culture to the diagnostic laboratory of the Institute for Infectious Diseases and Zoonoses (LMU Munich). Aerobic bacteriological culture was performed on a set of different plates including Columbia agar with and without 5% defibrinated sheep blood (BBL Columbia Agar Base; Becton Dickinson, Le Pont de Claix, France), Gassner agar (Sifin Diagnostics GmbH, Berlin, Germany), and Rambach agar (Chromocult Rambach Agar; Merck KGaA, Darmstadt, Germany). In addition, Columbia sheep-blood agar with colistin-naladixic acid (Becton Dickinson) was used for selective cultivation of Gram-positive bacteria, and Bordet-Gengou agar (Difco Bordet Gengou Agar Base; Becton Dickinson) for selective isolation of Bordetella spp. All plates were prepared in-house. Plates were incubated at 36–38°C under aerobic conditions and examined after 24 and 48 hours. Identification of every colony type was performed using a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometer (Microflex LT MALDI Biotyper; Bruker Daltonik GmbH, Bremen, Germany).

Statistical analysis

The number of animals that had a positive sample for each location and the number of animals that had a positive sample for Staphylococcus spp., were formatted descriptively in contingency tables. A bacterial culture result was defined as positive for each patient if the bacterial culture of either left, right or both sides of a sampling site yielded bacterial growth.

Given the small sample size and the number of potential confounders, differences in the bacterial flora of the nasal cavity between dogs and cats were just described qualitatively for this sample as simple proportions without CI or inferential statistical analysis.

The agreement between the three locations for positive bacterial growth and the agreement for the most prevalent bacterial species, Staphylococcus spp., was expressed using the kappa statistic for measuring agreement of a nominal variable between more than two assessment methods. A negative kappa statistic suggests a level of agreement worse than would be expected by chance, kappa near zero represents a level of agreement that would be expected by chance, and increasingly positive values represent greater agreement. The value of kappa is affected by the prevalence of the event, and the potential impact of this on the value of kappa can be measured by the prevalence index (Byrt et al. 1993). We suspected that where a positive result was defined as the isolation of any bacterial species, the proportion of positive agreement between sites would be higher than the proportion of negative agreement between sites. This will tend to reduce the unadjusted value of kappa, particularly at higher kappa values. Kappa can also be affected by disagreement between sites on the proportion of positive and negative cases. The magnitude of this effect can be estimated from the bias index and when bias is high, the unadjusted kappa value is elevated, with the greatest effect at low values of kappa (Byrt et al. 1993). Consequently, for each paired comparison, we have reported mean and 95% CI for the prevalence and bias indices, and the unadjusted and prevalence-bias adjusted values for kappa.

All analysis was conducted using the statistical software programme R (R Foundation for Statistical Computing, Vienna, Austria) and the epiR package (Stevenson and Sergeant 2024).

Results

Patient population

The study included 19 dogs and 21 cats, all privately owned. The median age for dogs was 6 (min 1, max 13) years, and for cats 7 (min 0.5, max 15) years. The 19 dogs comprised seven intact and seven neutered males, and one intact and four spayed females. The cats comprised three intact and eight neutered males and three intact and seven spayed females. The most common dog breeds were Parson Russell Terrier (n = 2), Poodle (n = 2), German Shepherd (n = 2) and Yorkshire Terrier (n = 2) and for cats, Domestic Shorthair (n = 12) and Norwegian Forest Cat (n = 2) (see Supplementary Table 1). Based upon results of the diagnostic imaging, endoscopy and histopathology, patients were categorised as having chronic rhinitis (n = 30), nasal neoplasia (n = 7), or aspergillosis (n = 3) (Table 1). Antibiotic pre-treatment within 4 weeks prior to sample collection had been administered in 12/40 (30%) patients included in the study (Supplementary Table 1).

Table 1. Distribution (number and percentage of each host species) for the underlying nasal diseases in dogs and cats included in a study comparing bacterial flora isolated from three different sampling locations.

Diagnosis	Dogs (n = 19)	Cats (n = 21)	Total (n = 40)
Chronic rhinitis	13 (68%)	17 (81%)	30 (75%)
Nasal neoplasia	3 (16%)	4 (19%)	7 (17%)
Sinonasal aspergillosis	3 (16%)	0	3 (8%)

Bacterial isolates

A total of 240 samples taken from 40 patients were submitted for bacterial culture. The numbers of animals with positive bacterial growth are presented in Table 2 and the numbers of animals positive for Staphylococcus spp. are reported in Table 3.

Table 2. Number and percentage of animals with a positive or negative culture for any aerobic bacteria from three different sampling locations in a study of 19 dogs and 21 cats with chronic nasal disease (n = 40).

Location	Positive	Negative
Nasal discharge	32 (80.0%)	8 (20.0%)
Nasal cavity	37 (92.5%)	3 (7.5%)
Nasal biopsy	30 (75.0%)	10 (25.0%)

Table 3. Number and percentage of animals with a positive or negative culture for Staphylococcus spp. from three different nasal sampling locations in a study of 19 dogs and 21 cats with chronic nasal disease (n = 40).

Location	Positive	Negative
Nasal discharge	21 (52.5%)	19 (47.5%)
Nasal cavity	31 (77.5%)	9 (27.5%)
Nasal biopsy	24 (60.0%)	16 (40.0%)

In cats, 18 Gram-positive and 10 Gram-negative bacterial species were isolated. In dogs, 17 Gram-positive and eight Gram-negative bacterial species were determined. The bacterial culture results for all patients are presented in Supplementary Table 1.

The most frequently isolated bacterial species in dogs in this sample were Staphylococcus pseudintermedius, Staphylococcus spp. and Streptococcus spp. In the enrolled cats, Pasteurella spp. and Staphylococcus felis were the bacterial species cultured most frequently. All bacterial species isolated from dogs and cats are presented in Supplementary Table 2.

The prevalence and bias indices, and the raw and prevalence-bias adjusted kappa values for agreement between each of the pairs of locations for positive bacterial growth and the agreement for when Staphylococcus spp. were isolated are reported in Table 4.

Table 4. Measures of prevalence, bias and agreement of microbiological culture results for any aerobic bacteria or for Staphylococcus spp., from three nasal sampling sites in a study of 19 dogs and 21 cats with chronic nasal disease. Interpretation of numeric values of kappa is based on the recommendations of Cohen (1960).

Agreementbetween sites	Prevalence index^a(95% CI)	Bias index^b(95% CI)	Raw kappa(95% CI)	Interpretation of levelof agreement fromraw kappa and CI^c	Prevalence andbias adjustedkappa (95% CI)	Interpretation ofprevalence and biasadjusted kappa and CI^c
Any aerobic bacteria
ND vs. NC	0.72 (0.58–0.87)	−0.13 (−0.27 to 0.02)	0.08 (−0.45 to 0.61)	Slight (poor to substantial)	0.55 (0.23–0.78)	Moderate (fair to substantial)
ND vs NB	0.55 (0.38–0.72)	0.05 (−0.13 to 0.23)	0.14 (−0.26 to 0.55)	Slight (poor to moderate)	0.4 (0.07–0.67)	Moderate (slight to substantial)
NC vs. NB	0.68 (0.52–0.83)	0.18 (0.02 to 0.33)	0.04 (−0.44 to 0.52)	Slight (poor to moderate)	0.45 (0.12–0.71)	Moderate (slight to substantial)
Staphlococcus spp.
ND vs. NC	0.3 (0.11–0.49)	−0.25 (−0.45 to −0.05)	0.28 (−0.02 to 0.58)	Fair (poor to moderate)	0.3 (−0.03 to 0.59)	Fair (poor to moderate)
ND vs. NB	0.13 (−0.08 to 0.33)	−0.07 (−0.29 to 0.14)	0.34 (0.05–0.64)	Fair (slight to substantial)	0.35 (0.02–0.63)	Fair (slight to substantial)
NC vs. NB	0.38 (0.18–0.57)	0.18 (−0.02 to 0.37)	0.27 (−0.06 to 0.60)	Fair (poor to moderate)	0.35 (0.02–0.63)	Fair (slight to substantial)

^a Prevalence index: where −1 implies both observers agree on all negative classifications, 0 implies observers agree on half the negative and half the positive classifications and +1 implies both observers agree on all positive classifications.

^b Bias index: where 0 implies no difference in the proportion of positive classifications and no difference in the proportion of negative classifications, and 1 implies all the positive classifications by one observer are classified as negative by the other or vice-versa.

^c Kappa < 0: no agreement; 0–0.2: slight agreement, 0.2–0.4: fair agreement, 0.4–0.6: moderate agreement, 0.6–0.8: substantial agreement, 0.8–1.0: almost perfect agreement.

This indicates that whilst our data are consistent with a wide range of agreement between sites for both the isolation of any bacterial species and for identification of Staphylococcus spp, the point estimates for agreement are never greater than moderate: there is no evidence from this study that there is likely to be agreement between sites of nasal culture. For the isolation of any bacterial species, the influence of prevalence bias can be seen in the difference between the unadjusted and adjusted values of kappa but the imprecision of our estimates of agreement remains. There was less evidence for bias, particularly for the isolation of any bacterial species.

Discussion

The present study shows that bacterial sampling of nasal discharge, the nasal cavity or nasal biopsies is associated with a high likelihood of obtaining a positive bacterial culture. In addition, the study frequently revealed positive bacterial growth for more than one species, in some cases for up to six different bacterial organisms. Our results were consistent with a wide range of agreement between sites both for the presence of any bacteria or the presence of Staphylococcus spp. and the mean agreement for this dataset was never more than moderate. Because the sample collection method likely influences culture results, some authors have recommended a deep swab or biopsy, stating that other sampling methods cannot be used as an alternative for most bacteria (Johnson et al. 2005; Johnson and Kass 2009; Lobetti 2014; Cohn 2020). However, this does not take into account that polymicrobial colonisation or infection seems to be common in the nasal cavity, as shown in the present study.

It has been suggested that culture results from the rostral nasal cavity may differ from those obtained from the caudal nasal cavity with a higher prevalence of positive samples in the former (Abramson et al. 1980). This theory could not be confirmed in the present study, in which a high prevalence of bacterial growth was detected in all three locations. Similarly, brush or lavage samples from superficial epithelium may not adequately reflect bacterial and fungal infiltration in the nasal mucosa. In addition, samples from the nasal cavity and nasal tissue biopsies could be contaminated by bacteria located in the nasal antrum and the skin surrounding the nares. In cases that require a culture from deeper nasal structures, using a protected catheter brush or protected biopsy forceps should be considered (Windsor and Johnson 2006). In this study, disinfection of the nares was performed before insertion of a swab into the nasal cavity for sampling. In addition, both nasal cavities were flushed with sterile saline before collection of the biopsy samples. Nevertheless, bacterial contamination could not be ruled out completely, because the nasal cavity is not sterile.

The present study evaluated samples from both dogs and cats in order to investigate potential differences in the prevalence of bacterial species in feline and canine patients with nasal disease. The results show that for this sample of animals, there were differences in the bacterial composition between dogs and cats. While staphylococci accounted for > 50% of the canine isolates, there was no evidence of a single predominant bacterial genus in enrolled cats. Most of these bacterial species have also been cultured from clinically healthy dogs and cats (Lee-Fowler and Reinero 2012). This emphasises that the distinction between the normal commensal flora and potentially pathogenic bacterial growth is probably impossible to make in most cases. Repeated nasal cultures revealing significant growth of the same bacterial species might indicate pathogenicity in certain cases; however, this has yet to be proven in prospective studies. In some cases, multi resistant bacteria or bacteria with a zoonotic potential, such as toxigenic Corynebacterium ulcerans, which can cause diphtheria-like disease in humans, have been cultured from dogs and cats with rhinitis (Saeki et al. 2015; Fungwithaya et al. 2017). Therefore, culture might be indicated for animals showing signs of rhinitis who share a household with immunosuppressed persons. However, nasal carriage of these potential pathogens has also been described in healthy animals. The International Society for Companion Animal Infectious Diseases (ISCAID) recommends culture and sensitivity testing for antibiotic selection in chronic feline rhinitis patients if there is recurrence of clinical signs, non-responsiveness to antibiotic treatment, or if pathogenic bacteria such as Pseudomonas spp. have been detected (Lappin et al. 2017). In the present study, only one cat with chronic rhinitis revealed positive growth for Pseudomonas sp., however, the pathogen could be detected in all three sampling locations in this patient. Evidence for a greater number of bacteria across nasal sites has also been described for dogs with lymphoplasmacytic and fungal rhinitis and nasal neoplasia (Norris and Laing 1985; Windsor et al. 2004, 2006; Greene and Calpin 2012).

There was no evidence from the current study for agreement between culture sites, either for the presence of any bacteria or the presence of Staphylococcus spp. However, our results were consistent with a wide range of agreement, although the mean agreement for this dataset was never more than moderate. Based on these results, no specific sampling site for bacterial culturing can be recommended, and the likelihood of obtaining a positive culture result is high in every location. Disagreement between sampling sites does not imply that the bacteria identified at any one site are necessarily responsible for the clinical disease, as the analysis measures agreement and not validity of the test. Sampling nasal discharges only cannot be seen as exchangeable with sampling more caudal locations and if sampling is performed in a clinical case to look for potential pathogenic organisms and guide antimicrobial therapy, our results suggest it might be advisable to sample all three locations.

Trying to identify an optimum sampling site may be even less relevant when looking at data from nasal microbiome studies in dogs and cats with different nasal diseases. Studies using next generation sequencing techniques were able to show species-rich bacterial communities in the canine and feline nasal cavity (Dorn et al. 2017; Tress et al. 2017). The majority of bacteria detected in these studies had never been isolated before with conventional culture techniques. Significant differences could be shown in the composition of microbiota colonising the nose of healthy dogs compared to dogs with nasal neoplasia or chronic rhinitis, indicating an influence of the primary disease process on the bacterial microflora (Dorn et al. 2017). Furthermore, an influence of age, facial conformation and environmental factors on the nasal microbial composition could be shown (Dorn et al. 2017; Tress et al. 2017; Vangrinsven et al. 2021). Similar findings are subject to discussion in human medicine, where current knowledge on the upper respiratory microbiome is mainly based on cultivation assays, targeting only a small fraction of microbial communities in comparison to next generation sequencing of the bacterial 16S rRNA gene (Kumpitsch et al. 2019).

This study has some limitations. Due to the prospective character, the study only included a limited number of cases. The small sample size resulted in wide CI, indicating a lack of precision for assessment of the level of agreement. Furthermore, obligatory anaerobic species could have been missed. Although sampling was performed using a standardised protocol and disinfection measures before swab or biopsy forceps were advanced into the nasal cavity, bacterial contamination of both sampling devices by the skin surrounding the nares, the nasal vestibule or by intranasal secretions could not be totally excluded and might have affected the results. The location of the sampling, which was standardised in this study, could also have had an impact on the culture results. In addition, pre-treatment with antibiotics and differences between host species could have influenced the bacterial culture results. In the context of this study, it was not possible to evaluate whether prior antimicrobial treatment might have influenced bacterial growth, species richness or resistance profile of bacteria. However, since comparison of different sampling sites for bacterial detection was the primary aim of the study and not description of the nasal microflora, antibiotic pre-treatment was not defined as an exclusion criterion.

In conclusion, this study indicates that bacterial culture results in feline and canine nasal disease seem to be site specific and inconsistent between sites within a patient. Sampling results are therefore not likely to be useful in most cases to indicate antimicrobial choice, given the lack of evidence that the isolated organism will be a pathogen of interest. Primary rhinosinusitis is considered rare in dogs and cats. Findings should always be interpreted in accordance with clinical signs, imaging, and histopathology to investigate potential factors predisposing to secondary bacterial infection. The lack of precision in the estimates of the agreement between sampling sites in this study suggests that there is no evidence to support nasal sampling as a useful diagnostic technique at a clinical level, and that results could be misleading in guiding treatment.

Acknowledgements

The authors would like to thank Prof. Dr. LFH Theyse for help with language editing and structuring of the paper.

Disclosure statement

No potential conflict of interest was reported by the author(s).