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Antibiotic susceptibility of bacterial isolates from 502 dogs with respiratory signs
  1. M. Rheinwald, DVM1,
  2. K. Hartmann, Professor, Dr. med. vet., Dr. med. vet. habil., Dipl. ECVIM-CA1,
  3. M. Hähner, DVM1,
  4. G. Wolf, Dr. med. vet.2,
  5. R. K. Straubinger, Professor, Dr. med. vet., Ph.D.2 and
  6. B. Schulz, Dr. med. vet., Dipl. ECVIM-CA1
  1. 1Clinic of Small Animal Medicine, Ludwig Maximilian University of Munich, Veterinaerstrasse. 13, Muenchen 80539, Germany
  2. 2Institute for Infectious Diseases and Zoonoses, Ludwig Maximilian University of Munich, Veterinaerstrasse. 13, Muenchen 80539, Germany
  1. E-mail for correspondence: M.Rheinwald{at}medizinische-kleintierklinik.de

Abstract

The aim of this study was to investigate the prevalence of bacterial species isolated from bronchoalveolar lavage fluid (BALF) samples taken from dogs with respiratory signs and to determine their antibiotic susceptibility. Clinical cases were included in the study if they showed signs of respiratory disease and data relating to bacterial culture and susceptibility of BALF samples were available. The medical records of 493 privately owned dogs that were presented between January 1989 and December 2011 were evaluated retrospectively. In 35 per cent of samples, no bacteria were cultured. Bacteria isolated from culture-positive samples included Streptococcus species (31 per cent of positive cultures), Enterobacteriaceae (30 per cent, including Escherichia coli (15 per cent)), Staphylococcus species (19 per cent), Pasteurella species (16 per cent) and Pseudomonas species (14 per cent). Bordetella bronchiseptica as a primary respiratory pathogen was isolated in 8 per cent of cases. Enrofloxacin showed the best susceptibility pattern; 86 per cent of all isolates and 87 per cent of Gram-negative bacteria were susceptible to this antibiotic. Amoxicillin/clavulanic acid yielded the best susceptibility pattern in Gram-positive bacteria (92 per cent). Therefore, these antibiotics can be recommended for empirical or first-line treatment in dogs with bacterial lower respiratory tract infections.

  • Internal medicine
  • Respiratory disease
  • Dogs
  • Antimicrobials
  • Resistance
  • Bacteriology
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Introduction

Infections of the lower respiratory tract are common in dogs presented to veterinary practices. Besides primary pathogenic bacteria such as Bordetella bronchiseptica and Streptococcus equi subspecies zooepidemicus (Vieson and others 2012), many different bacterial species can cause secondary infections of the lower respiratory tract (Harpster 1981, Thayer and Robinson 1984, Jameson and others 1995, Peeters and others 2000). These opportunistic infections are induced by a range of factors, such as viral or parasitic infection, inflammation, trauma, aspiration, neoplasia, anomalies, systemic immunodeficiency or any other cause of impaired local defence mechanisms (Cohn and Reinero 2007, Vieson and others 2012).

Although treatment should aim to solve the underlying problem if possible, controlling the infectious component is an important part of therapy (Vieson and others 2012). It is therefore important to identify the bacterial species involved and use antimicrobials to which the microbes present are most likely susceptible (Thayer and Robinson 1984, Lee-Fowler and Reinero 2012). However, in cases in which bacterial culture and susceptibility testing cannot be conducted or for first-line treatment in emergency situations, selection of antimicrobial agents must be based on empirical data on bacterial prevalence and antibiotic susceptibility (Ford 2009). In a previous study that looked at samples of transtracheal aspirates in dogs with respiratory signs, bacteria isolated most frequently were Enterobacteriaceae (Angus and others 1997); however, a more recent study investigating bacteria present in bronchoalveolar lavage fluid (BALF) of dogs with respiratory signs in Germany revealed Pasteurella species and B bronchiseptica as the two predominant species (Steinfeld and others 2012). In contrast to that, a study analysing data from dogs with confirmed bacterial lower respiratory tract infection detected Mycoplasma species in almost one-third of bacterial cultures (Johnson and others 2013). Bacterial species for which there were a high proportion of resistant isolates identified in earlier studies include Escherichia coli and Pseudomonas species (Angus and others 1997, Steinfeld and others 2012, Johnson and others 2013). The severity of respiratory disease and the types of antimicrobials that have been administered recently should also be taken into account when considering empirical antibiotic treatment. Epstein and others (2010) showed that animals with severe respiratory disease needing positive pressure ventilation were more likely to be infected with Gram-negative enteric bacteria. Additionally, isolates from patients with respiratory failure were less susceptible to commonly used antibiotics in that study (Epstein and others 2010). A recent study by Proulx and others (2014) showed that dogs with bacterial pneumonia frequently harboured bacteria that were resistant to antimicrobials administered during a 4-week period before tracheal wash sampling.

There are currently few published studies investigating the bacteria involved in canine lower respiratory tract infection and their susceptibility to common antibiotics in a large cohort of dogs. Therefore, the aim of the present study was to describe the distribution of bacteria isolated from the lower airways of a large number of dogs with respiratory signs, as well as their antibiotic susceptibility.

Materials and methods

Patient population

The medical records of 493 privately owned dogs that were presented to the Clinic of Small Animal Medicine of the University of Munich, Germany, between January 1989 and December 2011 were evaluated retrospectively. Dogs were included in the study if they were presented with clinical signs indicating a respiratory problem, and if results of an aerobic bacterial culture obtained from BALF were available. For inclusion, patients had to display one or more of the following clinical signs: nasal discharge (89 dogs), coughing (350), dyspnoea/tachypnoea (132) or abnormal findings on auscultation of the lungs (250). Patients with suspected or confirmed heart failure, pleural effusion and neoplasia as potential causes of respiratory signs were excluded. No information about previously administered antibiotic drugs or cytology results of BALF samples was available in most cases. Cases in which BALF cytology results indicated oropharyngeal contamination by the presence of Simonsiella species or squamous epithelial cells were excluded.

The dogs were aged between eight weeks and 16 years (median six years, data from 497 cases available). Information about sex and breed was recorded for 498 patients. There were 252 male dogs (50.6 per cent) and 246 female dogs (49.4 per cent). Of the 108 different breeds represented, the most common were 113 crossbreed dogs (22.7 per cent), 46 dachshunds (9.2 per cent), 29 German shepherd dogs (5.8 per cent), 18 poodles (3.6 per cent) and 16 Yorkshire terriers (3.2 per cent).

Sample collection

Samples were collected endoscopically from the lower respiratory tract under general anaesthesia. Samples were obtained through the working channel of the endoscope. For bronchoscopic examination of the respiratory tract and sampling, different endoscope models with different diameters appropriate to the patient's size were used. For sampling 1–2 ml/kg sterile isotonic saline solution (0.9 per cent sodium chloride) were delivered through the working channel followed by immediate suction. Recovered BALF was collected in a sterile tube and submitted for microbiological examination.

Bacteriological examination

A total of 502 BALF samples from 493 patients (nine patients were presented and sampled twice) were submitted for microbiological examination and antibiotic susceptibility testing. Aerobic bacterial cultivation was performed on different nutrient agars. For detection of bacteria of the family Enterobacteriaceae, agar–agar, sheep blood agar and Gassner/Rambach agar (Rambach agar since 1994) were used. In addition, colistin–nalidixic acid agar was used for selective culturing of Gram-positive bacteria and Bordet–Gengou agar (since 2003) for the selective detection of B bronchiseptica. Plates were incubated at 37°C under aerobic conditions and examined after 24 and 48 hours, followed by biochemical differentiation if necessary. Colonies that needed subcultivation were excluded if contamination was suspected.

Susceptibility testing

The agar disc diffusion method was used to test antimicrobial susceptibility. Samples of isolated bacteria were placed onto Mueller–Hinton agar, and several discs impregnated with defined amounts of antibiotic agents were placed onto the nutrient agar. During incubation, antibiotics built zones of inhibition of bacterial growth that were measured and compared with standardised limits corresponding to current Clinical and Laboratory Standards Institute guidelines (CLSI). According to the diameter of the inhibitory zones, bacteria were classified as ‘susceptible’, ‘intermediate’ or ‘resistant’ to a certain antibiotic. For interpretation of antibiotic susceptibility, intermediate isolates were considered resistant. Susceptibility test panels varied because of the large timespan. For analysis, antibiotic agents that are commonly used in small animal practice (including enrofloxacin, gentamicin, first-generation cephalosporins, ampicillin/amoxicillin, amoxicillin/clavulanic acid, sulphonamides with trimethoprim, cefotaxime and doxycycline) were selected and the antibiotic susceptibility of the most frequently isolated bacteria was evaluated for these antimicrobials.

Statistical analysis

A two-tailed Fisher's exact test was performed using QuickCalcs (GraphPad Software Inc, La Jolla, California, USA) for evaluation of antibiotic resistance. P<0.05 was considered statistically significant.

Results

Bacterial isolates

Of the 493 dogs included in the study, culture results of 502 samples were available for analysis. In 176 samples, no bacteria were cultured (35.1 per cent). A single bacterial species was isolated in 172 samples (34.3 per cent); two or more bacterial species were isolated in 154 samples (30.7 per cent). Gram-negative and Gram-positive species were co-cultured in 32.2 per cent (105/326) of all positive samples; Gram-positive and Gram-negative species were cultured separately in 19.9 per cent (65/326) and 44.5 per cent (145/326) of all samples, respectively. All isolated bacterial species and their detection rates are displayed in Table 1.

TABLE 1:

Bacterial isolates cultured from 502 lower respiratory tract samples (bronchoalveolar lavage fluid) of 493 dogs with respiratory signs

The most frequently isolated bacterial species were Streptococcus (30.7 per cent of all positive samples) and Staphylococcus (18.7 per cent), followed by Pasteurella (16.0 per cent) and Pseudomonas (14.4 per cent). B bronchiseptica was detected in 8.0 per cent of all positive samples. Gram-negative rods of the family Enterobacteriaceae were detected in 29.8 per cent of positive samples, including 48 E coli isolates (14.7 per cent of positive samples).

Bacterial susceptibility

Antibiotic susceptibilities of the most commonly isolated bacteria are displayed in Table 2. Most isolates (85.7 per cent) were susceptible to enrofloxacin, as well as 86.8 per cent of Gram-negative bacteria. However, only 82.6 per cent of Gram-positive isolates were susceptible to enrofloxacin. With 91.9 per cent and 85.9 per cent susceptible Gram-positive bacteria, amoxicillin/clavulanic acid and the first-generation cephalosporins showed a better susceptibility pattern concerning Gram-positive isolates, respectively. Because of the lower proportion of Gram-negative species susceptible to amoxicillin/clavulanic acid (59.0 per cent) and first-generation cephalosporins (60.0 per cent), only a moderate proportion of all isolates were susceptible to these antibiotics (68.0 per cent and 73.2 per cent, respectively). The third-generation cephalosporin cefotaxime was tested only three times against Gram-positive isolates; therefore, results for this antibiotic against Gram-positive bacteria cannot be interpreted. Furthermore, the proportion of isolates susceptible to a combination of the two most potent antimicrobials against Gram-positive and Gram-negative species were calculated. Of all isolates, 89.8 per cent were susceptible to enrofloxacin in combination with amoxicillin/clavulanic acid, whereas 93.0 per cent of Gram-positive isolates and 88.6 per cent of Gram-negative isolates were susceptible to this combination of antimicrobials.

TABLE 2:

Proportions of all isolated bacteria and most commonly cultured species (%) from bronchoalveolar lavage fluid of dogs with respiratory signs that were susceptible to selected antibiotics

The proportion of Gram-negative isolates susceptible to enrofloxacin, as well as the proportion of all cultured bacteria susceptible to enrofloxacin, were significantly higher than the corresponding proportions of isolates susceptible to amoxicillin/clavulanic acid (P<0.0001). No significant difference could be detected in the proportion of Gram-positive isolates susceptible to amoxicillin/clavulanic acid compared with enrofloxacin (P=0.1431). In addition, no significant differences were detected between the combination of enrofloxacin and amoxicillin/clavulanic acid and enrofloxacin alone if the proportions of all susceptible isolates were compared (P=0.1200) as well as if the proportions of susceptible Gram-positive (P=0.0604) or Gram-negative species (P=0.5780) were compared.

During the earlier years (1989–1999), there was a significantly higher proportion of bacterial species susceptible to enrofloxacin compared with the later years (2000–2011) (P=0.0061). There was also a significantly higher proportion of E coli isolates that were resistant to enrofloxacin in the later years (P=0.0144). No significant changes could be detected for the proportion of all isolates susceptible to amoxicillin/clavulanic acid, as well as for the proportion of E coli isolates susceptible to amoxicillin/clavulanic acid between the timeframes.

Discussion

This study investigated detection rates of bacterial isolates in BALF in a large number of dogs over a period of 23 years, as well as their antibiotic susceptibility. Bacterial species most frequently isolated were Streptococcus species and Staphylococcus species. In contrast to that, a recently published German study determined Pasteurella species and B bronchiseptica as the most common bacterial isolates in BALF from 84 dogs with respiratory signs (Steinfeld and others 2012). In an older study, the most prevalent species were E coli, Pasteurella species and Streptococcus species (Angus and others 1997). There are several explanations for the differences in the detection rates between the two previous studies and this one. First, there might be differences in the dog populations studied. Although all three studies included dogs with suspected lower respiratory disease, the two previous studies included much fewer cases. Because inclusion of patients was based on clinical signs and all three studies were designed retrospectively, it is impossible to distinguish between primary infectious and underlying non-infectious respiratory diseases that could influence the prevalence of bacterial isolates in the different investigations. Secondly, differences in the microbiological spectrum of the isolates could also be influenced by the different timeframes and different geographical locations in which the studies were carried out. In addition, information about antimicrobial treatment of dogs before sampling was not available for all three studies, but might have influenced bacterial detection rates and their antibiotic susceptibility.

In a recent study of bacterial isolates in the BALF of 105 dogs with confirmed lower respiratory tract infections, the most prevalent isolates in this study were Mycoplasma species and B bronchiseptica; but Pasteurella species, Enterobacteriaceae and Streptococcus species were isolated frequently as well. However, only dogs with cytologically confirmed septic inflammation with intracellular bacteria in BALF were evaluated in that study, therefore including a different patient population than in the present study (Johnson and others 2013).

In this study, B bronchiseptica was detected in 8 per cent of all positive samples and represented the only isolated species that is considered a primary pathogen in the lower respiratory tract of dogs (Thompson and others 1976, Bemis and others 1977b, Keil and Fenwick 1998). In contrast, Steinfeld and others (2012) detected B bronchiseptica in 20 per cent of positive samples. This difference cannot be explained by the use of specific culture media, since these were not used in the previous investigation, in contrast to the present study, in which Bordet–Gengou agar was used as a selective Bordetella culture medium (since 2003). Higher isolation rates of B bronchiseptica are to be expected in shelter dogs or dogs kept in large groups (Bemis and others 1977a, Chalker and others 2003, Radhakrishnan and others 2007). Therefore, variations in patient populations between the two studies might also have influenced the differences in detection rates of B bronchiseptica. In the study investigating bacterial isolates in the BALF of dogs with confirmed lower respiratory tract infection, B bronchiseptica was detected in 22 per cent of samples (Johnson and others 2013).

Johnson and others (2013) revealed that 57 per cent of dogs infected with B bronchiseptica were co-infected with additional bacteria, which represents a higher number of co-infections than in the present study (38 per cent). One reason for this difference could be the fact that additional bacteria in the previous study were frequently Mycoplasma species (69 per cent, 9/13) (Johnson and others 2013). In contrast to that, Mycoplasma species were only isolated from 3 per cent (11/326) of samples positive for bacterial growth in the present study, which can be attributed to the lack of specific selection media necessary for cultivation of this organism (Chalker 2005).

In the second part of the study, in vitro susceptibility of frequently isolated bacteria was evaluated. While enrofloxacin showed the best susceptibility pattern against Pasteurella species (100 per cent) and Enterobacteriaceae except for E coli (88 per cent) as well as a good susceptibility pattern against B bronchiseptica (91 per cent), only moderate proportions of E coli (73 per cent) and Pseudomonas species (72 per cent) were susceptible to enrofloxacin. This is similar to the results of Johnson and others (2013) except for low proportions of susceptible Pseudomonas species (<20 per cent) and B bronchiseptica (<70 per cent) in that study. In contrast to the findings of the present study, Steinfeld and others (2012) found out that all Pseudomonas species (100 per cent) but only a low proportion of E coli (29 per cent) were susceptible to enrofloxacin. Angus and others (1997) found a high proportion of E coli isolates that were susceptible to enrofloxacin (92 per cent). However, data in that study were obtained over the time period 1989–1995, potentially reflecting a more favourable resistance situation during earlier years. In the present study, E coli isolates cultured in earlier years were significantly more likely to be susceptible to enrofloxacin, indicating increasing resistance rates over time for this species. Furthermore, case numbers of these previous studies were lower and data were obtained over a shorter time period, potentially explaining differences in susceptibility data for enrofloxacin.

More than 90 per cent of Gram-positive bacteria were susceptible to amoxicillin/clavulanic acid. Data are comparable with previously published studies (Steinfeld and others 2012, Johnson and others 2013), taking into account that both studies tested only low numbers of Gram-positive bacteria for this antibiotic combination. Based on the data of the present study, the use of amoxicillin/clavulanic acid cannot be recommended for the treatment of Gram-negative bacteria with the exception of B bronchiseptica and Pasteurella species. In contrast to the resistance pattern shown for amoxicillin/clavulanic acid, non-potentiated ampicillin/amoxicillin cannot even be recommended for the empirical use against Gram-positive bacteria, since only a low proportion of isolated Staphylococcus species (50 per cent) and a moderate proportion of cultured Streptococcus species (76 per cent) were susceptible to this antimicrobial.

If a broad antibiotic coverage is indicated in a severely sick dog with a suspected lower respiratory tract infection, and airway sampling for culture and susceptibility testing is not possible or results are pending, the use of a combination of different antibiotic agents is recommended by some authors (Rozanski and Rondeau 2002, Brady 2004, Ford 2009, Cohn 2010, Lee-Fowler and Reinero 2012). However, the combination of the two most potent antibiotics in the present study, enrofloxacin and amoxicillin/clavulanic acid, revealed only a slight increase in the proportion of isolates susceptible to this combination (90 per cent) compared with the proportion of isolates susceptible to enrofloxacin alone (86 per cent), which was not significant.

No anaerobic cultivation was performed in this study. Since several studies have shown a significant anaerobic population in lower respiratory tract cultures (Angus and others 1997, Johnson and others 2013), additional anaerobic cultivation of samples should be considered in cases in which anaerobic bacterial infection is suspected. A recent study by Tenwolde and others (2010) revealed obligate anaerobic isolates in 48 per cent of cultures from dogs and cats with foreign body pneumonia. Since enrofloxacin has poor activity against anaerobic bacteria (Boothe 1990), this agent should not be administered for suspected or confirmed anaerobic infections.

Among the most frequently isolated bacteria, Pseudomonas species and E coli were associated with a poor susceptibility to commonly used antibiotics, which is in agreement with similar results obtained in previous studies (Angus and others 1997, Steinfeld and others 2012, Johnson and others 2013). Less than 35 per cent of the isolated Pseudomonas species were susceptible to β-lactam antibiotics, potentiated sulphonamides or doxycycline, whereas less than 50 per cent of all E coli isolates were susceptible to doxycycline, ampicillin/amoxicillin, amoxicillin/clavulanic acid or potentiated sulphonamides. The lowest grade of resistance was found in enrofloxacin and gentamicin. However, only moderate proportions of isolated E coli (73 per cent and 70 per cent, respectively) and Pseudomonas species (72 per cent for each antibiotic) were susceptible to these two antimicrobial agents. In particular, Pseudomonas aeruginosa is considered to be intrinsically resistant to several antibiotics (Olivares and others 2013). These organisms also showed a high level of resistance in several other studies investigating the susceptibility patterns of bacteria isolated from different locations other than the respiratory tract in small animals (Oluoch and others 2001, Clarke 2006).

Interestingly, only a low proportion of the majority of cultured bacteria was susceptible to doxycycline—a frequently used antibiotic for canine respiratory tract infections (Ford 2009, Schulz and others 2011)—with the exception of B bronchiseptica (100 per cent of isolates susceptible). In contrast to that, Steinfeld and others (2012) revealed a moderate proportion of Staphylococcus species and a high proportion of Streptococcus species that were susceptible to doxycycline, but had a low proportion of Gram-negative bacteria that were susceptible to this drug. Based on these results, doxycycline is not recommended as an empirical treatment option for dogs with respiratory tract infection.

This study has several limitations. Due to the retrospective nature of this study, only limited clinical data were available for the dogs included, and BALF cytology results could not be obtained for most cases. In addition, quantification of bacterial isolates and cultivation of obligate anaerobic bacteria were not performed. Furthermore, no standardised protocol was used for the sampling and the processing of sampling material. Since inclusion was solely based on the presence of clinical signs, it was impossible to differentiate between animals with bacterial infection of the lower respiratory tract and patients with other respiratory tract diseases with bacterial colonisation or oropharyngeal contamination during the sampling process. For that purpose, it would have been necessary to include information regarding quantitative bacterial cultures and/or cytology results confirming suppurative inflammation or detection of intracellular bacteria (Peeters and others 2000, Johnson and others 2013). Most bacterial isolates detected in the present study have also been found in lower respiratory tract samples from healthy dogs, indicating that they can be part of the physiological bacterial microflora of the lower airways (Lindsey and Pierce 1978, McKiernan and others 1984, Bauer and others 2003). Therefore, positive bacteriological results should be interpreted with caution and alongside with other diagnostic findings to differentiate between bacterial infection and colonisation.

Due to the fact that there was no antibiotic all isolated bacterial species were fully susceptible for, antimicrobial treatment should be guided by BALF culture results and susceptibility testing, if possible. In daily clinical practice, BALF cytology, and possibly Gram-staining (Peeters and others 2000), might help to differentiate between Gram-positive bacteria and Gram-negative bacteria, and therefore aid in the choice of a suitable antibiotic agent. Based on the data presented here, enrofloxacin can be recommended as a first-line treatment in patients with suspected lower respiratory tract infections because of Gram-negative or unknown aerobic bacterial pathogens as long as results of bacteriological examination and susceptibility testing are pending. Amoxicillin/clavulanic acid can be chosen if infection with Gram-positive bacteria is suspected.

References

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Footnotes

  • Provenance: not commissioned; externally peer reviewed

  • Correction notice This article has been corrected since it was published Online First. The corresponding e-mail address has been changed to M.Rheinwald@medizinische-kleintierklinik.de.

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