Diagnostic Microbiology and Infectious Disease 82 (2015) 20–25
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Microbiological and clinical aspects of respiratory infections associated with Bordetella bronchiseptica Celia García-de-la-Fuente a,⁎, Laura Guzmán a, María Eliecer Cano a, Jesús Agüero a,b, Carmen Sanjuán a, Cristina Rodríguez a, Amaia Aguirre a, Luis Martínez-Martínez a,b a b
Service of Microbiology, University Hospital Marqués de Valdecilla-IDIVAL, Santander, Spain Department of Molecular Biology, School of Medicine, University of Cantabria, Spain
a r t i c l e
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Article history: Received 29 October 2014 Received in revised form 20 January 2015 Accepted 24 January 2015 Available online 2 February 2015 Keywords: Bordetella bronchiseptica Respiratory infections Pulsed-field gel electrophoresis Veterinary pathogen Molecular identification
a b s t r a c t Bordetella bronchiseptica is a well-known veterinary pathogen, but its implication in human disease is probably not fully recognized. The purpose of this study was to determine the clinical significance of 36 B. bronchiseptica isolates from respiratory samples of 22 patients. Therefore, we describe microbiological characteristics, including phenotypic and genotypic identification as well as antimicrobial susceptibilities of the isolates. Clonal relatedness was evaluated using pulsed-field gel electrophoresis (PFGE). Most of the patients had some underlying immunosuppressive condition. Eighteen out of 22 (82%) patients had respiratory symptoms, and the death of 2 patients was associated with respiratory infection.All strains were correctly identified at species level by the simultaneous use of phenotypic methods and were confirmed by specific amplification of the upstream region of the fla gene. Tigecycline, minocycline, doxycycline, colistin, and meropenem were the most active agents tested. PFGE analysis revealed that repeated infections involving each patient had been caused by the same strain. Published by Elsevier Inc.
1. Introduction Bordetella bronchiseptica is a strict aerobic, nonfermentative, catalase- and oxidase-positive gram-negative coccobacillus. This bacterium was first isolated and identified in 1911 (Ferry, 1911) in dogs with distemper disease, and then it was named Bacillus bronchianis. It is currently a well-recognized pathogen in many domestic and wild animals. However, it is an uncommon cause of infection in humans. Sporadic cases of infection by B. bronchiseptica have been reported and are particularly associated with patients who are debilitated or immunosuppressed (Lorenzo-Pajuelo et al., 2002; Ner et al., 2003; Spilker et al., 2008). The organism is less commonly isolated from immunocompetent individuals (De la Torre et al., 2012; Rath et al., 2008). In the past, 2 reviews about human illnesses associated to B. bronchiseptica have been published, in 1991 and 1995, respectively (Woolfrey and Moody, 1991; Le Coustumier et al., 1995), but only in a few cases, microbiological data allowing a reliable identification of the considered microorganism as B. bronchiseptica were presented. Most recently, another review (Mattoo and Cherry, 2005) was published but did not provide enough clinical information on B. bronchiseptica infections. Finally, in 2011 a report presented data on 8 cases collected over a period of 15 years (Wernli et al., 2011), but in 4 of them, a molecular identification of the organism was not provided. Most human cases occur as a pulmonary disease manifested as pneumonia, bronchitis, or whooping-cough. Less frequently, B. bronchiseptica can be involved in systemic infections ⁎ Corresponding author. Tel.: +34-942203359; fax: +34-942203462. E-mail address: [email protected] (C. García-de-la-Fuente). http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.011 0732-8893/Published by Elsevier Inc.
such as meningitis (Belen et al., 2003). Transmission to humans from infected animals can occur through inhalation of infected aerosols or by direct contact with respiratory secretions. Transmission between humans is possible via respiratory droplets since nosocomial transmission of B. bronchiseptica has also been reported in the literature (Bose et al., 2008; Huebner et al., 2006). This suggests that the animal contact is not the only transmission route of B. bronchiseptica in immunocompromised patients. However, there are still unresolved issues concerning the true incidence and the clinical relevance of this organism. In this report, we present microbiological data of B. bronchiseptica, including antimicrobial susceptibilities and molecular typing of isolates obtained from 22 patients from whom clinical data are also evaluated. 2. Material and methods 2.1. Bacterial strains and patients This retrospective study was undertaken at the University Hospital Marqués de Valdecilla of Santander (Cantabria, Northern Spain), an 800-bed tertiary hospital. The computarized database of the Service of Microbiology was consulted to identify clinical samples from which B. bronchiseptica was isolated. A total of 36 isolates, involving 22 patients, were found from June 2004 to November 2011. Two patients have been included in a previous report (De la Torre et al., 2012). Clinical data were obtained from the medical charts of the patients. For each episode, we collected information about the age, sex, underlyng conditions, inpatient/outpatient status, symptoms, diagnosis, culture source and date of isolation, antibiotic treatment, and outcome.
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2.2. Identification and antimicrobial susceptibility testing The strains were stored frozen in trytic soy broth with 10% glycerol at −70 °C. The original identification of all isolates had been carried out with the WalkAway system using MicroScan Neg Combo 38 panel (Dade Behring, West Sacramento, CA, USA) and API20NE strips (bioMérieux, Marcy l′Etoile, France), following manufacturers' recommendations. For the present study, the identification of B. bronchiseptica isolates was reassessed. Frozen isolates were subcultured onto Muller–Hinton agar plates (bioMérieux) and were incubated aerobically at 35 °C for 48 h to ensure viability and purity. Four isolates (from 3 patients) out of 36 conserved organisms could be not recovered. Then, phenotypic identification and susceptibility of the 32 available isolates were performed using the Vitek 2 system with ID-GNB cards (bioMérieux). Addittionally, the MICs of 24 antibiotics were determined by broth microdilution according to the guidelines of the CLSI (2010) using cation-adjusted Mueller–Hinton II broth (DIFCO Laboratories, Detroit, MI, USA). Standard laboratory powders were supplied, as follows: penicillin G, ampicillin, amoxicillin, cefotaxime, amikacin, tobramycin, gentamicin, nalidixic acid, trimethoprim-sulfamethoxazole, erythromycin, rifampin, tetracycline, doxycycline, minocycline, ciprofloxacin, and colistin (Sigma-Aldrich, Tres Cantos, Spain); amoxicillin-clavulanate and cefuroxime (GlaxoSmithKline, Tres Cantos, Spain); moxifloxacin (Bayer, Barcelona, Spain); telithromycin (Aventis Pharma, S.A., Madrid, Spain); tigecycline (Wyeth Farma, San Sebastián de los Reyes, Spain); and imipenem, meropenem, azithromycin, and aztreonam (Discovery Fine Chemicals, Dorset, UK). They were reconstituted according to the manufacturers' instructions, serially diluted in cation-adjusted Mueller–Hinton II broth (DIFCO Laboratories), and dispensed into in house prepared microdilution panels The MIC was defined as the lowest concentration of antibiotic at which no visible growth at 24 h and 48 h was observed. Up to this date, the CLSI and EUCAST susceptibility breakpoints have not been available for B. bronchiseptica; for this reason, resistance and susceptibility criteria have not been defined in this study. 2.3. Molecular identification and typing The strains were identified as B. bronchiseptica by specific amplification of the upstream region of the fla gene using PCR, as described elsewhere (Hozbor et al., 1999). Molecular typing of 32 isolates from 19 patients was performed by pulsed-field gel electrophoresis (PFGE). After overnight digestion with 30 U of XbaI at 37 °C, the restriction fragments were separated using a CHEF-DRIII system (Bio-Rad, Hercules, CA, USA). Electrophoresis was performed in a 1% agarose gel at 6 V/cm and 14 °C with 0.5X TBE buffer. Pulse times were ramped from 7 to 20 s for 9 h and 30 to 50 s for 11 h. Cluster analysis was performed with Fingerprinting II v4.5 software (Bio-Rad) by using the Dice similarity coefficient and the unweighted pair group method with arithmetic means (UPGMA), with 0.5% of optimization and 1% of tolerance. Isolates from different patients showing the same or similar XbaI pattern were aditionally studied with SpeI-PFGE (pulse times: 7–20 s for 11 h and 30–50 s for 13 h) following the same analysis conditions. Isolates were classified as indistinguishable if they showed no band differences (100% similarity), as subtypes of the same strain if they showed 1–3 band differences (85–99% similarity), and as different strains if they showed 4 or more band differences (b85% similarity) (Binns et al., 1998). 3. Results 3.1. Conventional and molecular identification All the isolates were gram-negative coccobacilli, oxidase positive, and catalase positive. Agar plates with 5% sheep blood agar and MacConkey, incubated in 5% CO2, yielded visible colonies after 48 h.
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The API 20 NE system read at 48 h identified correctly 35/36 (97%) isolates. The remaining isolate was missidentified as Psychrobacter phenylpiruvicus (91.3% probability). Three numeric profiles were obtained, 1200067 (50%), 1200025 (27%), and 1200027 (23%) giving good or very good identification as B. bronchiseptica. All isolates produced positive results for nitrate reduction, urease and adipato, and citrate assimilation. MicroScan Neg Combo 38 panel identified 32/36 (89%) isolates as B. bronchiseptica with N98% probability and misidentified 1 isolate as Ralstonia paucula (89.06% probability); 2 isolates were identified with very low probability (b60%) as B. bronchiseptica and proved to be inconclusive in one. The Vitek 2 system identified 28/32 (87.5%) isolates properly, with N98% probability and misidentified 2 isolates as Acinetobacter lwofii (92–93% probability) and 2 as Oligella urethralis (90% probability). All isolates were subsequently confirmed as B. bronchiseptica by PCR-specific amplification of the upstream region of the fla gene. 3.2. Susceptibility testing MIC50 and MIC90 values of the 24 antimicrobial agents tested are presented in Table 1. The lowest MIC at which 90% of isolates tested are inhibited (MIC90) was that of tigecycline, minocycline, doxycycline, and meropenem, whereas the highest MIC90 were those of β-lactams as well as cephalosporins, macrolides, ketolides, and trimethoprim-sulfamethoxazole. On the other hand, moxifloxacin demonstrated lower MIC50 and MIC 90 compared to ciprofloxacin. For the rest of antibiotics tested, MIC values were variable. 3.3. Molecular typing PFGE analysis (Fig. 1) of XbaI restriction fragments revealed 16 different patterns, which became 17 after analyzing the results of SpeIPFGE. These 17 pulsotypes were grouped in 12 clusters based on ≥85% similarity of PFGE profiles. Isolates belonging to the same patients presented identical or very similar pulsotype, as well as isolates from patients 20 and 19 (pulsotype 4B), who were mother and son, respectively. Five of the 7 isolates from patient 7 had identical pattern, and 2 Table 1 MIC50 and MIC90 values of the antimicrobial agents tested for 32 B. bronchiseptica clinical isolates. Antimicrobial agent
Penicillin Ampicillin Amoxicillin/clavulanate Aztreonam Cefuroxime Cefotaxime Imipenem Meropenem Amikacin Gentamicin Tobramycin Nalidixic acid Ciprofloxacin Moxifloxacin Tigecycline Trimethoprim/sulfamethoxazole Erythromycin Azithromycin Telithromycin Rifampicin Minocycline Doxycycline Tetracycline Colistin
MIC50 (μg/mL)
MIC90 (μg/mL)
24 h
48 h
24 h
48 h
128 64 16/8 512 N512 128 1 0.06 16 2 2 16 1 0.5 ≤0.03 16/304 8 1 16 32 0.06 0.06 0.5 ≤0.03
N512 128 32/16 512 N512 N512 4 0.06 64 4 8 32 4 1 0.06 N64/1216 32 4 32 128 0.5 0.5 2 ≤0.03
N512 256 64/32 N512 N512 N512 2 0.5 64 4 8 32 4 1 1 N64/1216 32 4 32 64 0.25 0.125 2 ≤0.03
N512 N512 256/128 N512 N512 N512 8 4 128 8 32 64 4 1 2 N64/1216 32 8 64 256 1 1 4 ≤0.03
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C. García-de-la-Fuente et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 20–25 Dice (Opt:0.50%) (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
PFGE SpeI
100
90
PFGE XbaI 80
70
60
PFGE XbaI
PATIENT / STRAIN DATE OF ISOLATION PFGE PATTERN 11/2694
June/07
8
13/1689
Mar/09
8
21/310
Jan/11
11
5/524
Feb/05
3
18/3770
July/10
10
22/5596
Nov/11
12
15/3263
June/09
6B
8/3970
Oct/06
6A
1/1475
June/04
1
12/399
Jan/09
2B
17/4968
Aug/09
2B
2/2125
Aug/04
2A
9/4941
Dec/06
2B
14/3024
May/09
9
7/4221
Dec/05
5A
7/308
Jan/06
5A
7/1400
Apr/06
5A
7/3673
Sept/06
5A
7/5031
Oct/07
5A
7/982
Mar/06
5C
7/710
Feb/06
5B
10/5187
Dec/06
7
10/5170
Dec/06
7
19/5215
Sept/10
4B
19/6005
Oct/10
4B
19/6324
Nov/10
4B
20/5381
Sept/10
4B
20/6179
Nov/10
4B
20/6934
Dec/10
4B
6/3283
Sept/05
4A
6/1284
Mar/06
4A
6/910
Feb/07
4A
Fig. 1. PFGE of XbaI- and SpeI-157 digested chromosomal DNA from isolates of B. bronchiseptica. The dendrogram was obtained by XbaI-PFGE of 32 isolates from 19 patients and was constructed by using Dice similarity coefficients (0.5% optimization and 1.0% tolerance) and UPGMA algorithm. Patient and strain number, date of isolation, and PFGE pattern are included along each PFGE lane. PFGE patterns were assigned after analysis of pulsotypes obtained with both XbaI and SpeI digestion. Twelve PFGE groups were obtained based on ≥85% similarity of PFGE profiles.
were closely related with more than 95% of similarity. Other patients whose isolates showed identical or very similar PFGE pattern, but without any apparent epidemiological link, were patients 11 and 13 (pulsotype 8); patients 8 and 15 (pulsotypes 6A and 6B); and patients 12, 17, and 9 (pulsotype 2B) along with patient 2 (pulsotype 2A). The last 4 isolates have identical pattern when only considering the analysis of XbaI-PFGE. Susceptibility patterns among the pulsotypes from the same patients were identical.
3.4. Clinical data Table 2 summarizes demographic data, underlying diseases, clinical characteristics, and outcome of the 22 patients considered in this study. Four patients were diagnosed in 2004; 3, in 2005; 3, in 2006; 1, in 2007; 6, in 2009; 3, in 2010; and 2, in 2011. A total of 11 (50%) patients were female, and the median age was 60 years (range, b1–90). Nineteen patients were admitted to the hospital, and 4 patients (18%) required admission to the intensive care unit. Four patients had repetitive episodes, and they were the only ones who had documented exposure to dogs or cats. Seventeen patients (77%) had active comorbidities at the time of infection/colonization. In 9 of them, more than 1 of the considered underlying diseases was found. Furthermore, several patients were immunosuppressed due to chronic corticosteroid therapy (n = 6), other immunosupressive therapy (n = 3), hematological malignancies
(n = 2), or HIV infection (n = 1). Only 4 patients (18%) had no known underlying conditions. B. bronchiseptica, in these 22 patients, were isolated from sputum (n = 25), from nasopharyngeal lavages (n = 6), from traqueal aspirates (n = 2), from protected brush (n = 1), from bronchial aspirate (n = 1), and from middle ear effusion (n = 1). Twenty-seven (75%) samples were monomicrobial; in the remaining 9 (25%) samples, a copathogen was also cultured, including Haemophilus influenzae, patients 2 and 11; Staphylococcus aureus, patient 17; Aspergillus fumigatus, patients 6 and 7 (3 episodes and 1 episode, respectively); Corynebacterium pseudodiphteriticum, patient 15; and Mycobacterium avium complex, patient 7 (1 episode). Patients 6 and 7 had been in contact with dogs, and patients 19 and 20 had been in contact with cats. According to the clinical presentation at time of isolation of B. bronchiseptica, 5/22 (23%) had acute exacerbation of chronic obstructive pulmonary disease (COPD); 5/22 (24%), acute respiratory infection; 3/ 22 (14%), fever; 2/22 (9%), pneumonia; 2/22 (9%), chronic cough; 1/22 (5%), like syndrome; 1/22 (5%), respiratory distress; 1/22 (5%), acute bronchospasm; and 1/22 (5%), laryngotracheitis plus otitis media. Twenty out of 22 (91%) patients had signs and symptoms of infection, and 18/22 (82%) patients had respiratory symptoms. The most common clinical symptoms and signs at presentation included fever with a temperature ≥38 °C (61%), productive cough (46%), and shortness of breath and dyspnoea (45%). At physical examination, ronchi and basal crackles were detected in 64% of the patients.
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Table 2 Clinical data of B. bronchiseptica respiratory infections. Patient/strain number
Age (years)/ sex
Date of isolation
PFGE pattern
Underlying disease
Diagnosis
Clinical sample
Therapy/duration
Outcome
1/1475 2/2125 3 4/2699 5/524 63283 61284 6910 7/4221 7/308 7/710 7/982 7/1400 7/3673 7 7/5031 8/3970 9/4941 10/5187
80/F 69/M 19/F 69/M 62/M 32/M
1 2A Unworked Unworked 3 4A 4A 4A 5A 5A 5B 5C 5A 5A Unworked 5A 6A 2B 7
COPD/diabetes Prostate cancer No data CHD COPD/bladder cancer CF Lymphoma Lung transplant Kidney transplant, bronchiectasis
COPD exacerbation Ascites/fever Pneumonia Fever COPD exacerbation Prolonged fever
Sputum Sputum Sputum Sputum Sputum Sputum
Chronic cough/dyspnea
Sputum
81/F 63/F 32/F
June/04 Aug/04 Sept/04 Sept/04 Feb/05 Sept/05 Mar/06 Feb/07 Dec/05 Jan/06 Feb/06 Mar/0 Apr/06 Sept/06 Oct/06 Oct/07 Oct/06 Dec/06 Dec/06
COPD COPD/myeloma HIV/HCV
COPD exacerbation Acute bronchospasm Acute respiratory disease
Sputum Sputum PB
Azithrormycin 2 wk None Amoxicillin/clavulanate 10 d Ciprofloxacin 2 wk Moxifloxacin 3 wk TMP-SMX 2 d Azithromycin 2 wk Levofloxacin 8 d Amoxicillin/clavulanate 8 d Amoxicillin/clavulanate 8 d Amoxicillin/clavulanate 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Levofloxacin + TMP-SMX 10 d No data Telithromycin 5 d Levofloxacin 1 wk
Death Death Recovery Recovery Recovery Recurrence Recurrence Recovery Recurrence Recurrence Recurrence Recurrence Recurrence Recurrence Recurrence Recovery Unknown Recurrence Recovery
10/5170 11/2694
67/F
June/07
7 8
Diabetes
BAS Sputum
Levofloxacin 5 d
Recovery
12/399 13/1689
73/M 78/M
Jan/09 Mar/09
2B 8
COPD Pulmonary fibrosis
Sputum Sputum
Ceftriaxone 5 d TMP-SMX 5 d
Recovery Recovery
14/3024 15/3263
45/M 66/F
May/09 June/09
9 6B
Liver transplant/VHC CHD/diabetes
TA TA
Ciprofloxacin 1 wk Levofloxacin 10 d
Recovery Recovery
16/587 17/4968 18/3770 19/5215 19/6005 19/6324 20/5381 20/6179 20/6934 21/310 22/5596
73/M 73/F 80/F b1/M
July/09 Aug/09 July/10 Sept/10 Oct/10 Nov/10 Sept/10 Nov/10 Dec/10 Jan/11 Nov/11
Unworked 2B 10 4B 4B 4B 4B 4B 4B 11 12
None None COPD None
Acute respiratory disease COPD exacerbation Acute respiratory disease Respiratory distress Acute respiratory disease No data Laryngotracheitis/COM Pneumonia Pertussis-like syndrome
Sputum Ear media Sputum Nasopharyngeal lavate
No data Azithromycin 3 d Levofloxacin + TMP-SMX 2 wk Erythromycin 4 d Ciprofloxacin 7 d TMP-SMZ + rifampin 2 wk None Ciprofloxacin 7 d TMP-SMZ + rifampin 2 wk Levofloxacin 2 wk Amoxicillin/clavulanate 7 d
Unknown Recurrence Recovery Recurrence Recurrence Recovery Recurrence Recurrence Recovery Recovery Death
52/F
31/F
81/M 90/M
None
Chronic cough
Nasopharyngeal lavate
COPD/diabetes CHD/diabetes
COPD exacerbation Acute respiratory disease
Sputum Sputum
CHD = chronic heart disease; CF = cystic fibrosis; TMP-SMX = trimethroprim-sulfamethoxazole; PB = protected brush; BA = bronchial aspirate; TA = tracheal aspirate; COM = chronic otitis media. Unworked, isolate not available for PFGE.
Laboratory data were available from only 17 episodes, in 12 (71%) of which the white blood cell count ranged from 12,300 to 22,100/mm 3 with 75–95% neutrophils. In 5 patients in whom C-reactive protein was studied, values from 1.5 to 4.7 mg/dL were observed (normal range 0.0–0.5 mg/dL). Only 4 patients had blood cultures requested, with negative results in all cases. Chest x-ray was performed for 59% (13/22) of the patients, of whom 31% had significant alterations, including pneumonia (2/13) or subsegmental bronchiectasis (2/13). All patients had a Gram-stained smear of the specimens revealing neutrophils and abundant gram-negative coccobacilli, including intracellular forms. In 31 episodes, the patients received antibiotic therapy, including levofloxacin, ciprofloxacin, or moxifloxacin (n = 14); azithromycin, erythromycin, or telithromycin (n = 5); amoxicillin-clavulanate (n = 5); trimethroprim-sulfamethoxazole (n = 2); ceftriaxone (n = 1); levofloxacin plus trimethroprim-sulfamethoxazole (n = 2); or trimethroprim-sulfamethoxazole plus rifampicin (n = 2). Three patients died during their hospitalization. In patients 1 and 22, death was related to respiratory infection (both patients had been treated with azithromycin and amoxicillin-clavulanate respectively). In patient 2, death was related to other medical causes (this patient had not received antibiotic treatment).
4. Discussion Most reports on B. bronchiseptica infection/colonization in humans correspond to small case series or single case reports of debilitated or immunosuppressed patients (Berkowitz et al., 2007; Dworkin et al., 1999; Garcia San Miguel et al., 1988; Lorenzo-Pajuelo et al., 2002; Wernli et al., 2011). The reason why only a limited number of cases of B. bronchiseptica are reported could be due to the fact that it is considered a respiratory commensal. In this study, the majority of infections/ colonizations caused by B. bronchiseptica occurred in adults with underliyng diseases, and only 19% of the patients had no comorbid conditions. In all cases, B. bronchiseptica was recovered from the respiratory tract, which supports the previously described tropism of this bacterium for the respiratory tissue (Le Coustumier et al., 1995; Wernli et al., 2011). Most common clinical presentations of B. bronchiseptica infections were very similar from those caused by widely recognized pathogens, such as Haemophillus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Except for 3 patients, for which clinical data were lacking, the episodes, according to clinical records, were considered as infection and treated as such. However, as organisms were often recovered from the sputum, there can be no full convincing evidence for its clinical implication, and the possibility that they are just colonizers cannot be completely excluded.
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Four patients, 2 of them immunocompetent, who had repeated episodes of infection for extended periods of time, were the only ones with documented animal exposure. The persistence or recurrence of B. bronchiseptica infection had been previously described (De la Torre et al., 2012; Rath et al., 2008). Perhaps this can be due to the lack of clinical effectiveness of macrolides, used for other Bordetella infections, or because B. bronchiseptica may survive intracellularly for a long time for which they require adhesin expression as filamentous hemagglutinin and other virulence factors as adenylate cyclase-hemolysine (Gueirard et al., 1995). Furthermore, B. bronchiseptica (unlike B. pertussis) could persist intracellularly in infected macrophages due to their acid tolerance (Schneider et al., 2000). According to a more recent study, B. bronchiseptica are able to resist the macrophage attacks and remain viable for several days, although they do not multiply (Gueirard et al., 2005). However, according to these authors, once B. bronchiseptica invades respiratory epithelial cells, the bacteria could be protected from antibodies and the complement system and is resistant to phagocytosis in patients with a deficient host immune system. Therefore, B bronchiseptica could persist in cells for a longer time. Patients 9 and 17 had recurrent symptoms, but no microbiological cultures were performed. There is little information on molecular typing of B. bronchiseptica isolates from clinical origin. In the present study, PFGE was able to differenciate 17 pulsotypes, grouped in 12 clusters, among the 32 isolates affecting 19 patients. These results indicate that PFGE offers a high discriminatory power for genotyping B. bronchiseptica isolates of human origin, as has been demonstrated in previous studies in animals (Bose et al., 2008; Shin et al., 2007). In order to rule out a poor discriminative capacity of XbaI, SpeI was also used to study isolates from different patients showing the same or similar PFGE-pattern. The coincident results of the PFGE assay using XbaI or SpeI in practically all the evaluated isolates confirm the clonal relation of these isolates. Unfortunately, in the absence of additional epidemiological information due to the retrospective nature of this study, it is difficult to evaluate the relevance of this finding. Isolates recovered from the same single patient for almost 2 years with identical or very similar PFGE patterns indicated that the same strain has caused multiple episodes. This may be related to the fact that the patient owned a dog living with her and, although no cultures were performed on the pet, it seems relevant that no more episodes occurred after the dog was vaccinated (Bordetella bronchiseptica vaccine). Multiple isolates of B. bronchiseptica with the same PFGE patterns were also recovered from patients with confirmed animal contact. Misidentification of B. bronchiseptica isolates as other oxidase-positive, gram-negative rods can occur when only automated identification systems are used (Loeffelholz and Sanden, 2007). In our experience, B. bronchiseptica does not need special culture requirements, except for an extended incubation time of 48–72 h. We show, in agreement with other authors (Bose et al., 2008), that the identification of a large number of B. bronchiseptica isolates can be easily achieved by a set of conventional phenotypic methods as Gram stain, oxidase and urease test, API 20NE, or automated systems. In this study, none of the 2 automated systems evaluated were 100% reliable. The API 20NE system identified 97% of isolates, while MicroScan and Vitek 2 identified 89% and 87.5% of isolates, respectively. All strains were correctly identified at species level using simultaneously Vitek 2 and API 20NE. However, phenotypic analysis should be combined with molecular identification methods, such as PCR-specific amplification of the upstream region of the fla gene, as a reference method. Data on the antibiotic susceptibility patterns of B. bronchiseptica species are also very limited. Furthermore, few specific studies on the mechanisms of resistance in B. bronchiseptica clinical isolates have been published. A species-specific β-lactamase from B. bronchiseptica, BOR-1 class, has been described in a human isolate (Lartigue et al., 2005) what could explain the highest level of MICs of penicillins in our isolates. Efflux mechanism and/or reduced membrane permeability could also be related to the highest level of MICs for other β-lactams and cephalosporins.
In the absence of reference methods and breakpoints from CLSI or EUCAST for this organism, the results of susceptibility testing are difficult to interpret from a clinical point of view. No specific guidelines for the treatment of B. bronchiseptica infection are available. Antibiotic courses of 2–4 weeks have been recommended for treating the disease (Gueirard et al., 1995). Longer courses, of up to 6 months, have been required for treatment of some neutropenic or immunocompromised patients (Berkowitz et al., 2007). Although erythromycin is usually recommended for treating Bordetella spp., the high MIC90 value of erythromycin for our isolates suggests its ineffectiveness for B. bronchiseptica infections, which is reinforced with the results observed in patients 6 and 19 (Table 2). Both minocycline or moxifloxacin might represent reasonable treatment options for this organisms, although they were not used to treat any of our patients. Antibiotic combinations and longer treatment periods, as used in patients 7, 18, 19, and 20, are options which could also be taken into account. In sumary, our data suggest that B. bronchiseptica is capable of causing serious infections and is most likely an opportunistic pathogen, particularly in patients with previous history of respiratory disease and in those who are immunocompromised. We also recommend the search for a history of exposure to animals since this is rarely documented. Finally, we conclude that susceptibility testing should be routinely performed, when B. bronchiseptica is detected in clinical specimens. Interpretative criteria for clinical interpretation of MICs should be developed. References Belen O, Campos JM, Cogen PH, Jantausch BA. Postsurgical meningitis caused by Bordetella bronchiseptica. Pediatr. Infect. Dis. J. 2003;22(4):380–1. Berkowitz DM, Bechara RI, Wolfenden LL. An unusual cause of cough and dyspnea in an immunocompromised patient. Chest 2007;131(5):1599–602. Binns SH, Speakman AJ, Dawson S, Bennett M, Gaskell RM, Hart CA. The use of pulsedfield gel electrophoresis to examine the epidemiology of Bordetella bronchiseptica isolated from cats and other species. Epidemiol. Infect. 1998;120:201–8. Bose A, Javaid W, Ashame E, Kiska D, Riddell S, Blair D. Bordetella bronchiseptica: an emerging nosocomial pathogen in immunocompromised patients. Clin. Microbiol. Newsl. 2008;30:117–9. Clinical and Laboratory Standards Institute (CLSI). Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria: approved guideline. CLSI document M45-A2. Pennsylvania: CLSI; 2010. De la Torre MJ, de la Fuente CG, de Alegría CR, Del Molino CP, Agüero J, Martinez-Martinez L. Recurrent respiratory infection caused by Bordetella bronchiseptica in an immunocompetent infant. Pediatr. Infect. Dis. J. 2012;31:981–3. Dworkin MS, Sullivan PS, Buskin SE, Harrington RD, Olliffe J, MacArthur RD, et al. Bordetella bronchiseptica infection in human immunodeficiency virus-infected patients. Clin. Infect. Dis. 1999;28:1095–9. Ferry NS. Etiology of canine distemper. J. Infect. Dis. 1911;8:399–420. Garcia San Miguel L, Quereda C, Martinez M, Martin-Davila P, Cobo J, Guerrero A. Bordetella bronchiseptica cavitary pneumonia in a patient with AIDS. Eur. J. Clin. Microbiol. Infect. Dis. 1988;17:675–6. Gueirard P, Weber C, Le Coustumier A, Guiso N. Human Bordetella bronchiseptica infection related to contact with infected animals: persistence of bacteria in host. J. Clin. Microbiol. 1995;33:2002–6. Gueirard P, Bassinet L, Bonne IM, Prevost C, Guiso N. Ultrastructural analysis of the interactions between Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica and human tracheal epithelial cells. Microb. Pathog. 2005;38:41–6. Hozbor D, Fouque F, Guiso N. Detection of Bordetella bronchiseptica by the polymerase chain reaction. Res. Microbiol. 1999;150:333–41. Huebner ES, Christman C, Dummer S, Tang YW, Goodman S. Hospital-acquired Bordetella bronchiseptica infection following hematopoietic stem cell transplantation. J. Clin. Microbiol. 2006;44(7):2581–3. Lartigue MF, Poirel L, Fortineau N, Nordmann P. Chromosome-borne class A BOR-1 βlactamase of Bordetella bronchiseptica and Bordetella parapertussis. Antimicrob. Agents Chemother. 2005;49:2565–7. Le Coustumier A, Gueirard P, Guiso N. Epidémiologie des infections humaines à Bordetella bronchiseptica. Med. Mal. Infect. 1995;25(Suppl. 3):1243–7. Loeffelholz M, Sanden G. Bordetella pertussis. In: Murray P, Baron E, Jorgensen J, Landry M, Pfaller M, editors. Manual of clinical microbiology. 9th ed. Washington, DC: ASM Press; 2007. p. 803–14. Lorenzo-Pajuelo B, Villanueva JL, Rodríguez-Cuesta J, Vergara-Irigaray N, Bernabeu-Wittel M, Garcia-Curiel A, et al. Cavitary pneumonia in an AIDS patient caused by an unusual Bordetella bronchiseptica variant producing reduced amounts of pertactin and other major antigens. J. Clin. Microbiol. 2002;40(9):3146–54. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 2005;18:326–82. Ner Z, Ross LA, Horn MV, Keens TG, MacLaughlin EF, Starnes VA, et al. Bordetella bronchiseptica infection in pediatric lung transplant recipients. Pediatr. Transplant. 2003;7(5):413–7.
C. García-de-la-Fuente et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 20–25 Rath BA, Register KB, Wall J, Sokol DM, Van Dyke RB. Persistent Bordetella bronchiseptica pneumonia in an immunocompetent infant and genetic comparison of clinical isolates with kennel cough vaccine strains. Clin. Infect. Dis. 2008;46(6):905–8. Schneider B, Gross R, Haas A. Phagosome acidification has opposite effects on intracellular survival of Bordetella pertussis and B. bronchiseptica. Infect. Immun. 2000;68:7039–48. Shin EK, Seo YS, Han JH, Hahn TW. Diversity of swine Bordetella bronchiseptica isolates evaluated by RAPD analysis and PFGE. J. Vet. Sci. 2007;8:65–73.
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Spilker T, Liwienski AA, LiPuma JJ. Identification of Bordetella spp. in respiratory specimens from individuals with cystic fibrosis. Clin. Microbiol. Infect. 2008;14:504–6. Wernli D, Emonet S, Schrenzel J, Harbarth S. Evaluation of eight cases of confirmed Bordetella bronchiseptica infection and colonization over a 15-year period. Clin. Microbiol. Infect. 2011;17:201–3. Woolfrey BF, Moody JA. Human infections associated with Bordetella bronchiseptica. Clin. Microbiol. Rev. 1991;4:243–55.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Microbiological and clinical aspects of respiratory infections associated with Bordetella bronchiseptica Celia García-de-la-Fuente a,⁎, Laura Guzmán a, María Eliecer Cano a, Jesús Agüero a,b, Carmen Sanjuán a, Cristina Rodríguez a, Amaia Aguirre a, Luis Martínez-Martínez a,b a b
Service of Microbiology, University Hospital Marqués de Valdecilla-IDIVAL, Santander, Spain Department of Molecular Biology, School of Medicine, University of Cantabria, Spain
a r t i c l e
i n f o
Article history: Received 29 October 2014 Received in revised form 20 January 2015 Accepted 24 January 2015 Available online 2 February 2015 Keywords: Bordetella bronchiseptica Respiratory infections Pulsed-field gel electrophoresis Veterinary pathogen Molecular identification
a b s t r a c t Bordetella bronchiseptica is a well-known veterinary pathogen, but its implication in human disease is probably not fully recognized. The purpose of this study was to determine the clinical significance of 36 B. bronchiseptica isolates from respiratory samples of 22 patients. Therefore, we describe microbiological characteristics, including phenotypic and genotypic identification as well as antimicrobial susceptibilities of the isolates. Clonal relatedness was evaluated using pulsed-field gel electrophoresis (PFGE). Most of the patients had some underlying immunosuppressive condition. Eighteen out of 22 (82%) patients had respiratory symptoms, and the death of 2 patients was associated with respiratory infection.All strains were correctly identified at species level by the simultaneous use of phenotypic methods and were confirmed by specific amplification of the upstream region of the fla gene. Tigecycline, minocycline, doxycycline, colistin, and meropenem were the most active agents tested. PFGE analysis revealed that repeated infections involving each patient had been caused by the same strain. Published by Elsevier Inc.
1. Introduction Bordetella bronchiseptica is a strict aerobic, nonfermentative, catalase- and oxidase-positive gram-negative coccobacillus. This bacterium was first isolated and identified in 1911 (Ferry, 1911) in dogs with distemper disease, and then it was named Bacillus bronchianis. It is currently a well-recognized pathogen in many domestic and wild animals. However, it is an uncommon cause of infection in humans. Sporadic cases of infection by B. bronchiseptica have been reported and are particularly associated with patients who are debilitated or immunosuppressed (Lorenzo-Pajuelo et al., 2002; Ner et al., 2003; Spilker et al., 2008). The organism is less commonly isolated from immunocompetent individuals (De la Torre et al., 2012; Rath et al., 2008). In the past, 2 reviews about human illnesses associated to B. bronchiseptica have been published, in 1991 and 1995, respectively (Woolfrey and Moody, 1991; Le Coustumier et al., 1995), but only in a few cases, microbiological data allowing a reliable identification of the considered microorganism as B. bronchiseptica were presented. Most recently, another review (Mattoo and Cherry, 2005) was published but did not provide enough clinical information on B. bronchiseptica infections. Finally, in 2011 a report presented data on 8 cases collected over a period of 15 years (Wernli et al., 2011), but in 4 of them, a molecular identification of the organism was not provided. Most human cases occur as a pulmonary disease manifested as pneumonia, bronchitis, or whooping-cough. Less frequently, B. bronchiseptica can be involved in systemic infections ⁎ Corresponding author. Tel.: +34-942203359; fax: +34-942203462. E-mail address: [email protected] (C. García-de-la-Fuente). http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.011 0732-8893/Published by Elsevier Inc.
such as meningitis (Belen et al., 2003). Transmission to humans from infected animals can occur through inhalation of infected aerosols or by direct contact with respiratory secretions. Transmission between humans is possible via respiratory droplets since nosocomial transmission of B. bronchiseptica has also been reported in the literature (Bose et al., 2008; Huebner et al., 2006). This suggests that the animal contact is not the only transmission route of B. bronchiseptica in immunocompromised patients. However, there are still unresolved issues concerning the true incidence and the clinical relevance of this organism. In this report, we present microbiological data of B. bronchiseptica, including antimicrobial susceptibilities and molecular typing of isolates obtained from 22 patients from whom clinical data are also evaluated. 2. Material and methods 2.1. Bacterial strains and patients This retrospective study was undertaken at the University Hospital Marqués de Valdecilla of Santander (Cantabria, Northern Spain), an 800-bed tertiary hospital. The computarized database of the Service of Microbiology was consulted to identify clinical samples from which B. bronchiseptica was isolated. A total of 36 isolates, involving 22 patients, were found from June 2004 to November 2011. Two patients have been included in a previous report (De la Torre et al., 2012). Clinical data were obtained from the medical charts of the patients. For each episode, we collected information about the age, sex, underlyng conditions, inpatient/outpatient status, symptoms, diagnosis, culture source and date of isolation, antibiotic treatment, and outcome.
C. García-de-la-Fuente et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 20–25
2.2. Identification and antimicrobial susceptibility testing The strains were stored frozen in trytic soy broth with 10% glycerol at −70 °C. The original identification of all isolates had been carried out with the WalkAway system using MicroScan Neg Combo 38 panel (Dade Behring, West Sacramento, CA, USA) and API20NE strips (bioMérieux, Marcy l′Etoile, France), following manufacturers' recommendations. For the present study, the identification of B. bronchiseptica isolates was reassessed. Frozen isolates were subcultured onto Muller–Hinton agar plates (bioMérieux) and were incubated aerobically at 35 °C for 48 h to ensure viability and purity. Four isolates (from 3 patients) out of 36 conserved organisms could be not recovered. Then, phenotypic identification and susceptibility of the 32 available isolates were performed using the Vitek 2 system with ID-GNB cards (bioMérieux). Addittionally, the MICs of 24 antibiotics were determined by broth microdilution according to the guidelines of the CLSI (2010) using cation-adjusted Mueller–Hinton II broth (DIFCO Laboratories, Detroit, MI, USA). Standard laboratory powders were supplied, as follows: penicillin G, ampicillin, amoxicillin, cefotaxime, amikacin, tobramycin, gentamicin, nalidixic acid, trimethoprim-sulfamethoxazole, erythromycin, rifampin, tetracycline, doxycycline, minocycline, ciprofloxacin, and colistin (Sigma-Aldrich, Tres Cantos, Spain); amoxicillin-clavulanate and cefuroxime (GlaxoSmithKline, Tres Cantos, Spain); moxifloxacin (Bayer, Barcelona, Spain); telithromycin (Aventis Pharma, S.A., Madrid, Spain); tigecycline (Wyeth Farma, San Sebastián de los Reyes, Spain); and imipenem, meropenem, azithromycin, and aztreonam (Discovery Fine Chemicals, Dorset, UK). They were reconstituted according to the manufacturers' instructions, serially diluted in cation-adjusted Mueller–Hinton II broth (DIFCO Laboratories), and dispensed into in house prepared microdilution panels The MIC was defined as the lowest concentration of antibiotic at which no visible growth at 24 h and 48 h was observed. Up to this date, the CLSI and EUCAST susceptibility breakpoints have not been available for B. bronchiseptica; for this reason, resistance and susceptibility criteria have not been defined in this study. 2.3. Molecular identification and typing The strains were identified as B. bronchiseptica by specific amplification of the upstream region of the fla gene using PCR, as described elsewhere (Hozbor et al., 1999). Molecular typing of 32 isolates from 19 patients was performed by pulsed-field gel electrophoresis (PFGE). After overnight digestion with 30 U of XbaI at 37 °C, the restriction fragments were separated using a CHEF-DRIII system (Bio-Rad, Hercules, CA, USA). Electrophoresis was performed in a 1% agarose gel at 6 V/cm and 14 °C with 0.5X TBE buffer. Pulse times were ramped from 7 to 20 s for 9 h and 30 to 50 s for 11 h. Cluster analysis was performed with Fingerprinting II v4.5 software (Bio-Rad) by using the Dice similarity coefficient and the unweighted pair group method with arithmetic means (UPGMA), with 0.5% of optimization and 1% of tolerance. Isolates from different patients showing the same or similar XbaI pattern were aditionally studied with SpeI-PFGE (pulse times: 7–20 s for 11 h and 30–50 s for 13 h) following the same analysis conditions. Isolates were classified as indistinguishable if they showed no band differences (100% similarity), as subtypes of the same strain if they showed 1–3 band differences (85–99% similarity), and as different strains if they showed 4 or more band differences (b85% similarity) (Binns et al., 1998). 3. Results 3.1. Conventional and molecular identification All the isolates were gram-negative coccobacilli, oxidase positive, and catalase positive. Agar plates with 5% sheep blood agar and MacConkey, incubated in 5% CO2, yielded visible colonies after 48 h.
21
The API 20 NE system read at 48 h identified correctly 35/36 (97%) isolates. The remaining isolate was missidentified as Psychrobacter phenylpiruvicus (91.3% probability). Three numeric profiles were obtained, 1200067 (50%), 1200025 (27%), and 1200027 (23%) giving good or very good identification as B. bronchiseptica. All isolates produced positive results for nitrate reduction, urease and adipato, and citrate assimilation. MicroScan Neg Combo 38 panel identified 32/36 (89%) isolates as B. bronchiseptica with N98% probability and misidentified 1 isolate as Ralstonia paucula (89.06% probability); 2 isolates were identified with very low probability (b60%) as B. bronchiseptica and proved to be inconclusive in one. The Vitek 2 system identified 28/32 (87.5%) isolates properly, with N98% probability and misidentified 2 isolates as Acinetobacter lwofii (92–93% probability) and 2 as Oligella urethralis (90% probability). All isolates were subsequently confirmed as B. bronchiseptica by PCR-specific amplification of the upstream region of the fla gene. 3.2. Susceptibility testing MIC50 and MIC90 values of the 24 antimicrobial agents tested are presented in Table 1. The lowest MIC at which 90% of isolates tested are inhibited (MIC90) was that of tigecycline, minocycline, doxycycline, and meropenem, whereas the highest MIC90 were those of β-lactams as well as cephalosporins, macrolides, ketolides, and trimethoprim-sulfamethoxazole. On the other hand, moxifloxacin demonstrated lower MIC50 and MIC 90 compared to ciprofloxacin. For the rest of antibiotics tested, MIC values were variable. 3.3. Molecular typing PFGE analysis (Fig. 1) of XbaI restriction fragments revealed 16 different patterns, which became 17 after analyzing the results of SpeIPFGE. These 17 pulsotypes were grouped in 12 clusters based on ≥85% similarity of PFGE profiles. Isolates belonging to the same patients presented identical or very similar pulsotype, as well as isolates from patients 20 and 19 (pulsotype 4B), who were mother and son, respectively. Five of the 7 isolates from patient 7 had identical pattern, and 2 Table 1 MIC50 and MIC90 values of the antimicrobial agents tested for 32 B. bronchiseptica clinical isolates. Antimicrobial agent
Penicillin Ampicillin Amoxicillin/clavulanate Aztreonam Cefuroxime Cefotaxime Imipenem Meropenem Amikacin Gentamicin Tobramycin Nalidixic acid Ciprofloxacin Moxifloxacin Tigecycline Trimethoprim/sulfamethoxazole Erythromycin Azithromycin Telithromycin Rifampicin Minocycline Doxycycline Tetracycline Colistin
MIC50 (μg/mL)
MIC90 (μg/mL)
24 h
48 h
24 h
48 h
128 64 16/8 512 N512 128 1 0.06 16 2 2 16 1 0.5 ≤0.03 16/304 8 1 16 32 0.06 0.06 0.5 ≤0.03
N512 128 32/16 512 N512 N512 4 0.06 64 4 8 32 4 1 0.06 N64/1216 32 4 32 128 0.5 0.5 2 ≤0.03
N512 256 64/32 N512 N512 N512 2 0.5 64 4 8 32 4 1 1 N64/1216 32 4 32 64 0.25 0.125 2 ≤0.03
N512 N512 256/128 N512 N512 N512 8 4 128 8 32 64 4 1 2 N64/1216 32 8 64 256 1 1 4 ≤0.03
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C. García-de-la-Fuente et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 20–25 Dice (Opt:0.50%) (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
PFGE SpeI
100
90
PFGE XbaI 80
70
60
PFGE XbaI
PATIENT / STRAIN DATE OF ISOLATION PFGE PATTERN 11/2694
June/07
8
13/1689
Mar/09
8
21/310
Jan/11
11
5/524
Feb/05
3
18/3770
July/10
10
22/5596
Nov/11
12
15/3263
June/09
6B
8/3970
Oct/06
6A
1/1475
June/04
1
12/399
Jan/09
2B
17/4968
Aug/09
2B
2/2125
Aug/04
2A
9/4941
Dec/06
2B
14/3024
May/09
9
7/4221
Dec/05
5A
7/308
Jan/06
5A
7/1400
Apr/06
5A
7/3673
Sept/06
5A
7/5031
Oct/07
5A
7/982
Mar/06
5C
7/710
Feb/06
5B
10/5187
Dec/06
7
10/5170
Dec/06
7
19/5215
Sept/10
4B
19/6005
Oct/10
4B
19/6324
Nov/10
4B
20/5381
Sept/10
4B
20/6179
Nov/10
4B
20/6934
Dec/10
4B
6/3283
Sept/05
4A
6/1284
Mar/06
4A
6/910
Feb/07
4A
Fig. 1. PFGE of XbaI- and SpeI-157 digested chromosomal DNA from isolates of B. bronchiseptica. The dendrogram was obtained by XbaI-PFGE of 32 isolates from 19 patients and was constructed by using Dice similarity coefficients (0.5% optimization and 1.0% tolerance) and UPGMA algorithm. Patient and strain number, date of isolation, and PFGE pattern are included along each PFGE lane. PFGE patterns were assigned after analysis of pulsotypes obtained with both XbaI and SpeI digestion. Twelve PFGE groups were obtained based on ≥85% similarity of PFGE profiles.
were closely related with more than 95% of similarity. Other patients whose isolates showed identical or very similar PFGE pattern, but without any apparent epidemiological link, were patients 11 and 13 (pulsotype 8); patients 8 and 15 (pulsotypes 6A and 6B); and patients 12, 17, and 9 (pulsotype 2B) along with patient 2 (pulsotype 2A). The last 4 isolates have identical pattern when only considering the analysis of XbaI-PFGE. Susceptibility patterns among the pulsotypes from the same patients were identical.
3.4. Clinical data Table 2 summarizes demographic data, underlying diseases, clinical characteristics, and outcome of the 22 patients considered in this study. Four patients were diagnosed in 2004; 3, in 2005; 3, in 2006; 1, in 2007; 6, in 2009; 3, in 2010; and 2, in 2011. A total of 11 (50%) patients were female, and the median age was 60 years (range, b1–90). Nineteen patients were admitted to the hospital, and 4 patients (18%) required admission to the intensive care unit. Four patients had repetitive episodes, and they were the only ones who had documented exposure to dogs or cats. Seventeen patients (77%) had active comorbidities at the time of infection/colonization. In 9 of them, more than 1 of the considered underlying diseases was found. Furthermore, several patients were immunosuppressed due to chronic corticosteroid therapy (n = 6), other immunosupressive therapy (n = 3), hematological malignancies
(n = 2), or HIV infection (n = 1). Only 4 patients (18%) had no known underlying conditions. B. bronchiseptica, in these 22 patients, were isolated from sputum (n = 25), from nasopharyngeal lavages (n = 6), from traqueal aspirates (n = 2), from protected brush (n = 1), from bronchial aspirate (n = 1), and from middle ear effusion (n = 1). Twenty-seven (75%) samples were monomicrobial; in the remaining 9 (25%) samples, a copathogen was also cultured, including Haemophilus influenzae, patients 2 and 11; Staphylococcus aureus, patient 17; Aspergillus fumigatus, patients 6 and 7 (3 episodes and 1 episode, respectively); Corynebacterium pseudodiphteriticum, patient 15; and Mycobacterium avium complex, patient 7 (1 episode). Patients 6 and 7 had been in contact with dogs, and patients 19 and 20 had been in contact with cats. According to the clinical presentation at time of isolation of B. bronchiseptica, 5/22 (23%) had acute exacerbation of chronic obstructive pulmonary disease (COPD); 5/22 (24%), acute respiratory infection; 3/ 22 (14%), fever; 2/22 (9%), pneumonia; 2/22 (9%), chronic cough; 1/22 (5%), like syndrome; 1/22 (5%), respiratory distress; 1/22 (5%), acute bronchospasm; and 1/22 (5%), laryngotracheitis plus otitis media. Twenty out of 22 (91%) patients had signs and symptoms of infection, and 18/22 (82%) patients had respiratory symptoms. The most common clinical symptoms and signs at presentation included fever with a temperature ≥38 °C (61%), productive cough (46%), and shortness of breath and dyspnoea (45%). At physical examination, ronchi and basal crackles were detected in 64% of the patients.
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Table 2 Clinical data of B. bronchiseptica respiratory infections. Patient/strain number
Age (years)/ sex
Date of isolation
PFGE pattern
Underlying disease
Diagnosis
Clinical sample
Therapy/duration
Outcome
1/1475 2/2125 3 4/2699 5/524 63283 61284 6910 7/4221 7/308 7/710 7/982 7/1400 7/3673 7 7/5031 8/3970 9/4941 10/5187
80/F 69/M 19/F 69/M 62/M 32/M
1 2A Unworked Unworked 3 4A 4A 4A 5A 5A 5B 5C 5A 5A Unworked 5A 6A 2B 7
COPD/diabetes Prostate cancer No data CHD COPD/bladder cancer CF Lymphoma Lung transplant Kidney transplant, bronchiectasis
COPD exacerbation Ascites/fever Pneumonia Fever COPD exacerbation Prolonged fever
Sputum Sputum Sputum Sputum Sputum Sputum
Chronic cough/dyspnea
Sputum
81/F 63/F 32/F
June/04 Aug/04 Sept/04 Sept/04 Feb/05 Sept/05 Mar/06 Feb/07 Dec/05 Jan/06 Feb/06 Mar/0 Apr/06 Sept/06 Oct/06 Oct/07 Oct/06 Dec/06 Dec/06
COPD COPD/myeloma HIV/HCV
COPD exacerbation Acute bronchospasm Acute respiratory disease
Sputum Sputum PB
Azithrormycin 2 wk None Amoxicillin/clavulanate 10 d Ciprofloxacin 2 wk Moxifloxacin 3 wk TMP-SMX 2 d Azithromycin 2 wk Levofloxacin 8 d Amoxicillin/clavulanate 8 d Amoxicillin/clavulanate 8 d Amoxicillin/clavulanate 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Ciprofloxacin 8 d Levofloxacin + TMP-SMX 10 d No data Telithromycin 5 d Levofloxacin 1 wk
Death Death Recovery Recovery Recovery Recurrence Recurrence Recovery Recurrence Recurrence Recurrence Recurrence Recurrence Recurrence Recurrence Recovery Unknown Recurrence Recovery
10/5170 11/2694
67/F
June/07
7 8
Diabetes
BAS Sputum
Levofloxacin 5 d
Recovery
12/399 13/1689
73/M 78/M
Jan/09 Mar/09
2B 8
COPD Pulmonary fibrosis
Sputum Sputum
Ceftriaxone 5 d TMP-SMX 5 d
Recovery Recovery
14/3024 15/3263
45/M 66/F
May/09 June/09
9 6B
Liver transplant/VHC CHD/diabetes
TA TA
Ciprofloxacin 1 wk Levofloxacin 10 d
Recovery Recovery
16/587 17/4968 18/3770 19/5215 19/6005 19/6324 20/5381 20/6179 20/6934 21/310 22/5596
73/M 73/F 80/F b1/M
July/09 Aug/09 July/10 Sept/10 Oct/10 Nov/10 Sept/10 Nov/10 Dec/10 Jan/11 Nov/11
Unworked 2B 10 4B 4B 4B 4B 4B 4B 11 12
None None COPD None
Acute respiratory disease COPD exacerbation Acute respiratory disease Respiratory distress Acute respiratory disease No data Laryngotracheitis/COM Pneumonia Pertussis-like syndrome
Sputum Ear media Sputum Nasopharyngeal lavate
No data Azithromycin 3 d Levofloxacin + TMP-SMX 2 wk Erythromycin 4 d Ciprofloxacin 7 d TMP-SMZ + rifampin 2 wk None Ciprofloxacin 7 d TMP-SMZ + rifampin 2 wk Levofloxacin 2 wk Amoxicillin/clavulanate 7 d
Unknown Recurrence Recovery Recurrence Recurrence Recovery Recurrence Recurrence Recovery Recovery Death
52/F
31/F
81/M 90/M
None
Chronic cough
Nasopharyngeal lavate
COPD/diabetes CHD/diabetes
COPD exacerbation Acute respiratory disease
Sputum Sputum
CHD = chronic heart disease; CF = cystic fibrosis; TMP-SMX = trimethroprim-sulfamethoxazole; PB = protected brush; BA = bronchial aspirate; TA = tracheal aspirate; COM = chronic otitis media. Unworked, isolate not available for PFGE.
Laboratory data were available from only 17 episodes, in 12 (71%) of which the white blood cell count ranged from 12,300 to 22,100/mm 3 with 75–95% neutrophils. In 5 patients in whom C-reactive protein was studied, values from 1.5 to 4.7 mg/dL were observed (normal range 0.0–0.5 mg/dL). Only 4 patients had blood cultures requested, with negative results in all cases. Chest x-ray was performed for 59% (13/22) of the patients, of whom 31% had significant alterations, including pneumonia (2/13) or subsegmental bronchiectasis (2/13). All patients had a Gram-stained smear of the specimens revealing neutrophils and abundant gram-negative coccobacilli, including intracellular forms. In 31 episodes, the patients received antibiotic therapy, including levofloxacin, ciprofloxacin, or moxifloxacin (n = 14); azithromycin, erythromycin, or telithromycin (n = 5); amoxicillin-clavulanate (n = 5); trimethroprim-sulfamethoxazole (n = 2); ceftriaxone (n = 1); levofloxacin plus trimethroprim-sulfamethoxazole (n = 2); or trimethroprim-sulfamethoxazole plus rifampicin (n = 2). Three patients died during their hospitalization. In patients 1 and 22, death was related to respiratory infection (both patients had been treated with azithromycin and amoxicillin-clavulanate respectively). In patient 2, death was related to other medical causes (this patient had not received antibiotic treatment).
4. Discussion Most reports on B. bronchiseptica infection/colonization in humans correspond to small case series or single case reports of debilitated or immunosuppressed patients (Berkowitz et al., 2007; Dworkin et al., 1999; Garcia San Miguel et al., 1988; Lorenzo-Pajuelo et al., 2002; Wernli et al., 2011). The reason why only a limited number of cases of B. bronchiseptica are reported could be due to the fact that it is considered a respiratory commensal. In this study, the majority of infections/ colonizations caused by B. bronchiseptica occurred in adults with underliyng diseases, and only 19% of the patients had no comorbid conditions. In all cases, B. bronchiseptica was recovered from the respiratory tract, which supports the previously described tropism of this bacterium for the respiratory tissue (Le Coustumier et al., 1995; Wernli et al., 2011). Most common clinical presentations of B. bronchiseptica infections were very similar from those caused by widely recognized pathogens, such as Haemophillus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Except for 3 patients, for which clinical data were lacking, the episodes, according to clinical records, were considered as infection and treated as such. However, as organisms were often recovered from the sputum, there can be no full convincing evidence for its clinical implication, and the possibility that they are just colonizers cannot be completely excluded.
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Four patients, 2 of them immunocompetent, who had repeated episodes of infection for extended periods of time, were the only ones with documented animal exposure. The persistence or recurrence of B. bronchiseptica infection had been previously described (De la Torre et al., 2012; Rath et al., 2008). Perhaps this can be due to the lack of clinical effectiveness of macrolides, used for other Bordetella infections, or because B. bronchiseptica may survive intracellularly for a long time for which they require adhesin expression as filamentous hemagglutinin and other virulence factors as adenylate cyclase-hemolysine (Gueirard et al., 1995). Furthermore, B. bronchiseptica (unlike B. pertussis) could persist intracellularly in infected macrophages due to their acid tolerance (Schneider et al., 2000). According to a more recent study, B. bronchiseptica are able to resist the macrophage attacks and remain viable for several days, although they do not multiply (Gueirard et al., 2005). However, according to these authors, once B. bronchiseptica invades respiratory epithelial cells, the bacteria could be protected from antibodies and the complement system and is resistant to phagocytosis in patients with a deficient host immune system. Therefore, B bronchiseptica could persist in cells for a longer time. Patients 9 and 17 had recurrent symptoms, but no microbiological cultures were performed. There is little information on molecular typing of B. bronchiseptica isolates from clinical origin. In the present study, PFGE was able to differenciate 17 pulsotypes, grouped in 12 clusters, among the 32 isolates affecting 19 patients. These results indicate that PFGE offers a high discriminatory power for genotyping B. bronchiseptica isolates of human origin, as has been demonstrated in previous studies in animals (Bose et al., 2008; Shin et al., 2007). In order to rule out a poor discriminative capacity of XbaI, SpeI was also used to study isolates from different patients showing the same or similar PFGE-pattern. The coincident results of the PFGE assay using XbaI or SpeI in practically all the evaluated isolates confirm the clonal relation of these isolates. Unfortunately, in the absence of additional epidemiological information due to the retrospective nature of this study, it is difficult to evaluate the relevance of this finding. Isolates recovered from the same single patient for almost 2 years with identical or very similar PFGE patterns indicated that the same strain has caused multiple episodes. This may be related to the fact that the patient owned a dog living with her and, although no cultures were performed on the pet, it seems relevant that no more episodes occurred after the dog was vaccinated (Bordetella bronchiseptica vaccine). Multiple isolates of B. bronchiseptica with the same PFGE patterns were also recovered from patients with confirmed animal contact. Misidentification of B. bronchiseptica isolates as other oxidase-positive, gram-negative rods can occur when only automated identification systems are used (Loeffelholz and Sanden, 2007). In our experience, B. bronchiseptica does not need special culture requirements, except for an extended incubation time of 48–72 h. We show, in agreement with other authors (Bose et al., 2008), that the identification of a large number of B. bronchiseptica isolates can be easily achieved by a set of conventional phenotypic methods as Gram stain, oxidase and urease test, API 20NE, or automated systems. In this study, none of the 2 automated systems evaluated were 100% reliable. The API 20NE system identified 97% of isolates, while MicroScan and Vitek 2 identified 89% and 87.5% of isolates, respectively. All strains were correctly identified at species level using simultaneously Vitek 2 and API 20NE. However, phenotypic analysis should be combined with molecular identification methods, such as PCR-specific amplification of the upstream region of the fla gene, as a reference method. Data on the antibiotic susceptibility patterns of B. bronchiseptica species are also very limited. Furthermore, few specific studies on the mechanisms of resistance in B. bronchiseptica clinical isolates have been published. A species-specific β-lactamase from B. bronchiseptica, BOR-1 class, has been described in a human isolate (Lartigue et al., 2005) what could explain the highest level of MICs of penicillins in our isolates. Efflux mechanism and/or reduced membrane permeability could also be related to the highest level of MICs for other β-lactams and cephalosporins.
In the absence of reference methods and breakpoints from CLSI or EUCAST for this organism, the results of susceptibility testing are difficult to interpret from a clinical point of view. No specific guidelines for the treatment of B. bronchiseptica infection are available. Antibiotic courses of 2–4 weeks have been recommended for treating the disease (Gueirard et al., 1995). Longer courses, of up to 6 months, have been required for treatment of some neutropenic or immunocompromised patients (Berkowitz et al., 2007). Although erythromycin is usually recommended for treating Bordetella spp., the high MIC90 value of erythromycin for our isolates suggests its ineffectiveness for B. bronchiseptica infections, which is reinforced with the results observed in patients 6 and 19 (Table 2). Both minocycline or moxifloxacin might represent reasonable treatment options for this organisms, although they were not used to treat any of our patients. 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