Isolation of Bordetella species from unusual infection sites

Received 12 January 2015 Accepted 19 February 2015 Laboratorio de Bacteriologı́a, Departamento de Bioquı́mica Clı́nica, Hospital de Clı́nicas José de San Martı́n, Facultad de Farmacia y Bioquı́mica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina Laboratorio de Bacteriologı́a. Hospital Interzonal de Agudos Eva Perón, Provincia de Buenos Aires, Argentina Instituto de Microbiologı́a y Parasitologı́a Médica (IMPaM, UBA-CONICET), Facultad de Medicina, Universidad de Buenos Aires, Argentina Laboratorio de Bacteriologı́a, Hospital de Niños Ricardo Gutierrez, Ciudad Autónoma de Buenos Aires, Argentina Laboratorio de Microbiologı́a, Hospital Universitario Austral, Provincia de Buenos Aires, Argentina Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA


Introduction
Except for Bordetella petrii, which was initially found as a freeliving environmental bacterium (von Wintzingerode et al., 2001), all other bordetellae are found only in warm-blooded animals and humans.Bordetella avium is a bird pathogen causing coryza and rhinotracheitis in poultry (Rimler, 1985;Kersters et al., 1984).Bordetella bronchiseptica may cause respiratory infections in many animal species and, infrequently, also in humans.Bordetella ansorpii, Bordetella hinzii, Bordetella holmesii, B. petrii, and Bordetella.trematum are found rarely in human infections and mainly cause disease in immunocompromised patients.Bordetella parapertussis is found in sheep and humans, and Bordetella pertussis is thought to be a strictly human pathogen (Wirsing von Koning et al., 2011).
Herein we describe four cases of infections due to Bordetella species that have been recovered from unusual infection sites: two cases of B. hinzii infections, one recovered from the urine of a patient with chronic prostatitis and the other from a liver cyst in an immunocompetent patient; one B. trematum case from a bone biopsy of a patient with chronic osteomyelitis of the hip; and one B. holmesii case isolated from the joint fluid of an immunocompetent patient with diagnosed septic arthritis.

Case report 1
A 55-year-old male with a history of chronic prostatitis was seen in the Urology Department at Hospital Interzonal de Agudos Eva Pero ´n because of mictional urgency, dysuria and a long history of pelvic pain.A urine sample was taken for bacteriological culture, and empirical treatment with trimethoprim-sulfamethoxazole (TMP-SMX) was started.The urinalysis showed a pathological sediment with more than 25 leukocytes per field, pyuria and bacteriuria.The urinary culture yielded .10 5 c.f.u.ml 21 of a Gram-negative rod.The organism was identified using standard biochemical tests as B. hinzii.Identification results obtained by the VITEK 2 and API 20 NE systems are shown in Table 1.Identification through these two systems yielded the wrong result.Instead, identification carried out using matrixassisted laser desorption ionization-time-of-flight (MALDI TOF) MS (Bruker Daltonik) showed a spectral score of 2.292 for B. hinzii.
Additionally, the 16S rRNA sequencing results showed 99 % identity with the sequences corresponding to the 16S rRNA of B. hinzii (AF177667).
Based on the susceptibility test result (Table 2), a 1 month course of TMP-SMX therapy was prescribed.
Urine culture after treatment was negative.However, the patient returned to the hospital 2 weeks later, complaining of urinary symptoms.A new urine sample was collected, demonstrating 25 leukocytes per field.A pure growth of more than 10 5 c.f.u.ml 21 of a microorganism with the same phenotypic characteristics as the previous urine culture isolate was obtained.The isolate was again identified as B. hinzii.The antibiotic sensitivity was the same as that of the prior isolate.This time, the same therapy was established for 3 months with good clinical progress.

Case report 2
A 14-year-old male, from Formosa (Argentinean province), with a history of left hip septic arthritis caused by Staphylococcus aureus, not responding to antibiotic treatment, was referred to the Ricardo Gutierrez Children's Hospital owing to unfavourable progress.On admission he was febrile and haemodynamically unstable.Wide surgical toilette with bone hip curettage was performed.From the bone biopsy culture, growth of a micro-organism presumptively identified by standard biochemical tests as B. trematum together with Escherichia coli was obtained.The results of the microorganism identification by the VITEK 2 system and by the API 20 NE system are shown in Table 2.In addition, identification using MALDI TOF MS (Bruker Daltonik) showed a spectral score of 2.206 for B. trematum.Moreover, the 16S rRNA sequencing results showed that the isolate had 99 % identity with the 16S rRNA gene from B. trematum (GenBank accession no KF601906).
The patient required multiple surgical debridements because of his torpid outcome, and also Steinman nail placement and skeletal traction.The clinical diagnosis was hip pandiaphysitis and femur epiphysiolysis.The patient had hip septic arthritis caused by S. aureus with superinfection due to E. coli plus B. trematum.Pathological examination was consistent with chronic osteomyelitis.Based on antibiotic susceptibility (Table 2), the patient was treated intravenously with meropenem and TMP-SMX for 6 months.Once the antibiotic therapy was complete, the patient was discharged, with oral minocycline administered as an outpatient.

Case report 3
A 16-year-old male was admitted to the Emergency Department of the Austral Hospital, Buenos Aires, Argentina, in March 2009 because of severe pain and functional disability of his right knee and fever of 3 days' progress (102.2 u F).He reported having suffered increasing knee pain after a slight trauma that occurred while he was playing football 4 days before.
On admission, his right knee was oedematous, erythematous, and tender to touch, with limited motion range (with signs of inflammation).Ultrasound of the knee soft tissue showed a thin band of fluid adjacent to the distal insertion of the quadriceps tendon.Surgical drainage of the joint by arthrotomy was indicated.
Examination of the synovial fluid showed 10 4 white blood cells mm 23 with 80 % neutrophils, but no microorganisms were observed on Gram-stained smear.In cultures on solid media (5 % sheep blood agar plates and chocolate agar plates), incubated in 5 % CO 2 , no bacterial growth was obtained, however in subcultures in liquid medium (thioglycollate broth) on 5 % sheep blood agar and chocolate agar, grew a microorganism, grew a microorganism, showing small colonies surrounded by a brownish area.On trypticase soy agar, colonies showed a  1; identification using MALDI TOF MS (Bruker Daltonik) showed a spectral score of 2.073 for B. holmesii.Moreover, the amplification and sequence analysis of 16S rRNA showed a 99 % identity with the 16S rRNA from B. holmesii (GenBank accession no.DQ409136.1).
Based on assuming septic arthritis due to S. aureus, the patient was empirically treated with clindamycin (1 g every 6 h)/gentamicin (240 mg every 24 h) for 4 days.When the isolation of B. holmesii was reported, the patient was making favourable progress with the antibiotic therapy administered empirically; therefore this therapy was continued.Then, owing to having made good clinical progress, he was discharged from hospital with the indication to receive treatment with TMP-SMX 10 mg kg 21 day 21 for 17 days.

Case report 4
A 58-year-old female, with a history of hypothyroidism and open cholecystectomy 20 years before, consulted the Hospital de Clı ´nicas Jose ´de San Martı ´n Emergency Room due to abdominal pain in the right upper quadrant.The patient was afebrile and normotensive.On physical examination the abdomen was tender and depressible.
Laboratory findings on the peripheral venous blood sample on admission were: white blood cell count, 18 600 mm 23 (with 73.5 % neutrophils); haematocrit, 26.9 %; haemoglobin count, 8.6 g dl 21 ; and platelet count, 478 000 mm 23 .She  1).The growth of the same microorganism was obtained in both samples submitted.The culture result was reported 5 days after sample culture, but the patient had already been discharged because of good clinical progress (with haemodynamic stability and a soft, depressible and non-tender abdomen); therefore the isolation result was not taken into account.

Laboratory investigations
The organisms were identified using standard biochemical tests following the scheme proposed by Wauters & Vaneechoutte (2011)
Most of the isolates included in their study were from the respiratory tracts of chickens and turkeys.B. avium poultry infections (rhinotracheitis) have been well documented (Rimler, 1985;Kersters et al., 1984).However, although B. hinzii is capable of colonizing turkeys (Vandamme et al., 1995), experimental infections in turkey have shown that it is non-pathogenic (Jackwood et al., 1985).
B. hinzii has been reported in respiratory samples from three patients.Two of them had an immunosuppressive disease: a patient with cystic fibrosis and another with AIDS.In the first, B. hinzii was isolated from sputum repeatedly over a period of 3 years (Funke et al., 1996); in the second, the organism was recovered from a bronchoalveolar lavage together with Nocardia asteroides (Gadea et al., 2000).Recently Palacia ´n Ruiz et al. (2013) have reported the case of a respiratory infection with B. hinzii in an 85-year-old immunocompetent patient.In this case report the organism was isolated from sputum together with Klebsiella oxytoca (Palacia ´n Ruiz et al., 2013).B. hinzii has also been described as a cause of bacteraemia in immunosuppressed patients: in a patient with myelodysplastic syndrome (Fry et al., 2007), in another patient with Epstein-Barr virus-associated diffuse large B-cell lymphoma (Hristov et al., 2008) and in an AIDS patient (Cookson et al., 1994).Furthermore, a case of chronic cholangitis due to B. hinzii has been documented in a liver transplant recipient (Arvand et al., 2004).In this case the organism was isolated in four biliary specimens collected over 6 months from a 29-year-old male receiving immunosuppressive therapy.In addition, a case of fatal bactaeremia after endoscopic retrograde cholangiopancreatography in an immunocompetent patient with cholestasis has been described (Kattar et al., 2000).had participated in a cookout at a farm 2 weeks before his illness as the only epidemiological history; however, no further details regarding contact with poultry or birds were available.As suggested by these authors (Arvand et al., 2004;Kattar et al., 2000), gastrointestinal colonization by B. hinzii and a subsequent ascending infection to the liver could be hypothesized in our patient.
B. hinzii isolation from urine in a patient with prostatitis has not previously been described in the literature.The only remarkable epidemiological link for this case was the drinking of unsafe water: the patient documented that he had drunk water and had bathed in an Australian tank pool where often there were dead birds.Again in this case, gastrointestinal colonization could be hypothesized since the ascending path is the most common route of infection of the urinary tract.As gastrointestinal colonization typically occurs through oral transmission, B. hinzii could have been acquired by drinking water contaminated with dead poultry.
As regards antibiotic susceptibility, our results are consistent with those published by other authors (Kattar et al., 2000).Interestingly, cephalothin was more active than cefotaxime.Furthermore, as in our work, resistance to fourth-generation cephalosporins has been reported in B. hinzii.The organism was also susceptible to aminoglycosides, ciprofloxacin and TMP-SMX; however, quinolone resistance (Arvand et al., 2004) and TMP-SMX resistance (Palacia ´n Ruiz et al., 2013) have been described in this species.Unlike B. hinzii, B. avium retains sensitivity to most beta-lactam antibiotics: ampicillin, ampicillin-sulbactam, amoxicillin-clavulanic acid, piperacillin-tazobactam, cephalothin, ceftriaxone, cefotaxime, cefepime and imipenem (Kattar et al., 2000); consequently, this antibiotic sensitivity profile can be used to differentiate both species.
B. trematum was first described by Vandamme et al. (1996).They performed a comprehensive phenotypic and genotypic analysis of 10 atypical or unclassified Bordetella strains and found that such strains belonged to a new species of Bordetella.All of them had been isolated from ear infections or leg, arm and ankle wounds.Before the species B. trematum had been established, Dorittke et al. (1995) had described the isolation of a 'B.avium-like organism' from a patient with chronic otitis media, which was later reclassified as B. trematum (Vandamme et al., 1996).Although B. trematum has been isolated from skin and soft tissue infections, isolation from bone biopsy of a patient with chronic hip osteomyelitis has not previously been reported in the literature.In addition, B. trematum usually colonizes or infects ulcers in diabetic patients (Daxboeck et al., 2004), but our patient did not present this underlying disease.
Reports in the literature mention this Bordetella species as oxidase-negative (Vandamme et al., 1996;Daxboeck et al., 2004), but the fact that the oxidase activity in these works was tested with a freshly prepared 1 % solution of N,N''-dimethyl-p-phenylene monochloride, and not with the N,N,N',N'-tetramethyl-p-phenylenediamine derivative, should be taken into account.For our B. trematum isolate, the oxidase reaction performed with the dimethyl derivative was negative (reading time up to 1 min), whereas, when we used the tetramethyl-p-phenylenediamine derivative, an oxidase-positive result was obtained within 15 s.Regarding identification by API 20 NE, other authors (Daxboeck et al., 2004;Herna ´ndez-Porto et al., 2013) have reported that this system identified the organism as B. avium (biocode 0000063) because B. trematum was not present in their database.However, other biocodes could be obtained considering that the nitrate reduction in this species is variable and that the oxidase test result varies depending on the reagent used.We obtained biocode 1000067, which corresponds to Achromobacter denitrificans 78.3 %, B. bronchiseptica 18.3 %, because our strain reduced nitrate and was oxidase-positive by using the N,N,N',N'-tetramethyl-p-phenylenediamine derivative.
As regards the sensitivity to beta-lactam antibiotics and in agreement with Herna ´ndez-Porto et al. (2013), cefotaxime was not very active.Interestingly, as in B. hinzii cases, cephalothin was more active than cefotaxime (Table 2); ceftazidime, cefepime carbapenems and piperacillin-tazobactam were also active.Regarding other antibiotics and in accordance with previous reports (Daxboeck et al., 2004;Herna ´ndez-Porto et al., 2013), aminoglycosides and TMP-SMX were active.The lack of fluoroquinolone activity observed in our isolate has also been pointed out previously (Daxboeck et al., 2004;Herna ´ndez-Porto et al., 2013).
B. holmesii was described by Weyant et al. (1995)  and unaccompanied by bacteraemia.One of these patients was a 54-year-old woman who consulted owing to suspected right prosthetic knee infection but no history of frequent or unusual infections.As in our case, the other case was a previously healthy 15-year-old boy who developed post-traumatic knee arthritis with no preceding history of respiratory symptoms and no significant past medical history, including no previous invasive bacterial infections and no adverse reactions to vaccines (Abouanaser et al., 2013).Unlike our case, where the patient was initially treated with clindamycin plus gentamicin (and then switched to TMP-SMX, assuming septic arthritis due to S. aureus), in the two cases described by Abouanaser et al. (2013), initial treatment was with ceftriaxone, and then oral fluoroquinolone (ciprofloxacin in the first case; levofloxacin in the second case), as per literature reports documenting low fluoroquinolone MICs for most B. holmesii isolates.As in our case, in both Abouanaser cases, clinical progress was favourable.In our case, the organism was highly susceptible to beta-lactam (ampicillin, piperacillin-tazobactam, ceftriaxone, ceftazidime, cefepime, imipenem, meropenem), gentamicin, ciprofloxacin and TMP-SMX antibiotics.However, resistance to cefotaxime (Shepard et al., 2004;Panagopoulos et al., 2010;Monnier et al., 2010;Nguyen et al., 2013;Jonckheere et al., 2012;Bassetti et al., 2012), ceftriaxone (Monnier et al., 2010;Van Balen et al., 2012;Stoddard, 2013) and TMP-SMX has been reported (Monnier et al., 2010;Nguyen et al., 2013); resistance to third-generation cephalosporins should be considered since sometimes the initial treatment for septic arthritis by B. holmesii has been ceftriaxone (Abouanaser et al., 2013).
As has previously been noted, B. holmesii may be an underrecognized pathogen owing to its slow growth and difficult identification.In our case, the B. holmesii isolate was misidentified as Acinetobacter lwoffii by the VITEK 2 Compact system (bioMe ´rieux) using the GN Colorimetric Identification Card; using the NH Colorimetric Identification Card, the organism was erroneously identified as Neisseria sicca/Oligella urethralis.There have been reports of repeated misidentifications of this organism as A. lwoffii using standard automated laboratory systems (Abouanaser et al., 2013;Panagopoulos et al., 2010).The misidentification as Moraxella lacunata by API 20 NE (Table1) has already been reported (Moissenet et al., 2011).These misidentifications by the VITEK 2 automated and API 20 NE systems can be attributed to the fact that this species is not found in the databases.An excellent review about microbiology, epidemiology, clinical features, diagnosis and treatment of B. holmesii has recently been published by Pittet et al. (2014).
Cumulative evidence suggests that these Bordetella species can cause clinical diseases other than those recognized to date.Further studies are needed to establish pathogenicity of these Bordetella species in these infection sites.
We wish to highlight the importance of isolating Bordetella species from severe infections and unusual sites, and also the importance of polyphasic identification to address definitive identification.

Table 1 .
Phenotypic identification results of VITEK 2 and API 20 NE systems

Table 2 .
Antimicrobial susceptibility of Bordetella species (mg ml 21 ) in the right hepatic lobe was observed.Two samples of bilious fluid obtained from the punctured cyst were sent for culture.Direct examination of the material revealed the presence of moderate inflammatory reaction, and no bacteria were observed on Gram stain.
Weisburg et al. (1991)order to confirm the species.PCR products of the 16S rRNA, using the primers described byWeisburg et al. (1991), were obtained with Taq DNA polymerase (Promega) based on the manufacturer's specifications.Sequencing of the 1.4 kb PCR product was performed on both DNA strands at the Macrogen sequencing facility, Seoul, Korea.The sequences obtained for the four isolates