- Email: [email protected]
Infections caused by Gordonia species at a medical centre in Taiwan, 1997 to 2008
ORIGINAL ARTICLE
BACTERIOLOGY
Infections caused by Gordonia species at a medical centre in Taiwan, 1997 to 2008 C. C. Lai1, C. Y. Wang1, C. Y. Liu2, C. K. Tan3, S. H. Lin4, C. H. Liao2, C. H. Chou5, Y. T. Huang6, H. I. Lin1 and P. R. Hsueh6 1) Department of Internal Medicine, Cardinal Tien Hospital, Taipei County, 2) Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, 3) Department of Intensive Care Medicine, Chi-Mei Medical Center, Tainan, 4) Department of Internal Medicine, Taipei County Hospital, Taipei County, 5) Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin and 6) Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan
Abstract The inability of conventional identification systems to accurately identify Gordonia spp. often results in the misdiagnosis of infections by these rare pathogens, which require genomic sequencing for precise identification. In the present study, we describe nine cases of the various types of infection caused by Gordonia spp. From 1997 to 2008, 66 isolates (from 30 patients) initially identified as Rhodoccus spp. by conventional biochemical methods, by the Bacteriology Laboratory of National Taiwan University Hospital, were retrospectively analysed to assess the accuracy of species identification. Fifteen of these isolates (from nine patients) were later found to be Gordonia spp. by two molecular methods: PCR-restriction fragment length polymorphism for heat shock protein gene (hsp65) and the 16S rRNA gene sequencing analysis. Gordonia sputi (n = 8) was the most common species, followed by Gordonia terrae (n = 7). Most of the isolates were isolated from blood (n = 11), followed by soft tissue (n = 2) and eye (n = 2). Five patients presented with bacteraemia and two of these had catheter-related bloodstream infection. Two patients had soft tissue infections and another two patients had infective keratitis and conjunctivitis. The random amplified polymorphic DNA patterns for isolates from different patients were different, indicating that they were genetically unrelated. Accurate identification with molecular methods is required if the role of Gordonia spp. in causing infection is to be recognized. Keywords: Bacteraemia, catheter related infection, Gordonia spp., keratitis, molecular diagnosis Original Submission: 20 August 2009; Revised Submission: 30 September 2009; Accepted: 6 October 2009 Editor: D. Raoult Article published online: 14 October 2009 Clin Microbiol Infect 2010; 16: 1448–1453 10.1111/j.1469-0691.2010.03085.x Corresponding author: P. R. Hsueh, Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan E-mail: hsporen@ ntu.edu.tw
Introduction Gordonia spp., previously classified as Rhodococcus spp., are ubiquitous in the environment, and often are found in soil and water. Five Gordonia spp, Gordonia terrae, Gordonia bronchialis, Gordonia polyisoprenivorans, Gordonia sputi and Gordonia otitidis, have been implicated rarely as a cause of human infections [1–5] in both immunocompromised and immunocompetent patients. The limited number of reported cases included central venous catheter-related bloodstream
infection, endocarditis, brain abscess and cutaneous infection [1–12]. Three to four days were required to isolate the bacterium, owing to its slow growth. This delay may lead to false negative results and an underestimation of the incidence of infection caused by this genus. In addition, the inability of readily available identification systems to accurately identify Gordonia spp. leads to further difficulties. Precise identification of these microorganisms requires genomic sequencing, a method that is unavailable in most clinical laboratories. These problems, along with the difficulty of determining whether environmental bacteria are pathogens when they are isolated from human specimens, may contribute to the underdiagnosis of Gordonia spp. infections. A better understanding of the epidemiology and clinical characteristics of infections caused by these environmental or opportunistic pathogens is needed. In the present study, we describe nine cases of the various types of
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases
Lai et al.
CMI
infections caused by Gordonia spp., all of which were initially misidentified as Rhodococcus spp.
Infection Gordonia
1449
accession number and closest match observed were obtained [13,14]. Antimicrobial susceptibility testing
Materials and Methods Hospital setting and patient selection
National Taiwan University Hospital is a 2500-bed, academically affiliated medical centre providing both primary and tertiary care in northern Taiwan. A total of 66 isolates identified as Rhodococcus spp. by the Clinical Bacteriology Laboratory of National Taiwan University Hospital from January 1997 to June 2008 were preserved for further investigation. Those ultimately identified as Gordonia spp. defined the patients for inclusion. The clinical charts of all patients were retrospectively reviewed. Information on age, gender, underlying conditions, treatment and outcome was recorded. Catheter-related bacteraemia (CRBSI) was defined as the presence of bacteraemia and clinical manifestations of infection (i.e. fever, chills and/or hypotension) in a patient with an intravenous catheter, and no apparent source for the bloodstream infection except the catheter. Skin and soft-tissue infection (SSTI) caused by Gordonia spp. was defined as clinical soft tissue inflammation with positive culture for Gordonia spp. alone. If no primary focus could be identified, the bacteraemia was classified as primary.
The MICs for the Gordonia isolates of 20 antimicrobial agents were determined by the broth microdilution method (PMIC/ ID-30 Panel, Phoenix; Becton-Dickinson, Sparks, MD, USA) after incubation for 48 h [15]. For Gordonia spp. there are no MIC interpretive breakpoints of antimicrobial agents recommended by the CLSI. Random amplified polymorphic DNA (RAPD) analysis
Isolates were incubated in brain-heart infusion (Difco Laboratories, Detroit, MI, USA) for 36–48 h at 35C in ambient air. Bacterial genomic DNA was extracted by using a commercial kit (Puregene; Gentra Systems, Inc., Minneapolis, MN, USA). The RAPD assay, generated by arbitrarily primed PCR (APPCR), was performed using two arbitrary oligonucleotide primers: M13 (5¢-TTATGTAAAACGACGGCCAGT-3¢) and H4 (5¢-GGAAGTCGCC-3¢) (OPERON Technologies, Inc., Alameda, CA, USA). The conditions for PCR were in accordance with those used in a previous study [16].
Results Microbiological investigations
Bacterial isolates
A total of 66 isolates (from 39 patients) initially identified as Rhodococcus spp. during the study period were subjected to further testing to detect Gordonia spp., by conventional biochemical and two molecular methods. Initial identification of Gordonia spp. was based on Gram-stained appearances (Gram-positive coryneform bacilli), colonial morphology and conventional biochemical reactions, including hydrolysis of casein, xanthine, hypoxanthine and tyrosine [9]. Identification as Gordonia spp. was confirmed by two molecular methods. The first molecular method was a previously described PCRrestriction fragment length polymorphism (RFLP) identification scheme that used an amplified 440-bp segment of the 65-kDa heat shock protein gene (hsp65) performed using the two primers TB11 (5¢-ACCAACGATGGTGTGTCCAT-3¢) and TB12 (5¢-CTTGTCGAACCGCATACCCT-3¢) [13,14] and digestion by MspI and HinfI. The second molecular method was 16S rRNA gene sequencing using a pair of universal primers, 8FLP (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1492RPL (5¢-GGTTACCTTGTTACGACTT-3¢) [13]. The sequences (1080 bp) obtained were compared with published sequences in the GenBank database by using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/blast). The
Among the 66 isolates initially identified as Rhodococcus spp. by conventional biochemical methods during the 11-year study period, 15 isolates from nine patients were identified as Gordonia spp. by PCR-RFLP of hsp65 and 16S rRNA gene sequencing (Table 1). The unique fragments of the isolates digested by HinfI (245/150 bp for Gordonia spp.) and MspI (180/130 for G. terrae and 440 for G. sputi) identified these two Gordonia spp. [14]. Two patients had three and five Gordonia isolates, respectively. G. sputi (n = 8) was the most common pathogen, followed by G. terrae (n = 7). Most of the isolates were from blood (n = 11, from five patients), followed by soft tissue (n = 2, from two patients) and cornea (n = 2, from two patients). Clinical characteristics
The clinical characteristics of the nine patients with Gordonia infection are summarized in Table 1. The patients were in the age range 13–75 years, and six of the nine patients were male. Eight patients had various underlying diseases but the other patient did not have any evidence of comorbidity. Five patients presented with bacteraemia and two of them had catheter-related bloodstream infection. Although transoesophageal echocardiography was not performed in three
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
30/M
14/F
48/M
7 (2004)
8 (2006)
9 (2006)
Nil
Congenital Hirschsprung’s disease, short bowel syndrome, total parenteral nutrition
Acute lymphoblastic leukaemia, bone marrow transplant recepient Gastric cancer, diabetes mellitus COPD, chronic atrial fibrillation Gastroschisis with colon atresia receiving operation, Chronic hepatitis B virus infection
Diabetes mellitus
Acute myeloid leukaemia
Underlying diseases
G. terrae
G. sputi
G. terrae
G. sputi G. terrae G. sputi
G. terrae
G. sputi
G. terrae
Gordonia spp.
180/130
440
180/130
440 180/130 440
180/130
440
180/130
MspI
COPD, chronic obstructive pulmonary disease; RFLP, restriction fragment length polymorphism.
23/M
49/M 75/M 13 days/M
62/F
2 (1997)
3 (1999)
58/F
1 (1997)
4 (1999) 5 (1999) 6 (2000)
Age (years)/sex
Patient number (year)
245/150
245/150
245/150
245/150 245/150 245/150
245/150
245/150
245/150
HinfI
PCR-RFLP of hsp65
TABLE 1. Clinical characteristics of nine patients with Gordonia infection
EF538740.1/99%
EF538740.1/99%
FJ200387.1/99%
EF538740.1/99% FJ200387.1/99% EF538740.1/99%
FJ200387.1/99%
EF538740.1/99%
FJ200387.1/99%
GenBank (accession number)/ maximal identity
Cellulitis
Catheter-related bloodstream infection
Wound infection
Primary bacteraemia Primary bacteraemia Keratitis/conjunctivitis
Primary bacteraemia
Keratitis/conjunctivitis
Catheter- related bloodstream infection
Clinical diagnosis
Blood D (10/02/1999) Blood E (24/03/1999) Cornea ulcer F (18/01/2000) Right femoral traumatic wound G (15/01/2004) Blood (H1), Hickman Catheter (H2, H3, H4) H1 (08/11/2006) H2 (14/11/2006) H3 (21/11/2006) H4 (04/12/2006) H5 (10/12/2006) Abdominal wall abscess I (08/12/2006)
Blood (A1, A2) Port-A Cath (A3) A1 (04/08/1997) A2 (28/10/1997) A3 (18/12/1997) Cornea ulcer B (20/06/1997) Blood C (03/06/1999)
Site of isolations (isolate designation and date of isolation)
Amoxicillin–clavulanate
Imipenem and amikacin lock therapy
Amoxicillin–clavulanate
Cefoxitin Vancomycin, ciprofloxacin Topical sulfamethoxazole
Vancomycin + imipenem
Topical sulfamethoxazole
Vancomycin, imipenem, ciprofloxacin
Antimicrobialtherapy
Improved
Improved
Improved
Improved Improved Improved
Improved
Improved
Improved until removal of Port-A catheter
Outcome
1450 Clinical Microbiology and Infection, Volume 16 Number 9, September 2010
CMI
Lai et al.
CMI
bacteraemia patients, primary bacteraemia was diagnosed in these patients because they showed no murmur or peripheral stigmata of endocarditis. All outcomes were favourable despite variation in the antibiotic regimens. Bacteraemia in one patient (patient 1) with CRBSI (Port-A Cath) as a result of G. terrae lasted for 4 months despite prolonged antibiotic use, including vancomycin, imipenem and intravenous ciprofloxacin. The infection resolved after subsequent removal of the Port-A Cath 4 months later. By contrast, the catheter was not removed in patient 8 with persistent CRBSI as a result of G. sputi under management with systemic antimicrobial agents and antibiotic lock therapy with amikacin. One patient had G. terrae-related cellulitis and improved after treatment with amoxicillin/clavulanate. Another patient had traumatic wound infection as a result of G. terrae and was successfully managed with repeated surgical debridement and treatment with amoxicillin/clavulanate. In addition, corneal culture grew G. sputi alone in a newborn and in a diabetic woman who presented with ocular redness, swelling and corneal ulcer, leading to the diagnosis of infective keratitis and conjunctivitis, which responded to topical antibiotics. Drug susceptibilities
The MIC ranges of 20 antimicrobial agents for the nine nonduplicate isolates (one isolate per person) of Gordonia spp. are shown in Table 2. MIC ranges were similar in the two species tested. All isolates showed high MIC values of nitrofurantoin (£16–‡64 mg/L) and quinupristin–dalfopristin (>2 mg/L). One isolate recovered from patient 4 (Table 1)
TABLE 2. Susceptibilities of nine nonduplicate isolates of Gordonia spp. to 20 antimicrobial agents. by the broth microdilution method MIC range (mg/L)
Agent
Gordonia terrae (n = 5)
Gordonia sputi (n = 4)
Penicillin Ampicillin Amoxicillin–clavulanate Oxacillin Clindamycin Erythromycin Tetracycline Streptomycin Gentamicin Ciprofloxacin Levofloxacin Moxifloxacin Nitrofurantoin Chloramphenicol Trimethoprim–sulfamethoxazole Rifampin Vancomycin Teicoplanin Quinupristin–dalfopristin Linezolid
£0.125 £2 £1/0.5 £0.125 0.5–1 £0.25 £0.5 £1000 £2 £0.5 £1 £1 32–64 £1 £0.5/9.5 £0.5 £0.5 £0.5 >2 £0.25
£0.125 £2 £1/0.5 £0.125 £0.25–0.5 £0.25 £0.5 £1000 £2 £0.5 £1 £1 £16–64 £1 £0.5/9.5 £0.5 £0.5–1 £0.5 >2 £0.25–2
Infection Gordonia
1451
exhibited higher MICs of oxacillin (‡2 mg/L) vancomycin (1 mg/L) and linezolid (2 mg/L) than the other eight isolates. RAPD patterns
Distinct RAPD patterns, obtained using two random primers were found among the 15 isolates. Identical RAPD patterns were found for the three isolates (A1–3) from patient 1 and the five isolates (H1–5) from patient 8.
Discussion Gordonia spp. have occasionally been isolated as causative agents of human infections, including CRBSI, endocarditis, brain abscess and cutaneous infections [1–12]. Identification of Gordonia spp., as for most other Gram-positive bacilli, is time-consuming, labour-intensive and often difficult, owing to frequently inconclusive results being obtained in biochemical tests. The use of HPLC is required to determine the genus level and separate Gordonia from the related genera Dietzia, Corynebacterium, Rhodococcus and Tsukamurella. Furthermore, identification to the species level requires advanced molecular methods such as a comparison of 16S rRNA gene sequencing data with those available in GenBank. Some sequences in the GenBank database may be less reliable than those found in other validated databases, especially in the case of older sequences [13,14]. Recently, a PCR-RFLP analysis of hsp65 has been reported to be useful for the identification of clinically significant species and taxa of aerobic actinomycetes [13,14]. Previous studies have shown that Gordonia spp. may be misidentified as other gram-positive bacilli, such as Rhodococcus or Nocardia spp. [9,11]. In this survey of 66 isolates previously identified as Rhodococcus spp. by conventional microbiological culture and biochemical analysis, 15 isolates were found to be Gordonia spp. by 16S rRNA gene sequencing. These findings suggest that Gordonia spp. may be underreported and that 16S rRNA gene sequencing is helpful for accurate identification. Five patients in this series had Gordonia bacteraemia and three of them had various underlying malignancies. Three of these cases were caused by G. terrae and the other two cases were caused by G. sputi. All of these patients had an initial presentation of fever, and no specific infection focus was found in three of them. Despite the use of different combinations of antimicrobial agents in three cases with invasive Gordonia infection, all of them responded to antibiotic therapy. In previously reported cases [1–12], CRBSI was the most common type of the invasive Gordonia infection and immunocompromised status was the major risk factor. Patient 1 of this
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
1452
Clinical Microbiology and Infection, Volume 16 Number 9, September 2010
study suffered persistent G. terrae CRBSI despite antibiotic treatment and this resolved after catheter removal. In addition, antibiotic-lock therapy with systemic antibiotic treatment successfully cleared the infection and rescued the Hickman catheter in patient 8, who had G. sputi CRBSI. This limited experience might suggest that cases of CRBSI as a result of Gordonia spp. should be treated with catheter removal to clear the infection. As for antibiotic lock therapy, we cannot draw any conclusion regarding the efficacy for Gordonia CRBSI until more clinical experience is accumulated. SSTI as a result of Gordonia, such as breast abscess and sternal wound infections, have occasionally been reported [17–20]. All of the reported cases were caused by either G. terrae or G. bronchialis and most of them required combined therapy with prolonged antibiotic treatment and surgical debridement [17–20]. The two patients (patients 7 and 9) with G. terrae-related SSTI reported in the present study were immunocompetent and successfully cleared their infections after medical and surgical management. Thus, G. terrae should be considered as one of the pathogens causing SSTI, and surgical debridement might be crucial for the successful treatment of this rare clinical entity. In the present series, a newborn and a diabetic woman had G. sputi-related infective keratitis and conjunctivitis, which responded to topical antibiotics. Our review of the literature found only one report of ocular Gordonia infection [21]. A review of antibiotic susceptibility data on 24 clinical isolates by Blaschke et al. [5] found that carbapenems, aminoglycosides, linezolid and fluoroquinolones showed good activity against Gordonia spp., although the activity of ampicillin, trimethoprim-sulfamethoxazole, clarithromycin and penicillin was poor, with 56–70% of tested isolates showing higher MICs. The present study found that the MICs of most study agents for Gordonia were low, except in the case of nitrofurantoin and quinupristin–dalfopristin. Furthermore, the MICs of amoxicillin/clavulanic acid, ciprofloxacin and vancomycin for the Gordonia isolates were £1/0.5, £0.5, and £0.5 mg/L, respectively, and the outcomes in patients treated with these three antimicrobials were favourable. Because of the various susceptibility testing methods used in different studies and the limited number of cases tested, it is difficult to draw inferences about optimal antibiotic treatment [1–12]. RAPD pattern analysis-based arbitarily primed PCR has a high discriminatory power for molecular typing and the present study is the first to report its use in the typing of Gordonia spp. The analysis showed that isolates from different patients had different patterns, indicating that they were genetically unrelated and that nosocomial transmission had not occurred. Second, multiple isolates from two patients with persistent CRBSI had identical RAPD patterns, implying
CMI
incomplete treatment for an occult focus or persistent carriage of the same bacteria. In conclusion, infections caused by Gordonia spp. are difficult to diagnose in immunocompromised and immunocompetent patients. The clinical manifestations are protean, and include primary and secondary bacteraemia, soft tissue infection and infective keratitis. Accurate identification with molecular methods is required if these unusual pathogens are to be detected. Further clinical and laboratory investigation of Gordonia spp. infections is needed to establish treatment guidelines.
Transparency Declaration The authors declare that there are no conflicts of interest in relation to this study.
References 1. Drancourt M, McNeil MM, Brown JM et al. Brain abscess due to Gordonia terrae in an immunocompromised child: case report and review of infections caused by G. terrae. Clin Infect Dis 1994; 19: 258–262. 2. Riegel P, Ruimy R, de Briel D et al. Bacteremia due to Gordonia sputi in an immunocompromised patient. J Clin Microbiol 1996; 34: 2045– 2047. 3. Kempf VA, Schmalzing M, Yassin AF et al. Gordonia polyisoprenivorans septicemia in a bone marrow transplant patient. Eur J Clin Microbiol Infect Dis 2004; 23: 226–228. 4. Sng LH, Koh TH, Toney SR, Floyd M, Butler WR, Tan BH. Bacteremia caused by Gordonia bronchialis in a patient with sequestrated lung. J Clin Microbiol 2004; 42: 2870–2871. 5. Blaschke AJ, Bender J, Byington CL et al. Gordonia species: emerging pathogens in pediatric patients that are identified by 16S ribosomal RNA gene sequencing. Clin Infect Dis 2007; 45: 483–486. 6. Drancourt M, Pelletier J, Cherif AA, Raoult D. Gordonia terrae central nervous system infection in an immunocompetent patient. J Clin Microbiol 1997; 35: 379–382. 7. Lesens O, Hansmann Y, Riegel P et al. Bacteremia and endocarditis caused by a Gordonia species in a patient with a central venous catheter. Emerging Infect Dis 2000; 6: 382–385. 8. Buchman AL, McNeil MM, Brown JM, Lasker BA, Ament ME. Central venous catheter sepsis caused by unusual Gordonia rhodococcus species: identification with a digoxigenin-labeled rDNA probe. Clin Infect Dis 1992; 15: 694–697. 9. Gil-Sande E, Brun-Otero M, Campo-Cerecedo F, Esteban E, Aguilar L, Garcia-de-Lomas J. Etiological misidentification by routine biochemical tests of bacteremia caused by Gordonia terrae infection in the course of an episode of acute cholecystitis. J Clin Microbiol 2006; 44: 2645–2647. 10. Verma P, Brown JM, Nunez VH et al. Native valve endocarditis due to Gordonia polyisoprenivorans: case report and review of literature of bloodstream infections caused by Gordonia species. J Clin Microbiol 2006; 44: 1905–1908. 11. Pham AS, De I, Rolston KV, Tarrand JJ, Han XY. Catheter-related bacteremia caused by the nocardioform actinomycete Gordonia terrae. Clin Infect Dis 2003; 36: 524–527.
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
Lai et al.
CMI
12. Blanc V, Markarian A, Debunne MV et al. Gordonia terrae: a difficult to diagnose emerging pathogen? J Clin Microbiol 2007; 45: 1076–1077. 13. Sheng WH, Huang YT, Chang SC, Hseuh PR. Brain abscess caused by Tsukamurella tyrosinosolvens in an immunocompetent patient. J Clin Microbiol 2009; 47: 1602–1604. 14. Steingrube VA, Wilson RW, Brown BA et al. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J Clin Microbiol 1997; 35: 817–822. 15. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th edn. CLSI document M7-A7. Wayne, PA: CLSI, 2006. 16. Hsueh PR, Hung CC, Teng LJ et al. Report of invasive Rhodococcus equi infection in Taiwan, with an emphasis on the emergence of multidrug-resistant strains. Clin Infect Dis 1998; 27: 370–375.
Infection Gordonia
1453
17. Bakker XR, Spauwen PH, Volmans WM. Mycetoma of the hand caused by a case report. J Hand Surg [Br] 2004; 29: 188–190. 18. Zardawi IM, Hones F, Clark DA, Holland J. Gordonia terrae induced suppurative granulomatous mastitis following nipple piercing. Pathology 2004; 36: 275–280. 19. Richet HM, Craven PC, Brown JM et al. A cluster of Rhodococcus (Gordona) bronchialis sternal-wound infections after coronary artery bypass surgery. N Engl J Med 1991; 324: 104–109. 20. Werno AM, Anderson TP, Chambers ST, Laird HM, Murdoch DR. Recurrent breast abscess caused by Gordonia bronchialis in an immunocompetent patient. J Clin Microbiol 2005; 43: 3009–3010. 21. Aoyama K, Kang Y, Yazawa K, Gonoi T, Kamei K, Mikami Y. Characterization of clinical isolates of Gordonia species in Japanese clinical sample during 1998–2008. Mycopathologia 2009; 164: 175–183.
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
BACTERIOLOGY
Infections caused by Gordonia species at a medical centre in Taiwan, 1997 to 2008 C. C. Lai1, C. Y. Wang1, C. Y. Liu2, C. K. Tan3, S. H. Lin4, C. H. Liao2, C. H. Chou5, Y. T. Huang6, H. I. Lin1 and P. R. Hsueh6 1) Department of Internal Medicine, Cardinal Tien Hospital, Taipei County, 2) Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, 3) Department of Intensive Care Medicine, Chi-Mei Medical Center, Tainan, 4) Department of Internal Medicine, Taipei County Hospital, Taipei County, 5) Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin and 6) Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan
Abstract The inability of conventional identification systems to accurately identify Gordonia spp. often results in the misdiagnosis of infections by these rare pathogens, which require genomic sequencing for precise identification. In the present study, we describe nine cases of the various types of infection caused by Gordonia spp. From 1997 to 2008, 66 isolates (from 30 patients) initially identified as Rhodoccus spp. by conventional biochemical methods, by the Bacteriology Laboratory of National Taiwan University Hospital, were retrospectively analysed to assess the accuracy of species identification. Fifteen of these isolates (from nine patients) were later found to be Gordonia spp. by two molecular methods: PCR-restriction fragment length polymorphism for heat shock protein gene (hsp65) and the 16S rRNA gene sequencing analysis. Gordonia sputi (n = 8) was the most common species, followed by Gordonia terrae (n = 7). Most of the isolates were isolated from blood (n = 11), followed by soft tissue (n = 2) and eye (n = 2). Five patients presented with bacteraemia and two of these had catheter-related bloodstream infection. Two patients had soft tissue infections and another two patients had infective keratitis and conjunctivitis. The random amplified polymorphic DNA patterns for isolates from different patients were different, indicating that they were genetically unrelated. Accurate identification with molecular methods is required if the role of Gordonia spp. in causing infection is to be recognized. Keywords: Bacteraemia, catheter related infection, Gordonia spp., keratitis, molecular diagnosis Original Submission: 20 August 2009; Revised Submission: 30 September 2009; Accepted: 6 October 2009 Editor: D. Raoult Article published online: 14 October 2009 Clin Microbiol Infect 2010; 16: 1448–1453 10.1111/j.1469-0691.2010.03085.x Corresponding author: P. R. Hsueh, Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan E-mail: hsporen@ ntu.edu.tw
Introduction Gordonia spp., previously classified as Rhodococcus spp., are ubiquitous in the environment, and often are found in soil and water. Five Gordonia spp, Gordonia terrae, Gordonia bronchialis, Gordonia polyisoprenivorans, Gordonia sputi and Gordonia otitidis, have been implicated rarely as a cause of human infections [1–5] in both immunocompromised and immunocompetent patients. The limited number of reported cases included central venous catheter-related bloodstream
infection, endocarditis, brain abscess and cutaneous infection [1–12]. Three to four days were required to isolate the bacterium, owing to its slow growth. This delay may lead to false negative results and an underestimation of the incidence of infection caused by this genus. In addition, the inability of readily available identification systems to accurately identify Gordonia spp. leads to further difficulties. Precise identification of these microorganisms requires genomic sequencing, a method that is unavailable in most clinical laboratories. These problems, along with the difficulty of determining whether environmental bacteria are pathogens when they are isolated from human specimens, may contribute to the underdiagnosis of Gordonia spp. infections. A better understanding of the epidemiology and clinical characteristics of infections caused by these environmental or opportunistic pathogens is needed. In the present study, we describe nine cases of the various types of
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases
Lai et al.
CMI
infections caused by Gordonia spp., all of which were initially misidentified as Rhodococcus spp.
Infection Gordonia
1449
accession number and closest match observed were obtained [13,14]. Antimicrobial susceptibility testing
Materials and Methods Hospital setting and patient selection
National Taiwan University Hospital is a 2500-bed, academically affiliated medical centre providing both primary and tertiary care in northern Taiwan. A total of 66 isolates identified as Rhodococcus spp. by the Clinical Bacteriology Laboratory of National Taiwan University Hospital from January 1997 to June 2008 were preserved for further investigation. Those ultimately identified as Gordonia spp. defined the patients for inclusion. The clinical charts of all patients were retrospectively reviewed. Information on age, gender, underlying conditions, treatment and outcome was recorded. Catheter-related bacteraemia (CRBSI) was defined as the presence of bacteraemia and clinical manifestations of infection (i.e. fever, chills and/or hypotension) in a patient with an intravenous catheter, and no apparent source for the bloodstream infection except the catheter. Skin and soft-tissue infection (SSTI) caused by Gordonia spp. was defined as clinical soft tissue inflammation with positive culture for Gordonia spp. alone. If no primary focus could be identified, the bacteraemia was classified as primary.
The MICs for the Gordonia isolates of 20 antimicrobial agents were determined by the broth microdilution method (PMIC/ ID-30 Panel, Phoenix; Becton-Dickinson, Sparks, MD, USA) after incubation for 48 h [15]. For Gordonia spp. there are no MIC interpretive breakpoints of antimicrobial agents recommended by the CLSI. Random amplified polymorphic DNA (RAPD) analysis
Isolates were incubated in brain-heart infusion (Difco Laboratories, Detroit, MI, USA) for 36–48 h at 35C in ambient air. Bacterial genomic DNA was extracted by using a commercial kit (Puregene; Gentra Systems, Inc., Minneapolis, MN, USA). The RAPD assay, generated by arbitrarily primed PCR (APPCR), was performed using two arbitrary oligonucleotide primers: M13 (5¢-TTATGTAAAACGACGGCCAGT-3¢) and H4 (5¢-GGAAGTCGCC-3¢) (OPERON Technologies, Inc., Alameda, CA, USA). The conditions for PCR were in accordance with those used in a previous study [16].
Results Microbiological investigations
Bacterial isolates
A total of 66 isolates (from 39 patients) initially identified as Rhodococcus spp. during the study period were subjected to further testing to detect Gordonia spp., by conventional biochemical and two molecular methods. Initial identification of Gordonia spp. was based on Gram-stained appearances (Gram-positive coryneform bacilli), colonial morphology and conventional biochemical reactions, including hydrolysis of casein, xanthine, hypoxanthine and tyrosine [9]. Identification as Gordonia spp. was confirmed by two molecular methods. The first molecular method was a previously described PCRrestriction fragment length polymorphism (RFLP) identification scheme that used an amplified 440-bp segment of the 65-kDa heat shock protein gene (hsp65) performed using the two primers TB11 (5¢-ACCAACGATGGTGTGTCCAT-3¢) and TB12 (5¢-CTTGTCGAACCGCATACCCT-3¢) [13,14] and digestion by MspI and HinfI. The second molecular method was 16S rRNA gene sequencing using a pair of universal primers, 8FLP (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1492RPL (5¢-GGTTACCTTGTTACGACTT-3¢) [13]. The sequences (1080 bp) obtained were compared with published sequences in the GenBank database by using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/blast). The
Among the 66 isolates initially identified as Rhodococcus spp. by conventional biochemical methods during the 11-year study period, 15 isolates from nine patients were identified as Gordonia spp. by PCR-RFLP of hsp65 and 16S rRNA gene sequencing (Table 1). The unique fragments of the isolates digested by HinfI (245/150 bp for Gordonia spp.) and MspI (180/130 for G. terrae and 440 for G. sputi) identified these two Gordonia spp. [14]. Two patients had three and five Gordonia isolates, respectively. G. sputi (n = 8) was the most common pathogen, followed by G. terrae (n = 7). Most of the isolates were from blood (n = 11, from five patients), followed by soft tissue (n = 2, from two patients) and cornea (n = 2, from two patients). Clinical characteristics
The clinical characteristics of the nine patients with Gordonia infection are summarized in Table 1. The patients were in the age range 13–75 years, and six of the nine patients were male. Eight patients had various underlying diseases but the other patient did not have any evidence of comorbidity. Five patients presented with bacteraemia and two of them had catheter-related bloodstream infection. Although transoesophageal echocardiography was not performed in three
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
30/M
14/F
48/M
7 (2004)
8 (2006)
9 (2006)
Nil
Congenital Hirschsprung’s disease, short bowel syndrome, total parenteral nutrition
Acute lymphoblastic leukaemia, bone marrow transplant recepient Gastric cancer, diabetes mellitus COPD, chronic atrial fibrillation Gastroschisis with colon atresia receiving operation, Chronic hepatitis B virus infection
Diabetes mellitus
Acute myeloid leukaemia
Underlying diseases
G. terrae
G. sputi
G. terrae
G. sputi G. terrae G. sputi
G. terrae
G. sputi
G. terrae
Gordonia spp.
180/130
440
180/130
440 180/130 440
180/130
440
180/130
MspI
COPD, chronic obstructive pulmonary disease; RFLP, restriction fragment length polymorphism.
23/M
49/M 75/M 13 days/M
62/F
2 (1997)
3 (1999)
58/F
1 (1997)
4 (1999) 5 (1999) 6 (2000)
Age (years)/sex
Patient number (year)
245/150
245/150
245/150
245/150 245/150 245/150
245/150
245/150
245/150
HinfI
PCR-RFLP of hsp65
TABLE 1. Clinical characteristics of nine patients with Gordonia infection
EF538740.1/99%
EF538740.1/99%
FJ200387.1/99%
EF538740.1/99% FJ200387.1/99% EF538740.1/99%
FJ200387.1/99%
EF538740.1/99%
FJ200387.1/99%
GenBank (accession number)/ maximal identity
Cellulitis
Catheter-related bloodstream infection
Wound infection
Primary bacteraemia Primary bacteraemia Keratitis/conjunctivitis
Primary bacteraemia
Keratitis/conjunctivitis
Catheter- related bloodstream infection
Clinical diagnosis
Blood D (10/02/1999) Blood E (24/03/1999) Cornea ulcer F (18/01/2000) Right femoral traumatic wound G (15/01/2004) Blood (H1), Hickman Catheter (H2, H3, H4) H1 (08/11/2006) H2 (14/11/2006) H3 (21/11/2006) H4 (04/12/2006) H5 (10/12/2006) Abdominal wall abscess I (08/12/2006)
Blood (A1, A2) Port-A Cath (A3) A1 (04/08/1997) A2 (28/10/1997) A3 (18/12/1997) Cornea ulcer B (20/06/1997) Blood C (03/06/1999)
Site of isolations (isolate designation and date of isolation)
Amoxicillin–clavulanate
Imipenem and amikacin lock therapy
Amoxicillin–clavulanate
Cefoxitin Vancomycin, ciprofloxacin Topical sulfamethoxazole
Vancomycin + imipenem
Topical sulfamethoxazole
Vancomycin, imipenem, ciprofloxacin
Antimicrobialtherapy
Improved
Improved
Improved
Improved Improved Improved
Improved
Improved
Improved until removal of Port-A catheter
Outcome
1450 Clinical Microbiology and Infection, Volume 16 Number 9, September 2010
CMI
Lai et al.
CMI
bacteraemia patients, primary bacteraemia was diagnosed in these patients because they showed no murmur or peripheral stigmata of endocarditis. All outcomes were favourable despite variation in the antibiotic regimens. Bacteraemia in one patient (patient 1) with CRBSI (Port-A Cath) as a result of G. terrae lasted for 4 months despite prolonged antibiotic use, including vancomycin, imipenem and intravenous ciprofloxacin. The infection resolved after subsequent removal of the Port-A Cath 4 months later. By contrast, the catheter was not removed in patient 8 with persistent CRBSI as a result of G. sputi under management with systemic antimicrobial agents and antibiotic lock therapy with amikacin. One patient had G. terrae-related cellulitis and improved after treatment with amoxicillin/clavulanate. Another patient had traumatic wound infection as a result of G. terrae and was successfully managed with repeated surgical debridement and treatment with amoxicillin/clavulanate. In addition, corneal culture grew G. sputi alone in a newborn and in a diabetic woman who presented with ocular redness, swelling and corneal ulcer, leading to the diagnosis of infective keratitis and conjunctivitis, which responded to topical antibiotics. Drug susceptibilities
The MIC ranges of 20 antimicrobial agents for the nine nonduplicate isolates (one isolate per person) of Gordonia spp. are shown in Table 2. MIC ranges were similar in the two species tested. All isolates showed high MIC values of nitrofurantoin (£16–‡64 mg/L) and quinupristin–dalfopristin (>2 mg/L). One isolate recovered from patient 4 (Table 1)
TABLE 2. Susceptibilities of nine nonduplicate isolates of Gordonia spp. to 20 antimicrobial agents. by the broth microdilution method MIC range (mg/L)
Agent
Gordonia terrae (n = 5)
Gordonia sputi (n = 4)
Penicillin Ampicillin Amoxicillin–clavulanate Oxacillin Clindamycin Erythromycin Tetracycline Streptomycin Gentamicin Ciprofloxacin Levofloxacin Moxifloxacin Nitrofurantoin Chloramphenicol Trimethoprim–sulfamethoxazole Rifampin Vancomycin Teicoplanin Quinupristin–dalfopristin Linezolid
£0.125 £2 £1/0.5 £0.125 0.5–1 £0.25 £0.5 £1000 £2 £0.5 £1 £1 32–64 £1 £0.5/9.5 £0.5 £0.5 £0.5 >2 £0.25
£0.125 £2 £1/0.5 £0.125 £0.25–0.5 £0.25 £0.5 £1000 £2 £0.5 £1 £1 £16–64 £1 £0.5/9.5 £0.5 £0.5–1 £0.5 >2 £0.25–2
Infection Gordonia
1451
exhibited higher MICs of oxacillin (‡2 mg/L) vancomycin (1 mg/L) and linezolid (2 mg/L) than the other eight isolates. RAPD patterns
Distinct RAPD patterns, obtained using two random primers were found among the 15 isolates. Identical RAPD patterns were found for the three isolates (A1–3) from patient 1 and the five isolates (H1–5) from patient 8.
Discussion Gordonia spp. have occasionally been isolated as causative agents of human infections, including CRBSI, endocarditis, brain abscess and cutaneous infections [1–12]. Identification of Gordonia spp., as for most other Gram-positive bacilli, is time-consuming, labour-intensive and often difficult, owing to frequently inconclusive results being obtained in biochemical tests. The use of HPLC is required to determine the genus level and separate Gordonia from the related genera Dietzia, Corynebacterium, Rhodococcus and Tsukamurella. Furthermore, identification to the species level requires advanced molecular methods such as a comparison of 16S rRNA gene sequencing data with those available in GenBank. Some sequences in the GenBank database may be less reliable than those found in other validated databases, especially in the case of older sequences [13,14]. Recently, a PCR-RFLP analysis of hsp65 has been reported to be useful for the identification of clinically significant species and taxa of aerobic actinomycetes [13,14]. Previous studies have shown that Gordonia spp. may be misidentified as other gram-positive bacilli, such as Rhodococcus or Nocardia spp. [9,11]. In this survey of 66 isolates previously identified as Rhodococcus spp. by conventional microbiological culture and biochemical analysis, 15 isolates were found to be Gordonia spp. by 16S rRNA gene sequencing. These findings suggest that Gordonia spp. may be underreported and that 16S rRNA gene sequencing is helpful for accurate identification. Five patients in this series had Gordonia bacteraemia and three of them had various underlying malignancies. Three of these cases were caused by G. terrae and the other two cases were caused by G. sputi. All of these patients had an initial presentation of fever, and no specific infection focus was found in three of them. Despite the use of different combinations of antimicrobial agents in three cases with invasive Gordonia infection, all of them responded to antibiotic therapy. In previously reported cases [1–12], CRBSI was the most common type of the invasive Gordonia infection and immunocompromised status was the major risk factor. Patient 1 of this
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
1452
Clinical Microbiology and Infection, Volume 16 Number 9, September 2010
study suffered persistent G. terrae CRBSI despite antibiotic treatment and this resolved after catheter removal. In addition, antibiotic-lock therapy with systemic antibiotic treatment successfully cleared the infection and rescued the Hickman catheter in patient 8, who had G. sputi CRBSI. This limited experience might suggest that cases of CRBSI as a result of Gordonia spp. should be treated with catheter removal to clear the infection. As for antibiotic lock therapy, we cannot draw any conclusion regarding the efficacy for Gordonia CRBSI until more clinical experience is accumulated. SSTI as a result of Gordonia, such as breast abscess and sternal wound infections, have occasionally been reported [17–20]. All of the reported cases were caused by either G. terrae or G. bronchialis and most of them required combined therapy with prolonged antibiotic treatment and surgical debridement [17–20]. The two patients (patients 7 and 9) with G. terrae-related SSTI reported in the present study were immunocompetent and successfully cleared their infections after medical and surgical management. Thus, G. terrae should be considered as one of the pathogens causing SSTI, and surgical debridement might be crucial for the successful treatment of this rare clinical entity. In the present series, a newborn and a diabetic woman had G. sputi-related infective keratitis and conjunctivitis, which responded to topical antibiotics. Our review of the literature found only one report of ocular Gordonia infection [21]. A review of antibiotic susceptibility data on 24 clinical isolates by Blaschke et al. [5] found that carbapenems, aminoglycosides, linezolid and fluoroquinolones showed good activity against Gordonia spp., although the activity of ampicillin, trimethoprim-sulfamethoxazole, clarithromycin and penicillin was poor, with 56–70% of tested isolates showing higher MICs. The present study found that the MICs of most study agents for Gordonia were low, except in the case of nitrofurantoin and quinupristin–dalfopristin. Furthermore, the MICs of amoxicillin/clavulanic acid, ciprofloxacin and vancomycin for the Gordonia isolates were £1/0.5, £0.5, and £0.5 mg/L, respectively, and the outcomes in patients treated with these three antimicrobials were favourable. Because of the various susceptibility testing methods used in different studies and the limited number of cases tested, it is difficult to draw inferences about optimal antibiotic treatment [1–12]. RAPD pattern analysis-based arbitarily primed PCR has a high discriminatory power for molecular typing and the present study is the first to report its use in the typing of Gordonia spp. The analysis showed that isolates from different patients had different patterns, indicating that they were genetically unrelated and that nosocomial transmission had not occurred. Second, multiple isolates from two patients with persistent CRBSI had identical RAPD patterns, implying
CMI
incomplete treatment for an occult focus or persistent carriage of the same bacteria. In conclusion, infections caused by Gordonia spp. are difficult to diagnose in immunocompromised and immunocompetent patients. The clinical manifestations are protean, and include primary and secondary bacteraemia, soft tissue infection and infective keratitis. Accurate identification with molecular methods is required if these unusual pathogens are to be detected. Further clinical and laboratory investigation of Gordonia spp. infections is needed to establish treatment guidelines.
Transparency Declaration The authors declare that there are no conflicts of interest in relation to this study.
References 1. Drancourt M, McNeil MM, Brown JM et al. Brain abscess due to Gordonia terrae in an immunocompromised child: case report and review of infections caused by G. terrae. Clin Infect Dis 1994; 19: 258–262. 2. Riegel P, Ruimy R, de Briel D et al. Bacteremia due to Gordonia sputi in an immunocompromised patient. J Clin Microbiol 1996; 34: 2045– 2047. 3. Kempf VA, Schmalzing M, Yassin AF et al. Gordonia polyisoprenivorans septicemia in a bone marrow transplant patient. Eur J Clin Microbiol Infect Dis 2004; 23: 226–228. 4. Sng LH, Koh TH, Toney SR, Floyd M, Butler WR, Tan BH. Bacteremia caused by Gordonia bronchialis in a patient with sequestrated lung. J Clin Microbiol 2004; 42: 2870–2871. 5. Blaschke AJ, Bender J, Byington CL et al. Gordonia species: emerging pathogens in pediatric patients that are identified by 16S ribosomal RNA gene sequencing. Clin Infect Dis 2007; 45: 483–486. 6. Drancourt M, Pelletier J, Cherif AA, Raoult D. Gordonia terrae central nervous system infection in an immunocompetent patient. J Clin Microbiol 1997; 35: 379–382. 7. Lesens O, Hansmann Y, Riegel P et al. Bacteremia and endocarditis caused by a Gordonia species in a patient with a central venous catheter. Emerging Infect Dis 2000; 6: 382–385. 8. Buchman AL, McNeil MM, Brown JM, Lasker BA, Ament ME. Central venous catheter sepsis caused by unusual Gordonia rhodococcus species: identification with a digoxigenin-labeled rDNA probe. Clin Infect Dis 1992; 15: 694–697. 9. Gil-Sande E, Brun-Otero M, Campo-Cerecedo F, Esteban E, Aguilar L, Garcia-de-Lomas J. Etiological misidentification by routine biochemical tests of bacteremia caused by Gordonia terrae infection in the course of an episode of acute cholecystitis. J Clin Microbiol 2006; 44: 2645–2647. 10. Verma P, Brown JM, Nunez VH et al. Native valve endocarditis due to Gordonia polyisoprenivorans: case report and review of literature of bloodstream infections caused by Gordonia species. J Clin Microbiol 2006; 44: 1905–1908. 11. Pham AS, De I, Rolston KV, Tarrand JJ, Han XY. Catheter-related bacteremia caused by the nocardioform actinomycete Gordonia terrae. Clin Infect Dis 2003; 36: 524–527.
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453
Lai et al.
CMI
12. Blanc V, Markarian A, Debunne MV et al. Gordonia terrae: a difficult to diagnose emerging pathogen? J Clin Microbiol 2007; 45: 1076–1077. 13. Sheng WH, Huang YT, Chang SC, Hseuh PR. Brain abscess caused by Tsukamurella tyrosinosolvens in an immunocompetent patient. J Clin Microbiol 2009; 47: 1602–1604. 14. Steingrube VA, Wilson RW, Brown BA et al. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J Clin Microbiol 1997; 35: 817–822. 15. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th edn. CLSI document M7-A7. Wayne, PA: CLSI, 2006. 16. Hsueh PR, Hung CC, Teng LJ et al. Report of invasive Rhodococcus equi infection in Taiwan, with an emphasis on the emergence of multidrug-resistant strains. Clin Infect Dis 1998; 27: 370–375.
Infection Gordonia
1453
17. Bakker XR, Spauwen PH, Volmans WM. Mycetoma of the hand caused by a case report. J Hand Surg [Br] 2004; 29: 188–190. 18. Zardawi IM, Hones F, Clark DA, Holland J. Gordonia terrae induced suppurative granulomatous mastitis following nipple piercing. Pathology 2004; 36: 275–280. 19. Richet HM, Craven PC, Brown JM et al. A cluster of Rhodococcus (Gordona) bronchialis sternal-wound infections after coronary artery bypass surgery. N Engl J Med 1991; 324: 104–109. 20. Werno AM, Anderson TP, Chambers ST, Laird HM, Murdoch DR. Recurrent breast abscess caused by Gordonia bronchialis in an immunocompetent patient. J Clin Microbiol 2005; 43: 3009–3010. 21. Aoyama K, Kang Y, Yazawa K, Gonoi T, Kamei K, Mikami Y. Characterization of clinical isolates of Gordonia species in Japanese clinical sample during 1998–2008. Mycopathologia 2009; 164: 175–183.
ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1448–1453