Azlocillin

A 17-year-old male patient with cystic fibrosis (CF), with CF mutation homozygous (Delta)F508, was admitted to the Royal Belfast Hospital for Sick Children in August 2001 for a course of intravenous antibiotics to treat a chronic pulmonary infection. He had an 11-year history of pulmonary infection with both a mucoid and a non-mucoid Pseudomonas aeruginosa, which had remained relatively sensitive to several antibiotics used routinely in the treatment of P aeruginosa CF chest infections, including gentamicin, tobramycin, aztreonam, ceftazidime, ciprofloxacin, piperacillin/tazobactam, imipenim and meropenem. During that time he was not reported to have been colonised/infected with any other bacterial organism in his respiratory tract.

On admission, an unidentified Gram-negative rod was isolated in addition to his chronic strains of mucoid and non-- mucoid P aeruginosa. This new organism was isolated from freshly expectorated sputum as the sole organism type on Burkholderia cepacia selective agar (BCSA; MAST Diagnostics Ltd, Merseyside, UK) following incubation at 37 deg C for 48 h. As this was a new isolate growing on BCSA, it was sent for molecular identification and characterisation.

Subsequently, this organism was isolated from the patient's sputum on five occasions over a two-month period, and presently remains a part of the established resident bacterial flora. Semi-quantitative determination of organism numbers demonstrated that it was consistently present at the + + level, approximating to 10^sup 4^ - 10^sup 5^ colony forming units(cfu)/g sputum.

Clinically, since the first isolation of this organism from the patient's sputum, there has been deterioration in his clinical status, including increased cough, chest tightness, wheeze, shortness of breath, production of purulent sputum, fatigue and weight loss of 1.8 kg over this two-month period. In addition, his forced vital capacity (FVC) fell from 89% to 77% predicted. Drug therapy during this period included a two-- week course of intravenous (iv) antibiotics (ceftazidime and tobramycin), as well as an oral course of ciprofloxacin and nebulised colomycin. He also received a course of oral steroids and itraconazole during this period, as there was some evidence of allergic bronchopulmonary aspergillosis (ABPA).

Microbiologically, this organism was poorly identified (48% identification) phenotypically by the API 20NE scheme as Alcaligenes faecalis, with the profile 0000457. The organism was resistant in vitro by standard NCCLS disc diffusion assay to gentamicin, temocillin, ceftazidime, azlocillin, meropenem, aztreonam and colistin, and was sensitive to tobramycin, piperacillin/tazobactam, imipenem and ciprofloxacin.

Subsequently, in order to aid identification, the organism was examined using molecular techniques. All DNA isolation procedures were carried out in a Class II biological safety cabinet in a room geographically separate from that used to set up reaction mixes and also from the 'post PCR' room, in order to minimise false-positive results and in accordance with good molecular diagnostic practice (GMDP), as detailed in the guidelines of Millar et al.1 DNA was extracted from a single colony using the Roche High Purity PCR Template kit (Roche Diagnostics Ltd, UK), following the manufacturer's instructions. All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA, and from the amplification and post-PCR room, in order to minimise contamination.

Reaction mixes (50 p(mu)L) were set up as follows: 10 mmol/L Tris-HC1 (pH 8.3), 50 mmol/L KC1, 2.5 mmol/L MgC1^sub 2^, 200 (mu)mol/L (each) dATP, dCTP, dGTP and dTTP, 1.25 units Thermus aquaticus (Taq) DNA polymerase (Amplitaq; Perkin Elmer), 0.2 (mu)ol/L (each) of the 16S rRNA primers P11P (forward) 5' - GAG GAA GGT GGG GAT GAC GT -3' and P13P (reverse) 5' - AGG CCC GGG AAC GTA TTC AC -3', as previously described,2 and 4 (mu)L DNA template.

Following a hot start, the reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96 deg C for 3 min, followed by 40 cycles of 96 deg C for 1 min, 55 deg C for 1 min, 72"C for 1 min, and a final extension at 72"C for 10 min. During each run, molecular-grade water was included randomly as a negative control and appropriate DNA template from Staphylococcus aureus was included as a positive control. Following amplification, samples (15 (mu)L) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco, UK) in TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA [pH 8.31), stained with ethidium bromide (5 (mu)/100 mL). Gels were visualised under ultraviolet illumination, using a gel image analysis system (UVP Products, England), and all images archived as digital (*.bmp) graphic files.

Subsequently, amplicons were purified (particularly to remove dNTPs, polymerases, salts and primers) using a QlAquick PCR purification kit (Qiagen Ltd., UK) and eluted in Tris-HC1 (10 mmol/L [pH 8.51) prior to sequencing. Cy-5'-- labelled primer (PlP) was prepared and used for sequencing in the forward direction with the ALF Express II (Amersham-Pharmacia Ltd., Bucks, UK) employing the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, UK; cat no: RPN 2438). Thermal cycling parameters were: 96 deg C for 1 min, followed by 25 cycles of 96 deg C for 10 sec, 50 deg C for 5 sec, 60" deg C for 5 sec, followed by a 4 deg C hold. Sequences obtained were compared with those stored in the GenBank data system, using BLAST alignment software (www.blast.genome.ad.jp). Direct sequencing of the PCR amplicon identified the organisms as Pandoraea apista, with 183/183 bases called (100% homology with P. apista AF139173 and AF139172 - no other species matched), and subsequently this sequence was deposited in GenBank (accession number AF419312).

The genus Pandoraea was described recently by Coenye et al.3 and presently consists of five species. These organisms have been shown to form part of the microbial community in the lungs of CF patients; and diagnostically present several challenges to identification. The majority of isolates described to date have been cultured from BCSA. In the case described here, particular attention was given to the correct identification of the isolate, as the patient was negative for the B. cepacia complex (BCC) and it was considered important to rule out BCC, particularly for infection control purposes.

Extended phenotyping schemes, such as the API 20NE system, are unable to identify this organism, as presently the genus is not included in the profile listing, leading to potential misidentification as A. faecalis, or alternatively as A. denitrificans, CDC IV C2 and Acinetobacter spp., as recently described.4 More recently, this grouping has been renamed as three newly described Pandoraea genomospecies.5

One solution to aid absolute identification of such organisms would be to include molecular analyses in the diagnostic algorithm. As such organisms are capable of growth on BCSA, the primary consideration is to exclude BCC organisms. Thus, we performed several species-specific PCR assays for BCC, including those associated with the recA locus for BCC organisms, as previously described.6,7 All were negative, demonstrating the presence of a pure culture of Pandoraea spp.

Further consideration was given to the possibility of cross-- reactivity of other BCC PCR-specific assays, especially those based on 16S ribosomal RNA (rRNA), leading to the potential for molecular misidentification of Pandoraea spp. as BCC. Previously, we showed that the PSL1/PSR1 primers, described by Campbell et al,8 also amplify P norimbergensis.9 As a result, we further investigated other 16S rRNA BCC-- specific primer pairs and noted that the RHGF/RHGR primer set described by LiPuma et al.10 would also amplify Pandoraea spp. (Table 1).

At this stage, it is unclear whether or not the G1/G2 primer pair described by Whitby et al.11 would amplify Pandoraea spp., as 23S rRNA of Pandoraea spp. has yet to be described, nor was this primer set initially challenged with Pandoraea spp. Thus, we suggest that when a suspect colony resembling Pandoraea spp. is isolated in respiratory secretions from a CF patient, a molecular sequence-based approach be adopted, employing broad-range or universal 16S rRNA sequencing, as described here and previously.12

Where no (or limited) molecular facilities exist in the primary diagnostic laboratory, such isolates should be forwarded to a reference or specialty laboratory for further examination and confirmation. Recently, a specific PCR technique based on 16S rRNA was described for Pandoraea spp.,13 as well as species-specific primers for the respective species within this genus. Routine employment of such primer sets in diagnostic algorithms may help to avoid potential misidentification of Pandoraea spp. as BCC, and the various complications for infection control associated with Gram-negative organisms in CE

Clinically, there is limited data in the literature describing disease progression associated with the presence of this organism in the airway of CF patients, probably resulting from problems associated with its laboratory identification. In the case described here, it is difficult to separate disease progression resulting from chronic Pseudomonas aeruginosa infection with that associated with the Pandoraea sp.

In view of the relatively resistant nature of this organism's antibiogram, the patient's progressive clinical deterioration and the ecological establishment of this organism as part of the bacterial flora within the patient's respiratory tract, careful clinical consideration should be given to such cases until more information about disease progression and optimised management are known. However, the opportunity to perform such studies will depend on microbiologists employing extended identification schemes to confirm the presence of these species in patients' sputa.

The authors wish to thank Dr. Colin Graham, Northern Ireland Regional Genetics Centre, Belfast City Hospital, for scientific assistance in the determination of the CF genotype.

References

1 Millar BC, Xu J, Moore JE. Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 2002; 40: 1575-80.

2 Millar B, Moore J, Mallon P et al. Molecular diagnosis of infective endocarditis - a new Duke's criterion. Scand J Infect Dis 2001; 333: 673-80.

3 Coenye T, Falsen E, Haste B et al. Description of andoraea gen.

nov. with Pandoraea apista sp. nov., Pandoraea pulmonicola sp. nov., Pandoraea pnomenusa sp. nov., Pandoraea sputorum sp. nov. and Pandoraea norimbergensis comb. nov. Int J Syst Evol Microbiol 2000; 50: 887-99.

4 Henry DA, Mahenthiralingam E, Vandamme P Coenye T, Speert DP. Phenotypic methods for determining genomovar status of the Burkholderia cepacia complex. J Clin Microbiol 2001; 39: 1073-8.

5 Daneshvar MI, Hollis DG, Steigerwalt AG et al. Assignment of CDC weak oxidizer group 2 (WO-2) to the genus Pandoraea and characterization of three new Pandoraea genomospecies. J Clin Microbiol 2001; 39: 1819-26.

6 Moore JE, Millar BC, Jiru X, McCappin J, Crowe M, Elborn JS. Rapid characterization of the genomovars of the Burkholderia cepacia complex by PCR single-stranded conformational polymorphism (PCR-SSCP) analysis. J Hosp Infect 2001; 48: 129-34.

7 Mahenthiralingam E, Bischof J, Byrne SK et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis and Burkholderia cepacia genomovars I and III. J Clin Microbiol 2000; 38: 3165-73.

8 Campbell PW 3rd, Phillips JA 3rd, Heidecker GJ, Krishnamani MR, Zahorchak R, Stull TL. Detection of Pseudomonas (Burkholderia) cepacia using PCR. Pediatr Pulmonol 1995; 20: 44-9.

9 Moore JE, Coenye T, Vandamme P, Elborn JS. First report of Pandoraea norimbergensis isolated from food - potential clinical significance. Food Microbiol 2001; 18: 113-4.

10 LiPuma JJ, Dulaney BJ, McMenamin JD et al. Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients. J Clin Microbiol 1999; 37: 3167-70.

11 Whitby PW, Dick HL, Campbell PW 3rd, Tullis DE, Matlow A, Stull TL. Comparison of culture and PCR for detection of Burkholderia cepacia in sputum samples of patients with cystic fibrosis. J Clin Microbiol 1998; 36: 1642-5.

12 Millar BC, Jiru X, Moore JE, Earle JA. A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material. J Microbiol Methods 2000; 42: 139-47.

13 Coenye T, Liu L, Vandamme P LiPuma JJ. Identification of Pandoraea species by 16S ribosomal DNA-based PCR assays. J Clin Microbiol 2001; 39: 4452-5.

JOHN E. MOORE1, ALASTAIR REID2, BEVERLEY C. MILLAR1, XU JIRU1,3,4, JOHN MCCAUGHANS5, COLIN E GOLDSMITH1, JONATHAN COLLINS6,7,PHILIP G. MURPHY6,7 and J. STUART ELBORN3

1Northern Ireland Public Health Laboratory, Belfast City Hospital;

2Northern Ireland Paediatric Cystic Fibrosis Centre, Royal Belfast Hospital for Sick Children; Northern Ireland Regional Adult Cystic Fibrosis Centre, Belfast City Hospital; School of Biological and Life Sciences, University of Ulster, Coleraine; Department of Microbiology, Kelvin Building, The Royal Group of Hospitals, Grosvenor Road, Belfast, Northern Ireland; Department of Microbiology, The Adelaide & Meath Hospitals, Tallaght, Dublin; and 7Department of Microbiology, Trinity College, Dublin, Republic of Ireland.

Correspondence to: Dr John E. Moore,

Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland.

Email: jemoore@niphl.dnet.co.uk

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