1 Section of Clinical Microbiology, The University of Texas M.D. Anderson Cancer Center, Unit 84, 1515 Holcombe Boulevard, Houston, TX 77030, USA; 2 Division of Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA
Received 29 December 2004; returned 8 February 2005; revised 27 March 2005; accepted 29 March 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: Seven patients with infection and eight bacterial strains were studied. Tests included antibiotic susceptibility and analysis of the DNA gyrase and topoisomerase genes and the effect of efflux pump inhibitor Phe-Arg-ß-naphthylamide (PANA).
Results: The patients all had underlying disease of cancer. The infections involved bloodstream (one case), intravascular catheter (four cases), urinary tract (one case) and pleural space (one case of empyema). Fever up to 39.2°C characterized these infections, which resolved upon treatment by combination antibiotics. Microbiologically, all organisms were resistant to multiple fluoroquinolones and cefepime, but were susceptible to amikacin, imipenem and ticarcillin/clavulanate. These quinolone-resistant B. diminuta strains were probably selected out by the prophylactic use of a quinolone in six of these patients. Additionally, the B. diminuta type strain ATCC 11568T that was isolated before the quinolone era from water was also resistant to ciprofloxacin and intermediate to levofloxacin, suggesting intrinsic quinolone resistance. The DNA gyrase and topoisomerase of six analysed strains all contained GyrA Ala-83 and Met-87, GyrB Leu-466 or Thr-466, and ParC Gln-57, Val-66 and Ala-80 that were probably the cause of fluoroquinolone resistance. PANA had nearly negligible effect.
Conclusions: B. diminuta is intrinsically resistant to fluoroquinolones and can be selected out to cause infections.
Keywords: B. diminuta , fluoroquinolone resistance , quinolones
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cases occurred sporadically during 19982004 at The University of Texas M.D. Anderson Cancer Center in Houston, a 500 bed tertiary care cancer centre. All clinical data were obtained through review of the medical records. The bacteria were isolated from blood cultures in five cases, from urine culture in one case and from pleural catheter discharge in one case. Approximately 30 000 blood cultures were performed yearly at this institution using the Bactec 9240 automated culturing system (BD Diagnostic Systems, Sparks, MD, USA) and Isolator tubes (Wampole Laboratories, Princeton, NJ, USA). The Isolator tube, when positive, allowed quantification of bacterial colonies from 10 mL of blood cultured. In addition to the seven clinical strains, the B. diminuta type strain ATCC 11568T was also included in the study.
Bacterial identification
Two of the seven B. diminuta strains were identified by biochemical tests performed in our laboratory and a reference laboratory (Houston City Health Laboratory, Houston, TX, USA). The other five strains, being recent isolates, were identified presumptively as B. diminuta by biochemical tests and definitively by sequencing analysis of the 16S ribosomal RNA gene.7 Briefly, genomic DNA from pure culture colonies was extracted and subjected to amplification by a polymerase chain reaction (PCR) for a 593 bp fragment of the 16S rRNA gene. A set of universal bacterial primers was used for the amplification: 5'-TGCCAGCAGCCGCGGTAATAC-3' and 5'-CGCTCGTTGCGGGACTTAACC-3' (positions 5151107 of GenBank accession J01859 for Escherichia coli). The amplicon was sequenced by the dye-terminator method in an ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA), and sequence analysis was performed through a query to the GenBank Basic Local Alignment Search ToolTM (BLAST).8
Antibiotic susceptibility test and efflux pump inhibitor
The antibiotic susceptibility test was performed using Etest (AB Biodisk, Solna, Sweden) and interpreted according to the breakpoints set for Pseudomonas aeruginosa and non-Enterobacteriaceae by the NCCLS.9 The efflux pump inhibitor Phe-Arg-ß-naphthylamide dihydrochloride (PANA) (Sigma, St Louis, MO, USA) was used to assess the role of efflux pumps. A concentration of 20 mg/L PANA was incorporated into the agar for routine Etest.
Analysis of gyrA, gyrB and parC genes
Three sets of PCR primers were used to amplify the genes for DNA gyrase and topoisomerase IV. The primers for gyrA were: 5'-TACGCGATGAGCGTGATCGTC-3' and 5'-GTTGTGCGGCGGGATGTTGGT-3' (positions 334822 of Pseudomonas aeruginosa GI 459928).10 The primers for gyrB were: 5'-GAACGACAGCTACCACGAGAC-3' and 5'-TGCGCTATCACAAGATCATCCT-3' (positions 480 to 1196 of Brevundimonas vesicularis GI 19909566) (T. Hamada, 2002, unpublished data). The primers for parC were: 5'-GGCTTGAAGCCCGTGCACCG-3' (forward) and 5'-ATGTTGGTCGCCATGCC-3' (reverse) or 5'-CACGGCGATGCCCGACGATC-3' (reverse) (based on a few consensus sequences). Amplification of parE was attempted using the primers 5'-CGGCCTGCACGGCGT-3' and 5'-GCAGTCGGCCAGCTTG-3', which correspond to 618 and 1783 of Caulobacter crescentus AE005832.11
GenBank accessions
The partial gyrA and gyrB sequences of ATCC 11568T and the parC sequences of clinical strain MDA0824 were deposited as GenBank accessions AY654591, AY654592 and AY971356, respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The clinical features of the seven patients are summarized in Table 1. The patients were six men and one woman and ranged in age from 26 to 74 years (median 54). Five of them had haematological malignancy. The first patient, on the day of chemotherapy, exhibited a fever to 39°C and relative leucocytosis (6.9 x 109/L) from his baseline of 2.7 x 109/L despite prophylaxis with levofloxacin. From the cultures of the central venous catheter (CVC) and peripheral blood, B. diminuta and coagulase-negative Staphylococcus were isolated. The Isolator tubes were also positive, and from the 10 mL of blood cultured, 2130 and 1315 colonies of B. diminuta were obtained from the CVC and peripheral blood, respectively. The bacteraemia was further treated with vancomycin and cefepime (before the culture results) and the patient defervesced 3 days later without CVC removal.
|
The sixth patient who suffered from prostatic sarcoma had an episode of severe obstructive uropathy with significant urethral discharge. In spite of the levofloxacin use, B. diminuta and an Enterococcus sp. were isolated from the urine with 10 00050 000 cfu/mL for each organism. The possible urinary tract infection (UTI) was further treated with levofloxacin (before availability of the culture results) and the patient was discharged.
The last patient who suffered from angiosarcoma of the heart with complication of chronic pleural effusion and placement of a chest catheter presented with fever, shortness of breath, and purulent discharge from the catheter. Both B. diminuta and Staphylococcus aureus were isolated from discharge. The catheter was replaced and empyema drained with concurrent antibiotic therapy. The patient made a full recovery.
Antibiotic susceptibility
The susceptibility test results of the seven clinical strains and the type strain ATCC 11568T are summarized in Table 2. All eight strains were found to be susceptible to amikacin, imipenem and ticarcillin/clavulanate. They were infrequently susceptible to trimethoprim/sulfamethoxazole (three of eight strains tested), ceftriaxone (one of six) and ceftazidime (one of eight). They were all resistant to cefepime and ciprofloxacin and either resistant or intermediate to ampicillin. All five available clinical strains that were tested were also resistant to gatifloxacin and levofloxacin. The ATCC strain was intermediate to levofloxacin and susceptible to gatifloxacin.
|
A portion of the gyrA gene encompassing the potential quinolone resistance-determining region (QRDR) in B. diminuta was successfully amplified and sequenced for the six available strains, and up to 479 nucleotides (157 amino acids) were obtained and analysed. The region shared 65% identical residues with that of E. coli,13 and 79% with that of Caulobacter crescentus,11 a non-pathogenic free-living water bacterium (no antibiotic susceptibility data available) that is closest to B. diminuta in phylogeny.14 Among the six strains, the amino acid residues were conserved to 99100%. A limited alignment of the QRDR is shown in Figure 1. The region is conserved for members of the family Enterobacteriaceae and P. aeruginosa, and the B. diminuta strains contained six significantly different residues (underlined). Among them, Ala-83 and Met-87 were candidate residues responsible for or contributing to the quinolone resistance because substitutions at these positions are known to confer resistance in Enterobacteriaceae and P. aeruginosa (Table 3) and organisms in other genera.
|
|
A portion of gyrB gene was amplified successfully and sequenced for ATCC 11568T and four clinical strains. The region contained 220 amino acid residues and was more conserved than GyrA across species, from 68% identical with E. coli to 85% identical with C. crescentus. For the five B. diminuta strains, all residues were conserved to 99100%. In comparison with known quinolone-resistant residues for E. coli and P. mirabilis, one residue, Leu-466 or Thr-466 was found to be potentially important for resistance (Table 3).
The analysis of the parC gene is shown in Table 3. Of the 138 amino acids analysed, the strains were conserved to 99100%, and they shared 53% identical residues with that of E. coli and 86% with that of C. crescentus. Three residues, Gln-57, Val-66 and Ala-80, all identical among the six strains, were significantly different from usual quinolone-sensitive residues in other organisms.
Amplification of the parE gene was attempted, which yielded the same gyrB gene for two strains (0824 and 2271) while other strains were not amplified.
Effect of PANA
The potential effect of efflux pump inhibitor PANA was assessed (Table 4). The chemical showed a consistent, albeit small, reduction in MIC for ceftriaxone (1.5- to 2.7-fold) and ampicillin (2- to 6-fold). It had no effect on cefepime. The effects on ciprofloxacin, levofloxacin and gatifloxacin were none to small (1.3- to 2.7-fold) depending on strains, but overall nearly negligible.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The quinolone resistance of our B. diminuta strains prompted us to investigate the mechanisms. In most quinolone-sensitive bacteria, the two critical QRDR residues in the GyrA subunit are Ser or Thr at position 83 and Asp or Glu at position 87. Mutations in these positions lead to quinolone resistance in many species, such as several members of the family Enterobacteriaceae, P. aeruginosa, Neisseria gonorrhoeae, Campylobacter jejuni, Mycoplasma hominis, Vibrio parahaemolyticus and Ehrlichia spp.10,13,15,16,18,19,2329 Common mutations are Ser-83 or Thr-83 to Ile, Leu, Phe, Tyr or Arg, and Asp-87 or Glu-87 to Gly, Tyr, Asn, Lys, Ile or Ser (Table 3). In addition, a Ser-83 to Ala mutation has been shown to be associated with reduced susceptibility or resistance in several genera.21,26,3033 In our B. diminuta strains, these residues are Ala-83 and Met-87. The analogy for Ala-83 is apparent and the Met-87 is structurally conserved to the resistance residue Ile-87; thus, these residues are the likely cause of B. diminuta's quinolone resistance.
The GyrB subunit is less commonly associated with quinolone resistance; however, mutations of four residues are found causing resistance: Asp-426 to Asn, Lys-447 to Glu, Ser-464 to Tyr or Phe, and Glu-466 to Asp (Table 3). In our B. diminuta strains, Asp-426, Arg-447 and Ser-464 were identical or similar to those quinolone-sensitive sequences, and the only difference was Leu-466 or Thr-466. Thus, Leu-466 or Thr-466 might have further contributed to the resistance.
A few ParC mutations, i.e. Thr-57 to Ser, Thr-66 to Ile, Ser-80 to Ile or Arg, and Glu-80 to Lys, Val or Gly, also confer quinolone resistance.2022,34 All our strains contained three significantly different residues at these positions: Gln-57, Val-66 and Ala-80. Despite the current lack of analogy, these residues may potentially contribute to the quinolone resistance.
Efflux pumps have been shown to be important for the quinolone resistance of P. aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia.35 These medically important organisms are also environmental and closely related to B. diminuta by phylogeny. We assessed the effect of a broad-spectrum efflux pump inhibitor PANA and failed to show substantial impact on the quinolone resistance of our B. diminuta strains. Saenz et al. found that PANA significantly reduced the MIC of nalidixic acid, but not fluoroquinolones.36 With nalidixic acid not tested, our results on fluoroquinolones are consistent with that finding.
The B. diminuta ATCC strain was isolated from the environment (freshwater stream) in the 1950s before the quinolone era,2 yet it was resistant to ciprofloxacin and intermediate to levofloxacin, suggesting its intrinsic resistance to quinolones. Intrinsic resistance to quinolones is rare but has been reported for aquatic Aeromonas spp., i.e. A. caviae, A. hydrophila and A. sobria, in which the QRDR residue GyrA Ser-83 is mutated to Ile or Arg.25 In addition, B. diminuta has been found to degrade quinoline and 6-hydroxyquinoline37 compounds that are structurally similar to quinolones, especially oxolinic acid, an early form of quinolone. This finding, although it has not been reported by other groups, may warrant further investigation as to whether bacteria, particularly those naturally resistant ones, can actually degrade quinolones in addition to being resistant to them.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Lefson E, Hugh R. A new type of polar monotrichous flagellation. J Gen Microbiol 1954; 10: 6870.[ISI][Medline]
3 . Segers P, Vancanneyt M, Pot B et al. Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Busing, Doll, and Freytag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. Int J Syst Bacteriol 1994; 44: 499510.[Abstract]
4 . Hall G. Non-fermentative Gram-negative bacilli and miscellaneous gram-negative rods. In: Mahon C, Manuselis G, eds. Textbook of Diagnostic Microbiology. Philadelphia, PA, USA: W. B. Saunders Company, 2000; 53963.
5 . Gilad J, Borer A, Peled N et al. Hospital-acquired Brevundimonas vesicularis septicaemia following open-heart surgery: case report and literature review. Scand J Infect Dis 2000; 32: 901.[CrossRef][ISI][Medline]
6 . Koneman EW, Allen SD, Janda WM, et al. Color Atlas and Textbook of Diagnostic Microbiology, 5th edn. Philadelphia, PA, USA: Lippincott Williams & Wilkins Co., 1997; 253320.
7 . Han XY, Pham AS, Tarrand JJ et al. Rapid and accurate identification of mycobacteria by sequencing hypervariable regions of the 16S ribosomal RNA gene. Am J Clin Pathol 2002; 118: 796801.[CrossRef][ISI][Medline]
8
.
Altschul SF, Thomas LM, Alejandro AS et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389402.
9 . National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A6. MIC Testing Supplemental Tables. Wayne, PA, USA: NCCLS, 2003.
10 . Kureishi A, Diver JM, Beckthold B et al. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates. Antimicrob Agents Chemother 1994; 38: 194452.[Abstract]
11
.
Nierman WC, Feldblyum TV, Laub MT et al. Complete genome sequence of Caulobacter crescentus. Proc Natl Acad Sci USA 2001; 98: 413641.
12 . Han XY, Tarrand JJ. Moraxella osloensis blood and catheter infections during anticancer chemotherapy: clinical and microbiological studies of 10 cases. Am J Clin Pathol 2004; 121: 5817.[CrossRef][ISI][Medline]
13 . Hussain K, Elliott EJ, Salmond GP. The parD- mutant of Escherichia coli also carries a gyrAam mutation. The complete sequence of gyrA. Mol Microbiol 1987; 1: 25973.[ISI][Medline]
14 . Sly LI, Cox TL, Beckenham TB. The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications. Int J Syst Bacteriol 1999; 49: 4838.[Abstract]
15
.
Weigel LA, Steward CD, Tenover FC. gyrA Mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob Agents Chemother 1998; 42: 26617.
16
.
Weigel LA, Anderson GJ, Tenover FC. DNA gyrA and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis. Antimicrob Agents Chemother 2002; 46: 25827.
17 . Yamagishi J, Yoshida H, Yamayoshi M et al. Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet 1986; 204: 36773.[CrossRef][ISI][Medline]
18
.
Jalal S, Ciofu O, Hoiby N et al. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2000; 44: 7102.
19
.
Takenouchi T, Sakagawa E, Sugawara M. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrob Agents Chemother 1999; 43: 4069.
20
.
Eaves DJ, Randall L, Gray DT et al. Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica. Antimicrob Agents Chemother 2004; 48: 40125.
21
.
Saenz Y, Zarazaga M, Brinas L et al. Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans and animals. J Antimicrob Chemother 2003; 51: 10015.
22 . Everett MJ, Jin YF, Ricci V et al. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Agents Chemother 1996; 40: 23806.[Abstract]
23 . Bebear CM, Bove JM, Bebear C et al. Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones. Antimicrob Agents Chemother 1997; 41: 26973.[Abstract]
24 . Beckmann L, Muller M, Luber P et al. Analysis of gyrA mutations in quinolone-resistant and -susceptible Campylobacter jejuni isolates from retail poultry and human clinical isolates by non-radioactive single-strand conformation polymorphism analysis and DNA sequencing. J Appl Microbiol 2004; 96: 10407.[CrossRef][ISI][Medline]
25
.
Goni-Urriza M, Arpin C, Capdepuy M et al. Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance. Antimicrob Agents Chemother 2002; 46: 3509.
26
.
Maurin M, Abergel C, Raoult D. DNA gyrase-mediated natural resistance to fluoroquinolones in Ehrlichia spp. Antimicrob Agents Chemother 2001; 45: 2098105.
27
.
Okuda J, Hayakawa E, Nishibuchi M et al. Sequence analysis of the gyrA and parC homologues of a wild-type strain of Vibrio parahaemolyticus and its fluoroquinolone-resistant mutants. Antimicrob Agents Chemother 1999; 43: 115662.
28 . Ridley A, Threlfall EJ. Molecular epidemiology of antibiotic resistance genes in multiresistant epidemic Salmonella typhimurium DT 104. Microb Drug Resist 1998; 4: 1138.[ISI][Medline]
29 . Shigemura K, Shirakawa T, Okada H et al. Mutations in the gyrA and parC genes and in vitro activities of fluoroquinolones in 91 clinical isolates of Neisseria gonorrhoeae in Japan. Sex Transm Dis 2004; 31: 1804.[ISI][Medline]
30 . Bachoual R, Ouabdesselam S, Mory F et al. Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Microb Drug Resist 2001; 7: 25761.[CrossRef][ISI][Medline]
31 . Baucheron S, Imberechts H, Chaslus-Dancla E et al. The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar typhimurium phage type DT204. Microb Drug Resist 2002; 8: 2819.[CrossRef][ISI][Medline]
32 . Biedenbach DJ, Jones RN. Fluoroquinolone-resistant Haemophilus influenzae: frequency of occurrence and analysis of confirmed strains in the SENTRY antimicrobial surveillance program (North and Latin America). Diagn Microbiol Infect Dis 2000; 36: 2559.[CrossRef][ISI][Medline]
33
.
Tavio MM, Vila J, Ruiz J et al. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. J Antimicrob Chemother 1999; 44: 73542.
34 . Hiasa H. The Glu-84 of the ParC subunit plays critical roles in both topoisomerase IV-quinolone and topoisomerase IV-DNA interactions. Biochemistry 2002; 41: 1177985.[CrossRef][ISI][Medline]
35
.
Zhang L, Li XZ, Poole K. Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia. J Antimicrob Chemother 2001; 48: 54952.
36
.
Saenz Y, Ruiz J, Zarazaga M et al. Effect of the efflux pump inhibitor Phe-Arg-ß-naphthylamide on the MIC values of the quinolones, tetracycline and chloramphenicol, in Escherichia coli isolates of different origin. J Antimicrob Chemother 2004; 53: 5445.
37 . Bott G, Lingens F. Microbial metabolism of quinoline and related compounds. IX. Degradation of 6-hydroxyquinoline and quinoline by Pseudomonas diminuta 31/1 Fa1 and Bacillus circulans 31/2 A1. Biol Chem Hoppe Seyler 1991; 372: 3813.[ISI][Medline]
|