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Stenotrophomonas maltophilia and Burkholderia cepacia Complex

GEORG MASCHMEYER | ULF B. GBEL

Stenotrophomonas maltophilia and species of the Burkholderia cepacia

complex (Bcc) are important nosocomial pathogens in hospitalized patients, particularly those with prior broad-spectrum antibacterial therapy, primarily patients with cystic fibrosis (CF) (see Chapter 68). They are intrinsically resistant to most antimicrobial or disinfectant agents and are phenotypically unremarkable, and Bcc exhibits an extensive diversity of genotypes.

Microbiology, Taxonomy, and Identification

STENOTROPHOMONAS S. maltophilia, formerly named Pseudomonas and then Xanthomonas maltophilia, is the only species in the genus. S. maltophilia bacteria are motile, free-living, glucose-nonfermentative, gram-negative aerobic bacilli with multitrichous polar flagella. S. maltophilia grows readily on most bacteriologic media, typically appearing pale-yellow, grayish, or lavender-green when grown on blood agar. Preliminary identification may be facilitated by its ammonia-like odor. Most clinical isolates are oxidase negative and use maltose and usually dextrose and xylose.1 S. maltophilia may produce extracellular deoxyribonuclease on selected media, can hydrolyze esculin and orthonitrophenyl--dgalactopyranoside, and produces catalase and a strong acid reaction in oxidation-fermentation in maltose medium. Most strains require methionine for growth.2 In vitro resistance patterns differ markedly between institutions, and testing results may not correctly predict clinical treatment response. BURKHOLDERIA CEPACIA COMPLEX B. cepacia, previously known as Pseudomonas cepacia, now represents genomovar I of the large and diverse Bcc. Like Stenotrophomonas species, Bcc bacteria are motile, free-living, glucose-nonfermentative, gram-negative aerobic bacilli with multitrichous polar flagella. The appearance of Bcc colonies is variable, depending on the strain and the culture medium used. Identification of highly treatment-resistant small-colony variants of Bcc on selective media may be of clinical importance.3 Based on phenotypic and genotypic analyses, Bcc has been divided into currently 10 genomic species (genomovars): B. cepacia (genomovar I); B. multivorans (genomovar II); B. cenocepacia (genomovar III, with four recA clusters, IIIA- IIID); B. stabilis (genomovar IV); B. vietnamiensis (genomovar V); B. dolosa (genomovar VI); B. ambifaria (genomovar VII); B. anthina (genomovar VIII), B. pyrrocinia (genomovar IX), and B. ubonensis (genomovar X).4 Recently, five novel species, B. latens, B. diffusa, B. aboris, B. seminalis, and B. metallica, have been proposed as members of the Bcc using a polyphasic approach based on comparative 16S ribosomal RNA and recA sequencing, multilocus sequence typing (MLST), and intermediate DNA-DNA binding values.5 At present, MLST represents the most promising way for identifying species and strains of the Bcc.6 To isolate Bcc organisms, selective media have been developed that usually contain sucrose with or without lactose, and antibiotics, such as polymyxin, gentamicin, or vancomycin. Three media are in use: the

Pseudomonas cepacia agar (PCA), the oxidation-fermentation polymyxin bacitracin lactose agar (OFBL), and recently the B. cepacia selective agar (BCSA). The last is more selective by suppressing growth of non-Bcc bacteria, whereas Bcc members form visible pinpoint colonies within 24 hours. Colonies are smooth and slightly elevated. A comparison of all three media revealed a superior performance of the BCSA, achieving 43%, 93%, and 100% growth of Bcc organisms at 24, 48, and 72 hours, respectively.7 It has, hence, been recommended to include the use of selective media and extended incubation for CF respiratory tract specimens. Meanwhile, these recommendations have been implemented in clinical microbiology laboratory protocols of most CF care sites reviewed recently.8 Members of the Bcc can be identified by available commercial tests, such as API 20NE, Phoenix, MicroScan, or Vitek. However, misidentification occurs in a number of cases, and care should be taken to correctly differentiate Burkhold eria species from Achromobacter, Ralstonia, and other nonfermenting, gram-negative rods. A large polyphasic analysis of 1051 isolates from 115 CF treatment centers in 91 US cities conducted in 2000 revealed an overall misidentification rate of 11% for isolates identified as Bcc by referring laboratories. This rate was even higher (36%) for isolates not specifically identified or identified as a species other than Bcc.9 There is an increasing number of laboratories implementing semiautomated microbial identification systems. A recent comparison of the Phoenix automated microbiology system (BD Diagnostics, Sparks, Md) with the MicroScan WalkAway 96 system (Dade Behring, West Sacramento, Calif) revealed difficulties in correctly identifying at the species level five isolates representing B. cenocepacia, B. multivorans, and B. gladioli.10 The evaluation of the new Vitek 2 colorimetric card (ID-GN; bioMrieux) for identification of nonfermentative, gramnegative rods revealed accurate identification of most Bcc isolates.11

Virulence Factors and Pathogenesis

Both S. maltophilia and members of the Bcc uncommonly cause community-acquired infections in previously healthy individuals. Their resistance to most antimicrobial agents selects them in specific patient populations. S. maltophilia is a nosocomial pathogen that occurs in many of the same types of hospitalized patients as Bcc. In most clinical situations, however, isolation of S. maltophilia will represent colonization or contamination rather than true infection, and it has been difficult in many instances to substantiate a causative role of S. maltophilia because of its rather limited pathogenicity and the lack of obvious virulence factors.12 One candidate virulence factor may be a recently described alkaline serine protease, the StmPr1 protease, enabling S. maltophilia to degrade human serum and tissue proteins (e.g., the immunoglobulin G heavy chain).13 The gene of another putative virulence factor has been identified in a clinical S. maltophilia isolate harboring a phage genome containing an open reading frame that exhibited significant sequence similarity to the V. cholerae zonula occludens toxin Zot.14 A most remarkable property was its resistance to pan-protease inhibitors such as 1-antitrypsin and 2-macroglobulin. Biofilm formation contributes to succesful colonisation of abiotic surfaces, such as catheters and lung epithelia. A diffusible signal factor (DSF), methyl dodecenoic acid, regulates a number of virulence traits and antimicrobial resistance (e.g., motility, extracellular proteases,

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lipopolysaccharide (LPS) synthesis, microcolony formation, and tolerance toward antibiotics, and heavy metal ions).15 DSF has been shown to influence P. aeruginosa in mixed S. maltophiliaP. aeruginosa biofilms, conferring increased bacterial stress tolerance (e.g., resistance to cationic antimicrobial peptides), an effect associated with increased tolerance to polymyxins.16 In a neonatal mouse pneumonia model, S. maltophilia elicited a strong inflammatory response as measured by significant interleukin-8 (IL-8) and tumor necrosis factor- (TNF-) expression in respiratory epitehlial cells and macrophages, respectively. Low rates of pneumonia and sepsis (20%) in TNF receptor-1 (TNFR1)negative mice compared with wild-type mice (100%) suggested an important role of TNF- signaling.17 The most striking information drawn from the complete genome sequence of a clinical S. maltophilia strain (K279a) was the large number of genes conferring resistance to antimicrobials and heavy metals.18 There were nine resistance-nodulation-division (RND)type efflux pump genes. Patients with CF and those with chronic granulomatous disease are predisposed to infection by Bcc bacteria. Here, colonization by B. multivorans or B. cenocepacia of the respiratory tract is associated with significantly higher morbidity and mortality19 (Fig. 220-1), particularly after lung transplantation, increasing the mortality within the first 6 months after transplantation to up to 40%20 and to 85% after 10 years.21 Whether this is strictly attributable to the virulence of Bcc or rather represents the poor disease status of CF patients affected by Bcc colonization is controversial.22,23 In principle, all Bcc species may cause infections in CF patients. B. cenocepacia and B. multivorans are by far the most frequent Bcc bacteria in this patient population, causing up to 80% of Bcc infections, whereas other Bcc species account for less than 10% of cases. Drug resistance in Bcc is mediated by an immunodominant drug efflux pump (bcrA).24 Bcc species express a number of different virulence factors that variably contribute to the pathogenesis of Bcc infections. By using proteomic profiling, Chung and Speert identified a number of virulence factors associated with B. cenocepacia survival in mice.25 Virulence factors may be present in some but absent in other strains of a given species, enabling the respective organisms to colonize and invade lung tissue, to survive in intracellular compartments, and to induce and elicit robust inflammatory responses. This is particularly true for the epidemic E12 lineage of B. cenocepacia. Current knowledge about Bcc virulence determinants contributing to colonization, invasion, and intracellular survival has been summarized in a comprehensive review.26 B. cepacia ET12 contains a hybrid of two insertion sequences as well as a 1.4-kilobase open reading frame, which have
100

75

50

25 P. aeruginosa B. cenocepacia 0 0 100 Months 200

Figure 220-1 Survival of patients with cystic fibrosis infected by Burkholderia cenocepacia compared with Pseudomonas aeruginosa. (From Jones AM, Dodd ME, Govan JR, et al. Burkholderia cenocepacia and Burkholderia multivorans: Influence on survival in cystic fibrosis. Thorax. 2004;59:948-951.)

been demonstrated in transmissible B. cenocepacia species. Transmissibility is genetically related to esmR and cblA genes, and esmR (or the B. cepacia epidemic strain marker [BCESM]) is detected only in B. cenocepacia strains. It has been shown that the BCESM is part of a genomic island encoding virulence and metabolism-associated genes designated B. cenocepacia island (cii).27 Noteworthy is the presence of an N-acyl homoserine lactone (AHL) synthase gene and the corresponding regulator gene. One factor affecting the early stages of colonization is the scavenging of iron. Bcc possesses at least four iron-binding siderophores: salicylic acid, ornibactin, pyochelin, and cepabactin.26 Adherence in epidemic B. cenocepacia strains is conferred by the presence of long, flexible type II pili exhibiting cable morphology. These giant cable pili mediate attachment to respiratory epithelia.28 Genes necessary for cable biosynthesis are encoded by the cbl operon, consisting of at least seven genes: cblA, the major pilin subunit; cblB, a proposed chaperone; cblC, a proposed usher protein; cblD, a minor pilin protein; and the regulatory genes cblR, cblS, and cblT. The first four genes of the cbl operoncblA, cblB, cblC, and cblDwere shown to be sufficient for pilus assembly in Escherichia coli, but the regulatory genes are required for pilus biogenesis in B. cenocepacia.29-31 Bcc cells lacking Cbl pili still bind to cytokeratin 13 (CK13), the 55-kDa protein expressed in CF patients preferentially in bronchiolar and respiratory epithelium,28,29 indicating that other bacterial proteins, such as a 22-kDa protein, mediate adherence. Mutants not expressing cable pili (cblA or CblS mutants) showed reduced binding (50%). In mutants lacking the 22-kDa adhesin (adhA mutants), adhesion toward CK13 was almost completely abolished (0% to 8%). For optimal binding, both Cbl pili and the adhesin appear to be required.32 Other fimbrial structures, such as mesh (Msh), filamentous (Fil), spine (Spn), and spike (Spk), have been identified, but their pathogenetic relevance has yet to be determined.26,33 Nonfimbrial adhesins, a 37-kDa protein corresponding to the Bcc porin C, and an unidentified 66-kDa outer membrane protein have also been described, but detailed knowledge is still lacking.34 Nonpiliate strains use lipid receptors expressed mainly on the basolateral surface of resporatory epithelia and alveolar type II pneumocytes.35 Bcc bacteria have invasive potential. They can migrate across the epithelial barrier to invade lung parenchyma and capillaries.36,37 Invasiveness may be due to inhibition of natural pulmonary defense mechanisms such as human -defensins.38 Invasive clinical Bcc isolates have shown their ability to survive in macrophages and pulmonary epithelial cells, whereas environmental strains may lack this ability.39 Adhesion by cable pili and the 22-kDa adhesin appears to be required for transmigration across the squamous epithelium because mutants lacking either adhesin, the adhA mutant in particular, were shown to be compromized in their transmigration capacity.32 In contrast, B. cenocepacia expressing both pili and the 22-kDa adhesin invaded and migrated across the epithelial barrier, causing IL-8 release and epithelial damage. Bacteria were surrounded by filopodia and present in membrane-bound vesicles within the cells after 24 hours.40 Martin and Mohr39 compared the capacity of a clinical B. cenocepacia isolate and an environmental B. multivorans strain to invade cultured macrophages and pulmonary epithelial cells. Although both strains showed similar invasion frequencies for macrophages, the clinical strain was more invasive in epithelial cells and able to survive and replicate both in macrophages and in epithelial cells. Cable pili induce epithelial cell cytotoxicity and thus appear to play a major role in the pathogenesis of B. cenocepacia infection. Purified cable pili activate caspases and major cysteine proteinases involved in apoptosis.41 Besides transcytosis and and disruption of the epithelial barrier by pilus-mediated epithelial cell death described previously, Bcc species are able to transmigrate the respiratory epithelium through paracytosis by disrupting tight junctions by dephosphorylation and dissociation of occludin from the tight junction complex.42 It has also been shown that lipases produced by Bcc species may play a role in invasion because the lipase inhibitor Orlistat significantly decreased the invasion, affecting neither plasma membrane nor tight junction integrity.43 Invasiveness was impaired

Cumulative survival (%)

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when two genes (i.e., fliG, encoding a component of the motor-switch complex, and fliI, encoding an ATPase required for protein translocation) were disrupted.44 However, reduced invasion was not due to defective adherence. Invasion was inhibited by cytochalasin D, suggesting that the host-cell cytoskeleton may play a role in facilitating bacterial entry.45 This is consistent with the observation that Bcc mutants lacking the bscN gene, encoding an ATP-binding protein possibly representing part of a type III secretion system, exhibited attenuated virulence in a murine model.46 Invasion of B. cenocepacia can be strongly inhibited by bovine lactoferrin, an effect not influenced by its iron-binding activity.47 This may be of clinical importance because it has been demonstrated that recombinant human lactoferrin (rhL) inhibited growth and biofilm formation in several Bcc species. Susceptibility to rifampicin was enhanced in the presence of rhl.48 B. ceno cepacia and some other Bcc species express two metalloproteases, ZmpA and ZmpB, that may contribute to the spread of these organisms by degrading collagen and fibronecting. They are able to inactivate major host protease inhibitors, such as 2-macroglobulin, which may contribute to systemic dissemination and septicemia.49 In addition, immunoglobulins, transferrin, and lactoferrin are cleaved by ZmpB, allowing B. cenocepacia to evade host defences. Several mechanisms appear to enable intracellular survival. Reactive oxygen and nitrogen intermediates possess critical roles in the host defense against Bcc, as shown in p47phox(-/-) mice and in mice with a targeted disruption of the inducible nitric oxide synthase (iNOS) gene.50 Bcc-infected macrophages primed with interferon- produced less nitric oxide than interferon-primed, noninfected cells.51 It could be shown that an overexpressed azurin homologue, normally involved in electron transfer during denitrification, induced apoptosis in macrophages in a caspase-dependent manner.52 Several catalaseperoxidases of B. cenocepacia have been described that provide bacteria expressing these enzymes a significant advantage in resisting attack by macrophage-derived H2O2.53 Similarly, superoxide dismutases like the B. cenocepacia periplasmic superoxide dismutase SodC protect this bacterium from exogenous O2, thus contributing to intracellular survival in macrophages.54 Pigments, such as the pyomelanin produced by some B. cenocepacia strains, are capable of scavenging free radicals, thus attenuating the oxidative burst.55 Flagellum-mediated motility appears to facilitate adhesion to and penetration of epithelial barriers by Bcc.44 Biofilm formation followed by invasion and destruction of epithelial cells has been identified as a major pattern of invasiveness in B. cenocepacia.36 Strains producing abundant exopolysaccharide (EPS) persisted in a mouse model of pulmonary infection.56,57 Eighty to 90% of clinical Bcc isolates produce the EPS cepacian. Cepacian is required for the development of thick biofilms. The initiation of biofilm formation is unaffected. No clear correlation between the ability of various strains to produce EPS and to form biofilms and persistence and virulence has been found.58 In CF patients, pulmonary infection is characterized by an excessive accumulation of neutrophils. It has been shown that EPS from a clinical B. cenocepacia strain interfered with the function of neutrophils by inhibiting chemotactic migration and scavenging of ROS. This, together with the resistance of Bcc species to antibacterial peptides, a nonoxidative defense by neutrophils, explains the inability of CF patients to clear the bacteria from infected lungs.59 Fifty-three percent of clinical B. cenocepacia IIIA isolates showed a nonmucoid phenotype, whereas 100% of the isolates from the B. ceno epacia IIIB lineage and 82.8% of the B. multivorans isolates were frankly mucoid.60 Phenotypic switching, preferably mucoid-to-nonmucoid conversions, has been observed in sequential isolates from 15 patients, and it is presumed that nonmucoid isolates are associated with increased disease severity, whereas the mucoid phenotype is associated with persistence. Bcc LPS induces the release of proinflammatory cytokines (TNF-, IL-6, and IL-8) from blood monocytes and whole blood,61,62 thus contributing to the severe inflammatory response observed in CF patients, who are often showing a rapidly necrotizing course. Bcc lipopolysaccharides further induce increased surface expression of CR3 on neutrophils as well as the priming of respiratory burst activity.63

Most B. cenocepacia strains, isolates of the ET12 lineage in particular, are able to induce a strong and sustained inflammatory reaction by the interaction of bacterial ligands such as LPS or flagella with tolllike receptor (TLR). It could be shown that highly purified LPS from clinical Bcc strains induced a TLR4/CD14-mediated activation of mitogen-activated protein kinase pathways and activation of NFB.64 LPS from different clinical isolates elicited varied immune responses. Although LPS isolated from B. cenocepacia strains activates cells through MyD88-dependent pathways, LPS isolated from B. multiv orans acts through MyD88-independent pathways. This may be due to differences in the acylation patterns and may explain different clinical outcomes.65,66 B. cenocepacia flagella induce a a strong TLR5-mediated NFB activation and IL-8 secretion.67 Interaction of LPS and flagellins with TLR4 and TLR5, respectively, does significantly contribute to pathogenesis, but other signaling events have been proposed that explain the robust inflammatory response found in epidemic B. ceno cepacia infections. A recent report described direct binding of strain BC7 to TNFR1, activating the TNFR1 signaling pathway in a manner similar to TNF-. This resulted in strong IL-8 production.68 Another possibly important way of subverting the hosts immune response has been described,69 demonstrating that B. cenocepacia, but not B. multi vorans, disrupted maturation and induced necrosis in human dendritic cells. Expression of virulence factors and environmental adaptation in B. cenocepacia are regulated by two sets of quorum sensing (QS) genes, the cepIR and cciIR, the former present in all B. cenocepacia strains and the latter exclusively in the epidemic strains containing the cci island.70 CepI produces two AHL, primarily octanoyl-homoserine lactone, whereas CciI produces mainly hexanoyl-homoserine lactone. The transcriptional regulator CepR corresponds to the AHL signals by regulating respective target gene positively or negatively. CepR is also required for cciIR gene expression. The cepIR QS system regulates the expression of a variety of virulence factors, such as extracellular proteases, chitinase, ornibactin biosynthesis, biofilm maturation, and motility. QS mutants affect attachment and stability of B. cenocepacia biofilms.71 Flannagan and colleagues identified a six-gene cluster encoding a two-component regulatory system and an HtrA protease required for adaptation of B. cenocepacia to environmental stress (e.g., exposure to osmotic or thermal stress and survival in a rat model of chronic lung infection).72 Recently, a novel sensor kinase-response regulator, AtsR, has been described. Inactivation of the atsR gene resulted in overexpression of Hcp (hemolysin-coregulated protein) probably secreted by an upregulated type VI secretion system, increased biofilm production, stronger adherence to polystyrene and lung epithelial cells, and actin rearrangements in infected macrophages.73 At present, it is unclear whether the protrusions formed in macrophages contribute to bacterial escape from macrophages or delay phagosomelysosome fusion. Inhibition or delay of phagolysosomal fusion appears to play a crucial role in intracellular survival of B. cenocepacia. Here, mtgC, needed for growth of B. cenocepacia in magnesium-depleted medium, appears to play an important role.74 Two alternative sigma factors, RpoE and RpoN, were shown to be crucial for regulating genes required delaying phagolysosomal fusion and phagosome maturation.75,76 More information will result once complete fully annotated genomes will become available. So far, more than 30 Burkholderia genome-sequencing projects have been initiated. The sequence of B. cenocepacia J2315, a strain of the ET12 lineage, has been completed at the Sanger Institute, and sequences are available for searching. The genome is very large; it totals 8 Mb in three chromosomes of 3.8, 3.2, and 0.8 Mb, respectively, and a 92.7-kb plasmid. It encodes more than 7000 genes, 10% of which have been acquired through horizontal gene transfer.4

Epidemiology
S. maltophilia may be acquired from diverse environmental sources, such as tap water, ready-to-eat salads, or contaminated solutions. Among CF patients, colonization with S. maltophilia has been reported

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for 10% to 15% of them77,78 and has not been found to adversely influence patients prognosis.79 Bcc organisms are distributed ubiquitously and found most commonly on plant roots, the rhizosphere, soil, and moist environments. They are of increasing importance for agriculture and bioremediation because of their antinematodal and antifungal properties as well as their capability to degrade a wide range of toxic compounds.80,81 More than 20% of environmental Bcc strains are closely genetically related to clinical isolates,82 and outbreaks have been reported originating from diverse sources such as contaminated faucets, nebulizers, chlorhexidine solution, alcohol-free mouthwash, multidose albuterol vials used among multiple patients, indigo-carmine dye used in enteral feeding, tap water, bottled water, cosmetics, napkins, nasal sprays, and ultrasound gels. Patients may acquire Bcc either from the environment or through patient-to-patient transmission. B. cenocepacia and B. mul tivorans are more predominant among CF patients than non-CF patients.4 Some authors have detected B. cepacia more frequently among non-CF patients.83 Recent progress in molecular typing methods enabled hospital epidemiologists to correctly identify outbreak strains and, hence, to identify the source and trace transmission routes. Ribotype restriction fragment length polymorphism (RFLP) profiles and pulsed-field gel electrophoresis (PFGE)-resolved RFLPs were used to identify a single dominant and highly transmissible clone in a hospital outbreak involving CF and non-CF patients. Their risk for acquisition was linked to hospitalization, and thus infection control policies must consider the transmission between non-CF and CF patients.84 A comprehensive study evaluating multiple genomic typing systems including random amplified polymorphic DNA (RAPD) typing, PFGE, and BOX polymerase chain reaction (BOX-PCR) fingerprinting compared the results obtained by these different methods with each other as well as to data from previous studies with multilocus restriction typing (MLRT). The authors concluded that PFGE and RAPD fingerprinting were most suitable for small-scale studies (i.e., local outbreaks), whereas BOX-PCR fingerprinting appeared more appropriate for large-scale studies aimed at analyzing global epidemiology.85 In the same study, BOX-PCR fingerprinting was considered a rapid and easy alternative to MLRT. The availability of rapid and accurate tests for genomovar identification allowed for a comprehensive analysis of the prevalence of different Bcc species. By the age of 18 years, about 3.5% of CF patients harbor Bcc.86 Data from the United States, Canada, and Italy show that B. cenocepacia and B. multivorans are the most prevalent genomovars among CF patients, accounting for 95% of all infections. B. cenocepacia are predominant, ranging from 80% to 50% (mean, 67.5%) among different CF populations studied so far. Further analysis of the different recA lineages revealed significant geographic differences. Whereas type IIIB strains represented 75% of all US B. cenocepacia isolates, type IIIA strains are more prevalent in Canada and Europe, accounting for about 70% of all genomovar III isolates.4,87-89 The unique distribution of selected Bcc genomovars indicates the presence of epidemic strains exhibiting particular virulence and transmissibility. So far, two genetic elements have been identified that are associated with epidemic spread: cblA, a gene encoding a protein for cable pilus production, and esmR, also called BCESM, representing a 1.4-kb putative open reading frame with homology to negative transcriptional regulators. Analyzing all B. cenocepacia strains isolated from Canadian CF patients of different geographic origin, Speert and co-workers89 identified four genetic lineages defined by RAPD and PFGE. Only strains from RAPD type 02, representing the ET12 clonal lineage, harbored both BCESM and cblA. The predominance of this RAPD type in Ontario, Canada was correlated with a significantly higher prevalence, accounting for 22% of patients in Ontario versus 5% in Quebec. A population structure analysis of B. cenocepacia90 revealed that 86.7% of all restriction types clustered into three major clonal complexes, comprising epidemic clones ET12 (RT-6 complex), PHDC (RT-46 complex), and Midwest (RT-88 complex). These clones have a wide geographic distribution and exhibit varying degrees of genetic recombination. Infection with clone ET12 has been associated with

increased mortality and the so-called cepacia syndrome, characterized by rapid, often fatal respiratory failure and septicemia.91 PDHC is the clone responsible for almost all Bcc infections in the mid-Atlantic region of the United States.92 A strain belonging to this clonal lineage has been isolated from organic soils in four agricultural fields that had been planted with onions for several years. This indicates that environmental strains may play a pivotal role in the epidemiology of Bcc infections and could explain the ongoing human acquisition despite infection control measures.93 The third clone described is most prevalent in CF patients from the Midwestern region of the United States.94 In contrast to some reports advocating the identification of the cblA gene as a means of influencing patient segregation and infection control strategies,95 several authors90,92 provided evidence that the presence of putative transmissibility factors cblA or esmR varied significantly among established epidemic clones, leading to the conclusion that infection control measures should not be based on the presence or absence of these markers. An attempt to identify other genetic elements that may be specific for epidemic Bcc strains led to the identification of a novel insertion sequence, designated IS1363, in clone PHDC.96 IS1363 was also found in most isolates of clone ET12, but not in other Bcc species except B. ambifaria (genomovar VII). At present, it remains unclear whether this IS element contributes to the increased capacity of both clones to infect CF patients. However, together with other IS elements, it may contribute to the genomic plasticity of Bcc species. Considering the acquisition of environmental strains by CF patients, the observation of frequent genetic recombination in B. ceno cepacia populations may have important implications for the biotechnical use of Bcc species.90,97

Clinical Manifestations
STENOTROPHOMONAS MALTOPHILIA The most frequent clinical manifestations of S. maltophilia infection are bloodstream infections and pneumonia.12,98,99 S. maltophilia bloodstream infection results in a high fatality rate, particularly when not treated promptly with appropriate antibiotics.100 However, most S. maltophilia isolates from respiratory secretions represent colonization rather than infection.101 True S. maltophilia pneumonia is more likely to occur among intensive care or cancer patients and is associated with extensive use of broad-spectrum antibiotics, advanced age, mechanical ventilation, and a higher Acute Physiology and Chronic Health Evaluation II (APACHE II) score.98 S. maltophilia pneumonia is among the nosocomial infections most frequently treated inappropriately102 and is associated with a high mortality rate, particularly when associated with bacteremia or obstruction. It may be complicated by septic shock and multiple organ dysfunction syndrome. Chest radiographs may show lobar, nodular, or bronchopneumonic infiltrates, and computed tomography scans typically exhibit diffuse bilateral multifocal infiltrates and ground-glass attenuation (Fig. 220-2).103,104 The respiratory

Figure 220-2 Thoracic computed tomography scan in an allogeneic hematopoietic stem cell transplant recipient with Stenotrophomonas maltophilia pneumonia. (From Gasparetto EL, Bertholdo DB, Davaus T, et al. Stenotrophomonas maltophilia pneumonia after bone marrow transplantation: Case report with emphasis on the highresolution CT findings. Br J Radiol. 2007;80:e19-e20.)

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microbial flora is often mixed, even in cases in which S. maltophilia is considered to be a significant pathogen. However, S. maltophilia may represent indirect pathogenicity through the production of at least two inducible -lactamases, L1 and L2, hydrolyzing almost all classes of -lactam antimicrobials and thus supporting the growth of pathogens such as Serratia marcescens and Pseudomonas aeruginosa even in the presence of imipenem or ceftazidime.105 S. maltophilia bacteremias are typically central venous catheterrelated and may be polymicrobial.106,107 In cancer patients, antimicrobial treatment with fluoroquinolones, trimethoprim-sulfamethoxazole, carbapenems, or cephalosporins may predispose for S. maltophilia bacteremia.108,109 Single cases of other clinical manifestations to S. maltophilia infection, such as endocarditis on both native and prosthetic valves, endophthalmitis, sinusitis, cellulitis, meningitis, liver abscess, and myositis, have been described. Cellulitis develops around catheter insertion sites or arises hematogenously. Ecthyma gangrenosum may be a rare cutaneous complication of both Bcc and S. malto philia bacteremia. In contrast, hematogenous skin lesions of S. maltophilia present as firm, tender, erythematous nodules. S. malto philia isolated from the urinary tract often represent colonization in the presence of a Foley catheter rather than true infection. However, the urinary tract may be the focus of severe sepsis, particularly after instrumentation or surgery. BURKHOLDERIA CEPACIA COMPLEX Patients with CF and those with chronic granulomatous disease are predisposed to Bcc pneumonia.4,89,110-112 Apart from chronic asymptomatic carriage, rapid and fatal clinical deterioration with necrotizing granulomatous pneumonia, called cepacia syndrome,113 and bacteremia may occur (Fig. 220-3).114 Increased mortality has been observed in CF patients after colonization with Bcc.19,91,115,116 CF patients colonized with B. cepacia genom-

ovar III who are undergoing lung transplantation have been reported to carry an up to 50% risk for fatal post-transplantation complications,20,21,117 which has led several centers to avoid lung transplantation in CF patients colonized with this organism.118 Most CF patients colonized with Bcc, however, show little change in their clinical picture, and those who do may be effectively treated with adequate antimicrobial agents.23 Bcc bacteremia, most often catheter-related and polymicrobial, has been reported in cancer patients119 and in patients undergoing hemodialysis,120 and nosocomial pneumonia was observed in intensive care patients who were mechanically ventilated and pretreated with broadspectrum antibiotics such as fluoroquinolones and ceftazidime.121,122 Bcc skin and soft tissue infection may occur in patients with burns or surgical wounds and in soldiers with prolonged foot immersion in water. Genitourinary tract infection caused by Bcc has been reported after urethral instrumentation, after transrectal prostate biopsy, or through exposure to contaminated solutions.

Treatment
STENOTROPHOMONAS MALTOPHILIA Treatment recommendations for both S. maltophilia and B. cepacia complex infections vary profoundly, and an ideal treatment standard has not been established. It is essential to distinguish between a clinically significant S. maltophilia infection and colonization or poly microbial infection with other, potentially more pathogenic microorganisms involved. For treatment considerations, it should be kept in mind that prior use of fluoroquinolones, carbapenems, or third- or fourth-generation cephalosporins represents a major risk factor for the development of S. maltophilia bacteremia.107,108,123 In patients other than those with CF, who have bacteremia caused by S. maltophilia alone, an indwelling venous catheter is likely to be the source of infection. Removal of this foreign body hastens cure.109 Some authors have found that the attributable mortality in non-CF patients with S. maltophilia or Bcc bacteremia may be similar to that observed in other bacteremias caused by gram-negative bacilli.12,124 Trimethoprim-sulfamethoxazole (TMP-SMZ) and ticarcillin clavulanic acid, given alone or in combination, are agents with consistent therapeutic activity against S. maltophilia isolates.106,125,126 For ticarcillin-clavulanate, high rates of in vitro resistance among S. malto philia have been reported from single centers with extensive use of -lactam antibiotics, as has been the case for TMP-SMX.127-129 The in vitro sensitivity testing of S. maltophilia against antimicrobial drugs may show results contrasting to the clinical outcome in patients treated with these antibiotics.130 This is particularly relevant for TMP-SMZ. Ceftazidime, aztreonam, moxifloxacin, or ciprofloxacin may be suitable for treatment as well, depending on susceptibility.106,131-133 Minocycline has excellent in vitro activity; however, clinical experience with this agent is limited. Tigecycline has been reported to be a new potentially effective agent for clinical treatment of S. maltophilia infections but experience is limited.134,135 Antimicrobial therapy of S. maltophilia infection, pending in vitro susceptibility testing, may be initiated with a combination of TMP-SMZ and ticarcillin-clavulanate at high dosages (15 to 20 mg/kg/day of trimethoprim) for TMP-SMZ and 3.1 g every 4 hours of ticarcillinclavulanate. Removal of foreign material or necrotic tissue is a valuable adjunct to medical therapy. In cases in which removal of an indwelling venous catheter is not suitable, systemic antimicrobial therapy in combination with antibiotic-lock treatment of the catheter has been successfully performed.136 Some patients with a history of allergy to TMP-SMX may successfully undergo desensitization, enabling them to be treated effectively with TMP-SMX.137 In mechanically ventilated patients with S. maltophilia infection of their lower respiratory tract, systemic antibiotic treatment in combination with nebulized aminoglycoside application may be considered,106 provided that no previous application of this modality has caused the emergence of aminoglycoside-resistant S. maltophilia.

Figure 220-3 Chest radiograph showing typical appearances of the cepacia syndrome in a patient with cystic fibrosis. (From Jones AM, Dodd ME, Webb AK. Burkholderia cepacia: Current clinical issues, environmental controversies and ethical dilemmas. Eur Respir J. 2001;17:295-301.)

2866

PART III Infectious Diseases and Their Etiologic Agents

Multivariate analyses have shown that the presence of septic shock at onset of infection, profound neutropenia, and delay in appropriate antimicrobial therapy are significant predictors of poor outcome in patients with S. maltophilia bacteremia.101,105,119,126 BURKHOLDERIA CEPACIA COMPLEX Antimicrobial agents that are effective against Bcc include meropenem, TMP-SMZ, chloramphenicol, and minocycline. Other potentially active single agents include the ureidopenicillins, third-generation cephalosporins, and fluoroquinolones.138 Rates of in vitro resistance of Bcc to TMP-SMZ range from 5% in Quebec, Canada89 and Latin America to 10% in Europe.99 Combination antimicrobial treatment is recommended for patients with pulmonary Bcc infection.139-141 Successful treatment with combinations of meropenem with ciprofloxacin and tobramycin has been reported, as has been for ceftazidime and tobramycin, whereas the combination of TMP-SMZ with a -lactam may result in antagonism.142 Additional nebulization of antimicrobial agents such as meropenem or tobramycin has been reported to be effective as well.143 Among more recently developed antimicrobial agents, doripenem appears to have therapeutical potential against Bcc.144 In individual CF patients with life-threatening Bcc infection, shortterm adjunctive treatment with methylprednisolone may be beneficial.145 Future immunotherapeutic options, primarily prophylactic vaccination against Bcc146 or nasal immunization,147 appear to be promising, particularly in light of scarce new antimicrobial agents under development for the treatment of multidrug-resistant gramnegative bacilli.

Prevention and Control

Because both B. cepacia complex and S. maltophilia are commonly found in the environment, and patient-to-patient transmission,

although repeatedly reported,80,87,148-151 is less frequent than acquisition from other sources, strategies aiming at prevention of infections caused by these multidrug-resistant pathogens are difficult to design.89 Accepted prophylactic measures include (1) an appropriate antibiotic policy, particularly a critical use of ciprofloxacin, cefepime, and imipenem; (2) strict hand hygiene and institution of barrier techniques for colonized or infected patients; and (3) surveillance among CF patients and identification of potential nosocomial reservoirs such as the public water system,152 commercially available drinking water,153 sink drains,154 faucet aerators,155 contaminated handwash or mouthwash solutions,156 and medical equipment. Education of patients and health care workers is a cornerstone of such preventive measures. Isolation and segregation measures have been proved useful for prevention of transmission of Bcc between CF patients.157 For CF patients, living with a person colonized by Bcc, contact with a Bcc-colonized patient, hospitalization, and attending a summer CF camp have been shown to be associated with an increased risk for becoming colonized or infected with these organisms.158 Patient segregation and rigorous infection control measures should be reinforced to reduce or prevent transmission of Bcc. Patients infected with transmissible genomovar strains should not be cohorted with patients infected with B. multiv orans or other Bcc genomovars.157,159 However, continued efforts will be required to implement CF sputum surveillance with improved species and strain identification and to elucidate bacterial or host factors contributing to patient-to-patient transmission.93 A double-blind, placebo-controlled trial in adult CF patients infected with B. cenocepacia has not shown any benefit from nebulized taurolidine.160 In cases in which control measures did not prevent the infection of CF patients, acquisition from natural environments should be considered.161 The widespread use of Bcc in agriculture and bioremediation of contaminated environmental sites causes a conflict about its commercial use in light of its potentially life-threatening impact on CF patients.113,162

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