First Attempt of Sequential Living Donor Liver and Hematopoietic Stem Cell Transplantation in a Child With Advanced Hepatocellular Carcinoma: Case Report.

Effective therapeutic options for advanced hepatocellular carcinoma are limited. Hematopoietic stem cell transplantation may offer a graft-versus-tumor effect. Combined liver and hematopoietic stem cell transplantation from the same donor with preparatory conditioning may promote tolerogenicity to the liver allograft and offers the potential for immunosuppression withdrawal. We report our experience with the use of this approach in a pediatric patient with invasive hepatocellular carcinoma and pulmonary metastases who underwent a living-donor liver transplantation followed by reduced-toxicity myeloablative conditioning and hematopoietic stem cell transplant from the same parental donor. Neutrophil engraftment and full donor chimerism was achieved without liver allograft dysfunction. Despite normal liver function and marrow engraftment, the patient succumbed to multisystem organ failure from disseminated toxoplasmosis. At autopsy, there was no histologic evidence of tumor recurrence. No pulmonary nodules were found. Regardless of the unfortunate overall result, this case demonstrates preliminary feasibility of sequential living-donor liver transplantation and hematopoietic stem cell transplantation for unresectable and metastasized hepatic tumors. Future studies in select pediatric patients require evaluation of the optimal conditioning regimen and prevention strategies for opportunistic infections to determine both graft-versus-tumor effect on hepatic tumors and durability of tolerogenicity and possible immunosuppression withdrawal.

Adolescent and young adult cancer: principles of care.

Adolescents and young adults (ayas) with cancer in active treatment face a number of barriers to optimal care. In the present article, we focus on the 3 critical domains of care for ayas-medical, psychosocial, and research-and how changes to the system could overcome barriers. We summarize the current literature, outline recommended principles of care, raise awareness of barriers to optimal care, and suggest specific changes to the system to overcome those barriers in the Canadian context. Many of the recommendations can nevertheless be applied universally. These recommendations are endorsed by the Canadian Task Force on Adolescents and Young Adults with Cancer and build on outcomes from two international workshops held by that group.

Transcriptional response of mucoid Pseudomonas aeruginosa to human respiratory mucus.

UNLABELLED: Adaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogen Pseudomonas aeruginosa is capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential. IMPORTANCE: The basis for chronic colonization of patients with cystic fibrosis (CF) by the opportunistic pathogen Pseudomonas aeruginosa continues to represent a challenging problem for basic scientists and clinicians. In this study, the host-adapted, alginate-overproducing Pseudomonas aeruginosa 2192 strain was used to assess the changes in its transcript levels following growth in respiratory CF mucus. Several significant and unexpected discoveries were made: (i) although the alginate overproduction in strain 2192 was caused by a stable mutation, a mucus-derived signal caused reduction in the transcript levels of alginate biosynthetic genes; (ii) mucus activated the expression of the type VI secretion system, a mechanism for killing of other bacteria in a mixed population; (iii) expression of a number of genes involved in respiration was altered; and (iv) several small regulatory RNAs were identified, some being mucus regulated. This work highlights the strong influence of the host environment in shaping bacterial survival strategies.

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa.

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.

Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa.

We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa. FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus. A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the sigma(28) binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor sigma(28) was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus sigma(54) recognition sites and one sigma(28) site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either sigma(28) or sigma(54). A transcript was detected by primer extension that was 8 bp downstream of the consensus sigma(28)-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both sigma(28)-dependent and -independent mechanisms of transcription exist.

Interaction of the antiactivator FleN with the transcriptional activator FleQ regulates flagellar number in Pseudomonas aeruginosa.

Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption of fleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN(24K-->Q) and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

Evolution, incidence, and susceptibility of bacterial bloodstream isolates from 519 bone marrow transplant patients.

Bacteria remain an important cause of infection in bone marrow transplants. To examine shifts in the etiology and susceptibility of bacterial isolates from transplants, we reviewed the incidence and susceptibility of blood isolates during a 7-year period. The infection rate fell dramatically during this time. Gram-positive organisms were isolated more often than gram-negative organisms, but the trend is reversing. Streptococci surpassed staphylococci for 5 years as the leading pathogen. Increasing resistance to penicillin, ciprofloxacin, and imipenem was noted in Streptococcus species. With the exception of type 1 beta-lactamase-producing bacteria and Pseudomonas aeruginosa, gram-negative isolates remained overall susceptible to ceftazidime. Increased antibiotic prophylaxis coincided with the reduction in percentage of infected patients and increase in resistance to beta-lactam antibiotics. Mortality attributed to bacteremia was low except for infections caused by P. aeruginosa and the Enterobacter, Serratia, Citrobacter group. There was no mortality attributable to gram-positive organisms such as Staphylococcus aureus and viridans streptococci.

Recognition of Lewis x derivatives present on mucins by flagellar components of Pseudomonas aeruginosa.

Pseudomonas aeruginosa binds to human respiratory mucins by mechanisms involving flagellar component-receptor interactions. The adhesion of P. aeruginosa strain PAK is mediated by the flagellar cap protein, FliD, without the involvement of flagellin. Two distinct types of FliD proteins have been identified in P. aeruginosa: A type, found in strain PAK, and B type, found in strain PAO1. In the present work, studies performed with the P. aeruginosa B-type strain PAO1 indicate that both the FliD protein and the flagellin of this strain are involved in the binding to respiratory mucins. Using polyacrylamide-based fluorescent glycoconjugates in a flow cytometry assay, it was previously demonstrated that P. aeruginosa recognizes Le(x) (or Lewis x) derivatives found at the periphery of human respiratory mucins. The aim of the present work was therefore to determine whether these carbohydrate epitopes (or glycotopes) are receptors for FliD proteins and flagellin. The results obtained by both flow cytometry and a microplate adhesion assay indicate that the FliD protein of strain PAO1 is involved in the binding of glycoconjugates bearing Le(x) or sialyl-Le(x) determinants, while the binding of flagellin is restricted to the glycoconjugate bearing Le(x) glycotope. In contrast, the type A cap protein of P. aeruginosa strain PAK is not involved in the binding to glycoconjugates bearing Le(x), sialyl-Le(x), or sulfosialyl-Le(x) glycotopes. This study demonstrates a clear association between a specific Pseudomonas adhesin and a specific mucin glycotope and demonstrates that fine specificities exist in mucin recognition by P. aeruginosa.

A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation.

Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.

FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells.

Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by approximately 80% for the flhA mutant and approximately 50% for the fliC mutant, showing that only part of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997-1999.

During 1997-1999, a total of 70,067 isolates (6631 Pseudomonas aeruginosa isolates) were analyzed in the SENTRY program by geographic region and body site of infection. The respiratory tract was the most common source of P. aeruginosa. P. aeruginosa isolation rates increased during the study interval. Europe was the only region to show a significant decline in beta-lactam and aminoglycoside susceptibility rates. There was a reduction in the rates of susceptibility of Canadian isolates to imipenem and of Latin American isolates to meropenem. A total of 218 multidrug-resistant P. aeruginosa isolates (MDR-PSA; resistant to piperacillin, ceftazidime, imipenem, and gentamicin) were observed; MDR-PSA occurrence rates (percentages of all isolates) ranged from 8.2% (Latin America) to 0.9% (Canada). No antimicrobial inhibited >50% of MDR-PSA strains. Molecular characterization of selected, generally resistant strains was performed. Isolates showing unique ribogroups were found in Europe, Latin America, and the United States, but clonal spread was documented in several medical centers.

Randomized, double-blind, multicenter trial comparing clinafloxacin with imipenem as empirical monotherapy for febrile granulocytopenic patients.

In a double-blind, multicenter trial, 541 febrile granulocytopenic patients were randomized to receive either intravenous (iv) clinafloxacin (200 mg every 12 h) or i.v. imipenem (500 mg every 6 h) as empirical monotherapy. More baseline pathogens were susceptible to clinafloxacin (259 [99%] of 262 organisms) than to imipenem (253 [95%] of 265; P=.03). Initial favorable clinical response rates for clinafloxacin (88 [32%] of 272 patients) and imipenem (89 [33%] of 269) were similar. After addition of other antimicrobial agents, overall response rates were 259 (95%) of 272 for clinafloxacin and 251 (93%) of 269 for imipenem. During the study, only 13 clinafloxacin (5%) and 18 imipenem (7%) recipients died. Both drugs were generally well tolerated. Drug-related skin rash occurred more often with clinafloxacin (11% vs. 6%; P=.07), whereas nausea (2% vs. 5%; P=.16), Clostridium-difficile-associated diarrhea (3% vs. 8%; P=.02), and seizures (0% vs. 2%; P=.06) occurred more often with imipenem. These results suggest that clinafloxacin and imipenem have similar efficacy as empirical monotherapy in febrile granulocytopenic patients.

Recognition of mucin components by Pseudomonas aeruginosa.

Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis. This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs. The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms. While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g. increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF. The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins. Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens. There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known. However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.

In vitro activity of selected cephalosporins and erythromycin against staphylococci and pneumococci isolated at 38 North American medical centers participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

The SENTRY Antimicrobial Surveillance Program employs a worldwide network of hospitals to monitor the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with nosocomial and community-acquired bloodstream, respiratory tract, wound, and urinary tract infections. The purpose of this analysis of SENTRY data is to extract information on the current North American susceptibility patterns of pneumococci and oxacillin-susceptible staphylococci from the comprehensive SENTRY program database. Clinical isolates were provided by 30 centers in the United States (grouped into five regions) and eight centers in Canada. Susceptibility testing was performed at a central reference laboratory using broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. Of 34 530 North American bacterial isolates tested during 1997 and 1998, 565 (1.6%) were oxacillin-susceptible, coagulase-negative staphylococci (CoNS). Cefazolin, cefepime, and ceftriaxone all had excellent activity against these CoNS (97.3%-99. 3% susceptible), and 90.4% were susceptible to ceftazidime. A total of 4404 isolates (12.8%) were oxacillin-susceptible Staphylococcus aureus. Overall, 98.9% to 99.2% were susceptible to cefazolin, cefepime, and ceftriaxone; ceftazidime did not have acceptable activity against these S. aureus. Streptococcus pneumoniae accounted for 1665 (4.8%) of North American SENTRY isolates. A total of 1212 isolates (72.8%) were fully susceptible to penicillin (MIC /= 2 microg/ml). The rate of penicillin susceptibility was highest in Canada, and lowest in the South Central and South East regions of the United States. Cefepime, cefuroxime, ceftazidime, and erythromycin all demonstrated excellent efficacy (94%-99.8% susceptibility) against fully penicillin-susceptible isolates of S. pneumoniae. Among pneumococci with intermediate penicillin resistance, 88% were susceptible to cefepime, 92% to cefotaxime, and only 14% to ceftazidime. None of the antimicrobial agents in this analysis demonstrated adequate activity against fully penicillin-resistant pneumococci. In summary, the fourth-generation cephalosporin, cefepime, demonstrated consistently excellent efficacy against oxacillin-susceptible staphylococci and most pneumococci, and remains an appropriate choice for empiric therapy of serious infections.

In vitro efficacy of six cephalosporins tested against Enterobacteriaceae isolated at 38 North American medical centres participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

The SENTRY Antimicrobial Surveillance Program is an ongoing international collaboration that monitors the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial infections. SENTRY data on the current cephalosporin susceptibility patterns (1997-98) of North American isolates of clinically important Enterobacteriaceae were analyzed. Susceptibility to a selection of cephalosporins was assessed at a central laboratory using reference broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. The third- and fourth-generation cephalosporins tested demonstrated excellent activity against Escherichia coli and Klebsiella pneumoniae, whereas some of the older agents maintained good efficacy. Extended spectrum beta-lactamases were detected in all regions of the United States and Canada (1.8-10.7%). Cefepime was the most active agent tested against pathogens with the potential for enzyme-mediated resistance due to Amp C. The third-generation agents maintained acceptable efficacy against Serratia marcescens, but were less effective against Citrobacter and Enterobacter species. The older cephalosporins were generally inadequate against these pathogens, in contrast to cefepime, which was the widest spectrum cephalosporin overall. Some significant regional variations in spectrum were detected.

Pilus-mediated adherence of Haemophilus influenzae to human respiratory mucins.

Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeable H. influenzae bind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeable H. influenzae to human respiratory mucins. We used isogenic H. influenzae strains with a mutation in the structural gene for pilin (hifA), a laboratory H. influenzae strain transformed with a type b pilus gene cluster (from strain C54), antibodies raised against H. influenzae HifA, and Escherichia coli strains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliated H. influenzae to mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence. E. coli strains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding of H. influenzae to human respiratory mucins.

Comparison of the activity of two broad-spectrum cephalosporins tested against 2,299 strains of Pseudomonas aeruginosa isolated at 38 North American medical centers participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

Pseudomonas aeruginosa is an important nosocomial pathogen. Resistance to certain beta-lactam antimicrobial agents among P. aeruginosa is increasing. The SENTRY Antimicrobial Surveillance Program was designed to employ a network of hospitals in the United States, Canada, Latin America, and Europe to monitor the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial bloodstream, respiratory tract, wound, and urinary tract infections. The purpose of this analysis of SENTRY results was to extract information on the current North American susceptibility pattern of P. aeruginosa for two antipseudomonal cephalosporins, ceftazidime, and cefepime. Clinical isolates were provided by 30 centers in the United States (grouped into five regions) and eight centers in Canada. Susceptibility testing was performed at a central reference laboratory by using broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. Of the 34, 530 North American bacterial isolates tested during 1997 and 1998, 2299 (6.7%) were P. aeruginosa. There were no substantial differences in regional rates of P. aeruginosa susceptibility to ceftazidime (range 78.8-81.9%) or cefepime (range 80.0-83.4%) The percentage of resistant isolates among the 1784 United States isolates was 13.3% for ceftazidime versus 7.1% for cefepime (p < 0.05). It is essential to continue surveillance of the in vitro efficacy of these and other beta-lactam agents against P. aeruginosa because of the clinical importance of these safe and broad-spectrum cephems used alone or in combination in current clinical practice.

Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa.

Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.