Molecular epidemiology and risk factors for colonization by vancomycin-resistant Enterococcus in patients with hematologic malignancies.

OBJECTIVE: To study the molecular epidemiology of vancomycin-resistant Enterococcus (VRE) colonization and to identify modifiable risk factors among patients with hematologic malignancies. SETTING: A hematology-oncology unit with high prevalence of VRE colonization. PARTICIPANTS: Patients with hematologic malignancies and hematopoietic stem cell transplantation recipients admitted to the hospital. METHODS: Patients underwent weekly surveillance by means of perianal swabs for VRE colonization and, if colonized, were placed in contact isolation. We studied the molecular epidemiology in fecal and blood isolates by pulsed-field gel electrophoresis over a 1-year period. We performed a retrospective case-control study over a 3-year period. Cases were defined as patients colonized by VRE, and controls were defined as patients negative for VRE colonization. Case patients and control patients were matched by admitting service and length of observation time. RESULTS: Molecular genotyping demonstrated the primarily polyclonal nature of VRE isolates. Colonization occurred at a median of 14 days. Colonized patients were characterized by longer hospital admissions. Previous use of ceftazidime was associated with VRE colonization (P < .001), while use of intravenous vancomycin and antibiotics with anaerobic activity did not emerge as a risk factor. There was no association with neutropenia or presence of colonic mucosal disruption, and severity of illness was similar in both groups. CONCLUSION: Molecular studies showed that in the majority of VRE-colonized patients the strains were unique, arguing that VRE acquisition was sporadic rather than resulting from a common source of transmission. Patient-specific factors, including prior antibiotic exposure, rather than breaches in infection control likely predict for risk of fecal VRE colonization.

Characterization of P1-deficient isogenic mutant of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Haemophilus influenzae biogroup aegyptius (formerly H. aegyptius) is the etiologic agent of Brazilian purpuric fever (BPF). A surface-exposed epitope on the outer membrane protein P1 is present on most strains of H. influenzae biogroup aegyptius associated with BPF but is absent in almost all non-disease associated strains. The role of the outer membrane protein P1 in the pathogenesis of this disease was evaluated by utilizing an isogenic P1-deficient mutant. We compared the ability of the wild type and P1 isogenic mutant to grow under various conditions. The P1-deficient strain grew at a similar rate to the wild type in both complex and chemically defined medium. The P1-deficient mutant also had a similar growth rate to the wild type under anaerobic conditions. Anaerobic growth, however, resulted in up-regulation of the P1 protein in the wild type strain. Three assays were used to examine the pathophysiologic role of the P1 protein in BPF: 1) serum resistance; 2) sustained bacteremia in the infant rat model; and 3) the human microvascular endothelial cell (HMEC) cytotoxicity assay. Both the mutant and wild-type strains were resistant to killing in 95% normal human serum. The P1-deficient strain was also as virulent as the wild type in both the infant rat model of bacteremia and in the HMEC-1 tissue culture model. These results demonstrate that serum resistance, sustained bacteremia in the infant rat, and cytotoxicity of HMEC cells occur in the absence of P1. The P1 protein is not essential for the pathogenic potential identified by these assays. However, these results demonstrate that an anaerobic environment is a potent physiologic regulator of P1 protein expression. The impact of anaerobiosis on protein expression and pathogenesis will require further investigations.

Identification, genomic organization, and analysis of the group III capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5).

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps(K54)DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps(K54)M and Kps(K54)T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps(K54)E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM(truncated) and its 5' noncoding regulatory sequence were identified. In contrast to the complete kps(K54)M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsM(truncated) was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.

Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus.

The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.

Creation of an isogenic P1-deficient mutant of Haemophilus influenzae biogroup aegyptius.

Haemophilus influenzae biogroup aegyptius, the causative agent of Brazilian purpuric fever (BPF), expresses a heat-modifiable 48 kDa outer membrane protein, P1, which is conserved in most Brazilian case-clone isolates. To study the role of P1 in pathogenesis of BPF we constructed via homologous recombination an isogenic P1-deficient mutant of H. influenzae biogroup aegyptius. The procedure involved a modification of Hererot's method for development of competence. Modifications included variations in the growth conditions, use of cAMP, specific characteristics of the donor DNA, and antibiotic selection. P1-deficient mutants were confirmed by SDS-PAGE, loss of reactivity with a specific monoclonal antibody on Western blot, restriction analysis and Southern blot. Our results establish the first successful transformation of homologous DNA into H. influenzae biogroup aegyptius.

DNA sequence analysis and restriction fragment length polymorphisms of the P1 gene of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a fulminant pediatric disease caused by specific strains of Haemophilus influenzae biogroup aegyptius. A conserved epitope on the P1 protein of strains of H. influenzae biogroup aegyptius is seen on most virulent isolates. The P1 protein from a Brazilian case-clone strain of H. influenzae biogroup aegyptius was analyzed by cloning and sequencing the gene. Three major variable regions are present within the P1 gene of the BPF clone in an architecture similar to that of the previously sequenced P1 genes from H. influenzae. The DNA sequence data of the P1 gene provided information for restriction fragment length polymorphism analyses among strains of H. influenzae biogroup aegyptius. Using PCR for amplification of the P1 gene, we found that AlwI restriction of this gene allowed for a highly accurate segregation of virulent strains of H. influenzae biogroup aegyptius associated with BPF. The strong association of virulent phenotypes with specific AlwI restriction patterns of the P1 gene provides a basis for the convenient and accurate identification of strains of H. influenzae biogroup aegyptius which cause BPF.

Clinical and molecular epidemiology of Enterococcus faecalis bacteremia, with special reference to strains with high-level resistance to gentamicin.

The objective of this study was to characterize the clinical and molecular epidemiology of Enterococcus faecalis bacteremia, specifically that involving strains with high-level resistance to gentamicin (HLGR). Episodes of E. faecalis bacteremia at the Buffalo Veterans Affairs Medical Center from January 1986 to September 1989 were retrospectively identified. Of 94 episodes, 45 (48%) were due to strains with HLGR. Hemolytic activity was detected with greater frequency (85%) among the latter strains than among those without HLGR (P < .001). Of the 54 episodes for which medical charts were available for review, 94% were hospital acquired, and 46% were due to strains with HLGR. An examination of the plasmid DNA content of isolates revealed restriction-fragment-length polymorphism. One plasmid pattern was identified in 15 isolates with HLGR (P < .001), and chromosomal DNA digest patterns suggested a common clonality; there was no direct evidence for patient-to-patient spread of these strains. The use of antibiotics, the presence of invasive devices, surgery, and admission to an intensive-care unit were not significantly associated with HLGR bacteremia. Mortality during hospitalization was 65%, with no difference between figures for HLGR and non-HLGR infections. This high mortality regardless of gentamicin susceptibility status suggests that E. faecalis bacteremia is a marker for severe illness. In contrast to previous studies, this investigation identified no clinical factors associated with HLGR E. faecalis bacteremia.

Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid.

The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis.

The outer membrane of Branhamella catarrhalis contains a major, heat-modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium. The gene encoding CD was cloned and expressed in Escherichia coli. The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations. The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species. The CD protein contains a proline-rich region, which appears to account for its aberrant migration in gels. Restriction fragment-length analysis of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays. The major heat-modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B. catarrhalis.

Expression of an immunoreactive 72 kDa protein in strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a newly described pediatric syndrome that results in significant morbidity and mortality. BPF is caused by specific phenotypic strains of Haemophilus influenzae biogroup aegyptius that are capable of intravascular survival. Immunoblotting of outer membrane proteins of H. influenzae biogroup aegyptius with normal human serum showed that most virulent strains of H. influenzae biogroup aegyptius associated with BPF expressed an immunologically prominent protein at 72 kDa. A corresponding protein in avirulent isolates migrated at 79 kDa. Although a minor component on SDS-PAGE analysis of the outer membrane, specific antibody against this protein is present in high concentrations in normal human serum.

Stable, conserved outer membrane epitope of strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

Brazilian purpuric fever is a rapidly fatal childhood disease associated with a clonal strain of Haemophilus influenzae biogroup aegyptius. We describe a conserved, surface-exposed epitope present on 95% of H. influenzae biogroup aegyptius isolates that are associated with Brazilian purpuric fever. This epitope, defined by reaction with the monoclonal antibody 8G3, is on or associated with the 48-kDa heat-modifiable P1 protein. The epitope is absent on strains of H. influenzae biogroup aegyptius that are not associated with Brazilian purpuric fever but is present on one strain of H. influenzae biotype II. None of 81 other Haemophilus strains tested reacted with 8G3. The sensitivity and specificity of the 8G3 monoclonal antibody in detecting Brazilian case-clone strains of H. influenzae biogroup aegyptius associated with Brazilian purpuric fever are 95 and 99%, respectively. Immunoelectron microscopy revealed that the epitope is surface exposed, and N-terminal amino acid sequencing of an 8G3-reactive P1 protein from a strain of H. influenzae biogroup aegyptius showed 100% correlation with the published N-terminal amino acid sequence of a P1 protein of H. influenzae type b. The virulence of the organism in an infant rat model of bacteremia was not dependent on the expression of this epitope.

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

Lipooligosaccharide epitopes shared among gram-negative non-enteric mucosal pathogens.

The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the 'mini-gel' format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their Mr during electrophoresis and allowed estimation of Mr from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.

Haemophilus influenzae lipopolysaccharide-induced blood brain barrier permeability during experimental meningitis in the rat.

The factors responsible for blood-brain barrier (BBB) injury during bacterial meningitis are incompletely defined. We evaluated the role of Haemophilus influenzae type b (Hib) lipopolysaccharide (LPS) in the alteration of blood-brain barrier permeability (BBBP) in an adult, normal and leukopenic, rat model of meningitis. Intracisternal inoculation of Hib LPS resulted in (a) dose-dependent increases in BBBP from 2 pg to 20 ng, with significant attenuation in the peak response after challenge with 500 ng and 1 microgram; (b) time-dependent increases in BBBP, with a delayed onset of at least 2 h, maximum alteration at 4 h, and complete reversal at 18 h; (c) greater BBBP than after challenge with the live parent strain; (d) and a close correlation (r = 0.86) between CSF pleocytosis and BBBP at 4 h. The LPS effect was significantly inhibited by preincubation with Polymyxin B and neutrophil acyloxyacyl hydrolase, however two different oligosaccharide-specific monoclonal antibodies did not inhibit activity. No change in BBBP after inoculation with Hib LPS occurred in leukopenic rats. Hib LPS, in the setting of an intact leukocyte response, exerts profound effects on BBBP.

Role of cerebrospinal fluid pleocytosis and Haemophilus influenzae type b capsule on blood brain barrier permeability during experimental meningitis in the rat.

The influence of leukocytes and Haemophilus influenzae type b (Hib) capsule on blood brain barrier permeability (BBBP) to circulating 125I-albumin in normal and leukopenic rats was assessed after intracisternal inoculation of encapsulated (Rd-/b+/02) or unencapsulated (Rd-/b-/02) isogenic strains of Hib. Both normal and leukopenic animals had increased BBBP 18 h after inoculation, with normal rats demonstrating significantly increased BBBP after challenge with the encapsulated strain. Despite cerebrospinal fluid (CSF) pleocytosis in normal rats, CSF bacterial concentrations were not lower. Normal rats cleared unencapsulated Rd-/b-/02 more effectively than leukopenic rats, with BBBP correlating with CSF bacterial density and not leukocyte concentrations. Challenge with heat-killed Rd-/b+/02 resulted in increased BBBP in both normal and leukopenic rats, with greater BBBP at higher bacterial concentrations. The data suggest: (a) significant increases in BBBP occur in the near absence of CSF leukocytes; (b) CSF leukocytes can augment changes in BBBP; (c) type b capsule inhibits host clearance mechanisms within the CSF; and (d) BBBP appears to correlate with bacterial concentrations within the CSF.

Oral ciprofloxacin therapy for gram-negative bacillary osteomyelitis.

Gram-negative osteomyelitis frequently responds poorly to conventional therapy. Ciprofloxacin displays excellent in vitro activity against gram-negative bacilli and offers the potential for outpatient therapy. In this ongoing study, ciprofloxacin therapy is being evaluated for the treatment of gram-negative osteomyelitis. Twenty-three patients (16 men and seven women) have been treated under the protocol (750 mg orally twice daily for 1.5 to six months), and 14 patients have completed therapy. All patients had either growth on bone cultures from an open or percutaneous biopsy, or an arthrocentesis to confirm the diagnosis. Involved sites included ankle or tibia (seven patients), vertebra (four patients), hip (five patients), metatarsal (four patients), phalanx (two patients), and metacarpal (one patient); 16 patients had chronic disease, and seven patients had acute disease. Patients had a total of 28 gram-negative bacilli, 12 gram-positive cocci, and one anaerobic gram-negative rod, for an average of 1.8 pathogens per patient. Eighteen of the 28 gram-negative bacilli were Pseudomonas species. The geometric mean minimal inhibitory concentration for all the gram-negative bacilli was 0.15 microgram/ml. The geometric mean minimal inhibitory concentration for the gram-positive isolates was 0.41 microgram/ml. All patients who completed therapy experienced a cure, with a mean follow-up of 6.1 months. Infections in all patients, except for two who are still taking ciprofloxacin, are resolving, both clinically and radiologically. One patient who was not eligible for the protocol experienced a superinfection with methicillin-resistant Staphylococcus aureus. Side effects have included urticaria, lethargy, nausea, and transient elevations of liver and renal function test results. Overall, ciprofloxacin therapy was well tolerated. This study suggests that ciprofloxacin holds promise for the outpatient treatment of gram-negative osteomyelitis.