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The microbiome of the female reproductive tract has implications for women's reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence.

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Staphylococcus aureus is a common inhabitant of the human nasopharynx. It is also a cause of life-threatening illness, producing a potent array of virulence factors that enable survival in normally sterile sites. The transformation of S. aureus from commensal to pathogen is poorly understood. We analyzed S. aureus gene expression during adaptation to the lung using a mouse model of S. aureus pneumonia. Bacteria were isolated by bronchoalveolar lavage after residence in vivo for up to 6 hours. S. aureus in vivo RNA transcription was compared by microarray to that of shake flask grown stationary phase and early exponential phase cells. Compared to in vitro conditions, the in vivo transcriptome was dramatically altered within 30 minutes. Expression of central metabolic pathways changed significantly in response to the lung environment. Gluconeogenesis (fbs, pckA) was down regulated, as was TCA cycle and fermentation pathway gene expression. Genes associated with amino acid synthesis, RNA translation and nitrate respiration were upregulated, indicative of a highly active metabolic state during the first 6 hours in the lung. Virulence factors regulated by agr were down regulated in vivo and in early exponential phase compared to stationary phase cells. Over time in vivo, expression of ahpCF, involved in H(2)O(2) scavenging, and uspA, which encodes a universal stress regulator, increased. Transcription of leukotoxic α and β-type phenol-soluble modulins psmα1-4 and psmβ1-2 increased 13 and 8-fold respectively; hld mRNA, encoding δ-hemolysin, was increased 9-fold. These were the only toxins to be significantly upregulated in vivo. These data provide the first complete survey of the S. aureus transcriptome response to the mammalian airway. The results present intriguing contrasts with previous work in other in vitro and in vivo models and provide novel insights into the adaptive and temporal response of S. aureus early in the pathogenesis of pneumonia.

The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.

Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by high-performance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity.

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.

Group B streptococci (GBS) (Streptococcus agalactiae) are a major cause of sepsis and meningitis in neonates and infants and of invasive disease in pregnant women, nonpregnant, presumably immunocompromised adults, and the elderly. Nine GBS serotypes based on capsular polysaccharide antigens have been described. The serotype distributions among invasive and colonizing isolates differ between pediatric and adult populations and have changed over time. Thus, periodic monitoring of GBS serotype distributions is necessary to ensure the proper formulation and application of an appropriate GBS vaccine for human use and to detect the emergence of novel serotypes. Since the mid-1990s, the proportion of GBS isolates that are nontypeable by standard serologic methods has increased, creating a need for more sensitive typing methods. We describe a typing method that uses DNA dot blot hybridization with probes generated by PCR from the GBS capsular genes for serotypes Ia, Ib, and II to VIII. PCR primers were designed to amplify type-specific GBS capsular gene sequences. Gene probes were constructed from the PCR products and used to classify isolates based on hybridization profiles. A total of 306 previously serotyped invasive and colonizing isolates were used to compare our dot blot capsular typing (DBCT) identification method with Lancefield serotyping (LS). A dot blot capsular type was assigned to 99% (303 of 306) of the isolates, whereas 273 of 306 isolates (89%) were assigned a Lancefield serotype. The overall agreement between the methods was 95% (256 of 270 isolates typeable by both methods). We conclude that the DBCT method is a specific and useful alternative to the commonly used LS method.

Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.

The polysialic acid capsule of Escherichia coli K1 is an essential virulence determinant. The kps gene cluster, which encodes the proteins necessary for polymer synthesis and transport, is divided into three functional regions. In this report, we present evidence that the neuD gene from region 2 is involved in sialic acid synthesis. A non-polar chromosomal deletion in neuD was constructed. The defect was complemented by neuD in trans or by the addition of exogenous sialic acid. A NeuD homologue, Neu(III)D, from serotype III Streptococcus agalactiae (GBS) also restored capsule expression to the neuD deletion strain. These data confirm the role of neuD in E. coli sialic acid capsule synthesis and demonstrate that the neu(III)D homologue from GBS shares a similar enzymatic function.

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

A 10-year-old boy presented with nuchal rigidity and cerebrospinal fluid (CSF) leukocytosis initially and again on day 6 of intravenous cefuroxime therapy (200 mg/kg/day). Both CSF specimens yielded nontypable beta-lactamase-negative Haemophilus influenzae that were susceptible by disk tests but relatively resistant to cefuroxime (MIC, 8- to 16-fold greater than that of control isolates). To define the mechanism of resistance, the cefuroxime resistance marker was transformed to a susceptible H. influenzae recipient; inactivation and permeability of beta-lactam substrate were tested and the penicillin-binding protein (PBP) profiles were examined. Inactivation of beta-lactam substrate was not detected and reduced permeability was not found. However, reduced beta-lactam binding to PBPs 4 and 5 was observed; 18- to 27-fold more penicillin and 2.5-to 4-fold more cefuroxime was required to saturate or block 50% of the binding sites of these PBPs, respectively. Thus, reduced affinity of PBPs 4 and 5 for beta-lactam substrate appears to be the mechanism of cefuroxime resistance in this strain. The reduced affinity of these targets appears to have contributed to the bacteriologic and clinical failure in this patient.

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

Ampicillin-resistant, non-beta-lactamase-producing isolates of Haemophilus influenzae contain a variety of penicillin-binding protein (PBP) patterns that differ from the single pattern of eight PBPs characteristic of susceptible strains. During genetic transformation of resistance, only some of the anomalies in PBP pattern were transformed, specifically those relating to the penicillin-binding capacities of PBPs 4 (Mr of 62,000) and 5 (Mr of 59,000) and, in some transformations, PBP 3 (Mr of 71,000). Comparison of the binding of penicillin by PBPs 4 and 5 of three resistant transformants (derived with DNA from different donors) revealed a decrease in the rate of PBP acylation and no appreciable change in the rate of deacylation as compared to the susceptible recipient. Thus, rapid turnover of these PBPs does not play a role. Retransformation studies confirm that altered PBPs 3, 4, and 5 are associated with resistance and suggest that these PBPs are major targets for the beta-lactam antibiotics in H. influenzae.

We questioned whether the apparent rarity of ampicillin-resistant, non-beta-lactamase-producing (NBLP) Haemophilus influenzae was due to failure of detection. We identified ampicillin-resistant and ampicillin-susceptible H. influenzae by the agar dilution technique, using 10(5)- and 10(3)-CFU inocula. We compared the disk susceptibility of 18 ampicillin-resistant NBLP strains, 13 ampicillin-resistant, beta-lactamase-producing strains, and 10 ampicillin-susceptible strains by using standard 10- and 2-micrograms ampicillin disks on two different media. We also investigated the possibility that disks containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin could be used to distinguish between the two ampicillin-resistant populations. Using the disk containing 2 micrograms of ampicillin, we correctly differentiated all the ampicillin-resistant isolates from the ampicillin-susceptible isolates on both media (a zone diameter of less than or equal to 15 mm was considered resistant). In contrast, the 10-micrograms ampicillin disk failed to detect 44% (8 of 18) of the ampicillin-resistant NBLP strains (a zone diameter of less than or equal to 19 mm was considered resistant). The MIC of ampicillin with the 10(3)-CFU inoculum correlated better with zone diameters than with the 10(5)-CFU inoculum. A difference in zone diameters of greater than or equal to 8 mm between the disk containing 10 micrograms of clavulanic acid and 2 micrograms of ampicillin and the disk containing only 2 micrograms of ampicillin correctly identified all beta-lactamase-producing strains. We conclude that the 2-micrograms ampicillin disk tests more readily identify ampicillin resistance in H. influenzae than do the 10-micrograms ampicillin disk tests. Future investigation should determine whether this in vitro resistance correlates with clinical treatment failures.

Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.

We questioned if PBP analysis could differentiate strains of Haemophilus influenzae, H. aegyptius, and H. influenzae biogroup aegyptius associated with Brazilian Purpuric Fever. A relatively homogeneous PBP pattern was observed for all strains. The amount of penicillin bound to PBP 5 appeared to separate H. influenzae and H. aegyptius isolates, whereas PBP 5 of those strains associated with Brazilian Purpuric Fever bound an intermediate amount. We conclude that based on PBP profiles, the strains tested appear to be difficult to separate taxonomically and may represent a common species.

Penicillin-binding protein (PBP) alterations have been associated with non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 microgram ml-1 for the susceptible isolates at an inoculum of 100,000 c.f.u. when tested by broth dilution, and was 0.5 microgram ml-1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 micrograms ml-1 when tested by broth dilution, and from 1.5 to 16 micrograms ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotypic and phenotypic diversity among 16 penicillin G-resistant (Penr) isolates of Neisseria meningitidis recovered from human blood or cerebrospinal fluid in Spain was compared with that among 12 penicillin-susceptible (Pens) isolates by the use of multilocus enzyme electrophoresis, serotyping, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Thirteen distinctive multilocus enzyme genotypes (electrophoretic types [ETs] ) were identified among the 28 isolates. There was slightly less genetic diversity among the eight ETs of Penr isolates (H = 0.385) than among the eight ETs of Pens isolates (H = 0.431). Cluster analysis demonstrated two distinctive complexes of ETs and one ET that was not closely related to either complex. The possibility of a singular clonal origin of penicillin G-resistant isolates was excluded by the observations that resistance occurred in isolates of each of the two distantly related complexes of ETs, that three of the four ETs represented by multiple isolates included both susceptible and resistant strains, and that serotypes and growth requirements were not associated with the resistance phenotype. The 28 isolates showed a relatively homogeneous pattern of four PBPs, with apparently reduced penicillin G binding by PBP 3 of the Penr isolates.

Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-beta-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.

We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3.

Beginning in 1985, relatively penicillin G-resistant (Penr) meningococci which did not produce beta-lactamase were isolated from the blood and cerebrospinal fluid of patients in Spain. We identified 16 Penr (mean MIC, 0.3 microgram/ml; range, 0.1 to 0.7 microgram/ml) and 12 penicillin-susceptible (Pens; mean MIC, less than or equal to 0.06 microgram/ml) strains of Neisseria meningitidis by the agar dilution technique using an inoculum of 10(4) CFU and questioned which disk susceptibility test would best differentiate these two populations. We compared the disk susceptibility of these strains using disks containing 2 (P2) and 10 (P10) U of penicillin G, 2 (Am2) and 10 (Am10) micrograms of ampicillin, and 1 microgram of oxacillin (OX1). We also investigated susceptibility with disks containing 30 micrograms of each of cephalothin (CF30), cefoxitin (FOX30), cefuroxime (CXM30), and cefotaxime (CTX30) and 75 micrograms of cefoperazone (CFP75) and determined by cluster analysis any correlation with the zone diameters obtained with P2 disks. Using the P2 and AM2 disks (in contrast to the P10 and AM10 disks), we correctly differentiated all the Penr from Pens isolates. In addition, the zone diameters with the P2 disk gave the best correlation with the penicillin G MIC determinations. All 16 Penr strains and 3 of 12 Pens strains showed zone diameters of 6 mm around OX1 disks, limiting the usefulness of OX1 disks. The zone diameters obtained with CF30, CXM30, and OX1 disks correlated with those obtained with the P2 disk, which suggests that these antibiotics have similar effects on these strains. In contrast, the data obtained with FOX30, CTX30, and CFP75 disks did not cluster with those obtained with the P2 disk, which suggests that there was a difference in the bacterial target or reflects their greater activity. We conclude that the P2 disk tests more readily identify Penr meningococci than do the standard P10 disk tests.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.

We describe the application of generalized linear model methodology to the problem of testing differences among ligand-receptor interactions, and show that the method is analogous to weighted least squares regression methodology and F tests familiar to many investigators. The method accommodates incomplete block designs so that one can obtain kinetic parameter estimates directly comparable among samples analysed on incompletely overlapping sets of experimental runs. We demonstrate the method with data that compare saturation kinetics for a single penicillin-binding protein in isogenic ampicillin susceptible and resistant bacteria.