Resistance to antimicrobial chemotherapy: a prescription for research and action.

The growing problem of resistance to antimicrobial chemotherapy was discussed by participants at the February 1995 workshop at Emory University on population biology, evolution, and control of infectious diseases. They discussed the nature and source of this problem and identified areas of research in which information is lacking for the development of programs to control of the emergence and spread of resistant bacteria. Particular attention was given to theoretical (mathematical modeling) and empirical studies of the within and between-host population biology (epidemiology) and the evolution of microbial resistance to chemotherapeutic agents. Suggestions were made about the kinds of models and data needed, and the procedures that could be employed to stem the ascent and dissemination of resistant bacteria. This article summarizes the observations and recommendations made at the 1995 meeting and in the correspondence between participants that followed. It concludes with an update on the theoretical and empirical research on the between- and within-host population biology and evolution of resistance to antimicrobial chemotherapy most of which has been done since that meeting.

Origins and development of peptide antibiotic research. From extracts to abstracts to contracts.

That cationic proteins might be factors on the antimicrobial defenses of mammalian hosts and are apparently associated with the cytoplasmic granules of phagocytic leukocytes first became evident on the late nineteenth century. It remained, however, for development of sophisticated microanalytic techniques in microbiology, cell biology and protein biochemistry to place these hypotheses in the realm of established theory. This article is a brief summary of significant steps in the development of this theory. It also attempts to outline the firmly established scope and significance of these developments both for the theory of immunity to infection in the different phyla and for the now global quest for new antibiotics.

cis Elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.

Treatment of the failing implant: case reports.

Three case reports of treatment of the failing implant are presented. The implants were immobile but had lost a significant amount of osseous support. The cause of failure was determined to be a combination of bacterial and occlusal traumatogenic insult. The defects were debrided and the implant surface was detoxified with tetracycline. Decalcified freeze-dried bone allograft was implanted in the osseous defects and covered with expanded polytetrafluoroethylene material in accordance with principles of guided tissue regeneration. The barrier membrane was removed 6 to 8 weeks after placement. Eight months to 1 year posttreatment, all sites demonstrated a substantial reduction in probing depth, a gain in clinical attachment, and bone fill of the defects adjacent to the implant.

The regulation of leukotoxin production in Actinobacillus actinomycetemcomitans strain JP2.

Actinobacillus actinomycetemcomitans (A.a.) can produce a potent leukotoxin that is thought to be involved in evasion of the host immune response. In order to understand the role of A.a. and its leukotoxin in the initiation and progression of periodontal disease, it is important determine how the expression of A.a. virulence factors might be regulated by the local periodontal micro-environment. To facilitate the measurement of leukotoxin levels, a leukotoxin-beta-galactosidase gene fusion was constructed and recombined into the chromosome of A.a. strain JP2 at the leukotoxin locus. The resulting strain, AAM17, produces beta-galactosidase under control of the leukotoxin promoter. It also produces leukotoxin, since integration of the gene fusion into the chromosome was designed to produce a duplication of the leukotoxin gene. This strain was used to measure the change in leukotoxin level in response to alterations in two environmental signals: iron concentration and oxygen tension. When AAM17 was grown in iron-limited media that did not alter growth rate but did increase the levels of other iron-regulated proteins, the levels of the leukotoxin-beta-galactosidase were similar to those found in AAM17 grown in iron-replete media. These results were confirmed in strains AAM17 and JP2 by leukotoxicity assays and RNA blots. Aerobic growth of AAM17 resulted in a three-fold decrease in leukotoxin beta-galactosidase activity compared with anaerobically grown cells. These results indicate that the A.a. leukotoxin is regulated by some of the environmental signals that may vary in the gingival crevice.

Molecular genetics of polymyxin resistance in Salmonella typhimurium.

Resistance to Polymyxin and CAP are conferred by a point mutation, pmrA505, in position 81 that results in an Arg to His substitution in pmrA, a regulator protein. The protein, pmrB is expressed in the cell membrane. This is consistent with the hypothesis that it is a sensor/kinase protein. A newly discovered gene, pmrD, confers resistance to polymyxin B equal to that of pmrA505 when expressed on a multicopy plasmid, but such transformed strains are not resistant to CAP. Evidence is presented that pmrA505 can be expressed, but only suboptimally, in the absence of pmrD. However, there is an absolute requirement for pmrA+ in order for pmrD to express resistance to polymyxin B. It is therefore suggested that pmrD is regulated by the two component regulatory pair pmrAB.

Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies.

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment which contains a small open reading frame that could encode an 85-amino-acid peptide. When an omega-Tet insertion was made into the putative pmrD open reading frame (pmrD2::omega-Tet), the resulting plasmid no longer conferred polymyxin resistance, whereas an omega-Tet insertion into vector sequences had no effect. Maxicell analysis confirmed that a protein of the expected size is made in vivo. The PmrD protein shows no significant homology to any known protein, but it does show limited homology across the active site of the p15 acid protease from Rous sarcoma virus, indicating that the protein may have proteolytic activity. However, changing the aspartic acid residue at the putative active site to alanine reduced but did not eliminate polymyxin resistance. When pmrD2::omega-Tet replaced the chromosomal copy of pmrD, the resulting strain showed wild-type sensitivity to polymyxin and could be complemented to resistance by a plasmid that carried pmrD. The pmrA505 allele confers resistance to polymyxin when present in single copy on the chromosome or when present on a plasmid in pmrA+ pmrD+ cells. In combination with the pmrD(2)::-Tet mutation, the effect o the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present in the chromosome or on a plasmid. Conversely, a strain carrying an insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmrD. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene product and PmrD.

Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence.

We isolated spontaneous mutations (pmrA) in the smooth strain Salmonella typhimurium LT2 that show increased resistance to the cationic antibacterial proteins of human neutrophils and to the drug polymyxin B. The mutation in one strain, JKS5, maps to 93 min on the S. typhimurium chromosome, near the proP gene and the melAB operon. The mutation, designated pmrA505, confers a 1,000-fold increase in resistance to polymyxin B and a 2- to 4-fold increase in resistance to neutrophil proteins. We cloned both the pmrA505 and pmrA+ alleles and found that the pmrA+ gene is partially dominant over pmrA505. DNA sequence analysis of the pmrA505 clone revealed three open reading frames (ORFs). The deduced amino acid sequences indicated that ORF1 encodes a 548-amino-acid (aa) protein with a putative membrane-spanning domain and no significant homology to any known protein. ORF2 and ORF3, which encode 222- and 356-aa proteins, respectively, show strong homology with the OmpR-EnvZ family of two-component regulatory systems. ORF2 showed homology with a number of response regulators, including OmpR and PhoP, while ORF3 showed homology to histidine kinase-sensor proteins EnvZ and PhoR. Genetic analysis of the cloned genes suggested that ORF2 contained the pmrA505 mutation. Comparison of the pmrA505 and pmrA+ ORF2 DNA sequences revealed a single G-A transition, which would result in a His-to-Arg substitution at position 81 in the ORF2 mutant protein. We therefore designate ORF2 PmrA and ORF3 PmrB. The function of ORF1 is unknown.

Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes.

CAP37 (cationic antimicrobial protein of molecular mass 37 kDa) is a multifunctional protein isolated from the granules of human neutrophils. It is antibiotic and chemotactic and binds lipopolysaccharide. A synthetic peptide, amino acid sequence NQGRHFCGGALIHARFVMTAASCFQ, based on residues 20-44 of CAP37 protein mimics its antibiotic and lipopolysaccharide binding action. Peptide 20-44, at the concentrations tested, has antibacterial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. The bactericidal action of the peptide was pH dependent, with maximum activity at pH 5.0 and pH 5.5 and decreased activity at pH 7.0. Various truncations, substitutions, and other modifications in the sequence deteriorate its activity. Free sulfhydryl groups and/or disulfide bridge formation are required for optimum antibiotic activity, since substitution of serines for, or alkylation of, cysteine residues 26 and 42 eliminates bactericidal activity. Evidently amino acids 20-44 represent an important, perhaps principal, antibacterial domain of CAP37. This peptide should provide new insight into the mechanism of antimicrobial activity of CAP37 and may serve as a model for new, useful, synthetic antibiotics.

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes.

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.

Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, lktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt- strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lktCABD and lktCA. respectively. Lkt- strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the lkt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment.

Human neutrophil granule cationic protein CAP37 is a specific macrophage chemotaxin that shares homology with inflammatory proteinases.

Cationic antimicrobial protein CAP37 (Mr = 37 kD) is derived from the azurophilic granules of human PMN. In vitro and in vivo studies demonstrate that CAP37 is a novel monocyte-specific chemoattractant. The N-terminal amino acid sequence of CAP37 shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, substitutions in the catalytic triad (serine for a histidine at position 41 and glycine for a serine at position 175), may account for its lack of serine protease activity. A full length cDNA for CAP37 was identified in an HL60 cDNA library screened with oligonucleotide probes designed from the N-terminal amino acid sequence. Sequencing of the cDNA reveals a protein of 225 amino acids with significant nucleotide homology to cathepsin G and human neutrophil elastase.

Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase.

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte-specific chemotactic activity. CAP37 is a single-chain protein consisting of 222 amino acid residues. It has three N-glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn-114 alone, others at Asn-114 and Asn-110 or Asn-145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30-37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity.

CAP37, an antimicrobial protein of human neutrophil granules, is a specific chemoattractant for monocytes. Purified to homogeneity by sequential chromatography over carboxymethyl Sephadex, G-75 Sephadex, and hydrophobic interaction HPLC, demonstratively endotoxin-free CAP37 was maximally chemotactic over a range of 1.3 X 10(-9)-10(-8) M. Thus it was active in the same molar concentrations as formyl-methionyl-leucyl-phenylalanine. CAP37 lacked chemotactic activity for neutrophils and lymphocytes. In checkerboard assays CAP37 had some chemokinetic activity as well. It was also chemotactic for rabbit mononuclear cells. Higher concentrations (2.7 X 10(-8) M) were required for activity with rabbit cells than with human. Sequence analysis of the first 42 NH2-terminal amino acid residues of CAP37 showed strong homologies with known serine proteases that mediate various functions in inflammation. However, a critical substitution of a serine for a histidine at position 41 suggested that CAP37 lacked serine protease action. This impression was supported by the failure of CAP37 to bind tritiated diisopropyl fluorophosphate. 89% of total CAP37 was released extracellularly from human neutrophils while they phagocytized Staphylococcus aureus. We propose that CAP37 released from neutrophils during phagocytosis and degranulation may mediate recruitment of monocytes in the second wave of inflammation.

A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils.

We have isolated a Salmonella typhimurium (ST) mutant, JKS400, deficient in the production of a surface-exposed outer membrane protein (Omp) and phenotypically hypersensitive to the oxidative antimicrobial mechanism of polymorphonuclear leukocytes (PMNs). This Omp migrated at approximately 59 kiloDaltons (kD) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We found with P22 transduction that the capacities to produce the protein and to exert wild-type resistance to oxidative killing were tightly linked. Transduction of JKS400 with a P22(HT)int- bacteriophage grown on a Tn10 insertion library in LT2 yielded tetracycline-resistant isolates that had been returned to wild-type protein production. Further experiments showed that restoration of protein production was accompanied by restoration of the parental resistance phenotype to killing by PMNs and by restoration to wild-type resistance to H2O2. The map position of the Tn10 was determined to be at 96 minutes in the Salmonella chromosome. This protein appears to behave as a virulence factor, promoting the capacity of Salmonella typhimurium LT2 to survive oxygen-dependent killing mechanisms in neutrophils.

CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases.

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.

Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans.

A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.