The PBP 5 synthesis repressor (psr) gene of Enterococcus hirae ATCC 9790 is substantially longer than previously reported.

A reexamination of the nucleotide sequence of the psr gene of Enterococcus hirae revealed the presence of two additional nucleotides at residues 1190 and 1191. As a result, instead of a stop codon after 148 aa, the psr gene product would contain 293 aa residues. The revised size of the gene product was confirmed by subsequently cloning and expressing the psr gene in Escherichia coli. The derived amino acid sequence of the revised psr gene product was found to be similar to several other proteins in the combined GenBank/EMBL database. The protein products of some of these genes are thought to play regulatory role(s) in exo or capsular polysaccharide synthesis and/or in cell wall metabolism. All the putative homologs of the revised Psr appear to have a putative membrane-anchoring domain at their N-termini. Amino acid blocks with high degrees of similarity have been identified in the aligned sequences, and it is suggested that these common motifs could be of structural or functional importance.

Borderline methicillin-susceptible Staphylococcus aureus strains have more in common than reduced susceptibility to penicillinase-resistant penicillins.

Ten epidemiologically unrelated Staphylococcus aureus isolates with borderline levels of susceptibility to antistaphylococcal penicillinase-resistant penicillins (PRPs) were investigated together with appropriate S. aureus control strains. By a nitrocefin microplate assay, all borderline PRP-susceptible test strains were found to produce comparable amounts of beta-lactamase. Hydrolytic activity against another chromogenic substrate (PADAC) and against the PRPs was also demonstrated in membrane preparations from induced cells of 9 of the 10 borderline test strains. When bacterial membranes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two methicillin-inducible bands of about 32 and 31 kDa were detected, after Coomassie blue staining, in the membrane protein patterns of the same nine borderline test strains. By gel renaturation and zymographic detection of beta-lactamase activity, both methicillin-inducible membrane proteins were detected with nitrocefin as a substrate, whereas only one band (presumably the smaller protein) was detected with PADAC. With the remaining borderline test strain (a40), only the larger band was detected in the renatured gels with nitrocefin as a substrate. Plasmid DNA analysis revealed that the borderline susceptible test strains, again with the exception of a40, shared a 17.2-kb plasmid yielding four HindIII fragments of 7.0, 5.3, 3.5, and 1.4 kb. In Western blot (immunoblot) experiments using rabbit antiserum to penicillin-binding protein (PBP) 2a, test strain a40, which did not share a number of features characteristically associated with the other borderline test strains, was eventually shown to produce PBP 2a. Five other S. aureus strains, belonging to phage group 94/96, were found to display the borderline phenotype, including such distinguishing features as the membrane-associated PRP- and PADAC-hydrolyzing activity, the two methicillin-inducible membrane proteins, and the 17.2-kb plasmid. These results suggest that borderline susceptible S. aureus strains share more common features than reduced susceptibility to PRPs.

Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties.

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.

Borderline susceptibility to methicillin in Staphylococcus aureus: a new mechanism of resistance?

Staphylococcus aureus strains with borderline levels of susceptibility or resistance to antistaphylococcal penicillinase-resistant penicillins (PRPs) were initially reported as neither heteroresistant nor multiply resistant organisms, producing large amounts of beta-lactamase, and becoming fully susceptible to PRPs in the presence of beta-lactamase inhibitors. This borderline susceptibility or low-level resistance was suggested to be due to beta-lactamase hyperproduction: according to this hypothesis, the staphylococcal beta-lactamase, when hyperproduced, would succeed in partially hydrolyzing methicillin and related PRPs. However, later studies demonstrated that borderline PRP susceptibility cannot be explained soley on this basis, beta-lactamase hyperproduction being neither sufficient nor necessary to determine the borderline phenotype. Intrinsic mechanisms have also been reported to be associated with some borderline PRP susceptible S. aureus strains. The more recent discovery of a PRP-hydrolyzing beta-lactamase (methicillinase) produced, in addition to the conventional penicillinase, by borderline S. aureus strains suggests that this second, more specific beta-lactamase is more likely to be responsible for the borderline phenotype than an increased amount of the penicillinase. The slow kinetics of PRP hydrolysis by methicillinase is consistent with its association with reduced susceptibility rather than true resistance to PRPs. The combined effect of methicillinase plus penicillinase on some common substrates might explain the increased beta-lactamase activity often observed in borderline S. aureus strains.

Bacterial walls, peptidoglycan hydrolases, autolysins, and autolysis.

Knowledge of the chemistry, ultrastructure, biosynthesis, assembly, and function of bacterial cell walls has expanded enormously since the opening of this field of research approximately 40 years ago, primarily by the early work of Milton Salton. It has become abundantly clear that, in most environments, walls are essential to the survival and growth of bacteria and in many ways are structurally and functionally unique. A common but not universal feature of bacterial walls is the presence of peptidoglycan (PG; murein, or in the case of certain Archae the analogous structure-pseudomurein). PGs are considered to be primarily responsible for the protective and shape-maintaining properties of walls. They are a biologically unique class of macro-molecules in that they are not linear or even branched macromolecules. Instead they are two- or three-dimensional net like polymers that are linked together by three different chemical bonds (glycosidic, amide, and peptide). In addition, they contain the D-isomers of some amino acids and therefore may possess DL, LD, and DD linkages. Furthermore, the exact chemical structure of a PG may vary depending on environmental factors, however, retaining the essential protective and shape maintaining properties of the wall. Thus, the overall architectural plan of the wall may be more important than the exact shape of the bricks used for the construct. Another somewhat unique feature of PGs (and walls) is their final assembly in situ on the outside of the cellular permeability barrier. A broad variety of bacteria have been shown to possess enzymes that can hydrolyze bonds in the wall PG. Hydrolysis of a sufficient number of bonds can result in the weakening of, or serious damage to, the protective properties of the PG. Frequently, a bacterial strain may possess more than one PG hydrolase activity. A commonly believed, but as yet unproven, hypothesis is that PG hydrolases play one or more roles in PG assembly and/or surface growth and cell division. At a minimum, such potentially suicidal activities must be exquisitely well regulated. Currently we know little concerning the regulation of these activities, or how they communicate with, and integrate with, chromosome replication, synthesis of cytoplasmic macromolecules, cell growth, and division, although such, probably two-way, communications must occur in growing and dividing cells. Recent data indicate that the psr element in Enterococcus hirae described by Fontana and collaborators as a genetic element that is involved in the regulation of the synthesis of PBP 5, also is involved in the regulation of several other surface properties. These properties include (1) autolysis rates of exponential phase. cells, (2) the retention of this property after cells enter the stationary phase, (3) lysozyme sensitivity, and (4) the ratio of rhamnose-containing wall polysaccharide to PG in the walls. Thus the psr element may be a part of a "global" regulation and communication system in E. hirae.

Penicillin resistance and autolysis in enterococci.

Comparison of several cell wall-related properties of the ATCC 9790 strain and the R40 strain, a penicillin-resistant, PBP5 overproducing strain, and Rev14, a penicillin-hypersensitive, PBP5-deficient strain, is consistent with a role of the genetic element, psr, in the global regulation of lysozyme sensitivity, autolytic capacity, and wall-rhamnose-containing polysaccharide content. These parameters appear to be independently regulated by a system that involves psr in a currently unknown manner.

Two-dimensional gel electrophoresis and immunoblotting of human serum albumin modified by reaction with penicillins.

A two-dimensional gel electrophoresis and immunoblotting procedure has been developed to assess the level of modification by penicillins in human serum albumin. The procedure can be used in in vitro experiments and in clinical studies with sera from patients treated with penicillins.

Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli.

The blaZ gene contained in a single 17.2-kb beta-lactamase plasmid from a borderline methicillin-susceptible Staphylococcus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAH12) containing two different staphylococcal HindIII inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 micrograms ml-1, independently of the inoculum size used, and produced large amounts of beta-lactamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the beta-lactamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different beta-lactamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin.

In-vitro evaluation of cefpodoxime.

In-vitro antimicrobial activity of cefpodoxime was evaluated against several microbial species by both conventional tests and additional parameters which take into consideration some of the conditions likely to be encountered in infected tissues. MICs for 414 recent clinical isolates, including staphylococci, streptococci, Haemophilus influenzae, Moraxella catarrhalis, several Enterobacteriaceae, Aeromonas hydrophila and Campylobacter jejuni were determined. MIC values overall were similar to those observed for strains from other geographical areas. Inhibition of growth by cefpodoxime was virtually unaffected by the inoculum size, even using bacterial populations as large as 10(9) cfu of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, H. influenzae, beta-lactamase-negative M. catarrhalis, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Growth in the presence of human serum as the only source of nutrients did not significantly affect the inhibition exerted by cefpodoxime, even against large bacterial populations of S. aureus, S. pneumoniae, E. coli, and K. pneumoniae. For K. pneumoniae, E. coli, P. mirabilis and beta-lactamase-negative M. catarrhalis it was also found that sub-MIC concentrations of cefpodoxime were still able to inhibit the majority of cells in microbial populations as large as 10(9) cfu. Evaluation of bactericidal activity demonstrated that cefpodoxime concentrations comparable to those achievable in plasma or in the respiratory tract were able to kill rapidly large bacterial populations of S. pneumoniae and S. pyogenes. The bactericidal activity was apparently lower against M. catarrhalis, H. influenzae, S. aureus, E. coli, and K. pneumoniae.

High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance.

The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams.

Evidence for a methicillin-hydrolysing beta-lactamase in Staphylococcus aureus strains with borderline susceptibility to this drug.

A new beta-lactamase that hydrolyses methicillin was found in the membrane fraction of two clinical isolates of Staphylococcus aureus with borderline susceptibility to this drug. 'Methicillinase' activity was detected in renatured sodium dodecyl sulfate polyacrylamide gel electrophoretograms of staphylococcal membrane proteins. The enzyme activity appeared to be inducible and was more easily detected using penicillin G (or methicillin) rather than nitrocefin as substrate. Similar activity was not detected in the membrane fraction of a methicillin-susceptible strain. These results suggest that, in the two borderline susceptible strains, rather than a hyperproduction of the penicillinase a specific methicillin-hydrolysing activity is responsible for the borderline susceptible phenotype.

The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases.

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.

The mechanism of staphylococci resistance to methicillin: a critical analysis of dominant opinions.

Resistance to methicillin has been reported to be constantly associated with production of a novel penicillin-binding protein (PBP), indicated as PBP2a. This PBP is always present in strains that are homogeneously resistant to the drug, but is also constantly present in the strains heterogeneously resistant, where by far the vast majority of the cells are indeed sensitive to the antibiotic. This apparent lack of correlation between the presence of PBP2a and methicillin resistance has led to the idea, shared by most scientists, that the presence of PBP2a alone cannot explain resistance of staphylococci to methicillin. This confusion has implications also for clinical microbiology. In fact in in-vitro assays, not rarely, strains that are resistant to methicillin appear sensitive to other beta-lactams. However, clinical experience has demonstrated that when infections caused by methicillin-resistant staphylococci, which, in in vitro assays, become sensitive to other beta-lactams, are treated with the latter antibiotics, the incidence of failures is higher than that observed when the same antibiotics are used to treat infections caused by staphylococci sensitive to methicillin. Also in consideration of the fact that the mechanism of resistance to methicillin is not yet understood, many authors recommend to consider methicillin-resistant staphylococci as being in-vivo resistant also to the beta-lactams to which they may become sensitive in in-vitro assays. In the opinion of the authors, the mechanism of staphylococcal resistance to methicillin is reasonably explainable at least in its fundamental aspects. The present dominant opinions concerning methicillin-resistant staphylococci will be critically analyzed on the basis both of data presented in the literature and of data obtained in the authors' laboratory.

Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.