We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not of TEM or SHV lineage have recently been described. The presence of ESBLs carries tremendous clinical significance. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates. There is substantial debate as to the optimal method to prevent this occurrence. It has been proposed that cephalosporin breakpoints for the Enterobacteriaceae should be altered so that the need for ESBL detection would be obviated. At present, however, organizations such as the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) provide guidelines for the detection of ESBLs in klebsiellae and Escherichia coli. In common to all ESBL detection methods is the general principle that the activity of extended-spectrum cephalosporins against ESBL-producing organisms will be enhanced by the presence of clavulanic acid. ESBLs represent an impressive example of the ability of gram-negative bacteria to develop new antibiotic resistance mechanisms in the face of the introduction of new antimicrobial agents.

Extended-spectrum beta-lactamases: a clinical update.

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.

https://www.pnas.org/content/pnas/76/4/1648.full.pdf

Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

β-Lactamases continue to be the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum β-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum β-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated β-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these β-lactamases will present a host of therapeutic challenges as we head into the 21st century.

Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat

Publisher Summary β -lactamases are the enzymes that are capable of hydrolyzing the β -lactam bond of penicillins and cephalosporins. The reaction catalyzed by β -lactamases with penicillins as substrates includes the rupture of the β -lactam bond to form the corresponding penicilloic acid. However, there is complexity in breakdown of the cephalosporins after β -lactamase action as there is no sporin and the formation of any single product—a fact that invalidates the iodometric assay of cephalosporinase for absolute measurement. Despite the wide and varying specificity, β -lactamases often require the 4-membered azetidinone ring to be condensed either with a thiazolidine (penicillins) or a dihydrothiazine nucleus (cephalosporins). The molecular basis of the resistance of Gram-negative bacteria to β -lactam antibiotics is very different from their Gram-positive counterparts. Some of the possible physiological role recognized for β -lactamases includes—namely, (1) hydrolysis of β -lactam antibiotics when required and (2) destruction of β -lactam antibiotics. The role of protecting the organisms that produce β -lactamases against the deleterious effects of penicillins and cephalosporins can often provide a major obstacle to the successful use of β -lactam antibiotics for therapy.

The β-Lactamases of Gram-Negative Bacteria and their Possible Physiological Role

An R factor from Pseudomonas aeruginosa, which confers resistance to penicillins, kanamycin, and tetracycline, was studied in Escherichia coli K-12. The R factor could coexist with F-like or I-like plasmids and therefore constituted a novel compatibility group. The R factor was transferable from E. coli to bacterial genera outside the Enterobacteriaceae (Pseudomonas and members of the Rhizobiaceae) to which transfer of F-like and I-like plasmids could not be demonstrated.

Properties of an R Factor from Pseudomonas aeruginosa

Azthreonam (SQ 26,776) is a synthetic monocyclic beta-lactam antimicrobial agent belonging to the monobactam family (Sykes et al., Nature [London] 291:489-491, 1981), members of which are characterized by having the 2-oxoazetidine-1-sulfonic acid moiety. Azthreonam exhibits a high degree of stability to beta-lactamases and is specifically active against aerobic gram-negative bacteria, including Pseudomonas aeruginosa. Its activity against these organisms was in general equal or superior to that observed with the third-generation cephalosporins, cefotaxime and ceftazidime. Like penicillins and cephalosporins, azthreonam interacts with essential penicillin-binding proteins of gram-negative bacteria. Azthreonam protected mice against experimental infections produced by a range of gram-negative bacteria, exhibiting efficacy comparable to that of cefotaxime and ceftazidime.

Azthreonam (SQ 26,776), a synthetic monobactam specifically active against aerobic gram-negative bacteria.

A recent article1 described two novel monocyclic β-lactam antibiotics produced by bacteria. In the past few years, we have screened bacteria isolated from numerous ecosystems and habitats for their ability to produce β-lactam antibiotics, using a strain of Bacillus licheniformis which is specific for molecules containing a β-lactam and which is sensitive down to 100 ng ml−1. From over one million bacterial isolates screened, we have now identified seven related molecules containing β-lactams produced by a range of bacterial species. For this new family of monocyclic β-lactams we suggest the class name ‘monobactam’. Bacteria which produce β-lactams are, in reality, relatively common in nature.

Monocyclic beta-lactam antibiotics produced by bacteria.

RP4, an R factor derived from Pseudomonas aeruginosa S8 and specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline, could be isolated as an extrachromosomal satellite of covalently closed circular DNA (CCC-DNA) of molecular weight about 62 million and of buoyant density 1.719 g/cm3 (60% guanine plus cytosine). When RP4 was compared by hybridization with RP1, an R factor originating in another strain of P. aeruginosa, it was shown that RP4 contains most, if not all, of the base sequences of RP1, and also additional sequences not found in RP1.

Richard B. Sykes

Papers

The β-Lactamases of Gram-Negative Bacteria and their Possible Physiological Role

Properties of an R Factor from Pseudomonas aeruginosa

Azthreonam (SQ 26,776), a synthetic monobactam specifically active against aerobic gram-negative bacteria.

Monocyclic beta-lactam antibiotics produced by bacteria.

Properties of RP4, an R Factor Which Originated in Pseudomonas aeruginosa S8