Penetration of β-lactamase inhibitors into the periplasm of Gram-negative bacteria

The effectiveness of a beta-lactamase inhibitor/beta-lactam combination against Gram-negative pathogens depends on many interplaying factors, one of which is the penetration of the inhibitor across the outer membrane. In this work we have measured the relative penetrations of clavulanic acid, sulbactam, tazobactam and BRL 42715 into two strains of Escherichia coli producing TEM-1 beta-lactamase, two strains of Klebsiella pneumoniae producing either TEM-1 or K-1, and two strains of Enterobacter cloacae each producing a Class C beta-lactamase. It was shown that clavulanic acid penetrated the outer membranes of all these strains more readily than the other beta-lactamase inhibitors. For the strains of E. coli and K. pneumoniae clavulanic acid penetrated approximately 6 to 19 times more effectively than tazobactam, 2 to 9 times more effectively than sulbactam and 4 to 25 times more effectively than BRL 42715. The superior penetration of clavulanic acid observed in this study is likely to contribute to the efficacy of clavulanic acid/beta-lactam combinations in combating beta-lactam resistant bacterial pathogens.     

A numerical taxonomic study of non-motile non-fermentative Gram-negative bacteria from foods

A numerical taxonomic study using 155 unit characters was performed on 63 strains of Gram-negative non-motile non-fermentative bacteria isolated from proteinaceous foods. Similar bacteria from other sources and several Pseudomonas strains from meat were included for reference purposes. Three clusters were observed at 76% S which contained all the food strains. Cluster 1 was composed entirely of Acinetobacter strains including 17 isolated from foods that were provisionally identified with Acinetobacter johnsonii. Cluster 2 contained 22 strains identified as Psychrobacter immobilis , of which 20 were from food. Cluster 3 contained all the Pseudomonas reference strains and 26 non-motile strains isolated from meat. These were shown to be non-motile variants of Pseudomonas fragi. A simple identification scheme, based on five tests, is presented for the distinction of the three types of bacteria.     

Production of 5α- and 5β-dihydrotestosterone by isolated human axillary bacteria

Abstract The metabolism of testosterone by coryneform bacteria in vitro has been studied. Metabolites identified after derivatization by capillary gas chromatography and further by combined gas chromatography-mass spectrometry were 17β-hydroxy-5α-androstan-3-one and 17β-hydroxy-5β-androstan-3-one. The mass spectral characteristics of the methyl oxime trimethylsilyl ethers of all the 17-hydroxy-androstan-3-one and 3-hydroxy-androstan-17-one isomers are recorded.     

Molecular characterisation by PCR-restriction fragment length polymorphism of TEM β-lactamases

Abstract To rapidly characterise TEM-derived extended-spectrum β-lactamases a fast and easy method using polymerase chain reaction-restriction fragment length polymorphism was developed. This method was validated with ten reference TEM-type extended-spectrum β-lactamases. The mutations involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised. TEM-20 differed from TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an unreported position 660, involving an amino acid substitution, arginine for histidine, at position 153. Moreover, a new extended-spectrum β-lactamase conferring low resistance to ceftazidime (TEM-29), was described. TEM-29 derived from TEM-1, with an amino acid substitution, his-164. Finally, the combination of polymerase chain reaction-restriction fragment length polymorphism and plasmid analysis allowed us to investigate nosocomial outbreaks due to clinical isolates of multi-resistant Klebsiella pneumoniae in three hospitals.     

Two novel transferable extended-spectrum β-lactamases from Klebsiella pneumoniae in Tunisia

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.     

Molecular evolution of ubiquitous β-lactamases towards extended-spectrum enzymes active against newer β-lactam antibiotics

Production of beta-lactamases, and of the plasmid-encoded TEM- and SHV-type enzymes in particular, is the most common mechanism of resistance against beta-lactam antibiotics in Gram-negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first- and second-generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally 'beta-lactamase-stable' third-generation cephalosporins or by monobactams, which fall into the substrate range of the mutant or 'extended-spectrum' beta-lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the alpha-3 helix), 162 (adjacent to the alpha-7 helix) and 235, 236 and 237 (on the beta-3 strand) are located in close proximity to the active-site cavity and are thought to open up novel enzyme-substrate interactions, involving, in particular, the oxyimino moieties of the newer beta-lactam compounds.     

Biofilm formation and cell-to-cell signalling in Gram-negative bacteria isolated from a food processing environment

AIMS: To investigate the biofilm-forming capacity and the production of quorum signals in Gram-negative bacteria isolated from a food production environment, and the possible correlation between both phenotypes. METHODS AND RESULTS: Sixty-eight Gram-negative bacteria were isolated from equipment and working surfaces in a raw vegetable processing line, and tested for biofilm-forming capacity using an in vitro microplate assay. All isolates showed significantly higher biofilm-forming capacity than Escherichia coli laboratory strain DH5alpha, which was included as a negative control, and differed up to 56-fold in relative biofilm-forming capacity. Various assays based on reporter bacteria were used to detect quorum signals produced by the isolates. Twenty-six isolates produced autoinducer-2, five isolates produced N-acyl-homoserine lactones (AHLs), and none produced the Pseudomonas quinolone signal. CONCLUSIONS: No correlation was found between in vitro biofilm-forming capacity and production of quorum signalling molecules among the 68 strains isolated from the raw vegetable processing line. SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent studies have shown a role of AHL-based quorum sensing in biofilm formation of specific Gram-negative bacterial strains. The current work shows that production of AHL and other quorum signals is not widespread in Gram-negative isolates from a raw vegetable processing line, and is not a general requirement for biofilm formation, at least in vitro.     

Characterization of regulatory element Rp3 of regulation of β-lactamases from Ralstonia pickettii

The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many other aspects of bacterial physiology. Although it is known that the sensor kinases RetS and LadS feed into the GacS/GacA system, the mechanism through which this occurs is unknown, as are the protein–protein interactions in this system. To characterize and define these interactions, we utilized a bacterial two-hybrid system to study the interactions of GacS and GacA from Pseudomonas fluorescens CHA0. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in vivo was investigated. We found that the entire GacA molecule is required for GacA to interact with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. In addition, homologs of Pseudomonas aeruginosa hybrid sensor kinases, RetS and LadS, were identified in P. fluorescens , and shown to interact with GacS, but not GacA.     

Influence of various bile salts on ββ-glucuronidase activity of intestinal bacteria

While the decrease of the β-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.     

Sensitivity of Escherichia coli to various β-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic β-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, β-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic β-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the β-lactamases have been studied and their significance in creating β-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of β-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC β-lactamase, or a plasmid-coded TEM-type β-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter,‘target access Index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Biochemical characterization of the carbapenem-hydrolyzing β-lactamase AsbM1 from Aeromonas sobria AER 14M: a member of a novel subgroup of metallo-β-lactamases

Abstract AsbM1, a carbapenem-hydrolyzing β-lactamase produced by Aeromonas sobria AER 14M, was purified chromatographically, with anion exchange chromatography performed in the absence of Zn²⁺. The molecular mass of AsbM1 was approximately 34000; the isoelectric point was 9.1. AsbM1 had high hydrolytic specificity for carbapenems but low hydrolysis rates for penicillins and cephalosporins. AsbM1 was resistant to the commercially available β-lactamase inhibitors but was inhibited by pCMB and the chelators EDTA and o -phenanthroline. Zinc, an activator for many metallo-β-lactamases, inhibited AsbM1 with an IC₅₀ of 8 μM. Analysis of the N-terminal sequence (27 amino acids) showed 26% similarity to the CphA metallo-β-lactamase. Because of the high specificity for carbapenems and the sensitivity to inhibition by Zn²⁺, AsbM1 should be included in a new subgroup of metallo-β-lactamases.     

Separation of plasmid-mediated extended spectrum β-lactamases by fast protein liquid chromatography (FPLC system)

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.     

Molecular evolution of class A β-lactamases: phylogeny and patterns of sequence conservation

We present a multiple alignment of the amino acid sequences of eight class A beta-lactamases and utilized it to propose a phylogeny, based on the nucleotide sequences of their corresponding genes. We have also used the alignment, together with the alpha-carbon co-ordinates of the Staphylococcus aureus protein, to search systematically for neighbouring residues that share the same pattern of conservation among the different members of the protein family. The distribution of invariant residues and of groups of residues with co-ordinate changes map, predominantly, at the region of the active site and at interfaces between structural elements, respectively. We have also contrasted the distribution of conserved residues with the positions which are known to differ in mutants and variants of class A beta-lactamases.     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Production of chitinase and β-N-acetylglucosaminidase by intestinal bacteria of Pinnipedian animals

The chitinase- and β-N-acetylglucosaminidase(GlcNAcase)-producing ability of intestinal bacteria from Pinnipedian animals was determined using fluorogenic 4-methylumbelliferone glycosides of N-acetylglucosamine oligosaccharides. Intestinal microflora of a single Cape fur seal, three California sea lions and three South American sea lions were characterized by a predominance of isolates of the Bacteroidaceae and Enterobacteriaceae families and the genus Clostridium . Of the 711 isolates tested 26.0, 10.0 and 8.7% could hydrolyse 4-MU(GlcNAc)₁ 4-MU(GlcNAc)₂, and 4-MU(GlcNAc)₃, respectively. This result suggests that β-GlcNAcase producers occur at a higher density than do chitinase producers. Moreover, β-GlcNAcase, and to a lesser degree, chitinase seem to be efficiently produced by facultative anaerobes in the Cape fur seal and the California sea lion, and by both facultative and obligate anaerobes in the South American sea lion. To our knowledge, this is the first paper to report that isolates of the family Bacteroidaceae and the genus Streptococcus produce chitinase and/or β-GlcNAcase.