Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49.

By repeating the cycle of mutagenesis and selection, the Escherichia coli dnaQ49 mutator acquired high level resistance to ampicillin (30,000 micrograms ml-1), streptomycin (26,000 micrograms ml-1) and ofloxacin (3000 micrograms ml-1). Under the strong pressure of ofloxacin, dnaQ49 also followed the history of mutations in the gyrase and topoisomerase i.v. genes previously observed in clinical isolates of quinolone-resistant E. coli. The results of these in vitro experiments suggest that naturally existing mutators may participate in the rapid acquisition of resistance to various antibiotics in patients. A possible mechanism for the occurrence of this adaptability is discussed with special reference to the property of mutagenesis accompanying DNA replication.

Tazobactam is a potent inactivator of selected inhibitor-resistant class A beta-lactamases.

The beta-lactam beta-lactamase inhibitor combinations (ampicillin/clavulanate, ampicillin/sulbactam, ticarcillin/clavulanate and piperacillin/tazobactam) were tested against selected inhibitor-resistant class A beta-lactamases of the TEM and OHIO-1 varieties. Minimum inhibitor concentrations (MIC) revealed that the Escherichia coli DH5 alpha transconjugate strains that possessed the OHIO-1 beta-lactamase, Met69Ile mutant of the OHIO-1 beta-lactamase, TEM-30 (Arg244Ser) and TEM-31 (Arg244Cys) beta-lactamase were most susceptible to piperacillin and piperacillin/tazobactam. Kinetic experiments with each beta-lactamase demonstrated that tazobactam was 10-25-fold more potent than clavulanate or sulbactam against TEM-30 and TEM-31 beta-lactamase. Tazobactam was also as effective as clavulanate against the Met69Ile mutant of the OHIO-1 beta-lactamase. Among the clinically available beta-lactams and beta-lactamase inhibitors, piperacillin/tazobactam appears to be the most potent combination against selected inhibitor-resistant strains of TEM and OHIO-1 beta-lactamase.

Inhibition of ampicillin-induced emergence of resistance in intestinal coliforms by targeted recombinant beta-lactamase.

The aim of the present study was to determine whether oral targeted recombinant beta-lactamase (TRBL) administration could overcome the development of ampicillin-induced resistance in the gut microbiota. Eighteen laboratory beagles with permanent jejunal fistula were randomised to receive ampicillin + placebo, ampicillin + TRBL or placebo. A total of 982 coliform isolates, collected from jejunal and faecal samples before, during and after the treatment were tested against nine antimicrobials. The proportion of ampicillin resistance (multi-resistance) among coliform isolates increased from 20 to 36% in the ampicillin + placebo group but far less, 20-36%, in the ampicillin + TRBL group. These results indicate that TRBL may prevent the emergence of beta-lactam-associated resistance in coliforms in the gut.

[Characterization of Haemophilus influenzae's resistance to ampicillin]. / Caracterización de la resistencia de Haemophilus influenzae a la ampicilina.

BACKGROUND: The resistance of Haemophilus influenzae to ampicillin is nearly always dependent on plasmid-mediated betalactamase production. The betalactamase type most frequently identified was TEM-1; type ROB has occasionally been identified. The presence of plasmid-mediated betalactamase has been studied in H. influenzae strains moderately susceptible or resistant to ampicillin. METHODS: The test used in the detection of the presence of betalactamase was nitrocefin hydrolysis; the enzyme type was identified by analytic isoelectric focusing; the codifying gene of this betalactamase was identified by hybridisation with a TEM-1 probe labeled with digoxigenine. Strains studied were 110, obtained during the period 1987 to 1989 and isolated from four Hospitals of the Vallés area of Barcelona. RESULTS: Enzyme production was detected in 105 of 110 strains studied; TEM-1 type betalactamase was identified in 108 and positive hybridisation was obtained in these strains with the TEM-1 probe. CONCLUSIONS: Nitrocefin hydrolysis is a reliable test for the detection of betalactamase production, although it may be unable to detect it (falses negatives) when the production is low. The only betalactamase type identified was TEM-1. The frequency of the strains with non-enzymatic resistance was 1.8%.

Multidrug resistant typhoid fever: therapeutic considerations.

Forty six blood culture positive cases were studied during the current outbreak of multidrug resistant typhoid fever (MRTF). The present outbreak was caused by E1 phage type and organisms were resistant to all commonly used drugs for the treatment of typhoid fever, viz., chloramphenicol (78%), co-trimoxazole (76%) and ampicillin (68%). Treatment failures with chloramphenicol (45.5%) corroborated well with in vitro resistance. No treatment failure was seen with chloramphenicol and ceftriaxone, when these drugs were used in cases infected with sensitive strains. Among the alternative drugs used in cases with in vitro sensitivity, successful clinical response was seen with ceftriaxone (4/4) and cefotaxime (8/9) as compared to cephalexin (3/5) or a combination of cephalexin and furazolidone (9/12).

[Study of the inactivation of antibiotics by bacterial colonies]. / Opredelenie inaktivatsii antibiotikov koloniiami bakterii.

A procedure of bacteria application to disks from the colonies was used for determining antibiotic inactivation in the disks by the bacteria colonies after the disk direct contact with the colonies. Changes in the antibiotic activity in the disks were registered after incubation at 37 degrees C for 2 hours. It was shown that ampicillin resistant strains of E. coli K12 carrying R plasmids and strains of S. typhimurium and S. aureus inactivated the antibiotics in the disks and their population were homogenous in this respect. It is advisable to use the procedure in assaying drug resistance of bacterial populations.

Characterization of clinical isolates of beta-lactamase-negative, highly ampicillin-resistant Enterococcus faecalis.

We analyzed the penicillin-binding protein (PBP) profiles of two clinical isolates of Enterococcus faecalis for which ampicillin MICs were 32 and 64 micrograms/ml. Six PBPs were detected in both isolates, demonstrating an apparently increased amount of PBP 5 and decreased penicillin binding of PBPs 1 and 6. These results suggest that ampicillin resistance in the clinical isolates of E. faecalis described could be associated with alterations in different PBPs.

Ampicillin resistance of Shigella sonnei.

The ampicillin resistance of clinical isolates of Shigella sonnei is due to beta-lactamase production. Two kinds of resistance are found: low level, nontransmissible; and high level, tranmissible. The nontransmissible type of resistance results from a chromosomal mutation which increases the production of a beta-lactamase that hydrolyses cephalosporins relatively rapidly and gives cephalothin resistance. The transmissible type of resistance is due to an R factor mediating the synthesis of a different beta-lactamase that does not significantly hydrolyze cephalosporins or confer cephalothin resistance. One clinical isolate is shown to possess simultaneously both these mechanisms of ampicillin resistance.

Identification of beta-lactamase-negative, ampicillin-resistant strains of Haemophilus influenzae with four methods and eight media.

A challenge set of 143 non-beta-lactamase-producing strains of Haemophilus influenzae was tested for ampicillin susceptibility on two broth media and six agar media, using broth microdilution, agar dilution, disk diffusion, and E-test procedures. When beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were defined as those for which the ampicillin MIC was > or = 4.0 microg/ml, 5 to 44% of our selected strains were BLNAR depending on the medium and/or test method used. If nonsusceptible strains for which ampicillin MICs were intermediate were included in the BLNAR category, 32 to 50% of our isolates would be considered BLNAR. These data emphasize the need for a standardized testing procedure and a universal definition of BLNAR strains before the clinical relevance of such strains can be evaluated. NCCLS dilution tests with haemophilus test medium broth or agar are preferred for testing ampicillin against H. influenzae.

Susceptibilities of beta-lactamase-producing and -nonproducing ampicillin-resistant strains of Haemophilus influenzae to ceftibuten, cefaclor, cefuroxime, cefixime, cefotaxime, and amoxicillin-clavulanic acid.

In in vitro studies we evaluated the susceptibilities of beta-lactamase-producing and -nonproducing, ampicillin-resistant strains of Haemophilus influenzae and compared them with those of ampicillin-susceptible strains. Ampicillin, amoxicillin-clavulanic acid, ceftibuten, cefaclor, cefuroxime, cefixime, and cefotaxime were evaluated by broth microdilution tests and disk diffusion tests. The disk diffusion tests accurately categorized beta-lactamase-producing strains and ampicillin-susceptible strains as being susceptible to the study drugs other than ampicillin. Ampicillin-resistant, beta-lactamase-nonproducing strains were relatively resistant to all seven study drugs, but the disk diffusion test did not always predict that resistance. The clinical relevance of the decreased susceptibility to various agents remains unclear, but to be conservative, all ampicillin-resistant, beta-lactamase-nonproducing strains might be assumed to be resistant to other beta-lactams. After excluding that small group of isolates, reliable susceptibility test results were obtained with lots of Haemophilus Test Medium that met quality assurance criteria.

In vivo control of promoter and terminator efficiencies at a distance.

In pBR329, the genes providing resistance to ampicillin (beta-lactamase, bla) and chloramphenicol (chloramphenicol acetyl transferase, cat) are encoded on the same strand. The bla gene lies downstream of the cat gene, separated by an intergenic sequence of 414 bp. The transcription starts of the two genes are 1090 bp apart. We have probed, in vivo, the effect on transcription of the bla gene, of the introduction, in front of the cat gene, of a series of synthetic promoters covering a large (over 60-fold) range of efficiency. The rising efficiency of the cat promoter has several important consequences for transcription of the bla gene. First, a strong (up to sevenfold) stimulation of the bla promoter is observed, together with a shift of the main bla transcription start site, 10 bp upstream. Furthermore, the relative efficiencies of the bla transcription terminators are reduced. Finally, because of a lesser relative efficiency of the cat transcription terminators as well, we observe enhanced intrusion into the bla gene of transcripts initiated at the cat promoter, some of them extending to the bla transcription terminator and beyond. The operon-like expression of the cat-bla gene tandem is controlled by the efficiency of the cat terminator, which in turn depends on that of the cat promoter. This demonstrates a direct link between the efficiencies of promoter and terminator. Upon inhibition of bacterial gyrase activity, i.e. relaxation of negative supercoiling action, bla expression increases sharply in pBR329, but remains almost unchanged in a plasmid (pBRGC-1) in which cat is under the control of a 6.5-fold stronger promoter. Therefore, under normal gyrase activity, the stimulation of the bla promoter in pBRGC-1 (relative to pBR329) appears to be linked to topological relaxation of its template in situ, in keeping with earlier in vitro observations. We propose that the relaxed state of pBRGC-1 in situ could be due to the decrease in the plasmid linking number, introduced by the 10-12 RNA polymerases that simultaneously transcribe the cat gene in that plasmid, compared with only one or two in pBR329. We find that the negative superhelical densities of both plasmids are almost identical when extracted from the cell. Therefore gyrase would not correct for the relaxed state of plasmid pBRGC-1 observed in situ.

Role of penicillin-binding protein 5 in expression of ampicillin resistance and peptidoglycan structure in Enterococcus faecium.

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired beta-lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 microg/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 microg/ml), D344 (MIC, 24 microg/ml), and H80721 (MIC, 512 microg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 microg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.

Novel intercellular communication system in Escherichia coli that confers antibiotic resistance between physically separated populations.

AIMS: To determine whether intercellular signalling can occur between physically separated populations of Escherichia coli. METHODS AND RESULTS: Intercellular signalling between physically discrete populations of E. coli BL21 was analysed in bi-partite Petri dishes. Transfer of a growth-promoting signal resulted in induction of resistance to the antibiotic ampicillin. Optimal expression of the signal occurred when the signalling population was established as a bacterial lawn for 24 h. This represented an entry into the stationary phase of growth, as indicated by the expression profile of the RNA polymerase subunit sigma38 (sigmaS; sigma S). The growth-promoting effect was also observed when E. coli DH5alpha (luxS-) was used as the signalling population. Preventing passage of air between the two populations resulted in a complete cessation of the growth-promoting effect. CONCLUSIONS: A growth-promoting signal occurs between physically separated cultures of E. coli. The exact nature of the signal remains to be determined but does not involve the production of autoinducer-2 from the luxS gene. Signal transmission is likely to involve airborne transfer of a signal species. SIGNIFICANCE AND IMPACT OF THE STUDY: Intercellular signalling systems exist in bacteria that enable antibiotic resistance to be conferred between physically separated populations.

Membrane topology of penicillin-binding protein 3 of Escherichia coli.

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.

Ampicillin-resistant enterococcal species in an acute-care hospital.

A prospective review of all enterococcal isolates for 13 months showed that 9.0% were resistant to ampicillin (MIC, greater than or equal to 16 micrograms/ml; zone diameter, less than 15 mm), as determined by the Vitek system, disk diffusion, microdilution MIC testing, and macrodilution MIC testing. All were beta-lactamase negative. A total of 19 and 3 resistant isolates were from urine and intravascular sites, respectively. Ampicillin-resistant enterococci appear to be a growing clinical problem.

Antimicrobial resistance in Haemophilus influenzae isolated from blood, cerebrospinal fluid, middle ear fluid and throat samples of children. A nationwide study in Finland in 1988-1990.

A nation-wide survey of the prevalence of antimicrobial resistance in Haemophilus influenzae was conducted on isolates collected in 1988-90 from middle ear fluid (MEF), blood, or cerebrospinal fluid (CSF) in infected children or throat samples of healthy children. Altogether 885 strains were examined regarding capsular type b, beta-lactamase production and the minimal inhibitory concentration (MIC) of ampicillin, cefaclor, erythromycin, tetracycline, chloramphenicol, trimethoprim and trimethoprim-sulfamethoxazole for these strains was determined by the agar dilution method. 99% (578/585) of MEF isolates, 93% (112/121) of throat isolates, but only 6% (10/179) of blood/CSF isolates were not of type b (Hib). The rate of beta-lactamase production was 11.4% among Hib strains, 8.0% among non-type b MEF isolates, and 4.5% among non-type b throat isolates. No increase in the prevalence of beta-lactamase production in H. influenzae has taken place in Finland since the early 1980s. Resistance to ampicillin among strains that lacked beta-lactamase activity was rare (0.2%). Of the non-type b MEF and throat isolates, 5.9% and 2.7%, respectively, were resistant to trimethoprim and 3.6% and 2.7%, respectively, to trimethoprim-sulfamethoxazole. Resistance to other drugs was rare (< 2%) in all isolate groups.

Orally administered targeted recombinant Beta-lactamase prevents ampicillin-induced selective pressure on the gut microbiota: a novel approach to reducing antimicrobial resistance.

Antibiotics that are excreted into the intestinal tract promote antibiotic resistance by exerting selective pressure on the gut microbiota. Using a beagle dog model, we show that an orally administered targeted recombinant beta-lactamase enzyme eliminates the portion of parenteral ampicillin that is excreted into the small intestine, preventing ampicillin-induced changes to the fecal microbiota without affecting ampicillin levels in serum. In dogs receiving ampicillin, significant disruption of the fecal microbiota and the emergence of ampicillin-resistant Escherichia coli and TEM genes were observed, whereas in dogs treated with ampicillin in combination with an oral beta-lactamase, these did not occur. These results suggest a new strategy for reducing antimicrobial resistance in humans.