Metal Resistance and Its Association With Antibiotic Resistance.

Antibiotic resistance is recognised as a major global threat to public health by the World Health Organization. Currently, several hundred thousand deaths yearly can be attributed to infections with antibiotic-resistant bacteria. The major driver for the development of antibiotic resistance is considered to be the use, misuse and overuse of antibiotics in humans and animals. Nonantibiotic compounds, such as antibacterial biocides and metals, may also contribute to the promotion of antibiotic resistance through co-selection. This may occur when resistance genes to both antibiotics and metals/biocides are co-located together in the same cell (co-resistance), or a single resistance mechanism (e.g. an efflux pump) confers resistance to both antibiotics and biocides/metals (cross-resistance), leading to co-selection of bacterial strains, or mobile genetic elements that they carry. Here, we review antimicrobial metal resistance in the context of the antibiotic resistance problem, discuss co-selection, and highlight critical knowledge gaps in our understanding.

Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus.

In previous studies, the oligo-acyl-lysyl (OAK) C12(ω7)K-ß12 added to cultures of gram-positive bacteria exerted a bacteriostatic activity that was associated with membrane depolarization, even at high concentrations. Here, we report that multidrug-resistant Staphylococcus aureus strains, unlike other gram-positive species, have reverted to the sensitive phenotype when exposed to subminimal inhibitory concentrations (sub-MICs) of the OAK, thereby increasing antibiotics potency by up to 3 orders of magnitude. Such chemosensitization was achieved using either cytoplasm or cell-wall targeting antibiotics. Moreover, eventual emergence of resistance to antibiotics was significantly delayed. Using the mouse peritonitis-sepsis model, we show that on single-dose administration of oxacillin and OAK combinations, death induced by a lethal staphylococcal infection was prevented in a synergistic manner, thereby supporting the likelihood for synergism to persist under in vivo conditions. Toward illuminating the molecular basis for these observations, we present data arguing that sub-MIC OAK interactions with the plasma membrane can inhibit proton-dependent signal transduction responsible for expression and export of resistance factors, as demonstrated for ß-lactamase and PBP2a. Collectively, the data reveal a potentially useful approach for overcoming antibiotic resistance and for preventing resistance from emerging as readily as when bacteria are exposed to an antibiotic alone.

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids.

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.

Antibacterial & antiplasmid activities of Helicteres isora L.

BACKGROUND & OBJECTIVES: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. METHODS: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. RESULTS: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 microg/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. INTERPRETATION & CONCLUSIONS: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

[Plasmid elimination effect of usnic acid on antibiotic-resistant Staphylococcus aureus].

OBJECTIVE: To observe the influence of Usnic acid on the antibiotic-resistant plasmid in Staphylococcus aureus (Sa). METHODS: The antibiotic-resistant plasmid was abstracted from the clinical divided strain of Sa and plasmid elimination test was performed in vitro. RESULTS: Plasmid elimination test showed that Usnic acid could eliminate the resistant plasmid in Sa effectively. At 24th and 48th hour after the treatment of Usnic acid, the elimination rate of resistant plasmid was 5.2% and 16.4% respectively. CONCLUSION: Usnic acid can eliminate the antibiotic-resistant plasmid in Sa. It is possible to use Usnic acid to treat the infection of antibiotic-resistant Sa in clinic.

[Influence of increased partial argon and nitrogen pressure on the R-plasmid Escherichia coli transfer frequency].

Subject of the study was frequency of transfer of bacterial plasmids determining the antibiotic resistance (R-plasmid) in argon-containing gas mixtures. Investigated were reference strains of Escherichia coli K-12. Conjugation took place during incubation in pAr/pN2/pO2, ata (1 ata = 10(5) kPa): No. 1 - 0.0/0.8/0.2 (air control); No. 2 - 1.0/0.8/0/2; No. 3 - 0.0/1.8/0.2; No. 4 - 1.0/1.8/0.2. E.coli conjugation was found inhibited after 24 hrs. of incubation with pAr elevated to 1 ata, whereas elevated partial pressure of argon (1 ata) and nitrogen (1.8 ata) brought transfer frequency down to the minimum.

Prevalence of plasmid-mediated quinolone resistance.

Quinolone resistance encoded by the qnr gene and mediated by plasmid pMG252 was discovered in a clinical strain of Klebsiella pneumoniae that was isolated in 1994 at the University of Alabama at Birmingham Medical Center. The gene codes for a protein that protects DNA gyrase from quinolone inhibition and that belongs to the pentapeptide repeat family of proteins. The prevalence of the gene has been investigated by using PCR with qnr-specific primers with a sample of more than 350 gram-negative strains that originated in 18 countries and 24 states in the United States and that included many strains with plasmid-mediated AmpC or extended spectrum beta-lactamase enzymes. qnr was found in isolates from the University of Alabama at Birmingham only during 6 months in 1994, despite the persistence of the gene for FOX-5 beta-lactamase, which is linked to qnr on pMG252. Isolates from other locations were negative for qnr. The prevalence of mcbG in the same sample was also examined. mcbG encodes another member of the pentapeptide repeat family and is involved in immunity to microcin B17, which, like quinolones, targets DNA gyrase. A single clinical isolate contained mcbG on a transmissible R plasmid. This plasmid and one carrying the complete microcin B17 operon slightly decreased sparfloxacin susceptibility but had a much less protective effect than pMG252. Plasmid-mediated quinolone resistance was thus rare in the sample examined.

[Drug resistance of enterohemorrhagic Escherichia coli O157 and a possible relation of plasmids to the drug-resistance].

Antimicrobial susceptibility was examined using 89 enterohemorrhagic Escherichia coli O157 isolates obtained from diarrhea patients in Aichi Prefecture, Japan between June 1996 and June 1997. Among the 89 isolates, 15 (16.9%) were found to be resistant to 6 of 9 antibiotics examined. These 6 antibiotics were ampicillin (ABPC), cefaloridine (CER), chloramphenicol (CP), kanamycin (KM), streptomycin (SM), and tetracycline (TC). Among the 15 drug-resistant isolates, 7 were resistant to 4 drugs (ABPC, CER, SM, TC), 3 were resistant to 3 (ABPC and 2 of CER, SM, TC), 2 were resistant to 2 (SM, TC), one each to KM or SM. Another isolate showed resistance to 5 drugs (ABPC, CP, KM, SM, TC). Selected 13 drug-sensitive and selected 12 multi-drug resistant isolates were tested for the presence of plasmids. All of the drug-sensitive isolates had 54 MDa plasmid and the majority (8/13) had 2.0 MDa plasmids, whereas; all of the drug-resistant isolates except one (1/12) had 54 MDa plasmid and the majority had 8.0 MDa (9/12) and 4.2 MDa (11/12) plasmids. The first transformation test revealed that plasmids of 8.0 MDa (3/4) and 46 MDa (1/4) were transferred to a donor cell with ABPC resistance. 54 MDa plasmid was transferred to a donor cell with both of ABPC and TC resistance. In the second transformation test, only the 8.0 MDa plasmid was confirmed to be transferred to a donor cell with ABPC resistance. Accordingly, it was indicated that the ABPC resistant gene was carried on 8.0 MDa plasmid, and it was suggested that resistant genes for ABPC and TC, and ABPC were carried on 54 MDa, and on 46 MDa plasmids, respectively.

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems.

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.

[Means of bacterial resistance]. / Moyens de défense des bactéries.

Fifty years ago, the introduction of penicillin, followed by many other antibacterial agents, represented an often underestimated medical revolution. Indeed, until that time, bacterial infections were the prime cause of mortality, especially in children and elderly patients. The discovery of numerous new substances and their development on an industrial scale confronted us with the illusion that bacterial infections were all but vanquished. However, the widespread and sometimes uncontrolled usage of these agents has led to the selection of bacteria resistant to practically all available antibiotics. Bacteria utilize three main resistance strategies: (i) decrease in drug accumulation, (ii) modification of target, and (iii) modification of the antibiotic. Bacteria can decrease drug accumulation either by becoming impermeable to antibiotics, or by actively excreting the drug accumulated in the cell. As an alternative, they can modify the structure of the antibiotic's molecular target--usually an essential metabolic enzyme of the bacteria--and thus escape the drug's toxic effect. Lastly, they can produce enzymes capable of modifying and directly inactivating the antibiotics. In addition, bacteria have evolved extremely efficient genetic transfer systems capable of exchanging and accumulating resistance genes. Some pathogens, such as methicillin-resistant Staphylococcus aureus and enterococci are now resistant to almost all available antibiotics. Vancomycin is the only non-experimental drug left to treat severe infections due to such organisms. However, vancomycin resistance has already appeared several years ago in enterococci, and was also recently described in staphylococci, in Japan, France and the United-States. Antibiotics are precious drugs which must be administered to patients who need them. On the other hand, the development of resistance must be kept under control by a better comprehension of its mechanisms and modes of transmission and by abiding by the fundamental rules of anti-infectious chemotherapy, i.e.: (i) choose the most efficient antibiotic according to clinical and local epidemiological data, (ii) target the bacteria according to the microbiological data at hand, and (iii) administer the antibiotic at an adequate dose which will leave the pathogen no chance to develop any resistance.

Effects of halides on plasmid-mediated silver resistance in Escherichia coli.

Silver resistance of sensitive Escherichia coli J53 and resistance plasmid-containing J53(pMG101) was affected by halides in the growth medium. The effects of halides on Ag+ resistance were measured with AgNO3 and silver sulfadiazine, both on agar and in liquid. Low concentrations of chloride made the differences in MICs between sensitive and resistant strains larger. High concentrations of halides increased the sensitivities of both strains to Ag+.

Analysis of neomycin, kanamycin, tobramycin and amikacin resistance mechanisms in gentamicin-resistant isolates of Enterobacteriaceae.

Twenty-four gentamicin-resistant isolates of Enterobacteriaceae, obtained from the clinical laboratories of three health centres in Nablus, Palestine, were tested for susceptibility to neomycin, kanamycin, tobramycin and amikacin. Resistance rates were 29.2% for neomycin, 58.3% for kanamycin, 45.8% for tobramycin and 8.3% for amikacin. Fourteen (58.3%) isolates were noted to be multiresistant, i.e., resistant to gentamicin and two or more other aminoglycosides; resistance to gentamicin, kanamycin and tobramycin was the most common pattern of multiple resistance. This pattern implies the involvement of adenyltransferase ANT(")-I activity. Plasmid profiles and curing experiments suggested a plasmid localisation of gentamicin, neomycin, kanamycin and tobramycin resistance genes. However, a chromosomal location is proposed for plasmid-deficient strains. Cross-resistance in two isolates to all aminoglycosides tested suggested membrane impermeability to aminoglycosides as the mechanism of resistance.

[Effect of promethazine hydrochloride (pipolphen) on the stability of R plasmid resistance in Escherichia coli]. / Vliianie prometazina gidrokhlorida (pipol'fena) na stabil'nost' nasledovaniia R-plazmid shtammami Escherichia coli.

The influence of promethazin hydrochloride (pipolphen) on stability of R plasmid inheritance in Escherichia coli strains of various serogroups was studied. The strains were isolated from patients with acute intestinal infection and from healthy persons. It was shown that in subbacteriostatic concentrations (100 to 450 micrograms/ml) pipolphen promoted elimination of the R plasmids. Decreased stability of the R plasmid inheritance was not associated with the pipolphen concentration. No influence of the drug on the biochemical characteristics, antigenic properties and nutritional requirements of the plasmid-free derivatives was detected. The eliminating action of pipolphen and ethidium bromide in some strans of Escherichia coli was shown to be different.

[Experimental study of R plasmid eliminating action of Coptis chinensis on E. coli].

The R plasmid curing experiment was performed in vitro with E. coli strain E. 102 bearing R plasmid as target bacteria and Coptis chinensis as elimination agent. The influence of different time on R plasmid elimination was also observed. Results showed that when the acting time was 24 hours, the cure rate of R plasmid was 2.42% and when the acting time increased to 48 hours, the cure rate elevated to 22.57%. The Missing patterns of R plasmid might be occurred in disappearence of either multiple or single resistance.

Emergence of tetracycline resistance due to a multiple drug resistance plasmid in Vibrio cholerae O139.

Of the 173 clinical strains of Vibrio cholerae O139 isolated from India, Bangladesh, and Thailand tested, six strains from India were resistant to tetracycline, ampicillin, chloramphenicol, kanamycin, and gentamicin. These six strains harbored a self-transmissible plasmid that mediated resistance to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, sulfamethoxazole, trimethoprim, and O/129. The multiple drug resistance plasmids were 200 kb in size and belonged to the incompatibility group C. Although a majority of the O139 strains (94.8%) were highly resistant to streptomycin, sulfamethoxazole, trimethoprim, and O/129, the tetracycline-susceptible strains so far tested were plasmid-negative. The data suggest the existence of two distinct multiple antimicrobial agent resistance (MAR) patterns in V. cholerae O139.

Genetic analysis of tetracycline-resistant plasmids in enteropathogenic Escherichia coli isolated from patients in Nigeria.

Genetic analysis of antibiotic-resistant plasmids from 102 serologically defined strains of enteropathogenic Escherichia coli from Nigeria was carried out. All the isolates were screened for susceptibility to antibiotics, and 47 were found resistant to tetracycline. A total of 138 plasmids was isolated by agarose gel electrophoresis. Transformation and conjugation experiments showed that 57.4% of the resistant strains carried R-plasmids ranging in sizes from 2 to 46 x 10(6) daltons. Plasmid-determined resistance to tetracycline, ampicillin and streptomycin was found. Restriction endonuclease analysis of three of the commonest plasmids: p1679, p529 and p1479 revealed relatedness with respect to function and structure. The DNA segment on which TcR gene is located on each of them was identified by cloning into the vector plasmid pGL101. The recombinant plasmids pOADI and pOAD2 gave full expression of TcR gene when transformed into E. coli DHI. Furthermore, the tetracycline-resistant strains were examined for their phenotypic behaviour with respect to tetracycline and its lipophilic analogs.

Sequence identity with type VIII and association with IS176 of type IIIc dihydrofolate reductase from Shigella sonnei.

An uncommon dihydrofolate reductase (DHFR), type IIIc, was coded for by Shigella sonnei that harbors plasmid pBH700 and that was isolated in North Carolina. The trimethoprim resistance gene carried on pBH700 was subcloned and sequenced. The nucleotide sequence of the gene encoding type IIIc DHFR was identical to the gene encoding type VIII DHFR. The type IIIc amino acid sequence was approximately 50% similar to those of DHFRs commonly found in enteric bacteria. Furthermore, this gene was flanked by IS176 (IS26), an insertion sequence usually associated with those of aminoglycoside resistance genes. The gene for type IIIc DHFR was located by hybridization within a 1,993-bp PstI fragment in each of eight conjugative plasmids from geographically diverse strains of S. sonnei. Each plasmid also conferred resistance to ampicillin, streptomycin, and sulfamethoxazole and belonged to incompatibility group M. Plasmids carrying this new trimethoprim resistance gene, which is uniquely associated with IS176, have disseminated throughout the United States.

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases.

By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-beta-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum beta-lactamases.

A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter.

A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.