Acquisition Of Plasmids Research Articles

Mechanisms of bacterial resistance to biocides

Mechanisms of bacterial resistance to biocides can conveniently be considered as being of two types: (i) intrinsic, a natural property of an organism; (ii) acquired, resulting from genetic changes in a bacterial cell and arising either by mutation or by the acquisition of plasmids or transposons. Intrinsic resistance is demonstrated by bacterial spores, mycobacteria and Gram-negative organisms and is also shown by physiological (phenotypic) adaption as exemplified by cells growing as biofilms. Acquired resistance to biocides has been found with methicillin-resistant Staphylococcus aureus (MRSA) strains, but is most widely seen as bacterial resistance to mercury compounds and other metallic salts.

Transformation of Acinetobacter calcoaceticus RAG-1 by electroporation

A method is described for transforming Acinetobacter calcoaceticus RAG-1 with a transposon-delivery plasmid, pLOFPttKm, and the broad-host-range cloning vector pKT230. Kanamycin-resistant transformants isolated following electroporation with pLOFPttKm were shown to have lost the plasmid, and contained the transposon mini-Tn10PttKm in at least five unique, single sites. Transformation with pKT230 was confirmed by plasmid screening and acquisition of the kanamycin-resistant, streptomycin-resistant phenotype. By optimizing conditions of electric field strength and pulse length for electroporation, we obtained a maximum efficiency of 4.36 × 102/(μg DNA∙109 cells) for insertion of the mini-Tn10PttKm transposon, and 9.88 × 104/(μg DNA∙109 cells) for transformation with plasmid pKT230. Our results indicate that electroporation is a suitable method for introducing transposon and cloning vectors into A. calcoaceticus RAG-1, and possibly other Acinetobacter spp. The availability of a transformation system should enhance the molecular genetic and physiological analysis of these bacteria, particularly the vast majority of strains that are not competent for transformation with DNA.Key words: electroporation, Acinetobacter, transformation, plasmid, transposon.

Isolation from soil of symbiotic and nonsymbiotic Rhizobium leguminosarum by DNA hybridization

A procedure based upon DNA hybridization was developed for the specific detection of Rhizobium leguminosarum and its different biovars among bacteria isolated from soil. DNA colony hybridization and restriction fragment length polymorphism analysis with a R. leguminosarum chromosomal probe were found to be species specific for R. leguminosarum and Rhizobium etli. By using R. leguminosarum nod gene probes, biovar specificity was obtained. Of 302 soil isolates screened for their inability to grow on Luria-Bertani agar medium, 13 strains could be assigned to the R. leguminosarum species on the basis of DNA homology to the chromosomal probe and antibiotic resistance tests. Of these strains, three and two were assigned by colony hybridization and subsequent plant host specificity tests, respectively, to R. leguminosarum biovars viciae and trifolii. The eight other R. leguminosarum soil isolates lacked symbiotic information but were able to gain nodulation capacity with the acquisition of a conjugative symbiotic plasmid. They were thus considered as nonsymbiotic R. leguminosarum.Key words: Rhizobium leguminosarum, DNA hybridization, soil, symbiotic genes.

Interspecific conjugal transfer of antibiotic resistance among staphylococci isolated from the bovine mammary gland

Summary In vitro transferability of pemcillm. strepiomycin, tetracycline, and erythromycin resistance from coagulase-negative staphylococci to Staphylococcus aureus and among the former species of bovine mammary gland origin was examined by bacterial mating on filters and by mixed-culture matings in broth and in skim milk. One hundred twenty-six (42 each on filter, in broth, and in skim milk) matings were performed among 37 isolates of different Staphylococcus species. Transfer of resistance to penicillin, tetracycline, or erythromycin was not detected. Of 51 matings performed to determine streptomycin-resistance transfer, 9 (3 each on filters, in broth, and skim milk) were successful. Nine strains representing 3 species of coagulase-negative staphylococci were tested as prospective donors of streptomycin resistance. Of these, 2 strains could transfer streptomycin resistance. A double-resistant donor, Shominis, not only transferred its streptomycin resistance to an S chromogenes strain lacking resistance, but also to an S aureus strain already carrying penicillin and tetracycline resistance. The transfer of streptomycin resistance from the donor S hominis, harboring 2 plasmids, to a plasmidless S chromogenes recipient strain was associated with apparent acquisition of the smaller plasmid of the donor by the recipient. The single-resistant donor, S epidermidis 681A, transferred streptomycin resistance to a tetracycline-resistant. S aureus recipient. This strain however failed to transfer its streptomycin resistance to another S aureus, 2 S hyicus, and 1 S xylosus recipient. Frequency of transfer of streptomycin resistance ranged from 1.1 × 10−5 to 1 × 10−4. When transfer of resistance was successful, attempts were made to characterize the transfer process. Conjugation appeared to be the mode of streptomycin-resistance transfer. Transfer of resistance between staphylococci of bovine mammary gland origin appears to be fairly uncommon. However, in view of the limitations of the procedures used, additional in vitro and in vivo work is needed to further assess the role of coagulase-negative staphylococci in dissemination of antibiotic resistance.

Rapid Development of Ciprofloxacin Resistance in Methicillin-Susceptible and -Resistant Staphylococcus aureus

The fluoroquinolones, particularly ciprofloxacin, have been suggested to treat methicillin-resistant Staphylococcus aureus (MRSA) infections and colonization and methicillin-susceptible S. aureus (MSSA) infections. The development of ciprofloxacin resistance in MRSA and MSSA was prospectively evaluated. After 3 months of ciprofloxacin use, high-level resistance (MIC90, 64 micrograms/ml) developed in MRSA and increased at an alarming rate, from none to 79% over a 1-year period. High-level ciprofloxacin resistance also developed in MSSA, increasing to 13.6% over the same period. Antibiograms, phage typing, and plasmid profile analysis suggest that more than one clone of MRSA developed resistance and that ciprofloxacin resistance is not associated with the acquisition of a new plasmid. Most patients had nosocomial acquisition and about one-half had a history of previous ciprofloxacin use. Ciprofloxacin resistance can develop rapidly in S. aureus; thus, ciprofloxacin appears to have limited usefulness in treating staphylococcal infections and colonization, especially those due to MRSA.

Sorbose fermentation in relation to acquisition and maintenance of enterotoxin plasmids in Escherichia coli

Previous studies have shown that the majority of enterotoxigenic Escherichia coli (ETEC) strains isolated from humans failed to ferment sorbose. In an attempt to demonstrate if this phenomenon was due to enterotoxin (Ent) plasmid instability in sorbose fermenting strains, recipient E. coli strains were used in conjugation experiments with three donor Ent plasmids. No difference in the ability to uptake the Ent plasmid was shown between sorbose fermenting and non-fermenting recipient strains. In addition, no difference was seen in the stability of the Ent plasmids in recipient strains of each biotype. These results suggest that in-vivo selection may play a role in the predominant isolation of non-sorbose fermenting ETEC strains.

Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae

Klebsiella pneumoniae isolates from 11 patients at the Miriam Hospital were identified as resistant to cefoxitin and ceftibuten as well as to aztreonam, cefotaxime, and ceftazidime. Resistance could be transferred by conjugation or transformation with plasmid DNA into Escherichia coli and was due to the production of a beta-lactamase with an isoelectric point of 8.4 named MIR-1. In E. coli, MIR-1 conferred resistance to aztreonam, cefotaxime, ceftazidime, ceftibuten, ceftriaxone, and such alpha-methoxy beta-lactams as cefmetazole, cefotetan, cefoxitin, and moxalactam. In vitro, MIR-1 hydrolyzed cephalothin and cephaloridine much more rapidly than it did penicillin G, ampicillin, or carbenicillin. Cefotaxime was hydrolyzed at 10% the rate of cephaloridine. Cefoxitin inactivation could only be detected by a microbiological test. The inhibition profile of MIR-1 was similar to that of chromosomally mediated class I beta-lactamases. Potassium clavulanate had little effect on cefoxitin or cefibuten resistance and was a poor inhibitor of MIR-1 activity. Cefoxitin or imipenem did not induce MIR-1. The gene determining MIR-1 was cloned on a 1.4-kb AccI-PstI fragment. Under stringent conditions, probes for TEM-1 and SHV-1 genes and the E. coli ampC gene failed to hybridize with the MIR-1 gene. However, a provisional sequence of 150 bp of the MIR-1 gene proved to be 90% identical to the sequence of ampC from Enterobacter cloacae but only 71% identical to that of E. coli, thus explaining the lack of hybridization to the E. coli ampC probe. Plasmid profiles of the 11 K. pneumoniae clinical isolates were not identical, but each contained a plasmid from 40 to 60 kb that hybridized with the cloned MIR-1 gene. Both transfer-proficient and transfer-deficient MIR-1 plasmids belonged to the N incompatibility group. Thus, the resistance of these K. pneumoniae strains was the result of plasmid acquisition of a class I beta-lactamase, a new resistance determinant that expands the kinds of beta-lactam resistance capable of spread by plasmid dissemination among clinical isolates.

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.

Acquisition of β-lactamase and TetM-containing Conjugative Plasmids by Phenotypically Different Strains of Neisseria gonorrhoeae

beta-lactamase plasmids confer high-level penicillin resistance on strains of Neisseria gonorrhoeae and may be mobilized between strains by conjugative plasmids. The authors examined 32 isolates belonging to 23 auxotype/serovar (A/S) classes to determine the frequency with which they acquired the 4.4-, 3.2-, and 3.05-megadalton (Mdal) beta-lactamase plasmids, respectively. The 3.05-Mdal beta-lactamase plasmid was not acquired by any isolate. Each of 22 isolates possessing only the 2.6-Mdal plasmid or both the 2.6- and 24.5-Mdal plasmids acquired the 4.4- and 3.2-Mdal plasmids at similar frequencies, and 12 (55%) acquired the 4.4- or 3.2-Mdal plasmids at a frequency of greater than or equal to 10(-3) per input recipient; all transconjugants acquired the 24.5-Mdal conjugative plasmid. Among ten isolates that lacked the 2.6-Mdal plasmid (plasmidless), seven isolates that included five PAU/IB-2, -16 isolates acquired neither the 4.4- nor 3.2-Mdal plasmid; three non-PAU isolates acquired both the 4.4- and 3.2-Mdal plasmids (10(-2)-10(-5) per input recipient). All plasmidless isolates acquired the 25.2-Mdal TetM containing plasmid with frequencies ranging from 10(-1) to 10(-8) per input recipient. These data suggest that gonococcal strains possessing the 2.6-Mdal plasmid readily acquire the 4.4- and 3.2-Mdal beta-lactamase plasmids in vitro, whereas acquisition of these plasmids by strains lacking the 2.6-Mdal plasmid is strain dependent.

Transfer of beta-lactamase plasmids by conjugation in Neisseria gonorrhoeae.

Conjugation experiments in vitro have been used in an attempt to explain certain epidemiological phenomena seen amongst penicillinase producing Neisseria gonorrhoeae (PPNG) isolated at St Mary's Hospital, London. These include changes in plasmid profiles, a lack of beta-lactamase encoding plasmids in PAOU-requiring strains and a difference in the serological classification of strains of PPNG and non-PPNG isolated from the same clinic. It was shown that acquisition of the conjugative plasmid by beta-lactamase producing transconjugants does vary, but is not related to the auxotype/serovar (A/S) class of, or type of beta-lactamase plasmid carried by the donor. It was not possible to transfer either the 3.2MDa or 4.4MDa plasmids to PAOU-requiring strains of N gonorrhoeae. The conjugation frequency from a single donor to different recipients was shown to be variable, but was not influenced by the serovar of the recipients. It was shown that the transfer and acceptance of beta-lactamase encoding plasmids is variable but it was not possible to identify the factors responsible.

Correlation of in vitro and in vivo resistance development to antimicrobial agents

Abstract Preclinical in vitro and in vivo determinations of the likelihood of an antibiotic to develop resistance can and has proven predictive of their likelihood of resistance development in patients. Problematic antibiotic/bacterial species combinations are often associated with high frequencies of single-step resistance development in that species. Thus, treatment of organisms with rapid in vitro emergence of drug resistance should be monitored carefully. In vitro studies, however, are limited in predicting resistance mediated through acquisition of a resistance plasmid. The frequency of resistance development to a drug is dependent on factors such as the drug used for selections, the concentration (i.e., dosing) of the drug, the bacterium, and the site of infection. Organisms intrinsically less susceptible to an antibiotic develop resistance rapidly due to their low therapeutic ratios. Since cross-resistance often occurs within an antibiotic class, it may be desirable to initiate therapy with a drug with low resistance-selecting potential. Optimal dosing regimens are especially critical when treating bacterial species likely to develop drug resistance. Though combination drug therapies have proven affective in experimental animal infections and in man, they do not prevent resistant variants from emerging. Understanding of drug-resistance development will contribute to our management of infectious diseases.

Conjugal acquisition and stable maintenance of Ent plasmids in nontoxigenic wild-type strains of Escherichia coli.

In spite of the ability of the genetic determinants for enterotoxin production to be conjugally transferred, mobilized or transposed, enterotoxigenic Escherichia coli (ETEC) isolated from diarrheal patients is restricted to certain serotypes. Four conjugative enterotoxigenic plasmids (Ent plasmids) encoding either a heat-labile enterotoxin or a heat-stable enterotoxin or both and belonging to one of three incompatibility groups IncFI, IncHl, or IncX, were examined for their transferability to and stability in 157 nonenterotoxigenic Escherichia coli strains belonging to various serotypes and 89 clinical isolates nonenterotoxigenic but belonging to those serotypes in which ETEC from diarrheal patients are usually found. The serotypes of the strains to which Ent plasmids were efficiently transferred and in which they were maintained stably were not always the serotypes in which ETEC had usually been found and vice versa. The frequencies of transfer of four Ent and two R plasmids to each of the 157 recipients were correlated with each other, indicating that the frequency of transfer of the plasmid is not determined by a resident plasmid, if there is one, but by a recipient factor which commonly affects transferability to all donors. These results have led to the conclusion that the reason why only certain serotypes are found among ETEC isolated from diarrheal patients is not the ability of these strains specifically and preferentially to acquire and maintain the Ent plasmids.

Epidemiology of resistance in Neisseria gonorrhoeae.

In the face of almost unique selective pressures many strains of gonococci have become increasingly resistant to antibiotics. Both chromosomal mutation and acquisition of plasmids are involved. However, whereas there have probably been multiple selections of many mutation events determining chromosomal resistance, evidence suggests that a very limited number of plasmid acquisitions has occurred, with subsequent spread within members of the species.

Plasmid-mediated resistance to beta-lactam antibiotics in gram-negative bacteria: the role of in-vivo recyclization reactions in plasmid evolution.

Over 20 different plasmid-encoded beta-lactamases have so far been discovered. This paper considers genetic mechanisms by which beta-lactamase genes encoded by plasmids are disseminated across generic boundaries. Particular emphasis is placed on the evolution of plasmids carrying all or part of Tn3 and encoding TEM-1 beta-lactamase in Haemophilus and Neisseria species. Examples of the acquisition of broad host range plasmids carrying Tn3 sequences and of rescue of transposon sequences to indigenous plasmids or to the host chromosome have been found in these two genera. Studies on nonconjugative beta-lactamase plasmids in Neisseria and Haemophilus are consistent with the evolution of a family of plasmids originating from the insertion of Tn3 into an indigenous progenitor plasmid. A series of subsequent insertional and deletional events, most probably occurring as a consequence of genetic transfer, have given rise to the existing group of small, closely related ampicillin-resistance plasmids found currently in these genera. A general model for deletional and other rearrangements caused by recombinational recyclization during the evolution of resistance plasmids is described.

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac) transconjugants were selected from matings of Lac variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac; pTR2030, Hsp). For all of the S. cremoris strains examined, select Lac transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40 degrees C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.

Development of plasmid-mediated resistance in Vibrio cholerae during treatment with trimethoprim-sulfamethoxazole.

The persistence of Vibrio cholerae, biotype el tor, in a patient treated with trimethoprim-sulfamethoxazole was due to the acquisition of a conjugative resistance plasmid. The plasmid, with a molecular size of 72 megadaltons, belonged to incompatibility group 6-C and conferred resistance to ampicillin, chloramphenicol, sulfonamide, and trimethoprim.

Acquisition and Maintenance of Enterotoxin Plasmids in Wild-type Strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.

The origin of endosymbiotic nitrogen fixation by Rhizobium

It is proposed, that current Rhizobia, able to fix nitrogen symbolically, developed from a bacterium, that was the common ancestor of Rhizobium and Agrobacterium tumefaciens, by the acquisition of a small plasmid or a small part of a bacterial genome, carrying a large part (but not all) of the genes necessary for the expression of nitrogen fixation.

Parameters of acquired resistance and their role in the evaluation of new chemotherapeutic drugs

Acquired resistance can be defined as a qualitative alteration of the genetic material of a cell which is phenotypically correlated with a measurable decrease of the cell's sensitivity against one or several chemotherapeutic agents. There are two basic genetic mechanisms which can lead to the emergence of resistance: mutation and the acquisition of additional genetic material from another cell. Both forms of resistance play an important role in clinical situations: the emergence of resistance by mutation occurs in tumor cells and can also lead to therapeutic problems in antimicrobial chemotherapy. In bacteria, however, acquisition of resistance plasmids represents the dominating mechanism which is responsible for most therapeutic problems in the clinical environment. The different genetic mechanisms involved in the emergence of resistance are paralleled -- at least in bacteria -- by two principally different groups of biochemical mechanisms implementing resistance. Mutations lead to alterations of single cell constituents such as the cell membrane or cellular receptors necessary for the binding of the antimicrobial agent. This form of resistance is biochemically characterized by the inaccessibility of the cell interior for a particular compound or by the modification of an intracellular binding site which loses its affinity for the chemotherapeutic agent. Resistance plasmids on the other hand code for enzymes which inactivate the antibiotic (beta-lactamases, aminoglycosideinactivating enzymes, chloramphenicol-acetyltransferase); In some cases, they direct the synthesis of proteins which affect cell permeability (tetracycline) or isoenzymes which have a lower affinity for the inhibitor (trimethoprim). Resistance against antibiotics can be inducible; In these cases the regulatory mechanisms involved are stable genetical traits as resistance itself; Using chloramphenicol, beta-lactam-antibiotics and aminoglycosides as examples, it is demonstrated that resistance data gathered early in the development of a new drug are of little value in estimating the clinical potential of a new compound. Information on the rate at which resistance develops, on the pattern according to which it emerges ("single step" or "multi step") and on cross-resistance patterns is important in the characterization of a new drug but is often invalidated by later findings obtained in the clinical environment; The problem appears somewhat simpler if a new drug is a member of an already known class of compounds, e.g. a beta-lactam or an aminoglycoside. In such cases our knowledge of frequent enzymatic inactivation mechanisms provides a basis not only for the evaluation of an existing drug, but also for the synthesis of new derivatives.

Chemical composition of portland cement

Sequence type 11 (ST11) carbapenem-resistant Klebsiella pneumoniae (CRKP) has become the dominant clone in China. In this review, we trace the prevalence of ST11 CRKP in the China Antimicrobial Surveillance Network (CHINET), the key antimicrobial resistance mechanisms and virulence evolution. The recent emergence of ST11 carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) strains in China due to the acquisition of a pLVPK-like virulence plasmid, which may cause severe infections in relatively healthy individuals that are difficult to treat with current antibiotics, has attracted worldwide attention. There is a very close linkage among IncF plasmids, NTEKPC and ST11 K. pneumoniae in China. Hybrid conjugative virulence plasmids are demonstrated to readily convert a ST11 CRKP strain to a CR-hvKP strain via conjugation. Understanding the molecular evolutionary mechanisms of resistance and virulence-bearing plasmids as well as the prevalence of ST11 CRKP in China allows improved tracking and control of such organisms.