Characterisation of OXA-51, a novel class D carbapenemase found in genetically unrelated clinical strains of Acinetobacter baumannii from Argentina.

Acinetobacter baumannii is now one of the most frequently encountered nosocomial pathogens in intensive therapy units, and is renowned for being difficult to treat because of resistance to most antibiotics. Carbapenems are the remaining drugs of choice in many centres, but carbapenem resistance is now emerging in strains worldwide. Two subgroups of carbapenem-hydrolysing beta-lactamases, which differ in their amino-acid homology, have been found in some resistant strains. This report describes the emergence and characterisation of a novel carbapenemase (OXA-51) in genetically distinct carbapenem-resistant A. baumannii strains from Argentina. Enzyme kinetics and inhibitor studies were performed spectrophotometrically with purified beta-lactamase. Amplification of the gene was achieved with a two-step PCR method employing arbitrary partially degenerate and gene-specific primers. Transfer of imipenem resistance was attempted with the use of broth and membrane filter methods. Attempts to produce plasmid-cured variants were made in ethidium bromide curing experiments. OXA-51 was identified in two clones of A. baumannii, and was found to have < 63% amino-acid identity with subgroups 1 and 2. Enzyme kinetic studies confirmed that OXA-51 was a molecular class D enzyme with carbapenemase activity, and that it displayed the highest affinity for imipenem (Km value 11 microM). Sequence analysis of the gene identified distinct differences within conserved class D motifs when compared with subgroups 1 and 2. Attempts to transfer imipenem resistance and to determine a plasmid location for the gene failed. OXA-51 is the first of a new subgroup of carbapenemases to emerge in multiresistant clinical isolates of A. baumannii.

Analysis of the effect of progesterone in vivo on estrogen receptor distribution in the rat anterior pituitary and hypothalamus.

An acute injection of estradiol is known to cause a rapid redistribution of estrogen receptors in responsive cells, measurable as depletion of the cytosol receptor content with accompanying accumulation in the nucleus. We have examined the effects of progesterone on this process in the anterior pituitary and hypothalamus using an animal model in which sensitivity to steroidal feedback control of gonadotropin secretion has been defined. Ovariectomized immature rats were administered low dose estrogen replacement for 4 days. On the morning of the fifth day, groups of animals were injected according to one of the following protocols: 1) vehicle alone; 2) 5 or 10 micrograms estradiol; and 3) 0.8 or 3.2 mg progesterone, followed 1 h later by vehicle or 5 or 10 micrograms estradiol. All animals were killed 1 h after estradiol (or vehicle) injection, and levels of cytosol and nuclear estrogen receptors were measured. The only change occasioned by progesterone treatment was a decrease in anterior pituitary nuclear estrogen receptor levels. At the 5-microgram dose of estradiol, 0.8 and 3.2 mg progesterone were equally effective in diminishing nuclear estrogen receptor binding. When 10 micrograms estradiol were used to cause receptor redistribution, only the higher 3.2-mg dose of progesterone significantly depressed nuclear receptor binding. If ovariectomized animals were maintained in the absence of estrogen replacement, progesterone at either the 0.8- or 3.2-mg dosage was completely ineffective in altering the patterns of estradiol-induced cytosol or nuclear estrogen receptor levels. The results demonstrate a tissue-specific inhibitory action of progesterone on estrogen-induced enhancement of nuclear estrogen receptor binding. This inhibition can be partially overcome by increasing the level of estrogen used to effect receptor redistribution. The requirement for maintenance of a background level of estrogen suggests that the inhibitory action of progesterone is mediated through progesterone receptor interactions.

Do nonclinical uses of antibiotics make a difference?

An increasing range of antibacterial compounds is being used for nonclinical purposes, especially in the fields of animal husbandry and fish farming. As in human medicine, exposure to antibiotics has lead to the emergence of antibiotic-resistant bacteria in animal populations. The potential impact of antibiotic use in animals on human health and the management of clinical infections in humans is discussed in light of growing evidence to suggest that "new" resistance genes and multiresistant pathogens with increased pathogenicity are emerging in food animals as a direct consequence of antibiotic exposure.

Evaluation of nitrone spin-trapping agents as radioprotectors.

The focus of this investigation was to determine whether the nitrone spin-trapping compounds alpha-phenyl-N-tert-butylnitrone (PBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) are radioprotectors. Two methods were used to assess for radioprotection: measurement of oxidative damage to DNA bases and mammalian cell survival assays. Oxidative damage to DNA was quantified by measuring the relative amounts of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) produced by the reaction of hydroxyl radicals (OH.) with 2-deoxyguanosine (dG) after irradiation. PBN, DMPO and POBN, when dissolved in aqueous solutions of either dG or naked salmon sperm DNA, reduced the formation of 8-OH-dG by 137Cs gamma irradiation significantly. The spin-trapping agents, especially PBN at lower concentrations, were more effective in preventing radiation-induced formation of 8-OH-dG in naked DNA than in free dG. These data suggest that PBN, DMPO and POBN act as free radical scavengers which may associate with DNA and afford protection against gamma rays. However, no enhancement of survival was observed when Chinese hamster ovary (CHO) cells were exposed to high non-toxic concentrations of PBN or POBN prior to and during irradiation with 60Co gamma rays and scored for clonogenic survival. DMPO provided only minimal protection from radiation-induced cell killing.

Trimethoprim resistance in urinary pathogens in northern Scotland: epidemic spread of a resistance plasmid encoding the type Ib trimethoprim-resistant dihydrofolate reductase.

The prevalence of trimethoprim resistance in enterobacterial urinary pathogens from hospitalised patients in the Angus district of northern Scotland (22.8%) was twice that found in similar isolates from patients attending general practitioners (11.2%). Thirty-three of the 143 trimethoprim-resistant strains were shown to harbour transferable plasmids conferring high-level trimethoprim resistance. In total, 17 different plasmid types were distinguished. Two plasmids, pUK1184 and pUK1185, accounted for 36% of the trimethoprim resistance plasmids and were shown by restriction endonuclease digestion fingerprints to be closely related to plasmid pUK28, previously demonstrated to be endemic in urinary pathogens in the Edinburgh area. Only 21% of the plasmids were shown to encode the type Ia trimethoprim-resistant dihydrofolate reductase, whereas 70% of the trimethoprim resistance plasmids were found to encode the type Ib dihydrofolate reductase. Hybridisation of the trimethoprim resistance plasmids identified in this study with gene probes specific for the integrase genes of transposons Tn7 and Tn21 indicates that the dhfrIa is rarely present within Tn7 or related transposons in these plasmids and may be more prevalent within Tn21-like transposons. In contrast, with the exception of the two endemic plasmids that harboured the dhfrIb gene within a Tn7-like transposon, the majority of dhfrIb genes were not found to be associated with either Tn7- or Tn21-like structures.

The role of thymine starvation in the expression of type IV plasmid-encoded trimethoprim-resistant dihydrofolate reductase.

Hyperproduction of the type IV plasmid-encoded dihydrofolate reductase was studied in Escherichia coli J62-2 (pUK1123). Hyperproduction of the enzyme was shown to occur not simply as a response to a given concentration of trimethoprim but also to the presence of thymidine in the medium. Before hyperproduction occurred the bacteria began to elongate and die, thus showing the symptoms of thymine starvation. Hyperproduction also required the presence of L-methionine, adenine and glycine, suggesting that the elevated production of the enzyme was a response to the ability of trimethoprim to starve the cell of thymine metabolites.

The induction of trimethoprim resistance encoded by the type IV dihydrofolate reductase gene.

The effect of plasmid pUK1123, which confers low level resistance to trimethoprim when tested on solid minimal medium, but also no resistance when tested on IsoSensitest agar, was investigated in liquid media. The growth of Escherichia coli J62-2, harbouring pUK1123, was unaffected in liquid minimal medium containing trimethoprim 10 mg/L. However, in IsoSensitest broth, exposure to this drug concentration resulted in bacteriostasis. After an initial delay, resistance to trimethoprim was induced in IsoSensitest broth containing trimethoprim 10 mg/L, by the imposition of thymine starvation. This response was immediately reversible when trimethoprim was removed, confirming that resistance resulted from induction rather than selection of resistant mutants.

N-terminal amino-acid sequence and subunit structure of the type IV trimethoprim-resistant plasmid-encoded dihydrofolate reductase.

The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of Escherichia coli isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, E. coli J62-2 (pUK1123), and from E. coli C600 (pUK1150) containing a 2.6-kb HindIII fragment of pUK1123 cloned into plasmid pBR322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded E. coli enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from E. coli J62-2 harbouring the original clinical plasmid, pUK1123, also contained the E. coli DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHFR of Mr 33,000 coupled to a DNA-binding protein.

Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci.

The molecular and biochemical basis of resistance to high concentrations (MIC greater than or equal to 1000 mg/L) of trimethoprim (Tpr(H] was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(H) determinant (dfr A) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr(H) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(H) gene sequences among staphylococci.

Identification and cloning of the type IIIa plasmid-encoded dihydrofolate reductase gene from trimethoprim-resistant gram-negative bacteria isolated in Britain.

A clinical strain of Escherichia coli isolated in Nottinghamshire in 1980 was shown to harbour the type IIIa trimethoprim-resistant dihydrofolate reductase gene, previously identified on only one occasion, in New Zealand in 1979. The gene was identified by hybridisation with an 855-bp type III gene probe and its classification as a type IIIa dihydrofolate reductase was confirmed by detailed biochemical analysis of the enzyme product. The dihydrofolate reductase was identical in size and isoelectric point with the original type IIIa enzyme and shared similar inhibitory and kinetic profiles. The trimethoprim resistance gene was subsequently cloned and the type IIIa dihydrofolate reductase gene was localised to a 700-bp EcoRI-PstI fragment. This smaller fragment may prove to be a more specific DNA probe for the future identification of type IIIa dihydrofolate reductase genes.

The genetics of bacterial trimethoprim resistance in tropical areas.

Resistance to trimethoprim in Gram-negative bacteria is largely manifested by two trimethoprim resistant dihydrofolate reductases (types I and II) encoded by genes originally located on resistance plasmids. Although trimethoprim resistance increased markedly after the clinical introduction of trimethoprim in the West, its spread has slowed and, in Edinburgh at least, has actually been declining. This reduction has been accompanied by the migration of a transposon, encoding the type I plasmid resistance gene, into the bacterial chromosome. In tropical areas, the incidence of trimethoprim resistance is very much higher. In Tanzania, it has spilled over into other bacteria outside the Enterobacteriaceae, but it was in India where the major problem existed. The majority (64%) of the Indian Enterobacteriaceae studied were resistant to the drug and most of the resistance genes were located on very large plasmids which also conferred resistance to many other antibacterial drugs. Some Indian plasmids carried a new trimethoprim resistance gene which is not detectable by conventional sensitivity tests and may be spreading unnoticed elsewhere. The proportion of trimethoprim resistance has been related to the volume of antibacterial drugs used.

Antibiotic resistance in the tropics. 1. The genetics of bacterial ampicillin resistance in tropical areas.

Ampicillin and its derivatives are the most widely used beta-lactam antibiotics throughout the world. Ampicillin resistance in Gram-negative bacteria is usually manifested by plasmid-encoded beta-lactamases, which hydrolyse the beta-lactam ring of the antibiotic. There are at least 30 different plasmid-encoded beta-lactamases but almost all of them are found very infrequently. The one exception is the TEM-1 beta-lactamase which is found wherever transferable ampicillin resistance emerges and accounts for over 50% of all plasmid encoded ampicillin resistance. In India, the incidence of ampicillin resistance is high (82%) and, amongst Escherichia coli, a significant proportion of the plasmid-encoded beta-lactamases are different from those found in the United Kingdom. Although many Gram-negative species are able to accept the TEM-1 beta-lactamase, certain species have a pre-disposition to their own plasmid beta-lactamase types.

Outcome of noninvasive ventilation in children with neuromuscular disease.

OBJECTIVE: To assess the effect of institution of noninvasive ventilation (NIV) on clinical outcome and quality of life (QOL) in a cohort of children with severe neuromuscular disorders. METHODS: We reviewed records and obtained clinical data from the year prior to commencing NIV and annually thereafter. Data obtained included diagnosis, patient symptoms, mortality, NIV adverse effects, pulmonary function tests, polysomnographic data, length of hospitalizations, and health care costs. Patients and parents completed questionnaires assessing QOL with NIV and recalling QOL before NIV. RESULTS: Fourteen of 17 (82%) suitable patients were enrolled. Follow-up ranged from 6 to 84 months (median 30). Symptoms of daytime sleepiness (p = 0.003) and headache (p = 0.046) improved after initiation of NIV. Sleep quality assessed by polysomnography also improved. Hospitalization rates (p = 0.002) and health care costs (p = 0.003) decreased. QOL remained stable after NIV, despite disease progression. CONCLUSION: Treatment of respiratory failure, in children with neuromuscular disease, with noninvasive ventilation results in a reduction in symptoms, hospitalizations, and health care costs without adverse effects on quality of life.

Antimicrobial resistance spread in aquatic environments.

The increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the numbers of antibiotic resistant bacteria present in aquatic environments. The role of this environment to act, not only as a reservoir of clinical resistance genes, but also as a medium for the spread and evolution of resistance genes and their vectors, is discussed.

Identification and characterization of vanB2 glycopeptide resistance elements in enterococci isolated in Scotland.

Thirty-two vanB glycopeptide-resistant enterococci (28 Enterococcus faecium and 4 Enterococcus faecalis) were collected from hospitalized patients in Glasgow, Edinburgh, Dundee, and Aberdeen, Scotland, and the vanB element in each was compared to vanB1 of E. faecalis strain ATCC 51299. HhaI digestion of PCR fragments of the vanB ligase gene was used to identify vanB subtypes. All E. faecium isolates were vanB2, and all E. faecalis isolates were vanB1. Restriction fragment length polymorphism analysis of a 5,180-bp vanS(B)-vanX(B) long-PCR fragment of the vanB cluster showed the loss of HaeII restriction sites in vanS(B), vanW, and vanX(B) in strains containing a vanB2 ligase gene. Partial sequences of genes in the vanB2 cluster for two genomically distinct Scottish isolates were >99.8% identical to each other. vanS(B2), vanX(B2), and vanB2 sequences differed at the nucleotide level from those of vanS(B), vanX(B), and vanB by 4.2, 4.6, and 4.8%, respectively. The vanB2 resistance element appears to be widespread among VanB glycopeptide-resistant E. faecium strains isolated in Scottish hospitals.

Cloning of the type Ib trimethoprim-resistant dihydrofolate reductase gene and preparation of a specific biotinylated DNA probe.

A 3.0-kb PstI/HindIII fragment encoding the type Ib trimethoprim-resistant dihydrofolate reductase (DHFR) was cloned into pUC18. Following successive subcloning, the type Ib DHFR gene was identified within a 500 bp HincII fragment. The 500 bp fragment was purified, biotinylated and tested for its suitability as a specific DNA probe in non-isotopic hybridisation experiments with standard plasmids carrying genes for the available known Gram-negative DHFR types. Under conditions of high stringency the probe was specific for the type Ib gene and hence will be useful for monitoring the spread of this particular trimethoprim resistance gene.

Plasmid trimethoprim resistance in Vibrio cholerae: migration of the type I dihydrofolate reductase gene out of the Enterobacteriaceae.

Two strains of Vibrio cholerae biotype el tor were isolated in Tanzania. Each possessed a single resistance plasmid of 113 kbases belonging to the C incompatibility group. Both plasmids conferred resistance to sulphamethoxazole, ampicillin, streptomycin, chloramphenicol, tetracycline and trimethoprim. They are the first plasmids derived from V. cholerae strains, isolated in Southern Africa, to confer trimethoprim resistance. The trimethoprim resistance resulted from the plasmid-mediated production of a trimethoprim resistant dihydrofolate reductase which was very similar to the type I plasmid enzyme. This is the first example of movement of the gene encoding the type I dihydrofolate reductase into genera of other families besides the Enterobacteriaceae.

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase.

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.