Diversity of methicillin-resistantStaphylococcus aureus isolated in a Canadian hospital

Three neonates and three other patients located elsewhere in the hospital became infected withStaphylococcus aureus. Initial automated microdilution susceptibility testing with oxacillin and disk diffusion testing with amoxicillin-clavulanic acid indicated the isolates had borderline oxacillin resistance (MICs 4 µg/ml), presumably due to hyperproduction of -lactamase. Chromosomal DNA restriction fingerprinting and phage typing revealed the neonatal isolates to be identical; whereas, the other patients were infected with three different strains. Further analysis of the four strains by Southern hybridization with amecA specific oligoprobe and a quantitative -lactamase assay demonstrated that two strains carried themecA gene (coding for low affinity penicillin-binding protein 2a), and two strains were hyperproducers of -lactamase, including one which wasmecA gene positive. One strain neither carried themecA gene nor hyperproduced -lactamase. The twomecA gene positive strains displayed oxacillin MICs of 16 µg/ml on dilution susceptibility testing in 4% NaCl supplemented Mueller-Hinton agar. Hence, they were considered intrinsically methicillin-resistantStaphylococcus aureus. Both oxacillin and amoxicillin-clavulanic acid MICs were increased on NaCI supplementation. Results of amoxicillin-clavulanic acid disk diffusion susceptibility testing did not correlate with quantitative -lactamase production. It is recommended that clinical laboratories do not use amoxicillin-clavulanic acid disk diffusion assays to differentiate suspected borderline resistance due to -lactamase hyperproduction frommecA gene expression of PBP-2a since additional mechanisms may account for resistance.     

Emergence of resistance to beta-lactam agents inPseudomonas aeruginosa with group I beta-lactamases in Spain

The contribution of induction and stable derepression of chromosomal class I -lactamases to -lactam antibiotic resistance was studied in clinical isolates ofPseudomonas aeruginosa collected from patients treated with -lactam antibiotics. Multiple isolates from the same patient were characterized by O-serotyping as a primary screen, combined with pyocin typing. Sonicated extracts of cells were assayed for chromosomal and plasmid-mediated -lactamases by isoelectric focusing and cloxacillin inhibition studies. The specific -lactamase activity, basal and induced, with cefoxitin was determined to differentiate strains with inducible or derepressed production of the enzyme. Beta-lactamase induction was performed in each strain against the -lactam agents used in the therapy of each patient. The observations showed that induction against older penicillins such as penicillin, amoxicillin, and amoxicillin/clavulanate resulted in a moderate to strong increase in -lactamase activity, whereas the results obtained with first-generation cephalosporins varied with the -lactam agent tested. Third-generation cephalosporins were weak inducers of -lactamases, and their use as therapy preceded the appearance of strains that produce chromosomal group I -lactamases constitutively. These strains showed a remarkable reduction in sensitivity to ureidopenicillins, carboxipenicillins, third-generation cephalosporins, and monobactams, but not to carbapenems.     

Beta-lactamase types and beta-lactam resistance ofEscherichia coli strains with chromosomally mediated ampicillin resistance

On the basis of isoelectric focusing six -lactamase types could be distinguished in ampicillin-resistant and ampicillin-sensitive strains ofEscherichia coli. More than 90% of the ampicillin-resistant strains produced the same -lactamase type. The serotypes found in a group of ampicillin-resistant urinary tract infection strains did not represent the distribution usually found in urinary tract isolates. Chromosomal ampicillin resistance was always associated with high cephalothin MIC values and increased resistance to other -lactam antibiotics of the cephalosporin group.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

Use of a DNA hybridization method to verify results of screening for methicillin resistance in Staphylococci

Tests were performed by the disk diffusion method, agar dilution method and the E test to determine the susceptibility to methicillin and oxacillin of clinical isolates and control strains ofStaphylococcus aureus (n=106) and coagulase-negative species (n=131). Results were compared with those of a dot blot DNA hybridization test, in which themecA gene was detected using an oligonucleotide probe selected from themecA gene. Among theStaphylococcus aureus strains themecA gene was found in all but two strains inhibited by 8 mg/l of methicillin and all but two strains inhibited by 4 mg/l of oxacillin. A disk test using either 1 µg oxacillin or 10 µg methicillin and a tentative resistance breakpoint of 10 mm gave the best agreement with the hybridization test. For coagulase-negative staphylococci 34 of 35 strains inhibited by 8 mg/l methicillin hybridized with the probe as well as 58 of 82 strains inhibited by 1–4 mg/l; 93 of 97 strains inhibited by 0.5 mg/l oxacillin were also positive in the probe test. Using the 1 µg oxacillin disk and a resistance breakpoint of 10 mm good agreement was obtained between results of the disk diffusion and DNA hybridization tests. It is suggested that this genotypic method for detection of methicillin resistance is used as a reference method for routine methods.     

Activity of beta-lactamase inhibitor combinations onEscherichia coli isolates exhibiting various patterns of resistance to beta-lactam agents

The efficacy of the clinically available -lactam/-lactamase inhibitor combinations (amoxicillin/clavulanic acid (CA), ticarcillin/CA, amoxicillin/sulbactam, and piperacillin/ tazobactam) was evaluated on 300 amoxicillin-resistantEscherichia coli isolates having the main patterns of -lactam resistance. The patterns, which reflect the production of various -lactamase enzymes, were analyzed by a principal component analysis of susceptibility to 11 -lactam antibiotics or -lactam/-lactamase inhibitor combinations. Sixty-two percent of strains were not very susceptible to penicillins, cephalothin, or any -lactam/-lactamase inhibitor combinations except for piperacillin/tazobactam; these strains may represent high-level broad-spectrum -lactamase (so-called penicillinase) production phenotype or inhibitor-resistant TEM-like enzyme production phenotype. Of the strains, 14.7 % were resistant to amoxicillin and ticarcillin compatible with low-level broad-spectrum -lactamase production phenotype; 5.7 % were cefoxitin resistant and were postulated to present a high-level cephalosporinase production phenotype; and 2.6 % were resistant to cephalothin only, attributable to a low-level cephalosporinase production phenotype. Three percent of strains were intermediate or resistant to cefotaxime and may produce an extended-spectrum -lactamase, and the remaining strains (12 %), resistant to all tested antibiotics except for cefotaxime and piperacillin/tazobactam, were hypothesized to produce both broad-spectrum -lactamase plus cephalosporinase. The minimal inhibitory concentration (MIC) for these phenotype patterns indicated that combinations of CA plus amoxicillin or ticarcillin, or sulbactam plus amoxicillin, restored the activity of penicillins against phenotype 1 strains, whereas these combinations remained inactive against the other phenotype strains. Piperacillin plus tazobactam showed the best in vitro effect against the strains of all resistance phenotypes.     

Isolation ofEnterobacter aerogenes susceptible to beta-lactam antibiotics despite high level beta-lactamase production

This report describes a patient with nosocomial meningitis from whom four distinct isolates ofEnterobacter aerogenes were recovered over a complicated course of chemotherapy. The initial isolate was susceptible to expanded spectrum -lactams despite constitutive production of high levels of -lactamase. Resistant isolates recovered during antibiotic therapy had lost a 42,000 outer membrane protein. These data suggest that b-lactam susceptibility in the original isolate was due to hyperpermeability mediated by the 42,000 Dalton protein.     

Dissemination of cephalosporin-resistantSerratia marcescens strains producing a plasmidic SHV type beta-lactamase in Greek hospitals

The resistance to third generation cephalosporins in nineSerratia marcescens strains isolated in Greek hospitals was studied. Eight of the strains transferred resistance toEscherichia coli by means of large plasmids that encoded for an extended-spectrum -lactamase. Hybridization, isoelectric focusing and hydrolysis studies showed that the enzyme resembled the SHV-5 -lactamase. In the eight isolates that possessed the SHV type enzyme, cephalosporinase expression was inducible, whereas the remaining strain was a cephalosporinase hyperproducing strain. Introduction of a plasmid coding for the regulatoryampD gene in the latter strain eliminated -lactamase production and rendered the strain susceptible to cephalosporins.     

Beta-lactamase production and susceptibility of US and European anaerobic gram-negative bacilli to beta-lactams and other agents

The susceptibility of 1,476 US and European strains of anaerobic gram-negative bacilli to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was determined. All of theBacteroides fragilis group and 51 % of the non-Bacteroides fragilis group were -lactamase positive. Amongst the non-Bacteroides fragilis group, -lactamase positivity rates were higher for US strains (58 %) than for European strains (39 %). All strains were susceptible to imipenem and metronidazole. MIC90s of amoxicillin and ticarcillin for all -lactamase negative strains were 0.5 and 2 µg/ml, respectively. The addition of clavulanate reduced the MIC90s of amoxicillin ( 256 µg/ml) and ticarcillin ( 64 µg/ml) to 16 and 8 µg/ml, respectively, for theBacteroides fragilis group, and to 4 µg/ml for both agents for the non-Bacteroides fragilis -lactamase producing group. Twenty-nine cefoxitin-resistant strains were found, mainly in theBacteroides fragilis group, while 95 -lactamase producing strains (predominantlyBacteroides fragilis group and fusobacteria) did not show synergy between -lactams and clavulanate. Of the newer agents tested, meropenem and piperacillin-tazobactam were the most active (100 % of strains susceptible), followed by amoxicillin-BRL 42715 (99 % of strains susceptible); 94 to 98 % of the strains were susceptible to cefoperazone-sulbactam, tosufloxacin, tempafloxacin and clindamycin. Only 73 % of the strains were susceptible to cefotetan, compared to 91 % to cefoxitin; 88 % of the strains were susceptible to trospectomycin. Overall, all of the -lactam/-lactamase inhibitor combinations, imipenem, meropenem, cefoxitin, tosufloxacin, temafloxacin and clindamycin had good activity against -lactamase producing strains, while all agents tested had good activity against -lactamase negative strains.     

Susceptibility ofAlcaligenes denitrificans subspeciesxylos-oxydans to beta-lactam antibiotics

The susceptibility of 56 clinical isolates and two reference strains ofAlcaligenes denitrificans subsp.xylosoxydans to -lactam agents was tested and related to -lactamase activity of the strains. The MICs of 12 -lactams determined by an agar dilution method showed that all the strains were sensitive to imipenem and moxalactam. Forty-one cloxacillin-sensitive -lactamase producing strains were highly susceptible to azlocillin, piperacillin and ticarcillin, and less susceptible to several cephalosporins (cefamandole, cefoperazone, ceftazidime). The 17 remaining -lactamase-producing strains, which were sensitive to clavulanic acid and to a lesser extent cloxacillin, had variable resistance to the penicillins tested and synergy was obtained when these penicillins were combined with clavulanic acid or tazobactam. The choice of agents for treatment of infections with this organism must take into account the susceptibility phenotype of clinical isolates.     

Selection and properties ofPseudomonas aeruginosa variants resistant to beta-lactam antibiotics

The relation between basal and inducible-lactamase production and resistance to-lactam compounds was studied in five clinicalPseudomonas aemginosa isolates and their corresponding resistant variants selected in the presence of either piperacillin, ceftazidime or aztreonam. In all wild-type strains enzyme levels were barely detectable in the uninduced state and most-lactams, including sulbactam and clavulanic acid, exhibited poor induction potency. Imipenem proved to be the most potent inducer in both these strains and their resistant variants. In the variants selected by either piperacillin or ceftazidime enzyme production amounted to 1.28 units/mg protein of the cell-free supernatants following the addition of-lactams as inducers. Additionally, these variants exhibited the phenomenon of non-specific induction, i.e. the increase of enzyme production by either a complex nutrient medium or by addition of vitamins. Enzyme production in the aztreonam-resistant variants was identical to that in the wild-type strains with a single exception, where the entire derepression of-lactamase production in one of the variants took place. Derepression of the chromosomally mediated enzyme affects the susceptibility to ureidopenicillins more than that to carboxy-penicillins and cephalosporins, whereas the-lactamase-independent resistance results in increased resistance to all-lactams with the single exception of imipenem.     

Changes in the susceptibility ofBacteroides fragilis group organisms to various antimicrobial agents 1979–1989

The in vitro activity of metronidazole, chloramphenicol, clindamycin and 11 -lactam antibiotics against 135 clinical isolates of theBacteroides fragilis group was compared. In addition, changes in the resistance patterns of members of theBacteroides fragilis group isolated at the Hospital Universitario San Carlos in Madrid, Spain, between 1979 and 1989 were documented. The most active -lactam drugs were imipenem and -lactamase inhibitor combinations. In 1989, however, two strains were found to be resistant to imipenem and to all other -lactam agents tested. There was no emergence of resistance to metronidazole. Chloramphenicol was very effective: only one resistant strain was detected in 1979 and no chloramphenicol-resistant isolates were found during the rest of the study period. An outbreak of clindamycin resistance was noted in 1982, and the first cefoxitin resistant strains were recovered in 1985. The changing patterns of susceptibility of anaerobic bacteria to antimicrobial agents and the emergence ofBacteroides fragilis strains resistant to new -lactam agents suggest that periodic antimicrobial susceptibility tests should be performed in order to guide the selection of antimicrobial agents for therapy.     

Estradiol attenuation of beta-amyloid-induced toxicity: a comparison o.

17-estradiol (E2) has been shown to attenuate the toxicity of -amyloid peptides (A) in neuronal cultures with the effective concentration of E2 ranging from low nM to high M. This study compares the effective neuroprotective concentration of E2 against both A-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective E2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum E2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM E2 conferring significant protection in the calcein AM assay but 1 M E2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic A concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM E2 against H_{2 2} toxicity. These results indicate that low concentrations of E2 can attenuate A and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of E2-mediated attenuation of A toxicity.     

Moraxella catarrhalis: Clinical significance, antimicrobial susceptibility and BRO beta-lactamases

Moraxella catarrhalis is an important pathogen of humans. It is a common cause of respiratory infections, particularly otitis media in children and lower respiratory tract infections in the elderly. Colonisation of the upper respiratory tract appears to be associated with infection in many cases, although this association is not well understood. Nosocomial transmission is being increasingly documented and the emergence of this organism as a cause of bacteremia is of concern. The widespread production of a-lactamase enzyme rendersMoraxella catarrhalis resistant to the penicillins. Cephalosporins and-lactamase inhibitor combinations are effective for treatment of-lactamase producers, and the organism remains nearly universally susceptible to the macrolides, fluoroquinolones, tetracyclines and the combination of trimethoprim and sulfamethoxazole. Two major-lactamase forms, BRO-1 and BRO-2, have been described on the basis of their isoelectric focusing patterns. The BRO-1 enzyme is found in the majority of-lactamase-producing isolates and confers a higher level of resistance to strains than BRO-2. The BRO enzymes are membrane associated and their production appears to be mediated by chromosomal determinants which are transmissible by an unknown mechanism. The origin of these novel proteins is unknown.     

Susceptibility ofBacteroides non-fragilis and fusobacteria to amoxicillin,amoxicillin/clavulanate,ticarcillin, ticarcillin/clavulanate,cefoxitin, imipenem and metronidazole

The susceptibility of 234Bacteroides non-fragilis strains and 56 fusobacteria from 12 European centers to amoxicillin, amoxicillin/clavulanate, ticarcillin, ticarcillin/clavulanate, cefoxitin, imipenem and metronidazole was tested and related to -lactamase production. Beta-lactamase production was detected in 42.3 % of theBacteroides strains and 26.8 % of the fusobacteria. The MIC90 of amoxicillin for -lactamase-negative strains was 0.5 µg/ml and the MIC90 of ticarcillin 2.0 µg/ml. In the case of -lactamase-positive strains the MIC90 of amoxicillin (32 µg/ml) and ticarcillin (16 µg/ml) dropped to 1.0 µg/ml upon addition of clavulanate; 65.8 % of these strains were susceptible to amoxicillin and 98.2 % to ticarcillin, but all were susceptible when clavulanate was added. All strains were susceptible to imipenem and metronidazole, and 99.3 % to cefoxitin.     

Haemoglobin D-β-thalassaemia in a German family

Summary This report concerns a young woman, whose stained blood films revealed a hypochromic and microcytic red cell morphology. It could be shown by haemoglobin analysis that this atypical blood film was due to a double heterozygote disorder of haemoglobin D (Hb D) and-thalassaemia. The thalassaemia trait was inherited from her father and the Hb D from her mother. This is the first observation of this rate disorder in a German family. Comparable cases reported in the literature are mentioned.Abbreviations ⁺ residual-chain synthesis - ⁰ no residual-chain synthesis - Glu glutaminic acid - Glu (NH₂) or Gln glutamine - Hb Haemoglobin - MCH mean corpuscular haemoglobin - MCV mean corpuscular volume - MCHC mean corpuscular haemoglobin concentration - Lys lysine     

The ultrastructural effects of beta-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features

SummaryThe fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of - 1–40 (-AP). PC12 cells treated with -AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of -AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and -AP treated cells had two major P-face IMP populations, small-diameter (4–8 nm) IMPs, and large-diameter ( 9nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the -AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the -AP. These results demonstrate that -AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.     

Molecular epidemiology of an outbreak due to extended-spectrum beta-lactamase-producing enterobacteria in a French hospital

Between June 1992 and June 1993, 128 extended-spectrum -lactamase-producing enterobacteria (123Klebsiella pneumoniae, 3Escherichia coli, 1Enterobacter aerogenes, and 1Citrobacter diversus) were collected in a French university hospital. These isolates were recovered mainly from patients hospitalized in intensive care and neurosurgery units. The 128 strains were divided into 14 antibiotypes (ATBs; ATB1 to ATB14); 102 of 103 nonredundant isolates were shown to produce an SHV-4-related extendedspectrum -lactamase (pl 7.8, hybridization with abla _SHV probe); the remaining strain (Kp 2108) produced a TEM-3-related extended-spectrum -lactamase (pI 6.3, hybridization with abla _TEM probe). For representative isolates, five plasmid profiles (PI to PII4), eight ribotypes (E1 to E8), and seven arbitrarily primed polymerase chain reaction profiles (A1 to A7) were obtained. The results suggest the spread of an epidemic strain ofKlebsiella pneumoniae (E1, A1, PI, various ATBs) from an intensive care unit throughout the hospital. Another epidemic strain (E2, A2, PI, ATB4) was confined to the neurosurgery unit. Other extended-spectrum -lactamase-producingKlebsiella pneumoniae of ribotypes distinct from E1 or E2 might result from the spread of an epidemic plasmid, such as reported for isolates of other enterobacterial species. Conversely, they might represent imported cases, such as the strain Kp 2108, which produced a TEM-3-related -lactamase.     

In vitro antibacterial activity and beta-lactamase stability of the new carbapenem SM-7338

The in vitro activity of the new carbapenem SM-7338 was tested in comparison with imipenem, ceftazidime, cefotaxime, flomoxef, cefuzonam and cefmetazole against 2850 clinical bacterial isolates. SM-7338 showed good activity against a broad spectrum of grampositive and gram-negative bacteria. SM-7338 was very active against gram-negative bacteria, inhibiting allEnterobacteriaceae, except 25 % ofSerratia marcescens isolates, at a concentration of 0.78 mg/l. SM-7338 inhibited the majority ofPseudomonas spp. at concentrations of 3.13 mg/l, its activity being twofold higher than that of imipenem. However, the activity of SM-7338 against gram-positive cocci was about one-fourth that of imipenem. Against anaerobes, SM-7338 also had the best activity of the -lactams tested. The compound was inactive against methicillin-resistant staphylococci,Enterococcus faecium, Xanthomonas maltophilia andFlavobacterium spp., as were the other -lactams. SM-7338 was quite stable in the presence of various types of -lactamase, but was hydrolyzed byXanthomonas maltophilia -lactamase, as was imipenem. This high degree of stability was responsible for the potent activity of SM-7388 against -lactamase-producing species such asEnterobacter cloacae, Citrobacter freundii andProteus vulgaris. SM-7338 also showed bactericidal activity against clinical isolates at the MIC or at concentrations slightly above the MIC.     

In vitro activity of biapenem against beta-lactamase producingEnterobacteriaceae

The activity of biapenem was compared with that of imipenem and cefotaxime against 108 strains of -lactamase producingEnterobacteriaceae. Biapenem and imipenem were very active, inhibiting 90 % of the strains at a concentration of 0.5 µg/ml. Both carbapenems were very active against plasmidic -lactamase producers, with MIC90s below 1 µg/ml. However, the MIC90 of biapenem for cephalosporinase producers was 1 µg/ml. Against strains producing extended-spectrum -lactamases, biapenem exhibited better activity against TEM-type producers (MIC90 0.25 µg/ml) than against SHV-type producers (MIC90 0.5 µg/ml). Overall, the in vitro antibacterial activity of biapenem is similar to that of imipenem.