First isolations of KPC-2-carrying ST258 Klebsiella pneumoniae strains in Finland, June and August 2009.

The first two Klebsiella pneumoniae carbapenemase-producing (KPC) type 2 strains carrying ST258 were detected in Finland in June and early August 2009. They were found colonising two patients transferred from the Mediterranean; one patient referred from a hospital in Greece where isolates were first found in 2007 and another from Italy where the first isolates have been described only very recently.

Relapsing sepsis episodes of Escherichia coli with CTX-M ESBL or derepressed ampC genes in a patient with chronic autoimmune pancreatitis complicated by IgG4 hypergammaglobulinaemia.

Bloodstream recurrent infections have been reported for a variety of opportunistic bacteria. These are often either catheter related or are caused by indwelling devices. A case of relapsing sepsis with two Escherichia coli strains carrying extended-spectrum ß-lactamase and derepressed ampC genes is reported. The patient had seven episodes of bloodstream infections within 1 year and was diagnosed with chronic autoimmune pancreatitis and IgG4 hypergammaglobulinaemia. Abscesses were found in his spleen and pancreas cauda, which was finally resected. Relapses of bacteraemia with resistant enterobacteria should be considered during perioperative protection. Surgical removal of the infective focus could be curative.

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells.

In order to study the processing of rubella virus (RV) structural proteins (capsid protein, of 33 kDa; E2 of 42-47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.

The effect of a combination of Iso-Sensitest agar and ampicillin on the detection of enterobacter beta-lactamase.

The ampicillin MICs for faecal strains of E. cloacae (n = 11) and Enterobacter species not identified to species level (n = 3) were noticed to be two-fold to four-fold lower on Iso-Sensitest than on Mueller-Hinton II agar. The levels of inducible beta-lactamase on Iso-Sensitest, Mueller-Hinton, and a selective MacConkey-based agar, with and without ampicillin, were determined. The level produced on Iso-Sensitest with ampicillin was comparable to that induced by Mueller-Hinton alone. We conclude that when routine procedures for the detection of beta-lactamase-based resistance are evaluated, not only should care be taken to choose the right antibiotics, but also different combinations of beta-lactams and agar should be tested.

Screening for antimicrobial resistance in fecal samples by the replica plating method.

Replica plating can be used for the detection of antibiotic resistance in normal flora. We have evaluated this application of the replica plating method by comparing it with a five-colony method. The replica plating method uses a single plate for each antibiotic, with a concentration just above that for borderline resistance. By the five-colony method, five colonies per sample were picked, chosen to represent all different colony morphologies present, and MICs were determined by a standard agar dilution method. The gram-negative, aerobic floras of 131 fecal samples were screened for resistance to ampicillin, cefuroxime, nalidixic acid, trimethoprim, sulfamethoxazole, and tetracycline by both methods. The rate of resistance detection by the two methods did not differ statistically for any of the antibiotics tested. The breakpoint concentrations used for the replica plates in the study gave results similar to those produced by the agar dilution method and the breakpoint values of the National Committee for Clinical Laboratory Standards and can thus be recommended. As the only currently used resistance detection method, replica plating facilitates an exact determination of the percentage of resistant colonies/total number of colonies (between 1 and 100%) in a sample. This revealed an uneven distribution, with only 23% of the samples having resistance frequencies in the range of 10 to 85%; usually, the resistant flora either was a small minority or was very dominant in samples with resistance. This phenomenon was present for all of the antibiotics.

Typing of group A streptococci by random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) analysis was evaluated in comparison with restriction endonuclease analysis (REA) of genomic DNA and serotyping in the typing of 160 epidemiologically unrelated group A streptococci (GAS). Amplification of genomic DNA of GAS was performed with a single primer with an arbitrarily selected nucleotide sequence of 12 nucleotides. In total, 31 RAPD patterns and 15 REA patterns were observed among the isolates studied. The results of RAPD analysis were in accordance with the results of REA for 86% of the isolates, as both methods identified 15 different strains among 138 isolates. However, RAPD analysis differentiated 16 additional strains among 22 isolates. RAPD analysis was somewhat better than REA for differentiation of isolates of the same and different serotypes. However, not all of the serotypes were differentiated by RAPD analysis either. In conclusion, RAPD analysis provides a practical alternative for genomic typing of GAS. It can be recommended for the typing of GAS, especially if used in parallel with serotyping.

Antibiotic resistance. How wild are wild mammals?

In bacteria associated with humans, antimicrobial resistance is common, both in clinical isolates and in the less-studied commensal flora, and it is thought that commensal and environmental bacteria might be a hidden reservoir of resistance. Gilliver et al. have reported that resistance is also prevalent in faecal bacteria from wild rodents living in northwest England. Here we test the faeces of moose, deer and vole in Finland and find an almost complete absence of resistance in enterobacteria. Resistance is thus not a universal property of enterobacterial populations, but may be a result of the human use of antibiotics.

Antimicrobial and mercury resistance in aerobic gram-negative bacilli in fecal flora among persons with and without dental amalgam fillings.

Antimicrobial resistance is more widespread than can be accounted for as being a consequence of the selection pressure caused by the use of antibiotics alone. In this study, we tested the hypothesis that a high mercury content in feces might select for mercury-resistant bacteria and thus for antimicrobial resistance linked to mercury resistance. Three subject groups with different exposures to dental amalgam fillings were compared. None of the subjects had taken antimicrobial agents during the three preceding months or longer. The group exposed to dental amalgam (n = 92) had 13 times more mercury in feces than the group that had never been exposed to amalgam (n = 43) and the group whose amalgam fillings had been removed (n = 56). No significant differences in either mercury resistance or antibiotic resistance in the fecal aerobic gram-negative flora of these subject groups were seen. The following antimicrobial resistance frequencies were detected with a replica plating method: > or = 1% resistance was seen in 40% of the subjects for ampicillin, 14% of the subjects for cefuroxime, 6% of the subjects for nalidixic acid, 14% of the subjects for trimethoprim, 19% of the subjects for sulfamethoxazole, and 25% of the subjects for tetracycline. The amount of mercury in feces derived from amalgam was not selective for any resistance factors in aerobic gram-negative bacteria, but antimicrobial resistance was widespread even among healthy subjects with no recent exposure to antibiotics.

Problem of antimicrobial resistance of fecal aerobic gram-negative bacilli in the elderly.

In this study, we assessed the magnitude of risk (odds ratio [OR]) of patients being colonized with fecal aerobic gram-negative bacilli in two geriatric hospitals compared with the community, and we associated the use of antimicrobial agents with bacterial resistance. One fecal sample was collected from each of 341 patients, aged 60 years or older, during the hospital stay or when visiting the outpatient service. Samples were collected in 1988 and 1993 to 1994. The aerobic gram-negative bacilli from all samples were examined for resistance to seven antimicrobials by a replica plating method. The long-term-hospitalized patients had a significantly higher risk of being colonized with bacilli resistant to ampicillin (OR, 14.3; 95% confidence interval [95% CI], 6.0 to 34.1), cefuroxime (OR, 7.5; 95% CI, 2.7 to 20.8), trimethoprim (ORs, 22.3; 95% CI, 8.6 to 57.8), and tetracycline (OR, 5.2; 95% CI, 2.4 to 10.9) than the outpatients. The respective ORs among the short-term-hospitalized patients compared with the outpatients were 4.0 (95% CI, 1.9 to 8.4), 7.5 (95% CI, 2.7 to 20.8), 5.5 (95% CI, 2 to 14), and 2.0 (95% CI, 1 to 4). In 1993 to 1994 compared with 1988, in both hospitals there was a significantly increased risk of colonization by bacilli resistant to ampicillin (OR, 3.1; 95% CI, 1.9 to 5.1), cefuroxime (OR, 3.8; 95% CI, 2.1 to 6.7), and tetracycline (OR, 1.6; 95% CI, 1.0 to 2.5). However, the total use of antimicrobial agents increased only among the patients of the short-term-care hospital.

Dental amalgam fillings and the amount of organic mercury in human saliva.

We studied differences in the amounts of organic and inorganic mercury in saliva samples between amalgam and nonamalgam human study groups. The amount of organic and inorganic mercury in whole saliva was measured in 187 adult study subjects. The mercury contents were determined by cold-vapor atomic absorption spectrometry. The amount of organic and inorganic mercury in paraffin-stimulated saliva was significantly higher (p<0.001) in subjects with dental amalgam fillings (n = 88) compared to the nonamalgam study groups (n = 43 and n = 56): log(e) (organic mercury) was linearly related to log(e) (inorganic mercury, r(2) = 0.52). Spearman correlation coefficients of inorganic and organic mercury concentrations with the number of amalgam-filled tooth surfaces were 0.46 and 0.27, respectively. Our results are compatible with the hypothesis that amalgam fillings may be a continuous source of organic mercury, which is more toxic than inorganic mercury, and almost completely absorbed by the human intestine.

Screening for antimicrobial resistance in normal bacterial flora of the skin using the replica plating method.

The replica plating method was evaluated for detection of the antimicrobial resistance of normal bacterial flora of the skin and was compared with the results of a ten-colony method. If > or = 10% of the colonies from the master plate grew on a plate containing an antibiotic, the sensitivity of replica plating was comparable to that of a ten-colony method for samples containing resistant bacteria. However, this method classified significantly more samples as resistant to all eight antibiotics tested if the detection breakpoint was lowered to > or = 1% of the original colonies. Replica plating is an effective and practical tool for screening skin flora for resistance, also in samples with a low proportion of resistant strains.

Colonization of resistant faecal aerobic gram-negative bacilli among geriatric patients in hospital and the community.

Among the elderly most infections are caused by organisms of faecal origin. The study of the resistance of such Gram-negative bacilli should therefore be a priority. In this study, we determine the occurrence of resistance to five antimicrobials commonly used in geriatric outpatient care, and compare it with long-term and short-term hospitalized geriatric patients treated and not treated with antimicrobials.

Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables.

There is potential for the normal faecal flora of humans to be augmented by resistant strains of bacteria, acquired from food. The frequency of resistance in the aerobic Gram-negative faecal flora is often very high. The purpose of this study was to find out whether food strains contribute to this resistance. One hundred and thirty-seven vegetable samples were studied, 48 of Finnish origin, and 89 imported. From these samples, 535 different strains of bacteria belonging to the family Enterobacteriaceae were isolated. Enterobacter spp. were most frequent, Escherichia coli was rare. Sensitivity testing was undertaken only for isolates with different biotypes and antibiograms. No resistance was found to cefotaxime, aztreonam, imipenem, gentamicin, nalidixic acid or ciprofloxacin. The frequency of trimethoprim resistance was 0.2%, sulphamethoxazole resistance 1.3%, and tetracycline resistance 5.5%. These frequencies were much lower than those found in faecal flora. Chloramphenicol and cefuroxime resistance was found in 12% and 14% of isolates, respectively. The only statistically significant differences between the Finnish and imported strains were for these two; the Finnish isolates were more resistant to cefuroxime, whereas the imported ones were more resistant to chloramphenicol. Consequently, bacteria from vegetables are not responsible for the high prevalence of resistant Enterobacteriaceae in faecal flora in Finland; they are in fact unusually susceptible to the antibiotics studied. Multiresistance profiles, typical of strains associated with human activities, were not identified in these isolates.

Resistance to mercury and antimicrobial agents in Streptococcus mutans isolates from human subjects in relation to exposure to dental amalgam fillings.

Resistance to cefuroxime, penicillin, tetracycline, and mercury is reported for 839 Streptococcus mutans isolates from 209 human study subjects. The MICs of these drugs did not differ for isolates from one dental amalgam group and two nonamalgam subsets: a group with no known exposure to amalgam and a group whose members had their amalgam fillings removed.

A between-species comparison of antimicrobial resistance in enterobacteria in fecal flora.

Enterobacteria in fecal flora are often reported to be highly resistant. Escherichia coli is the main species; resistance data on other species are rare. To assess the effect of the host's environment, antimicrobial resistance was determined in fecal species of the family Enterobacteriaceae from three populations: healthy people (HP)(n = 125) with no exposure to antimicrobials for 3 months preceding sampling, university hospital patients (UP) (n = 159) from wards where the antibiotic use was 112 defined daily doses (DDD)/bed/month, and geriatric long-term patients (LTP) (n = 74) who used 1.8 DDD/bed/month. The mean length of hospital stay was 5 days for the UP and 22 months for the LTP. The isolates were identified to at least genus level, and MICs of 16 antimicrobials were determined. From the university hospital, resistance data on clinical Enterobacteriaceae isolates were also collected. Resistance data for on average two different isolates per sample (range, 1 to 5) were analyzed: 471 E. coli isolates and 261 other Enterobacteriaceae spp. Resistance was mainly found among E. coli; even in HP, 18% of E. coli isolates were resistant to two or more antimicrobial groups, with MIC patterns indicative of transferable resistance. Other fecal enterobacteria were generally susceptible, with little typically transferable multiresistance. Clinical Klebsiella and Enterobacter isolates were significantly more resistant than fecal isolates. The resistance patterns at both hospitals mirrored the patterns of antibiotic use, but LTP E. coli isolates were significantly more resistant than those from UP. Conditions permitting an efficient spread may have been more important in sustaining high resistance levels in the LTP. E. coli was the main carrier of antimicrobial resistance in fecal flora; resistance in other species was rare in the absence of antimicrobial selection.

Evaluation of methods for epidemiologic typing of group A streptococci.

Serotyping is widely used for epidemiologic investigation of group A streptococci (GAS). To evaluate molecular typing methods of GAS, restriction endonuclease analysis (REA) of genomic DNA and analysis of DNA restriction fragment length polymorphism of rRNA genes (ribotyping) were used in parallel with serotyping. The genomic DNA of 239 epidemiologically unrelated GAS isolates from human invasive infections was digested with HindIII restriction enzyme. Both REA and ribotyping differentiated subclasses within serotypes. However, they did not consistently differentiate between isolates of different serotypes. Ribotyping was less discriminatory than REA. Within the T1M1 serotype, often associated with invasive GAS infections, 92% of the REA patterns were identical, suggesting a common origin for these isolates. Most other serotypes studied were more heterogenic. Among 32 isolates nontypeable by serotyping, 11 distinct REA patterns and 5 ribotypes were identified. REA and ribotyping are useful supplementary tools for classification of GAS and can add to the discriminatory power of serotyping.

Antimicrobial resistance of fecal aerobic gram-negative bacilli in different age groups in a community.

We measured the occurrence of antimicrobial resistance in fecal aerobic gram-negative bacilli by age in community subjects. For none of the eight antimicrobial agents studied were there any statistically significant differences in the carriage rates of resistance in different age groups. Bacterial resistance was common in all age groups, including the children, and occurred for all antimicrobial agents tested.

Increase of antimicrobial resistance of faecal aerobic gram-negative bacteria in a geriatric hospital.

Antimicrobial resistance of faecal aerobic Gram-negative bacteria to eight different antimicrobials was determined by a velvet replica-plating method in 1988 and 1933. Faecal samples were taken from 131 geriatric inpatients in the Turku City Hospital with a hospitalization of more than 7 days. From 1987 to 1992 the use of first and second generation cephalosporins and ciprofloxacin increased from 3.32 defined daily doses (DDD) per bed to 24.25 DDD/bed and from 0.63 DDD/Bed to 28.11 DDD/bed, respectively. A statistically significant increase was observed in the frequency of samples resistant (with >= 1% of resistant colonies) to cefuroxime (p = 0.0004) and ceftazidime (p = 0.037) in patients who received antimicrobial therapy and to ampicillin (p = 0.046) in patients who had not received antimicrobial therapy. In addition, despite the decreased use of sulphonamides and trimethoprim (from 17.11 DDD/bed to 5.54 DDD/bed) no significant changes in the frequency of resistant faecal samples were observed. Use of ciprofloxacin has been found to cure resistance plasmids from bacteria in vitro. However, despite the increased use of ciprofloxacin, no decrease in faecal bacteria resistant to any of the other antimicrobials (i.e. trimethoprim) studied was observed.

Immunoaffinity purification of baculovirus-expressed rubella virus E1 for diagnostic purposes.

Three monoclonal antibodies, termed 4E10, 1E11:10, and 2D9:1, were generated against rubella virus. Immunoblot analysis with purified authentic rubella virus or recombinant baculovirus-expressed rubella virus structural proteins E1, E2, and C demonstrated that they were directed against the E1 envelope glycoprotein of the rubella virus particle. By using the yeast Ty virus-like particle system, it was possible to map the binding site of 1E11:10 within amino acids 236 to 286 of the E1 protein and the binding sites of 2D9:1 and 4E10 outside this region. Immunoaffinity purification with these monoclonal antibodies made it evident that they are useful for obtaining large quantities of pure baculovirus-expressed rubella virus envelope protein E1. The diagnostic potential of this immunoaffinity-purified recombinant rubella virus E1 protein compared with that of authentic rubella virus is demonstrated.