Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain

Sixteen amikacin-resistant clinical Acinetobacter baumannii isolates from nine different hospitals in Spain were investigated to determine whether the high incidence of amikacin-resistant A. baumannii was due to the dissemination of an amikacin-resistant strain or to the spread of an amikacin resistance gene. The epidemiological relationship studied by repetitive extragenic palindromic PCR and low-frequency restriction analysis of chromosomal DNA showed that the same clone was isolated in eight of nine hospitals, although other clones were also found. The strains were studied for the presence of the aph(3')-VIa and aac(6')-I genes, which encode enzymes which inactivate amikacin, by PCR. All 16 clinical isolates had positive PCRs with primers specific for the amplification of the aph(3')-VIa gene, whereas none had a positive reaction for the amplification of the aac(6')-I gene. Therefore, the high incidence of amikacin resistance among clinical A. baumannii isolates in Spain was mainly due to an epidemic strain, although the spread of the aph(3')-VI gene cannot be ruled out.     

In vitro activities of antimicrobial agents, alone and in combination, against Acinetobacter baumannii isolated from blood

In vitro activities of 15 antimicrobial agents against 90 strains of Acinetobacter baumannii isolated from blood cultures from hospitalized patients were determined using the agar dilution method. Imipenem, ofloxacin, and ciprofloxacin had the best antimicrobial activity with minimum inhibitory concentrations (MIC50s) of 0.25 mu g/ml and MIC90s of 0.5-1 mu g/ml. beta-lactam antibiotics other than imipenem had poor activity, with MIC50s ranging from 8 to 64 mu g/ml and MIC90s from 32 to > or = 256 mu g/ml. The checkerboard titration method was used to study the effects of combination of two antimicrobial agents. Combinations of ceftazidime, aztreonam, imipenem, or ciprofloxacin with amikacin showed either synergistic effects or partial synergistic effects for 40.9%-86.4% of 22 tested strains. The best in vitro activity was observed with the combination of imipenem and amikacin. No antagonistic effects were observed with the combination of imipenem and amikacin. Synergistic effects were confirmed by time-kill curve studies. In conclusion, imipenem, ofloxacin, and ciprofloxacin were the three most active agents against human blood isolates of A. baumannii. The combination of a beta-lactam or ciprofloxacin with amikacin was synergistic for some of the isolates.     

Antimicrobial resistance and epidemiological study of Haemophilus influenzae strains isolated in Portugal. The Multicentre Study Group

In the course of a multicentric surveillance study, nine laboratories sent 375 isolates of Haemophilus influenzae to the Sector de Resistência aos Antibióticos (SRA) from the National Institute of Health in Lisbon, between 1 January and 31 December 1992. The majority of the H. influenzae isolates were from the respiratory tract (84.8%); only 5.1% were of invasive origin. Overall resistance for ampicillin was 11.7%, tetracycline 3.7%, and chloramphenicol 2.4%. All isolates tested were fully susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Multiresistance was rare, occurring only in 2.4% of the isolates, although 50% of the ampicillin resistant strains had at least one additional resistance marker. Forty two isolates (11.2%) produced a TEM-1 type beta-lactamase, as shown by isoelectric focusing. beta-lactamase production was not detected in two of the ampicillin resistant strains. Fifteen of the 42 beta-lactamase producing strains (35.7%) contained detectable DNA plasmid: nine harboured large plasmids with an apparent molecular mass of 45 or 54 kb depending on their resistance phenotype and six harboured a small plasmid of 5 kb. In order to study transfer of resistance in both ampicillin and multiresistant strains conjugation experiments were performed for 14 isolates, seven of which harboured a large plasmid and seven had no detectable plasmid DNA. All 14 transferred their resistance phenotype but only a single large plasmid could be demonstrated in ten transconjugants. Restriction endonuclease analysis of plasmids from six representative transconjugants, isolated in different hospitals, revealed that there was no dissemination of a single R plasmid, which suggests an independent process of acquisition of resistance genes.     

E-test to study small inhibitor concentrations, bacterial diversity and to identify presumptively beta-lactamases in strains of Pseudomonas aeruginosa and Acinetobacter baumannii associated with +nosocomial infections

The objectives of the present study were to characterize the surface expression of low density lipoprotein receptor (LDL-R) in Epstein-Barr virus transformed lymphocytes (EBV-L) and to determine the applicability of the cellular system for the study of familial hypercholesterolemia. The EBV-L subsets and LDL-R expression were determined by immuno-cytofluorimetry. The LDL-R expression in EBV-L which consisted of mostly B cells was no different among antigenic subsets. EBV-L cultured in lipoprotein deficient serum demonstrated a 9.3-fold higher LDL-R expression than primary lymphocytes. Lovastatin caused an additional 1.9-and 1.4-fold increase in EBV-L and primary lymphocytes respectively. This difference in lovastatin response is statistically significant (paired t-test, P < 0.001). 54% of the high LDL-R expression in EBV-L was related to the changes in proliferation measured as stimulation index (SI). LDL and lovastatin modulated the LDL-R expression without affecting SI. FH subjects demonstrated 2% (homozygote, n = 1) and 44.6 +/- 12.3% (heterozygotes, n = 35) in LDL-R expression of controls (n = 30). This maintenance of the FH phenotype and the intrinsically high LDL-R expression in EBV-L make the cellular system suitable for the study of FH as well as the regulation of LDL-R.     

Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived beta-lactamase in Acinetobacter baumannii

A clinical strain of Acinetobacter baumannii (strain Ab41) that was resistant to all beta-lactam antibiotics tested except ceftazidime, ceftriaxone, ceftizoxime, and imipenem produced three beta-lactamases: a presumptive chromosomal cephalosporinase, a TEM-1-like beta-lactamase (pI 5.4), and a novel OXA-derived beta-lactamase named OXA-21 (pI 7.0). The gene encoding OXA-21 was located in an integron. The nucleotide sequence showed three mutations compared with the sequence of OXA-3, with two being silent; the nonsilent mutation generated a substitution of Ile-217 to Met.     

Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes

A series of 74 Yersinia enterocolitica clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with HindIII and BglI and using an rrn operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0. 56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3-biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that Y. enterocolitica O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.     

Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii

The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47,423 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 2-ketoglutaric acid<-->L-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence. Therefore, the gene and its product were named dat and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively. Feeding experiments of A. baumannii with L-[U-14C]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane. Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting. These results indicate that the dat gene (as well as the ddc gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1,3-diaminopropane synthesis is unknown.     

Clinical importance and antibiotic resistance of Acinetobacter spp. Proceedings of a symposium held on 4-5 November 1996 at Eilat, Israel

Members of the genus Acinetobacter, particularly multiresistant strains of A. baumannii, are implicated in a wide spectrum of nosocomial infections, including bacteraemia, secondary meningitis and urinary tract infection, but have now assumed a particularly important role as agents of nosocomial pneumonia in intensive care units (ICUs). Rapid genotyping methods for the identification and typing of these organisms have allowed a better appreciation of the epidemiology and survival of these organisms in the hospital environment. Their emergence as significant pathogens seems to be related partly to their survival ability and partly to their ability to develop resistance rapidly to the major groups of antibiotics, resulting in a considerable selective advantage in environments (such as ICUs) with widespread and heavy uses of antibiotics. Molecular and biochemical mechanisms of resistance to the major beta-lactam, aminoglycoside and quinolone groups of antibiotics have now been elucidated in some detail for these organisms, and experimental models, including a mouse model of A. baumannii pneumonia, have been developed to examine the efficacy of different therapeutic regimens for difficult-to-treat-infections caused by these bacteria. 'Non-classic' antibiotic combinations--such as ticarcillin with clavulanic acid and sulbactam--seem to show promise for treating systemic infections caused by otherwise multiresistant strains, but revised screening procedures in the pharmaceutical industry may be required in the near future to select novel compounds with activity against multiresistant Acinetobacter spp. and other emerging gram-negative, non-fermentative bacilli in general.     

Inhibition of cyclin D1 expression and phosphorylation of retinoblastoma protein by phosmidosine, a nucleotide antibiotic

In this report, we studied the effect of phosmidosine, a proline-containing nucleotide on the serum-induced cell cycle progression in human lung fibroblast WI-38 cells. Phosmidosine suppressed S-phase entry and arrested cell cycle progression at the G1 phase. In serum-stimulated cells, phosmidosine did not affect the activation of the mitogen-activated protein kinase cascade. However, phosmidosine inhibited hyperphosphorylation of retinoblastoma (RB) protein by RB-kinases such as cyclin-dependent kinase 4 and cyclin-dependent kinase 2, probably as a result of the inhibition of cyclin D1 expression. Furthermore, in tsFT210 cells, a temperature-sensitive cdc2 mutant isolated from the mouse mammary carcinoma cell line FM3A, phosmidosine, irreversibly inhibited the cell cycle progression at G1 without affecting the G2 to M transition. Phosmidosine acts at an earlier point in G1 compared with mimosine or aphidicolin, well-known cell cycle blockers at the G1-S boundary. Taken together, phosmidosine arrested cells at a specific point between the start point and restriction point in G1 and is a useful drug that may contribute to the understanding of the regulatory mechanisms of G1 progression.     

Detection and treatment of psychiatric illness in a general medical ward: a modified cost-benefit analysis

This article describes a prospective, randomized, controlled trial of screening and treatment for psychiatric disorder in medical in-patients. The study has assessed whether increased recognition of psychiatric disorder among medical in-patients improves clinical outcome and reduces the costs of care, and whether routine involvement of a psychiatrist in the assessment and care of medical in-patients with probable psychiatric disorder is superior to the efforts of the physicians alone. A total of 218 medical in-patients who scored over the screening threshold for psychiatric disorder on the General Health Questionnaire were randomly allocated to one of two intervention groups or a control group. Six months later their mental health, subjective health status, quality of life, and costs of care was reassessed. Mental health and quality of life at 6 months were similar in the two intervention groups and the control group. Patients whose physicians were told the results of the screening test had lower costs for subsequent admissions, but this was probably due to differences between the groups in terms of employment status. Treatments recommended by psychiatrists broke down when patients were discharged home, leading to inadequate treatment of psychiatric disorders. We have not been able to show that routine screening for psychiatric disorder produces any benefit, either in better outcome for patients or reduced costs for the NHS. Further research should: consider examining a more homogeneous group in terms of costs of care; screen only for disorders likely to respond to a specific treatment; and ensure that treatment recommendations are carried out.     

Genetic diversity of nifH gene sequences in paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic profile of the dolastatin 15 analog LU103793 when administered daily for 5 days every 3 weeks. PATIENTS AND METHODS: Fifty-six courses of LU103793 at doses of 0.5 to 3.0 mg/m2 were administered to 26 patients with advanced solid malignancies. Pharmacokinetic studies were performed on days 1 and 5 of course one. Pharmacokinetic variables were related to the principal toxicities. RESULTS: Neutropenia, peripheral edema, and liver function test abnormalities were dose-limiting at doses greater than 2.5 mg/m2 per day. Four of six patients developed DLT at 3.0 mg/m2 per day, whereas two of 12 patients treated at 2.5 mg/m2 per day developed DLT. Pharmacokinetic parameters were independent of dose and similar on days 1 and 5. Volume of distribution at steady-state (Vss) was 7.6 +/- 2.0 L/m2, clearance 0.49 +/- 0.18 L/h/m2, and elimination half-life (t1/2) 12.3 +/- 3.8 hours. Peak concentrations (Cmax) on day 1 related to mean percentage decrement in neutrophils (sigmoid maximum effect (Emax) model). Patients who experienced dose-limiting neutropenia had significantly higher Cmax values than patients who did not, whereas nonhematologic DLTs were more related to dose. CONCLUSION: The recommended dose for phase II evaluations of LU103793 daily for 5 days every 3 weeks is 2.5 mg/m2 per day. The lack of prohibitive cardiovascular effects and the generally acceptable toxicity profile support the rationale for performing disease-directed evaluations of LU103793 on the schedule evaluated in this study.     

PspA, a protection-eliciting pneumococcal protein: immunogenicity of isolated native PspA in mice

PspA is a surface exposed virulence factor of S. pneumoniae that can elicit protective immunity to pneumococcal sepsis in mice. It can be released from pneumococci by washing them with a solution containing 2% choline chloride, by growing pneumococci in media containing 1.2% choline chloride, or by growing pneumococci in media in which the choline has been replaced by ethanolamine. Our results indicate that PspA is the major protection-eliciting antigen in each of these preparations. Two injections of < or = 1 microgram of native PspA purified by use of a choline-Sepharose column are highly immunogenic in BALB/c and CBA/N mice, and even in the absence of adjuvant can elicit protection against otherwise fatal sepsis with 100 times the LD50 of S. pneumoniae. Fragments comprising the N-terminal 115 and 245 amino acids of PspA were able to elicit protection but only in the presence of complete Freund's adjuvant (CFA). In the absence of CFA the 245 amino acid fragment was less than 1/100 as immunogenic as native PspA.     

Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic marker (bioluminescence) for monitoring the environmental fate of rhizobacterium

Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant estimating the wild-type population. Seed and root samples were taken 0, 7, 14, 21, or 28, 35 or 42, and 70 days after planting in each year and processed for enumeration by spiral plating or immunofluorescent colony staining (IFC). In both years, the populations of 89B-27, R34, and GEM-8, as measured by IFC, were not significantly different (P > 0.05) from each other at each sampling time. However, the populations of R34 and GEM-8, as measured by spiral plating and differentiation based on their respective phenotypes, were significantly lower (P < 0.05) than the wild-type populations and their IFC-determined populations. These data indicate that traditional marker systems may underestimate populations and hence the survival and colonization of genetically marked bacteria.     

Typing, resistance behavior and occurrence of methicillin-resistant Staphylococcus aureus strains in a surgical intensive care unit

Over a period of three years, the frequency of the appearance of methicillin-resistant S. aureus strains (MRSA) was observed on a surgical intensive care unit. During this above-mentioned period of investigation it came to a heaped occurrence of nosocomial infections on this ICU with altogether 332 S. aureus-stems being isolated from different patient specimen. 204 (61.5%) of these were resistant against methicillin and could be divided into 48 first- and 156 follow-up-isolates. The thereupon accomplished differentiation of the 48 MRSA-first isolates by means of lysotyping and the pioneered GenePath Strain Typing System for a standardized pulsed-field-gel-electrophoresis (PFGE) gave the proof of 7 different MRSA-types. Around 7 different, in part parallel chains of infection on this ICU were observed, which could be led back to different strains. In reference to all analyzed S. aureus, an especially high rate (90%) of MRSA on this ICU could be isolated in taken wound-swabs, followed by 83.3% MRSA at catheter tips and 71,9% in tracheal and bronchial secretion. A consideration of the antibiotic susceptibility yielded, that also gentamicin and the quinolones showed an in-vitro resistance against MRSA, while fosfomycin, fusidic acid, chloramphenicol and trimethoprim/sulfamethoxazole reached positive responding rates between 80 and 100%. On the other hand, presently still 100% of the explored MRSA-strains are susceptible for glycopeptides such as vancomycin and teicoplanin. Because of intensive hospital hygienic measures the number of newly isolated MRSA could be reduced clearly on this ward.     

Analysis of clonal diversity of the peach-potato aphid, Myzus persicae (Sulzer), in Scotland, UK and evidence for the existence of a predominant clone

Clones of the peach-potato aphid, Myzus persicae (Sulzer), mostly from Scotland, UK were examined using an rDNA fingerprinting technique. Eighty patterns (genotypes) were found amongst the 276 clones. A large number of clones (30%) from all sample areas in Scotland exhibited the same simple pattern, suggesting the presence of a single M. persicae clone. There was no difference in genotype distributions between M. persicae collected from brassica or potato crops, suggesting that host-adapted genotypes have no advantage in the field. Different fingerprints were randomly distributed in the environment, although clones taken from the same leaf were more often the same fingerprint. Highly distinctive fingerprints, which were more widely distributed, suggest that this technique could be used to follow individual clones. In addition to the common clonal type, multiple fingerprint bands were found over successive years, implying that, in Scotland, local overwintering asexual populations are the most common source of M. persicae in the following year.     

Proxy informant reliability and bias in epidemiological research: analysis of a screening questionnaire for mental disorders

OBJECTIVES: To evaluate the reliability, magnitude and direction of the resulting bias in the application of a screening instrument for mental disorders by considering proxy informants in comparison to primary informants. METHODS: Data are taken from a general morbidity community-based survey carried out in 520 randomly selected households of an industrial area of the Metropolitan Region of Salvador, the capital of Bahia state, Brazil. During the pilot phase, the first 70 families of the total sample were asked to participate in the evaluation of research instruments. The Questionnaire of Adult Psychiatric Morbidity, QAPM, consists of 44 questions about psychiatric symptoms widely used in Brazil. The husbands and wives of the selected families answered QAPM questions regarding themselves and their respective partners. One family refused to participate. The Kappa index was estimated for each QAPM question. To assess the magnitude and direction of bias, the proportional variation of prevalence was estimated from proxy and primary respondents. Each informant was analyzed as a primary informant when answering about his/her own symptoms and as a proxy informant when answering those about his/her partner. RESULTS: Proxy informants as compared to primary informants show weak reliability, as measured by the Kappa Index, particularly when husbands reported on their wives' symptoms. An overall underestimation of prevalence estimates was found, which reveals the potential negative bias with the use of proxy informants for psychological symptoms. No bias was found for only two questions (lack of appetite and globus hystericus) when women were taken as proxy informants for their husbands. In addition, departures of proxy informants from primary informant-based estimates were greater among men than to women. CONCLUSIONS: Proxy informants underestimate the occurrence of psychological symptoms in this community-based study. When the feasibility of a research project, based on the QAPM depends on the use of proxies, wives may be recommended as better informants than their husbands.     

Dose-dependent plasma clearance of MK-826, a carbapenem antibiotic, arising from concentration-dependent plasma protein binding in rats and monkeys

After intravenous administration of MK-826, a new carbapenem antibiotic, the compound exhibited nonlinear pharmacokinetics in rats and monkeys. In both species, time-averaged plasma clearance (based on total concentrations) increased about 5-fold over the 10- to 180-mg/kg dose range. MK-826 was extensively plasma protein bound in rat and monkey plasma, and the extent of binding was concentration dependent at plasma concentrations achieved after administration of these doses. Rosenthal analysis of the plasma protein binding indicated that there were two classes of binding sites. The binding capacity of the primary site was comparable to the plasma albumin concentration, which suggested that this primary site consisted of a single site on albumin. The extent of binding of MK-826 to rat albumin was similar to that in whole plasma. Clearance values based on unbound concentrations appeared independent of dose from 10 to 180 mg/kg, which is consistent with saturation of protein binding as the primary cause of the nonlinear pharmacokinetic behavior.