The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition

The new beta-lactamase SHV-72 was isolated from clinical Klebsiella pneumoniae INSRA1229, which exhibited the unusual association of resistance to the amoxicillin-clavulanic acid combination (MIC, 64 microg/ml) and susceptibility to cephalosporins, aztreonam, and imipenem. SHV-72 (pI 7.6) harbored the three amino acid substitutions Ile8Phe, Ala146Val, and Lys234Arg. SHV-72 had high catalytic efficiency against penicillins (k(cat)/K(m), 35 to 287 microM(-1) x s(-1)) and no activity against oxyimino beta-lactams. The concentration of clavulanic acid necessary to inhibit the enzyme activity by 50% was 10-fold higher for SHV-72 than for SHV-1. Molecular-dynamics simulation suggested that the Lys234Arg substitution in SHV-72 stabilized an atypical conformation of the Ser130 side chain, which moved the O gamma atom of Ser130 around 3.5 A away from the key O gamma atom of the reactive serine (Ser70). This movement may therefore decrease the susceptibility to clavulanic acid by preventing cross-linking between Ser130 and Ser70.     

EPCAM Germ Line Deletions as Causes of Lynch Syndrome in Spanish Patients

The standard genetic test for Lynch syndrome (LS) frequently reveals an absence of pathogenic mutations in DNA mismatch repair genes known to be associated with LS. It was recently shown that germ line deletions in the last exons of EPCAM are involved in the etiology of LS. The aim of this study was to evaluate the prevalence of EPCAM deletions in a Spanish population and the clinical implications of deletion. Probands from 501 families suspected of having LS were enrolled in the study. Twenty-five cases with MSH2 loss were identified: 10 had mutations of MSH2, five had mutations of MSH6, and 10 did not show MSH2/MSH6 mutations. These 25 cases were analyzed for EPCAM deletions using multiplex ligation-dependent probe amplification, and deletions were mapped using long-range PCR analysis. One subject with no MSH2/MSH6 mutations had a large deletion in the EPCAM locus that extended for 8.7 kb and included exons 8 and 9. The tumor exhibited MSH2 promoter hypermethylation. EPCAM deletion analysis followed by MSH2 methylation testing of the tumor is a fast low-cost procedure that can be used to identify mutations that cause LS. We propose that this procedure be incorporated into clinical genetic analysis strategies and present a decision-support flow diagram for the diagnosis of LS.Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early-onset cancers of the colorectum and endometrium and tumors of the stomach, pancreas, small intestine, ovary, bladder, and bile duct.¹ In the Spanish population, about 2.5% of colorectal cancers are associated with LS.² The carcinogenetic etiology of this syndrome involves a DNA mismatch repair (MMR) inactivation caused by a germ line mutation of an MMR gene (MLH1, MSH2, MSH6, or PMS2) followed by somatic inactivation of the second allele.¹ As a consequence of MMR inactivation, these tumors exhibit microsatellite instability (MSI) and loss of expression of the mutated MMR gene.¹ It was recently shown that germ line deletions involving the last exon of the non-MMR gene, EPCAM (OMIM#185535), may silence its neighboring gene, MSH2 (OMIM#609309), which is located 17 kb downstream of EPCAM, via promoter hypermethylation. This epigenetic inactivation seems to be effective only in tissues in which EPCAM is expressed.^3,4 The EPCAM gene codes for the epithelial cell adhesion molecule also known as CD326, which is expressed in all normal epithelial cells and in carcinoma tumors.⁵ Thus, deletions of the last exon of EPCAM constitute a distinct class mutation associated with LS.Currently, the standard genetic test for LS (point mutation and large-rearrangement analysis of MLH1, MSH2, MSH6, and PMS2) frequently fails to detect a pathogenic mutation. For this reason, we evaluated the association between EPCAM deletions and LS in a Spanish population and its clinical implications.     

Pneumocystis jiroveci dihydropteroate synthase gene mutations among colonized individuals and Pneumocystis pneumonia patients from Spain

Cotrimoxazole, an association of trimethoprim and sulfamethoxazole, and dapsone, are mainstays for the prophylaxis and treatment of Pneumocystis pneumonia (PcP). The inability to culture Pneumocystis prevents routine susceptibility testing and detection of drug resistance. Instead, molecular techniques have been used to detect Pneumocystis jiroveci dihydropteroate synthase (DHPS) mutations that cause sulfa resistance in other microorganisms. The most frequent DHPS mutations occur at nucleotide positions 165 and 171, which lead to an amino acid change at positions 55 and 57. Several studies suggest that these mutations are associated with the failure of chemoprophylaxis for PcP. The aim was to establish the frequency and characteristics of P jiroveci DHPS mutations among colonized individuals and PcP patients from Spain. A total of 50 colonized individuals and 25 PcP patients were studied. DHPS polymorphisms were identified by restriction fragment length polymorphism assay. The analysis provided a rate of 28% of DHPS gene mutations in our population, with the presence of all possible polymorphisms described. The presence of mutations was higher in PcP patients than in colonized subjects (40% vs 22%), probably because of the chemoprophylaxis used in PcP patients. The comparison between patients with and without DHPS mutations did not show statistical differences due to age, sex, steroid use, sulfa drug exposure, or smoking. A high rate of DHPS mutations in our area of Spain, not only confined to patients previously exposed to sulfa drugs, is shown in this study. As well as PcP patients, colonized individuals who harbor P jiroveci strains with DHPS mutations could play a major role in the transmission cycle of these mutations, representing a reservoir and source of infection for susceptible individuals. Further research is thus warranted to assess the true scope of the problem and to design rational preventive strategies.     

Effect of adverse drug reactions on length of stay in surgical intensive care units

OBJECTIVE: To determine the frequency of adverse drug reactions in surgical intensive care units and evaluate their effect on the length of stay. DESIGN: Prospective cohort study. Between May 1997 and December 1999, while the patients were staying in the surgical intensive care unit, data were gathered regarding suspected adverse drug reactions and on different variables related to the length of stay. SETTING: Surgical intensive care units of our hospital. PATIENTS: A total of 401 patients hospitalized in the surgical intensive care unit. MAIN RESULTS: In 37 of the 401 patients seen (9.2%; 95% confidence interval, 6.6-12.5), 39 different adverse drug reactions were detected. The adverse drug reactions were most frequently caused by the following drugs: morphine hydrochloride (n = 13), meperidine hydrochloride (n = 9), and metamizole (n = 7). Five adverse drug reactions were severe, the suspected medication had to be discontinued in 14 cases, and new drugs were necessary to manage the adverse drug reaction in 28 cases. The crude estimation of the effect of adverse drug reactions performed on the length of stay with a bivariant regression model indicated that each adverse drug reaction was related to an increase of 3.39 days (95% confidence interval, 1.47-5.31) in the length of stay. This estimation was reduced to 2.31 days (95% confidence interval, 0.64-3.99) when considering other variables that might cause confusion for analysis, although it is still important. CONCLUSIONS: Adverse drug reactions are a significant clinical and economic problem in surgical intensive care units.     

In Vitro Activity of Daptomycin in Combination with β-Lactams,Gentamicin, Rifampin,and Tigecycline against Daptomycin-Nonsusceptible Enterococci

Enterococci that are nonsusceptible (NS; MIC > 4 μg/ml) to daptomycin are an emerging clinical concern. The synergistic combination of daptomycin plus beta-lactams has been shown to be effective against vancomycin-resistant Enterococcus (VRE) species in vitro. This study systematically evaluated by in vitro time-kill studies the effect of daptomycin in combination with ampicillin, cefazolin, ceftriaxone, ceftaroline, ertapenem, gentamicin, tigecycline, and rifampin, for a collection of 9 daptomycin-NS enterococci that exhibited a broad range of MICs and different resistance-conferring mutations. We found that ampicillin plus daptomycin yielded the most consistent synergy but did so only for isolates with mutations to the liaFSR system. Daptomycin binding was found to be enhanced by ampicillin in a representative isolate with such mutations but not for an isolate with mutation to the yycFGHIJ system. In contrast, ampicillin enhanced the killing of the LL-37 human antimicrobial peptide against daptomycin-NS E. faecium with either the liaFSR or yycFGHIJ mutation. Antagonism was noted only for rifampin and tigecycline and only for 2 or 3 isolates. These data add support to the growing body of evidence indicating that therapy combining daptomycin and ampicillin may be helpful in eradicating refractory VRE infections.     

Statins and Voriconazole Induce Programmed Cell Death in Acanthamoeba castellanii

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC₅₀s) and IC₉₀s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.     

Dietary exclusion for childhood atopic dermatitis

Question Atopic dermatitis (AD) (eczema) is very common in childhood. Parents are often concerned that food allergy plays a role in their child’s eczema presentation and practise elimination diets for symptom control. Should I recommend that children with AD eliminate cow’s milk or eggs from their diets?Answer There is insufficient evidence about the benefit of eliminating cow’s milk in unselected patients with AD. Some evidence suggests that egg elimination might benefit those children with AD who are suspected of being allergic to eggs and who are sensitized to eggs; however, more research is needed in this population to guide the use of food elimination. Prolonged elimination diets that are not physician guided also put patients at risk of nutritional deficiencies and failure to thrive, and might actually contribute to the development of clinical food allergy.