Isolates of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae causing invasive infections in Spain remain susceptible to cefotaxime and imipenem.

OBJECTIVES: The epidemiology of invasive Haemophilus influenzae has changed in recent years. ß-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to ß-lactam antibiotics indicated in the treatment of invasive infections. METHODS: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven ß-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins. RESULTS: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to ß-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to ß-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively. CONCLUSIONS: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of ß-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.

Frequent carriage of resistance mechanisms to ß-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.

OBJECTIVES: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to ß-lactams. METHODS: A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. RESULTS: In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to ß-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by ß-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). CONCLUSIONS: These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of ß-lactam resistance mechanisms, particularly PBP3 mutations.

Parallel increase in community use of fosfomycin and resistance to fosfomycin in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

OBJECTIVES: To document fosfomycin susceptibility of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC), analyse trends in fosfomycin use and investigate fosfomycin resistance in ESBL-EC isolated from urinary tract infections (UTIs). METHODS: Twenty-seven Spanish hospitals participating in the European Antimicrobial Resistance Surveillance Network were requested to collect up to 10 sequential ESBL-EC for centralized susceptibility testing and typing. EUCAST guidelines were followed for antibiotic susceptibility testing, and bla(ESBL) type, phylogroups and O25b serotype were determined by PCR and sequencing. In addition, the trend in fosfomycin resistance among ESBL-EC causing UTIs was determined in 9 of the 27 hospitals. Total fosfomycin use for ambulatory care was established by WHO-recommended methods. RESULTS: A total of 231 ESBL-EC (42.4% CTX-M-15, 34.2% SHV-12 and 23.4% CTX-M-14) were collected. The overall rate of fosfomycin resistance was 9.1%, but varied according to ESBL type (5.6% of CTX-M-14 isolates, 5.1% of SHV-12 and 15.3% of CTX-M-15). Of 67 O25b/B2 isolates, 11 (16.4%) were fosfomycin resistant. Predictors of infection with fosfomycin-resistant ESBL-EC were O25b/phylogroup B2 isolates, female gender and nursing home residence. Among 114 197 UTIs caused by E. coli 4740 (4.2%) were due to ESBL-EC. Fosfomycin resistance increased in these isolates from 4.4% (2005) to 11.4% (2009). The use of fosfomycin grew from 0.05 defined daily doses per 1000 inhabitants per day (1997) to 0.22 (2008), a 340% increase. CONCLUSIONS: Key factors related to increased fosfomycin resistance in ESBL-EC causing UTIs could be the rapid growth in community use of fosfomycin, the widespread distribution of the 025b/B2 E. coli clone and the existence of a susceptible population comprising women residing in nursing home facilities.

Emergence of extended-spectrum beta-lactamases and AmpC-type beta-lactamases in human Salmonella isolated in Spain from 2001 to 2005.

OBJECTIVES: To study the resistance to third-generation cephalosporins in Salmonella strains isolated from humans in a 5 year period in Spain, and to identify the responsible genes and their dissemination. METHODS: Twenty-seven isolates were analysed by PCR and sequencing to identify the genes responsible for the beta-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli K12J53, plasmid detection with S1-PFGE, hybridization and PCRs of the transconjugants. The genetic relationship was determined by PFGE. RESULTS: We found bla(CTX-M-9) and bla(CTX-M-10) in Salmonella Virchow PT19. bla(CTX-M-14) was detected in Salmonella (IV) 44:z(4),z(23):-, Salmonella Enteritidis PT6a, Salmonella Typhimurium DT193 and Salmonella Typhimurium DT104B. bla(CTX-M-1) was found in Salmonella Litchfield. bla(CTX-M-15) and bla(CTX-M-32) were found in Salmonella Enteritidis PT1. bla(SHV-12) was found in Salmonella Blockley, Salmonella Hadar PT2, Salmonella Enteritidis PT21, Salmonella Enteritidis PT1 and Salmonella Bredeney. bla(SHV-2) was found in Salmonella Livingstone. bla(CMY-2) was detected in Salmonella Bredeney, Salmonella Newport, Salmonella Enteritidis PT5b and Salmonella Heidelberg. bla(DHA-1) was detected for the first time in Spain in Salmonella Newport. One strain of Salmonella Senftenberg harboured two extended-spectrum beta-lactamases, bla(SHV-12) and bla(CTX-M-9). We have found a large variety of beta-lactamase families as well as several members of major relevance, such as CTX-M-15, CTX-M-32, CMY-2 and DHA-1. XbaI-PFGE, conjugation assays and S1-PFGE hybridization showed that all these beta-lactamases were mediated by plasmids. CONCLUSIONS: This study demonstrates the emergence of a public health risk related to resistance to beta-lactams in Salmonella. The resistance trends need to be monitored carefully.

CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin.

OBJECTIVES: To describe trends in fosfomycin resistance in urinary isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBLs) in relation to fosfomycin consumption and to characterize representative fosfomycin-resistant isolates. METHODS: In 2007-08, an unexpected increase in fosfomycin resistance in ESBL-producing urinary E. coli was observed. Laboratory records were reviewed and a prospective surveillance study was initiated on all urinary tract infections caused by ESBL-producing, fosfomycin-resistant E. coli. bla(ESBL) types, phylogroups, genetic environment and afa/dra operon were determined by PCR and sequencing. Molecular epidemiology was analysed by PFGE and multilocus sequence typing. To elucidate possible mechanisms of fosfomycin resistance, uhpT, glpT, uhpA, ptsI, cyaA and murA genes were analysed. Fosfomycin consumption was determined as recommended by WHO. RESULTS: From 2004 to 2008, fosfomycin consumption increased by 50%, while fosfomycin resistance in ESBL producers increased from 2.2% to 21.7%. Of 26 isolates studied, 24 produced CTX-M-15 and belonged to the O25b-ST131-phylogroup B2 clonal strain. PFGE revealed two clusters. Cluster I included 18 isolates, 16 of them indistinguishable from strains producing CTX-M-15 previously described in Madrid. The five isolates of Cluster II had the IS26 linked to bla(CTX-M-15) and the afa/dra operon. In Cluster I isolates, no mutations in glpT, uhpT, uhpA, ptsI, cyaA and murA were detected. Cluster II isolates showed a 15 bp deletion (A(169)-C(183)) in uhpA. CONCLUSIONS: Fosfomycin resistance in urinary E. coli has increased due to the acquisition of this resistance by a previously circulating CTX-M-15-producing E. coli O25b-ST131-phylogroup B2 strain. This happened during a period when the use of fosfomycin increased by 50%.

Emergence of CTX-M-15-producing Klebsiella pneumoniae of multilocus sequence types 1, 11, 14, 17, 20, 35 and 36 as pathogens and colonizers in newborns and adults.

OBJECTIVES: To characterize the population structure and resistance mechanisms of Klebsiella pneumoniae isolates that are highly resistant to third-generation cephalosporins, collected from five Spanish hospitals. METHODS: A total of 162 K. pneumoniae isolates from five hospitals located in three geographical areas of Spain were characterized. The number of isolates from each hospital ranged from 3 to 82. The genetic relationship between isolates was established by PFGE and multilocus sequence typing (MLST). bla(ESBL) types and other antibiotic resistance genes were analysed by PCR and sequencing. Plasmids were classified according to their incompatibility group by a PCR-based replicon-typing scheme. RESULTS: All 162 isolates carried the bla(CTX-15) gene. Fifty-eight isolates (35.8%) caused clinical infections and 104 (64.2%) were colonizers. Sixty-nine (42.6%) isolates were collected from newborns and 93 (57.4%) from adults. Using PGFE, the 162 isolates were grouped into seven clusters that were further identified as members of the MLST types 1, 11, 14, 17, 20, 35 and 36. Two hospitals each had two different clones and the remaining three hospitals had a single CTX-M-15-producing K. pneumoniae clone. All clones carried different antibiotic resistance genes, including bla(OXA-1), aac(3)-IIa, aac(6')-Ib-cr, qnrS1 and qnrB. In four of the seven (57.1%) clones the bla(CTX-M-15) gene was transferred by conjugation; in all cases plasmids of the incompatibility group IncF were identified by PCR. CONCLUSIONS: This study shows that multiresistant K. pneumoniae producing CTX-M-15 of MLST types 1, 11, 14, 17, 20, 35 and 36 are spreading as pathogens and colonizers among newborns and adult patients in Spain.

Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes.

Salmonella serotypes are defined on the basis of somatic (O) antigens which define the serogroup and flagellar (H) factor antigens, both of which are present in the cell wall of Salmonella. Most Salmonella organisms alternatively express phase-1 or phase-2 flagellar antigens encoded by fliC and fljB genes, respectively. Our group previously published two multiplex PCRs for distinguishing the most common first- and second-phase antigens. In this paper we describe a third multiplex PCR to identify the most common serogroups (O:B; O:C1; O:C2; O:D and O:E). The combination of these three PCRs enabled us to completely serotype organisms belonging to the Salmonella species. This multiplex PCR includes 10 primers. A total of 67 Salmonella strains belonging to 32 different serotypes were tested. Each strain generated one serogroup-specific fragment ranging between 162 and 615bp. Twenty-eight strains belonging to 21 serotypes, with a serogroup different from those tested in this work, did not generate any fragments. To compare molecular serotyping with traditional serotyping, 500 strains, received according to the order of arrival in the laboratory, were serotyped using both methods. The three multiplex PCRs were able to serotype 84.6% of the tested strains. This method was found to be very helpful in our laboratory as an alternative method for typing strains causing outbreaks, and it can be used to supplement conventional serotyping, since it is also applicable to motionless and rough strains.

[The EPICARDIAN project, a cohort study on cardiovascular diseases and risk factors among the elderly in Spain: methodological aspects and major demographic findings]. / El proyecto EPICARDIAN: un estudio de cohortes sobre enfermedades y factores de riesgo cardiovascular en ancianos españoles: consideraciones metodológicas y principales hallazgos demográficos.

BACKGROUND: Despite a greater incidence of ischemic heart disease among individuals over age 65, most cardiovascular research has been focused on the middle-aged adult population. To date no cohort study on this population have been made in Spain. This study is aimed as reviewing the role and methodology of cohort studies as an epidemiological tool absolutely essential for researching the prevalence and incidence of angina, AMI, stroke and the major cardiovascular risk factors. METHODS: Cohort study in three areas of Spain (Lista district in Madrid), Arevalo (Avila) and Begonte (Lugo). Age and sex stratified random sample by based on the municipal censuses of each area and municipality (n = 5.079). Two-stage initial cohort assessment: home survey structured for the screening ischemic heart disease and classic risk factors (hypertension, dyslipemia, diabetes and smoking habit) and clinical assessment for case confirmation. In the follow-up phase the MONICA project "cold pursuit" method modified for pinpointing and investigating indicent cases was used, employing all of the hospital and primary care clinical records for confirming the cardiovascular event. Data was also requested from the Spanish National Institute of Statistics as to the cause and date of death of the deceased individuals in the cohort. RESULTS: The overall AMI prevalence was 4% (95% CI: 3.4%, 4.5%); definite plus probable AMI being 6.2% (95% CI: 5.5-6.9). The definite AMI prevalence was higher among the mean 6.7% (95% CI: 5.63-7.79) than among the women, 2% (95% CI: 1.51-2.55) (p < 0.001). Hypertension prevalence according to JNCV1 criteria was 68%, hypercholesterolemia 26.4% according to NCEP criteria, diabetes prevalence 13.4% according to WHO criteria, and 11.3% were smokers. The cumulative incidence for a 3.2-year period for nonfatal definite AMI was 1.4% (95% CI: 1.1-1.8); 1.1% (95% CI: 0.74-1.37) probable AMI: 1.17 (IC95%: 0.824-1.48) for fatal definite AMI or death due to AMI and 1.13% (IC 95%: 0.824-1.48) for sudden death. CONCLUSIONS: The elderly population included in this study shows a high prevalence of cardiovascular risk factors, as well as ischemic heart disease incidence rates three times higher than those of the middle-aged adult population in Spain. The risk profile for women is significantly worse than for men, which may be due to the higher death rate at earlier ages among men.

Magnesium supplementation during cardiopulmonary bypass to prevent junctional ectopic tachycardia after pediatric cardiac surgery: a randomized controlled study.

OBJECTIVES: We analyzed the role of magnesium sulfate (MgSO(4)) supplementation during cardiopulmonary bypass in pediatric patients undergoing cardiac surgery, assessing the incidence of hypomagnesemia and the incidence of junctional ectopic tachycardia. METHODS: We performed a randomized, double-blind, controlled trial in 99 children. MgSO(4) or placebo was administered during the rewarming phase of cardiopulmonary bypass: group 1, placebo group (29 patients); group 2, 25 mg/kg of MgSO(4) (30 patients); and group 3, 50 mg/kg of MgSO(4) (40 patients). RESULTS: At the time of admission to the cardiac intensive care unit, groups receiving MgSO(4) had significantly greater levels of ionized magnesium (group 1, 0.51 + or - 0.07; group 2, 0.57 + or - 0.09; group 3, 0.59 + or - 0.09). Hypomagnesemia before bypass was common (75%-86.2%) and not significantly different among the groups. The proportion of hypomagnesemia decreased significantly at admission to the cardiac intensive care unit in groups receiving MgSO(4) (group 1, 77.8%; group 2, 63%; group 3, 47.4%). Patients receiving placebo (group 1) had a significantly greater occurrence of junctional ectopic tachycardia than groups receiving MgSO(4) (group 1, n = 5 [17.9%]; group 2, n = 2 [6.7%]; group 3, n = 0 [0%]). Age (<1 month), Aristotle score (>4), and history of cardiac failure were associated with junctional ectopic tachycardia. None of the patients with those characteristics in group 3 had junctional ectopic tachycardia. No association was found between study groups and the Pediatric Risk of Mortality score or length of stay in the cardiac intensive care unit. CONCLUSIONS: Supplementation with MgSO(4) during cardiopulmonary bypass seems to reduce the incidence of hypomagnesemia and junctional ectopic tachycardia at admission to the cardiac intensive care unit. This effect seems to be dose related.

[Serotype and phage type distribution of human Salmonella strains isolated in Spain, 1997-2001]. / Distribución de los serotipos y fagotipos de Salmonella de origen humano aislados en España en 1997-2001.

INTRODUCTION: Salmonellosis is one of the most frequent causes of gastroenteritis in Spain. Serotyping is the gold standard epidemiological marker for subdividing Salmonella spp. strains. A small number of serotypes are very frequently isolated, reducing the discriminatory power of serotyping. Thus, to increase our knowledge of Salmonella spp. epidemiology, additional epidemiological markers, such as phage typing, should be used for this purpose. METHODS: Salmonella spp. strains of human origin sent to the Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE, Spanish Reference Laboratory for Salmonella and Shigella) between 1997 and 2001 were serotyped using conventional agglutination methods, and Enteritidis, Typhimurium, Hadar, Virchow and Typhi serotypes were additionally phage typed according to internationally-developed schemes. RESULTS: A total of 30,856 Salmonella spp. strains, isolated in the majority of Spanish Autonomous Communities, were analyzed. Enteritidis (51%) and Typhimurium (24%) were the most frequently isolated serotypes. The following were the most frequent serotype/phage type combinations: Enteritidis/PT1 (18%), Enteritidis/PT4 (15%), Enteritidis/PT6a (5%), Typhimurium/DT104 (5%) and Enteritidis/PT6 (3%). The serotype Enteritidis/PT1 showed the greatest increase over the period studied, from 11.61% in 1997 to 24.74% in 2001. CONCLUSIONS: A hierarchical typing approach for Salmonella spp., using serotyping coupled with phage typing allowed a higher level of discrimination among Salmonella serotypes. Application of this approach in epidemiological studies could be highly useful for early characterization of related strains.

Mechanism of resistance to several antimicrobial agents in Salmonella Clinical isolates causing traveler's diarrhea.

The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding beta-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.

Evaluación de la organización administrativa y de la calidad de atención que brinda el sub-centro de la Ferroviaria Baja / Evaluation of the administrative organization and the quality of attention that offers the sub-center of Railway Loss

La presente investigación se realizó en el Subcentro de la Ferroviaria Baja correspondiente al segundo nivel de atención del Sistema Regionalizado de Salud del Ministerio de Salus Pública. Es una Institución adscrita a la Universidad Central del Ecuador a través de la Facultad de Ciencias Médicas y en la actualidad depende de la Escuela Nacional de Enfermería constituyendo la primera Unidad Operativa de Salud que se dirige por una enfermera profesional. Como primera actividad se realizó un diagnóstico de Salud de esta Unidad Operativa identificando como su principal problema la organización administrativa de este Sub Centro y surge la necesidad de evaluar la calidad de atención que ofrece esta Institución y determinar que factores estan favoreciendo o limitando el cumplimiento de sus labores. Por esta razón es que a través de éste trabajo hemos querido profundizar en el análisis de "Como influye la organización Administrativa del Sub Centro de Salud de la Ferroviaria Baja en la atención integral de Salud de la población del área de influencia de éste, en Quito durante el mes de abril de 1986...