Antimicrobial activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa recovered during the Study for Monitoring Antimicrobial Resistance Trends (SMART) program in Spain (2016-2018).

OBJECTIVE: To analyse the susceptibility to ceftolozane-tazobactam and comparators in Enterobacterales and Pseudomonas aeruginosa isolates recovered from intraabdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream infection (BSI) in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study. METHODS: The susceptibility of 5,351 isolates collected in 11 Spanish hospitals (2016-2018) were analysed (EUCAST-2020 criteria) by broth microdilution and were phenotypically studied for the presence of extended-spectrum beta-lactamases (ESBL). Ceftolozane-tazobactam and/or carbapenem resistant isolates were genetically characterized for ESBL and carbapenemases. RESULTS: Escherichia coli was the most frequent pathogen (49.3% IAI, 54.9% UTI, 16.7% RTI and 50% BSI), followed by Klebsiella pneumoniae (11.9%, 19.1%, 13.1% and 15.4%, respectively). P. aeruginosa was isolated in 9.3%, 5.6%, 32% and 9%, respectively. The frequency of isolates with ESBLs (2016-2017) was: 30.5% K. pneumoniae, 8.6% E. coli, 2.3% Klebsiella oxytoca and 0.7% Proteus mirabilis. Ceftolozane-tazobactam was very active against non-ESBL-(99.3% susceptible) and ESBL-(95.2%) producing E. coli being less active against K. pneumoniae (98% and 43.1%, respectively) isolates. CTX-M-15 was the most prevalent ESBL in E. coli (27.5%) and K. pneumoniae (51.9%) frequently associated with OXA-48-like carbapenemase. Overall, 93% of P. aeruginosa isolates were susceptible to ceftolozane-tazobactam, preserving this activity (>75%) in isolates resistant to other beta-lactams except in those resistant to meropenen or ceftazidime-avibactam. GES-5, PER-1, VIM-1/2 were the most prevalent enzymes in isolates resistant to ceftolozane-tazobactam. CONCLUSIONS: Ceftolozane-tazobactam showed high activity rates against isolates recovered in the SMART study although it was affected in K. pneumoniae and P. aeruginosa isolates with ESBL and/or carbapenemases.

Nationwide sentinel surveillance of bloodstream Candida infections in 40 tertiary care hospitals in Spain.

Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Spain to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for acquiring a Candida infection. Prospective laboratory-based surveillance was conducted from June 2008 to June 2009 in 40 medical centers located around the country. A case of candidemia was defined as the isolation of a Candida species from a blood culture. Incidence rates were calculated per 1,000 admissions. Antifungal susceptibility tests were performed by using broth microdilution assay according to the guidelines of the Clinical and Laboratory Standards Institute. We detected 984 cases, for an overall incidence of 1.09 cases per 1,000 admissions. The crude mortality was 20.20%. Candida albicans was the most common species (49.08%), followed by C. parapsilosis (20.73%), C. glabrata (13.61%), and C. tropicalis (10.77%). Overall, decreased susceptibility to fluconazole occurred in 69 (7.01%) incident isolates. Antifungal resistance was rare, and a moderate linear correlation between fluconazole and voriconazole MICs was observed. This is the largest multicenter candidemia study conducted to date and shows the substantial morbidity and mortality of candidemia in Spain.

Antimicrobial susceptibility trends and evolution of isolates with extended spectrum ß-lactamases among Gram-negative organisms recovered during the SMART study in Spain (2011-2015).

OBJECTIVE: The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study monitors antimicrobial susceptibility and extended spectrum ß-lactamases (ESBLs) in Gram-negative bacilli recovered from intra-abdominal infections (IAI). METHODS: Antimicrobial susceptibility of 5,343 isolates from IAI recovered in 11 centres during the 2011-2015 SMART-Spain program was analysed by standard microdilution (EUCAST criteria) and compared with that from 2002-2010. ESBLs were phenotypically detected. RESULTS: Escherichia coli, the most common isolate, significantly decreased in community acquired IAI (60.9% 2002-2010 vs. 56.1% 2011-2015, P=0.0003). It was followed in prevalence by Klebsiella pneumoniae that increased both in the community (8.9% vs. 10.8%, P=0.016) and nosocomial (9.2% vs. 10.8%, P=0.029) IAI and P. aeruginosa, which significantly increased in community acquired IAI (5.6% vs. 8.0%, P=0.0003). ESBLs were more prevalent in K. pneumoniae (16.3%) than in E. coli (9.5%) of nosocomial origin and were more frequently isolated from elderly patients (>60 years). Considering all Enterobacteriaceae, ertapenem (92.3-100%) and amikacin (95.5%-100%) were the most active antimicrobials. Ertapenem activity, unlike amoxicillin-clavulanate or piperacillin-tazobactam, remained virtually unchanged in ESBL (100%) and non-ESBL (98.8%) E. coli producers. Its activity decreased in ESBL-K. pneumoniae (74.7%) but was higher than that of amoxicillin-clavulanate (14.0%) and piperacillin-tazobactam (24.0%). Interestingly, ertapenem susceptibility was maintained in >60% of ESBL isolates that were resistant to amoxicillin-clavulanate, piperacillin-tazobactam or fluoroquinolones. CONCLUSIONS: SMART-Spain results support current guidelines which include ertapenem as empiric treatment in mild-moderate community-acquired IAI, particularly with ESBL producers. These recommendations will need to be updated with the recently introduction of new antimicrobials.

Characterization of hepatitis B virus genotypes in chronically infected patients.

Genomic mutations occurring during reverse transcription of hepatitis B virus (HBV) could explain its genetic diversity and account for 8 genetically distinct genotypes that are geographically distributed quite differently. The main objectives of this study were to determine the prevalence of hepatitis B virus genotypes in patients with chronic hepatitis B and to see if there was a relationship between genotypes and risk factors for transmission based on HBeAg status. A total of 14 serum samples were analyzed using INNO-LIPA HBV genotyping assay. Genotype D was the most prevalent (64.3%) followed by genotype A (28.6%). There was one case of co-infection (D/E genotypes) that was confirmed by PCR sequencing. All patients except one were HBeAg-negative and anti-HBe-positive. The risk factors for HBV transmission were unknown in half of the cases; in the other half, sexual, transfusion, maternal or interfamilial transmission were observed. The results show that genotype D is the most prevalent genotype in our hospital, followed by genotype A. On the other hand, no relationship was found between HBeAg status and genotype.

[Patterns of susceptibility to antibiotics of Enterobacteriaceae causing intra-abdominal infection in Spain: SMART 2003 study outcomes]. / Patrones de sensibilidad a antimicrobianos de Enterobacteriaceae causantes de infecciones intraabdominales en España: resultados del estudio SMART 2003.

SMART (Study for Monitoring Antimicrobial Resistance Trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intra-abdominal infections. The objective of this subanalysis was to assess antimicrobial susceptibility patterns among Entero-bacteriaceae recovered at 13 participating Spanish sites during 2003. Antimicrobial susceptibility testing was performed using broth microdilution techniques according to the CLSI (formerly NCCLS) guidelines for MIC testing. The presence of extended-spectrum beta-lactamases (ESBL) was confirmed in isolates with a MIC of ceftriaxone, ceftazidime, or cefepime>or=2 mg/l by comparing cefepime MICs with and with-out clavulanate. A total of 981 Enterobacteriaceae recovered from 840 patients were tested, of which 398 (41%) were community-acquired. Escherichia coli was the most common isolate (571 isolates; 58%), followed by Klebsiella spp. (153; 16% Enterobacter spp. (97; 10%), and Proteus spp. (63; 6%). A total of 191 isolates (19%) from 176 patients produced inducible beta-lactamases. The carbapenems and amikacin were the most consistently active agents against the Enterobacteriaceae (susceptibility>or=99%). Resistance rates for ceftazidime, cipro-floxacin, and levofloxacin exceeded 10%. ESBLs were detected phenotypically in 61 (6%) isolates, being the most common E. coli (61%), Klebsiella spp. (20%), and Enterobacter spp. (8%). Antimicrobial resistance among Enterobacteriaceae isolated from intra-abdominal infections is a problem in Spain. A significant proportion of inducible beta-lactamase and ESBL-producing Enterobacteriaceae causing intra-abdominal infection were acquired in the community. The carbapenems ertapenem, imipenem and meropenem and the aminoglycoside amikacin were highly active in vitro against Enterobacteriaceae isolated from intra-abdominal sites, including ESBL-producing organisms.

Sentinel surveillance of invasive candidiasis in Spain: epidemiology and antifungal susceptibility.

In order to know the epidemiology and the changes of antifungal resistance in invasive candidiasis (IC) we carried out this prospective study of Candida strains belonging to patients admitted to 26 Spanish hospitals from June 2011 to June 2012 diagnosed with IC. Clinical information and the identity of the Candida species were collected and antifungal susceptibility was tested using broth microdilution in five agents: amphotericin B, fluconazole, voriconazole, caspofungin and anidulafungin. A total of 705 cases-isolates were documented. Most of the patients suffered from candidemia and several underlying diseases and more than half of them were neutropenic or under immunosuppressive therapy, factors associated with higher mortality. Thirty percent of global mortality was documented. C. albicans was the most frequently isolated species, although an increase of non-C. albicans species was observed. Resistance to fluconazole was of 3.4%, lower than in previous years (6.3%). C. parapsilosis presented a higher MIC90 of echinocandins compared to other species.

Comparison of three tests for measuring footpad swelling in the mouse.

Three tests, footpad thickness, footpad weight and footpad dye accumulation have been compared for measuring the local inflammatory reaction developed in the hind footpads of the mouse after injection of increasing doses of heat killed Candida albicans yeasts. The 3 tests showed high correlation indicating that any one of them may be used to evaluate such inflammatory reactions.

Five-year survey of cefotaxime resistance in Spain.

During 1991-1995 a Spain collaborative study group surveyed the resistance to cefotaxime both in community as well as in hospital isolates of bacteria. The isolates tested during the study period of 5 years were 813, 875, 3631, 3184, and 3050 strains, respectively. Antimicrobial activity of cefotaxime was assayed by broth or agar microdilution, in accordance with criteria of the National Committee of Clinical Laboratory Standards (NCCLS). Cefotaxime resistance included 2.5% of all isolates: 2.6% Enterobacteriaceae, 1.7% Streptococcus pneumoniae, 0.5% Haemophilus influenzae, 0.0% Haemophilus spp., and 0.0% Moraxella catarrhalis. The overall incidence of resistance to cefotaxime decreased fro member of Enterobacteriaceae from 3.6% in 1991 to 2.5% in 1995. The incidence of resistance varied with the species and was highest in Enterobacter and in Citrobacter freundii.

Emergence of resistance to beta-lactam agents in enterobacteriaceae species with group I beta-lactamases in Spain.

The contribution of induction and stable derepression of chromosomal group I beta-lactamases to beta-lactam antibiotics resistance was studied in clinical isolates of Enterobacteriaceae, collected from patients treated with these antibiotics. Multiple isolates of the same species from the same patient were characterized by different typing methods. Sonicated extracts of cells were assayed for chromosomal and plasmid-mediated beta-lactamases by isoelectric focusing and cloxacillin inhibition studies. The specific beta-lactamase activity, basal and induced with cefoxitin, was determined to differentiate strains with inducible or derepressed production of the enzyme. Induction of beta-lactamases was performed in each strain against the beta-lactams used in the therapy of each patient. Older penicillins resulted in a moderate to strong increase in beta-lactamase activity, whereas the results obtained with first-generation cephalosporins were species dependent. Expanded-spectrum cephalosporins were weak inducers of beta-lactamases. Indeed, the use of cefotaxime for treatment preceded the appearance of strains that produced chromosomal group I beta-lactamases constitutively. These strains showed a remarkable reduction in sensitivity to ureidopenicillins, carboxipenicillins, expanded-spectrum cephalosporins, and monobactams, but not to carbapenems.

Detection of circulating antigens in the diagnosis of acute toxoplasmosis.

Using an enzyme-linked immunosorbent assay, we have found circulating antigens of Toxoplasma gondii in three models of murine toxoplasmosis: mice infected with trophozoites of the RH strain (acute toxoplasmosis), the Beverley strain (subacute toxoplasmosis), and the T626 strain (chronic toxoplasmosis). Circulating antigens were detected 48 hr after infection in the mice infected with the RH strain, and all mice had antigenemia by the fourth day. In those infected with the Beverley strain, circulating antigens were detected from the second day after inoculation until the end of the study, with a peak (71% of the infected mice) on day 10. Of those infected with the T626 strain, 40% had antigenemia at 13 days after infection. The detection of circulating antigens in serum is directly related to the presence of toxoplasmosis in the acute phase in the three models studied and, therefore, may prove very useful in the rapid diagnosis of this disease.

Specific and rapid identification of multiple-antibiotic resistant Pseudomonas aeruginosa clones isolated in an intensive care unit.

Seventeen multiple-antibiotic-resistant Pseudomonas aeruginosa isolates were collected from two patients hospitalized in the same intensive care unit. They showed a parallel acquisition of resistance to antibiotics and they were, therefore, thought to have a common clonal origin. These strains were typed by biotyping, serotyping, plasmid profile, three different PCR-based techniques, and macrorestriction analysis to determine their relationship. Only the use of PCR techniques and macrorestriction analysis allowed an accurate identification of the clones and revealed that each patient was infected by A unique multidrug-resistant strain. Therefore, there was no cross-infection or reinfection with a new strain.

Candidal infection of bone. Assessment of serologic tests in diagnosis and management.

In this case report, 30 sera from a 25-year-old heroin abuser with intervertebral candidosis were treated for the presence of anti-Candida albicans antibodies by agglutination, counterimmunoelectrophoresis, and indirect immunofluorescent assay. Sera were also adsorbed with heat-killed blastospores to remove antibodies against yeast-phase cells and tested by indirect immunofluorescent assay for anti-C. albicans germ tube antibodies (CAGTAs). Humoral responses to candidal 47-kD antigen were studied by immunoblotting in 23 unadsorbed sera. Anti-C. albicans antibodies were found in high titers by the three procedures but correlated poorly with the clinical evolution of the disease. CAGTAs were present from the beginning of the infection: Titers decreased in association with antifungal treatment and the patient's improvement, eventually becoming negative. Only class IgG antibodies to the 47-kD antigen were detected. These were present during the full course of the infection, failing to disappear at the end of the study. In this case, detection of CAGTAs appeared to be an important aid to diagnosis of the bony candidal infection, as they are detected early during the illness and seemed to have a prognostic significance.

Antibodies against Trichosporon beigelii in vaginal washings from asymptomatic women.

Trichosporon beigelii was isolated from vaginal washings from three asymptomatic women. All three women had IgG or IgA anti-T. beigelii antibody titres greater than or equal to 20 when tested by an indirect immunofluorescence assay against the three strains isolated. Titres greater than or equal to 160 were found when each patient was tested against her own isolate. Patients with Candida albicans vulvovaginitis, or from whom C. albicans or Toruloposis glabrata was isolated from vaginal washings, or who had negative cultures for yeasts, had titres less than or equal to 20.

Detection of anti-interferon-gamma autoantibodies in subjects infected by Mycobacterium tuberculosis.

SETTING: Among the cytokines involved in defensive mechanisms against Mycobacterium tuberculosis infection, special attention has been given to interferon-gamma (IFN-gamma); a local synthesis of this cytokine as well as IL-2 (type 1 cytokines) at the site of disease in patients with tuberculous pleuritis has been demonstrated. Moreover, high levels of IgG autoantibodies against IFN-gamma have been shown in several clinical situations. It has been suggested that these antibodies could serve to limit the intensity or duration of the immune response or be able to interfere with the pathophysiological effects of IFN-gamma. OBJECTIVE: To investigate the potential role of anti-IFN-gamma antibodies in the course of M. tuberculosis infection. DESIGN: Investigation of the presence of these antibodies in sera from healthy and ill subjects infected with M. tuberculosis in relation to the extent of the disease and the presence of IFN-gamma in sera by enzyme-linked-immunosorbent assay (ELISA). In order to investigate the presence of these antibodies at the site of infection we included 12 pleural fluids from tuberculosis patients and 9 pleural fluids from other origins. RESULTS: In the course of M. tuberculosis infection the production of anti-IFN-gamma IgG antibodies is induced, being particularly higher in healthy skin test converters. Among tuberculosis patients, the presence of anti-IFN-gamma autoantibodies is significantly associated with detectable levels of the cytokine in sera. Levels of anti-IFN-gamma antibodies in moderately advanced and far advanced tuberculosis patients are significantly greater than in healthy individuals. These antibodies increase at the site of infection. CONCLUSION: Anti-IFN-gamma antibodies must be considered as a new element in the immune response to M. tuberculosis. It would be of great interest to investigate this point especially at the site of infection.

Detection of HGV in serum and peripheral blood mononuclear cells of maintenance haemodialysis patients.

The aim of the present study was to investigate the prevalence of hepatitis G virus (HGV) and also hepatitis C virus (HCV) infections in maintenance haemodialysis patients, and to identify extrahepatic sites as HGV reservoirs. HGV RNA was detected in the serum of 6/61 (10%) patients and in the peripheral blood mononuclear cells of 2/61 (3%) patients (one of whom was serum negative). These findings suggest that lymphoid cells constitute an extrahepatic HGV reservoir. HCV RNA was detected in 7/61 (11%) patients. Five of these patients (71%) were identified as carrying HCV genotype 1b. Co-infection with HCV and HGV was detected only in one patient. Haemodialysis patients are at risk for HGV infection, by nosocomial routes or via transfusions. HGV itself does not seem to be an important cause of hepatitis since all six HGV RNA positive patients not co-infected by HCV or HBV showed normal ALT values.

Seroprevalence of hepatitis B and C, and human immunodeficiency type 1 viruses in a rural population from the Republic of Equatorial Guinea.

The seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) markers was evaluated in a group of 2042 subjects from a rural area in the Republic of Equatorial Guinea, to obtain a better understanding of the transmission patterns of these viruses. Antibodies to HIV-1 were detected in 12 subjects (0.6%); the seroprevalence did not differ significantly by age or gender. Overall seroprevalence for HCV was 1.7% (in patients aged > 40 years, 5.6%). Hepatitis B surface antigen was detected in 8.8% of subjects, with the higher seroprevalence in children aged < or = 18 years of 13.4% contrasting with the higher seroprevalence of HCV in older subjects of the Equatoguinean studied population. These results indicate differences in the distribution of the viruses and, probably, different routes of transmission. The study demonstrates the existence of a high HBV carrier rate in children, concluding that hepatitis B vaccine should be incorporated into the Expanded Programme on Vaccination in Equatorial Guinea.

Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli.

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.

Effect of erythromycin treatment on specific immunologic response in mice.

Six week old Swiss mice were sensitized by subcutaneous injection of 10(7) sheep red blood cells without adjuvant. One hour after sensitization, the mice were treated with erythromycin lactobionate for ten days. The minimal (15 mg/kg/day) and maximal (57 mg/kg/day) doses clinically used were assayed. The daily dose of erythromycin was administered intraperitoneally, in two injections, one every 12 hours. The kinetics of delayed type hypersensitivity reaction, measured by means of the foot-pad test, was evaluated by challenging different groups of fourteen mice with an eliciting dose of 10(8) SRBC injected into the foot-pad on days 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 after sensitization. Total and 2-mercaptoethanol resistant haemagglutinating antibody titres were determined in sera obtained from mice immediately after measuring the delayed type hypersensitivity reaction. Treatment with maximal erythromycin dose gave rise to a significant enhancement of the cellular immune response, and also to an acceleration of the humoral antibody response. On the other hand, treatment with minimal erythromycin dose gave rise to a slight depression of the immune cellular response and also to a depression of the antibody production at the beginning of the humoral response.

Effects of fasting and immobilization stresses on the phagocytic capacity of rat polymorphonuclear phagocyte monolayers.

The effect of two different types of acute stress (immobilization and fasting) on the polymorphonuclear leukocyte phagocytic function has been studied in male and female rats. With this aim, a subgroup of rats was under immobilization and fasting, another under complete energy deprivation and a third one (controls), exposed to the normal activity of the animal room, for 15 hours. The stress induction was assessed by controlling weight variations and gastric ulcers generation. Both stressors induced weight loss but only immobilization resulted in the development of gastric ulcer in all the animals studied. Phagocytosis was increased in male rats stressed by fasting and in immobilized female rats. In the remaining subgroups polymorphonuclear leukocyte cells showed a phagocytic capacity within the range of control values. Only comparison of the males group stressed by fasting with the male group stressed by fasting and immobilization showed a significant depression in phagocytosis.