Search PubMed⌕ Search

Biomedical subjects

Fernando Baquero

Publications and source records attributed to Fernando Baquero.

At least 55 records · Page 3Linked to original sources
PubMed2004

Hypermutation and the preexistence of antibiotic-resistant Pseudomonas aeruginosa mutants: implications for susceptibility testing and treatment of chronic infections.

Whether or not resistant mutants will be present before the start of antibiotic treatment of an initially susceptible population of bacteria depends on the size of the infecting population, the rate of mutation to resistance, and the amount of time that the population has been maintained. In the present investigation, we argue that for the treatment of chronic infections caused by hypermutable Pseudomonas aeruginosa of the sort frequently found in cystic fibrosis patients, mutants resistant to all single antipseudomonal drugs will almost invariably be present in a high proportion at the onset of treatment, and consequently, these strains should be considered resistant to all agents when they are used as monotherapy. Using a construct of P. aeruginosa strain PAO1 with a mutS deletion (strain PAODeltamutS), we show that when in vitro populations of less than 5 x 10(4) seemingly susceptible hypermutable bacteria are confronted with any of 11 antipseudomonal agents, mutants for which the MICs and the minimum bactericidal concentrations are in the range of clinical resistance will almost invariably ascend to dominance within 24 to 36 h. This does not occur for PAO1 without the mutS deletion. The results of our detailed analysis of this evolution of acquired resistance to two of these antibiotics, imipenem and ciprofloxacin, indicate that although the rates of mutation to resistance in PAODeltamutS are on the order of 1 x 10(-6) per generation, resistant mutants are very likely to either be present in cultures of between 2 x 10(4) and 4 x 10(4) bacteria or arise after the bacterial populations are confronted with antibiotics. We also demonstrate with in vitro experiments that the problem of acquired resistance to treatment with single antibiotics can be thwarted by combination therapy with pairs of antibiotics of different classes with synergistic activities. We discuss the clinical implications of our analysis of these observations.

Anti-Bacterial Agents↗
PubMed2004

Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal.

Eight pulsed-field gel electrophoresis subtypes and six Tn1546 variants were identified among Enterococcus faecalis isolates of a single clone recovered in three geographically separate Portuguese hospitals. Some clonal subtypes were found in particular hospitals, and Tn1546 variants were either widespread or confined to some of them. We also report on the first Tn1546 transposon containing an ISEf1 insertion.

Anti-Bacterial Agents↗
PubMed2004

Polymorphic mutation frequencies in Escherichia coli: emergence of weak mutators in clinical isolates.

Polymorphisms in the rifampin resistance mutation frequency (f) were studied in 696 Escherichia coli strains from Spain, Sweden, and Denmark. Of the 696 strains, 23% were weakly hypermutable (4 x 10(-8) < or = f < 4 x 10(-7)), and 0.7% were strongly hypermutable (f > or = 4 x 10(-7)). Weak mutators were apparently more frequent in southern Europe and in blood isolates (38%) than in urinary tract isolates (25%) and feces of healthy volunteers (11%).

Anti-Bacterial Agents↗
PubMed2004

Dramatic increase in prevalence of fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain.

The occurrence of extended-spectrum beta-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 microg/ml) and cefotaxime (1 microg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (P < 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of Escherichia coli. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-beta-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.

Electrophoresis, Gel, Pulsed-Field↗
PubMed2004

Trends in antimicrobial resistance in 1,968 invasive Streptococcus pneumoniae strains isolated in Spanish hospitals (2001 to 2003): decreasing penicillin resistance in children's isolates.

To address the public health problem of antibiotic resistance, the European Union (EU) founded the European Antimicrobial Resistance Surveillance System. A network of 40 hospitals that serve approximately 30% of the Spanish population (about 12 million) participated. Each laboratory reported data on antimicrobial susceptibility testing using standard laboratory procedures that were evaluated by an external quality control program. The antibiotic consumption data were obtained from the National Health System. We compared the antibiotic susceptibility of Spanish isolates of invasive Streptococcus pneumoniae (2001 to 2003) with antibiotic consumption. Invasive S. pneumoniae was isolated from 1,968 patients, 20% of whom were children at or below the age of 14 years. Of non-penicillin-susceptible strains (35.6%; 95% confidence interval, 34 to 37.2), 26.4% were considered intermediate and 9.2% were considered resistant. Between 2001 and 2003, penicillin resistance decreased from 39.5 to 33% overall and from 60.4 to 41.2% in children at or below the age of 14 years (P = 0.002). Resistance to erythromycin was at 26.6%, and coresistance with penicillin was at 19.1%. Of total isolates, the ciprofloxacin MIC was >2 mug/ml for 2.1%, with numbers increasing from 0.4% (2001) to 3.9% (2003). Total antibiotic use decreased from 21.66 to 19.71 defined daily doses/1,000 inhabitants/day between 1998 and 2002. While consumption of broad-spectrum penicillins, cephalosporins, and erythromycin declined, use of amoxicillin-clavulanate and quinolones increased by 17.5 and 27%, respectively. The frequency of antibiotic resistance in invasive S. pneumoniae in Spain was among the highest in the EU. However, a significant decrease in penicillin resistance was observed in children. This decrease coincided with the introduction of a heptavalent conjugate pneumoccocal vaccine (June 2001) and with a global reduction in antibiotic consumption levels.

Adolescent↗
PubMed2004

High genetic diversity among Stenotrophomonas maltophilia strains despite their originating at a single hospital.

The levels of genetic relatedness of 139 Stenotrophomonas maltophilia strains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct XbaI PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an S. maltophilia strain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of S. maltophilia isolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.

Bacteremia↗
PubMed2004

Dynamics of long-term colonization of respiratory tract by Haemophilus influenzae in cystic fibrosis patients shows a marked increase in hypermutable strains.

The persistence and variability of 188 Haemophilus influenzae isolates in respiratory tract of 30 cystic fibrosis (CF) patients over the course of 7 years was studied. Antibiotic susceptibility testing, DNA fingerprinting, and analysis of outer membrane protein profiles were performed on all isolates. A total of 115 distinct pulsed-field gel electrophoresis profiles were identified. Ninety percent of patients were cocolonized with two or more clones over the studied period. A third of the patients were cross-colonized with one or two H. influenzae strains; 11% of the clones persisted for 3 or more months. Biotype, outer membrane protein profiles, and resistance profiles showed variation along the studied period, even in persisting clones. Four isolates (2.1%) recovered from 3 patients were type f capsulate, with three of them belonging to the same clone. beta-Lactamase production was detected in 23.9% of isolates while 7% of the beta-lactamase-negative isolates presented diminished susceptibility to ampicillin (beta-lactamase-negative ampicillin resistance phenotype). Remarkably, 21.3% of the H. influenzae isolates presented decreased susceptibility to ciprofloxacin, which was mainly observed in persisting clones. Of the H. influenzae isolates from CF patients, 18 (14.5%) were found to be hypermutable in comparison with 1 (1.4%) from non-CF patients (P < 0.0001). Ten patients (33.3%) were colonized by hypermutable strains over the study period. A multiresistance phenotype and long-term clonal persistence were significantly associated in some cases for up to 7 years. These results suggest that H. influenzae bronchial colonization in CF patients is a dynamic process, but better-adapted clones can persist for long periods of time.

Adolescent↗
PubMed2004

Fosfomycin and rifampin disk diffusion tests for detection of Escherichia coli mutator strains.

A simple method, using commercial disks to detect Escherichia coli mutator strains, is proposed. The breakpoint for detecting strains with a mutation frequency >/=5 x 10(-7) was established at >/=70 and >/=20 colonies in the inhibition zone of fosfomycin and rifampin disks, respectively, after seeding 100 microl of an overnight culture. Strains with <30 and <10 colonies in fosfomycin and rifampin inhibition zones are presumptively non-mutators.

Anti-Bacterial Agents↗
PubMed2004

[Seroepidemiologic study of hepatitis A in the community of Madrid during the year 2002].

INTRODUCTION: The aim of this study was to investigate the prevalence and risk factors associated with hepatitis A virus (HAV) infection in a representative population sample to determine who can benefit from vaccination strategies and to investigate the age limit at which previous HAV antibody screening is not required. METHODS: From April to September 2002, we studied a total of 557 patients, 90 children and 467 blood donors, aged 1-65 years. Information on demographic variables (age, gender, place of residence and education level) was recorded. Patients with history of hepatitis or other liver diseases were excluded. Anti-HAV antibodies (IgG) were determined with an automated enzyme immunoassay (AxSYM, Abbott Diagnostics). The chi-square and Mantel-Haenszel tests were used for the statistical analysis. RESULTS: The overall prevalence of HAV infection was 41.5%. There was a significant increase in prevalence with age (chi-square TL:205, P < .0001), with rates from the youngest to oldest groups of 5.5%, 23.5%, 28.1%, 64.2% and 93.2%, respectively. Apart from age, the only other risk factor independently associated with prevalence was the level of education, with higher prevalence at the lower education levels (OR 5 2.7; chi-square = 32.11, P < .0001). CONCLUSIONS: The prevalence of anti-HAV antibodies has decreased in recent years in the community of Madrid. Among the population less than 35 years of age, 75% of individuals are susceptible to the infection and could benefit from universal vaccination without previous screening for anti-HAV antibodies.

Adolescent↗
PubMed2003

Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model.

A pathogenesis model based on the interaction between Caenorhabditis elegans and bacterial opportunistic pathogens has recently been developed. In the case of Pseudomonas aeruginosa, the model is based on three different modes of nematode killing (fast killing, slow killing and lethal paralysis) by virulent bacteria that has been incubated in different nutrient media. Using parametric statistics and Probit analysis, we test the reliability of the three different killing systems with respect to bacterial virulence. To accomplish this, we use three P. aeruginosa strains, each with a different level of virulence and one strain of non-virulent Escherichia coli. Probit function proved to be effective in quantifying the virulence of P. aeruginosa. The results of the killing curve analysis using the Probit function demonstrates that the slow-killing test is the most reliable method for quantifying virulence using the C. elegans model of bacterial pathogenesis. Although the greatest virulence differences are observed after long periods of incubation, the Probit analysis clearly shows that the death kinetics of C. elegans depend on the first hours of nematode/bacteria interaction. In contrast, fast killing seems to be non-specific, at least under our experimental conditions, since the killing rates of virulent P. aeruginosa and non-virulent E. coli strains were indistinguishable.

Animals↗
PubMed2003

Streptococcus pyogenes isolates with characterized macrolide resistance mechanisms in Spain: in vitro activities of telithromycin and cethromycin.

The in vitro activities of telithromycin and cethromycin (ABT-773) against 412 Streptococcus pyogenes isolates, consecutively collected in 17 Spanish hospitals from different geographical areas, were evaluated and compared with those of erythromycin A, penicillin G, clindamycin and quinupristin-dalfopristin. With a susceptibility testing breakpoint of < or = 1 mg/L for both compounds, 96.1% of isolates were susceptible to telithromycin and 99.8% to cethromycin. Erythromycin non-susceptible isolates (intermediate plus resistant, MIC > or = 0.5 mg/L) comprised 23% of those tested, and were analysed for the genetic basis of their resistance by PCR. Among these isolates (n = 95), 72.6% harboured mef(A), 8.4%, erm(B), and 3.2%, erm(A), as sole macrolide resistance gene, whereas the presence of mef(A) plus erm(A) (11.6%) or mef(A) plus erm(B) (4.2%) was also observed. Both ketolides displayed a significant in vitro activity against S. pyogenes regardless of the macrolide resistance mechanisms. Nevertheless, in the case of telithromycin, 11 out of 19 of the erm(B)-positive isolates (2.7% of total population) exhibited an MIC range of 4-32 mg/L. According to the present results, telithromycin and cethromycin offer a wide coverage against S. pyogenes isolates in a geographic area with a high incidence of resistance to currently used macrolides.

Anti-Bacterial Agents↗
PubMed2003

Antimicrobial susceptibility profile of molecular typed cystic fibrosis Stenotrophomonas maltophilia isolates and differences with noncystic fibrosis isolates.

Multiresistance in Stenotrophomonas maltophilia limits the effectiveness of antimicrobial therapy for infections due to this organism. It can be of special concern in cystic fibrosis (CF) patients due to frequent antimicrobial administration. The in vitro activity of 41 antimicrobial agents against 76 epidemiologically defined CF S. maltophilia isolates by pulsed-field-gel electrophoresis (PFGE) technique under XbaI and SpeI restriction was compared with that obtained with 51 non-CF strains recovered from respiratory sources. Minimal inhibitory concentrations (MICs) were determined with the standard National Committee for Clinical Laboratory Standards agar dilution technique, but with 24-hr incubation. Forty-seven different PFGE profiles were observed within 76 S. maltophilia CF isolates. Minocycline (resistance rate, 0%; MIC(90), 1 microg/ml), doxycycline (6.4%; 8 microg/ml), trovafloxacin (4.2%; 2 microg/ml), moxifloxacin (6.3%; 2 microg/ml), clinafloxacin (6.3%; 2 g/ml), and moxalactam (17.0%; 64 g/ml) displayed low resistance rates. On the contrary, resistance rates were higher with ceftazidime (70.0%; 256 microg/ml), cefepime (83.0%; 128 microg/ml), piperacillin (87.2%; >1,024 microg/ml), ticarcillin (87.2%; >512 microg/ml), and aztreonam (95.7%; >1,024 microg/ml). Clavulanate reverted resistance to ticarcillin and aztreonam in 40.4% and 31.7% of strains, respectively. Aminoglycosides displayed reduced activities with susceptibility rates lower than 20% and MIC(90) higher than 128 microg/ml. With the exception of trimethoprim-sulfamethoxazole (25.4 vs. 31.3%), CF isolates were more resistant than non-CF isolates. Remarkably, resistance was enhanced in S. maltophilia isolates persistently recovered in chronically colonized patients. Susceptibility analysis demonstrated higher resistance rates among CF S. maltophilia isolates when compared with respiratory isolates from non-CF patients. Moreover, persistently recovered CF S. maltophilia isolates were more resistant than sporadic non-CF isolates.

Adolescent↗
PubMed2003

[Antibiotic resistance in 622 Streptococcus pneumoniae isolated from blood and cerebrospinal fluid in 33 Spanish hospitals participating in the European Antimicrobial Resistance Surveillance System (2000)].

INTRODUCTION: In 1998 the European Union founded the European Antimicrobial Resistance Surveillance System (EARSS) for the control of antimicrobial resistance in invasive pathogens. This report present the results obtained for S. pneumoniae in the year 2000 in Spain. METHODS: Participants included 33 hospitals covering approximately 25% of the Spanish population. The laboratories carried out isolation, identification and sensitivity studies with their usual methods. Quality control was performed by the UK National External Quality Assurance Scheme (NEQAS). A form was filled out with clinical information on the patient and data on the hospital, department and specimen isolated. This information was entered in a central database for analysis and validation by the Whonet.5 program. RESULTS: Invasive S. pneumoniae was isolated in 622 patients. Overall incidence was 6.4 cases/100,000 inhabitants. Among the strains isolated, 33.1% (CI 95%: 29.6-37) showed resistance to penicillin (22.2% intermediate; 10.9% resistant); 11% showed resistance to cefotaxime (10.1% intermediate; 0.9% resistant); and 21.1% were resistant to erythromycin. Erythromycin resistance was 7% in penicillin-sensitive strains and 46.4% in penicillin-resistant strains (p < 0.001). A total of 51.7% of strains were resistant to penicillin in children < or = 4 years old, as compared to 29% in patients > 4 years old (p < 0.001). A ciprofloxacin MIC > 2 mg/L was found for 2.4% of isolates. CONCLUSIONS: Penicillin resistance in invasive S. pneumoniae in Spain is among the highest in the European Union. The decrease in sensitivity to penicillin was significantly associated with cefotaxime and erythromycin resistance. Children < or = 4 years old have a significantly higher risk than adults of acquiring infection with strains resistant to penicillin, cefotaxime and erythromycin.

Adolescent↗
PubMed2003

[Comparative in vitro activity of garenoxacin (BMS-284756). Sentry program, Spain (1999-2000)].

INTRODUCTION: To evaluate the in vitro activity of the new des-fluoro quinolone, garenoxacin (BMS-284756), compared to activities of ciprofloxacin, levofloxacin, and gatifloxacin in clinical isolates recovered over 1999 and 2000 within the SENTRY antimicrobial surveillance program. METHODS: Quinolone-MICs were performed using the standard NCCLS microdilution technique in 2599 isolates recovered from Hospital Ramón y Cajal (Madrid), Virgen Macarena (Sevilla), and Bellvitge (Barcelona). RESULTS: The modal MIC range value exhibited by garenoxacin ( < or = 0.03-0.12 mg/L) for Enterobacteriaceae was similar to that of the other quinolones tested. A total of 70% of Pseudomonas aeruginosa isolates were susceptible to garenoxacin and 85% to ciprofloxacin and levofloxacin. Garenoxacin exhibited the highest activity in Staphylococcus aureus, including both methicillin-susceptible and -resistant isolates, with MIC90 values of < or = 0.03 and 2 mg/L, respectively. All Streptococcus pneumoniae isolates were susceptible to garenoxacin, regardless of their penicillin susceptibility status; in terms of MIC90, garenoxacin was 16 times more active than ciprofloxacin and levofloxacin and 4-8 times more active than gatifloxacin. All 6 ciprofloxacin-resistant S. pneumoniae strains showed garenoxacin MIC values ranging from < or = 0.03 to 0.5 mg/L. In Haemophilus influenzae and Moraxella catarrhalis, garenoxacin displayed excellent in vitro activity (MIC < or = 0.06 mg/L), similar to that of the other quinolones tested. CONCLUSIONS: Garenoxacin activity was similar to the activity of other quinolones in Enterobacteriaceae, but was lower in P. aeruginosa. Garenoxacin activity was clearly higher than that of other quinolones in gram-positive isolates, including methicillin-resistant S. aureus and S. pneumoniae with reduced penicillin susceptibility.

Anti-Infective Agents↗
PubMed2003

Mycoplasma pneumoniae: a reduced-genome intracellular bacterial pathogen.

Mycoplasma pneumoniae has classically been considered an extracellular (or membrane-associated) organism. Nevertheless, the recently elucidated genomic structure of this pathogen strongly suggest that this organism may have been subjected to the process of reductive genetic evolution which is characteristic of intracellular bacteria. We studied the Mycoplasma pneumoniae RYC15989 strain, recovered from a pericardial biopsy sample from a patient with atypical pneumonia and acute pericarditis. The interaction of this strain with human hepatocytes Hep-G2 and mouse neuroblastoma N2-A cell lines was investigated. Confocal laser scanning microscopy and electronic microscopy evidence is presented of the intracellular location of fluorochrome-labelled Mycoplasma pneumoniae in cell lines infected with the organism in vitro. This finding provides preliminary evidence of cellular invasive capacity of Mycoplasma pneumoniae and casts some new light on the pathogenic potential of Mycoplasma pneumoniae in host infection.

Animals↗
PubMed2003

Multi-resistant Gram-negative bacilli: from epidemics to endemics.

PURPOSE OF REVIEW: Infections due to multi-drug resistant Gram-negative bacilli represent a worrying situation for the management of hospitalized patients. In addition, these bacteria are increasingly involved in epidemics throughout the world. This review focuses on recent data that may help to understand the emergence and dissemination of multi-drug resistant bacilli and the current trend from epidemic to endemic situations. RECENT FINDINGS: Well-established clones enhance their resistance phenotype by the acquisition of new resistant genes, via gene capture genetic units (plasmids, transposons or integrons), thus facilitating the co-selective process under different antimicrobial selective pressures and therefore the long-term persistence of organisms in selective environments. Not only resistant bacterial clones are selected, but also their genetic structures carrying resistance genes. Therefore, current epidemiology of multi-drug resistant bacilli is not only focused on bacterial clones but also on any kind of resistance gene capture units. In this scenario a multiclonal population structure of bacterial organisms corresponds to a collection of different strains sharing resistance genes carried by horizontally transferred genetic structures. As different strains tend to prefer different environments, this concept helps understand why the epidemiology of multi-drug resistant Gram-negative bacilli is moving from epidemics to endemics. SUMMARY: The emergence and spread of multi-drug resistant bacilli in the nosocomial setting should be understood in terms of a complex interplay of bacterial clonality, resistance genes and genetic structures promoting rapid dissemination of antimicrobial resistance. Intervention strategies in the forthcoming scenario should identify existing epidemic and/or endemic situations involving clonal organisms or resistance genes carried by epidemic gene capture units.

Cross Infection↗
PubMed2003

Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation.

The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 x 10(-7) (20% of isolates) and 2.5 x 10(-8). The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and "normomutable" strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.

Bacterial Proteins↗