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J Melo-Cristino

Publications and source records attributed to J Melo-Cristino.

At least 19 recordsLinked to original sources
PubMed2006

DNA methylase activity as a marker for the presence of a family of phage-like elements conferring efflux-mediated macrolide resistance in streptococci.

Recently, two related chimeric genetic elements (Tn1207.3 and Phi10394.4) were shown to carry the macrolide efflux gene mef in Streptococcus pyogenes (group A streptococci [GAS]). The dissemination of elements belonging to the Tn1207.3/Phi10394.4 family in recent isolates of GAS, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus pneumoniae, and Streptococcus agalactiae recovered in Portugal was surveyed. In total, 149 GAS, 18 S. pneumoniae, 4 S. dysgalactiae subsp. equisimilis, and 5 S. agalactiae isolates from infections, presenting the M phenotype of macrolide resistance and containing the mef gene, were screened for the presence of Tn1207.3/Phi10394.4 by PCR targeting open reading frames (ORFs) specific for these related elements. All the GAS isolates tested and one of the S. dysgalactiae subsp. equisimilis isolates carried Tn1207.3. However, neither of these elements was found in the isolates of the other streptococcal species. It was also noted that the DNAs of the isolates carrying Tn1207.3 were resistant to cleavage by the endonuclease SmaI. Cloning and expression of ORF12 of Tn1207.3 in Escherichia coli showed that it encoded a methyltransferase that rendered DNA refractory to cleavage by SmaI (M.Spy10394I). Using this characteristic as a marker for the presence of the Tn1207.3/Phi10394.4 family, we reviewed the literature and concluded that these genetic elements are widely distributed among tetracycline-susceptible GAS isolates presenting the M phenotype from diverse geographic origins and may have played an important role in the dissemination of macrolide resistance in this species.

Anti-Bacterial Agents↗
PubMed2006

Heterogeneity of pneumococcal phase variants in invasive human infections.

BACKGROUND: Streptococcus pneumoniae can be carried asymptomatically in the nasopharynx of its human host but can also cause a wide range of infections. A role for pneumococcal phase variants in the different lifestyles of this bacterium has been suggested but no systematic survey of the colony phenotypes of isolates associated with human infections has been undertaken. RESULTS: We report the colony opacity phenotypes of a genetically diverse set of 304 invasive isolates representing 10 serotypes. Over half of the isolates (52%) presented the opaque phenotype whereas transparent variants accounted for only 26% of the total. However, the frequency of recovery of each phase variant was not uniform, while serotypes 1, 4, 12B and 23F presented the opaque phenotype more frequently than expected by chance, serotypes 3 and 14 where less frequently associated with this phenotype. CONCLUSION: The opaque phenotype was the most frequent phenotype found among invasive isolates. An unexpected and equally important finding is the variability of the dominant opacity phenotype found among serotypes. This observation highlights the heterogeneity of opacity phenotypes in invasive isolates and lends further support to the proposal that other factors, in addition to the site of isolation, determine the opacity phenotype of a given isolate. The association between serotype and colonial opacity could help explain epidemiological differences observed among pneumococcal serotypes such as a higher invasive disease potential.

Animals↗
PubMed2006

Emergence of optochin resistance among Streptococcus pneumoniae in Portugal.

In most clinical microbiology laboratories optochin susceptibility is used in the screening and identification of Streptococcus pneumoniae. We report the characterization of 32 optochin-resistant S. pneumoniae strains from 10 laboratories that constituted 3.2% of all isolates recovered in 2005 in 30 laboratories in Portugal. Resistant isolates consisted of bile-soluble optochin-susceptible and optochin-resistant subpopulations with identical antimicrobial susceptibility patterns, capsular types and pulsed-field gel electrophoresis (PFGE) profiles. The most frequent serotypes--1, 19A, 11A, 3, 8, and 15A--were all common serotypes present in infection and colonization isolates in the country. The PFGE profiles of the 32 isolates corresponded to those of previously identified clones and confirmed that the emergence of these strains could not be attributed to clonal expansion. Clinical laboratories must be aware that optochin-resistant pneumococci are presently circulating in the community. Because accurate identification of S. pneumoniae is essential for correct diagnosis and adequate therapy of patients, we recommend that at least the bile solubility test should be routinely performed in cases of suspected pneumococcal etiology, even if the isolates are optochin-resistant.

Anti-Bacterial Agents↗
PubMed2006

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal.

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.

Bacitracin↗
PubMed2006

Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes.

The studies that correlate the results obtained by different typing methodologies rely solely on qualitative comparisons of the groups defined by each methodology. We propose a framework of measures for the quantitative assessment of correspondences between different typing methods as a first step to the global mapping of type equivalences. A collection of 325 macrolide-resistant Streptococcus pyogenes isolates associated with pharyngitis cases in Portugal was used to benchmark the proposed measures. All isolates were characterized by macrolide resistance phenotyping, T serotyping, emm sequence typing, and pulsed-field gel electrophoresis (PFGE), using SmaI or Cfr9I and SfiI. A subset of 41 isolates, representing each PFGE cluster, was also characterized by multilocus sequence typing (MLST). The application of Adjusted Rand and Wallace indices allowed the evaluation of the strength and the directionality of the correspondences between the various typing methods and showed that if PFGE or MLST data are available one can confidently predict the emm type (Wallace coefficients of 0.952 for both methods). In contrast, emm typing was a poor predictor of PFGE cluster or MLST sequence type (Wallace coefficients of 0.803 and 0.655, respectively). This was confirmed by the analysis of the larger data set available from http://spyogenes.mlst.net and underscores the necessity of performing PFGE or MLST to unambiguously define clones in S. pyogenes.

Anti-Bacterial Agents↗
PubMed2006

Clonal relationships between invasive and noninvasive Lancefield group C and G streptococci and emm-specific differences in invasiveness.

Lancefield group G and group C streptococci (GGS and GCS, respectively) are pathogens responsible for a number of life-threatening infections. A collection of 116 recent (1998 to 2004) invasive (n = 28) and noninvasive (n = 88) GGS and GCS clinical isolates from Portugal were characterized. All isolates were identified as Streptococcus dysgalactiae subsp. equisimilis and characterized by emm typing and DNA macrorestriction profiling using pulsed-field gel electrophoresis (PFGE). emm typing revealed the presence of 22 distinct types, including 3 novel types. PFGE identified 14 clones with more than two isolates, but over half of the isolates were concentrated in 3 large clones. Individual clones and emm types showed a low level of association, since the majority of the clones included more than one emm type and the same emm type was found among diverse genetic backgrounds. Two emm types, stg2078 and stg10, were significantly more frequent among invasive isolates, and another two, stg6792 and stg166b, were present only in noninvasive isolates, suggesting a correlation between emm type and invasive disease potential.

Bacterial Typing Techniques↗
PubMed2005

Rapid inversion of the prevalences of macrolide resistance phenotypes paralleled by a diversification of T and emm types among Streptococcus pyogenes in Portugal.

In Portugal erythromycin resistance of 26.6% (n = 352) remained constant during 1998 to 2003, however in 1998 the MLS(B) phenotype dominated (85%), whereas in 2003 the M phenotype prevailed (77%). A decline in T12/emm22 MLS(B) isolates could partially explain the drop in this phenotype, but the rise of the M phenotype was not due to clonal expansion.

Anti-Bacterial Agents↗
PubMed2005

Characterization of the genetic lineages responsible for pneumococcal invasive disease in Portugal.

The availability of a conjugate vaccine has the potential to reduce the disease burden of pneumococci and to alter the serotype frequency in the disease-causing population through immunoselection. These changes will probably be reflected in the distributions of individual genetic lineages within the population. We present a characterization of a collection of recent (1999 to 2002) invasive isolates from Portugal (n = 465) by macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. During this time, serotypes 14, 1, 3, 4, 8, 9V, 23F, 7F, 19A, and 12B were the 10 most prevalent overall by decreasing rank order. By combining the PFGE data with the sequence types (STs) of 104 isolates, we were able to identify the genetic lineages of the majority of the isolates. We found 66 STs, including 20 novel STs, corresponding to 47 different lineages by e-BURST analysis. We found in our collection a number of previously identified internationally disseminated lineages, especially among macrolide-resistant and penicillin-resistant isolates, and these accounted for most of the isolates. Most of the major lineages (17 of 25) were identified in all years of the study, suggesting that the pneumococcal population associated with invasive disease was stable. This study provides a characterization of the pneumococcal population associated with invasive disease that will be useful for detecting potential selective effects of the novel conjugate vaccine.

Alleles↗
PubMed2004

Streptococcus agalactiae in a large Portuguese teaching hospital: antimicrobial susceptibility, serotype distribution, and clonal analysis of macrolide-resistant isolates.

Group B streptococci are emerging as a cause of serious infection worldwide. The capsular polysaccharides are not only important virulence factors but also the target of vaccine development efforts. Serotypes III (24.6%), V (23.4%), Ia (17.8%), and II (16.3%) were the most prevalent among 252 Streptococcus agalactiae isolates collected during 1999-2002 in the largest hospital of Lisbon, Portugal. The substantial proportion of bacteremic patients (17 neonates and 21 adults) in this period illustrates the present importance of S. agalactiae as a cause of invasive disease. All isolates were fully susceptible to penicillin (MIC(50) = 0.064 microg/ml; MIC(90) = 0.094 microg/ml, range 0.008-0.094), cefotaxime, chloramphenicol, ofloxacin, and vancomycin. Resistance was found to tetracycline (75.4%), erythromycin (10.7%), and clindamycin (9.9%). Of the 27 erythromycin-resistant isolates, 70.4% had the cMLS(B), 22.2% the iMLS(B), and 7.4% the M phenotype. All isolates presenting the M phenotype carried the mef(A) gene, whereas the erm(B) gene was found in a large fraction of MLS(B) isolates (n = 17) and only a small proportion (n = 7) the erm(A) gene [erm(TR) variant]. All isolates carried a single macrolide-resistance determinant. Macrolide resistance was not attributable to a single clone as evidenced by distinct serotype and pulsed-field gel electrophoretic profiles. Careful surveillance of S. agalactiae invasive infections in Portugal is essential, and the treatment or intrapartum prophylaxis of patients who are allergic to penicillin should be guided by contemporary resistance patterns observed in the country.

Anti-Bacterial Agents↗
PubMed2004

Case of aortic endocarditis caused by Lactobacillus casei.

A case of Lactobacillus aortic valve endocarditis in a 53-year-old immunocompetent patient with past history of rheumatic fever is reported. Clinical symptoms began after a dental extraction and the patient's diet included several yogurts per day. Blood, bone marrow cultures and the replaced aortic valve were positive for Lactobacillus: The clinical isolate was identified as Lactobacillus casei by 16S rDNA sequencing.

Aortic Valve↗
PubMed2004

Invasive Streptococcus pneumoniae from Portugal: implications for vaccination and antimicrobial therapy.

The distribution of pneumococcal serotypes among 465 invasive isolates recovered from 1999 to 2002 in Portugal was analysed by age group. Serotype 14 was either the most prevalent or the second most prevalent in all age groups. Among children aged < 2 years, serotypes 6B and 23F, which are usually associated with children, together with serotypes 19A and 14, accounted for more than half of the isolates. In contrast, in older adults (> or = 60 years), serotypes 3, 14, 1, 8 and 4 were the most prevalent. The potential coverage of the seven-valent conjugate vaccine is 63.2% among infants, and does not change significantly if children aged < 6 years are considered, which is a lower coverage than in other European countries. The potential coverage of the 23-valent polysaccharide vaccine is high in all age groups, particularly among older adults (80.7%). All isolates were tested for their susceptibility to penicillin, cefuroxime, cefotaxime, vancomycin, erythromycin, clindamycin, levofloxacin, gatifloxacin, moxifloxacin, linezolid, quinupristin-dalfopristin, tetracycline, chloramphenicol and trimethoprim-sulphamethoxazole. Most isolates collected from children aged < 6 years had decreased susceptibility to at least one antibiotic class, whereas isolates from patients aged > or = 6 years were mostly susceptible to all antimicrobial agents tested. Overall, 23% of isolates showed reduced susceptibility to penicillin. Most (98.5%) isolates remained fully susceptible to cefotaxime, and a single isolate was resistant to quinolones.

Age Distribution↗
PubMed2003

Macrolide resistance in Streptococcus pneumoniae isolated from patients with community-acquired lower respiratory tract infections in Portugal: results of a 3-year (1999-2001) multicenter surveillance study.

A nationwide multicenter study (including 31 laboratories) of the antimicrobial susceptibility of 1210 Streptococcus pneumoniae isolates from patients with community-acquired lower respiratory tract infections (LRTI) was carried out over 3 years (1999-2001) in Portugal. Testing of all isolates was undertaken in a central laboratory. Overall macrolide resistance was 13.1%. Decreased susceptibility to penicillin was 24.5% (15.5% low-level and 9.0% high-level resistance). Taken into consideration, the resistance rates reported in a previous surveillance study of 1989-1993, a six-fold increase of erythromycin resistance in the last decade was documented. Resistance to erythromycin, clarithromycin, and azithromycin was higher in pediatric patients than in adults. The overwhelming majority (82.3%) of macrolide-resistant isolates were multidrug resistant, although 44.9% were fully susceptible to penicillin. Most macrolide-resistant isolates (80.4%) showed the MLSB phenotype (76.6% MLSB-constitutive resistance, and 3.8% MLSB-inducible resistance) and were also resistant to clindamycin, tetracycline, and co-trimoxazole. The M phenotype was seen in 19.6% isolates and these had MIC90 values of 8 mg/L for erythromycin and clarithromycin, and of 12 mg/L for azithromycin. The clinical significance of macrolide resistance in the management of LRTI is discussed. Because of the specific situation concerning macrolide resistance described in S. pneumoniae, careful use of macrolide antibiotics in therapy and cautious monitoring of macrolide resistance should be continued in Portugal.

Adult↗
PubMed2001

A multicenter study of the antimicrobial susceptibility of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis isolated from patients with community-acquired lower respiratory tract infections in 1999 in Portugal.

A nationwide multicenter study (including 25 laboratories) of the antimicrobial susceptibility of bacterial pathogens commonly associated with community-acquired lower respiratory tract infections (LRTI), with testing undertaken in a central laboratory, was conducted in Portugal in 1999. Antimicrobial resistance in Haemophilus influenzae has not increased in the last decade. Of the 498 isolates tested, 12.4% produced beta-lactamase and >95% were susceptible to all antimicrobials except ampicillin. In contrast, there was a rapid increase of resistance in Streptococcus pneumoniae. Of the 312 isolates tested, 24.7% exhibited decreased susceptibility to penicillin (13.5% showed low-level and 11.2% high-level resistance), 13.8% were resistant to erythromycin, clarithromycin and azithromycin, and 13.6% to cefuroxime and to tetracycline. Of the 38 Moraxella catarrhalis tested, 81.6% produced beta-lactamase. Resistance to penicillin, cefuroxime, erythromycin, clarithromycin, and azithromycin in S. pneumoniae and beta-lactamase production in H. influenzae were significantly higher in pediatric patients than in adults. Overall, amoxycillin/clavulanate was the most active antimicrobial agent in vitro against H. influenzae, S. pneumoniae, and M. catarrhalis isolated from patients with community-acquired LRTI in Portugal.

Adult↗
PubMed1999

Streptococcus pyogenes isolated in Portugal: macrolide resistance phenotypes and correlation with T types. Portuguese Surveillance Group for the Study of Respiratory Pathogens.

From January 1998 to June 1999, 302 clinical isolates of Streptococcus pyogenes were collected from 10 microbiology laboratories in Portugal. All strains were highly sensitive to penicillin (MIC90 = 0.012 mg/liter). The prevalence of erythromycin resistance was 35.8% and of tetracycline resistance 41.4%. The majority (79.6 %) of erythromycin-resistant strains were of the MLSB constitutive resistance (CR) phenotype with high-level resistance to erythromycin (MIC90 >256 mg/liter) and to clindamycin (MIC90 >256 mg/liter), 16.7% showed the M phenotype with low-level erythromycin-resistance (MIC90 = 24 mg/liter) and susceptibility to clindamycin, and four isolates showed a phenotype characterized by low-level erythromycin resistance (MIC90 = 8 mg/liter) and high-level clindamycin resistance (MIC90 >256 mg/liter), not previously described. Erythromycin resistance was not associated with invasive strains. Only minor discrepancies between disk diffusion and E-test methods were observed. T serotyping was very useful for the epidemiological characterization of the strains. The most prevalent T types were T1, T4, T9, T12, T13, and T28. A statistically significant association with resistance patterns was found: T12 with erythromycin resistance MLS(B) CR phenotype (p< 0.001), T4 with erythromycin resistance M phenotype (p<0.001), and T13 with tetracycline resistance (p<0.01). Because of the high prevalence of resistance, careful surveillance of S. pyogenes isolates in Portugal is essential, routine antimicrobial susceptibility testing in clinical microbiology laboratories should be strongly encouraged, antibiotic prescription should be reviewed, and macrolides should no longer be used in the empirical therapy of acute pharyngitis.

Anti-Bacterial Agents↗
PubMed1998

Antimicrobial resistance in staphylococci and enterococci in 10 Portuguese hospitals in 1996 and 1997. POSGAR. Portuguese Study Group of Antimicrobial Resistance.

During a 2-year period, 10 Portuguese hospitals located throughout the country studied antimicrobial susceptibilities of clinically relevant staphylococci and enterococci. Of more than 12,000 Staphylococcus aureus isolates tested, two main patterns were found, methicillin-sensitive organisms most of them resistant only to penicillin but a few to other antimicrobials and methicillin-resistant S. aureus (MRSA) strains (prevalence 48.2%) resistant to most of the antimicrobials tested and uniformly susceptible to vancomycin. Among coagulase-negative staphylococci (CNS), 71% of S. epidermidis (approximately 5,000 isolates tested) and 84% S. haemolyticus (approximately 1,000 isolates tested) were also resistant to methicillin as well as most other antimicrobials except vancomycin. Most of the 5,000 Enterococcus faecalis isolates tested were susceptible to ampicillin and vancomycin, in contrast to 650 E. faecium isolates, 70% of which were resistant to ampicillin and 20% to vancomycin and all other antibiotics. A high prevalence of aminoglycoside resistance occurred in both Enterococcus species. This survey showed that resistance profiles of staphylococci and enterococci hospital isolates have not changed in the last 5 years in Portugal, with the exception of the rise in vancomycin resistance in E. faecium. The high prevalence of methicillin resistance in S. aureus and in the CNS remains an issue of medical concern.

Anti-Bacterial Agents↗
PubMed1995

Unusually large number of methicillin-resistant Staphylococcus aureus clones in a Portuguese hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hospital de Santa Maria, a 1,300-bed teaching hospital in Lisbon, Portugal, since the mid-1980s with a prevalence of 30% in 1993. A total of 54 MRSA and 93 methicillin-susceptible S. aureus (MSSA) isolates recovered during the first 3 months of 1993 were analyzed for the particular mecA polymorphs and Tn554 attachment sites (in the case of MRSA) and for pulsed-field gel electrophoretic patterns. While all MRSA isolates shared a very similar multidrug resistance antibiogram, molecular methods allowed the identification of an unusually large number of genetic backgrounds (24 different pulsed-field gel electrophoresis patterns in 54 isolates) and three different mecA polymorphs among the MRSA strains. Similar large variation in the genetic backgrounds of MSSA was observed. The most frequent mecA polymorph (mecA type I) was found in association with three different Tn554 patterns. Among the MRSA strains of Hospital Santa Maria, we found two clonal types previously described in Portugal: one corresponding to the dominant clone in an MRSA outbreak at the pediatric ward of the Lisbon Hospital Dona Estefânia and another one identical to the Iberian epidemic clone identified in several Portuguese hospitals and in MRSA outbreaks in Barcelona and Madrid. This suggests that MRSA clones of Hospital de Santa Maria may have been a reservoir for staphylococcal strains over the past decade.

Bacterial Typing Techniques↗
PubMed1994

Methicillin-resistant Staphylococcus aureus disease in a Portuguese hospital: characterization of clonal types by a combination of DNA typing methods.

Fifteen pediatric patients as well as the five nursing staff of the Burn Unit of the Hospital D. Estefania in Lisbon, Portugal, were assayed at weekly intervals over a five-month period in order to identify the nature and number of methicillin-resistant Staphylococcus aureus (MRSA) clones associated with colonization and wound infection. Methicillin resistance was confirmed by a mec-specific DNA probe. MRSA isolates were classified into chromosomal types (clones) on the basis of a variety of techniques: (i) ribotyping; (ii) restriction digestion by the endonuclease ClaI followed by Southern hybridization with the mecA-specific DNA probe and (iii) by hybridization with Tn554; and (iv) pulsed-field electrophoresis (PFE) of SmaI digests followed by (v) Southern hybridization with the mecA DNA probe. A sixth, physiological technique (population analysis) was used to define the mode of phenotypic expression of methicillin resistance in each isolate. All isolates carried a single, common polymorph (ClaI type III) of the mecA gene. Hybridization with Tn554 resolved these isolates to two novel patterns (alpha and beta), of which one (Tn554 alpha) was predominant (90%). This pattern could be further resolved to four closely related PFE types (A through D). In contrast, all isolates with the Tn554 beta pattern belonged to an additional, grossly different PFE type E. The Tn554 beta class was also unique in that these bacteria carried the mecA gene in a SmaI fragment smaller (about 170 kb) than that found in the alpha type strains (194 kb). Most isolates (83%) showed a single heterogeneous (population analysis Class 3) mode of resistance expression. The data demonstrate the full capacity of the globally rare (ClaI type III) MRSA clone for colonization and virulence. The results also document the stability of the complex heterogeneous resistance phenotype as well as the stability of the chromosomal types under conditions of in vivo carriage over a period of several months. In a few isolates the same mecA polymorph was present in several, grossly different genetic backgrounds, suggesting horizontal transfer of the mecA gene.

Adult↗