Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6.

Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(ß) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(ß) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.

Invasive pneumococcal diseases in Uruguayan children: comparison between serotype distribution and conjugate vaccine formulations.

An on-going Streptococcus pneumoniae surveillance in Uruguay furnished national information on serotype distribution of isolates from invasive infections in children

Evolution of Streptococcus pneumoniae serotypes and penicillin susceptibility in Latin America, Sireva-Vigía Group, 1993 to 1999. PAHO Sireva-Vigía Study Group. Pan American Health Organization.

BACKGROUND: Since 1993 the Pan American Health Organization has coordinated a surveillance network with the National Reference Laboratories of Argentina, Brazil, Chile, Colombia, Mexico and Uruguay aimed at monitoring capsular types and antimicrobial susceptibility of Streptococcus pneumoniae causing invasive disease in children <6 years of age. METHODS: The surveillance system included children 6 years of age and younger with invasive disease caused by S. pneumoniae. The identification, capsular typing and susceptibility to penicillin of the isolates were conducted using a common protocol, based on standard methodologies. RESULTS: By June, 1999, 4,105 invasive pneumococcal isolates had been collected mainly from pneumonia (44.1%) and meningitis (41.1%) cases. Thirteen capsular types accounting for 86.1% of the isolates (14, 6A/6B, 5, 1, 23F, 19F, 18C, 19A, 9V, 7F, 3, 9N and 4) remained the most common types during the surveillance period. Diminished susceptibility to penicillin was detected in 28.6% of the isolates, 17.3% with intermediate and 11.3% with high level resistance. Resistance varied among countries and increased during this period in Argentina, Colombia and Uruguay. Serotypes 14 and 23F accounted for 66.6% of the resistance. CONCLUSION: These surveillance data clearly demonstrate the potential impact of the introduction of a conjugate vaccine on pneumococcal disease and the need for more judicious use of antibiotics to slow or reverse the development of antimicrobial resistance.

Three-year assessment of methicillin-resistant Staphylococcus aureus clones in Latin America from 1996 to 1998.

Four hundred ninety-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America-Argentina, Brazil, Chile, Uruguay and Mexico-were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal SmaI digests (ClaI-mecA::ClaI-Tn554::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::kappa::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E'::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.

Antibiotic resistance in Streptococcus pneumoniae in six Latin American countries: 1993-1999 surveillance.

The impact of invasive pneumococcal invasive disease is increased by the emergence of antibiotic resistance. We report regional and temporal variations in antibiotic resistance for 4,105 invasive Streptococcus pneumoniae isolates collected from Latin American children <5 years, between 1993 and 1999. Reduced susceptibility to penicillin was detected in 1,182 isolates (28.8%); 36% of these were resistant (> or = 2 microg/ml), including 12.6% with MIC > or = 4 microg/ml, occurring primarily in serotypes 14 and 23F. Reduced susceptibility to third-generation cephalosporins was detected in 12.1% of the collection. Mexico had the highest proportion of reduced susceptibility to penicillin (51.6%) and to third-generation cephalosporins (22%), whereas Brazil had the lowest at 20.9% and 0.7%, respectively. Isolates cultured from patients with pneumonia were more likely to have reduced susceptibility to third-generation cephalosporins than isolates from patients with meningitis (p < 0.0001). Susceptibility to trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, and vancomycin was tested by disk diffusion for 2.899 isolates. Reduced susceptibility was observed for 45.6%, 11.5%, 6.9%, and 0%, respectively. Thirty-one percent of the strains were resistant to > or = 2 drugs. High levels of antibiotic resistance in Latin America emphasize the need for the development of and adherence to rational antibiotic use guidelines. On-going surveillance will monitor the impact of these programs.

[Impact of Streptococcus pneumoniae on pneumonia in Latin American children. SIREVA-Vigía Group]. / Impacto de Streptococcus pneumoniae en las neumonías del niño latinoamericano. Grupo SIREVA-Vigía.

Community-acquired pneumonia is one of the leading causes of infant morbidity and mortality. Studies conducted in developing countries indicate that the most serious symptoms of pneumonia are associated with bacterial causes, mainly Streptococcus pneumoniae, followed by Haemophilus influenzae type b. Managing those infections in children under two years of age is hindered by the lack of appropriate vaccines and by the decreased susceptibility of S. pneumoniae to penicillin and other antibiotics. In 1993, at the initiative of the Regional System for Vaccines of the Pan American Health Organization, and with funding from the Canadian International Development Agency, a study was designed to identify the S. pneumoniae capsular types that cause invasive disease in Latin American children under 5 years of age. The objective of the study was to determine the ideal composition of a conjugate vaccine that could be used in Latin America, and the penicillin susceptibility of the S. pneumoniae isolates. The initiative was undertaken in Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay. This report analyzes the information that the participating countries generated on pneumococcal pneumonia. A total of 3,393 children were found with systemic S. pneumoniae infections, of which 1,578 corresponded to pneumonias. The analysis focused on 1,409 cases of pneumonia in Argentina, Brazil, Colombia, Mexico, and Uruguay. Of the children, 63.8% of them were under two years of age. Twelve prevalent capsular types were identified, of which serotypes 14, 5, and 1 were the three most common in the majority of the countries. At the beginning of the study the highest level of penicillin resistance was found in Mexico (47.0%), and the lowest in Colombia (12.1%). Over the 1993-1998 period, resistance to penicillin increased in the five countries. Penicillin resistance was associated with a small number of capsular serotypes, mainly 14 and 23F. The first of those serotypes was resistant to penicillin and to trimethoprim-sulfamethoxazole, and the second was multiresistant. The frequency of resistance to trimethoprim-sulfamethoxazole was high in all of the countries; Argentina had the highest level, 58.0%. A decrease in susceptibility to chloramphenicol was uncommon, except in Colombia, where there was a resistance level of 23.4%. Resistance to erythromycin was low in all the countries, and all the isolates were susceptible to vancomycin.

Ten-year review of invasive pneumococcal diseases in children and adults from Uruguay: clinical spectrum, serotypes, and antimicrobial resistance.

OBJECTIVES: Since 1987, the Reference Laboratory of the Ministry of Health of Uruguay has been monitoring infections due to Streptococcus pneumoniae in patients under 5 years of age, in those between 5 to 14 years of age, and in adults. The purpose of the present study was to retrospectively analyze a 10-year collection of invasive S. pneumoniae isolates from children 5 to 14 years of age and adults. METHODS: The Reference Children's Hospital, Pasteur Hospital, and two private hospitals in Montevideo as well as four hospitals located in other representative areas of the country participated in the pneumococcal surveillance program. Based on the information available at the Microbiology Department of the Central Public Health Laboratory (demographic data, date and site of isolate, and clinical diagnosis), all patients with an invasive pneumococcal disease were recorded. Pneumonia was clinically and radiologically diagnosed and etiology was assessed by isolation of S. pneumoniae from blood or pleural fluid. All specimens were collected at the Emergency Service. Capsular serotyping and antimicrobial susceptibilities were determined for each isolate. RESULTS: During the 10-year period, 228 invasive S. pneumoniae were identified and included in the study (blood, n = 129; cerebrospinal fluid [CSF], n = 73; pleural fluid, n = 20; peritoneal fluid, n = 3; synovial fluid, n = 1; pericardic fluid, n = 1; abscess, n = 1). The most frequent clinical presentations were pneumonia (n = 71) and meningitis (n = 69). Thirty-five adults had an underlying condition including, four with malignancies, four with lupus, two with human immunodeficiency virus (HIV)-infected, and two patients in hemodialysis among others. Eighteen of the 228 patients died (7.9% fatality rate), but only four of these had an underlying condition. Eleven fatal cases were attributable to meningitis (2 children, 9 and 11 years old; 9 adults, mean age, 59 y). Four patients with pneumonia and three with sepsis died, including a splenectomized woman. Nine different capsular serotypes (1, 5, 7, 9, 12, 15, 19A, 20, and 23A) were identified among the 18 fatal cases. Resistance to penicillin, generally combined with trimethoprim-sulfamethoxazole, fluctuated annually, not surpassing 10%. CONCLUSIONS: The study results indicated that 96% of the serotypes involved in severe pneumococcal diseases were included in the 23-valent vaccine and that S. pneumoniae resistance to penicillin was moderate.

Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. PAHO/Rockefeller University Workshop. Pan American Health Organization.

The Pan American Health Organization (PAHO) has conducted a study of Streptococcus pneumoniae in six Latin-American countries: Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay. Sterile site isolates from children aged < or =5 years showing clinical symptoms of pneumonia (as defined by the clinical criteria of WHO), meningitis, sepsis or bacteremia (without infectious foci), arthritis, and peritonitis were the source of most of the invasive pneumococcal isolates collected between the end of 1993 and 1996 in the six participating countries. Partial characterization of these isolates (antibiotic resistance and serotyping) have already been described (Microbial Drug Resistance 3:(2):131-163, 1997). In the next phase of the study, 326 S. pneumoniae isolates with reduced penicillin susceptibility were transferred to the Laboratory of Microbiology at The Rockefeller University for molecular characterization, and a summary and overview of the findings is described in this article. Some of the most interesting findings were as follows: (1) There was a surprisingly high representation of two internationally spread clones, which made up >80% of the strains with penicillin MIC of 1 microg/ml or higher; most of these isolates were recovered in large cities, supporting the likelihood that the source of these clones is through international travel. (2) The frequency of resistance to trimethoprim/sulfamethoxazole was extremely high (present in 85% of all isolates with decreased penicillin susceptibility). (3) None of these isolates was resistant to ofloxacin, and macrolide resistance was rare (present in 6.4% of the isolates). (4) There was an apparent inverse relationship between level of penicillin resistance and genetic diversity. (5) There were striking differences in the "microbiologic profiles" of the six different Latin-American countries.

The apparent importation of penicillin-resistant capsular type 14 Spanish/French clone of Streptococcus pneumoniae into Uruguay in the early 1990s.

Since 1987, Streptococcus pneumoniae (SPN) has been monitored in pediatric and adult populations. In 1994, the SIREVA/PAHO surveillance allowed the assessment of a dramatic increase of penicillin resistance, mainly associated to SPN type 14 also resistance to trimethoprim/sulfamethoxazole. To determine the genetic relatedness of the resistant isolates, analysis of SmaI digests of 61 isolates was performed by pulsed field gel electrophoresis (PFGE). Twelve different profiles were identified. The type 14 resistant SPN (n = 47) displayed a common B pattern, with 15 genetically related subtypes (1-6 bands of difference), with B1 the predominant one (51%). One of the subtypes (B12) was indistinguishable from a French isolate (M13P). This strain represented a South European clone, identified in the late 1980s, that also included serogroup 9 isolates. The type 23F isolates (n = 3) were identical with a representative strain of the well-characterized intercontinental type 23F clone. Neither the type 14 penicillin-susceptible (n = 4) nor the resistant SPN of other serotypes (n = 10) belonged to the B pattern. In summary, the results suggest that the dramatic increase of penicillin resistance in Uruguay depends on the introduction and spread of a type 14 clone, apparently imported from the south of Europe. Follow-up of this phenomenon is mandatory from the point of view of epidemiology and is also a priority for biologic research.

Frequency, Type and Associated Diseases of Bacteria and Virus in The Oropharynx of Children Born to Human Immunodeficiency Virus-Infected Mothers.

HIV-infected children are more likely than other children to develop pneumonia, which in these children is often recurrent or persistent. The main reservoir of the major pathogens is the nasopharynx, but to date no data has been published on the frequency and biologic characteristics of S.pneumoniae, H.influenzae and respiratory viruses found in the upper respiratory tract of children born to human immunodeficiency virus-infected mothers. To document these aspects, 105 children was monitored by pharyngeal swab (PS) and nasopahryngeal aspirates (NPA) who attended an outpatient clinic for HIV-infection evaluation. Bacterial identification was performed by standard procedures. Serotype, biotype and beta-lactamase production was investigated in H.influenzae isolates. S.pneumoniae serotypes were recognized by "quellung" and the susceptibility to 4 antibiotics was assessed. Respiratory syncytial viruses, parainfluenza, influenza A and B, and adenoviruses were diagnosed by indirect immunofluorescence and/or viral isolation in cell cultures. Twenty-nine children were identified as infected by HIV as a result of maternal-child transmission. Seventy children born to HIV-positive mothers but who were not HIV-infected served as controls. Of 269 PS, 110 110 S. pneumoniae and 92 H.influenzae were identified. Also 31 viruses were detected in 188 NPA. After stratifying by age no differences were observed in the frequency of bacterial colonization or in the presence of viruses in the upper respiratory tract of the two groups. Some biologic characteristics of the agents were noteworthy such as the frequency of colonization by S.pneumoniae serotype 14, the predominance of H.influenzae biotype I and the high frequency of viruses in NPA of asymptomatic children. Of note, although colonization frequencies were similar, children presenting with acute respiratory illness (ARI) were more likely to have bacteria isolated if they also had HIV-infection than if they were HIV-negative. It is concluded that HIV-infection in infants as a result of maternal virus transmission have a similar frequency of bacteria and virus colonization of their respiratory tract, but a higher frequency of ARI and perhaps a higher frequency of types of bacteria with special characteristics.

Capsular type distribution and susceptibility to antibiotics of Streptococcus pneumoniae clinical strains isolated from Uruguayan children with systemic infections. Pneumococcus Study Group.

Children under 24 months of age are at high risk for serious infection with Streptococcus pneumoniae but they do not elicit effective immune responses to the currently available capsular polysaccharide vaccines. A polysaccharide protein conjugated vaccine involving the most frequent types has become an urgent need. To produce such a vaccine for Latin America, information on type distribution is required. Recently, Uruguay was 1 of the 6 countries in Latin America where surveillance for invasive pneumococcal infections in children under the age of 5 years was carried out. Seventy percent of the 182 invasive S. pneumoniae isolates were recovered from patients under 24 months of age, and 19% were recovered from infants under 6 months. The 7 most frequent types were 14, 5, 1, 6B, 3, 7F, and 19A; representing 80% of invasive isolates. Twenty-one types were identified, 16 in pneumonia and 14 in meningitis. Resistance to penicillin increased during the study period, from 29% in 1994, to 40% in 1995-1996, mainly because of the spread of type 14 strains resistant to penicillin and trimethoprim/sulphamethoxazol (89% of resistant isolates). The high proportion of systemic pneumococcal infections recorded in patients under 24 months of age and the increasing resistance of these agents to first-choice antibiotics point to an urgent need for a capsular polysaccharide protein conjugated vaccine.

Colonizing and invasive strains of Streptococcus pneumoniae in Uruguayan children: type distribution and patterns of antibiotic resistance.

For the first time in Uruguay the frequency of moderate to heavy colonization of the upper respiratory tract by Streptococcus pneumoniae was investigated in children with acute respiratory infections (n = 717) and in healthy controls (n = 564). Serotypes of S. pneumoniae were identified as colonizing and invasive strains and their susceptibility to antibiotics was determined. Semiquantitative cultures of nasopharyngeal aspirates yielded 42.1% of positives in ambulatory patients and 15.2% in controls. Throat swabs from hospitalized children and matched controls revealed, respectively, 18.4 and 11.5% colonization. Different sampling and culture procedures were evaluated. Seasonal variations in colonization were also detected. Geographic variations in serotype frequency and distribution were assessed. Serotype 14 was predominant among invasive and colonizing strains in patients. Ten serotypes, included in the 23-valent S. pneumoniae vaccine, were not detected. Intermediate resistance to penicillin was seldom observed, but 37.2% of the invasive strains were resistant to cotrimoxazole, indicating the need for a permanent surveillance of isolates to antibiotic susceptibility.