Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae colonizing children with community-acquired pneumonia and children attending day-care centres in Fortaleza, Brazil

Bart Wolfa,*, Luis C. Reyb, Sylvain Brissec, Luciano B. Moreirad, Dana Milatovicc, Andre Fleerc, John J. Roorde and Jan Verhoefc

a Department of Paediatrics, St Lucas Andreas Hospital, Amsterdam, PO Box 9243, 1006 AE, The Netherlands; b Department of Paediatrics, University Hospital of the Federal University of Ceara and Albert Sabin Children's Hospital, Fortaleza, Brazil; c Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical Centre, Utrecht, The Netherlands; d Department of Microbiology, Federal University of Ceara, Fortaleza, Brazil; e Department of Paediatrics, Free University Hospital, Amsterdam, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To study clonal diversity of penicillin-resistant Streptococcus pneumoniae, 161 randomly selected isolates with reduced susceptibility to penicillin, collected from the nasopharynx of children under 5 years of age with community-acquired pneumonia and healthy controls from public day-care and immunization centres in Fortaleza, Brazil, were characterized by microbiological and serological techniques and automated ribotyping. Also included were 44 randomly selected penicillin-susceptible strains and three international reference strains. With automated ribotyping 75 ribopatterns were observed: 50 ribogroups were unique and 25 ribogroups were represented by two or more isolates. Genetic diversity was extensive but some degree of genetic homogeneity was found in strains from children with pneumonia, strains from children in day-care centres, isolates with reduced susceptibility to penicillin and isolates expressing ‘paediatric’ serogroups. Fourteen (56%) clusters contained both isolates with reduced penicillin susceptibility and penicillin-susceptible isolates, suggesting emergence of penicillin resistance. In general, there was a good correlation between ribogroups and serogroups, but 12 (48%) clusters contained isolates with alternative serogroups. Isolates with such alternative serogroups were more often encountered in penicillin-susceptible strains (41%) than in strains with reduced susceptibility to penicillin (7%). Thirty-eight (19%) isolates (including seven penicillin-susceptible strains) showed ribotypes indistinguishable from those of two international epidemic clones of S. pneumoniae: ribogroup 54-S-1 (15 isolates) with a ribopattern characteristic of the 23F multiresistant ‘Spanish/USA’ clone and ribogroup 74-S-3 (23 isolates) with a pattern similar to that of the 6B multiresistant ‘Spanish’ clone.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is the major cause of pneumonia worldwide, and in non-industrialized countries is estimated to cause 1.2 million deaths a year in children under 5.1,2 Penicillin-resistant strains of S. pneumoniae, first isolated in the late 1960s in Papua New Guinea and Australia,3 are now prevalent throughout the world and are often associated with resistance to other antimicrobial agents.4–6 The emergence and spread of pneumococcal resistance to penicillin seems to involve at least four processes: (i) the remodelling of genes encoding penicillin-binding proteins (PBP); (ii) the horizontal transfer of penicillin-resistance PBP genes; (iii) the geographic spread of resistant clones; and (iv) increases in MICs presumably through point mutations in PBP genes.7 Several examples of the international spread of resistant pneumococcal clones have been reported: the spread of a multiresistant clone of serotype 6B from Spain to Iceland in the late 1980s was reported by Soares and coworkers,8 and Munoz and colleagues reported evidence for the intercontinental spread of a multiresistant clone of serotype 23F from Spain to children attending a day-care centre in the USA.9 This clone subsequently spread throughout the United States.9 In contrast to the clonal spread of resistant pneumococci of serotypes 6B and 23F, an extensive genetic diversity has been observed among penicillin-resistant pneumococci in Africa, where the increasing emergence of penicillin resistance is thought to be due to horizontal transfer of altered PBP genes among the pneumococcal population.10,11 There is little information on the genetic relationship among penicillin-sensitive and penicillin-resistant isolates recovered within countries, although penicillin-resistant pneumococci have been reported from many countries.5,6,8,9,12,13 The nasopharyngeal flora of young children is known to be the main ecological reservoir of S. pneumoniae, and although nasopharyngeal isolates are not useful for predicting the cause of invasive disease in individual patients, they are useful in predicting resistance rates and serotype distribution of invasive isolates in a given population.12,14–18 In addition, the majority of antibiotic-resistant strains belong to only a limited number of capsular serotypes, which correspond to serotypes carried by children of pre-school age.4,5 Thus, children are not only primary targets of pneumococcal disease but are also the most important carriers of antibiotic-resistant strains.19

Phenotypic methods, like antimicrobial susceptibility profiles and serotyping, are often used to distinguish bacterial isolates for epidemiological studies. However, in recent years DNA-based typing techniques have emerged as the methods of choice for typing bacterial isolates, as they are less subject to natural variation.20 Since 1993, antimicrobial resistance in pneumococcal strains isolated from patients with invasive disease from different regions of Brazil has been monitored through the Regional System for Vaccines (SIREVA), established by the Pan American Health Organization. Little is known, however, of the epidemiology of antibiotic-resistant pneumococci colonizing children in Brazil. The purpose of the present study was to investigate the clonal spread of penicillin-resistant pneumococci and to define the degree of genetic variability among strains isolated from the nasopharynx of children with community-acquired pneumonia and healthy children from day-care and immunization centres in Fortaleza, Northeastern Brazil. These strains were also compared with penicillin-susceptible isolates from the same paediatric population, and with representative multiresistant strains recovered from different parts of the world.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients

From 15 March 1998 to 15 December 1998, a single nasopharyngeal specimen was collected from children between 2 months and 5 years of age who presented at the Emergency Department of one of the three major Children's Hospitals in Fortaleza and who fulfilled the WHO criteria for community-acquired pneumonia.21 The control group consisted of children who were recruited randomly from day-care centres (DCC), and public immunization centres (IC) in the eight health districts of Fortaleza. Children were enrolled in the study only after informed consent was obtained orally from their parents.

Bacterial isolates

Nasopharyngeal specimens were collected on cotton-tipped swabs (Transwab, MW 173P, Medical Wire & Equipment Co., Wiltshire, UK) and transported in Amies medium within 4 h to the Microbiology Laboratory of the Department of Pathology of the Federal University of Ceara and processed immediately. S. pneumoniae was identified by standard laboratory procedures and stored at –70°C in Microbank vials (Pro-Lab Diagnostics, Neston, UK) until transported in dry-ice by air to the Clinical Microbiology Laboratory of the University Medical Centre Utrecht in The Netherlands, where the identity of each isolate was confirmed.22 From a total of 435 S. pneumoniae isolates, a group of 161 randomly selected isolates with reduced susceptibility to penicillin (MIC >= 0.12 mg/L), and 44 randomly selected penicillin-susceptible strains (MIC <=; 0.06 mg/L), collected from 97 children with community-acquired pneumonia and 108 healthy controls from DCC and IC, was characterized by microbiological, serological and molecular techniques

Susceptibility testing.
MICs for penicillin, ceftriaxone, erythromycin, clindamycin, rifampicin, cotrimoxazole and chloramphenicol were determined by a microdilution method (Sensititre, AccuMed Int., Sussex, UK) and interpreted according to the National Committee for Clinical Laboratory Standards.23 S. pneumoniae ATCC 49619 was used as a control strain. Strains with an M-phenotype were resistant to erythromycin (MIC >= 1 mg/L) but sensitive to clindamycin (MIC <=; 0.25 mg/L).24 Isolates were considered multiresistant if they were fully resistant to >=3 antibiotics.4

Serotyping.
Serotyping was performed by the coagglutination method using 12 pooled antisera (Statens Seruminstitute, Copenhagen, Denmark) and protein A from Staphylococcus aureus (Cowan I strain NCTC 8530) in a disposable tray (LIP Equipment & Services Ltd, Shipley, West Yorkshire, UK) and, if necessary with the Quellung reaction using type-specific pneumococcal antisera (Statens Seruminstitute).25

Ribotyping.
Automated ribotyping was performed with the RiboPrinter Microbial Characterisation System (Qualicon Europe Ltd, Warwick, UK) according to the manufacturer's instructions. Briefly, the pneumococcal isolates were removed from storage at –70°C, subcultured overnight at 37°C in 5% CO2 on Columbia agar (Oxoid, Unipath Ltd, Basingstoke, UK) supplemented with 5% sheep blood, placed in tubes containing lysis buffer and loaded into the RiboPrinter Microbial Characterization Unit (MCU). Within the MCU, bacterial DNA was digested with EcoRI and the restriction fragments were separated by electrophoresis and transferred directly on to nylon membranes. A pattern of the restriction fragments containing rRNA genetic information was created through hybridization with a chemiluminescently-labelled DNA probe containing the rRNA operon (rrnB) from Escherichia coli. The chemiluminescence patterns were then electronically imaged and stored. For each membrane image, the software located the lane positions, reduced the background noise, scaled each lane's image intensity and used the data from the lanes containing DNA standards of known sizes to normalize the band positions.26 Similarity coefficients were calculated on the basis of the overall profile, based upon both band position and relative intensity. Output patterns were merged into a single ribogroup using an initial threshold similarity value of 0.93. Subsequently, the threshold value converged, as a function of the size of the ribogroup, until the minimal similarity value of 0.90. Reproducibility of ribogroup assignment was ascertained by running a set of control strains at regular time intervals over the study period. Thus, no inter-gel variation was detected.

A cluster was defined as a ribopattern shared by at least two S. pneumoniae isolates. Three international reference strains, including the 23F ‘Spanish/USA’ clone, the 6B ‘Spanish’ clone and the 19A ‘South African’ clone, which were obtained from Dr Keith Klugman (Pneumococcal Diseases Research Unit, Johannesburg, South Africa), were also tested with the same method. 9,11,27 The research protocol was approved by the Ethics Committee of the Medical Council of the State of Ceara, Brazil.

Statistics

The data were analysed with SPSS-PC for Windows (version 8.0) software. Associations between categorical variables were tested by using the {chi}2 test or Fischer's exact test, when appropriate. The t-test for independent samples and one-way ANOVA was used to compare group means. The discriminatory power of a typing method is its ability to distinguish between unrelated strains. It is determined by the number of types defined by the test method and the relative frequencies of these types. These two facets of discrimination are not generally presented as a single numerical value and therefore cannot be used for a straightforward comparison of different methods. For discriminatory power of the different typing methods the Simpson's index (SI) of diversity, based on the probability that two unrelated strains sampled from the test population will be placed into different typing groups characterized as the same type, was used.28 The degree of genetic clustering was defined as the percentage of strains displaying ribogroups that were observed twice or more.29 A P value <0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenotypic characterization

Antibiogram.
The study group consisted of 44/205 (21%) penicillin-susceptible (MIC <=; 0.06 mg/L), 149/205 (73%) intermediate penicillin-resistant (MIC 0.1–1 mg/L) and 12/205 (6%) highly penicillin-resistant (MIC >= 2 mg/L) isolates. The prevalence of isolates with reduced susceptibility to penicillin was similar in children with pneumonia and in healthy controls. Reduced susceptibility to cotrimoxazole (MIC >= 1/19 mg/L), erythromycin (MIC >= 0.5 mg/L), clindamycin (MIC >= 0.5 mg/L), chloramphenicol (MIC >= 8 mg/L), rifampicin (MIC >= 2 mg/L) and ceftriaxone (MIC >= 1 mg/L) was found in 52, 32, 25, 6, 3 and 2% of the isolates, respectively. Twelve of the 65 erythromycin-resistant pneumococci showed an M-phenotype. Thirty-two (16%) children carried multiresistant strains. Multiresistant strains and highly penicillin-resistant strains were more common, albeit not significantly, in children with pneumonia and children from DCC than in children from IC. Resistance to antibiotics other than penicillin was more frequent in strains with a reduced susceptibility to penicillin and only one multiresistant strain was penicillin susceptible. In addition, strains resistant to >=4 antibiotics were only encountered in highly penicillin-resistant strains, suggesting co-transfer of antibiotic resistance genes among penicillin-resistant pneumococci.

Serogroups.
The most common serogroup was serogroup 6 (38%), followed by serogroup 19 (19%), 23 (12%) and 14 (10%). A higher variability of serogroups was observed among penicillin-susceptible isolates compared with isolates with reduced susceptibility to penicillin: the 44 penicillin-susceptible strains expressed 13 capsular polysaccharides, whereas the 161 strains with reduced susceptibility to penicillin expressed only 12 different serogroups (FigureGo). ‘Paediatric’ serogroups were more often encountered in strains with reduced susceptibility to penicillin (87%) than in penicillin-susceptible strains (50%) (P < 0.0001).



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Figure. Distribution of serogroups (%) among isolates with reduced penicillin susceptibility (n = 161) and penicillin-susceptible (n = 44) isolates ({blacksquare}, penicillin-susceptible;{square} , penicillin-intermediate/resistant).

 
Genotypic characterization

All strains proved to be typeable by automated ribotyping. Depending on the strain, 5–15 hybridizing fragments were observed, ranging from 1 to >20 kb. Automated ribotyping of the 205 isolates yielded 75 different genotypes: 50 ribogroups were unique, whereas 25 distinct ribogroups were shared by two or more isolates (range 2–23).

The SI of diversity was lower, and the degree of genetic clustering higher, in strains isolated from children with pneumonia and children from DCC compared with strains isolated from children from IC. In addition, SI was lower and the degree of genetic clustering higher in strains with reduced susceptibility to penicillin than in penicillinsusceptible strains (Table IGo). The 32 multiresistant strains were represented by 24 ribogroups (SI = 98%), 12 highly penicillin-resistant strains by 10 ribogroups (SI = 97%) and 12 strains with an M-phenotype included 10 ribogroups (SI = 97%), indicating a high genetic heterogeneity in these strains. Fourteen of the 25 (56%) clusters contained both isolates with reduced penicillin susceptibility and penicillin-susceptible isolates (Table IIGo). The discrimination index of ribotyping was higher (SI = 96.2%) than that of serotyping (SI = 79.6%). The clustering of pneumococcal strains observed by serotyping did not always correlate with the genetic relatedness observed by ribotyping and strains with an identical serogroup were distributed across different ribogroups; 12 of 25 (48%) genetic clusters contained isolates with alternative serogroups. Isolates with such alternative serogroups were more often encountered in penicillin-susceptible strains (41%) than in strains with reduced susceptibility to penicillin (7%) (P < 0.0001). The discrimination index of ribotyping was lower in the ‘paediatric’ serogroups (SI = 95.2%) compared with the index in the non-paediatric serogroups (SI = 97.2%), and serogroup 14 showed the highest degree of genetic homogeneity (SI = 61%). Comparison of strains within DCC showed that strains with an identical phenotype (e.g. antibiogram and serogroup) only proved to be genetically indistinguishable in 67% of the cases. Thirty-eight (19%) isolates (including seven penicillin-susceptible strains) showed the same ribotype as the strains representative of two international epidemic clones of S. pneumoniae: ribogroup 54-S-1 (15 isolates), with a ribopattern characteristic of the 23F multiresistant ‘Spanish/USA’ clone widely spread in Europe, the United States, Asia, South Africa and Latin America; and ribogroup 74-S-3 (23 isolates) with a ribopattern indistinguishable from that of the 6B multiresitant ‘Spanish’ clone (Table IIGo). All isolates of ribogroup 54-S-1 were also resistant to cotrimoxazole, but nearly all of them were chloramphenicol susceptible and only a few were highly penicillin resistant, with occasional isolates showing resistance to macrolides. None of the isolates of ribogroup 54-S-1 isolates expressed serogroup 23: penicillin-susceptible isolates expressed serogroup 7 and isolates with reduced susceptibility to penicillin expressed serogroup 14. Most isolates of ribogroup 74-S-3 expressed serogroup 6 but three penicillin-susceptible isolates expressed serogroups 3 and 9. Most isolates of ribogroup 74-S-3 were also resistant to cotrimoxazole but hardly any showed ceftriaxone and chloramphenicol resistance, and none was highly penicillin-resistant, with occasional isolates showing resistance to macrolides.


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Table I. Ribogroups in 205 isolates according to penicillin susceptibility and clinical status
 

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Table II. Genotypic and phenotypic properties of 155 Brazilian and two international reference strains
 
The ribogroup of a third international 19A ‘South African’ clone was not encountered in the Brazilian study group. Genetically related strains showed wide dispersal both within and between DCC in Fortaleza (Table IIIGo).


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Table III. Distribution of genetically related strains among 66 children attending 10 DCC (A–J) in Fortaleza, Brazil
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first molecular epidemiological study of penicillin-resistant S. pneumoniae colonizing children in Northeastern Brazil. The majority of our isolates showed a very large variation of genetic background with some degree of genetic clustering in strains from children with pneumonia and in strains from healthy children in DCC. Some degree of genetic homogeneity was also found in strains with a reduced susceptibility to penicillin and strains expressing paediatric serogroups, in particular serogroup 14. Certain genotypes, however, appear to have prevailed in the pneumococcal population irrespective of these factors and the mechanism for this is poorly understood. Moreover, the variety of ribogroups found in this study suggests that pneumococcal resistance is not merely the intrusion of a few resistant clones. High genetic diversity was also shown in sterile site isolates collected between 1993 and 1996 from five geographic regions in Brazil.30 Pulsed-field gel electrophoresis has shown to be a highly discriminating molecular typing method for pneumococci but genetic clustering with ribotyping proved to be comparable and, moreover, is very suitable for computerized analysis of the fingerprints.13,20,31 Automated ribotyping, which can be applied to virtually all bacteria, not only standardizes the technical and interpretative aspects of the procedure, but also makes use of an expandable database of electronic fingerprints suitable for rapid inter-laboratory communication.26 Although the discriminatory power of ribotyping is lower than that of pulsed-field gel electrophoresis, ribotyping appears satisfactory to draw first-line epidemiological conclusions. Indeed, isolates belonging to a given ribogroup most probably share a common ancestral strain since it is very unlikely that the same ribotype banding pattern would originate independently in different strains. However, documentation of recent clonal spread needs confirmation using more discriminatory typing methods.32 The higher variability of genetic background among penicillin-susceptible strains and the lower variation in strains with reduced susceptibility to penicillin has also been described in other reports, but the exact mechanism for this relationship is not clear.8,33 Our data clearly show that strains from different serogroups can be genetically closely related whereas strains with the same serogroup may be genetically different. The frequently encountered shifts in capsular serogroup within genetically closely related strains can be explained either by horizontal transfer of capsular genes or by the fact that ribotyping, using only one restriction endonuclease, was not discriminatory enough. However, these data do confirm reports in the literature that capsular type is not a good criterion for estimating the relatedness of different isolates of pneumococci. 31,34,35 Moreover, our finding that strains with an identical phenotype in children from DCC were genetically indistinguishable in only two-thirds of the cases also indicates that identity of serogroup and MIC are insufficient criteria for defining similarity between pneumococci, even when the bacteria are isolated from the same DCC.

Another interesting feature of our study was the spread among children in Fortaleza of two genotypes closely related to the international epidemic clones. Although ribotyping revealed that 15 Brazilian isolates shared the 23F ‘Spanish/ USA’ clone characteristics and 23 isolates the 6B ‘Spanish’ international clone's genetic features, significant phenotypic differences were found. The appearance of isolates that express serogroups not typical for the particular clone in multiresistant isolates has been described repeatedly and Barnes, Corso and Nesin and coworkers in the USA and Tomasz and colleagues in Latin-America reported capsular transformation of the international clone 23F from serogroup 23 to serogroup 14 on various occasions.33,35–37

In addition, the changes in antimicrobial resistance patterns suggest that antibiotic susceptibility may have evolved in response to differing antibiotic pressures in Northeastern Brazil, as chloramphenicol is rarely used in the outpatient treatment of children in Fortaleza, in contrast to erythromycin. Erythromycin-resistant variants closely related to the 23F ‘Spanish/USA’ clone have also been reported from France, the USA and South Africa.13,37,38

Two hypotheses could account for our observations: (i) the international clones have been introduced into Brazil through intercontinental spread but have undergone phenotypic changes as compared with the original Spanish clones; or (ii) strains sharing the same ribotype as the international clones have evolved toward penicillin resistance in Brazil, independently from their Spanish counterparts. Distinguishing between these hypotheses will need further investigation, using more discriminatory typing methods, of the genetic relationships between penicillin-susceptible and penicillin-resistant Brazilian isolates and comparison with the Spanish isolates, as well as determination of the genes conferring penicillin resistance in Brazil.

Finally, the data indicate the epidemic potential of certain penicillin-resistant pneumococcal ribotypes. Continuous surveillance of S. pneumoniae is urgently needed to monitor the antimicrobial resistance level and spread of penicillin-resistant clusters, and intervention studies should be planned in which the effect of reduced antibiotic pressure on the prevalence of resistant strains in the nasopharyngeal flora can be evaluated.


    Acknowledgments
 
The authors are grateful to Dr Keith Klugman from Johannesburg, South Africa, for generous donation of the three reference strains, Mrs Karlijn Kusters for assistance with the ribotyping and Dr Nijs Lagerweij for statistical advice. The study was supported by a grant from the Medical Research Board of the St Lucas Hospital in Amsterdam, The Netherlands.


    Notes
 
* Corresponding author. Fax: +31-20-5108168; E-mail: m.j.wolf{at}amc.uva.nl Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 6 March 2000; returned 8 June 2000; revised 12 July 2000; accepted 2 August 2000