Leila Carvalho Campos

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups: a review

The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

Prevalence of Streptococcus pneumoniae serotype 6C among invasive and carriage isolates in metropolitan Salvador, Brazil, from 1996 to 2007.

The newly described Streptococcus pneumoniae serotype 6C accounted for 2.3% (16/709) of meningitis cases and 3.2% (3/95) of nasopharyngeal isolates from healthy individuals in Brazil. The strains were multidrug resistant (18.8%) and genetically diverse. Despite low serotype 6C prevalence, continuous surveillance is necessary to guide vaccine strategies.

Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae prior to introduction of the 10-valent pneumococcal conjugate vaccine in Brazil, 2000–2007

This study describes the serotype distribution and antibiotic resistance patterns among 397 S. pneumoniae meningitis case isolates recovered in Salvador, Brazil, during the period of 2000-2007, before introduction of the 10-valent pneumococcal conjugate vaccine. The active hospital-based surveillance showed a decline in the annual incidence rates of pneumococcal meningitis during the period of study, from 1.12 cases to 0.83 cases/100,000 persons for all age groups (P<0.001), with an overall case-fatality rate of 28.6% (113 of 395) for all patients and 41.9% (57 of 136) for those <5 years of age. Serotypes 14 (n=55; 13.9%), 3 (n=32; 8.1%), 23F (n=32; 8.1%), 19F (n=31; 7.8%), 6B (n=30; 7.6%), 18C (n=28; 7.1%), and 6A (n=20; 5%) were the most prevalent serotypes. In patients <5 years the estimated projected coverage of 7-, 10- and 13-valent conjugate vaccines was 74.3%, 75.7% and 83.1%, respectively. Antimicrobial susceptibility testing revealed that 22.1% (n=88) of isolates were non-susceptible to penicillin, 56% were non-susceptible to trimethoprim/sulphamethoxazole, and 29.6% were non-susceptible to tetracycline. Nonsusceptibility to penicillin and cefotaxime was detected solely among serotype 14 isolates (n=4; 1%). This study provides an important baseline to assess the impact of conjugate vaccine implantation on the epidemiology of meningitis due to Streptococcus pneumoniae in Salvador, Brazil.

The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli

The presence of the enteroaggregative Escherichia coli (EAEC) virulence genes aatA, aap, and aggR was assayed in strains of different diarrheagenic E. coli pathotypes and nonpathogenic E. coli. The dispersin-encoding gene (aap) was detected in EAEC, diffusely adherent E. coli, and nonpathogenic E. coli, demonstrating that molecular diagnostics of EAEC based on aap detection may identify non-EAEC strains.

Genetic diversity and antibiotic resistance of clinical and environmental Vibrio Cholerae suggests that many serogroups are reservoirs of resistance.

Vibrio cholerae is an important human pathogen and the cause of cholera. Since genetic variation and antibiotic resistance of strains have implications for effective treatment of the disease, we examined the genetic diversity and antibiotic resistance profile in 92 clinical strains (serogroup O1) and 56 environmental strains (O1 antigen, 42 strains; non-O1 antigen, 14 strains) isolated in Brazil between 1991 and 1999. Clinical and environmental O1 strains showed greater drug resistance compared to environmental non-O1 strains. Nearly all clinical O1 strains were resistant to one or more antibiotics while half of the environmental O1 and non-O1 strains were resistant to one or more antibiotics. No plasmids or class 1 integrons were detected in the strains by PCR analysis. Multilocus enzyme electrophoresis analysis (MLEE) suggests most of the O1 strains belong to a single (South American) clone that is related but different to seventh-pandemic strains isolated from other parts of the world. Our results show that there is a close genetic relationship between clinical and environmental O1 strains and that many serogroups and the environment can be a reservoir for antibiotic resistance.

Diffusely Adhering Escherichia coli (DAEC) Strains of Fecal Origin Rarely Express F1845 Adhesin

A total of 398 diffusely adhering Escherichia coli (DAEC) strains of fecal origin were analyzed for the presence of sequences homologous to the structural subunit gene (daaE) of the F1845 fimbria. For that purpose, a DNA fragment homologous to daaE, obtained by PCR, was used as a probe in colony hybridization assays. Only two strains carried daaE and expressed F1845, suggesting that this fimbria is rare among DAEC strains.

Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil.

OBJECTIVE The present study was undertaken to characterize CTX-M ESBL-producing Klebsiella pneumoniae collected from hospitals in different cities of Brazil. MATERIAL AND METHODS Eighty-five K. pneumoniae strains isolated from hospitalized patients in six different hospitals of three cities of Brazil were analyzed. ESBL production was confirmed by the standard double-disk synergy test and the Etest®. The MIC50 and MIC90 for ESBL-producing isolates were determined by the Etest® method. The antimicrobial susceptibilities of bacterial isolates were determined using the agar diffusion method according to the CLSI. Screening for blaTEM, blaSHV, blaCTX-M genes and class 1 integron was performed by PCR amplification. To determine the genomic diversity of CTX-M-producers, isolates were analyzed by macrorestriction profile analysis following PFGE. RESULTS AND DISCUSSION Seventy-one K. pneumoniae isolates were ESBL-producing. PCR and sequencing experiments detected 38 CTX-M-producing K. pneumoniae belonged to groups CTX-M 1, CTX-M 2, CTX-M 8 and CTX-M 9. The association of different types ESBL (CTX-M, SHV and TEM) was frequent. All K. pneumoniae isolates carried class 1 integron. PFGE analysis revealed thirty-one clonal types among CTX-M-producing isolates. The data presented herein illustrate the diversity of genotypes of CTX-M producing K. pneumoniae among Brazilians hospitals.

Antimicrobial resistance among Salmonella serovars isolated from different sources in Brazil during 1978-1983

A total of 748 Salmonella strains (97 serovars) isolated from human (291), animal (119), environmental (141), food (102) and animal feed (95) sources were examined for resistance to 9 antimicrobial agents. Most of the human isolates were from hospitalized patients (282). An overall resistance rate of 98.8% was determined with 100% for human and environmental isolates. Resistance to sulfadiazine (87.7%) was most common, followed by streptomycin (61.2%), ampicillin (39%) and trimethoprim-sulphamethoxazole (37.9%). Fifty one different resistance patterns were identified with Su (164 strains), Su-Sm (122) and Su-Sm-Tc-Cm-Km-Ap-Nx-Gm-Tm (95) predominating, the latter occurring only in human isolates. Multiple resistance was most frequently found among human isolates, particularly in S. derby and S. typhimurium strains. The relationship between antibiotic resistance, serovar and source of isolation of the Salmonella strains is discussed.

Evaluation of a Multiplex PCR for Identification of Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) has emerged as an important pathogen associated with endemic and epidemic diarrheal diseases in both industrialized and developing countries (6). EAEC strains are defined by their characteristic “stacked brick” aggregative adherence (AA) pattern to cultured epithelial cells (9), and this is the basis of the assay considered the gold standard for EAEC identification. However, this technique requires specialized facilities and can therefore be performed only in reference laboratories. As alternative to this technique, a variety of phenotypic and molecular assays have been proposed (1, 2, 4, 7, 15, 17). Recently, a multiplex PCR assay for EAEC detection has been developed (4). This assay detects simultaneously three EAEC plasmid-borne genes: aggR, which encodes a central regulator involved in the expression of several EAEC virulence genes (6, 10); aap, which encodes the antiaggregation protein dispersin (16); and aatA, which is part of a gene cluster that codes for a specific ATP-binding cassette transporter system (11). The latter corresponds to the EAEC probe fragment (CVD432) previously described (2). In order to determine the specificity and sensitivity of multiplex PCR in detecting EAEC, a total of 379 fecal E. coli isolates were examined by this assay and the results subsequently correlated with the adherence pattern to HEp-2 cells. These strains were isolated from children under 5 years of age, with symptoms of gastrointestinal disorders, admitted to the Hospital Jesus, Rio de Janeiro, Brazil, between August 2003 and September 2004. PCR conditions and the primers used were the same as those described by Cerna et al. (4). All E. coli isolates were subjected to the adherence assay with cultured epithelial cells, employing a 3-h incubation of HEp-2 cells with bacterial cultures (5). Strains displaying noncharacteristic adherence (NC) or nonadherence (NA) were tested by employing an additional 3-h incubation of cells with bacteria (6-h assay). At least one of the three loci sought was detected in 199 (52.5%) strains, while 180 (47.5%) strains were negative for them. The most prevalent was aap, detected in 153 (40.4%) strains, followed by aggR, detected in 109 (28.8%), and aatA, detected in 101 (26.6%). Concerning the adherence patterns observed, 158 (41.7%) strains showed the AA pattern and were therefore classified as EAEC (Table ​(Table1).1). Only four (1.1%) strains showed localized adherence (LA), which is a characteristic of enteropathogenic E. coli (14), while 156 (41.2%) showed an NC pattern and 61 (16.1%) were NA. Considering the AA pattern as the gold standard for EAEC identification, the detected loci were more frequent among EAEC strains, and the sensitivity and the specificity of the multiplex PCR were 93.5% and 81%, respectively. In addition, the positive predictive value was 75.2%, and the negative predictive value was 95.3%. None of the strains displaying the LA pattern amplified any of the primers tested. The three genes were also detected in strains displaying the NC pattern, and aap was detected in 10 NA strains. These NC and NA strains should be further analyzed in regard to the presence of additional plasmid and chromosome-associated EAEC virulence factors, since there is a possibility that they are EAEC strains that have lost the capacity to express the AA phenotype. The simultaneous detection of the three genes was most prevalent among the strains characterized as EAEC. Single or different combinations of two genes were also found preferentially in EAEC strains but also among the NC strains (Table ​(Table2).2). The detection of any of the three genes by multiplex PCR defines a strain as EAEC (4). TABLE 1. Detection of EAEC plasmid-borne virulence factors by multiplex PCR in fecal E. coli strains in regard to their HEp-2 cell adherence patterns TABLE 2. Combinations of EAEC plasmid-borne virulence factors detected by multiplex PCR in fecal E. coli strains These three plasmid-borne genes detected by the multiplex PCR have been shown to be specific and appropriate markers for EAEC detection (3, 4, 7). In this study, multiplex PCR was shown to be sensitive and specific in detecting EAEC among fecal E. coli strains. Other previous published studies have also demonstrated that this technique is sensitive (3, 4). However, only strains previously characterized as EAEC, as defined by HEp-2 adherence, were evaluated in these studies. Additionally, two other multiplex PCRs employing different gene targets have been described for EAEC detection (8, 12), demonstrating that this technique is a useful tool in the detection of EAEC. However, both of the latter studies included the detection of astA (enteroaggregative heat-stable enterotoxin gene), which has been demonstrated to be present in other diarrheagenic E. coli pathotypes and nonvirulent E. coli (13). This may raise the possibility of the detection of E. coli strains that are not EAEC. Since the technique evaluated in this study detects genes located on the high-molecular-weight virulence plasmid (pAA) of EAEC (4, 6), the inclusion of a chromosome-associated EAEC gene in the multiplex reaction, as performed by Jenkins et al. (8), may improve its sensitivity/specificity, including the detection of EAEC strains that do not harbor pAA. In conclusion, our results demonstrated that the multiplex PCR technique is a suitable alternative in detecting EAEC among E. coli strains isolated from feces, since PCR-based diagnostic tests are commonly employed in clinical laboratory practice.

Burden of group A streptococcal meningitis in Salvador, Brazil: report of 11 years of population-based surveillance.

BACKGROUND Over recent decades, a resurgence of invasive group A streptococcal (GAS) infections has been observed; GAS remains a rare cause of pyogenic meningitis. We report herein population-based findings of long-term surveillance for GAS meningitis in Salvador, Brazil, and estimate the overall burden of invasive GAS infections. METHODS From February 1996 to February 2007 we conducted active surveillance for GAS meningitis in the state reference hospital for infectious diseases in Salvador, Brazil. Data on clinical presentation, laboratory records, and outcome were collected through interviews and chart review. GAS isolates were evaluated for antimicrobial susceptibility and emm type. RESULTS We identified 20 cases of GAS meningitis, which accounted for 0.9% of all culture-proven bacterial meningitis in the study period. The mean annual incidence of GAS meningitis was 0.03 cases per 100,000 population in metropolitan Salvador and peaked in children <1 year of age (0.67 cases per 100,000 population). Among 17 cases with clinical information available, 41% required intensive care unit support and 25% died. Tested isolates were susceptible to penicillin and exhibited large emm type diversity. Based on the incidence of GAS meningitis, we estimate that the annual incidence of GAS infection is 3 cases per 100,000 population in metropolitan Salvador. CONCLUSIONS Although rare, GAS is a life-threatening cause of bacterial meningitis. Knowledge of the incidence and emm type variability of the disease is necessary for planning immunization strategies.