Antonio Fernando Pestana de Castro

Bundle-Forming Pili and EspA Are Involved in Biofilm Formation by Enteropathogenic Escherichia coli

Microcolony formation is one of the initial steps in biofilm development, and in enteropathogenic Escherichia coli (EPEC) it is mediated by several adhesins, including the bundle-forming pilus (BFP) and the EspA filament. Here we report that EPEC forms biofilms on plastic under static conditions and a flowthrough continuous culture system. The abilities of several EPEC isogenic mutants to form biofilms were assessed. Adhesins such as BFP and EspA, important in microcolony formation on epithelial cells, are also involved in bacterial aggregation during biofilm formation on abiotic surfaces. Mutants that do not express BFP or EspA form more-diffuse biofilms than does the wild type. We also determined, using gfp transcriptional fusions, that, consistent with the role of these adhesins in biofilms, the genes encoding BFP and EspA are expressed during biofilm formation. Finally, expression of espA is controlled by a quorum-sensing (QS) regulatory mechanism, and the EPEC qseA QS mutant also forms altered biofilms, suggesting that this signaling mechanism plays an important role in EPEC biofilm development. Taken together, these studies allowed us to propose a model of EPEC biofilm formation.

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

ABSTRACT Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates.

ABSTRACT Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of E. coli is unclear. This study was designed to characterize diarrheagenic E. coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis. E. coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E. coli and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of eae+E. coli were serotyped and examined by PCR for genes encoding EPEC virulence markers. The eae+E. coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.

Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR

Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined. The ERIC-PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC-PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)-PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates.

Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers

Department of Microbiology, Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Department of Clinical Analysis, School of Pharmacy, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Institute of Biological Sciences, Universidade Federal do Para, Belem, PA, Brazil; School of Public Health, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Environmental Company of Sao Paulo State (CETESB), Sao Paulo, SP, Brazil

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil.

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.

Sorotipagem de amostras de Streptococcus suis isoladas de suínos em granjas dos Estados de São Paulo, Minas Gerais e Paraná

Streptococcus suis infection in swine is common in all countries where hog production is well developed. This infection has been associated with bronchopneumonia, meningitis, arthritis, pericarditis, myocarditis, endocarditis, fibrinous polyserositis, septicaemia, rhinitis, and abortion. Streptococcus suis has also been described as a pathogen for ruminants and humans. In Brazil there are several clinical evidences about the existence of S. suis disease in pigs affecting more than 50% of farms in States of Sao Paulo, Minas Gerais and Parana. In the present research 51 strains of S. suis isolated from piggeries of the States of Sao Paulo, Minas Gerais and Parana were collected from different pathologies such as septicaemia, meningitis, arthritis and pneumonia and been recovered either in pure culture or as the predominant organism from porcine tissues. Culture of specimens was carried out on 5% bovine blood agar plates incubated at 37°C for 24 hr. For the biochemical identification the a-hemolytic colonies of all capsulated isolates were submitted to various conventional tests, such as hydrolysis of arginine, Voges-Proskauer Test, and production of acid from various carbohydrates (inulin, salicin, trehalose, lactose, sucrose, sorbitol, mannitol and glycerol). The strains were also tested for their ability to grow in the presence of 6,5% NaCl and for the amylase production. In addition strains were tested by Api Strep 20 to confirm the identification of S.suis. For capsular typing only capsulated strains were typed by co-agglutination test, using antisera raised in rabbits against all reference strains from serotypes 1 to 8. Strains belonging to other serotypes were also typed. The co-agglutination was used for serotyping and the capsular reaction test was carried out for measuring the potency of the prepared antisera. From the total of 51 examined strains the following results were obtained, with regard to serotyping: 30 (58,8%) were serotype 2, 11 (21,6%) were serotype 3, seven (13,72%) were serotype 7, two (3,92%) were serotype 1 and one strain belonged to serotype 14 (1,96%). As far as we are concerned, this is the first report on the isolation of a large number of S. suis strains in Brazil, from cases of illness caused by this bacterium among piglets. Also it was carried out serotyping of the isolates, showing a high prevalence of serotype 2, as compared to other known serotypes of S. suis.

Identification of fluoroquinolone-resistant extended-spectrum β-lactamase (CTX-M-8)-producing Escherichia coli ST224, ST2179 and ST2308 in buffalo (Bubalus bubalis)

D2 differed from one another by fewer than five single nucleotide differences, but only the WM98 sequence was not interrupted by large insertions or deletions (positions of insertions/deletions are indicated in Figure 1). AB307-0294 (GenBank accession number CP001172) was also identical over most of this span, but contained patches that differed and lacked a large span. The AB0057 sequence (GenBank accession number CP001182), for which the ampC gene was previously corrected, differed at 33 more positions (single base substitutions or additions or deletions; mainly the absence of an A or a T in a run of As or Ts) and many of these differences may be errors caused by the sequencing technology used. Contigs containing ampC and its surrounds were retrieved from the whole genome sequence of G7 reported previously 6 and joined using the manually determined sequence described above. Comparison with the WM98 sequence revealed a segment of 31.8 kb, defined as between the first and last base differences surrounding the ISAba1-ampC in G7, which differed from the corresponding region in WM98 by 2.2% (Figure 1). This indicates that this segment has been replaced by a segment imported from another A. baumannii strain that included an ISAba1 upstream of the ampC gene. Hence, it appears that a DNA segment that included an ISAba1-activated ampC gene was introduced into an isolate belonging to the GC1 clonal complex, possibly by conjugation , and that homologous recombination incorporated it into the chromosome displacing the resident copy. Examination of the regions on either side of the 31.8 kb diverged segment revealed the presence of two smaller replaced patches of 4.7 and 2.8 kb in G7, which differed from the corresponding regions in WM98 by 4.5% and 2.1%, respectively (Figure 1). Outside these patches, WM98 and G7 differed by only 3 bp. This is the first study providing evidence for horizontal transfer of a DNA segment that contains an ISAba1-activated ampC gene between two A. baumannii strains. The findings highlight the significance of the horizontal transfer of chromosomal DNA segments in the generation of cephalosporin resistance in A. baumannii. 3 Hamidian M, Hall RM. Tn6168, a transposon carrying an ISAba1-activated ampC gene and conferring cephalosporin resistance in Acinetobacter baumannii. 4 Hamidian M, Hall RM. ISAba1 targets a specific position upstream of the intrinsic ampC gene of Acinetobacter baumannii leading to cephalosporin resistance. ISAba125-activated ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance. A …

Typing of Intimin (eae) Genes in Attaching and Effacing Escherichia coli Strains from Monkeys

Attaching and effacing Escherichia coli (AEEC) strains cause histopathological alterations termed “attaching and effacing (A/E) lesions” (8). The ability to cause A/E lesions is encoded on a large bacterial chromosomal pathogenicity island, the locus of enterocyte effacement (LEE). The central portion of LEE encodes intimin (Eae, 94- to 97-kDa outer membrane protein) and Tir, the intimin receptor, which is translocated into the host cell membrane by the type III system (8). Differentiation of intimin alleles represents an important tool for AEEC typing in pathogenesis and epidemiological, clonal, and immunological studies, and it may also be a potential tool in routine diagnostics (1, 2, 3, 4, 12, 15). The 5′ regions of eae genes are conserved, whereas the 3′ regions are heterogeneous. This observation led to the construction of universal PCR primers and allele-specific PCR primers, which made it possible to differentiate, at present, 15 variants of the eae gene that encode 15 different intimin types and subtypes (2, 4). Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains causing A/E lesions in the intestinal mucosa are considered AEEC (8, 14). In contrast to STEC, EPEC strains do not produce Shiga toxins. EPEC strains are a major cause of infant diarrhea in developing parts of the world and are pathogenic to several animal species (rabbits, calves, dogs, sheep, pigs, and primates) (3, 4, 5, 14). However, the serotypes of human and animal EPEC strains are usually different. Typical human EPEC strains present the bfp gene, which encodes the fimbriae called bundle-forming pili (BFP) (7, 14). Although enteric diseases, specifically diarrhea, are frequently associated with morbidity and mortality in nonhuman primates in captivity, studies of the role of different diarrheagenic E. coli strains in these diseases are lacking. Thomson and Scheffler (13) reported an outbreak of diarrhea caused by a Shiga toxin-negative AEEC isolate of serogroup O26 in marmosets maintained at the Primatology Center. Mansfield et al. (9, 10) associated a Shiga toxin-negative AEEC O156:H−, intimin ɛ-positive strain with a simian immunodeficiency virus opportunistic infection in rhesus monkeys (10) and a Shiga toxin-negative AEEC O26:H− ɛ intimin-positive strain with ulcerative colitis in cotton-top tamarins (9). Recently, Carvalho et al. (5) found that AEEC strains harboring genes for intimin production (eae positive) and lacking genes for Shiga toxin production (stx1 and stx2 negative) were the only group of diarrheagenic E. coli strains isolated from fecal samples of diarrheic and healthy marmosets. Eighteen of 56 (32%) animals carried E. coli strains with the eae gene, including 8 of 17 (47%) with diarrhea and/or enteritis and 10 of 39 (26%) healthy animals. All monkey AEEC strains isolated by Carvalho et al. (5) were able to cause the A/E lesion, as determined by the FAS test and confirmed by electron microscopy of infected HEP-2 cells. Monkey AEEC strains isolated by Carvalho et al. (5) were also examined for intimin subtypes α, β, δ, and γ, as described previously (1). Because the number of intimin subtypes studied was very limited, the majority of monkey strains showed nontypeable intimins (5). In order to ascertain whether these intimin subtypes were actually new ones, some of these strains were examined again by PCR using a set of new primers described by Blanco et al. (2, 4) for the already known intimins as well as for new eae variants β2, μ, ν, and ξ. For comparison studies, the monkey strains were serotyped by the method described by Guinee et al. (6), and the previous results obtained for bfp by PCR, as well as BFP expression by Western blotting, were reconsidered in this study (5). All 15 monkey E. coli strains assayed were positive with universal primers EAE-1 and EAE-2 that generated PCR products obtained from the amplified 5′-conserved region of the eae gene. Six monkey AEEC strains presented identical serotypes and intimins (two O142:H6 α1, two O128:H2 β1, and two O127:H40 γ2/θ strains) to human enteropathogenic E. coli (EPEC), whereas eight strains showed new serotypes not previously found in human or animal AEEC with β1 (two O132:H31 strains), β2 (one O139:H14 strain and one O167:H6 strain), ɛ (one O26:H7 strain), ι (two O49:H46 strains), and λ (one O33:H−) intimins. The remaining monkey strain, which belonged to serotype O167:H9 (β1), although it was not included among human EPEC serotypes, was characterized as an AEEC strain that caused an outbreak of gastroenteritis involving a large number (256 patients) of schoolchildren (11) (Table ​(Table1).1). The intimins α2, γ1, δ/κ, ζ, η, μ, ν, and ξ were not found among the AEEC strains isolated from marmosets in Brazil. However, considering that only 15 strains were studied, the diversity of intimins found among these strains was relatively high. TABLE 1. Serotypes and intimin types of monkey AEEC strains isolated in Brazil In conclusion, this study indicates that nonhuman primates may represent a natural resevoir of EPEC serotypes pathogenic for humans.