Search

Polymyxins are still used mainly in treating infections caused by carbapenem-resistant Klebsiella pneumoniae worldwide. The most frequent mechanism of acquired resistance to polymyxins in Gram-negative bacilli is the occurrence of mutations in chromosomal genes regulating operons responsible for lipopolysaccharide modification. As we observed at Santa Casa de São Paulo hospital the occurrence of infections caused by isolates resistant to polymyxins in patients previously treated with this antimicrobial, and new infections caused by the same polymyxin-susceptible species, in this study, we aimed to determine the clonality of consecutive K. pneumoniae isolates from the same patients and characterize the molecular determinants of polymyxin resistance in paired or clonal isolates. A total of 24 pairs and one trio of K. pneumoniae isolates were included in this study. Species identification was achieved by mass spectrometry and multiplex PCR. Polymyxin B minimal inhibitory concentrations were determined by broth microdilution. Clonality was evaluated using pulsed-field gel electrophoresis. The presence of insertions in mgrB gene was tested by PCR, and mutations on pmrA, pmrB, phoP, and phoQ were evaluated by PCR and complete nucleotide sequencing. A fraction of 23.8% of strains resistant to polymyxin B had an insertion in mgrB. Amino acid substitution F204L in PmrB may be implicated in polymyxin resistance. Substitutions T246A and R256G in PmrB were not implicated in polymyxin resistance. In this study, polymyxin resistance after a first susceptible isolate was detected was most frequently due to an infection caused by a distinct clone. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00600-5.

We studied 70 pediatric clinical isolates of Staphylococcus aureus to perform a comparison of different methods for detection of methicillin-resistant S. aureus (MRSA) using polymerase chain reaction for mecA as the “gold standard” assay. The isolates were tested with oxacillin and cefoxitin discs, oxacillin agar screening plate and E-test. Of the 70 strains, 43 were mecA-positive and 27 mecA-negative. Oxacillin agar screening plate and E-test had 100% sensitivity and specificity for the presence of mecA gene. We found a specificity of 96% for both disc-diffusion methods, and sensitivities of 95% (oxacillin disc) and 93% (cefoxitin disc). However, with the new Clinical and Laboratory Standards Institute 2007 cefoxitin breakpoints the sensitivity of this method would be increased to 100%, without any decrease in specificity. These results show how important is the evaluation of the available microbiological methods by different laboratories in different settings.

Childhood diarrheal diseases remain highly endemic in northeastern Brazil. The attributable fraction of all diarrheal diseases among children less than 2 years of age due to Escherichia coli was examined in a 2-year prospective study in two large urban centers of Brazil. Between May 1997 and June 1999, fecal E. coli isolates from 237 children with diarrhea (217 acute and 20 persistent cases) and 231 children without diarrhea (controls) attending two hospitals in Northeast Brazil were tested for their pattern of adherence to HEp-2 cells and for colony hybridization with DNA probes specific for the six pathotypes of diarrheagenic E. coli. Enteroinvasive E. coli, enterotoxigenic E. coli and enterohemorrhagic E. coli were not isolated from any children. Diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC) were the most frequent isolates with similar frequencies from children with or without diarrhea. Atypical EPEC (EAF-negative) strains were isolated with similiar frequency from both cases (5.5%) and controls (5.6%). Enteropathogenic E. coli (typical EPEC) strains, characterized by localized adherence pattern of adherence, hybridization with the EAF probe, and belonging to the classical O serogroups, were significantly associated with diarrhea (P = 0.03). These E. coli strains associated with diarrhea accounted for 9% of all children with diarrhea. Collectively, in Northeast Brazil, E. coli strains comprise a small proportion of severe diarrhea prevalence in children.

Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic Escherichia coli [EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of E. coli adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent E. coli (HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used ( daaC and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently ( P = 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%).

Twenty seven S. aureus isolates were obtained from cystic fibrosis (CF) patients at a tertiary care hospital in Brazil. Nineteen (70.4%) were methicillin-susceptible S. aureus (MSSA) and eight (29.6%) methicillin-resistant S. aureus (MRSA). Of the MRSA isolates, four had SCCmec type III and four had SCCmec type IV. PVL genes were not detected in any of the MSSA or MRSA isolates. New studies are necessary to evaluate the exact impact of these different MRSA clones in CF patients.

In a prospective study carried out in two urban centers in northeastern Brazil, 195 HEp-2-adherent Escherichia coli strains were isolated; 110 were identified as the only pathogen in stools of children with diarrhea, and 85 were from controls. Enteropathogenic E. coli isolates were identified in 21 children with diarrhea (8.9%) and 7 children without diarrhea (3.0%), and they were significantly associated with diarrhea ( P < 0.01). Enteroaggregative E. coli strains were isolated from 40 children with diarrhea (16.9%) and 38 children without diarrhea (16.4%) and showed no correlation with diarrhea ( P > 0.5). In 49 children with diarrhea (20.7%) and 40 children without diarrhea (17.3%), diffusely adherent E. coli (DAEC) isolates were detected and were not found to be associated with diarrhea ( P = 0.41). However, after stratification, for children older than 12 months of age a significant correlation between DAEC infection and diarrhea was detected ( P = 0.01). These results suggest that DAEC isolates should be considered potential pathogens in northeastern Brazil and also confirm the association of DAEC with age-dependent diarrhea.