Resolving taxonomic confusion: establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae.

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family.

Dissemination of blaOXA-370 is mediated by IncX plasmids and the Tn6435 transposon.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

Frequency of BKC-1-Producing Klebsiella Species Isolates.

BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1 The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.

Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli.

The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process.

Potential effect of 6 versus 12-weeks of physical training on cardiac autonomic function and exercise capacity in chronic obstructive pulmonary disease.

BACKGROUND: Exercise is an important part of chronic obstructive pulmonary disease (COPD) treatment. However, it is not know about the minimum effective time of physical training that could beneficially modify the cardiac autonomic modulation (CAM) and exercise capacity in these patients. AIM: To contrast the potential effects of a physical training program (PTP), for 6 versus 12 weeks, on CAM by linear and nonlinear heart rate variability (HRV) indices and exercise capacity in COPD patients. DESIGN: Prospective randomized controlled trial. SETTING: Outpatient pulmonary rehabilitation. POPULATION: Twenty moderate-to-severe COPD patients were randomly assigned to either a training group (N.=10) or a control group (N.=10). METHODS: HRV at rest and during submaximal test was determined by linear (rMSSD and SDNN) and non-linear indices (SD1, SD2 and sample entropy [SE]). In addition, key responses were obtained during cardiopulmonary exercise testing (CPET), the walking distance (WD) during the six minute walking test and submaximal constant speed testing (CST). PTP consisted of 30 minutes of aerobic exercise training on a treadmill, 3 times per week at 70% of CPET peak speed rate. Patients were evaluated on baseline, 6 and 12 weeks. RESULTS: Significant improvements in HRV indices, WD, as well as, other physiological responses were observed after 6 weeks of the PTP and maintained until 12 weeks (P<0.05). However, after 12 weeks, the SD1 index demonstrated an additional improvement compared with 6 weeks (P<0.05). Peak oxygen uptake and dioxide carbon production improved only after 12 weeks (P<0.05). Interestingly, the 6th week-baseline delta (6th week-baseline) of WD, SDNN and SE were significantly higher than 12th week-6th week delta (P<0.05). CONCLUSION: These results indicate that beneficial changes on cardiac autonomic modulation in conjunction with improvement in submaximal functional capacity occur in the first 6 weeks of PTP in moderate to severe COPD. CLINICAL REHABILITATION IMPACT: Short-term rehabilitation (6 weeks) is an effective sufficient time to beneficially modify important outcomes as cardiac modulation and exercise capacity in COPD patients.

Complete Genome Sequence of an F8-Like Lytic Myovirus ({varphi}SPM-1) That Infects Metallo-ß-Lactamase-Producing Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important cause of infection, especially in immunocompromised patients. In this regard, strains producing carbapenemases, mainly metallo-ß-lactamases (MBLs), have become a significant public health concern. Here, we present the complete annotated genome sequence (65.7 kb) of an F8-related lytic myovirus (Pbunalikevirus genus) that infects MBL-producing P. aeruginosa strains.

Multi-institutional outbreak of Burkholderia cepacia complex associated with contaminated mannitol solution prepared in compounding pharmacy.

BACKGROUND: Burkholderia cepacia complex (BCC) has been described as a cause of nosocomial outbreaks. We describe an outbreak of and identify risk factors for nosocomial BCC infections associated with intrinsically contaminated mannitol 3% solution. METHODS: Urinary and bloodstream infection caused by BCC were identified in hospitalized patients who underwent urologic surgery and received intraoperative irrigation of 3% mannitol solution in February 2009. The investigation included retrospective chart review, case control study, procedural review, and culture of mannitol solution. RESULTS: Seven BCC infections were identified. BCC isolates were recovered from blood and/or urine from patients and lots of mannitol in use during the outbreak period. Mannitol solution was produced by a compounding pharmacy. Receipt of larger volumes of contaminated solution was identified as a significant risk factor for infection (odds ratio, 1.5; P value < .05). BCC was also cultured in lots of mannitol in use in other hospitals. CONCLUSION: Manipulated mannitol solution is a potential source of infection. Contamination with paraben-degrading organisms can occur at the time of manufacture. Our findings suggest that contamination of mannitol at a compounding pharmacy occurred. Prompt communication to other hospitals and implementation of infection control measures were effective in avoiding further cases of infection.

Distinct Interaction of Two Atypical Enteropathogenic Escherichia coli Strains with Enterocytes In Vitro.

Typical and atypical Enteropathogenic Escherichia coli (EPEC) promote attaching-effacing lesions in intestinal cells but only typical EPEC carry the EPEC adherence factor plasmid. Atypical EPEC (aEPEC) are emerging agents of acute and persistent diarrhea worldwide. We aimed at comparing the ability of two aEPEC strains, 1711-4 (serotype O51:H40) and 3991-1 (serotype O non-typeable:non-motile) to invade, persist inside Caco-2 and T84 cells, and to induce IL-8 production. Typical EPEC strain E2348/69 was used for comparisons. The strains associated more significantly with T84 than with Caco-2 cells, with 3991-1 being the most adherent (P < 0.001). In contrast, aEPEC 1711-4 was significantly more invasive than the other strains in both cell lines, and was found within vacuoles near the basolateral cell surfaces. Strains persisted within both cell lines for at least 48 hours, but the persistence index was higher for 3991-1 in Caco-2 cells. IL-8 production was significantly higher from Caco-2 cells infected with 1711-4 for at least 48 hours (P < 0.001), and from T84 cells after 24 and 48 h than with the other strains (P = 0.001). We demonstrated that aEPEC are heterogeneous in various aspects of their interaction with enterocytes in vitro.

Involvement of pmrAB and phoPQ in polymyxin B adaptation and inducible resistance in non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

During investigation of susceptibility testing methods for polymyxins, 24 multidrug-resistant clinical isolates of Pseudomonas aeruginosa were observed to have a distinct, reproducible phenotype in which skipped wells were observed during broth microdilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated. The effects of various concentrations of polymyxin B on growth, the expression of resistance genes, and outer-membrane permeability were observed. Real-time PCR was performed to compare the expression, in response to selected concentrations of polymyxin B, of genes related to the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems in polymyxin B-susceptible isolate PAO1, polymyxin B-resistant isolate 9BR, and two isolates (19BR and 213BR) exhibiting the skipped-well phenotype. 19BR and 213BR appeared to have similar basal levels of expression compared to that of PAO1 for phoQ, arnB, and PA4773 (from the pmrAB operon), and in contrast, 9BR had 52- and 280-fold higher expression of arnB and PA4773, respectively. The expression of arnB and PA4773 increased in response to polymyxin B in a concentration-dependent manner for 9BR but not for 19BR and 213BR. For these isolates, expression was significantly increased for arnB and PA4773, as well as phoQ, only upon exposure to 2 mug/ml polymyxin B but not at a lower concentration of 0.125 microg/ml. The sequencing of the pmrAB and phoPQ operons for all three isolates revealed a number of unique mutations compared to that for PAO1. 1-N-phenylnaphthylamine (NPN) was used to study the effect of preincubation with polymyxin B on the self-promoted uptake of polymyxin B across the outer membrane. The preincubation of cells with 2 microg/ml polymyxin B affected baseline membrane permeability in 19BR and 213BR and also resulted in a reduced rate of NPN uptake in these isolates and in PAO1 but not in 9BR. The results presented here suggest that the skipped-well isolates have the ability to adapt to specific concentrations of polymyxin B, inducing known polymyxin B resistance genes involved in generating alterations in the outer membrane.

The flagella of an atypical enteropathogenic Escherichia coli strain are required for efficient interaction with and stimulation of interleukin-8 production by enterocytes in vitro.

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.

Antibiotic resistance and trend of urinary pathogens in general outpatients from a major urban city.

OBJECTIVE: We assessed the antimicrobial resistance patterns of pathogens responsible for urinary tract infections (UTI) in outpatients in São Paulo, Brazil, as well as the Escherichia coli antimicrobial resistance trend. MATERIALS AND METHODS: Outpatients urine cultures were collected from January 2000 to December 2003. Statistical analysis considered positive results for one bacterial species with colony count >or= 100,000 CFU/mL. Stratification was done on age group and gender. Statistical tests used included chi-square and the chi-square test for trend to evaluate differences between susceptibility rates among age groups and ordering in the E. coli resistance rates per year, respectively. RESULTS: There were 37,261 positive results with Enterobacteriaceae isolated in 32,530 (87.3%) and Gram-positive cocci in 2,570 (6.9%) cultures. E. coli had the highest prevalence (71.6%). Susceptibility tests were performed in 31,716 cultures. E. coli had elevated resistance rates (> 30%) to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Significant differences between age groups and ordering among years were observed. CONCLUSIONS: The use of trimethoprim-sulfamethoxazole is precluded in the population studied due to elevated resistance rates (> 30%) among most prevalent pathogens. Significant resistance rate differences among age groups and years were observed, particularly for fluoroquinolones. Fluoroquinolones should be used with caution. Nitrofurantoin should be used as empirical therapy for primary, non-complicated urinary tract infections.

Antibiotic resistance and trend of urinary pathogens in general outpatients from a major urban city

OBJECTIVE: We assessed the antimicrobial resistance patterns of pathogens responsible for urinary tract infections (UTI) in outpatients in São Paulo, Brazil, as well as the Escherichia coli antimicrobial resistance trend. MATERIALS AND METHODS: Outpatients urine cultures were collected from January 2000 to December 2003. Statistical analysis considered positive results for one bacterial species with colony count > 100,000 CFU/mL. Stratification was done on age group and gender. Statistical tests used included chi-square and the chi-square test for trend to evaluate differences between susceptibility rates among age groups and ordering in the E. coli resistance rates per year, respectively. RESULTS: There were 37,261 positive results with Enterobacteriaceae isolated in 32,530 (87.3 percent) and Gram-positive cocci in 2,570 (6.9 percent) cultures. E. coli had the highest prevalence (71.6 percent). Susceptibility tests were performed in 31,716 cultures. E. coli had elevated resistance rates (> 30 percent) to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Significant differences between age groups and ordering among years were observed. CONCLUSIONS: The use of trimethoprim-sulfamethoxazole is precluded in the population studied due to elevated resistance rates (> 30 percent) among most prevalent pathogens. Significant resistance rate differences among age groups and years were observed, particularly for fluoroquinolones. Fluoroquinolones should be used with caution. Nitrofurantoin should be used as empirical therapy for primary, non-complicated urinary tract infections.

In vitro synergy test of meropenem and sulbactam against clinical isolates of Acinetobacter baumannii.

Meropenem and imipenem are often the drugs of choice for the treatment of infections due to multidrug-resistant Acinetobacter baumannii. The present study aimed at evaluating the interaction between meropenem and sulbactam through microdilution and checkerboard methods against 48 clinical isolates of A. baumannii collected from Brazilian hospitals. All the isolates presented elevated minimum inhibitory concentration (>or=2 microg/mL) to either meropenem or sulbactam. The checkerboard method with the combination of meropenem and sulbactam demonstrated 29.2% (14/48) synergism, 47.9% (23/48) partial synergism, 10.5% (5/48) additive, 6.2% (3/48) indifference, and 6.2% (3/48) antagonism (SigmaFIC(min)=0.09 and SigmaFIC(max)=8). Thus, combinations of meropenem and sulbactam may show synergism or partial synergism for most A. baumannii isolates. Further studies may help identify treatment options for patients with infections caused by these organisms, particularly with this combination, where both drugs have time-dependent activities and might be suitable for therapy optimization studies.