In Planta Colonization and Role of T6SS in Two Rice Kosakonia Endophytes.

Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant-bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant-bacteria interaction.

LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348.

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization.IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

Virulence factors and mechanisms of antimicrobial resistance in Shigella strains from periurban areas of Lima (Peru).

The study was aimed to describe the serotype, mechanisms of antimicrobial resistance, and virulence determinants in Shigella spp. isolated from Peruvian children. Eighty three Shigella spp. were serogrouped and serotyped being established the antibiotic susceptibility. The presence of 12 virulence factors (VF) and integrase 1 and 2, along with commonly found antibiotic resistance genes was established by PCR. S. flexneri was the most relevant serogroup (55 isolates, 66%), with serotype 2a most frequently detected (27 of 55, 49%), followed by S. boydii and S. sonnei at 12 isolates each (14%) and S. dysenteriae (four isolates, 5%). Fifty isolates (60%) were multi-drug resistant (MDR) including 100% of S. sonnei and 64% of S. flexneri. Resistance levels were high to trimethoprim-sulfamethoxazole (86%), tetracycline (74%), ampicillin (67%), and chloramphenicol (65%). Six isolates showed decreased azithromycin susceptibility. No isolate was resistant to nalidixic acid, ciprofloxacin, nitrofurantoin, or ceftriaxone. The most frequent resistance genes were sul2 (95%), tet(B) (92%), cat (80%), dfrA1 (47%), blaOXA-1like (40%), with intl1 and intl2 detected in 51 and 52% of the isolates, respectively. Thirty-one different VF profiles were observed, being the ipaH (100%), sen (77%), virA and icsA (75%) genes the most frequently found. Differences in the prevalence of VF were observed between species with S. flexneri isolates, particularly serotype 2a, possessing high numbers of VF. In conclusion, this study highlights the high heterogeneity of Shigella VF and resistance genes, and prevalence of MDR organisms within this geographic region.

Diarrheagenic Escherichia coli phylogroups are associated with antibiotic resistance and duration of diarrheal episode.

Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (P < 0.05). There was a tendency (P = 0.06) for higher proportion of persistent diarrhea (≥ 14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (P > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

Oral lactoferrin administration does not impact the diversity or composition of the infant gut microbiota in a Peruvian cohort.

IMPORTANCE: Previous studies have suggested that oral lactoferrin enhances diversity in the gut microbiota in infants while inhibiting the growth of opportunistic pathogens. However, the effect of lactoferrin on infant gut microbiota over time has yet to be thoroughly studied. Our study suggests that lactoferrin oral treatment in infants aged 12-18 months does not affect gut microbiome diversity and composition over time. To our knowledge, this is the first study to report the effect of lactoferrin on infant gut microbiome composition over time and helps elucidate its impact on infant health and its therapeutic potential.

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains.

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.

Characterization of Escherichia coli D7111 producing the ß-LACTAMASE TEM-176. / Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176.

The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and ß-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new ß-lactamase bla TEM-176.

El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a ß-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva ß-lactamasa, con el nombre de bla TEM-176.

Effect of bovine lactoferrin in Salmonella ser. Typhimurium infection in mice.

Lactoferrin (LF) has in vitro antimicrobial activity against Gram-negative bacteria. Salmonella enterica subsp. enterica serovar Typhimurium causes systemic infection and acute diarrhea in humans, mainly in children younger than 2 years of age. The aim of the study was to determine the in vivo effect of bovine LF in Salmonella ser. Typhimurium infection in mice. 58 BALB/c mice were employed. Two hours before the infection with 300 microl of 10(7) CFU of Salmonella ser. Typhimurium, 29 mice received LF (2 mg) and 29 placebo (buffer). After the infection, the mice received LF (10 mg/ml) ad libitum or buffer, respectively, for 7 days. Mortality, weight and clinical signs (piloerection, hunched position and reduced movement) were monitored daily. The degree of inflammation and necrosis in the intestine, liver, spleen and brain were studied with a blinded observer. The mortality in the control group (8/29) was higher than in the LF group (1/29) (Kapplan Meier P < 0.05). From the third day post-infection the control group were significantly more symptomatic (P < 0.05). The blood culture for Salmonella spp. was positive for all mice studied in the control group (17/17), but positive in the LF group in only 6/17 animals (P < 0.05). In the LF group, the pathologic studies show less inflammation and focal necrosis in the four organs studied, with the greatest difference found in the intestine. Bovine LF protects against Salmonella ser. Typhimurium infection in mice, reducing the severity, mortality and the degree of inflammation of this infection.

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASA TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASE TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

pic gene of enteroaggregative Escherichia coli and its association with diarrhea in Peruvian children.

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea among children, HIV-infected patients, and travelers to developing countries. We have searched for 18 genes-encoding virulence factors associated with aggregative adherence, dispersion, biofilm, toxins, serine protease autotransporters of Enterobacteriaceae (SPATEs) and siderophores, analyzed in 172 well-characterized EAEC strains (aggR(+)) isolated from stool samples of 97 children with diarrhea and 75 healthy controls from a passive surveillance diarrhea cohort study in Peru. Eighty-one different genetic profiles were identified, 37 were found only associated with diarrhea and 25 with control samples. The most frequent genetic profile was aggC(+)aatA(+)aap(+)shf(+)fyuA(+), present in 19 strains, including diarrhea and controls. The profile set1A(+)set1B(+)pic(+) was associated with diarrhea (P < 0.05). Of all genes evaluated, the most frequent were aatA (CVD 342) present in 159 strains (92.4%) and fyuA in 157 (91.3%). When EAEC strains were analyzed as a single pathogen (excluding co-infections), only pic was associated with diarrhea (P < 0.05) and with prolonged diarrhea (diarrhea ≥ 7 days) (P < 0.05). In summary, this is the first report on the prevalence of a large set of EAEC virulence genes and its association with diarrhea in Peruvian children. More studies are needed to elucidate the exact role of each virulence factor.

[Effect of bovine lactoferrin biofilms in the formation of clinical strains of enteroaggregative E. coli]. / Efecto de lactoferrina bovina en la formación de biofilms en cepas clínicas de E. coli enteroagregativa.

OBJECTIVES: To determine the effect of bovine lactoferrin (BLF) in the formation of biofilms in clinical enteroaggregative Escherichia coli (EAEC) strains and whether this effect is independent of iron. MATERIALS AND METHODS: Two methods were used: (a) qualitative, by direct observation of optical microscopy, and (b) quantitative readings of the absorbance values using ELISA reader in the presence of bLf in concentrations of 0.01 mg mL and 1 mg/mL, with and without iron and no bLf (control). Analysis occurred in 122 strains of EAEC (60 strains from children with diarrhea and 62 healthy children) previously collected in a previous study of passive surveillance of diarrhea in the Southern Cone Lima. 31 strains of the same method were used for the qualitative study. RESULTS: (A) Qualitative method: 31 strains were evaluated with and without iron. Without iron biofi formation was 77% (24/31) in the control group versus 58% (14/31) with bLf of 0.01 mg/mL and 4% (1/31) with 1 mg/ml. Iron biofilm formation was 90% (28/31) in the control group versus 55% (17/31) with bLf of 0.01 mg/mL and 4% (1/31) with 1 mg/mL. (B) Quantitative method: without iron absorbance measured at OD 560 nm of the control group was 0.7 ± 0.5 versus 0.4 ± 0.3 with bLf 0.01 mg/mL and 0.3 ± 0.2 with bLf of 1 mg/mL (p<0.0001). This decrease in the presence of bLf included iron. CONCLUSIONS: bLf tends to decrease the formation of biofilms, showing an inhibitory effect in clinical isolates of EAEC; this effect is not iron-dependent.

Which mechanisms of azithromycin resistance are selected when efflux pumps are inhibited?

The aim of this study was to develop in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella spp. in the presence of Phe-Arg ß-naphthylamide (PAßN) and to observe which AZM resistance mechanisms other than efflux pumps were inhibited by PAßN emerge. The frequency of mutation ranged between <6.32 × 10(-10) and 5.22 × 10(-7) for E. coli and between <5.32 × 10(-10) and 1.69 × 10(-7) for Shigella spp. The E. coli mutants showed an increase in the AZM minimum inhibitory concentration (MIC) up to 128-fold, whilst the Shigella spp. mutants presented increases in MIC levels of up to 8-fold. In one mutant, the insertion of nucleotides encoding the amino acid sequence IMPRAS was found in the rplV gene. Increases in OmpW expression were observed in all E. coli mutants compared with their respective parental isolates. The combination of antibiotics and efflux pump inhibitors appears to be a good option to reduce the frequency of mutation in clinical isolates.

In vitro development and analysis of Escherichia coli and Shigella boydii azithromycin-resistant mutants.

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-ß-Naphthylamide (PAßN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAßN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAßN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.

Molecular mechanisms of antibiotic resistance in diarrhoeagenic Escherichia coli isolated from children.

Diarrhoeagenic Escherichia coli (DEC) are an important cause of diarrhoea in children and are associated with high antibiotic resistance. However, there are few studies on the molecular mechanisms of resistance in this group of bacteria. The aim of this study was to determine the mechanisms associated with antibiotic resistance in the most common phenotypes of DEC. A total of 369 E. coli strains [commensal strains and DEC from children with ('DEC-diarrhoea') or without ('DEC-control') diarrhoea] isolated from children aged <1 year in periurban districts of Lima, Peru, were analysed. In total, 154 ampicillin-resistant strains (36 commensals, 33 DEC-control and 85 DEC-diarrhoea) were studied by PCR for the most prevalent resistance mechanisms to ampicillin, trimethoprim/sulfamethoxazole (SXT), tetracycline and chloramphenicol as well as for integrase types 1 and 2. In addition, restriction fragment length polymorphism was performed for SXT-resistant strains. Commensal strains were more frequently resistant to nalidixic acid and ciprofloxacin (68% and 28%, respectively) than DEC strains (23% and 2%, respectively) (P<0.05). DEC-diarrhoea strains were more frequently SXT-resistant (78%) compared with DEC-control strains (65%) and commensal strains (60%) (P<0.05). The most frequent mechanisms of antibiotic resistance in DEC strains were: for ß-lactams, bla(TEM) (31%; 37/118); for SXT, sul2 (48%; 49/103); for tetracycline, tetA (27%; 23/84); and for chloramphenicol, cat (80%; 28/35). The genes sul1 and dfrA1, related to SXT resistance, were more frequent in the DEC-diarrhoea group (41% and 28%, respectively) than in the other two groups (P<0.05). There was a high diversity of resistance genes in DEC, including symptomatic strains.

[Levels of quinolones resistance and other antimicrobial in non-pathogenic Escherichia coli strains in children from the periurban area of Lima, Peru]. / Niveles de resistencia a quinolonas y otros antimicrobianos en cepas de Escherichia coli comensales en niños de la zona periurbana de Lima, Perú.

The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area.

[Molecular mechanisms of antibiotic resistance in Escherichia coli- associated diarrhea]. / Mecanismos moleculares de resistencia antibiótica en Escherichia coli asociadas a diarrea.

Antibiotic resistance is an emerging problem worldwide present in many bacteria, specially in Escherichia coli, which has high percentages of resistance to ampicilline, thrimethoprim-sulfamethoxazole, tetracycline, chloramphenicol and nalidixic acid, which implies important complications in antibiotic treatment when required. The increasing antibiotic resistance is due to the acquisition of different molecular mechanisms of resistance through point chromosomal mutations and /or horizontal transfer of genetic material between related or different species facilitated by some genetic elements such as integrons. This review discusses the effects of the most common molecular mechanisms of antibiotic resistance in E. coli: enzymatic inactivation, changes in the target site and permeability disturbances. Getting to know the mechanisms of resistance which are involved, as the World Health Organization recommends, will allow us to improve the surveillance of the antibiotic resistance, the control policies and the antibiotic utilization at a national level.

[Frequency and pathotypes of diarrheagenic Escherichia coli in Peruvian children with and without diarrhea]. / Frecuencia y patotipos de Escherichia coli diarrogénicas en niños peruanos con y sin diarrea.

UNLABELLED: INTRODUCTION; Diarrheagenic E. coli (DEC) are a major cause of diarrhea in children in developing countries. However, they are not part of routine diagnosis in clinical laboratories. OBJECTIVES: To determine the DEC prevalence in Peruvian children and to describe the genetic variability of these strains. MATERIALS AND METHODS: A total of 8 003 E. coli strains previously isolated from eight different studies of diarrhea in children, mainly from peri-urban areas of Lima, were analyzed. Diagnosis of DEC was done with Multiplex real-time PCR using genes for each of the 6 DEC groups. Conventional PCR was performed for the detection of additional virulence genes. RESULTS: Globally, the mean prevalence in diarrhea samples (n=4,243) was: enteroaggregative E. coli (EAEC) 9.9%, enteropathogenic E. coli (EPEC) 8.5%, enterotoxigenic E. coli (ETEC) 6.9%, diffusely adherent E. coli (DAEC) 4.8%, Shiga toxin-producing E. coli (STEC) 0.8% and enteroinvasive E. coli (EIEC) 0.6%. The relative frequency of each pathogen varies according to the age and the type of study. The main pathotypes in control samples (n=3,760) were EPEC (10.9%) and EAEC (10.4%). An important variability in the virulence genes frequency and molecular resistance mechanisms for each pathotype was found, without differences between diarrhea and control groups. CONCLUSIONS: DEC are a major cause of diarrhea in Peruvian children. These pathogens are highly heterogeneous. Additional studies are required to determine the prevalence in rural areas of Peru and in severe diarrhea cases.

[Adhesion patterns in diffusely adherent Escherichia coli (DAEC) strains isolated from children with and without diarrhea]. / Patrones de adherencia de cepas de Escherichia coli Difusamente adherente (DAEC) provenientes de niños con y sin diarrea.

INTRODUCTION: Diffusely adherent E. coli (DAEC) is the sixth recognized group of diarrheagenic E. coli. However, its association with diarrhea remains controversial. Variability in the adherence patterns of clinical strains is unknown. OBJECTIVES: To compare the adherence patterns between strains isolated from children with and without diarrhea. MATERIALS AND METHODS: A total of 31 DAEC strains were analyzed, 25 from children with diarrhea and 6 from asymptomatic (control) children, isolated from a cohort study of children under one year of age in the southern districts of Lima. DAEC were identified by PCR (daaD gene). The pattern and adherence score in HEp-2 cell culture were evaluated, Actin polimerization was determined by fluorescence actin staining (FAS) and motility was evaluated by conventional microbiology methods. RESULTS: Diffuse adherence pattern was found in 88% of diarrhea samples and in the total of control strains. The number of bacteria adhered per cell was significantly lower in diarrhea samples (p<0.05). However, actin polymerization was greater in diarrhea samples (60% vs. 17%). Motility test was positive in 60% of the diarrhea samples and in all control samples. CONCLUSIONS: Our findings suggest a difference between adherence patterns, actin polymerization and motility between DAEC strains corresponding to diarrhea and control groups. The significance of these results must be confirmed with a bigger number of strains and determining the presence of virulence genes in the strains.

[Enteroviral central nervous system infections in children treated at a hospital in Lima, Peru]. / Infecciones del sistema nervioso central por enterovirus en niños atendidos en un hospital de Lima, Perú

OBJECTIVES: To determine the frequency and clinical features of central nervous system infections caused by enterovirus in children treated at the Hospital Nacional Cayetano Heredia in Lima, Peru. MATERIALS AND METHODS: A prospective, descriptive study was performed from April 2008 to March 2010. Patients aged 1 month - 14 years with clinical diagnosis of encephalitis or aseptic meningitis were included. We investigated the presence of enterovirus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) and varicella-zoster virus (VZV) by polymerase chain reaction (PCR). RESULTS: 97 patients were included, out of which 69 % had acute encephalitis and 31 % acute meningitis. Enteroviruses were identified in 52,6% of all acute non-bacterial central nervous system infections; corresponding to 83,3 % of meningitis and 38,8 % of encephalitis. There were no cases of infection due to HSV-1, HSV-2 or VZV. Enterovirus infections reached 82,9 % in the warm months (November-January) and 28,6 % in the colder months (May-July). CONCLUSIONS: Enteroviruses are the principal etiologic agents in acute aseptic meningitis and encephalitis in pediatric patients in Lima, Peru. Enteroviruses have a seasonal epidemiological pattern with a clear increase in the number of cases during the summer months. It is useful to have this rapid diagnostic method available as an aid in the management of acute central nervous system infections.