Antibiotic resistance in environmental Escherichia coli - a simple screening method for simultaneous typing and resistance determination.

We describe a simple and standardised screening system (AREB) for surveillance of antibiotic resistant bacteria in the environment. The system consists of 96 well microplates containing eight sets of breakpoint amounts of 10 different antibiotics. The incubated microplates are read by a desktop scanner and the plate images are analysed by special software that automatically presents the resistance data. The AREB method is combined with a rapid typing method, the PhenePlate system, which yields information on the diversity of the bacteria in the studied samples, and on the possible prevalence of resistant clones. In order to demonstrate the usage of AREB, a comparative study on the resistance situation among 970 Escherichia coli isolates from sewage and recipient water in Sweden, Norway and Chile, was performed. Resistance rates to all antibiotics were markedly higher in hospital sewage than in other samples. Our data indicate that the AREB system is useful for comparing resistance rates among E. coli and other environmental indicator bacteria in different countries/regions. Simple handling and automatic data evaluation, combined with low cost, facilitate large studies involving several thousands of isolates.

Antibiotic resistance patterns of intestinal Escherichia coli isolates from Nicaraguan children.

In developing countries, diarrhoeal diseases are one of the major causes of death in children under 5 years of age. It is known that diarrhoeagenic Escherichia coli (DEC) is an important aetiological agent of infantile diarrhoea in Nicaragua. However, there are no recent studies on antimicrobial resistance among intestinal E. coli isolates in Nicaraguan children. The aim of the present study was to determine the antimicrobial resistance pattern in a collection of 727 intestinal E. coli isolates from the faeces of children in León, Nicaragua, between March 2005 and September 2006. All samples had been screened previously for the presence of DEC by multiplex PCR. Three hundred and ninety-five non-DEC isolates (270 from children with diarrhoea and 125 from children without diarrhoea) and 332 DEC isolates (241 from children with diarrhoea and 91 from children without diarrhoea) were analysed in this study. In general, antimicrobial resistance among the 727 intestinal E. coli isolates was high for ampicillin (60 %), trimethoprim-sulfamethoxazole (64 %) and chloramphenicol (11 %). Among individual E. coli categories, enteroaggregative E. coli isolates from children with and without diarrhoea exhibited significantly higher levels of resistance (P<0.05) to ampicillin and trimethoprim-sulfamethoxazole compared to the other E. coli categories. Resistance to ceftazidime and/or ceftriaxone and a pattern of multi-resistance was related to CTX-M-5- or CTX-M-15-producing E. coli isolates. The results suggest that E. coli isolates from Nicaraguan children have not reached the high levels of resistance to the most common antibiotics used for diarrhoea treatment as in other countries.

Asymptomatic norovirus infections in Nicaraguan children and its association with viral properties and histo-blood group antigens.

BACKGROUND: It has been previously reported that histo-blood group antigens (HBGAs) and particularly secretor status provides protection against symptomatic norovirus infection, but it remains unclear to what extent this includes asymptomatic infections in children. METHODS: To explore whether HBGAs or certain viral genotypes are associated with asymptomatic norovirus infections in a pediatric population in Nicaragua, we investigated 163 children ≤5 years of age, without a recent history of diarrhea (≤10 days). RESULTS: Asymptomatic norovirus infections were observed in 11.7% (19/163), with children ≤6 months of age being most frequently infected (16%). Of the 19 norovirus-positive children, 4 (21%) and 10 (53%) were infected with genogroups GI and GII, respectively, and 4 children (21%) were infected with viruses of both genogroups. Most children had ≥10 viral genomes per gram of feces. Nucleotide sequence analysis (15/19) revealed uncommon genotypes, such as, GII.7 (n = 5) and GII.2 (n = 3). An interesting observation was the low frequency of norovirus GII.4 strains among the asymptomatic children. AB blood type, Lewis a (Le) phenotype and nonsecretor genotype (sese) were not found among the asymptomatic children, but they occurred in population controls. CONCLUSIONS: Frequency of asymptomatic norovirus infections was similar to that observed in symptomatic children from Nicaragua. Norovirus GII.2 and GII.7 were frequently detected but the globally dominating GII.4 was infrequent. Host genetic factors previously observed to be associated with protection against symptomatic norovirus infection were not found in this study.

Diarrheagenic Escherichia coli markers and phenotypes among fecal E. coli isolates collected from Nicaraguan infants.

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

Caracterización fenotípica y genotípica de la resistencia a meticilina de cepas de Staphylococcus aureus aisladas en Antofagasta / Phenotypic and genotypic characterization of methicillin resistance in strains of Staphylococcus aureus isolated in Antofagasta

Staphylococcus aureus resistant to methicillin (SARM) has been associated with nosocomial infections due to its capacity to develop resistance to multiple antibiotics. There is little information about the SARM which are found in the hospital services of Antofagasta. We studied the phenotypic and genotypic characteristics of methicillin resistance in 38 strains of S. aureus isolated in Antofagasta, identified by coagulase and API Staph tests and by a biochemical test (Ph-system). The susceptibility to antibiotics was studied using the agar dilution technique, identifying SARM strains with discs of oxacillin. Beta-lactamase with nitrocephine, and the gene mecA by means of PCR. Eighty nine percent (34 strains) were SARM with a high resistance to ampicillin, penicillin, erythromycin, claritromycin. gentiamycin, amikacine and ciprofloxacine. All isolates were susceptible to vancomycin and rifampicin. Beta-lactamase was demonstrated in 79% of the SARM strains. Strain typing and resistance patterns revealed a great diversity of PhP-types and antibiotypes in the isolates. Ninety seven percent of the SARM strains had the gene mecA. One PhP-type (C6) was dominant (5 SARM strains) all had the mecA gene, produced beta lactamase and had the same pattern of antibiotic resistance. We conclude that the dominant phenotypes of SARM strains which have the mecA gene and multiple resistance to antibiotics are present in the hospitals of Antofagasta, and sound the alert on the risk of nosocomial transmission of epidemic clones of SARM.

Staphylococcus aureus resistentes a meticilina (SARM) han sido asociados con infecciones nosocomiales por su capacidad para desarrollar resistencia a múltiple antibióticos, existiendo escasa información acerca de SARM que están circulando en los servicios hospitalarios de Antofagasta. Se estudió características fenotIpicas y genotípicas de la resistencia a meticilina en 38 cepas de S. aureus aisladas en Antofagasta, identificadas por tests de coagulasa y API Staph y por tipificación bioquímica (Ph-Sistem). La susceptibilidad a antibióticos se realizo por técnica de dilución en agar, las cepas SARM fueron identificadas con discos de oxacilina, beta-lactamasa por nitrocefina y gen mecA fue detectado pot PCR. El 89% (34 cepas), fueron SARM con una alta resistencia a ampicilina, penicilina, eritromicina, gentamicina, amikacina y ciprofloxacino. Todos los aislados fueron susceptibles a vancomocina y rifampicina. Beta lactamasa fue demostrada en 79% de las cepas SARM. La tipificación y los patrones de resistencia revelaron una alta diversidad de PhP tipos y antibioticos en los aislamientos. El 97% de las cepas SARM albergaban el gen mecA. Un PhP tipo (C6) fue dominante. (5 cepas SARM), todos presentando el gen mecA, produciendo beta lactamasa y mostrando el mismo patrón de resistencia antibiótica. Se concluye que los fenotipos dominantes de cepas SARM que albergan el gen mecA y resistencia múltiples alos antibióticos están circulando en los hospitales de Antofagasta, alertando sobre el riesgo de transmisión intranosocomial de clones epidémicos de SARM.

Diversity of intestinal Escherichia coli populations in Nicaraguan children with and without diarrhoea.

Escherichia coli remains an important aetiological agent of infantile diarrhoea in Nicaragua. However, little is known about whether there is a high prevalence of endemic strains or whether infection is due to the epidemic spread of virulent clones. This study was undertaken to determine the diversity and distribution of clonal groups in a population of intestinal E. coli isolated from the faeces of children from León, Nicaragua, with (n=381) and without (n=145) diarrhoea, between March 2005 and September 2006. All samples had been screened previously for the presence of diarrhoeagenic E. coli (DEC) markers by multiplex PCR. From each sample, 8 E. coli colonies (where available) were analysed by biochemical fingerprinting (PhP-RE system), yielding a total of 4009 tested isolates. On average, three different biochemical phenotypes (BPTs) were found among the eight colonies analysed from each sample. The total diversity, measured as Simpson's diversity index (Di), was 0.97 among all 4009 isolates studied. Cluster analysis of data from all 4009 isolates revealed 24 common BPTs (identified in at least 1 % of the isolates) and 234 less common BPTs. Similar Di values were obtained among isolates from infants with and without diarrhoea, indicating that no widespread outbreak of DEC had occurred. Moreover, among samples that were positive for the DEC types enteroaggregative E. coli, enteropathogenic E. coli and enterotoxigenic E. coli (ETEC) carrying the eltB gene, the diversities were almost as high as among non-DEC samples, whereas samples positive for ETEC carrying estA, enteroinvasive E. coli and enterohaemorrhagic E. coli showed lower diversities, indicating the prevalence of virulent clonal groups among these samples. The PhenePlate patterns of the 24 common BPTs identified here were compared with those obtained from E. coli isolated in a cohort infant study performed in 1991-1992 in the same area. Only 4 % of the isolates from the 1990s were similar to any of the common BPTs found in the present study.

Prevalence of diarrhoeagenic Escherichia coli in children from León, Nicaragua.

Diarrhoeal disease is a public health problem worldwide, mostly affecting children in developing countries. In Nicaragua, diarrhoea is the second greatest cause of infant mortality. During the period March 2005 to September 2006, a total of 526 faecal samples from children aged 0-60 months (381 with and 145 without diarrhoea) from León, Nicaragua, were studied. In order to detect five different diarrhoeagenic Escherichia coli pathotypes simultaneously [enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and enteroinvasive E. coli (EIEC)], a mixture of eight primer pairs was used in a single PCR. At least one diarrhoeagenic E. coli pathotype was detected in 205 samples (53.8%) of the diarrhoea group and in 77 samples (53.1%) in the non-diarrhoea group. ETEC was detected significantly more often in children with diarrhoea (20.5%) than in children without diarrhoea (8.3%) (P=0.001). Atypical EPEC, EIEC and EAEC were detected with slightly lower frequencies in children with (16.0, 0.8 and 27.8%, respectively) than in children without (20.7, 1.4 and 33.1%, respectively) diarrhoea. EHEC was only detected in children with diarrhoea (2.1%). In conclusion, ETEC continues to be an important agent associated with diarrhoea in children from León, Nicaragua. Although not very frequent, the only findings that were 100% associated with diarrhoea were ETEC estA (4.7%) and EHEC (2.1%). Nevertheless, EAEC and EPEC were also frequent pathotypes in the population under study. In children with severe diarrhoea, more than half had EAEC, ETEC or EPEC, and EAEC was the most prevalent pathotype.

[Prevalence of antibiotic resistant Enterococcus spp in waste waters in the north of Chile]. / Prevalencia de Enterococos resistentes a antibióticos en aguas servidas en el norte de Chile.

BACKGROUND: There is little information available in Chile on the distribution of Enterococcus spp in waste water and its implications in transmission of antibiotic resistance through the water cycle. Enterococcus spp are common in nosocomial infections and may spread antibiotic resistance through the food chain. AIM: To determine the presence of antibiotic resistant Enterococcus spp in the sewage of Antofagasta, Chile. MATERIAL AND METHODS: Samples of sewage from two sewage treatment plants and from the Public Hospital of Antofagasta collector were obtained. Enterococcus spp were isolated on m-Enterococcus agar containing ampicillin, vancomycin and streptomycin. The isolates were identified and subjected to biochemical typing (PhPlate). Minimal inhibitory concentration determination was performed by agar dilution technique. RESULTS: High counts of resistant Enterococcus spp were found on the streptomycin plates, lower on ampicillin and very low on vancomycin plates. A total of 63 Enterococcus spp strains were typed and the identification showed 5 different species; E faecalis (65%), E faecium (14%), E hirae (13%), E durans (6%) and E gallinarum (2%). The typing revealed a high diversity among the isolates. Two biochemical phenotypes were predominant, C1 (21 strains) and C6 (7 strains). Both were highly resistant to gentamycin and streptomycin; moderately resistant to ampicillin, chloramphenicol, tetracycline and ciprofloxacin, and with intermediate susceptibility to vancomycin. Both phenotypes were found in the sewage of the hospital collector and in the treatment plants. CONCLUSIONS: In the sewage of Antofagasta we found dominating phenotypes of multiresistant Enterococcus spp. Sewage could be an important way of transmission of these microorganisms.

Prevalencia de Enterococos resistentes a antibióticos en aguas servidas en el norte de Chile / Prevalence of antibiotic resistant Enterococcus spp in waste waters in the north of Chile

Background: There is little information available in Chile on the distribution of Enterococcus spp in waste water and its implications in transmission of antibiotic resistance through the water cycle. Enterococcus spp are common in nosocomial infections and may spread antibiotic resistance through the food chain. Aim: To determine the presence of antibiotic resistant Enterococcus spp in the sewage of Antofagasta, Chile. Material and Methods: Samples of sewage from two sewage treatment plants and from the Public Hospital of Antofagasta collector were obtained. Enterococcus spp were isolated on m-Enterococcus agar containing ampicillin, vancomycin and streptomycin. The isolates were identified and subjected to biochemical typing (PhPlate). Minimal inhibitory concentration determination was performed by agar dilution technique. Results: High counts of resistant Enterococcus spp were found on the streptomycin plates, lower on ampicillin and very low on vancomycin plates. A total of 63 Enterococcus spp strains were typed and the identification showed 5 different species; E faecalis (65%), E faecium (14%), E hirae (13%), E durans (6%) and E gallinarum (2%). The typing revealed a high diversity among the isolates. Two biochemical phenotypes were predominant, C1 (21 strains) and C6 (7 strains). Both were highly resistant to gentamycin and streptomycin; moderately resistant to ampicillin, cloramphenicol, tetracycline and ciprofloxacin, and with intermediate susceptibility to vancomycin. Both phenotypes were found in the sewage of the hospital collector and in the treatment plants. Conclusions: In the sewage of Antofagasta we found dominating phenotypes of multiresistant Enterococcus spp. Sewage could be an important way of transmission of these microorganisms.

[Biochemical phenotypes and phage types of Salmonella enteriditis strains isolated in Antofagasta during the period 1997-2000]. / Fenotipos bioquímicos y fagotipos de cepas de Salmonella enteritidis aisladas en Antofagasta, 1997-2000.

BACKGROUND: PhP-S48 (Phene Plate Techniques AB), a method based on biochemical phenotypes has been developed and used successfully to typify S enteritidis strains in epidemiological studies. AIM: To identify phenotypes of S enteritidis isolated from eggs, chicken meat and infected humans in Antofagasta during the period 1997-2000. MATERIAL AND METHODS: PhP-S48 and phage typing were used to identify phenotypes of 33 S enteritidis strains, sixteen isolated from poultry and 17 from clinical sources. S enteritidis ATCC17036 was used as control strain. RESULTS: Twelve biochemical phenotypes (BTs) including 4 common (C) and 8 single (S) were identified. BTs C1 y C3 containing 16 and 5 strains, respectively, accounted for 63.6% of the isolates. BT C1 was found in poultry and human sources in the period 1997-2000, and BT C3 was isolated from humans, in the period 1999-2000. Using phage typing, 5 phage types (PT) and 3 strains could be not typed (NTs). PT1 and PT21 were the dominant phage types, with 14 and 13 strains respectively. Strains of PT1 were isolated from poultry and human sources in the period 1997-2000. PT21 was found in poultry samples in the period 1997-1998 and in clinical samples, in the period 1997-1998. Combination of biochemical phenotypes and phage typing divided the strains into 5 phenotypes (BT:PT). Two phenotypes were the most frequently isolated, phenotype C1:1 with 8 isolates found in eggs and humans in 1999, and phenotype C1:21 with 5 strains isolated in 1997-1999. CONCLUSIONS: These results indicate the presence of one persistent and one recently emerged phenotype among S enteritidis in Antofagasta, Chile. PhP-S48 also provided information about a relationship among the strains.