Candidemias en pediatría: distribución de especies y sensibilidad a los antifúngicos / Candidemia in Pediatrics: Species distribution and antifungal susceptibility

Las infecciones graves causadas por levaduras del género Candida son frecuentes en la población hospitalaria. Debido a las diferencias en la distribución de especies y la sensibilidad a los antifúngicos según el área geográfica y el tipo de paciente, resulta importante estudiar la epidemiología de cada institución. Con este propósito, hemos realizado un estudio retrospectivo y descriptivo sobre las candidemias ocurridas en el Hospital de Ninos «Superiora Sor María Ludovica¼ de la ciudad de La Plata. En un período de 6 años (2010-2015) se registraron 177 episodios de candidemia. Las especies predominantes fueron Candida albicans (45%) y Candida parapsilosis (28%). Las salas de internación con mayor cantidad de episodios fueron las unidades de terapia intensiva de pediatría, la neonatal y la cardiovascular (58%). En los casos donde se realizaron pruebas de sensibilidad a los antifúngicos, no se observó resistencia a la anfotericina B en todo el período y la resistencia a azoles se limitó a 4 aislamientos de especies menos frecuentes.

Serious infections caused by Candida yeasts are frequent in the hospital population. Due to differences in species distribution and antifungal susceptibility testing depending on the geographic area and the type of patient, it is important to study the epidemiology of each institution. For this purpose, we conducted a retrospective, descriptive study on the occurrence of candidemia in the Children's Hospital "Superiora Sor María Ludovica" of the city of La Plata. In a 6-year period (2010-2015), 177 candidemia episodes were recorded. The predominant species were Candida albicans (45%) and Candida parapsilosis (28%). The hospital wards with the highest number of candidemia episodes were the pediatric, neonatal and cardiovascular intensive care units (58%). No resistance to amphotericin B was observed throughout the period whereas resistance to azoles was limited to 4 strains of less frequent species.

[Candidemia in Pediatrics: Species distribution and antifungal susceptibility]. / Candidemias en pediatría: distribución de especies y sensibilidad a los antifúngicos.

Serious infections caused by Candida yeasts are frequent in the hospital population. Due to differences in species distribution and antifungal susceptibility testing depending on the geographic area and the type of patient, it is important to study the epidemiology of each institution. For this purpose, we conducted a retrospective, descriptive study on the occurrence of candidemia in the Children's Hospital "Superiora Sor María Ludovica" of the city of La Plata. In a 6-year period (2010-2015), 177 candidemia episodes were recorded. The predominant species were Candida albicans (45%) and Candida parapsilosis (28%). The hospital wards with the highest number of candidemia episodes were the pediatric, neonatal and cardiovascular intensive care units (58%). No resistance to amphotericin B was observed throughout the period whereas resistance to azoles was limited to 4 strains of less frequent species.

A cultivation-independent PCR-RFLP assay targeting oprF gene for detection and identification of Pseudomonas spp. in samples from fibrocystic pediatric patients.

Species-specific genetic markers are crucial to develop faithful and sensitive molecular methods for the detection and identification of Pseudomonas aeruginosa (Pa). We have previously set up a PCR-RFLP protocol targeting oprF, the gene encoding the genus-specific outer membrane porin F, whose strong conservation and marked sequence diversity allowed detection and differentiation of environmental isolates (Agaras et al., 2012). Here, we evaluated the ability of the PCR-RFLP assay to genotype clinical isolates previously identified as Pa by conventional microbiological methods within a collection of 62 presumptive Pa isolates from different pediatric clinical samples and different sections of the Hospital de Niños "Sor María Ludovica" from La Plata, Argentina. All isolates, but one, gave an oprF amplicon consistent with that from reference Pa strains. The sequence of the smaller-sized amplicon revealed that the isolate was in fact a mendocina Pseudomonas strain. The oprF RFLP pattern generated with TaqI or HaeIII nucleases matched those of reference Pa strains for 59 isolates (96%). The other two Pa isolates (4%) revealed a different RFLP pattern based on HaeIII digestion, although oprF sequencing confirmed that Pa identification was correct. We next tested the effectiveness of the PCR-RFLP to detect pseudomonads on clinical samples of pediatric fibrocystic patients directly without sample cultivation. The expected amplicon and its cognate RFLP profile were obtained for all samples in which Pa was previously detected by cultivation-dependent methods. Altogether, these results provide the basis for the application of the oprF PCR-RFLP protocol to directly detect and identify Pa and other non-Pa pseudomonads in fibrocystic clinical samples.

Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection.

The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans -the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.

Epidemiological study of shigellosis in an urban area of Argentina.

INTRODUCTION: Shigellosis represents one of the main causes of bloody diarrhoea in South America. This study aimed to establish the incidence of shigellosis in an urban zone of Buenos Aires, Argentina, by examining the type of Shigella and living conditions associated with this infection. METHODOLOGY: Between January 2009 and December 2010 we analyzed shigellosis in children admitted to the public health service with bloody diarrhoea from La Plata, the capital of Buenos Aires, Argentina. A total of 372 children under 15 years old with Shigella present in their stool samples were admitted to the study. Variables studied were patient age, type of Shigella, family economic status, and access to sewage services and safe drinking water. RESULTS: Shigella flexneri was found to be present in 66.8% of the cases. Incidence was 187 cases/year/100,000 children under 15 years old. Cases were mainly observed during the summer (38.5%) in the population of under 5 years old (69.1% of all cases). The risk of shigellosis increased 12 times in those children who lacked safe drinking water and this risk increased 1.5 times in the population without sewage services. Fewer cases of shigellosis were noted in downtown areas, while hot spots were identified in the suburbs. Treating one case of shigellosis has a local cost of US $976 while assuring safe drinking water and sewage services for one family costs US $634.  CONCLUSION: Incidence of shigellosis in urban areas is associated with quality of water and sewage services. Policies aimed at providing education and improving public utilities networks can help to reduce the incidence of shigellosis.

Identification of a new protective antigen of Bordetella pertussis.

Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.

Evaluación de los sistemas comerciales automatizados VITEK 2 y API 20NE para la identificación de organismos del complejo Burkholderia cepacia aislados de muestras clínicas / Evaluation of commercial systems VITEK 2 and API 20NE for identification of Burkholderia cepacia complex bacteria from clinical samples

Las especies del complejo Burkholderia cepacia (CBC) son capaces de causar infecciones crónicas del tracto respiratorio en pacientes con fibrosis quística y en otros individuos inmunocomprometidos. La mayoría de estas especies exhiben alta resistencia a la terapia antibiótica, lo que genera la necesidad de una detección rápida y precisa para poder implementar estrategias de control adecuadas. En este trabajo se utilizó la técnica de reacción en cadena de la polimerasa (PCR) para amplificar el gen recA (PCR-recA), con el fin de identificar microorganismos pertenecientes al CBC. Con este método molecular como referencia, se evaluó la sensibilidad (S) y la especificidad (E) de dos sistemas de identificación comerciales automatizados, VITEK 2 y API 20NE (bioMérieux®), así como también el valor de las pruebas bioquímicas manuales más representativas para la identificación de estos microorganismos. El método VITEK 2 presentó una S del 71,1 % y una E del 100 %; para el método API 20NE, estos valores fueron 69,7 % y 90,2 %, respectivamente. En cuanto a las pruebas fenotípicas manuales, los resultados obtenidos fueron más heterogéneos, lo que posiblemente se deba a que estas bacterias podrían sufrir presión selectiva para sobrevivir en pacientes crónicos y perder factores fenotípicos característicos. La técnica de PCR-recA resultó de fácil implementación, por lo que cabe considerar a esta técnica de identificación como una opción viable, aun en laboratorios de diagnóstico clínico de mediana complejidad.

Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as wel as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this wok, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.

[Evaluation of commercial systems VITEK 2 and API 20NE for identification of Burkholderia cepacia complex bacteria from clinical samples]. / Evaluación de los sistemas comerciales automatizados VITEK 2 y API 20NE para la identificación de organismos del complejo Burkholderia cepacia aislados de muestras clínicas.

Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as well as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this work, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Informe breve: evaluación del lavado de manos en un equipo de salud / Brief report: evaluating hand/washing in a health - caring group

El lavado de manos previo y posterior a la atención de cada paciente es el mejor método para prevenir la transmisión de microorganismos de un paciente a otro y así disminuir la infección hospitalaria. Se evaluó el conocimiento de la norma de lavado de manos y se observó el cumplimiento de la misma al iniciar la investigación y luego la intervención (concientización). La muestra estuvo integrada por el personal de salud de una sala del sector de Clínica II del Hospital de Niños de La Plata. Se realizó el cultivo de los pulpejos de los dedos sobre agar sangre antes y después de lavarse las manos a todo el personal que se presto voluntariamente; con estos cultivos se realizaron talleres de mostración. El porcentaje de conocimiento de la norma fue alta, el cumplimiento de lavado de mano pasó de 38,7% a 61,3% luego de la intervención. La educación continua es la manera de mejorar el cumplimiento de la norma del lavado de manos y así contribuir a disminuir la infección hospitalaria.

Informe breve: evaluación del lavado de manos en un equipo de salud / Brief report: evaluating hand/washing in a health - caring group

El lavado de manos previo y posterior a la atención de cada paciente es el mejor método para prevenir la transmisión de microorganismos de un paciente a otro y así disminuir la infección hospitalaria. Se evaluó el conocimiento de la norma de lavado de manos y se observó el cumplimiento de la misma al iniciar la investigación y luego la intervención (concientización). La muestra estuvo integrada por el personal de salud de una sala del sector de Clínica II del Hospital de Niños de La Plata. Se realizó el cultivo de los pulpejos de los dedos sobre agar sangre antes y después de lavarse las manos a todo el personal que se presto voluntariamente; con estos cultivos se realizaron talleres de mostración. El porcentaje de conocimiento de la norma fue alta, el cumplimiento de lavado de mano pasó de 38,7% a 61,3% luego de la intervención. La educación continua es la manera de mejorar el cumplimiento de la norma del lavado de manos y así contribuir a disminuir la infección hospitalaria.

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients.

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.

Analisis de la resistencia a los antibacterianos en los aislamientos de pacientes internados en el Hospital de Niños Superiora Sor María Ludovica durante el año 2005 / An analysis of the resistance to antibacterials in germs isolated from in patients at the Superiora Sor María Ludovica children's Hospital, 2005

El tratamiento empírico con antibióticos adecuados requiere del conocimiento de la epidemiología bacteriana local. Para ello se realizo un estudio retrospectivo de la sensibilidad a los antibióticos de los microorganismos recuperados durante 2005 de los pacientes internados.Los microorganismos mas frecuentemente aislados fueron: Staphylococcus aureus (S. aureus) (24.1 %), Escherichia coli (E. coli) (15.8 %), Klebsiella pneumoniae (K. Pneumoniae) (12.4%) y Pseudomonas aeruginosa (P. aeruginosa) (10.1 %). En S. aureus la resistencia a meticilina supero el 40 %, con alto porcentaje de resistencia acompañante a otros antibióticos y total sensibilidad a TMS. La resistencia en E. coli y K. pneumoniae a cefalosporinas de tercera generación llego a niveles alarmantes (34.1% y 60%, respectivamente). P. aeruginosa mostró altos niveles para ceftazidima, carbapenemes y gentamicina (entre el 10% y 20%), mantenido total sensibilidad al colistin.Resulta evidente que el control de la infección Hospitalaria y el uso racional de los antimicrobianos son las únicas alternativas para impedir el incremento de la residencia de las bacterias.

Analisis de la resistencia a los antibacterianos en los aislamientos de pacientes internados en el Hospital de Niños Superiora Sor María Ludovica durante el año 2005 / An analysis of the resistance to antibacterials in germs isolated from in patients at the Superiora Sor María Ludovica childrens Hospital, 2005

El tratamiento empírico con antibióticos adecuados requiere del conocimiento de la epidemiología bacteriana local. Para ello se realizo un estudio retrospectivo de la sensibilidad a los antibióticos de los microorganismos recuperados durante 2005 de los pacientes internados.Los microorganismos mas frecuentemente aislados fueron: Staphylococcus aureus (S. aureus) (24.1 %), Escherichia coli (E. coli) (15.8 %), Klebsiella pneumoniae (K. Pneumoniae) (12.4%) y Pseudomonas aeruginosa (P. aeruginosa) (10.1 %). En S. aureus la resistencia a meticilina supero el 40 %, con alto porcentaje de resistencia acompañante a otros antibióticos y total sensibilidad a TMS. La resistencia en E. coli y K. pneumoniae a cefalosporinas de tercera generación llego a niveles alarmantes (34.1% y 60%, respectivamente). P. aeruginosa mostró altos niveles para ceftazidima, carbapenemes y gentamicina (entre el 10% y 20%), mantenido total sensibilidad al colistin.Resulta evidente que el control de la infección Hospitalaria y el uso racional de los antimicrobianos son las únicas alternativas para impedir el incremento de la residencia de las bacterias.

Pertussis in Argentina and France.

We have studied the epidemiology of pertussis in two countries, Argentina and France, which have similar histories of long-term mass vaccination with a whole-cell vaccine. Both countries display a comparable epidemiology, with an increase of the incidence of the disease in non-vaccinated newborns. We used pulsed-field gel electrophoresis (PFGE) analysis and genotyping to compare Bordetella pertussis clinical isolates recovered in Argentina and France in 2001-2004. The majority of the isolates harbors prn2 allele and belongs to PFGE IVbeta group. Isolates were found to be very similar genetically suggesting a common evolution of the disease in these two countries using the same vaccine.

Stenotrophomonas maltophilia, bacilo gram negativo no fermentador aislados de muestras clínicas de pacientes pediátricos / Stenotrophomas maltophilia gram negativ rod isloted ferment with nosocomial of patients infants

Stenotrophomonas maltophilia (SMA) es un bacilo gram negativo no fermentador que causa infecciones en pacientes con defensas disminuidas y/o instrumentados. Se realizó un análisis retrospectivo de los aislamientos obtenidos en el año 2003 en el laboratorio de Bacteriología del Hospital de Niños Sor María Ludovica. Se obtuvieron 64 aislamientos de los cuales 36 fueron de origen respiratorio y 24 de hemocultivos. Tanto su resistencia a carbapenemes como su sensibilidad a TMS son orientadores para su diagnóstico microbiológico. Este perfil asociado a resistencia a otros antibióticos suele plantear problemas terapéuticos severos

Stenotrophomonas maltophilia, bacilo gram negativo no fermentador aislados de muestras clínicas de pacientes pediátricos / Stenotrophomas maltophilia gram negativ rod isloted ferment with nosocomial of patients infants

Stenotrophomonas maltophilia (SMA) es un bacilo gram negativo no fermentador que causa infecciones en pacientes con defensas disminuidas y/o instrumentados. Se realizó un análisis retrospectivo de los aislamientos obtenidos en el año 2003 en el laboratorio de Bacteriología del Hospital de Niños Sor María Ludovica. Se obtuvieron 64 aislamientos de los cuales 36 fueron de origen respiratorio y 24 de hemocultivos. Tanto su resistencia a carbapenemes como su sensibilidad a TMS son orientadores para su diagnóstico microbiológico. Este perfil asociado a resistencia a otros antibióticos suele plantear problemas terapéuticos severos

Bacterias aisladas de exudados óticos en pacientes pediátricos / Bacteria isolated from otic exudates of pediatrics patients

En el período comprendido entre el 1 de junio de 1989 y el 30 de diciembre de 1992, se estudiaron 290 exudados óticos en pacientes, cuyas edades oscilaban entre 2 meses y 16 años. De acuerdo con los microorganismos aislados, se dividieron en tres grupos: grupo 1 :microorganismos provenientes de orofaringe (90 casos); grupo 2: bacilos Gram negativos no fermentadores y fermentadores de glucosa (166 casos) y grupo 3: flora mixta (38 casos). En el grupo 1 se encontraron los niños con diagnóstico de otitis media aguda con efusión y los microorganismos más halados fueron Haemophilus influenzae, Streptococcus pneumoniae y Streptococcus pyogenes. En el grupo 2 los microorganismos más hallados fueron Pseudomonas aeruginosa y proteus mirabilis solos o acompañados. En el grupo 3 se aisló flora mixta (se entiende cocos Gram positivos, bacilos Gram positivos, Bacilos Gram negativos por lo menos tres tipos de microorganismos