Evolution of Drug Resistance: Insight on TEM β-Lactamases Structure and Activity and β-Lactam Antibiotics

Since the discovery of the first penicillin bacterial resistance to β-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of β-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. β-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the β-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM β-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

A 3-YEAR FOLLOW-UP OF A BRAZILIAN AIDS PATIENT WITH PROTRACTED DIARRHEA CAUSED BY Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most prevalent microsporidian parasite that causes gastrointestinal infection in persons with AIDS. Microsporidia are increasingly recognized as important opportunistic pathogens all over the world but in Brazil only few cases have been reported due either to the non awareness of the clinical presentation of the disease or to difficulties in the laboratory diagnosis. We report a 3-year follow-up of a Brazilian HIV-positive patient in whom microsporidial spores were detected in stools and were identified as E. bieneusi using electron microscopy and PCR. The patient presented with chronic diarrhea, CD4 T-lymphocytes count below 100/mm3 and microsporidial spores were consistently detected in stools. Albendazole was given to the patient in several occasions with transient relief of the diarrhea, which reappeared as soon as the drug was discontinued. Nevertheless, a diarrhea-free period with weight gain up to 18 Kg occurred when a combination of nucleoside and protease inhibitors was initiated as part of the antiviral treatment.

INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index). Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

Cyclospora cayetanensis in sputum and stool samples

We report the observation of acid-fast Cyclospora cayetanensis oocysts in a sputum sample. The patient, a 60 year-old, HIV negative man, was successfully treated for pulmonary tuberculosis during 1997. On February 1998, he was admitted to our center due to loss of weight, cough with purulent expectoration, dysphonia and a radiological picture of pulmonary fibrosis. Bacilloscopic study of sputum (negative for acid-fast bacilli) stained with Ziehl-Neelsen technique showed large (8-10 µm) spherical, acid-fast Cyclospora cayetanensis oocysts. No other pathogens were isolated on cultures from this sample or from laryngeal biopsy. Serial parasitologic studies showed C. cayetanensis and also eggs of Trichuris trichiura, Ascaris lumbricoides and Hymenolepis nana and of Entamoeba coli cysts. The patient lives in the outskirts of Buenos Aires in a brick-made house with potable water and works as builder of sewers. He travelled in several occasions to the rural area of province of Tucumán which has poor sanitary conditions. C. cayetanensis is an emergent agent of diarrhea and as far as we know this is the first time the parasite is observed in respiratory samples.

Bacterial inter-species communication mediated by the autoinducer-2 signal

Dissertation presented to obtain the Ph.D degree in Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Instituto Gulbenkian de Ciência.

Etiology of acute diarrhea among children in Ribeirão Preto-SP, Brazil

To study the main enteropathogens causing diarrhea in the region of Ribeirão Preto regarding serogroups and serotypes, the feces of 1836 children under 10 years old, from both sexes, attack of acute gastroenteritis, were analysed during a period of 4 years in Adolfo Lutz Institute - Ribeirão Preto, SP. The pathogens identified by standard methods were the following: Escherichia coli, Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., and Cryptosporidium spp. Positive samples were 22.8% (419) with 1.7% association of pathogens. Larger isolates were mainly from children 0 to 11 months old. Enteropathogenic E. coli (EPEC) was most frequent (8.7%) with predominance of serogroup O119 (40.2%), followed by Shigella (6.2%), 63.6% of which S. sonnei.

Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil

The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

Importance of hand germ contamination in health-care workers as possible carriers of nosocomial infections

The importance of hands in the transmission of nosocomial infection has been world wide admitted. However, it is difficult to induce this behavior in health-care workers. The aim of the present work was to point out the importance of hand bacteria colonization, the influence of hand washing and of patient physical examination. One hundred health-care workers were randomly divided in two groups: Group A without hand washing previous to patient physical examination or handling (PPE); group B with hand washing previous to PPE. Direct fingerprint samples in Columbia agar before and after PPE were obtained. The colonies were counted and identified by conventional techniques, and antibiograms according to NCCLS were performed. Before PPE group A participants showed a high number of bacteria regarding group B participants (73.9 Vs 20.7; p < 0.001); 44 out of 50 participants were carriers of potentially pathogen bacteria. No group B participants were carriers of potential pathogen bacteria before PPE. The latter group showed an increase in number of bacteria after PPE (20.7 CFU (before) Vs 115.9 CFU (after); p < 0.001). Sixteen group B participants were contaminated after PPE with potential pathogens such as S. aureus (50% of them meticillin resistant); Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis, half of them multiresistant. We can conclude on the importance of these results to implement educational programs and to provide the health-care workers with the proper commodities to fulfill this practice.

The epidemiology of acute viral gastroenteritis in hospitalized children in Cordoba city, Argentina: an insight of disease burden

Information concerning the disease burden of viral gastroenteritis has important implications for the use and monitoring the impact of public health policies. The present study, carried out in Córdoba city, Argentina, documents the epidemiology of severe viral diarrhea as well as the burden of viral gastrointestinal disease in the hospital children admission. A total of 133 stools were collected from hospitalized children (Town Childhood Hospital) suffering from acute diarrhea and studied for the presence of Group A rotavirus, astrovirus and adenovirus 40/41 by enzyme-immuno assay, between November 1997 and October 1998. Enteric viruses accounted for 42.1% of the total diarrheal cases analyzed. Group A rotaviruses, astroviruses, adenoviruses 40/41 and mixed infections were found in 35.3, 4.5, 1.5, and 0.8% studied specimens respectively. We estimated that 1 in 27 children in the 0-35 month-old cohort/range would be annually hospitalized for a viral gastroenteritis illness. The major impact on viral diarrhea lies on rotaviral infection, accouting for 84.0% of the viral diarrheal cases analyzed and for approximately one third of severe diarrheas requiring hospital admission in Córdoba City, Argentina.

Group B streptococcal neonatal infections in Rio Grande do Sul, Brazil

Group B Streptococcus is the most common pathogen found in neonatal sepsis in North America. OBJECTIVES: We describe 15 cases of neonatal infections by Group B Streptococcus (Streptococcus agalactiae) at a Neonatal Intensive Care Unit of a public and teaching hospital. METHODS: We conducted a study at Hospital de Clínicas de Porto Alegre, from January 1st, 1996 to June 30, 1999. Diagnosis of neonatal infection was established according to the findings of Group B Streptococcus in blood culture associated with alterations resembling sepsis on the basis of clinical picture and laboratory findings. RESULTS: Fifteen cases of neonatal infections by Group B Streptococcus were detected. Eleven cases consisted of early-onset sepsis, 2 cases of occult bacteremia and 2 cases of late-onset sepsis. Eight cases had septic shock (53%), 8 cases had pneumonia (53%), and 4 cases had meningitis (27%). Fourteen cases were diagnosed from a positive blood culture, and 1 case from evidence of these bacteria in pulmonary anatomopathological examination. Thirteen cases (87%) were diagnosed before 72 hours of life. We had 3 deaths (20%), and 3 cases of meningitis developing neurological deficits. CONCLUSIONS: Streptococcus Group B is one of the most important pathogens in the etiology of early-onset neonatal sepsis at our hospital, with high mortality and morbidity. However, we do not know the incidence of GBS neonatal infections at other hospitals. More data are needed to establish a basis for trials of different strategies to reduce these infections.

Prevalence of putative periodontopathogens from periodontal patients and healthy subjects in São Paulo, SP, Brazil

A. actinomycetemcomitans, B. forsythus, P. gingivalis, C. rectus, E. corrodens, P. intermedia, F. nucleatum, and T. denticola were identified from subgingival plaque from 50 periodontal patients and 50 healthy subjects. PCR products from each species showed a specific band and could be used to identify periodontal organisms from clinical specimens. Identical negative or positive results between PCR and culture occurred in 66% (A. actinomycetemcomitans) to 93% (F. nucleatum) of the samples. PCR detection odds ratio values for A. actinomycetemcomitans, B. forsythus, C. rectus, E. corrodens, P. intermedia, and T. denticola were significantly associated with disease having a higher OR values for B. forsythus (2.97, 95% CI 1.88 - 4.70). Cultures showed that A. actinomycetemcomitans, B. forsythus and P. intermedia were associated with periodontitis, however, P. gingivalis, C. rectus, E. corrodens and F. nucleatum were not significantly associated with the disease.

Isolation of Salmonella enterica and serologic reactivity to Leptospira interrogans in opossums (Didelphis virginiana) from Yucatán, México

The presence of Salmonella enterica and serologic evidence of infection by Leptospira interrogans, were detected in the opossum Didelphis virginiana in a semi-urban locality of the Yucatán State, México. Ninety-one opossums were captured during the period April 1996 and May 1998. From a total of 17 feces samples, four Salmonella enterica subsp. enterica serotypes (Sandiego, Newport, Anatum, and Minnesota), and one Salmonella enterica subsp. arizonae serovar O44:Z4,Z23:- were isolated. Some opossums presented mixed infections. From 81 sera samples, four (4.9%) were positive to antibodies to Leptospira serovars pomona and wolfii. Both animals infected with Salmonella enterica and those serologically positive to Leptospira interrogans were captured in peridomestic habitat. Opossums infected with Salmonella enterica, were captured in dry season, and those seropositive to Leptospira interrogans during the rainy season. The implications of infection and reactivity of these zoonotic pathogens in D. virginiana in the Yucatan state are briefly discussed.

Immune response and tissue cytoprotection: two sides of the same coin in immunopathology

Dissertation presented to obtain the Ph.D degree in Biochemistry

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Update on the population structure of MRSA causing infection in a central hospital and in healthcare centers in Portugal

RESUMO:Staphylococcus aureus é um dos principais agentes patogénicos humanos, sendo frequentemente associado a infecções nosocomiais e infecções na comunidade. A prevalência de S. aureus resistentes à meticilina (MRSA) em hospitais portugueses é uma das mais elevadas da Europa e tem sido caracterizada extensivamente; contrariamente, a prevalência e epidemiologia de MRSA na comunidade em Portugal não tem sido devidamente seguida. Com o objectivo de compreender as causas possíveis do aumento na frequência de MRSA num dos maiores hospitais centrais portugueses (HSM) ao longo de 17 anos, isolados de MRSA recolhidos em 1993 (n=54) e 2010 (n=180) de pus, sangue e urina foram analisados por PFGE, MLST, tipagem do spa e tipagem de SCCmec. Os resultados mostraram que ocorreu uma mudança global nos tipos clonais predominantes, onde o clone ST22-IVh substituiu os clones, ST239-IIIvar e ST247-I, representando mais de 70% da população actual. Além disso, entre 1993 e 2010 verificou-se um aumento na diversidade genética dos tipos clonais de MRSA. Para determinar a frequência e a natureza clonal de MRSA e S. aureus sensíveis à meticilina (MSSA) isolados de infecções de pele e tecidos moles (SSTI) em pessoas que frequentam centros de saúde em Portugal, 73 amostras foram recolhidas em nove centros de saúde (Rede Médicos Sentinela). Isolou-se um total de 40 S. aureus (55%), dos quais 17,5% eram MRSA. Os isolados de MRSA pertenciam aos clones ST22-IVh (n=4), ST5-IVc (n=2) e ST105-II (n=1), que foram descritos neste estudo como sendo clones de origem hospitalar. Os nossos resultados sugerem que o aumento da frequência de MRSA no HSM pode estar associado à emergência de um clone de MRSA com maior capacidade epidémica. Além disso, verificámos que a principal causa de SSTI em pessoas que frequentam centros de saúde em Portugal são MRSA de origem hospitalar e não MRSA associados à comunidade.------ABSTRACT: Staphylococcus aureus is one of the most important human pathogens, being a major cause of infections worldwide both in the hospital and in the community. In Portugal, the prevalence of methicillin resistant S. aureus (MRSA) in hospitals is one of the highest in Europe and has been characterized extensively; contrarily the prevalence and epidemiology of MRSA in the community has not been followed in a meaningful way. To understand the epidemiological events that could explain a steep increase in MRSA frequency in a major Portuguese central hospital (HSM) within a 17 year period, two MRSA collections recovered in 1993 (n=54) and 2010 (n=180) from pus, blood and urine were analyzed by PFGE, MLST, spa and SCCmec typing. The results showed that a major clonal shift occurred, wherein ST22-IVh clone has replaced the previous ST239-IIIvar and ST247-I clones and accounts for more than 70% of the present population. Moreover, an increase in genetic diversity of MRSA clonal types was observed between the two study periods. With the aim of determining the frequency and clonal nature of MRSA and methicillin-susceptible S. aureus (MSSA) causing skin and soft tissue infections (SSTI) in patients attending healthcare centers in Portugal, 73 samples were collected from nine healthcare centers (Medicos Sentinela Network). A total of 40 S. aureus were isolated, accounting for 55% of the SSTI, of which 17.5% were MRSA. MRSA isolates belonged to ST22-IVh (n=4), ST5-IVc (n=2) and ST105-II (n=1) that have also been described in the hospital in an equivalent period. Our results suggest that the increase in MRSA frequency in HSM may be associated to the emergence of a MRSA clone with higher epidemic potential. Moreover, we propose that the spillover of MRSA from the hospital rather than community-associated-MRSA was the main cause of SSTI in persons attending healthcare centers in Portugal.