Whole-Genome Analysis of Extensively Drug-Resistant Enterobacter hormaechei Isolated from a Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Currently, the Enterobacteriaceae species are responsible for a variety of serious infections and are already considered a global public health problem, especially in underdeveloped countries, where surveillance and monitoring programs are still scarce and limited. Analyses were performed on the complete genome of an extensively antibiotic-resistant strain of Enterobater hormaechei, which was isolated from a patient with non-Hodgkin's lymphoma, who had been admitted to a hospital in the city of Manaus, Brazil. METHODS: Phenotypical identification and susceptibility tests were performed in automated equipment. Total DNA extraction was performed using the PureLink genomic DNA mini-Kit. The genomic DNA library was prepared with Illumina Microbial Amplicon Prep and sequenced in the MiSeq Illumina Platform. The assembly of the whole-genome and individual analyses of specific resistance genes extracted were carried out using online tools and the Geneious Prime software. RESULTS: The analyses identified an extensively resistant ST90 clone of E. hormaechei carrying different genes, including blaCTX-M-15, blaGES-2, blaTEM-1A, blaACT-15, blaOXA-1 and blaNDM-1, [aac(3)-IIa, aac(6')-Ian, ant(2″)-Ia], [aac(6')-Ib-cr, (qnrB1)], dfrA25, sul1 and sul2, catB3, fosA, and qnrB, in addition to resistance to chlorhexidine, which is widely used in patient antisepsis. CONCLUSIONS: These findings highlight the need for actions to control and monitor these pathogens in the hospital environment.

Molecular epidemiology of methicillin resistant Staphylococcus species in healthcare workers of a blood bank in the Brazilian Amazon.

BACKGROUND: Healthcare workers are susceptible to colonization by multiresistant bacteria, which can increase the risk of outbreaks. METHODS: Samples were collected from the nasopharynx, hands, and lab coats of healthcare workers. The phenotypic identification was carried out using a VITEK®2 rapid test system. PCR tests for the mecA gene and the sequencing of the amplicons were performed. Staphylococcus epidermidis and Staphylococcus aureus phylogenies were reconstructed using the Bayesian inference. RESULTS: A total of 225 healthcare workers participated in this study. Of these, 21.3% were male and 78.7% female. S. epidermidis and S.aureus showed high levels of resistance to penicillin, ampicillin, erythromycin, tetracycline and cefoxitin. The prevalence of methicillin resistant S. aureus was 3.16% and methicillin resistant S. epidermidis was 100%. Multilocus sequence typing identified 23 new S. epidermidis sequence types, and one new allele and sequence type for S. aureus. The frequency of methicillin-resistant S. epidermidis in nursing and hemotherapy technicians as a percentage of the total number of healthcare workers was 5.8-3.1%, while the frequency of methicillin resistant S. aureus in hemotherapy technicians and biomedics, as a percentage of the total number of healthcare workers was 4.2-8.9%%. CONCLUSIONS: The healthcare workers at the city's blood bank, even when taking the necessary care with their hands, body and clothes, harbour methicillin-resistant S. aureus and S. epidermidis sequence types, which, as a potential source of multidrug resistant bacteria, can contribute to nosocomial infections among hematological patients.

New ST623 of Cryptococcus neoformans isolated from a patient with non-Hodgkin's lymphoma in the Brazilian Amazon.

BACKGROUND: Cryptococcosis is a disease of wide geographic distribution. It is most critical when it affects immunocompromised patients, with AIDS, tuberculosis or other diseases that require prolonged hospitalization. METHODS: This study described a case report, molecular epidemiology, the phylogenetic relationship, along with antifungal susceptibility test of a new ST 623 of C. neoformans isolated in a patient with non-Hodgkin's Lymphoma, from Manaus, Brazil. RESULTS: The new C. neoformans was susceptible to all antifungal drugs tested. Our results showed that ST623 new clone has no evident evolutionary proximity to any other ST of the VNI subtype group identified in Brazil. CONCLUSIONS: In the context of phylogenetic analysis, this new genotype belongs to VNI subtype, and subsequencing complete genome studies are necessary to better understand the phylogenetic relationships amongst STs in this group.

Molecular Characterization of Chryseobacterium indologenes with Multidrug Resistance in the Brazilian Amazon Region.

Chryseobacterium indologenes is an emerging nosocomial pathogen that produces IND-type chromosomal metallo-beta-lactamase. The phenotype and molecular aspects of two multidrug resistant C. indologenes strains and the analysis of the tertiary structure of the IND enzyme were studied. Identification of species and susceptibility tests were performed using the Vitek-2 compact. Chromosomal and plasmid DNA were extracted using PureLink™ Genomic DNA Mini Kit and PureLink Quick Plasmid Miniprep Kit, and the sequencing was performed using ABI 3130 genetic analyzer. Two strains were isolated and are registered as P-23 and P-113. Of the two, P-113 was sensitive to ciprofloxacin and cefepime only, whereas the P-23 showed reduced sensitivity to ceftazidime, ciprofloxacin, and tigecycline. The genetic analysis of both isolates identified the presence of the blaIND-like gene, with similarity to IND-3 and IND-8 alleles. The IND-3 identified in the P-133 sample presented a single mutation at position T355G, which corresponds to a nonsynonymous substitution of the amino acid at position 119 (Ser→Ala). The phylogenetic analysis of INDs showed lineages that are circulating in Asian and European countries. These results emphasize the need for effective preventive actions to avoid the dissemination of this type of pathogen in the hospital environment.

Role of peripheral blood minimum residual disease at day 8 of induction therapy in high-risk pediatric patients with acute lymphocytic leukemia.

Risk stratification and treatment intensification, based on minimal residual disease (MRD) mensurement, changed the prognosis of pediatric patients with acute lymphocytic leukemia (ALL). The main aim of this study was to investigate whether peripheral blood (PB) MRD measurement at day 8 (D8) could predict the risk stratification category determined by bone marrow (BM) MRD at day 15 (D15). The study was performed prospectively, in a cohort of 40 children with B-lineage ALL, adopting the protocol of the Brazilian Cooperative Group of the Treatment Childhood Leukemia (GBTLI-2009). MRD was detected by flow cytometry (FC) using a simplifed panel that can reliably identify MRD at early phases of induction therapy. Upon diagnosis, the proportion of low and high-risk patients, was 24:16 (60%:40%). The main result of our study demonstrated the potential of D8 MRD in anticipating of week the risk stratification of high-risk patients as determined by D15 BM MRD. In these patients D8 MRD level of 1% was able to segregate high risk fast responders from high risk slow responders (p = 0.0097). This result could represent an opportunity for early treatment intensification, as already performed in some protocols.

Molecular epidemiology of ß-lactamase-producing Neisseria gonorrhoeae strains in Manaus, AM, Brazil.

We report new sequence types of 14 penicillinase-producing Neisseria gonorrhoeae, isolated from sexually transmitted disease clinic attendees in Manaus, Brazil. They were characterized by WI/WII/WIII groups, susceptibility testing and Multi-Antigen Sequencing Typing/Mutilocus Sequence Typing protocols. Twelve were classified as WII/III and 2 as WI and were presented resistance to penicillin and tetracycline. New alleles for por and AroE genes and novel sequence types were identified, revealing molecular characteristics not described previously. ST1590 is the common ancestor after eBURST analysis, and these findings represent an important contribution of molecular epidemiology approach in gonococci's research in Amazonas.

Novel methicillin-resistant coagulase-negative Staphylococcus clone isolated from patients with haematological diseases at the Blood Bank Centre of Amazon, Brazil.

Methicillin-resistant Staphylococcus remains a severe public health problem worldwide. This research was intended to identify the presence of methicillin-resistant coagulase-negative staphylococci clones and their staphylococcal cassette chromosome mec (SCCmec)-type isolate from patients with haematologic diseases presenting bacterial infections who were treated at the Blood Bank of the state of Amazonas in Brazil. Phenotypic and genotypic tests, such as SCCmec types and multilocus sequence typing (MLST), were developed to detect and characterise methicillin-resistant isolates. A total of 26 Gram-positive bacteria were isolated, such as: Staphylococcus epidermidis (8/27), Staphylococcus intermedius (4/27) and Staphylococcus aureus (4/27). Ten methicillin-resistant staphylococcal isolates were identified. MLST revealed three different sequence types: S. aureus ST243, S. epidermidis ST2 and a new clone of S. epidermidis, ST365. These findings reinforce the potential of dissemination presented by multi-resistant Staphylococcus and they suggest the introduction of monitoring actions to reduce the spread of pathogenic clonal lineages of S. aureus and S. epidermidis to avoid hospital infections and mortality risks.

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region.

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 µg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.

Extended-spectrum beta-lactamase-producing bacteria isolated from hematologic patients in Manaus, State of Amazonas, Brazil

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17). All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried blaTEM, blaCTX-M, blaOXA, blaSHV genes, (1/12) blaTEM gene and (3/12) blaTEM, blaCTX-M, blaOXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the blaTEM, blaSHV, blaCTX-M and blaOXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 µg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.

Extended-spectrum beta-lactamase-producing bacteria isolated from hematologic patients in Manaus, State of Amazonas, Brazil.

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17). All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried bla TEM, bla CTX-M, bla OXA , bla SHV genes, (1/12) bla TEM gene and (3/12) bla TEM, bla CTX-M, bla OXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the bla TEM, bla SHV, bla CTX-M and bla OXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.

Prevalência de Staphylococcus aureus meticilina resistente (MRSA) em pacientes atendidos em Ambulatório de Dermatologia Geral em Manaus-Amazonas / Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in patients attended at a General Dermatology Ambulatory in Manaus, Amazon State, Brazil

O Staphylococcus aureus meticilina resistente (MRSA), de origem hospitalar ou comunitária, tem sido relatado como um dos principais problemas graves de saúde por apresentar resistência aos antibióticos beta lactâmicos e a outros como macrolídeos, lincosaminas, clindamicina, aminoglicosídeos, tetracilinas e sulfas. Diante desse problema, buscou-se identificar a prevalência de MRSA nos casos registrados pela Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta em Manaus, Amazonas, Brasil. Os dados foram obtidos do livro de registro de resultados de exames de cultura geral e antibiograma do laboratório de bacteriologia clínica, no período de setembro de 1998 a outubro de 2007. Foram realizados 1.494 exames em 1.500 amostras , das quais 783 (52,2por cento) eram de pacientes do gênero feminino e 717 (47,8por cento) do masculino. Das 239 amostras de S. aureus isoladas, 232 foram submetidas ao testes de suscetibilidade; dessas 44,0por cento (102/232) apresentaram resistência à oxalina, portanto a taxa de prevalência de MRSA foi de 15,5por cento nas amostras estudadas. O fato de a resistência à oxalina/meticilina inviabilizar a utilização de vários antibióticos justifica a preocupação com as taxas de prevalência ou incidência de infeccções causadas por estafilococos MRSA, ante as possíveis consequências para a terapia dos processos infecciosos, muitas vezes complicados e de elevado potencial de morbidade e mortalidade tanto em adultos quanto em crianças. Ações de vigilância e controle são necessárias para minimizar os riscos de infecção ou colonização dos profissionais de saúde ou pacientes e assegurar a continuidade das pesquisas. Desse modo, será possível caracterizar genotipicamente a origem hospitalar ou comunitária dessas cepas.

Infecção pulmonar por Strongyloides stercoralis em paciente tratado com imunosupressores / Case report: Pulmonary infection by Strongyloides stercoralis in a patient treated with immunosupressors

Strongyloides stercoralis é um parasito predominantemente intestinal. Nos casos graves, pode evoluir para a forma disseminada, principalmente se houver utilização prolongada de glicocorticóides, medicamentos imunodepressores ou radioterapia. Relata-se, neste estudo, o caso de P.F.C.S., do sexo feminino, 16 anos, proveniente da comunidade Menino de Deus na ilha de Pratari, município de Manacapuru, estado do Amazonas, Brasil, com diagnóstico de anemia hemolítica auto-imune desde agosto de 2005. Nenhum dos exames parasitológicos de fezes apresentou positividade para Strongyloides stercoralis, tendo sido detectado apenas no exame de escarro. Foi tratada com tiabendazol, via oral, sem resultado favorável e evoluiu para óbito. Ficou evidenciada a importância da realização de exames complementares, como o parasitológico de fezes, antes da utilização de medicamentos indicados para o tratamento de anemia hemolítica.

Anticorpos anti-eritrocitários em pacientes com Coombs direto positivo infectados com malária por P. vivax e P. falciparum / Anti-erithrocytes antibodies in patients with positive direct Coombs infected with P. vivax and P. falciparum malaria

Para se determinar a prevalência de anticorpos anti-eritrocitários de grupo sanguíneo foram analizadas 247 amostras de sangue de pacientes com malária vivax e falciparum com teste de Coombs direto positivo atendidos na Fundação de Medicina Tropical Manaus-Amazonas no período entre setembro/99 a março/2000.Realizaram-se os testes laboratoriais de Coombs direto, dosagens de hemoglobina, bilirrubina e eletroforese de proteínas.Das amostras testadas, 13,3 apresentaram Coombs direto positivo, sendo o anticorpo da classe IgG (33,3) o mais freqüente. Dos pacientes com malária vivax e Coombs direto positivo, 17 apresentaram anemia possivelmente devido a hemólise por auto-imunidade com o envolvimento da gamaglobulina IgG.Não foram detectados anticorposcontra antígenos de grupos sanguíneos nem aloanticorpos séricos.Torna-se necessário a realização de outras pesquisas para avaliação da existência de associação entre a positividade do Coombs direto e anemia ou se a mesma interfere ou não com o curso da doença.