Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Antibacterial activity of the Antarctic bacterium Janthinobacterium sp: SMN 33. 6 against multi-resistant Gram-negative bacteria

Background The increment of resistant strains to commonly used antibiotics in clinical practices places in evidence the urgent need to search for new compounds with antibacterial activity. The adaptations that Antarctic microorganisms have developed, due to the extreme environment that they inhabit, promote them as a potential new source of active compounds for the control of microorganisms causing infections associated with health care. The aim of this study was to evaluate the antibacterial activity of an ethanol extract of the Antarctic bacterium Janthinobacterium sp., strain SMN 33.6, against nosocomial multi-resistant Gram-negative bacteria. Results Inhibitory activity against human Gram-negative bacterial pathogens, with concentrations that varied between 0.5 and 16 µg ml- 1, was demonstrated. Conclusions The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

The use of PCR-RFLP to genetically distinguish the morphologically close species: Ceriodaphnia dubia Richard, 1894 and Ceriodaphnia silvestrii Daday, 1902 (Crustacea Cladocera) / Utilização do PCR-RFLP para diferenciar geneticamente as espécies Ceriodaphnia dubia Richard, 1894 e Ceriodaphnia silvestrii Daday, 1902 (Crustacea Cladocera)

The cladocerans are important components of planktonic and benthic freshwater and good indicators of the trophic state of water bodies. The morphological taxonomy of many species of Cladocera is considered complex with minor differences separating some species. Nowadays, molecular techniques provide a powerful tool to identify and classify different taxonomical levels, using mainly ribosomal RNA genes (rRNA) as molecular markers. In the present work we performed PCR-RFLP to separate Ceriodaphnia dubia, an exotic species in Brazil and the native species Ceriodaphnia silvestrii, widely distributed in Brazilian freshwater. The RFLP analysis of the ITS1-5.8S-ITS2 region of rRNA genes showed to be different between C. dubia and C. silvestrii when using enzymes EcoRI, ApaI and SalI. Thus, the ITS1-5.8S-ITS2 region proved to be a useful molecular marker to differentiate the studied Ceriodaphnia species, which makes the task easier of telling apart species that are morphologically very similar. Also, this methodology might be interesting in determining the distribution of the exotic species C. dubia in Brazilian freshwaters, particularly in cases when C. dubia occurs in the absence of C. silvestrii, a particularly difficult task for ecologists who are not taxonomy specialists.

Os cladóceros são considerados importantes componentes de comunidades bentônicas e planctônicas de água doce e bons indicadores do estado trófico da água. A taxonomia morfológica de muitas espécies de Cladocera é considerada complexa com pequenas diferenças que separam algumas espécies. Atualmente, as técnicas moleculares são consideradas uma ferramenta importante para identificar e classificar diferentes níveis taxonômicos, com a utilização, principalmente, de genes de rRNA como marcadores moleculares. No presente trabalho foi utilizada PCR-RFLP para diferenciar geneticamente Ceriodaphnia dubia, uma espécie exótica no Brasil, e a espécie nativa Ceriodaphnia silvestrii, amplamente distribuída em corpos d'água brasileiros. A análise por RFLP da região ITS1-5.8S-ITS2 dos genes de rRNA mostrou diferenças entre C. dubia e C. silvestrii para os sítios das enzimas de restrição EcoRI, ApaI e SalI. Dessa forma, a região ITS1-5.8S-ITS2 mostrou-se um marcador molecular útil para diferenciar as espécies de Ceriodaphnia estudadas, o que facilita a separação de espécies muito similares morfologicamente. Também, os resultados apresentados parecem ser interessantes na determinação da distribuição da espécie exótica C. dubia em corpos d'água brasileiros, principalmente nos casos onde C. dubia ocorre na ausência de C. silvestrii, uma tarefa difícil para ecologistas não especialistas em taxonomia.

Cytogenetic characterization of Rhomboplites aurorubens and Ocyurus chrysurus, two monotypic genera of Lutjaninae from Cubagua Island, Venezuela, with a review of the cytogenetics of Lutjanidae (Teleostei: Perciformes)

Lutjanidae, commonly known as snappers, includes 105 species, grouped in four subfamilies. In spite of the high number of species and of its worldwide distribution, the family has been little investigated and the phylogenetic relationships among some of its genera and species are still cause for debate. Only a small number of the species has been cytogenetically analysed. This study reports the first description of the karyotype of Rhomboplites aurorubens as well as data concerning the distribution of the constitutive heterochromatin and the location of the 18S rRNA and the 5S rRNA genes. Specimens of Ocyurus chrysurus from Venezuela were also investigated for the same cytogenetic features. Both species have a 48 uniarmed karyotype, but R. aurorubens has a single subtelocentric chromosome pair, the smallest of the chromosome complement, among the other acrocentric chromosomes. The C-positive heterochromatin is limited to the pericentromeric regions of all chromosomes. Both species show a single chromosome pair bearing the Nucleolus Organizer Regions, but NORs are differently located, in a terminal position on the short arms of the smallest chromosomes in R. aurorubens and in a paracentromeric position in a chromosome pair of large size in O. chrysurus. In O. chrysurus, the 5S rDNA gene cluster is located on a medium-sized chromosome pair, whereas in R. aurorubens it is syntenic with the 18S rDNA gene cluster on chromosome pair number 24. The obtained cytogenetic data, along with previous cytogenetic, morphological and molecular data for the family, reinforce the proposal to synonymize genus Ocyurus with Lutjanus. A review of Lutjanidae cytogenetics is also included.

Lutjanidae, comumente conhecidos como snappers, inclui 105 espécies, reunidas em quatro subfamílias. A despeito do grande número de espécies e de sua distribuição mundial, a família tem sido pouco estudada e as relações filogenéticas entre alguns de seus gêneros e espécies ainda é motivo de debates. Apenas um pequeno número de espécies foi citogeneticamente analisada. Esse estudo apresenta a primeira descrição do cariótipo de Rhomboplites aurorubens assim como dados relativos à distribuição de heterocromatina constitutiva e localização dos genes 18S rRNA e 5S rRNA. Espécimes de Ocyurus chrysurus da Venezuela foram também analisados quanto às mesmas características citogenéticas. Ambas as espécies têm cariótipos compostos de 48 cromossomos com um único braço, entretanto R. aurorubens tem um único par de cromossomos subtelocêntrico, o menor do complemento cromossômico, entre os outros cromossomos acrocêntricos. A heterocromatina C-positiva é limitada à região pericentromérica de todos os cromossomos. Ambas as species apresentam um único par com Regiões Organizadoras de Nucléolo, mas as RONs são localizadas em posições diferentes, em posição terminal no braço curto dos menores cromossomos de R. aurorubens e em posição paracentromérica no braço longo de um par de cromossomos grandes de O. chrysurus. Em O. chrysurus, os genes 5S rDNA estão localizados em um par de cromossomos de tamanho médio, enquanto em R. aurorubens eles são sintenicamente localizados com os genes 18S rDNA no par de cromossomos número 24. Os dados citogenéticos obtidos, junto com os dados morfológicos e moleculares disponíveis para a família reforçam a proposta de sinonimizar o gênero Ocyurus com Lutjanus. Uma revisão da citogenética dos Lutjanidae é também apresentada

Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl- Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.