Miliane Moreira Soares de Souza

Mapeamento do perfil de resistência e detecção do gene mecA em Staphylococcus aureus e Staphylococcus intermedius oxacilina-resistentes isolados de espécies humanas e animais

Antimicrobial resistant Staphylococcus species represent an important cosmopolitan problem, and its spreading control is a significative challenge. Resistance pattern of Staphylococcus aureus and Staphylococcus intermedius species isolated from animals and humans clinical samples to different antibiotics was evaluated through disk diffusion method, where ampicillin and penicillin presented the highest level of resistance. The evaluation of the resistance to oxacillin, due to the heterogeneity of the response of the studied genus was carried out through the following tests: modified agar diffusion, agar screen and microdilution, and further correlation with the detection of mecA gene in samples that showed resistance in at least one of the susceptibility tests used. The correlation between the results obtained from phenotypic methods and the detection of resistance gene, considered as a reference method, was used in order to validate its sensitivity. Eighty clinical staphylococcal isolates (29 human and 51 animal isolates) were evaluated, 28 were oxacillin-resistant, mecA gene being detected in 12 samples. Susceptibility assessment tests to oxacillin presented above 50% of specificity, disk diffusion and agar screen being the most sensitive one, while modified disk diffusion presented the lowest sensibility rate. Ampicillin and penicillin presented the highest level of resistance.

Aerobic bacterial microbiota in stomoxys calcitrans: preliminary studies in Brazil

The stable fly, Stomoxys calcitrans, has a great importance in medical and veterinary health due to its feeding and reproductive habits, which can disseminate some pathogenic agents among hosts even at long distances. This study aimed to describe, for the first time in Brazil, the bacterial microbiota in segments of the stable fly. Bacterial species were isolated from three different segments (cuticle, mouth parts and abdominal alimentary tract) of the stable fly. Twenty dairy farms were visited in four municipalities: Barra Mansa, Quatis, Resende and Rio Claro in the State of Rio de Janeiro in order to collect 20 flies in each site. Dissection of the flies and procedures of isolation and identification of bacterial species were performed. A total of 161 colonies of 33 distinct species were isolated, such as Escherichia coli, Staphylococcus aureus, and S. intermedius. Stomoxys calcitrans may harbor bacterial agents on their cuticle, mouth parts and abdominal alimentary tract and these bacteria may be pathogenic to their hosts.

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina

Mastitis is an inflammation of one or more mammary glands caused mainly by bacteria, among which the genus Staphylococcus plays an important role. Bacteria belonging to this genus are known to express virulence factors which allow their persistence and spread in the host. This study aimed to evaluate the phenotypic and genotypic aspects of virulence factors in Staphylococci spp. isolates from bovine mastitis clinical cases. A total of 272 milk samples from 8 farms in the South-Fluminense region of Rio de Janeiro were analyzed. The samples underwent conventional bacterial identification, yielding 250 Staphylococci spp. isolates. These were tested for the phenotypic detection of slime production by the microplate and Congo Red Agar methods. The hemolysins production, hemolytic synergism, caseinase and DNase production were also evaluated. The isolates were then assayed through the Polymerase Chain Reaction method to detect genes associated with virulence factors such as: capsule (cap5, cap8), fibronectin (fnbA, fnbB), slime (icaA, icaD) and hemolysins (hla e hlb). Regarding the number of isolates assessed, 58% (145/250) were identified as coagulase-negative Staphylococcus spp. and 42% (105/250) as coagulase-positive Staphylococcus spp. The latter comprised 36.2% (38/105) of isolates identified as S. aureus, 11.4% (12/105) as S. intermedius and 3.8% (4/105) belonging to the SIG group. The hemolisin production was not significant, whereas only 6,4% (16/250) produced alfa hemolysis, 4,8% (12/250) produced beta hemolysis and 1,6% (4/250) was able to produce both. Caseinase production was observed in 66.4% (166/250) and slime production assayed through the microplate method was positive in 76,8% (192/250). DNAse was detected in coagulase-negative Staphylococcus spp. (38/145) and in S. aureus (14/38). Low association between genetic detection of icaA (38/250) and icaD (54/250) and slime phenotypic expression (192/250) suggest that others genetic markers can be involved in this expression. Regarding gene amplification, the isolates did not show significant correlation between the genetic detection of icaA (38/250) and icaD (54/250) and slime production (192/250), indicating that other genetic markers may be involved in this trait expression. The frequency of the occurrence of the others studied genes was of 4% (10/250) for cap5 and cap8, 32,8% (82/250) for fnbA, 4,4% (11/250) for fnbB, 19,2% (48/250) for hla and 18% (45/250) for hlb. The major circulating strain profile on the farms encompassed slime and caseinase producer strains. The spaA gene was found in all of the S. aureus isolates, presenting varying amplicons sizes, with 300bp being the prevalent size. The amplification of the coa gene showed nine polymorphic variants, with 600bp being the prevalent amplicon. The agr gene was also detected in every S. aureus isolate, with an amplicon of 200bp. It was noticed that the presence or absence of the virulence genes assayed in this study were not correlated with the 6 distinct electrophoretic profiles obtained by PFGE.

Impairments of mecA gene detection in bovine Staphylococcus spp.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecALGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecALGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

Caracterização fenotípica da resistência a antimicrobianos e detecção do gene mecA em Staphylococcus spp. coagulase-negativos isolados de amostras animais e humanas

Coagulase-negative staphylococci (SCN) make part of the normal microbiota skin and although they have been considered saprophytics for years, nowadays their clinical significance as an etiologic agent has increased. In this study, 72 SCN isolates obtained from external ear canals of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal samples and S. cohnii subsp. cohnii in human samples. SCN isolates were evaluated in order to establish a phenotypical resistance pattern towards the most indicated antibiotics for staphyloccocal infections. A high level of resistance to penicillin and ampicillin was detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association between ampicillin and sulbactam. To certify the heterogeneous resistance pattern, oxacillin resistance was phenotypically detected by a modified-disc-diffusion test, agar screen, broth micro-dilution and agar dilution. The presence of the mecA gene was detected in 5.6% of the SCN isolates by Polimerase Chain Reaction (PCR).

Occurrence of Shiga-toxigenic Escherichia coli in Stomoxys calcitrans (Diptera: Muscidae)

This study aimed to verify the occurrence of Shiga toxin-producing Escherichia coli (STEC) strains in three distinct anatomic parts of the stable fly Stomoxys calcitrans by multiplex polymerase chain reaction (PCR Multiplex). According to the results obtained, E. coli was identified in 19.5% of the stable flies. Shiga toxin genes were detected in 13% of the E. coli isolated, most frequently from the surface, followed by abdominal digestive tract and mouth apparatus of insects, respectively. This is the first study to detect presence of STEC in Stomoxys calcitrans in Brazil; it has also revealed the potential role of stable flies as carriers of pathogenic bacterial agents.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil.

Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n = 62) and human (n = 67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6′)-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6′)-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000 pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored.

Characteristics of Quinolone Resistance in Salmonella spp. Isolates from the Food Chain in Brazil

Salmonella spp. is an important zoonotic pathogen related to foodborne diseases. Despite that quinolones/fluoroquinolones are considered a relevant therapeutic strategy against resistant isolates, the increase in antimicrobial resistance is an additional difficulty in controlling bacterial infections caused by Salmonella spp. Thus, the acquisition of resistance to quinolones in Salmonella spp. is worrisome to the scientific community along with the possibility of transmission of resistance through plasmids. This study investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. We evaluated 129 isolates, 39 originated from food of animal sources, and 14 from environmental samples and including 9 from animals and 67 from humans, which were referred to the National Reference Laboratory of Enteric Diseases (NRLEB/IOC/RJ) between 2009 and 2013. These samples showed a profile of resistance for the tested quinolones/fluoroquinolones. A total of 33 serotypes were identified; S. Typhimurium (63) was the most prevalent followed by S. Enteritidis (25). The disk diffusion test showed 48.8% resistance to enrofloxacin, 42.6% to ciprofloxacin, 39.53% to ofloxacin, and 30.2% to levofloxacin. According to the broth microdilution test, the resistance percentages were: 96.1% to nalidixic acid, 64.3% to enrofloxacin, 56.6% to ciprofloxacin, 34.1% to ofloxacin, and 30.2% to levofloxacin. Qnr genes were found in 15 isolates (8 qnrS, 6 qnrB, and 1 qnrD), and the aac(6′)-Ib gene in 23. The integron gene was detected in 67 isolates with the variable region between ±600 and 1000 bp. The increased detection of PMQR in Salmonella spp. is a serious problem in Public Health and must constantly be monitored. Pulsed-field gel electrophoresis was performed to evaluated clonal profile among the most prevalent serovars resistant to different classes of quinolones. A total of 33 pulsotypes of S. Typhimurium were identified with a low percentage of genetic similarity (≤65%). This result demonstrates the presence of high diversity in the resistant clones evaluated in this study.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment.

Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI–TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI–TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.