Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.

Molecular characterization and evaluation of antimicrobial susceptibility of enteropathogenic E. coli (EPEC) isolated from minas soft cheese

Foodborne disease caused by microorganisms is a problem of public health. Minas soft cheese is a national product manufactured using simple technology; it has high level of acceptance in the country making its production an important economic activity. Many microorganisms may be present in foods including the bacterium Escherichia coli (E. coli). Overall, E. coli is a harmless commensal bacterium; however, some strains may have a pathogenic potential. Several outbreaks of foodborne diseases associated with consumption of contaminated cheese have been reported, and the presence of pathogenic strains of E. coli has increased. The objective of this study was to isolate, evaluate the antimicrobial susceptibility, and characterize, by Multiplex PCR, the pathogenic E. coli strains isolated from Minas cheese commercialized in Rio de Janeiro. Thirty samples were analyzed and five strains of E. coli (EPEC) were identified. The assessment of antimicrobial susceptibility revealed 40% of the isolates resistant to ampicillin and 40% with intermediate resistance to ampicillin-sulbactam combination. These findings are a warning signal to health authorities since Minas cheese is a ready to eat food product, and therefore should not pose health risks to the population.

Streptococcus agalactiae in Brazil: serotype distribution, virulence determinants and antimicrobial susceptibility

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Modulação do estado redox em bactérias por quinoxalinas

As quinoxalinas são compostos heterocíclicos que têm, entre outras, capacidades antimicrobianas, inclusivamente contra bactérias resistentes aos antimicrobianos convencionais. Os mecanismos pelos quais estes compostos exercem a sua atividade ainda não está completamente esclarecido. O objetivo do presente estudo é avaliar o efeito redox em sinergismo/antagonismo com as quinoxalinas em modelos de bactérias com e sem resistências a antimicrobianos. No que se refere aos compostos foram utilizados a quinoxalina 1,4-dióxido (QNX), 2-metil-3-benzilquinoxalina-1,4-dióxido (2M3BQNX), 2-metilquinoxalina-1,4-dióxido (2MQNX) e a 2-amino-3-cianoquinoxalina-1,4-dióxido (2A3CQNX). Quanto aos modelos procariotas, foram utilizados a Salmonella enterica, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus saprophyticus, Enterobacter aerogenes, Enterobacter cloacae, Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli TEM 201 e Escherichia coli TEM 180. Nos compostos químicos em que se verificou a Concentração Mínima Inibitória (CMI), realizou-se o estudo do comportamento do crescimento bacteriano. Relativamente ao estado redox, foi avaliado para cada estirpe sensível, através do rácio GSH/GSSG, nas doses inibitórias e não inibitórias de cada composto. Os resultados apresentam que todos os compostos testados, à exceção do 2M3BQNX, têm atividade antimicrobiana na maioria das estirpes, excetuando a E. faecalis e a S. saprophyticus. Os rácios GSH/GSSG apontam para o efeito oxidante em K. pneumoniae e S. enterica e antioxidante na E. aerogenes. A conclusão do estudo sugere que os compostos apresentam elevada capacidade antibacteriana e influência no equilíbrio redox das bactérias, podendo contribuir para o esclarecimento do mecanismo de ação dos derivados das quinoxalinas 1-4 dióxido, nas bactérias.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from patients attending a public referral center for sexually transmitted diseases in Belo Horizonte, State of Minas Gerais, Brazil

Introduction The aim of this study was to determine the antimicrobial susceptibility of Neisseria gonorrhoeae isolates obtained from patients attending a public referral center for sexually transmitted diseases and specialized care services (STD/SCS) in Belo Horizonte, Brazil. Methods Between March 2011 and February 2012, 201 specimens of Neisseria gonorrhoeae were consecutively obtained from men with symptoms of urethritis and women with symptons of cervicitis or were obtained during their initial consultation. The strains were tested using the disk diffusion method, and the minimum inhibitory concentrations of azithromycin, cefixime, ceftriaxone, ciprofloxacin, chloramphenicol, penicillin, tetracycline and spectinomycin were determined using the E-test. Results The specimens were 100% sensitive to cefixime, ceftriaxone and spectinomycin and exhibited resistances of 4.5% (9/201), 21.4% (43/201), 11.9% (24/201), 22.4% (45/201) and 32.3% (65/201) to azithromycin, ciprofloxacin, chloramphenicol, penicillin and tetracycline, respectively. Intermediate sensitivities of 17.9% (36/201), 4% (8/201), 16.9% (34/201), 71.1% (143/201) and 22.9% (46/201) were observed for azithromycin, ciprofloxacin, chloramphenicol, penicillin and tetracycline, respectively. The specimens had plasmid-mediated resistance to penicillin PPNG 14.5% (29/201) and tetracycline TRNG 11.5% (23/201). Conclusions The high percentage of detected resistance to penicillin, tetracycline, chloramphenicol and ciprofloxacin indicates that these antibiotics are not appropriate for gonorrhea treatment at the Health Clinic and possibly in Belo Horizonte. The resistance and intermediate sensitivity of these isolates indicates that caution is recommended in the use of azithromycin and emphasizes the need to establish mechanisms for the surveillance of antimicrobial resistance for the effective control of gonorrhea.

Contributions of culture and antimicrobial susceptibility tests to the retreatment of patients with pulmonary tuberculosis

Introduction This study evaluated the efficacy of retreatment of pulmonary tuberculosis (TB) with regard to treatment outcomes and antimicrobial susceptibility testing (ST) profiles. Methods This retrospective cohort study analyzed 144 patients treated at a referral hospital in Brazil. All of them had undergone prior treatment, were smear-positive for TB and received a standardized retreatment regimen. Fisher's 2-tailed exact test and the χ2 test were used; RRs and 95% CIs were calculated using univariate and multivariate binary logistic regression. Results The patients were cured in 84 (58.3%) cases. Failure was associated with relapsed treatment and abandonment (n=34). Culture tests were obtained for 103 (71.5%) cases; 70 (48.6%) had positive results. ST results were available for 67 (46.5%) cases; the prevalence of acquired resistance was 53.7%. There were no significant differences between those who achieved or not therapeutic success (p=0.988), despite being sensitive or resistant to 1 or more drugs. Rifampicin resistance was independently associated with therapeutic failure (OR: 4.4, 95% CI:1.12-17.37, p=0.034). For those cases in which cultures were unavailable, a 2nd model without this information was built. In this, return after abandonment was significantly associated with retreatment failure (OR: 3.59, 95% CI:1.17-11.06, p=0.026). Conclusions In this cohort, the general resistance profile appeared to have no influence on treatment outcome, except in cases of rifampicin resistance. The form of reentry was another independent predictor of failure. The use of bacterial culture identification and ST in TB management must be re-evaluated. The recommendations for different susceptibility profiles must also be improved.

Performance of the Vitek 2 system software version 5.03 in the bacterial identification and antimicrobial susceptibility test: evaluation study of clinical and reference strains of Gram-positive cocci

Introduction. The genera Enterococcus, Staphylococcus and Streptococcus are recognized as important Gram-positive human pathogens. The aim of this study was to evaluate the performance of Vitek 2 in identifying Gram-positive cocci and their antimicrobial susceptibilities. Methods. One hundred four isolates were analyzed to determine the accuracy of the automated system for identifying the bacteria and their susceptibility to oxacillin and vancomycin. Results. The system correctly identified 77.9% and 97.1% of the isolates at the species and genus levels, respectively. Additionally, 81.8% of the Vitek 2 results agreed with the known antimicrobial susceptibility profiles. Conclusion. Vitek 2 correctly identified the commonly isolated strains; however, the limitations of the method may lead to ambiguous findings.

NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

Antimicrobial susceptibility of Campylobacter jejuni subsp. jejuni assessed by E-test and double dilution agar method in Southern Chile

The susceptibility patterns of 108 Campylobacter jejuni subsp. jejuni clinical strains, to six antimicrobial agents was determined by using the E-test and the double dilution agar methods. Using both metods, no strain was found to be resistant to ciprofloxacin, erythromycin and gentamicin, but two (1.8%) were resistant to tetracycline and all to aztreonam. Seven (6.5%) strains were resistant to ampicillin by the E-test and five (4.6%) by the double dilution agar method and by both meyhods. No great discrepancies were observed between both methods.

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional Löwenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.

Oxacillin Resistance and Antimicrobial Susceptibility Profile of Staphylococcus saprophyticus and Other Staphylococci Isolated from Patients with Urinary Tract Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular identification and antimicrobial susceptibility of Nocardia spp. isolated from bovine mastitis in Brazil

Nocardia spp. infections can cause severe damage to the mammary gland due to suppurative pyogranulomatous lesions and lack of clinical cure in response to conventional antimicrobial therapy. Although Nocardia infections are considered relatively uncommon in cows, there has been an apparent worldwide increase in the incidence of bovine mastitis caused by Nocardia spp, perhaps due to environmental transmission of this ubiquitous pathogen. The objectives of present study were to determine: (i) species distribution of 80 Nocardia isolates involved in bovine mastitis (based on molecular methods); and (ii) antimicrobial susceptibility pattern of all isolates from three geographical areas in Brazil. In this study, Nocardia nova (80%) was the most frequently isolated species, followed by Nocardia farcinica (9%). Additionally, Nocardia puris, Nocardia cyriacigeorgica, Nocardia veterana, Nocardia africana, and Nocardia arthritidis were detected using 16S rRNA sequencing. This is apparently the first report of N. puris, N. veterana, N. cyriacigeorgica, N. arthritidis and N. africana in association with bovine mastitis. Based on the disk diffusion test, isolates were most frequently resistant to cloxacillin (75%), ampicillin (55%) and cefoperazone (47%), whereas few Nocardia spp. were resistant to amikacin, cefuroxime or gentamicin. © 2013 Elsevier B.V. All rights reserved.

Exogenous bacterial osteomyelitis in 52 dogs: a retrospective study of etiology and in vitro antimicrobial susceptibility profile (2000-2013)

Background: Most clinical cases of osteomyelitis in dogs involve infectious agents, especially bacteria and fungi. The characterization of these microorganisms may aid in the prevention and treatment of disease.Objective: The aim of this study was to evaluate retrospectively microbiological cultures and in vitro antimicrobial susceptibility profile of isolates from 52 cases of bacterial osteomyelitis in long bones of dogs over 2000-2013. In 78% of the cases injuries were caused by a motor vehicle accident, but there were a few cases of dog bites (17%) and ascending infection due to pododermatitis (5%).Animals and methods: The isolated microorganisms were identified based on conventional phenotypic methods. In vitro disk diffusion test was performed using 30 different antimicrobials.Results: The isolates were obtained from femur (28%), humerus (16%), tibia (31%), and radius/ulna (25%). Among 52 cases, culture was positive in 88% of cases. Thirteen genus of different species of microorganisms were isolated. The most common microorganisms isolated were Staphylococcus spp. and Escherichia coli followed by Streptococcus spp., enteric bacteria, Corynebacterium sp. and anaerobic bacteria. In 42% of cases cultures were mixed. The most effective drugs against isolated bacteria were amoxicillin and clavulanate potassium (79%) followed by ceftriaxone (69%). High-resistance rates were documented against azithromycin (80%), penicillin (59%), and clindamycin (59%).Conclusions: The present study highlights diverse etiologic agents in cases of infectious bacterial osteomyelitis, with predominance of Staphylococcus genus, and reinforces the importance of obtaining cultures and susceptibility profiles given the high rates of antimicrobial resistance.

Antimicrobial susceptibility to b-lactams and metronidazole of microorganisms isolated from chronic and aggressive periodontitis

The objective of this study was to evaluate the microbial susceptibility to ß-lactams and metronidazole, and evaluate the production of ß-lactamases by microorganisms isolated from patients with chronic or aggressive periodontitis. The samples were obtained from 50 patients with periodontitis and microorganisms were isolated onto selective and nonselective culture media, identified by biochemical methods and tested for susceptibility to antimicrobial agents (amoxicillin, amoxicillin/clavulanate, cefoxitin, imipenem, metronidazole, penicillin G). The isolates were resistant to at least 1 mg/ml of any drug tested were evaluated to verify the production of ß-lactamases by the method of double layer (or biological) and chromogenic cephalosporin using nitrocefin. The results evidenced resistance to amoxicillin and penicillin G, while the susceptibility to association amoxicillin/clavulanate, imipenem and cefoxitin was widely disseminated among the organisms. Resistance to these drugs showed a clear correlation with the production of ß-lactamase in the majority of microbial groups.