Vigilância da resistência a antibióticos contra o Helicobacter pylori na região do Biobío (Chile) em uma década / Antibiotic resistance surveillance of helicobacter pylori at the biobío region (chile) in a decade

ABSTRACT BACKGROUND: Helicobacter pylori infection in Chile remains as a public and private health-care system's challenge, with a prevalence of the infection over 70%. Nowadays, antibiotic treatment of the infection is mandatory to prevent the arising of severe associated diseases but failures in the eradication therapy mainly due to clarithromycin resistance has been observed worldwide and first line eradication therapy seems to be not effective anymore in several geographical areas. Thus, health-care systems are committed to maintain an epidemiological surveillance upon the evolution of the antibiotic resistance of this priority 2 pathogen. OBJECTIVE: This work reports a 10 years surveillance of the primary antibiotic resistance of H. pylori clinical isolates at the Biobío region-Chile, and the evolution of resistance toward amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline among the species. METHODS: H. pylori strains were investigated during the periods 2005-2007 (1435 patients analysed) and 2015-2017 (220 patients analysed) by inoculating a saline homogenate biopsy onto the surface of Columbia agar (Oxoid, Basingstoke, UK) - supplemented with 7% horse red blood cells plus DENT inhibitor (Oxoid, Basingstoke, UK) - following by incubation at 37ºC under 10% CO2 atmosphere for five days. Antibiotic resistance pattern of the isolates was assessed using the disk diffusion test in Müeller-Hinton agar supplemented with 7% horse red blood cells followed by incubation for further three days under 10% CO2 atmosphere. Statistical analysis was done using the SPSS v22 software and P values <0.05 were considered statistically significant. RESULTS: A total of 41% of 1435 patients were detected to be infected with H. pylori by bacteriological culture in 2005-2007 period, meanwhile 32.7% from 220 patients were also infected in 2015-2017 period. The clinical isolates of H. pylori are mostly susceptible to amoxicillin and tetracycline (both over 98% of strains), but less susceptible to levofloxacin in both periods analysed (over 79% of the strains). On the other hand, metronidazole continuous showing the highest score of resistant isolates (over 40% of resistant strains), although an 18% fewer resistant strains were observed in 2015-2017 period. Clarithromycin, the key antibiotic in eradication therapies, has an increased frequency of resistant strain isolated in the decade (22.5% in 2005-2007 and 29.2% in 2015-2017). Multidrug resistant strains (two, three and four antibiotics) were also detected in both periods with the highest scores for simultaneous resistance to clarithromycin-metronidazole (18%) and clarithromycin-metronidazole-levofloxacin (12.5%) resistant strains. According to gender, the isolates resistant to amoxicillin, clarithromycin and metronidazole were more frequent in female, with a specific increment in amoxicillin and clarithromycin resistance. CONCLUSION: The frequency of clarithromycin resistance (29.2%) detected in 2015-2017 suggests that conventional triple therapy is no longer effective in this region.

RESUMO CONTEXTO: A infecção por Helicobacter pylori no Chile permanece como um desafio do sistema de saúde público e privado, com prevalência da infecção acima de 70%. Hoje em dia, o tratamento antibiótico da infecção é obrigatório para prevenir o surgimento de graves doenças associadas, mas falhas na terapia de erradicação, principalmente devido à resistência à claritromicina, têm sido observadas em todo o mundo, e a terapia de erradicação de primeira linha parece não ser mais eficaz em várias áreas geográficas. Assim, os sistemas de saúde estão comprometidos em manter uma vigilância epidemiológica sobre a evolução da resistência aos antibióticos deste patógeno prioritário tipo 2. OBJETIVO: Este trabalho relata uma vigilância de 10 anos da resistência antibiótica primária de isolados clínicos de H. pylori na região do Biobío-Chile, e a evolução da resistência em relação à amoxicilina, claritromicina, levofloxacina, metronidazol e tetraciclina entre as espécies. MÉTODOS: As cepas de H. pylori foram investigadas durante os períodos 2005-2007 (1435 pacientes analisados) e 2015-2017 (220 pacientes analisados) inoculando uma biópsia de homogeneizado fisiológico na superfície do agar Columbia (Oxoid, Basingstoke, Reino Unido) - suplementado com 7% de glóbulos vermelhos do cavalo mais o inibidor de DENTE (Oxoid, Basingstoke, Reino Unido) - seguindo pela incubação em 37ºC a atmosfera de 10% de CO2 por cinco dias. O padrão de resistência aos antibióticos dos isolados foi avaliado utilizando-se o teste de difusão em disco em agar Müeller-Hinton suplementado com 7% de glóbulos vermelhos de cavalo seguidos de incubação por mais três dias a atmosfera de 10% de CO2. A análise estatística foi realizada utilizando-se o software SPSS V22 e os valores de P<0,5 foram considerados estatisticamente significantes. RESULTADOS: Um total de 41% dos 1435 pacientes foram detectados como contaminados por H. pylori pela cultura bacteriológica no período 2005-2007, ao mesmo tempo 32,7% de 220 pacientes foram contaminados igualmente no período 2015-2017. Os isolados clínicos de H. pylori são principalmente suscetíveis à amoxicilina e tetraciclina (tanto mais de 98% das cepas), mas menos suscetíveis à levofloxacina em ambos os períodos analisados (mais de 79% das cepas). Por outro lado, o metronidazol permaneceu mostrando a maior pontuação de resistentes isolados (mais de 40% de cepas resistentes), embora tenham sido observados 18% menos cepas resistentes no período de 2015-2017. A claritromicina, o antibiótico-chave em terapias de erradicação, tem uma frequência aumentada de cepa resistente isolada na década (22,5% em 2005-2007 e 29,2% em 2015-2017). Cepas multirresistentes (dois, três e quatro antibióticos) também foram detectadas em ambos os períodos com os maiores escores de resistência simultânea à claritromicina-metronidazol (18%) e claritromicina-metronidazol-levofloxacina (12,5%) cepas resistentes. De acordo com o sexo, os isolados resistentes à amoxicilina, claritromicina e metronidazol foram mais frequentes no sexo feminino, com incremento específico em amoxicilina e resistência à claritromicina. CONCLUSÃO: A frequência de resistência à claritromicina (29,2%) detectada em 2015-2017 sugere que a terapia tripla convencional não é mais efetiva nesta região.

Susceptibilidad antimicrobiana en muestras urogenitales de ureaplasma urealyticum, el principal agente en infección intramaniótica / Antimicrobial susceptibility in urogenital samples of ureaplasma urealyticum, the main agent in intramaniotic infection

RESUMEN OBJETIVO: Ureaplasma urealyticum es el agente más frecuentemente aislado en infección intraamniótica. Los macrólidos son los antimicrobianos de primera elección en embarazadas. Se ha descrito el aumento de resistencia, pudiendo limitar las opciones terapéuticas durante la gestación. El propósito del estudio es evaluar susceptibilidad antimicrobiana de Ureaplasma urealyticum aislado en mujeres en edad fértil, que se atienden en Clínica Alemana Temuco, Araucanía, Chile. METODO: Se estudian todas las muestras de orina y flujo vaginal para cultivo de U. urealyticum, de pacientes entre 18 y 40 años, recibidas en el Laboratorio de Microbiología Clínica Alemana Temuco, en período Abril 2013 a Enero 2015. Se procesan las muestras con kit Mycoplasma IST 2 de Biomerieux. En las que resultan positivas, se estudia susceptibilidad a macrólidos, tetraciclinas y quinolonas. RESULTADOS: 426 muestras de orina y flujo vaginal (390 pacientes). 197 pacientes resultaron positivas para U. urealyticum. (50,5%). La susceptibilidad fue 88,4% (174 pctes) a Eritromicina, 87,9% (173 pctes) a Claritromicina y 91,9% (181 pctes) a Azitromicina (NS). 15 de 197 pacientes (7,6%) fueron resistentes a los 3 macrólidos. La susceptibilidad a Quinolonas fue 55,3% a Ciprofloxacino, y 94% a Ofloxacino. El 100% resultó susceptible a Tetraciclinas. CONCLUSIONES: Cerca del 10% de U. urealyticum aislados en nuestra serie son resistentes a macrólidos, contribuyendo a la no erradicación de la infección en tratamientos empíricos. Dentro de ellos, azitromicina aparece con la mayor efectividad. El aumento de resistencia limitará opciones terapéuticas, con gran impacto perinatal en futuro. La vigilancia de susceptibilidad en cada hospital es fundamental para elección terapéutica.

ABSTRACT INTRODUCTION: Ureaplasma urealyticum is the most frequently isolated microorganism in intra-amniotic infection. The macrolides are the first choice antimicrobials for treat this infection in pregnancy. The increasing resistance has been described worldwide, seriously limiting therapeutic options in pregnancy. The aim of the study is to evaluate antimicrobial susceptibility of U. urealyticum aislated in fertile-age women in Clínica Alemana Temuco, Araucania region, Chile. METHOD: Urine and vaginal samples were analyzed for U. urealyticum, from every 18 to 40 years old patients, received at Microbiology Laboratory of Clínica Alemana Temuco, between April 2013 to January 2015. The samples are processed with Mycoplasma IST 2 kit of Biomerieux. If they became positives, susceptibility to macrolides, tetracyclines and quinolones was studied. RESULTS: 426 urine and vaginal samples were collected (390 patients). 197 patients were positive for U. urealyticum (50.5%). The susceptibility was 88.4% (174 pts) to Erythromicyn, 87.9% (173 pts) to Clarithromycin and 91.9% (181 pts) to Azithromycin (NS). Resistance to all macrolides was observed in 15 out of 197 patients (7.6%). The susceptibility to Quinolones was 55.3% to Ciprofloxacin, and 94% to Ofloxacin. The 100% was susceptible to Tetracyclines. DISCUSSION: Near to 10% of isolated Ureaplasma spp in our serie were resistant to some macrolide, being a factor for failing to eradicate the infection in empirical treatment. Azithromycin was the most effective. The increasing resistance will limit therapeutic options, with great perinatal impact in the future. Susceptibility surveillance in each hospital is very important for therapeutic options.

Uso de marcadores fluorescentes na engenharia tecidual óssea: estudo piloto / Use of fluorescent markers in bone tissue engineering: pilot study

Introdução: a regeneração e o reparo de tecidos ósseos perdidos é objeto de estudo da Bioengenharia Tecidual. O uso de biomateriais substitutos ósseos biomiméticos visa estimular os sistemas celulares e bioquímicos para restabelecer de modo mais eficiente o tecido ósseo nos casos de sua reconstrução. Ao investigar o processo de remodelação, é vital identificar áreas de novo crescimento para avaliar a eficácia dos biomateriais implantados e respectivos regimes de tratamento. A avaliação qualitativa e quantitativa da regeneração óssea pode ser realizada através da aplicação de marcadores como o Xilenol, a Tetraciclina, a Calceína e a Alizarina. A administração desses marcadores de forma associada possibilita ainda marcar sequencialmente camadas de nova deposição e remodelação durante o reparo. Objetivo: estabelecer um protocolo para utilização dos marcadores fluorescentes de reparo ósseo xilenol, tetraciclina, calceína e alizarina, em ratos. Metodologia: foram utilizados 35 ratos da linhagem Wistar, machos adultos, com massa corpórea entre 350 e 400g, e idade aproximada de 4 a 5 meses, distribuídos randomicamente em 5 grupos experimentais, submetidos à confecção de defeito ósseo circular de 8 mm em região de calvária, e administração dos diferentes marcadores segundo os grupos; XO ­ Xilenol; Ca ­ Calceína; Al ­ Alizarina; Te ­ Tetraciclina; C ­ Controle. Após 15 dias de experimento, os animais foram eutanasiados e as calvárias processadas e analisadas por histomorfometria, microscopia de epifluorescência e microscopia de fluorescência. Resultados: todos protocolos empregados para utilização dos marcadores fluorescentes xilenol, calceína, alizarina e tetracicilina foram úteis para identificar área de deposição mineral durante o período analisado de regeneração óssea em ratos. As imagens obtidas pela microscopia de fluorescência revela a presença dos marcadores incorporados à matriz óssea neoformada, no entanto a utilização da Alizarina e Calceína dentro dos protocolos testados mostraram-se mais eficientes. Conclusão: os protocolos testados nesse estudo apresentaram-se viáveis para utilização em pesquisas envolvendo marcadores de regeneração óssea, com resultados superiores para Alizarina e Calceína

Introduction: The regeneration and repair of lost bone tissues is the subject of a study of Tissue Bioengineering. The use of biomimetic biomaterial bone substitutes aims to stimulate the cellular and biochemical systems to restore more efficiently the bone tissue in the cases of its reconstruction. When investigating the remodeling process, it is vital to identify areas of new growth to evaluate the efficacy of implanted biomaterials and their treatment regimens. The qualitative and quantitative evaluation of bone regeneration can be performed through the use of markers such as Xylenol, Tetracycline, Calcein and Alizarin. The administration of such markers in an associated manner also makes it possible to sequentially mark layers of new deposition and remodeling during the repair. Objective: to establish a protocol for the use of fluorescent xylenol, tetracycline, calcein and alizarin bone repair markers in rats. Metodology: thirtyfive male adult Wistar rats with a body mass ranging from 350 to 400 g and approximately 4 to 5 months old were randomly assigned to 5 experimental groups submitted to a circular bone defect of 8 mm in the region of calvaria, and administration of the different markers according to the groups; XO ­ Xylenol; Ca ­ Calcein; Al-Alizarin; Te ­ Tetracycline; C ­ Control. After 15 days of experiment, the animals were euthanized and the calvaria processed and analyzed by histomorphometry, epifluorescence microscopy and fluorescence microscopy. Results: all protocols used for fluorescence markers xylenol, calcein, alizarin and tetracycline were useful to identify area of mineral deposition during the analyzed period of bone regeneration in rats. The images obtained by fluorescence microscopy revealed the presence of the markers incorporated into the neoformed bone matrix, however the use of Alizarin and Calcein within the protocols tested were more efficient. Conclusion: the protocols tested in this study were feasible for use in research involving markers of bone regeneration, with superior results for Alizarin and Calcein.

Prevalence, serotyping and antimicrobials resistance mechanism of Salmonella enterica isolated from clinical and environmental samples in Saudi Arabia

Abstract Salmonella is recognized as a common foodborne pathogen, causing major health problems in Saudi Arabia. Herein, we report epidemiology, antimicrobial susceptibility and the genetic basis of resistance among S. enterica strains isolated in Saudi Arabia. Isolation of Salmonella spp. from clinical and environmental samples resulted in isolation of 33 strains identified as S. enterica based on their biochemical characteristics and 16S-rDNA sequences. S. enterica serovar Enteritidis showed highest prevalence (39.4%), followed by S. Paratyphi (21.2%), S. Typhimurium (15.2%), S. Typhi and S. Arizona (12.1%), respectively. Most isolates were resistant to 1st and 2nd generation cephalosporin; and aminoglycosides. Moreover, several S. enterica isolates exhibited resistance to the first-line antibiotics used for Salmonellosis treatment including ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In addition, the results revealed the emergence of two S. enterica isolates showing resistance to third-generation cephalosporin. Analysis of resistance determinants in S. enterica strains (n = 33) revealed that the resistance to β-lactam antibiotics, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, was attributed to the presence of carb-like, dfrA1, floR, tetA gene, respectively. On the other hand, fluoroquinolone resistance was related to the presence of mutations in gyrA and parC genes. These findings improve the information about foodborne Salmonella in Saudi Arabia, alarming the emergence of multi-drug resistant S. enterica strains, and provide useful data about the resistance mechanisms.

Modulation of drug resistance and biofilm formation of Staphylococcus aureus isolated from the oral cavity of Tunisian children

Abstract Objectives: This study aims to investigate the antimicrobial and the anti-biofilm activities of Lactobacillus plantarum extract (LPE) against a panel of oral Staphylococcus aureus (n = 9) and S. aureus ATCC 25923. The in vitro ability of LPE to modulate bacterial resistance to tetracycline, benzalchonium chloride, and chlorhexidine were tested also. Methods: The minimum inhibitory concentrations (MICs) and the minimal bactericidal concentrations of Lactobacillus plantarum extract, tetracycline, benzalchonium chloride and clohrhexidine were determined in absence and in presence of a sub-MIC doses of LPE (1/2 MIC). In addition, the LPE potential to inhibit biofilm formation was assessed by microtiter plate and atomic force microscopy assays. Statistical analysis was performed on SPSS v. 17.0 software using Friedman test and Wilcoxon signed ranks test. These tests were used to assess inter-group difference (p < 0.05). Results: Our results revealed that LPE exhibited a significant antimicrobial and anti-biofilm activities against the tested strains. A synergistic effect of LPEs and drug susceptibility was observed with a 2–8-fold reduction. Conclusion: LPE may be considered to have resistance-modifying activity. A more detailed investigation is necessary to determine the active compound responsible for therapeutic and disinfectant modulation.

effect of four commonly used root conditioner agents in different time periods applied on periodontally diseased and healthy teeth

Statement of Problem: Root surface contamination or infection can potentially change the consequences of regenerative periodontal therapies and therefore the modification and disinfection of the contaminated root surfaces are necessary

Objectives: This study aimed to compare the surface characteristics of the extracted human teeth after exposure to four root conditioners in different time periods

Materials and Methods: The study samples were prepared from 40 freshly extracted teeth including 20 affected teeth with periodontal diseases and 20 healthy teeth. After performing root planning, 240 dentinal block samples were prepared and each affected and healthy sample was randomly allocated to receive one of the following root conditioners; Ethylenediaminetetraaceti acid [EDTA], citric acid, doxycycline, and tetracycline or rinsed with normal saline as the control agent. The prepared specimens were evaluated using scanning electron microscope and the inter-group differences and changes in study indices; dentin [%], tubular spaces [%], and diameter of dentinal tubules [micro m[2]] were compared using one-way ANOVA test

Results: In the control group receiving normal saline, the changes in the indicators of dentin, tubular spaces, and diameter of dentinal tubules remained insignificant in all time periods. EDTA, citric acid, and tetracycline had chelating effects on the study indices; however, doxycycline led to gradual decrease of the tubular space and diameter as well as increase in dentin percentage

Conclusions: In different time intervals and when considering healthy or affected tooth surfaces, the effect of conditioning agents could be different. Amongst the four agents used, EDTA and tetracycline consistently increased the diameter of tubules and percentage of patent tubules in both healthy and diseased teeth

Increasing Incidence of High-Level Tetracycline-Resistant Neisseria gonorrhoeae due to Clonal Spread and Foreign Import

PURPOSE: The detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea. MATERIALS AND METHODS: The antimicrobial susceptibilities of 601 isolates of N. gonorrhoeae from 2004 to 2011 were tested by standard Clinical and Laboratory Standards Institute methods. To determine the molecular epidemiological relatedness, N. gonorrhoeae multi-antigen sequence typing was performed. RESULTS: The incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted. CONCLUSION: The increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey

INTRODUCTION: Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS: H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS: A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS: We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey. .

Detection of the Efflux-Mediated Erythromycin Resistance Transposon in Streptococcus pneumoniae

BACKGROUND: The present analysis focuses on phenotypic and genotypic characterizations of efflux-mediated erythromycin resistance in Streptococcus pneumoniae due to an increase in macrolide resistance in S. pneumoniae worldwide. METHODS: We investigated the prevalence of efflux-mediated erythromycin resistance and its relevant genetic elements from 186 specimens of S. pneumonia isolated from clinical and normal flora from Tehran, Iran. The presence of erythromycin resistance genes was tested by PCR with two sets of primers, specific for erm(B) and mef(A/E), and their genetic elements with tetM, xis, and int genes. Isolates were typed with the BOX PCR method and tested for resistance to six antibiotics. RESULTS: Antibiotic susceptibility tests revealed that 100% and 47% isolates were resistant to tetracycline and erythromycin, respectively. The erythromycin and clindamycin double-disc diffusion test for macrolide-lincosamide-streptograminB (MLSB) resistance phenotype showed 74 (84%) isolates with the constitutive MLSB phenotype and the remaining with the M phenotype. BOX PCR demonstrated the presence of 7 types in pneumococci with the M phenotype. Fourteen (16%) isolates with the M phenotype harbored mef(A/E), tetM, xis, and int genes. CONCLUSIONS: The present results suggest dissemination of polyclonal groups of S. pneumoniae with the M phenotype carrying resistance genes attributed to transposon 2009.

Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Conjugative transfer of resistance determinants among human and bovine Streptococcus agalactiae

Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10-7 and 9.10-7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.

Evaluación del método epsilométrico Etest para la determinación de la sensibilidad a tetraciclina en Paenibacillus larvae, agente causal de la loque americana de las abejas / [Evaluation of the Epsilometer (Etest) method for the detection of tetracycline susceptibility in Paenibacillus larvae, the causal agent of American foulbrood disease of honeybees].

American foulbrood (AFB) is a bacterial disease caused by the spore-forming, grampositive bacterium Paenibacillus larvae, which affects honeybee broods worldwide. The aim of this work was to compare the Epsilometer test (Etest) to the agar dilution method for testing a collection of 22 P. larvae strains to tetracycline by using MYPGP and Iso- Sensitest agars. Results showed that a categorical agreement of 100

was found when using Iso-Sensitest, while a categorical agreement of 86.36

was found (with 3 minor errors) when MYPGP was tested. In conclusion, the Etest could be a rapid and reliable method for testing MIC values of tetracycline in P. larvae only when used in combination with Iso-Sensitest agar. Nevertheless, these results should be confirmed with future studies involving a larger number of isolates.

Tetracycline and trimethoprim/sulfamethoxazole at clinical laboratory: Can they help to characterize Staphylococcus aureus carrying different SCCmec types?

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) can be difficult to detect at the clinical practice. METHODS: We analyzed 140 MRSA isolates from inpatients to correlate the antimicrobial susceptibility with the SCCmec types. RESULTS: Type III (n = 63) isolates were more resistant to ciprofloxacin, clindamycin, cloramphenicol, erythromycin, gentamicin, and rifampin than type IV (n = 65) ones (p < 0.05). Moreover, type IV isolates were susceptible to tetracycline (100%) and trimethoprim/sulfamethoxazole (98%), while type III isolates presented resistance to them. CONCLUSIONS: In regions where these SCCmec types are prevalent, the detection of specific resistant phenotypes could help to predict them, mainly when there are no technical conditions to SCCmec typing.

Emergence of decreased susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae in India.

Background. In the past, Neisseria gonorrhoeae has developed resistance to antimicrobial agents used for its treatment. Consequently, extended-spectrum cephalosporins form the mainstay of treatment for gonorrhoea. Methods. Samples from 88 patients attending the sexually transmitted diseases clinics from December 2009 to January 2011 in two referral hospitals in New Delhi were studied. Antimicrobial susceptibility testing was done using the disc diffusion method as per the calibrated dichotomous sensitivity technique against the following antibiotics: penicillin (0.5 i.u.), tetracycline (10 μg), nalidixic acid (30 μg), ciprofloxacin (1 μg), spectinomycin (100 μg), ceftriaxone (0.5 μg) and cefpodoxime (10 μg) (Oxoid UK). Azithromycin (15 μg) (Oxoid, UK) was tested as per the guidelines of the Clinical and Laboratory Standards Institute. Minimum inhibitory concentrations were determined using the Etest for penicillin, tetracycline, ciprofloxacin, ceftriaxone, spectinomycin and azithromycin as per the manufacturer’s instruction (Biomerieux, France). Results. Eighteen isolates of Neisseria gonorrhoeae were obtained. Three of these had decreased susceptibility to ceftriaxone and cefpodoxime by the disc diffusion method. The minimum inhibitory concentrations of ceftriaxone for two isolates were 0.064 μg/ml and for one isolate it was 0.125 μg/ml. Conclusion. Higher minimum inhibitory concentrations to extended-spectrum cephalosporins is of concern as it has been shown to precede treatment failure. This may warrant its use in increased/multiple dosages alone or possibly in combination (dual therapy), thereby complicating effective disease control. Our report is in accordance with earlier reports from different parts of the world. Therefore, a continuous surveillance of antimicrobial resistance is crucial to tailor treatment schedules for Neisseria gonorrhoeae in a particular geographical region.

Surveillance of Antimicrobial Susceptibility Patterns among Shigella Species Isolated in China during the 7-Year Period of 2005-2011

BACKGROUND: Shigella is a frequent cause of bacterial dysentery in the developing world. Treatment with antibiotics is recommended for shigellosis, but the options are limited due to globally emerging resistance. This study was conducted to determine the frequency and pattern of antimicrobial susceptibility of Shigella in China. METHODS: We studied the antimicrobial resistance profiles of 308 Shigella spp. strains (260 S. flexneri, 40 S. sonnei, 5 S. boydii, and 3 S. dysenteriae) isolated from fecal samples of patients (age, from 3 months to 92 yr) presenting with diarrhea in different districts of Anhui, China. The antimicrobial resistance of strains was determined by the agar dilution method according to the CSLI guidelines. RESULTS: The most common serogroup in the Shigella isolates was S. flexneri (n=260, 84.4%), followed by S. sonnei (n=40, 13.0%). The highest resistance rate was found for nalidixic acid (96.4%), followed by ampicillin (93.2%), tetracycline (90.9%), and trimethoprim/sulfamethoxazole (80.8%). Among the isolates tested, 280 (91.0%) were multidrug resistant (resistant to > or =2 agents). The most common resistance pattern was the combination of ampicillin, tetracycline, and trimethoprim/sulfamethoxazole (70.8%). Resistance to ampicillin and tetracycline were more common among S. flexneri than among S. sonnei isolates. CONCLUSIONS: S. flexneri is predominant in Anhui, China, and its higher antimicrobial resistance rate compared with that of S. sonnei is a cause for concern. Continuous monitoring of resistance patterns is necessary to control the spread of resistance in Shigella. The recommendations for antimicrobial treatment must be updated regularly based on surveillance results.

Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig

This study aimed to evaluate by the intra-osseous implant technique the most commonly used materials for pulp therapy in pediatric dentistry: calcium hydroxide (CH), Guedes Pinto paste and CTZ paste, according to FDI (1980) and ANSI/ADA (1982) recommendations. Thirty guinea pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks received one implant on each side of the lower jaw symphysis. The external lateral tube wall served as control for the technique. At the end of the observation periods, the animals were euthanized and specimens were prepared for routine histological examination. It was observed that CH and CTZ paste induced severe inflammation, a large amount of necrotic tissue, lymphocytes, foreign body cells and bone resorption, while Guedes Pinto Paste induced little or no inflammation in the 4-week observation period. After 12 weeks, the reactions to CH and Guedes Pinto paste were also absent/mild, presenting a general pattern of replacement by recently formed bone tissue while a moderate to severe inflammatory response was observed with CTZ paste. Guedes Pinto paste presented acceptable biocompatibility levels in both analyzed periods; CH only showed acceptable biocompatibility in the 12-week period while CTZ paste showed no biocompatibility in both periods. Among the tested materials, only Guedes Pinto paste presented an acceptable biocompatibility.

A pesquisa teve como objetivo avaliar a biocompatibilidade através da técnica de implantes intra-ósseos dos materiais utilizados em odontopediatria para tratamento pulpar: hidróxido de cálcio, pastas Guedes Pinto e CTZ, de acordo com as recomendações da FDI (1980) e ANSI/ADA(1982). Trinta guinea pigs, dez para cada material, divididos em períodos experimentais de 4 e 12 semanas receberam um implante em cada lado da sínfise mandibular. A parede lateral externa do copo serviu como controle para a técnica. No final dos períodos experimentais, os animais foram sacrificados e os espécimes preparados para o exame histológico de rotina. Observou-se que o hidróxido de cálcio e a pasta CTZ mostraram reação inflamatória severa, grande quantidade de tecido necrosado, linfócitos, células de corpo estranho e reabsorção óssea; enquanto a pasta Guedes Pinto induziu pouca ou nenhuma inflamação no período de 4 semanas. Após 12 semanas as reações para o hidróxido de cálcio e pasta Guedes Pinto foram ausentes/suaves apresentando um padrão geral de substituição por tecido ósseo neoformado, enquanto uma resposta inflamatória de moderada a severa foi observada para a pasta CTZ. A pasta Guedes Pinto apresentou níveis aceitáveis de biocompatibilidade nos dois períodos analisados; hidróxido de cálcio apresentou biocompatibilidade aceitável somente no período de 12 semanas e a pasta CTZ não mostrou biocompatibilidade em ambos os períodos. Entre estes, apenas a pasta Guedes Pinto apresentou níveis de biocompatibilidade nos dois períodos analisados.

Comparison among four commonly used demineralizing agents for root conditioning: a scanning electron microscopy

Dental roots that have been exposed to the oral cavity and periodontal pocket environment present superficial changes, which can prevent connective tissue reattachment. Demineralizing agents have been used as an adjunct to the periodontal treatment aiming at restoring the biocompatibility of roots. OBJECTIVE: This study compared four commonly used demineralizing agents for their capacity of removing smear layer and opening dentin tubules. METHODS: Fifty fragments of human dental roots previously exposed to periodontal disease were scaled and randomly divided into the following groups of treatment: 1) CA: demineralization with citric acid for 3 min; 2) TC-HCl: demineralization with tetracycline-HCl for 3 min; 3) EDTA: demineralization with EDTA for 3 min; 4) PA: demineralization with 37 percent phosphoric acid for 3 min; 5) Control: rubbing of saline solution for 3 min. Scanning electron microscopy was used to check for the presence of residual smear layer and for measuring the number and area of exposed dentin tubules. RESULTS: Smear layer was present in 100 percent of the specimens from the groups PA and control; in 80 percent from EDTA group; in 33.3 percent from TC-HCl group and 0 percent from CA group. The mean numbers of exposed dentin tubules in a standardized area were: TC-HCl=43.8±25.2; CA=39.3±37; PA=12.1±16.3; EDTA=4.4±7.5 and Control=2.3±5.7. The comparison showed significant differences between the following pairs of groups: TC-HCl and Control; TC-HCl and EDTA; CA and Control; and CA and EDTA. The mean percentages of area occupied by exposed dentin tubules were: CA=0.12±0.17 percent; TC-HCl=0.08±0.06 percent; PA=0.03±0.05 percent; EDTA=0.01±0.01 percent and Control=0±0 percent. The CA group differed significantly from the others except for the TC-HCl group. CONCLUSION: There was a decreasing ability for smear layer removal and dentin tubule widening as follows: AC>TC-HCl>PA>EDTA. This information can be of value as an extra parameter for choosing one of them for root conditioning.

Comparison of antibiotic resistance and virulence factors in pigmented and Non-pigmented Pseudomonas aeruginosa / Comparación de la resistencia antibiótica y los factores de virulencia en las Pseudomonas aeruginosa pigmentadas y no pigmentadas

OBJECTIVES: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However, pigment production appeared to be more significantly associated with multi-drug resistance, presence ofvirulence-associated genes, and expression ofcertain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status ofan isolate.

OBJETIVO: Pseudomonas aeruginosa produce múltiples factores de virulencia que han estado implicados en patogénesis y detección de quórum (quorum sensing). El objetivo de este estudio fue determinar las diferencias en los factores de virulencia de aislados de P aeruginosa pigmentada y no pigmentada. MÉTODO: Se evaluaron las asociaciones entre la producción de pigmentos (piocianina y pioverdina) y la producción de Dnasa, elastasa, lipasa, proteasa, sideróforos, motilidad asociada a superficies (twitching), patrones de resistencia antibiótica, y genes asociados con virulencia en 57 aislados de P aeruginosa no duplicados, de heridas, esputo, orina, exudado vaginal, exudados de oídos, ojos, y vías respiratorias, y aspirados de peritoneo y úlceras. RESULTADOS: La mayor parte (82.5%) de los aislados produjeron uno de los pigmentos. Los aislados pigmentados produjeron con mayor frecuencia y más significativamente (p < 0.05). Dnasa, elastasa, lipasa, proteasa, y siderósforos. Imipenem fue el único antibiótico al que todos los aislados eran susceptibles (p < 0.05), mientras que el 93% y el 32% fueron resistentes a la tetraciclina y a la norfloxacina, respectivamente. Sin embargo, no hubo diferencia significativa entre los aislados pigmentados y los no pigmentados cuando se comparaba la resistencia antibiótica. Si bien los aislados tenían múltiples genes asociados con la virulencia, exoS (51%), rhlA (37%) y rhlB (46%) fueron los genes predominantes detectados. Con excepción de exoY, los genes estuvieron presentes en aislados pigmentados con mayor frecuencia que en los aislados no pigmentados. CONCLUSIÓN: Los resultados de este estudio sugieren que la resistencia antibiótica per se podría no estar asociada con la producción de pigmentos en P aeruginosa. Sin embargo, la producción de pigmentos parecía estar asociada más significativamente con la resistencia a las multidrogas, la presencia de genes asociados con la virulencia, y la expresión de ciertos factores de virulencia, en particular la actividad de la elastasa, la proteasa, los sideróforos, y la Dnasa. Puesto que la producción de pigmentos es fácil de determinar, esto podría ser un buen punto de partida para identificar el estado de virulencia de un aislado.

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser83-->Arg83 or Ser83-->Asn83. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.

Regional Difference of Antibiotic Resistance of Helicobacter pylori Strains in Korea / 대한소화기학회지

BACKGROUND/AIMS: This study was performed to compare the prevalence rates of primary antibiotic resistance in Helicobacter pylori (H. pylori) isolates among different regions of Korea. METHODS: H. pylori were isolated from gastric mucosal biopsy specimens of 99 Koreans who lived in Gyeonggi (n=40), Kangwon province (n=40) and Busan (n=19) from April to August in 2008. All the patients had no history of H. pylori eradication therapy. The susceptibilities of the H. pylori isolates to amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin, and moxifloxacin were tested according to the agar dilution method. RESULTS: There was a difference in resistance to clarithromycin in three institutes located among Gyeonggi (32.5%), Kangwon province (12.5%) and Busan (42.1%) by One way ANOVA test (p=0.027) and nonparametric Kruskal Wallis test (p=0.027). However, by post-hoc analysis, there was no statistically significant difference among three regions. Similarly, the other 7 antibiotics (amoxicillin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin) did not show any significant difference. CONCLUSIONS: There was no significant regional difference of the primary antibiotic resistance of H. pylori. However, the included patient number might not be enough for this conclusion demanding further evaluations.