Quinolones resistance in Salmonella spp. isolated from broilers and chickens carcasses under federal inspection / Resistência à quinolonas em Salmonella spp. isoladas de frangos vivos e carcaças sob inspeção federal

We analyzed 77 Salmonella spp. strains, from which 20 were isolated from broilers (cloacal swabs) and 57 from chickens from slaughterhouses under federal inspection. The following serotypes were identified: Salmonella Saint Paul (29), Salmonella Heidelberg (27), Salmonella Anatum (9), Salmonella Cerro (5), Salmonella Senftenberg (5), Salmonella enterica (O: 4,5) (1) and Salmonella enterica (O: 9.12) (1). Fifteen strains (19.5%) were resistant to enrofloxacin, six (7.8%) to ciprofloxacin, and 26 (33.8%) to nalidixic acid in the Disk Diffusion Test. The fifteen enrofloxacin resistant strains were selected for the PCR to detect the genes gyrA, gyrB, parC, and parE, and genetic sequencing to identify mutations in these genes. Five strains (33.3%) had point mutations in the gyrA gene, and one (6.7%) presented a point mutation in the parC gene. None of the 15 strains had mutations in the gyrB and parE genes, and none had more than one mutation in the gyrA gene or the other genes. The presence of point mutations in the strains studied corroborates with the phenotypic resistance observed to nalidixic acid. However, it did not explain the resistance to fluoroquinolones found in the 15 strains. Other mechanisms may be related to the fluoroquinolones resistance, highlighting the need for additional mutation screening.(AU)

Foram analisadas neste estudo 77 estirpes de Salmonella spp., 20 isoladas de frangos vivos (suabes de cloaca) e 57 isoladas de carcaças, provenientes de abatedouros frigoríficos sob Inspeção Federal. Foram identificados os seguintes sorotipos: Salmonella Saint Paul (29), Salmonella Heidelberg (27), Salmonella Anatum (9), Salmonella Cerro (5), Salmonella Senftenberg (5), Salmonella enterica (O: 4,5) (1) e Salmonella enterica (O: 9,12) (1). Do total de estirpes estudadas, 15 (19,5%) se mostraram resistentes à enrofloxacina, seis (7,8%) à ciprofloxacina e 26 (33,8%) ao ácido nalidíxico no Teste de Difusão em Disco. Foram selecionadas as 15 estirpes resistentes à enrofloxacina para a realização da PCR para detecção dos genes gyrA, gyrB, parC e parEe para sequenciamento genético do produto da PCR para identificação de mutações nesses genes. Cinco estirpes (33,3%) apresentaram mutações pontuais no gene gyrA e uma (6,7%) apresentou mutação pontual no gene parC. Nenhuma das 15 estirpes apresentou mutações nos genes gyrB e parE e nenhuma apresentou mais de uma mutação no gene gyrA ou nos outros genes. A existência apenas de mutações pontuais em alguns genes das estirpes analisadas está de acordo com a resistência fenotípica observada ao ácido nalidíxico, mas não explica a resistência às fluoroquinolonas encontrada nas 15 estirpes. Outros mecanismos de resistência podem estar relacionados à resistência encontrada às fluoroquinolonas e estudos adicionais são necessários para investigar sua presença.(AU)

Quinolones resistance in Salmonella spp. isolated from broilers and chickens carcasses under federal inspection

ABSTRACT: We analyzed 77 Salmonella spp. strains, from which 20 were isolated from broilers (cloacal swabs) and 57 from chickens from slaughterhouses under federal inspection. The following serotypes were identified: Salmonella Saint Paul (29), Salmonella Heidelberg (27), Salmonella Anatum (9), Salmonella Cerro (5), Salmonella Senftenberg (5), Salmonella enterica (O: 4,5) (1) and Salmonella enterica (O: 9.12) (1). Fifteen strains (19.5%) were resistant to enrofloxacin, six (7.8%) to ciprofloxacin, and 26 (33.8%) to nalidixic acid in the Disk Diffusion Test. The fifteen enrofloxacin resistant strains were selected for the PCR to detect the genes gyrA, gyrB, parC, and parE, and genetic sequencing to identify mutations in these genes. Five strains (33.3%) had point mutations in the gyrA gene, and one (6.7%) presented a point mutation in the parC gene. None of the 15 strains had mutations in the gyrB and parE genes, and none had more than one mutation in the gyrA gene or the other genes. The presence of point mutations in the strains studied corroborates with the phenotypic resistance observed to nalidixic acid. However, it did not explain the resistance to fluoroquinolones found in the 15 strains. Other mechanisms may be related to the fluoroquinolones resistance, highlighting the need for additional mutation screening.

RESUMO: Foram analisadas neste estudo 77 estirpes de Salmonella spp., 20 isoladas de frangos vivos (suabes de cloaca) e 57 isoladas de carcaças, provenientes de abatedouros frigoríficos sob Inspeção Federal. Foram identificados os seguintes sorotipos: Salmonella Saint Paul (29), Salmonella Heidelberg (27), Salmonella Anatum (9), Salmonella Cerro (5), Salmonella Senftenberg (5), Salmonella enterica (O: 4,5) (1) e Salmonella enterica (O: 9,12) (1). Do total de estirpes estudadas, 15 (19,5%) se mostraram resistentes à enrofloxacina, seis (7,8%) à ciprofloxacina e 26 (33,8%) ao ácido nalidíxico no Teste de Difusão em Disco. Foram selecionadas as 15 estirpes resistentes à enrofloxacina para a realização da PCR para detecção dos genes gyrA, gyrB, parC e parEe para sequenciamento genético do produto da PCR para identificação de mutações nesses genes. Cinco estirpes (33,3%) apresentaram mutações pontuais no gene gyrA e uma (6,7%) apresentou mutação pontual no gene parC. Nenhuma das 15 estirpes apresentou mutações nos genes gyrB e parE e nenhuma apresentou mais de uma mutação no gene gyrA ou nos outros genes. A existência apenas de mutações pontuais em alguns genes das estirpes analisadas está de acordo com a resistência fenotípica observada ao ácido nalidíxico, mas não explica a resistência às fluoroquinolonas encontrada nas 15 estirpes. Outros mecanismos de resistência podem estar relacionados à resistência encontrada às fluoroquinolonas e estudos adicionais são necessários para investigar sua presença.

Fecal peritonitis in aging rat model. Therapeutic response to different antibiotic strategies

Purpose: To evaluate the response of aging rats with sepsis to two different antibiotic regimens. Methods: The study was conducted with 30 aging rats (18 month-old) with autologous feces peritonitis. The animals were divided into three groups: Group 0 received no therapeutic intervention (control), while Group 1 received a single dose of 40 mg/kg meropenem and Group 2 received a single dose of 20 mg/kg moxifloxacin. The intervention in both Groups was made 6 hours after induction of peritonitis. The animals were followed up to 15 days for evaluating morbidity and mortality. The weights at baseline were similar in all groups. Results: At the end of follow-up, weight loss was significantly greater (p=0.0045) in Group 0 (non-intervention controls). Culture from a blood sample at the end of follow-up was positive in all the animals in Group 0, in two animals in Group 1 and in four animals in Group 2. Morbidity/mortality was significantly higher in Group 0 compared to both Groups 1 and 2 (p=0.003) but the scores were not significantly different between Groups 1 and 2 (p=0.6967). Conclusion: Both antibiotic regimens rendered promising results for the treatment of fecal peritonitis.(AU)

Antimicrobial susceptibility of Salmonella Gallinarum and Salmonella Pullorum isolated from ill poultry in Brazil / Suscetibilidade a antimicrobianos de Salmonella Gallinarum e Salmonella Pullorum isolados de aves doentes no Brasil

Salmonella Gallinarum (S. Gallinarum) and Salmonella Pullorum (S. Pullorum) are poultry host-specific, agents of fowl typhoid and pullorum disease, respectively. These biovars cause septicemic infections, resulting in high mortality. Outbreaks are frequently reported worldwide, causing losses due to the elimination of infected flocks and treatments. The use of antimicrobial agents is frequent in poultry farms to prevent or treat gastrointestinal infections. In the present research it was evaluated the antimicrobial susceptibility of 50 S. Gallinarum and S. Pullorum isolates, from outbreaks that occurred between 1987 to 1991 and 2006 to 2013. The comparison of the susceptibility profiles showed that all isolates were susceptible to -lactams. All isolates from 1987-1991 were susceptible to all antibiotics tested except NAL and CIP (78%). The susceptibility profile of S. Gallinarum (2006 - 2013 period) was the following NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) and SXT (96%). S. Pullorum isolates (2006 - 2013 period) showed the following susceptibility rates to NAL (65%), CIP (71%), ENR (94%) and TET (94%). All isolates were susceptible to -lactams tested, however, resistance to quinolones and fluoroquinolones increased over time. Furthermore, low levels of resistance to other antibiotics were found in recent isolates, such as tetracyclines.(AU)

Salmonella Gallinarum (S. Gallinarum) e Salmonella Pullorum (S. Pullorum) são patógenos hospedeiro-específico de aves, agentes do tifo aviário e pulorose, respectivamente. Estes biovares causam infecções septicêmicas, resultando em alta mortalidade. Surtos são frequentemente relatados em diversos países, causando prejuízos devido à eliminação de lotes infectados e tratamentos. Agentes antimicrobianos são utilizados frequentemente em granjas avícolas para prevenir ou tratar infecções gastrointestinais. No presente trabalho, foi avaliada a susceptibilidade antimicrobiana de 50 isolados de S. Gallinarum e S. Pullorum, obtidos durante surtos que ocorreram entre 1987 a 1991 e entre 2006 a 2013. A comparação dos perfis de sensibilidade mostrou que todas as amostras são sensíveis aos -lactâmicos. Todos os isolados de 1987-1991 foram sensíveis a todos os antibióticos testados, exceto NAL e CIP (78%). O perfil de susceptibilidade de S. Gallinarum (surtos de 2006 a 2013) foi NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) e SXT (96%). Isolados de S. Pullorum (surtos de 2006 a 2013) apresentaram as seguintes taxas de sensibilidade: NAL (65%), CIP (71%), ENR (94%) e TET (94%). Todas as amostras foram sensíveis ao -lactâmicos testados, no entanto, a resistência às quinolonas e fluoroquinolonas aumentou ao longo do tempo. Além disso, baixos níveis de resistência a outros antibióticos foram encontrados em isolados recentes, tais como as tetraciclinas.(AU)

Detection of extended spectrum beta-lactamases and resistance in members of the Enterobacteriaceae family isolated from healthy sheep and dogs in Umuarama, Paraná, Brazil / Detecção de beta-lactamases de espectro estendido e resistência as quinolonas mediada por plasmídeos em membros da família Enterobacteriaceae isolados de ovinos e cães sadios de propriedades rurais da região de Umuarama, Paraná, Brasil

Bacterial resistance is a primary public health concern worldwide. Within this context, pets and breeding animals act as reservoirs for multidrug-resistant bacteria (MR), such as those producing extended spectrum beta-lactamases (ESBL) and those presenting plasmid-mediated quinolone resistance (PMQR). The aim of this study was to detect the presence of ESBL and PMQR in members of the Enterobacteriaceae family, isolated from healthy sheep and dogs from non-intense farming rural properties in the Umuarama region of Paraná, Brazil. A total of 81 oral and rectal swabs from dogs and sheep from 11 small rural properties were analyzed. These swabs were inoculated in tubes containing brain heart infusion broth (BHI), and the resulting cultures were inoculated on MacConkey agar (MAC) supplemented with 10 μg/mL cefotaxime for the selection of ESBL producers. The cells were also plated on MAC supplemented with 50 μg/mL nalidixic acid for selecting quinolone-resistant enterobacteria. The bacterial isolates were subjected to biochemical identification tests, antibiograms, double-disk synergic tests, and polymerase chain reaction analysis for resistance-inducing genes (blaESBL, qnr, and genes encoding efflux pump and acetylases). Four (5.00%) bacterial isolates (3 Escherichia coli and 1 Morganella morganii) resistant to cephalosporins and/or quinolones were identified; of these, three (75%) isolates were from sheep and one (25%) from a dog. These findings indicate the presence of MR bacteria in the normal microbiota of the animals studied. Animals colonized with such bacteria can contribute to the dissemination of antimicrobial resistance to other animals, environment, and/or human beings and can harbor endogenous infections in unfavorable conditions, which have poor prognosis due to the limited therapeutic options.(AU)

Resistência bacteriana é considerado o maior problema de saúde pública mundial da atualidade, sendo os relatos de infecções e surtos causados por bactérias multirresistentes cada vez mais frequentes na clínica veterinária e humana. Neste contexto, animais de companhia e criação podem atuar como reservatório de bactérias multirresistentes, como as produtoras de beta-lactamases de espectro estendido (ESBL) e as que apresentam resistência as quinolonas mediada por plasmídeos (PMQR). O objetivo deste trabalho foi detectar beta-lactamases de espectro estendido e resistência as quinolonas mediada por plasmídeos em membros da família Enterobacteriaceae isolados de ovinos e cães de propriedades rurais não tecnificadas da região de Umuarama, Paraná, Brasil. Foram analisados 81 swabs de cães e ovinos provenientes de 11 pequenas propriedades rurais da região de Umuarama (PR). Os swabs foram inoculados em caldo Brain Heart Infusion (BHI) e o crescimento obtivo foi em seguida semeado em placas de Petri contendo ágar MacConkey (MC) acrescido de cefotaxima 10 μg/mL para seleção de bactérias gram-negativas produtoras de ESBL; e placas contendo MC acrescido de ácido nalidíxico 50 g/mL para seleção de bactérias gram-negativas resistentes as quinolonas. Os isolados bacterianos obtidos foram submetidos a testes de antibiograma pelo método de disco-difusão em ágar, teste sinérgico do duplo-disco e reação em cadeia da polimerase (PCR) para genes que conferem resistência do tipo ESBL e para quinolonas. Dos 81 swabs coletados foi possível detectar quatro (4,97%) isolados bacterianos (3Escherichia coli e 1 Morganellamorganii) resistentes a cefalosporinas e/ou quinolonas. Destes isolados, três (75%) eram de ovinos e um (25%) de cão. Os resultados encontrados indicam a presença de cepas multirresistentes na microbiota normal dos animais estudados.[...](AU)

Brain damage and congenital cataract due to autogenously fecal peritonitis in pregnant Wistar rats

PURPOSE: To investigate the morphological aspects of brain and eyes in newborn rats whose mother underwent autogenously fecal peritonitis. METHODS: Four pregnant rats that underwent fecal peritonitis, with a 10% fecal suspension in dose of 4 ml per kilogram received two antimicrobial treatments: 1. intraperitoneal moxifloxacin and dexamethazone; and 2. Intravenous meropenem. After head inspection, the brain consistencies and the eyes belonging to all offspring were analyzed. RESULTS: The brains of newborn from rats that received 4 ml/kg of 10% suspension of feces showed, significantly smaller and less than the firm consistency of those in the control group. Congenital cataract was observed in 9 (34.6%). No cataract was observed in the 20 newborn rats from the mothers that received the combination of moxifloxacin and dexamethasone. Cataract could be observed in three (13.6%) offspring from mothers that received meropenem. CONCLUSIONS: Peritonitis can produce brain damage and congenital cataract in rats. The translation to humans is that intra abdominal infection in pregnant women may be associated with damage in brain and eye structures of their concepts. This can be averting using the adequate early therapeutically approach. .(AU)

Severe autogenously fecal peritonitis in Wistar rats with permanent bilateral carotid occlusion. Response to intra peritoneal moxifloxacin combined with dexamethasone

To evaluate the treatment outcome of severe peritonitis in rats submitted to permanent bilateral carotid occlusion (PBCO). METHODS: Sixteen Wistar rats (mean age of 8.5 months) with PBCO underwent autogenously fecal peritonitis, and were treated with moxifloxacin combined with dexamethasone, and followed-up for 45 days. Ten rats (mean age five months) without PBCO were used as a control group. The variables were expressed by their mean and standard error of the mean (SEM). p<0.05 was used for rejecting the null hypothesis. The study was approved by the Ethics Committee. RESULTS: There was a significant increase (p=0.0002) in the mortality and morbidity in older rats that underwent PBCO (study group). However, even among the survival rats presenting with severe residual abscesses both in the abdomen and thorax cavities, they present an almost normal life. CONCLUSIONS: The treatment of severe autogenously fecal peritonitis with intraperitoneal moxifloxacin combined with dexamethasone was very effective in young rats without permanent bilateral carotid occlusion. The treatment reached reasonable results in older rats with PBCO, even considering residual abscesses on abdomen and thorax. Older age was the greater risk factor for the outcome of the treatment of severe peritonitis. Sepsis remains a challenging situation, especially in elderly.(AU)

Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates

To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.(AU)

Determinantes emergentes de resistência antimicrobiana em Escherichia coli de origem clínica, fecal e de carne de aves e suínos

As grandes quantidades de antimicrobianos utilizados na produção de aves e suínos é um problema de saúde pública em todo mundo. O surgimento e disseminação de novos mecanismos de resistência a antibióticos de importância clínica em medicina humana e veterinária é fato que tem apontado a entrada de uma era pós-antibióticos. Considerando a importância da avicultura e suinocultura na economia brasileira e os riscos para saúde humana e dos animais, o objetivo deste estudo foi determinar a ocorrência de genes emergentes de resistência aos antibióticos das classes dos -lactâmicos, quinolonas, fosfomicina e colistina. Foram selecionados 1.152 isolados de Escherichia coli de origem clínica, fecal e de carne de suínos, frangos de corte, poedeiras e perus proveniente dos principais estados produtores e exportadores do Brasil (SP, RS, SC, PR, MG e GO). Através de métodos clássicos de biologia molecular e sequenciamento do genoma completo foi realizada a caracterização genotípica dos isolados, assim como a caracterização dos elementos genéticos móveis que carreavam esses genes. Dentre os isolados de aves (n=773), 103 (13,4%) foram produtores de ESBL: CTX-M-2 (n=61); CTX-M-55 (n=26); CTX-M-8 (n=12); CTX-M-2 + CTX-M-55 (n=3); CTX-M-2+CTX-M-8 (n=1); CTX-M-8+CTX-M-55 (n=1). 44 (5,7%) apresentaram CMY-2, presente em plasmídeos IncI1/ST12 e IncK. Em relação à resistência às quinolonas e colistina mediada por plasmídeos, os genes encontrados foram qnrA1 (1%), qnrB19 (6%) e qnrS1 (0,8%), além de 41 isolados positivos para mcr-1. A ocorrência de genes plasmidiais de resistência à fosfomicina (fosA3) foi de 3,4%, que estiveram relacionados a plasmídeos epidêmicos IncN/F33:A-:B-. Dentre os isolados de E. coli patogênicas (ETEC, STEC), comensais e de carne de suínos (n=378) foi encontrada uma alta prevalência de cepas positivas para mcr-1 (33,8%). Foram encontrados três isolados positivos para mcr-3 dentre os isolados de ETEC e comensais. O gene qnrS1 também teve alta prevalência (24,6%). Nos isolados de aves e suínos, o gene mcr-1 esteve presente em plasmídeos IncX4. Análises da sequência completa de DNA de vários plasmídeos mostrou relação com plasmídeos que circulam em criações animais na Ásia. Este estudo contribui para o estabelecimento de um cenário em relação à resistência antimicrobiana na produção animal no Brasil. Levando em conta que o Brasil está entre os maiores exportadores de carne de aves e suínos do mundo, é urgente a elaboração de estratégias para o controle da disseminação de bactérias resistentes a antibióticos clinicamente importantes.

The large amounts of antimicrobials used in the poultry and swine production is a public health concern worldwide. The emergence and spread of novel resistance mechanisms to important antibiotics in human and veterinary medicine is a fact that has pointed to entry into a "postantibiotic" era. Considering the importance of poultry and pig farming in the Brazilian economy and the risks to human and animal health, the aim of this study was to determine the occurrence of emerging resistance genes to -lactam, quinolones, fosfomycin and colistin antibiotics. A total of 1.152 of clinical, fecal and retail meat isolates of Escherichia coli from from swine, broilers, laying hens and turkeys from the main producing and exporting states of Brazil (SP, RS, SC, PR, MG and GO) were selected. Through classical molecular biology methods and whole genome sequencing, the characterization of the isolates was performed, as well as the characterization of the mobile genetic elements that carried these genes. Among avian isolates (n = 773), 103 (13.4%) were ESBL-producers: CTX-M-2 (n = 61); CTX-M-55 (n = 26); CTXM-8 (n = 12); CTX-M-2 + CTX-M-55 (n = 3); CTX-M-2 + CTX-M-8 (n = 1); CTX-M-8 + CTX-M-55 (n = 1). 44 (5.7%) showed pAmpC CMY-2, present in IncI1/ST12 and IncK plasmids. An isolate belonging to ST117 harboring blaCMY2 and blaCTX-M-2 integrated into the chromosome. Regarding plasmid-mediated resistance to quinolones and colistin, the genes found were qnrA1 (1%), qnrB19 (6%) and qnrS1 (0.8%), in addition to 41 mcr-1 positive isolates. The occurrence of plasmid-mediated fosfomycin resistance (fosA3) was 3.4%, which were related to the IncN/F33: A-: B- epidemic plasmids. A high prevalence of mcr-1 positive strains (33.8%) was found among pathogenic (ETEC, STEC) and commensal porcine E. coli isolates (n = 378). Three mcr-3 positive isolates were found among the ETEC and commensal isolates. The qnrS1 gene also had a high prevalence (24.6%). In avian and porcine isolates, the mcr-1 gene was present in IncX4 plasmids. Analyzes of the complete DNA sequence of various plasmids showed relation to plasmids circulating in animal production in Asia. This study contributes to the determination of a national scenario of antimicrobial resistance in animal production in Brazil. Taking into account that Brazil is among the largest exporters of poultry and pork in the world, it is urgent to devise strategies to control the spread of multidrug resistant bacteria to clinically important antibiotics.

Efficacy evaluation of some antibiotics against syrian brucella spp isolates, in vitro

Brucellosis is an endemic zoonosis in Syria, affecting large numbers of animals and there are an increasing number of cases in humans. The aim of this study is to investigate the in vitro efficacy of various traditional and new antibiotics against89 Brucella isolates (isolated from domestic animals) collected from different Syrian regions. Minimum inhibitory concentrations (MICs) of seventeen antibiotics were determined. Ciprofloxacin and ofloxacin were the most effective antibiotics, whereas sparfloxacin, levofloxacin, doxycycline and tetracycline had a moderate activity. In contrast, moxifloxacin and rifampicin had a low activity, while streptomycin, spiramycin and cephalosporines were ineffective. As a result, we come to the conclusion that a combination between one effective quinolone and doxycycline has a good efficacy against Brucella. Further in vivo studies are necessary to support this suggestion.(AU)

Detection of quinolone-resistance mutations in Salmonella spp. strains of epidemic and poultry origin

Mutations into codons Aspartate-87 (62%) and Serine-83 (38%) in QRDR of gyrA were identified in 105 Salmonella strains resistant to nalidixic acid (94 epidemic and 11 of poultry origin). The results show a high incidence of mutations associated to quinolone resistance but suggest association with others mechanisms of resistance.

Ciprofloxacin susceptibility reduction of Salmonella strains isolated from outbreaks

The antimicrobial susceptibility of 212 Salmonella strains isolated from patients and foods was evaluated and 45% were found to be resistant to nalidixic acid. Nalidixic acid resistant strains showed a higher minimal inhibitory concentration for ciprofloxacin than sensitive strains. During the study an increase of strains with reduced susceptibility to ciprofloxacin was also observed.

DISSEMINAÇÃO DE CLONES DE SALMONELLA SPP. RESISTENTES A DIFERENTES GERAÇÕES DE QUINOLONAS NA CADEIA ALIMENTAR.

As salmonelas são reconhecidas como causa comum de doenças transmitidas por alimentos (DTA) em humanos e representam um grave problema de saúde pública. Uma dificuldade adicional no controle das infecções bacterianas decorrentes deste gênero é a crescente resistência antimicrobiana. As quinolonas/fluoroquinolonas aparecem como uma estratégia terapêutica frente a estes isolados resistentes. A aquisição de resistência às quinolonas por Salmonella spp. são um alerta para comunidade cientifica assim como a possibilidade de transmissão de resistência pelo envolvimento de plasmídeos. O presente trabalho buscou avaliar os mecanismos envolvidos com a resistência às quinolonas e fluorquinolonas em isolados de Salmonella spp. Foram avaliados 152 isolados, sendo 39 oriundos de fontes alimentares de origem animal, 14 de origem ambiental, 32 de origem animal e 67 de origem humana, encaminhados ao Laboratório de Referência Nacional de Enteroinfecções Bacterianas (LRNEB) no período de 2009 a 2013 que apresentaram perfil de resistência às quinolonas/fluoroquinolonas testadas. Foi possível reconhecer 33 sorovares sendo os 6 prevalentes S.Typhimurium (63/152), seguido de S. Enteritidis (25/152), S. Gallinarum (23/152), S. Muenchen (4/152), S. Heidelberg (3/152), S. Infantis (3/152) e S. Saintpaul (3/152). No teste de difusão em disco foram detectados 83,5% de resistência ao acido nalidixico, 52% à enrofloxacina, 39,5% a ciprofloxacina, 36,8% a ofloxacina e 31,6% a levofloxacina. Outras classes de antimicrobianos foram testadas para avaliação de resistência. Elevadas taxas de resistência em isolados resistentes às quinolonas/fluorquinolonas foram evidenciadas frente as tetraciclinas, beta-lactâmicos e aminoglicosideos. No teste de microdiluição em caldo para determinação da CIM percentuais de resistência foram observados para: ácido nalidixico (94,7%), enrofloxacina (66,4%), ciprofloxacina (58,5%), ofloxacina (28,9%) e levofloxacina (25,7%). Quanto a detecção de genes de resistência foi possível verificar a presença de 18 isolados positivos para os genes qnr (8 qnrS, 6 qnrB e 4 qnrD), e 23 isolados aac(6)-Ib positivos. O gene integron foi detectado em 63 isolados com regiões variáveis entre +/- 600pb a >1000pb. O aumento de isolados de Salmonella spp. carreando genes PMQR é um sério problema que deve ser cuidadosamente monitorado. A avaliação da relação clonal entre os sorovares prevalentes que apresentaram resistência a diferentes classes de quinolonas, através da técnica de eletroforese em campo pulsado (PFGE), revelou que alguns pulsotipos encontrados foram relacionados à circulação de cepas resistentes as quinolonas, sendo estas encontradas nas diferentes fontes de isolamento e algumas circulantes em todo o território nacional.

Salmonellosis are recognized as a common cause of foodborne disease (FBD) in humans and represent a serious public health problem. The increasing of antimicrobial resistance is an additional difficulty in controlling bacterial infections from Salmonella spp. Quinolones/fluoroquinolones appear as a therapeutic strategy against these resistant isolates. The acquisition of resistance to quinolones in Salmonella spp. are a warning to the scientific community as well as the possibility of transmission of resistance by the involvement of plasmids. This study aimed to evaluate the mechanisms involved in resistance to quinolones and fluoroquinolones in isolates of Salmonella spp. We evaluated 152 isolates, 39 originated from food animal sources, 14 of environmental origin, 32 animal and 67 of human origin, referred to the National Reference Laboratory of Enteric Diseasis (LRNEB/IOC/RJ) in the period 2009-2013 that showed resistance profile of the quinolones/fluoroquinolones tested. It was possible to recognize 33 serotypes being the sixth most prevalent S.Typhimurium (63/152), followed by S. Enteritidis (25/152), S. gallinarum (23/152), S. Muenchen (4/152), S. Heidelberg (3/152), S. Infantis (3/152) and S. Saintpaul (3/152). In disk diffusion test were detected 83.5% resistance to nalidixic acid, 52% to enrofloxacin, ciprofloxacin 39.5%, 36.8% ofloxacin and 31.6% levofloxacin. Other classes of antimicrobials were tested to evaluate resistance. Tetracyclines, aminoglycosides and beta-lactams showed high rates of resistance in isolates with resistance to quinolones/fluoroquinolones. The broth microdilution test for MIC determination the following percentages of resistance were obtained: 94.7% to nalidixic acid, 66.4% for enrofloxacin, 58.5% to ciprofloxacin, 28.9% to ofloxacin and 25,7% to levofloxacin. As the detection of resistance genes was possible to verify the presence of 18 isolates positive for qnr genes (8qnrS, 6 qnrB and 4 qnrD), and 23 isolates aac(6')-Ib positive. The integron gene was detected in 63 isolates the variable regions of the +/- 600pb > 1000pb. The increase of isolates of Salmonella spp. silted PMQR genes is a serious problem that must be carefully monitored. The isolates were subjected to molecular typing method by pulsed field gel electrophoresis which were evaluated clonally among the most prevalent serovars that are resistant to different classes of quinolones. Some found pulsotypes were related movement of the strains resistant to quinolones, which are found in different sources of isolation and some circulating throughout the national territory.

DETECÇÃO DE BETA-LACTAMASES DE ESPECTRO ESTENDIDO E RESISTÊNCIA AS QUINOLONAS MEDIADA POR PLASMÍDEOS EM MEMBROS DA FAMÍLIA ENTEROBACTERIACEAE ISOLADOS DE OVINOS E CÃES SADIOS DE PROPRIEDADES RURAIS DA REGIÃO DE UMUARAMA, PARANÁ, BRASIL.

A resistência bacteriana é considerada o maior problema de saúde pública mundial da atualidade. Neste contexto, animais de companhia e criação atuam como reservatório de bactérias multirresistentes (MR), como as produtoras de beta-lactamases de espectro estendido (ESBL)e as que apresentam resistência as quinolonas mediada por plasmídeos (PMQR). O objetivo deste estudo foidetectar enterobactérias produtoras de beta-lactamases de espectro estendido (ESBL) e/ou resistentes as quinolonas em isolados de ovinos e cães sadios de propriedades rurais da região de Umuarama, Paraná, Brasil.Foram analisados 81 swabs de cães e ovinos de 11 pequenas propriedades rurais. Tubos contendo caldo Brain Heart Infusion (BHI) foram inoculados com os swabs coletados e o crescimento obtido foi semeado em ágar MacConkey (MC) acrescido de cefotaxima (CTX) 10 g/mLpara seleção de enterobactérias produtoras de ESBL; e placas contendo MC acrescido de ácido nalidíxico (NAL) 50 g/mL para seleção de enterobactérias resistentes as quinolonas. Os isolados bacterianos foram submetidos a testes de identificação bioquímica, antibiograma, teste sinérgico do duplo-disco e reação em cadeia da polimerase (PCR) para genes de resistência (blaESBL, qnr, bomba de efluxo e acetilases). Dos 81 swabs analisados foi possível identificar quatro isolados bacterianos (3 Escherichia coli e 1 Morganella morganii) resistentes a cefalosporinas e/ou quinolonas. Destes isolados, três (75%) eram de ovinos e um (25%) de cão. Os resultados encontrados indicam a presença debactérias MR na microbiota normal dos animais estudados. Animais colonizados com este tipo de bactérias podem contribuir para disseminação daresistência para outros animais, ambiente e/ou homem, ou em uma situação desfavorável, adquirir uma infecção endógena, com prognóstico ruim, devidoopções terapêuticas reduzidas.

Bacterial resistance is considered one of the main problems in current global public health. Within this context, pets and breeding animals act as reservoirs for multi-resistant bacteria (MR), such as the extended spectrum beta-lactamases (ESBL) producers, and those presenting plasmid-measured quinolone resistance (PMQR). The aim this study was to detect producing enterobacteria of beta-lactamases of extended spectrum (ESBL) and / or resistant to quinolones in isolated sheep and healthy dogs from rural properties in Umuarama region, Paraná, Brazil.A total of 81 swabs from dogs and sheep from 11 small rural properties were analyzed. Tubes containing Brain Heart Infusion (BHI) broth were inoculated with the swabs collected and the growth obtained was sowed in MacConkey (MC) agar added with 10-g/mL cefotaxime (CTX) for the selection of ESBL producing enterobacteria; and plates containing MC added with 50-g/mL nalidixic acid (NAL) for selecting enterobacteria resistant to quinolones. The bacterial isolates were submitted to biochemical identification tests, antibiogram, double-disk synergic test and polymerase chain reaction (PCR) for resistance genes (blaESBL, qnr, outflow pump and acetylases). From the 81 swabs analyzed, it was possible to identify four bacterial isolates (3 Escherichia coli and 1 Morganella morganii) resistant to cephalosporins and/or quinolones. From these isolates, three (75%) were from sheep and one (25%) from a dog. The findings indicate the presence of MR bacteria in the normal biota of the animals studied. Animals colonized with this type of bacteria can contribute to the dissemination of resistance to other animals, environments and/or human beings, or in an unfavorable situation, acquire an endogenous infection, with a bad prognosis, due to reduced therapeutic options.

Uso da orbifloxacina em piodermites caninas / Use of orbifloxacin in canine pyoderma

A orbifloxacina é uma fluoroquinolona de terceira geração indicada para o tratamento de várias infecções. Estudos recentes realizados in vitro demonstraram sua eficácia contra S. intermedius isolado de infecções de pele canina. No atual estudo, não foi observada diferença significativa entre os grupos de animais. No entanto, 65,6% dos animais obtiveram remissão completa dos sintomas com a dose média de 5,0 mg/kg administrada a cada 24 horas, durante 30 dias. Não foram relatados efeitos colaterais em nenhum dos animais. A orbifloxacina, na dose de 5,0 mg/kg, mostrou-se um fármaco seguro, prático e eficaz para o tratamento de piodermites na clínica veterinária.(AU)

Orbifloxacin is a third generation fluoroquinolone indicated for the treatment of many infections. Recent studies demonstrated its efficacy in vitro against S. intermedius isolated from canine skin infections. In the current study, there was no significant difference between groups of animals. However, 65.6% of the animals had complete remission of symptoms with 5.0 mg/kg administered every 24 hours for 30 days. No side effects were reported. Orbifloxacin 5.0 mg/kg demonstrated safe, practical and effective drug for treatment of pyoderma in veterinary practice.(AU)

Uso da orbifloxacina em piodermites caninas / Use of orbifloxacin in canine pyoderma

A orbifloxacina é uma fluoroquinolona de terceira geração indicada para o tratamento de várias infecções. Estudos recentes realizados in vitro demonstraram sua eficácia contra S. intermedius isolado de infecções de pele canina. No atual estudo, não foi observada diferença significativa entre os grupos de animais. No entanto, 65,6% dos animais obtiveram remissão completa dos sintomas com a dose média de 5,0 mg/kg administrada a cada 24 horas, durante 30 dias. Não foram relatados efeitos colaterais em nenhum dos animais. A orbifloxacina, na dose de 5,0 mg/kg, mostrou-se um fármaco seguro, prático e eficaz para o tratamento de piodermites na clínica veterinária.

Orbifloxacin is a third generation fluoroquinolone indicated for the treatment of many infections. Recent studies demonstrated its efficacy in vitro against S. intermedius isolated from canine skin infections. In the current study, there was no significant difference between groups of animals. However, 65.6% of the animals had complete remission of symptoms with 5.0 mg/kg administered every 24 hours for 30 days. No side effects were reported. Orbifloxacin 5.0 mg/kg demonstrated safe, practical and effective drug for treatment of pyoderma in veterinary practice.

Determination of quinolones by high performance liquid chromatography using a monolitic reversed-phase column / Determinação de quinolonas por cromatografia líquida de alta eficiência empregando coluna monolítica de fase reversa

Quinolones are synthetic antibacterial agents used for treating a wide variety of infections of urinary tract, gastrointestinal tract, and skin. The aim of this study was to develop a method for performing the determination of ciprofloxacin, ciprofloxacin ethylenediamine analog (a related compound), and norfloxacin by means of high performance liquid chromatographic (HPLC) technique. The compounds were separated on a Chromolith RP18 column, with isocratic system. The mobile phase was a mixture of phosphoric acid 0.042 mol/L, pH 3.0 ± 0.1: acetonitrile (87:13), flow rate of 1.6 mL/min and UV detection at 275nm. The range of calibration curve was 20.0-125.0 µg/mL for ciprofloxacin and 20.0-100.0 µg/mL for norfloxacin. The correlation coefficient was 0.9999 for ciprofloxacin and 0.9999 for norfloxacin. The retention time for ciprofloxacin was 3.4 min, for ethilenodiamine ciprofloxacin was 2.4 min, and 2.9 min for norfloxacin. Resolution between ciprofloxacin and ciprofloxacin ethylenediamine analog was 1.1. The proposed HPLC method is rapid, simple, accurate, reproducible, and it can be applied in analyzing raw-materials, as well as in determining ciprofloxacin, ciprofloxacin ethylenediamine and norfloxacin in diverse formats as capsules, tablets, and in solution for infusion.

As quinolonas são potentes antibacterianos sintéticos de terceira geração, que são indicadas para tratamento de diversas infecções do trato urinário, gastrointestinal e da pele. Este trabalho tem como objetivo propor um método por cromatografia líquida de alta eficiência (CLAE) para determinação das quinolonas: ciprofloxacina, análogo da ciprofloxacina etilenodiamina (substância relacionada) e norfloxacina. Estes compostos foram separados em coluna monolítica de fase reversa, sistema isocrático, detecção no comprimento de onda 275 nm, fase móvel contendo uma mistura de ácido fosfórico 0,042 mol/L, pH 3,0 ± 0,1 (ajustado com trietilamina): acetonitrila (87:13), vazão 1,6 mL/min, a temperatura ambiente. A curva analítica para ciprofloxacina compreendeu a faixa de 20,0-125,0 µg/mL e para norfloxacina de 20,0-100,0 µg/mL com coeficientes de correlação de 0,9999 e 0,9999, respectivamente. O tempo de retenção obtido para a ciprofloxacina foi de 3,4 min, para o análogo ciprofloxacina etilenodiamina, 2,4 min, e para a norfloxacina, 2,9 min. A resolução entre a ciprofloxacina e o análogo foi de 1,1. O método proposto é rápido, simples, exato, preciso e reprodutível e pode ser aplicado na análise de matérias-primas e na determinação de ciprofloxacina, ciprofloxacina etilenodiamina e norfloxacina, em cápsulas, comprimidos e soluções para infusão.

Detection of extended spectrum beta-lactamases and resistance in members of the Enterobacteriaceae family isolated from healthy sheep and dogs in Umuarama, Paraná, Brazil / Detecção de beta-lactamases de espectro estendido e resistência as quinolonas mediada por plasmídeos em membros da família Enterobacteriaceae isolados de ovinos e cães sadios de propriedades rurais da região de Umuarama, Paraná, Brasil

Bacterial resistance is a primary public health concern worldwide. Within this context, pets and breeding animals act as reservoirs for multidrug-resistant bacteria (MR), such as those producing extended spectrum beta-lactamases (ESBL) and those presenting plasmid-mediated quinolone resistance (PMQR). The aim of this study was to detect the presence of ESBL and PMQR in members of the Enterobacteriaceae family, isolated from healthy sheep and dogs from non-intense farming rural properties in the Umuarama region of Paraná, Brazil. A total of 81 oral and rectal swabs from dogs and sheep from 11 small rural properties were analyzed. These swabs were inoculated in tubes containing brain heart infusion broth (BHI), and the resulting cultures were inoculated on MacConkey agar (MAC) supplemented with 10 μg/mL cefotaxime for the selection of ESBL producers. The cells were also plated on MAC supplemented with 50 μg/mL nalidixic acid for selecting quinolone-resistant enterobacteria. The bacterial isolates were subjected to biochemical identification tests, antibiograms, double-disk synergic tests, and polymerase chain reaction analysis for resistance-inducing genes (blaESBL, qnr, and genes encoding efflux pump and acetylases). Four (5.00%) bacterial isolates (3 Escherichia coli and 1 Morganella morganii) resistant to cephalosporins and/or quinolones were identified; of these, three (75%) isolates were from sheep and one (25%) from a dog. These findings indicate the presence of MR bacteria in the normal microbiota of the animals studied. Animals colonized with such bacteria can contribute to the dissemination of antimicrobial resistance to other animals, environment, and/or human beings and can harbor endogenous infections in unfavorable conditions, which have poor prognosis due to the limited therapeutic options.

Resistência bacteriana é considerado o maior problema de saúde pública mundial da atualidade, sendo os relatos de infecções e surtos causados por bactérias multirresistentes cada vez mais frequentes na clínica veterinária e humana. Neste contexto, animais de companhia e criação podem atuar como reservatório de bactérias multirresistentes, como as produtoras de beta-lactamases de espectro estendido (ESBL) e as que apresentam resistência as quinolonas mediada por plasmídeos (PMQR). O objetivo deste trabalho foi detectar beta-lactamases de espectro estendido e resistência as quinolonas mediada por plasmídeos em membros da família Enterobacteriaceae isolados de ovinos e cães de propriedades rurais não tecnificadas da região de Umuarama, Paraná, Brasil. Foram analisados 81 swabs de cães e ovinos provenientes de 11 pequenas propriedades rurais da região de Umuarama (PR). Os swabs foram inoculados em caldo Brain Heart Infusion (BHI) e o crescimento obtivo foi em seguida semeado em placas de Petri contendo ágar MacConkey (MC) acrescido de cefotaxima 10 μg/mL para seleção de bactérias gram-negativas produtoras de ESBL; e placas contendo MC acrescido de ácido nalidíxico 50 g/mL para seleção de bactérias gram-negativas resistentes as quinolonas. Os isolados bacterianos obtidos foram submetidos a testes de antibiograma pelo método de disco-difusão em ágar, teste sinérgico do duplo-disco e reação em cadeia da polimerase (PCR) para genes que conferem resistência do tipo ESBL e para quinolonas. Dos 81 swabs coletados foi possível detectar quatro (4,97%) isolados bacterianos (3Escherichia coli e 1 Morganellamorganii) resistentes a cefalosporinas e/ou quinolonas. Destes isolados, três (75%) eram de ovinos e um (25%) de cão. Os resultados encontrados indicam a presença de cepas multirresistentes na microbiota normal dos animais estudados.[...]