Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine / Caracterização fenotípica e molecular de isolados de Pseudomonas aeruginosa resistentes a fluoroquinolonas na Palestina

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima’s D test and Fu’s Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them [...].

Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de β-lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene β-lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de β-lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para [...].

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine

Abstract Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of -lactamases were detected by phenotypic methods, while presence of -lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different -lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajimas D test and Fus Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, -lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Resumo Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de -lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene -lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de -lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para fluoroquinolonas, produtores de -lactamase, genes codificadores de exotoxina de secreção tipo III, a maioria deles tinha o gene integrase I e tinha alto nível de mutações nas regiões QRDR nos genes gyrA, parC e parE. Todos esses fatores podem desempenhar um papel importante na invasão dessas cepas e torná-las difíceis de tratar. O isolamento dessas cepas em diferentes centros médicos, indica a necessidade de uma aplicação estrita de medidas de controle de infecção em centros médicos da Cisjordânia-Palestina que visam reduzir despesas e danos causados por infecções por P. aeruginosa. As análises moleculares mostraram que os haplótipos de P. aeruginosa resistentes à fluoroquinolona palestina não são geneticamente diferenciados; no entanto, mais mutações podem existir nessas cepas.

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine / Caracterização fenotípica e molecular de isolados de Pseudomonas aeruginosa resistentes a fluoroquinolonas na Palestina

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of ß-lactamases were detected by phenotypic methods, while presence of ß-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different ß-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima's D test and Fu's Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, ß-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Fluoroquinolonas são agentes antimicrobianos importantes para o tratamento de infecções por Pseudomonas. Um total de 11 bacilos isolados de P. aeruginosa foram coletados de diferentes amostras clínicas provenientes de diferentes centros médicos na Cisjordânia-Palestina durante o ano de 2017. Neste estudo, resistência a fluoroquinolonas e secreções de ß-lactamases foram detectadas por métodos fenotípicos, enquanto a presença de sequências do gene ß-lactamase e outros fatores de virulência foram detectados pela técnica de PCR (Proteína C-reativa). O produto de PCR para os genes gyrA, parC e parE foram sequenciados para análises posteriores. As análises filogenéticas, os índices de diversidade populacional e a determinação de haplótipos foram realizados utilizando os softwares MEGA versão 6, DnaSP 5.1001 e o algoritmo de junção de mediana do programa Network 5, respectivamente. Os resultados deste estudo mostraram que a MIC para ciprofloxacina e norfloxacina tinha um intervalo de 32-256 µg/ml. Além disso, todos os bacilos isolados carregavam genes exoT ou exoT e exoY, genes de ß-lactamase diferentes e 82% desses isolados continham integrons de classe 1. As análises das sequências gyrA, parC e parE foram consideradas polimórficas, com alta diversidade de haplótipos (0,945-0,982), baixa diversidade de nucleotídeos (0,01225-0,02001) e o número de haplótipos foi de 9 para cada gene de gyrA e parE e 10 haplótipos para o gene parC. Os haplótipos fundadores são Hap-1 (18%), Hap-2 (27,3%) e Hap-6 (9,1%) para os genes gyrA, parC e parE, respectivamente. Dois dos haplótipos parE foram detectados como haplótipos InDel. As redes Median-joining (MJ) construídas a partir de haplótipos desses genes mostraram uma expansão semelhante à de uma estrela. Os testes de neutralidade (teste D de Tajima e teste Fs de Fu) para esses genes apresentaram valores negativos. As cepas palestinas de P. aeruginosa resistentes a fluoroquinolonas mostraram alto nível de MIC para fluoroquinolonas, produtores de ß-lactamase, genes codificadores de exotoxina de secreção tipo III, a maioria deles tinha o gene integrase I e tinha alto nível de mutações nas regiões QRDR nos genes gyrA, parC e parE. Todos esses fatores podem desempenhar um papel importante na invasão dessas cepas e torná-las difíceis de tratar. O isolamento dessas cepas em diferentes centros médicos, indica a necessidade de uma aplicação estrita de medidas de controle de infecção em centros médicos da Cisjordânia-Palestina que visam reduzir despesas e danos causados por infecções por P. aeruginosa. As análises moleculares mostraram que os haplótipos de P. aeruginosa resistentes à fluoroquinolona palestina não são geneticamente diferenciados; no entanto, mais mutações podem existir nessas cepas.

Frequency of Chromosomally Encoded gyrA and parC Genetic Determinants of Fluoroquinolone Resistance in A. baumannii

Fluoroquinolones are administered as routine drugs of choice for treating complicated urinary tract infections caused by multidrug resistant Acinetobacter baumannii strains. It is now a world-wide issue that gyr and par induced quinolone resistance as one of the major drug resistance mechanisms. This investigation is thus aimed to assess the prevalence of quinolone resistance and to characterize the gyrA and parC producing strains of A. baumannii. Genomic DNA from 50 fluoroquinolone resistant A. baumannii were screened for gyrA and parC by PCR for the genetic relatedness with fluoroquinolone resistance, with sequencing of the representative strains. All the strains were positive for gyrA(100%) and 82% (n=41)for parC. Presence of parC was observed in 56.09% (n=23) ciprofloxacin resistant A. baumannii with 43.90% (n=18) in levofloxacin resistant A baumannii. The findings of the present study showed the prevalence of fluoroquinolone resistance among A. baumannii in urinary tract infections and the frequency of gyrA and parC in inducing the resistance

Efficacy of desloratadine citrate disodium in the treatment of chronic urticaria and its effect on serum interleukin-23, interleukin-33 and PARC/CCL-18 / 中华皮肤科杂志

Objective To compare the efficacy of desloratadine citrate disodium versus loratadine in the treatment of chronic urticaria (CU),and to evaluate their effect on serum interleukin (IL)-23,IL-33 and pulmonary and activation-regulated chemokine/CC chemokine ligand 18 (PARC/CCL-18).Methods From January 2013 to December 2016,120 CU patients treated in Department of Dermatology,Wuwei Oncology Hospital were enrolled into this study,and divided into study group and control group by using a random number table.Patients in the study group took oral desloratadine citrate disodium tablets 8.8 mg once a day,and patients in the control group took loratadine tablets 10 mg once a day.The treatment lasted 28 days.The therapeutic effect was compared between the two groups,and changes in serum levels of IL-23,IL-33 and PARC/CCL-18 were compared before and after treatment.Statistical analysis was carried out by using two-sample t test and chi-square test for comparing indices between the two groups.Results The response rate was significantly higher in the study group (88.33％,53/60) than in the control group (61.67％ [37/60],x2 =15.352,P ＜ 0.01).After the treatment,the serum levels of IL-23,IL-33 and PARC/CCL-18 in the study group significantly decreased to 87.72 ± 22.16 ng/L,95.94 ± 18.27 ng/L,85.93 ±27.34 μg/L respectively,which were all lower than those in the control group (104.21 ± 32.05 ng/L,106.27 ±20.93 ng/L,95.72 ± 30.28 μg/L,respectively;t =3.264,4.034,3.934,respectively,P =0.020,0.006,0.015,respectively).No significant difference was observed in the incidence of adverse reactions between the study group and control group (P =0.298).Conclusion Desloratadine citrate disodium can markedly improve the clinical symptoms of CU with favorable safety,likely by inhibiting the immune response of the body and reducing the effect of chemokines on the chemotaxis of inflammatory cells.

E36 experiment at J-PARC / Experimento E36 en J-PARC

Summary The E36 experiment has been carried out at J-PARC to search for Lepton Universality violation in the kaon two body decays K l2 . A precise value of the decay width ratio R K = Γ ( K e 2 ) = Γ ( K μ 2 ) will be extracted and compared with the standard model (SM) value. Any sizable difference would suggest new physics beyond the SM. Simultaneously, E36 is sensitive to light U(1) gauge bosons which could be associated with dark matter or explain established muon-related anomalies (g-2). The experiment as well as the status of the analysis will be presented.

Resumen El experimento E36 se ha llevado a cabo en J-PARC para buscar anomalías a la universalidad leptónica en las desintegraciones en dos leptones del kaón K l2 . Se extraerá un valor preciso de la fracción de anchos de desintegración R K = Γ ( K e 2 ) = Γ ( K μ 2 ) y se comparará con el valor del modelo estándar (SM). Cualquier diferencia considerable sugeriría una nueva física más allá del SM. Simultáneamente, E36 es sensible a los bosones de gauge ligeros U(1) que podrían estar asociados con la materia oscura o explicar anomalías relacionadas con el muón (g-2). El experimento y el estado del análisis serán presentados.

Alarming resistance of Neisseria gonorrhoeae in a tertiary care hospital of North India

Background: This study attempted to elucidate the spectrum of sexually transmitted infections in a tertiary care centre in North India and to assess the antimicrobial resistance in Neisseria gonorrhoeae. Materials and Methods: Antimicrobial resistance pattern of N. gonorrhoeae was determined by the standard techniques. Genotypic detection of gyrA, parC and blaTEM genes was also carried out. The results of gyrA gene by polymerase chain reaction were confirmed by DNA sequencing. Results: N. gonorrhoea was identified in 10 (4.98%) patients, and antimicrobial sensitivity was performed in seven patients. All the seven patients tested were quinolone-resistant N. gonorrhoeae (QRNG), 5/7 were penicillinase-producing N. gonorrhoeae, 1/7 was chromosomally mediated penicillin-resistant N. gonorrhoeae and 3/7 were tetracycline-resistant N. gonorrhoeae. Minimal inhibitory concentration (MIC) by E-test was performed in five strains, and we observed that MIC90 for ciprofloxacin was ?4 ?g/ml, for penicillin was ?6 ?g/ml and for tetracycline was 12 ?g/ml, which clearly brackets them as resistant isolates. The presence of TEM gene was confirmed genotypically in six out of seven cases. In all seven cases, gyrA and parC were observed, thus confirming their QRNG status. Conclusion: Alarming increase in the resistance to commonly used antimicrobials for gonorrhoea in our study, especially of fluoroquinolones, is a clarion call for the urgent need for prudence in prescribing them. Observing the rampant resistance exhibited by N. gonorrhoeae, it is clear that the day is not far when it will acquire a superbug status and become intractable to treatment by the available antibiotics.

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran

Abstract The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 → Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 → Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

Molecular characterization and antimicrobial resistance properties of uropathogenic Escherichia coli isolated from elderly patients in a district tertiary hospital in shanghai / 中华传染病杂志

Objective To investigate the molecular epidemiology and antimicrobial resistance status of uropathogenic Escherichia coli (UPEC) in senior population in Putuo District ,Shanghai .Methods A total of 72 UPEC strains were isolated from elderly inpatients with urinary tract infections in Putuo Hospital ,Shanghai University of Traditional Chinese Medicine from January 2013 to March 2015 .The strains were characterized by multi‐locus sequence typing (MLST ) .The β‐lactamase gene and the plasmid mediated quinolone resistance (PMQR) gene were detected ,and the mutations of quinolone resistance‐determining regions (QRDR) in gyrA and parC genes were demonstrated .In vitro drug susceptibility test was performed .Continuous variables were compared using t test and categorical variables were compared using chi‐squared test or Fisher exact test .Results The UPEC strains showed different resistance rates to ciprofloxacin ,cefotaxime and trimethoprim‐sulfamethoxazole ,which were 76 .4% ,73 .6% and 65 .3% , respectively .UPEC still remained highly sensitive to imipenem ,meropenem ,amikacin and piperacillin‐tazobactam .Among 72 isolates ,55 (76 .4% ) of 49 (68 .1% ) extended‐spectrum β‐lactamase (ESBL )‐positive strains harbored blaCTX‐M genes .Among the 55 ciprofloxacin resistant strains ,51 (92 .7% ) had three or four mutations in QRDR of gyrA and parC genes .The “hot‐spot” mutations of QRDR were located at amino acid position 83 and 87 in gyrA gene and at positions 80 and 84 in parC gene .Forward analysis by MLST showed that the most frequent sequence types (ST ) were ST131 (18/72 ,25 .0% ) , ST1193(7/72 ,9 .7% ) ,ST405 (7/72 ,9 .7% ) ,ST38 (5/72 ,6 .9% ) and ST648 (3/72 ,4 .2% ) .ST131 isolates were predominant in ST which caused community‐onset urinary tract infections .Multiple drug‐resistance were detected in ST 131 ,ST405 ,ST38 and ST648 which were mainly producing blaCTX‐M ESBL .Conclusions Community‐acquired multiple drug‐resistant UPEC strains such as ST131 clone are prevalent in elderly patients .Thus ,monitoring of molecular epidemiology would be beneficial to prevent the prevalence of multiple drug‐resistant UPEC .

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Caracterización molecular de la región determinante de resistencia a quinolonas (QRDR) de la topoisomerasa IV de Bartonella bacilliformis en aislados clínicos

Bartonella bacilliformis es el agente etiológico de la Enfermedad de Carrión, endémica del Perú. Pocas investigaciones han sido realizadas acerca de los genes asociados a la resistencia antimicrobiana en aislados clínicos de este patógeno. Estos genes no están caracterizados molecularmente, ni se conoce la región asociada a dicha resistencia. Por ello, el objetivo del este trabajo fue caracterizar molecularmente la región determinante de la resistencia a las quinolonas (QRDR) en la topoisomerasa IV, que está codificada por los genes parC y parE, así como también desarrollar una prueba de susceptibilidad antimicrobiana para B. bacilliformis. Las muestras sanguíneas de 65 pacientes procedentes de La Libertad, Cusco, Ancash y Piura, se sembraron en placas de agar sangre e incubaron a 30 °C con 5% CO2. Luego se procedió a: (1) determinar la susceptibilidad antimicrobiana y (2) extraer el DNA genómico, amplificar los genes mencionados, secuenciarlos y analizarlos mediante herramientas bioinformáticas. Se obtuvieron 6 cultivos positivos. Los aislados fueron sensibles a la ciprofloxacina (excepto uno procedente de Quillabamba-Cusco, que presentó susceptibilidad disminuida) y resistentes al ácido nalidíxico. Del análisis de las secuencias aminoacídicas de ParC y ParE de B. bacilliformis se concluye que presentan diferencias aminoacídicas en comparación con las secuencias de las proteínas respectivas de E. coli K12 MG1655, que probablemente confieran resistencia al ácido nalidíxico pero no a la ciprofloxacina. Se determinó que las QRDR de las proteínas ParC y ParE de B. bacilliformis están comprendidas entre los aminoácidos 67 al 118 y 473 al 530, respectivamente. El antibiograma y la concentración mínima inhibitoria se evalúan mejor usando inóculos a escala 1 de McFarland y a los 6 días de incubación

Bartonella bacilliformis is the etiologic agent of Carrion's disease, which if endemic to Peru. Studies on antimicrobial resistance genes from clinical isolates of this pathogen are scarce, and the molecular characteristics of these genes and their region resistance-associated are currently unknown. In this work we made the molecular characterization of the quinolone-resistance, and establish the region (QRDR) for the topoisomerase IV, which is encoded by the parC and parE genes, as well as develop an antimicrobial susceptibility test for B. bacilliformis. 65 Blood samples from La Libertad, Cusco, Ancash and Piura were processed on Blood Agar plates and incubated at 30 °C, 5% CO2. The antimicrobial susceptibility was determined, then the genomic DNA extracted, aforementioned genes amplified, their sequence determined and it analyzed using bioinformatics tools. Six positive cultures were obtained. The isolates were susceptible to Ciprofloxacin (except one strain from Quillabamba _ Cusco, which showed decreased susceptibility) and were resistant to Nalidixic Acid. From the sequence analysis of B. bacilliformis ParC and ParE there have been shown amino acid differences compared to the respective protein sequences from E. coli K12 MG1655, which is likely to confer resistance to Nalidixic Acid but not to Ciprofloxacin. It was determined that B. bacilliformis ParC and ParE proteins QRDRs are comprised between amino acids 67 to 118 and 473 to 530, respectively. The antibiogram and the minimal inhibitory concentration are best assessed using the #1 McFarland standards after a 6-day incubation period

Mechanisms of quinolones resistance in Acinetobacter baumannii and homology analysis among the strains / 国际检验医学杂志

Objective To explore the mechanisms of quinolones resistance in Acinetobacter baumannii and homology analysis a-mong the strains .Methods 25 strains of quinolones-resistant Acinetobacter baumannii isolated clinically were collected .Kirby-Bauer(K-B) detection was utilized to detect the sensitivity of conventional drugs ,and polymerase chain reaction (PCR) was em-ployed to detect quinolone resistance-related genes gyrA and parC which were verified by restriction enzyme digestion and sequen-cing ,repetitive extragenic palindrome(REP)-PCR was adopted to analyze the strain homology .Results Multiple resistances to 12 kinds of antibacterial agents were found among the 25 strains of Acinetobacter baumannii which were sensitive only to minocycline and amikacin ,with sensitive rates were 48 .0% and 32 .0% ,respectively ,and were all sensitive to polymyxin B [minimal inhibitory concentration(MIC)≤2 μg/mL] .gyrA and parC genes were found in the all strain .Mutation TCA→TTA(Ser→Leu) at coden 83 in gyrA gene existed in 25 strains ,mutation TCG→TTG(Ser→Leu) at coden 80 in parC gene existed in 23 strains ,mutation GAA→GGA(Glu→Gly) at coden 84 in parC gene existed in 2 strains .REP-PCR showed that the strains had high degree of homology . Conclusion Quinolone-resistant Acinetobacter baumannii has high degree of homology ,existing gyrA and parC gene mutations .

Emergence of novel variants of gyrA and parC genes in a group of multiple resistant Klebsiella / 中华临床感染病杂志

Objective To investigate the distribution and variety of quinolone-resistance genes in a group of multiple resistant Klebsiella.Methods A total of 20 strains of multiple resistant Klebsiella were collected from inpatients in Huai' an First People' s Hospital of Nanjing Medical University during February 2010 and March 2012.Strains were identified by molecular identification,and quinolone target genes (gyrA,ParC) and quinolone-resistance genes mediated by mobile genetic elements (qnrA,qnrB,qnrS,aac (6′)-Ⅰ b-Cr,qepA) were analyzed with polymerase chain reaction (PCR).Results In 20 strains of Klebsiella,19 were Klebsiella pneumonia,and 1 was Klebsiella variicola.gyrA and parC genes were found in all 20 strains,and gyrA mutation was found in 11 strains (55.0％),andparC mutation was also found in 11strains (55.0％).aac(6′)-Ⅰ b-cr was positive in 10 strains (50.0％),qnrA was positive in 1 strain (5.0％),and qnrB was positive in 3 strains (15.0％).gyrA and parC in strain No.6 and No.10 were both novel variants (GenBank registration number:JX123016,JX123017,JX144393,JX144394).Conclusion Quinolone-resistance-determining region plays a key role in resistance to quinolones in this group of Klebsiella,and it is the first report on the emergence of novel variants of gyrA and parC in one Klebsiella pneumonia in China.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Detection of Mutation in gyrA and parC Gene on Resistant Salmonella enterica Serovars. Isolated from Two Hospitals in South East Nigeria.

Aim: To detect the resistant pattern and existence of the genes responsible for floroquinolone-resistant in the quinolone-resistant determining regions (QRDR’s) of S. enterica serovars.Typhi. Study Design: The Stool samples from Patients with symptoms of enteric fever, from different units and wards from two hospitals in Southeast region of Nigeria, were used for the surveillance. Place and Duration of Study: The study was carried out in the Department of Microbiology and Biotechnology, Nigerian Institute for Medical Research, Lagos, between July and December, 2011. Methodology: 50 isolates of Salmonella enterica serovar.Typhi were screened for the antibiotics susceptibility pattern, using multidisc agar diffusion and E-test. Double disc synergy Test (DDST) reported the presence of ESBL’s strains. The DNA amplification was performed by PCR using HOT FIREPol ® DNA polymerase with 25mMMgcl2. DNA – sequencing of the (QRDR’S) of gyrA (n= 32) and parC (n=3), was performed using sanger sequencing ABI 3730 x l, Applied Biosystems. Results: A total of 39(78%) of the S. enterica produced β-lactamase. ESBL’s positive strains were 17(34%) and 46(92%) isolates were Multi-Drug Resistant S. typhi (MDRST). Sequencing of the mutations in gyrA gene of the (QRDR’s) was at Asp-87- Gly and Asp- 87- Asn or at Ser-83- Tyr, while mutations in parC 3(6%), were at Asp-87- Gly. Conclusion: Chromosomal encoded ESBL’s and mutations were found to be responsible for the MDRST. Ceftriazone and levofloxacine were found to be significant alternatives in treating S. enterica serovar.Typhi. This is the first report of mutation in both gyrA and parC genes in S. enterica serovar.Typhi in Southeast Nigeria.

A novel variant of topoisomerase Ⅳ gene in a group of pan-drug resistant Acinetobacter baumannii / 中华微生物学和免疫学杂志

Objective To investigate the existence and variance of quinolone-resistance genes in a group of pan-drug resistant of Acinetobacter baumannii ( A.baumannii ).Methods Twenty strains of pandrug resistant A.baumannii were isolated from patients registered in Affiliated Hospital of Nantong University from January 2011 to April 2011.Drug target genes to quinolone (gyrA,parC) and quinolone-resistance genes mediated by mobile genetic elements [ qnrA,qnrB,qnrS,aac(6')-Ⅰ b-cr,qepA] were analyzed by PCR and verified by DNA sequencing.Results In all 20 strains of A.baumannii,the sense mutation was found in the quinolone resistance-determining region of the gyrA gene in the form of TCA to TTA at codon 83 (Ser-83-Leu).Moreover,in the quinolone resistance-determining region of the parC gene sense mutation was found in the form of TCG to TTG at codon 80 (Ser-80-Leu) and 3 synonymous mutations were CCC to CCT at codon 40,GTA to GT]T at codon 41 and CGT to CGC at codon 44.And parC gene was a new mutation.However,mutations were not found in quinolone-resistance genes mediated by mobile genetic elements [ qnrA,qnrB,qnrS,aac( 6 ' )-Ⅰ b-cr,qepA ].Conclusion Quinolone-resistance-determining region play a key role in resistance to quinolones in this group of A.baumannii.To our knowledge,this is first report about the emergence of the new mutation of parC gene in China.

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 µg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.

Clinical application of genotyping detection of human Mycoplasma urealyticum based on parC gene sequences / 中华微生物学和免疫学杂志

Objective To establish a novel PCR method that can differentiate the two biotype of Mycoplasma urealytium rapidly based on the disparities of parC gene sequences for clinical routine examination.Methods Design two pairs of specific primers and probes for the targeted gene according to the differences of the parC gene sequences from 14 standard serotypes.The specificity of this amplification was verified by detecting two Mycoplasma urealyticum standard strains including serovar1 and serovar4,50 clinical isolated strains ( 12 are Uu strains and 38 are Up strains) and 7 common bacterias from vagina.Collected 70 swab specimens from patients of Sexually Transmit Department with urogenital inflammation symptom and 71 swab specimens from the gynecology health examination population.All those specimens were detected by culture and our method respectively.The sensitivity of this method was evaluated by comparing with the culture.Differences of the infection rate and distribution of biotypes between different populations were analyzed using statistical software.Results Standard strains of Mycoplasma urealyticum and clinical isolates can be typed into two species by the PCR method without nonspecific amplification.The sensitivity of PCR method is much higher than that of culture ( P＜0.05).The infection rates of Uu,Up and the mixing were 8.57％,61.40％,24.30％ respectively for the patients with vaginitis.However,it was 8.80％,67.60％,8.45％ respectively for the gynecology health examination population.There is a significant difference of the total infection rate between the two population(P＜0.05).It showed no significant difference with the distribution of the two types of simple infection in the two groups ( P＞0.05).But the rate of mixing infection is much higher in the patients with vaginitis ( P＜0.05 ).Conclusion The pathology of Mycoplasma urealyticum may be related to the mixed infection of different biotypes.

Mutación en el gen gyrA de aislamientos hospitalarios de Acinetobacter baumannii en Montería, Colombia / Mutation of the gyrA Gen in Acinetobacter baumannii nosocomial isolates in Monteria, Colombia

Objetivo: Determinar las mutaciones delgen gyrA asociadas con la resistencia a fluoroquinolonas en Acinetobacter baumannii. Materiales y métodos: Entre agosto de 2005 y febrero de 2007 se recolectaron 23 aislamientos de A. baumannii de una clínica privada de tercer nivel de Montería. Se investigaron los genes gyrA, parC y adeB; este último codifica para la bomba de salida. Se realizó secuenciación del ADN y, para elanálisis de las secuencias, se usaron la base de datos GenBank y el motor de búsqueda BLASTX. Resultados: La amplificación del gen gyrA en aislamientos de A. baumannii generó unfragmento de 343 pb, el cual presentó pérdida del sitio de restricción con la enzima Hinfl en 12/23 (52,1%) de los aislamientos resistentes a fluoroquinolonas. La secuenciacióndel fragmento mostró mutación puntual con el cambio de Ser-83 a Leu, código de acceso GenBank EU886740. No se encontraron mutaciones en el gen parC, ni la presencia de la bomba de salida Ade. Conclusión: Los aislamientos de A. baumannii resistentes a fluoroquinolonas sugieren que la mutación del gen gyrA que codifica elcambio del aminoácido serina a leucina en el codón 83 de estos aislamientos, es responsable o, al menos, contribuye con la resistencia expresada a las fluoroquinolonas.

Objective: Determine mutations in thegyrA gene associated to resistance tofluoroquinolones in A. baumannii. Materials and methods: From August,2005 to February, 2007, 23 A. baumanniiisolates were collected in a third level private clinic in Monteria. Research on gyrA, parC and AdeB genes was carried out, and the latter encodes for the efflux pump. DNA sequencing was performed, and the GenBank database and BLASTX search engine were used for sequence analysis. Results: Amplification of the gyrA gene in A. baumannii isolates generated a 343pb fragment which presented loss of restriction site with the enzyme HinfI in 12/23 (52.1%) of the isolates resistant to fluoroquinolones. The fragment sequencing showed mutationcharacterized by the change of Ser-83 to Leu GenBank acces code EU886740. None of the isolates showed mutations in the parC gene or presence of the AdeB efflux pump. Conclusion: The A. baumannii isolates resistant to fluoroquinolones suggest thatmutation of the gyrA gene encoding the serine amino acid change to leucine at codon 83 of these isolates is responsible or at least contributes to the mentioned resistance to fluoroquinolones.