Detection of beta-lactamase-producing Enterobacteriaceae in a veterinary hospital environment / Detecção de Enterobactérias produtoras de beta-lactamases em ambiente hospitalar veterinário

Due to the strong selective pressure resulting from the misuse of antibiotics, the natural process of bacterial resistance has been accelerated, leading to the increasingly constant appearance of multiresistant isolates. The high number of multi-resistant bacteria is a one health problem. Enterobacteriaceae are usually commensal bacteria of the gastrointestinal tract. However, they can cause infections, and the most important resistance characteristic among them is the production of ß-lactamases. This study aimed to identify ESBL-producing Enterobacteriaceae of types of TEM, SHV, and the CTX-Mgroups. To isolate the enterobacteria, swabs were collected by swiping objects that had contact with the patients and professionals, and the water of the hospital environment. Ten collections were carried out, yielding 306 samples, from which 118 enterobacteria were identified: Escherichia coli, Enterobacter spp., Klebsiella spp., Proteus mirabilis, Serratiaspp., and Citrobacter spp. Isolates. The genes TEM and CTX-M, for the production of ß-lactamases, were detected in 12.7% of the 118 enterobacterial isolates. It is very important to know the bacterial population circulating in the veterinary hospital environment and its resistance to antimicrobials so that professionals can take appropriate measures to minimize the risks of transmission, especially from cages and consultation tables. In addition, the correct control of the microbiological quality of the supply water, as well as environmental cleaning procedures, are essential to prevent the transmission of these microorganisms.(AU)

Devido à grande pressão seletiva decorrente do uso indevido de antibióticos, tem se acelerado o processo natural de resistência das bactérias, levando ao aparecimento cada vez mais constante de isolados multirresistentes. O elevado número de bactérias multirresistentes identificadas é um problema da saúde única. As enterobactérias são bactérias geralmente comensais do trato gastrointestinal, entretanto podem causar infecções, e a característica de resistência mais importante entre elas é a produção de ß-lactamases. Buscando caracterizar melhor os microrganismos circulantes e potencialmente causadores de infecções em ambiente hospitalar veterinário, este estudo objetivou identificar as enterobactérias produtoras de ESBL do tipo TEM, SHV e os cinco grupos de CTX-M presentes em isolados circulantes em hospital veterinário. Foi realizada coleta de suabes de arrasto de objetos que entram em contato com os pacientes e com os profissionais que ali trabalham, bem como de água, para a identificação das enterobactérias. Foram realizadas 10 coletas, obtendo-se 306 amostras, dessas, 118 enterobactérias foram identificadas: Escherichia coli, Enterobacter, Klebsiella, Proteus mirabilis, Serratia e Citrobacter. Dentre as enterobactérias identificadas, alguns isolados possuíam genes para a produção de ß-lactamases, do tipo TEM e CTX-M. É de grande importância conhecer a população bacteriana circulante no ambiente hospitalar veterinário, e a sua resistência aos antimicrobianos, para que os profissionais possam tomar medidas apropriadas para minimizar os riscos de transmissão, principalmente a partir de gaiolas e mesas de atendimento. Além disso, o correto controle da qualidade microbiológica da água de abastecimento, bem como dos procedimentos de higienização do ambiente, são fundamentais para evitar a transmissão destes microrganismos.(AU)

Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil / Detecção da bomba de efluxo CmeABC em cepas de Campylobacter spp. resistentes à enrofloxacina isoladas de frangos de corte (Gallus gallus domesticus) no Estado do Rio de Janeiro, Brasil

Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers' meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine ß-Naphthylamide (PAßN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5µg/ml and 64µg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAßN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.(AU)

As aves são os principais reservatórios de Campylobacter spp., importante patógeno de origem alimentar e a carne de frango e produtos derivados são os principais veículos desse microrganismo. O aumento de cepas de Campylobacter spp. resistentes às fluorquinolonas, uma classe antimicrobiana frequentemente empregada na avicultura e na medicina humana, tornou-se uma grande preocupação para os produtores de aves e vários estudos avaliaram o aumento da resistência bacteriana a esses antimicrobianos. O papel do sistema de efluxo CmeABC tem sido enfatizado entre os mecanismos de resistência em Campylobacter spp. à fluorquinolonas. O presente estudo investigou a ocorrência da bomba de efluxo CmeABC em 81 cepas de Campylobacter jejuni e 78 cepas de Campylobacter coli resistentes à enrofloxacina, isoladas de frangos de corte coletados em seis abatedouros situados no polo avícola de São José do Rio Preto/RJ e de dois abatedouros comerciais situados na Região Metropolitana do Rio de Janeiro, de 2013 a 2016. A resistência à enrofloxacina foi avaliada pelo método de diluição em ágar para determinar a concentração inibitória mínima (CIM). O sistema de efluxo CmeABC foi investigado através da detecção dos genes cmeA, cmeB e cmeC por PCR. A atividade da bomba de efluxo CmeABC foi investigada em 20 cepas utilizando o inibidor da bomba de efluxo Phenylalanine-Arginine ß-Naftilamida (PAßN). Os três genes cmeA, cmeB e cmeC foram detectados em 94,3% das cepas (C. jejuni = 80 e C. coli = 70), enquanto o sistema estava ausente ou incompleto em 5,7% das cepas (C. jejuni = 1 e C coli = 8). A CIM variou entre 0,5µg/ml e 64µg/ml e 88,7% das cepas foram resistentes à enrofloxacina, enquanto 11,3% apresentaram resistência intermediária. A inibição da bomba de efluxo pelo PAßN reduziu a CIM da enrofloxacina até oito vezes em quinze cepas (75%). Estes resultados indicam que este sistema é frequente e ativo em cepas resistentes de Campylobacter spp. na presença de enrofloxacina.(AU)

Implicancias estructurales y fisiológicas de la célula bacteriana en los mecanismos de resistencia antibiótica / Structural and physiological implications of bacterial cell in antibiotic resistance mechanisms

RESUMEN: La alta capacidad de adaptación de las bacterias a ambientes hostiles ha permitido el desarrollo de resistencia a antibacterianos, causando problemas de impacto mundial en la salud hospitalaria y de la comunidad, limitando las opciones terapéuticas lo que afecta el control de enfermedades, elevando las tasas de morbi-mortalidad. Esta capacidad de resistencia es mediada por factores estructurales y fisiológicos de las bacterias que actúan a diferentes niveles tanto extracelular como intracelular. A niveles extracelulares se destaca la capacidad de las poblaciones bacterianas en la formación de biopelículas y la regulación de señales celulares quorum sensing, permitiendo la evasión de la acción antibiótica. A nivel de envoltura celular se destaca el funcionamiento y comportamiento de la pared celular y de la membrana celular, principalmente por medio de la regulación de la expresión de canales de entrada o porinas y/ o bombas de expulsión que impiden el acceso o inducen la salida de antibióticos; otros mecanismos integran la modificación de la actividad de drogas por medio de la hidrólisis o modificación del sitio activo del fármaco. A nivel intracelular, las bacterias pueden cambiar los procesos de óxido/reducción, modificar los sitios objetivos del antibiótico e inactivar los grupos transfer, y modificar las subunidades ribosomales afectando la acción de los antibióticos que inhiben la síntesis de proteínas. A esto se añaden las modificaciones en la expresión génica y del código genético, que regula todos los anteriores, y es capaz de generar cambios adaptativos, resistencia a fármacos y desinfectantes, entre otros. La presente revisión tiene como objetivo describir las implicancias estructurales y fisiológicas de la célula bacteriana en los mecanismos de resistencia antibiótica considerando la organización estructural y fisiológica involucrada en los principales mecanismos de resistencia a antibióticos presentes en bacterias de importancia clínica que conllevan a fallas terapéuticas con alto costo en salud humana.

SUMMARY: The high adaptability of bacteria to hostile environments has favored antibacterial resistance development, impacting hospital and community healthcare worldwide. It has also affected disease control, limited therapeutic options and raised morbiditymortality rate. This resistance ability is mediated by structural and physiological factors of bacteria acting at both extracellular and cellular levels. The ability of bacterial populations in biofilm formation and regulation of cellular signal quorum sensing at the extracellular level, allows for the evasion of antibiotic action. At a cellular level, the performance and behavior of the cell wall and cell membrane is emphasized, mainly by regulating the expression of inlet channels or porins and/or expulsion pumps preventing access to, or inducing the outflow of antibiotics. Other mechanisms integrate modification of drug activity by hydrolysis or modification of the active site of the drug. Further into intracellular level, bacteria can change the oxidation/reduction processes; modify the target sites of the antibiotic and inactivate transfer groups. Bacteria can also modify the ribosomal subunits affecting the antibiotics which inhibit protein synthesis, and cause modifications of gene expression and genetic code that regulate the above mechanism. These may also generate adaptive changes and resistance to drugs and disinfectants. The aim of the present review is to describe the structural and physiological implications of bacterial cell in the mechanisms of antibiotic resistance. The study also considered the structural and physiological organization involved in the main mechanisms of antibiotic resistance in bacteria relevant to clinical healthcare.

Habituation of enterotoxigenic Staphylococcus aureus to Origanum vulgare L. essential oil does not induce direct-tolerance and cross-tolerance to salts and organic acids

Enterotoxigenic Staphylococcus aureus strains that were isolated from foods were investigated for their ability to develop direct-tolerance and cross-tolerance to sodium chloride (NaCl), potassium chloride (KCl), lactic acid (LA) and acetic acid (AA) after habituation in sublethal amounts (1/2 of the minimum inhibitory concentration - 1/2 MIC and 1/4 of the minimum inhibitory concentration - 1/4 MIC) of Origanum vulgare L. essential oil (OVEO). The habituation of S. aureus to 1/2 MIC and 1/4 MIC of OVEO did not induce direct-tolerance or cross-tolerance in the tested strains, as assessed by modulation of MIC values. Otherwise, exposing the strains to OVEO at sublethal concentrations maintained or increased the sensitivity of the cells to the tested stressing agents because the MIC values of OVEO, NaCl, KCl, LA and AA against the cells that were previously habituated to OVEO remained the same or decreased when compared with non-habituated cells. These data indicate that OVEO does not have an inductive effect on the acquisition of direct-tolerance or cross-tolerance in the tested enterotoxigenic strains of S. aureus to antimicrobial agents that are typically used in food preservation.

Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs

Antibiotics are important adjuncts in the treatment of infectious diseases, including periodontitis. The most severe criticisms to the indiscriminate use of these drugs are their side effects and, especially, the development of bacterial resistance. The knowledge of the biological mechanisms involved with the antibiotic usage would help the medical and dental communities to overcome these two problems. Therefore, the aim of this manuscript was to review the mechanisms of action of the antibiotics most commonly used in the periodontal treatment (i.e. penicillin, tetracycline, macrolide and metronidazole) and the main mechanisms of bacterial resistance to these drugs. Antimicrobial resistance can be classified into three groups: intrinsic, mutational and acquired. Penicillin, tetracycline and erythromycin are broad-spectrum drugs, effective against gram-positive and gram-negative microorganisms. Bacterial resistance to penicillin may occur due to diminished permeability of the bacterial cell to the antibiotic; alteration of the penicillin-binding proteins, or production of β-lactamases. However, a very small proportion of the subgingival microbiota is resistant to penicillins. Bacteria become resistant to tetracyclines or macrolides by limiting their access to the cell, by altering the ribosome in order to prevent effective binding of the drug, or by producing tetracycline/macrolide-inactivating enzymes. Periodontal pathogens may become resistant to these drugs. Finally, metronidazole can be considered a prodrug in the sense that it requires metabolic activation by strict anaerobe microorganisms. Acquired resistance to this drug has rarely been reported. Due to these low rates of resistance and to its high activity against the gram-negative anaerobic bacterial species, metronidazole is a promising drug for treating periodontal infections.

High prevalence rate of extended-spectrum beta-lactamases (ESBL) among Enterobacteriaceae in a small Brazilian public hospital

The production of extended-spectrum beta-lactamases (ESBL) is considered one of the most important resistance mechanisms that impair antimicrobial treatment of infections caused by Enterobacteriaceae. Data on culture and susceptibility tests were collected from the Clinical Analyses and Research Laboratory charts reporting on patients admitted to the University Hospital of Maringá (HUM) from January 2004 to December 2009. The following Enterobacteriaceae were selected: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp. and Proteus mirabilis. All tests were performed according to the recommendations of the Clinical and Laboratory Standards Institute (biochemical identification; susceptibility tests; initial screening and phenotypic confirmatory tests for ESBL). For Enterobacter spp. isolates, a disk approximation test was carried out, adding a cefepime disk. Seven hundred samples were analyzed, and E. coli was the most prevalent bacteria (n= 356). ESBLs were detected phenotypically in 7.3 percent of E. coli, 61.7 percent of K. pneumoniae, 33.3 percent of K. oxytoca, 7.1 percent of P. mirabilis, and 13.4 percent of Enterobacter spp samples. Overall ESBL prevalence reached 22 percent when all producers were taken together. Although HUM is considered a small-sized hospital, it showed high levels of resistance to antimicrobial agents, similar to those observed in bigger hospitals, which demonstrated the need for careful epidemiological surveillance.

A produção de beta-lactamases de espectro ampliado (ESBL) é considerada um dos mais importantes mecanismos de resistência aos antimicrobianos, o que dificulta o tratamento de infecções causadas por enterobactérias. Dados sobre cultura e testes de sensibilidade foram coletados das fichas do Laboratório de Ensino e Pesquisa de Análises Clínicas de pacientes atendidos no Hospital Universitário de Maringá (HUM), de janeiro de 2004 a dezembro de 2009. As enterobactérias escolhidas foram: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp. e Proteus mirabilis. Os testes foram realizados de acordo com as recomendações do CLSI (identificação bioquímica; testes de suscetibilidade; triagem e confirmação fenotípica de produção de ESBL). Para isolados de Enterobacter spp., utilizou-se a técnica de disco aproximação, acrescentando um disco de cefepima. Setecentas amostras foram analisadas e E. coli foi a bactéria mais prevalente (n=356). ESBLs foram detectadas fenotipicamente em 7,3 por cento das amostras de E. coli, 61,7 por cento das de K. pneumoniae, 3,3 por cento das de K. oxytoca, 7,1 por cento das de P. mirabilis e em 13,4 por cento das de Enterobacter spp. A prevalência geral de ESBL chegou a 22 por cento, somando-se todos os isolados produtores. O HUM, mesmo sendo considerado um hospital de pequeno porte, apresenta níveis altos de resistência a antimicrobianos, semelhantes àqueles observados em hospitais maiores, demonstrando a necessidade de cuidadosa vigilância epidemiológica.

Resistencia a drogas antituberculosas en Caracas, Venezuela; 2001-2006 / Antituberculosis drug resistance in Caracas, Venezuela; 2001-2006

Para establecer la sensibilidad de las cepas de Mycobac-terium tuberculosis aisladas en Caracas, entre 2001 y 2006, fueron probadas utilizando el método colorimétrico para determinar las Concentraciones Inhibitorias Mínimas (CIM). De las 324 cepas, 46 (14,2%) mostraron resistencia a una o más drogas. Encontramos resistencia de alto nivel (CIM 8 µg/ml) y bajo nivel (CIM 1-4 µg/ml) a Estreptomicina en 6 (1,8%) y 25 (7,7%) de las cepas, respectivamente. Se encontró resistencia a Isoniacida de bajo nivel (MIC 0,125 - 0,5 µg/ml) en 8 (2,5%) y de alto nivel (MIC 1,0 µg/ml) en 15 (4,6%) de las cepas estudiadas. Hallamos 13 (4,0%) cepas resistentes a Rifampicina (RIF) (5 µg/ml) y 11 (3,4%) a Etambutol (10 µg/ml). De los 17 (5,2%) aislamientos resistentes a dos o más drogas, 12 (3,7%) fueron resistentes a INH y RIF (definido como multirresistencia, MDR). De las 12 cepas MDR, 11 fueron aisladas a partir de esputo y una de líquido pleural. Este estudio muestra un incremento en la prevalencia de la resistencia a las drogas antituberculosas en Caracas, especialmente las cepas MDR. Este aumento apunta hacia la necesidad de una encuesta na-cional, para evaluar el panorama real de la resistencia.

To asses drug susceptibility of Mycobacterium tuberculosis strains isolated from 2001 to 2006 in Caracas. Available strains were tested using colorimetric method to determine the Minimal Inhibitory Concentrations (MIC). Of 324 strains, 46 (14,2%) showed resistance to one or more drugs. High-resistance (8 µg/ml) and low-resistance (1-4 µg/ml) to Strep omycint was found in 6 (1,8%) and 25 (7,7%) strains, respectively. Isoniazid (INH) low-resistance (MIC 0.125 - 0.5 µg/ml) were found in 8 (2,5%) and high-resistant (MIC at 1.0 µg/ml) in 15 (4,6%), Rifampicin resistance (RMP) (5 µg/ml) in 13 (4%), and Ethambutol resistance (10 µg/ml) in 11 (3,4%) of the strains. Of the 17 (5,2%) isolates resistant to two or more drugs, 12 (3,7%) were resistant to INH and RMP (defined as multidrug resistance, MDR). Of these 12 MDR strains, 11 were isolated from sputum and one from pleu ral fluid. This study shows an increased prevalence of resistance to anti-tuberculosis drugs in Caracas, especially the prevalence of MDR strains, raises an urgent need of a proper nationwide survey to evaluate the true picture of resistance.

Perfil de resistência de Mycobacterium tuberculosis no estado de Mato Grosso do Sul, 2000-2006 / Drug resistance profile of Mycobacterium tuberculosis in the state of Mato Grosso do Sul, Brazil, 2000-2006

OBJETIVO: Determinar o perfil de resistência a drogas de Mycobacterium tuberculosis no estado de Mato Grosso do Sul no período entre 2000 e 2006. MÉTODOS: Estudo descritivo de casos notificados de tuberculose no Sistema de Informação de Agravos de Notificação, com cultura positiva para M. tuberculosis e testes de sensibilidade a rifampicina, isoniazida, estreptomicina e etambutol. Para as culturas, utilizaram-se os meios sólidos Lõwenstein-Jensen e Ogawa-Kudoh, assim como um sistema automatizado com meio líquido; para os testes de sensibilidade, o método das proporções. RESULTADOS: De 783 casos, 69,7 por cento eram de pacientes masculinos, na faixa etária de 20-49 anos (70 por cento), com forma pulmonar (94,4 por cento) e sorologia positiva para HIV (8,6 por cento); 645 (82,4 por cento) eram casos novos, e 138 (17,6 por cento) eram casos tratados. Identificou-se qualquer resistência em 143 casos (18,3 por cento). A taxa de resistência primária (RP) foi, respectivamente, 8,1 por cento, 1,6 por cento, 2,8 por cento e 12,4 por cento, para monorresistência, multirresistência (MR), outros padrões de associação de drogas e qualquer resistência, ao passo que a taxa de resistência adquirida (RA) foi, respectivamente, 14,5 por cento, 20,3 por cento, 10,9 por cento e 45,7 por cento, e a taxa de resistência combinada (RC) foi, respectivamente, 9,2 por cento, 4,9 por cento, 4,2 por cento e 18,3 por cento. A estreptomicina foi a droga mais comum na RP (3,4 por cento), e a isoniazida foi a mais comum na RA e RC (7,2 por cento e 3,7 por cento, respectivamente). CONCLUSÕES: Os níveis de resistência são elevados, prejudicando o controle da tuberculose em Mato Grosso do Sul. A MR adquirida, 12,7 vezes superior à MR primária, evidencia o uso prévio de medicamentos como indicativo de resistência. Os níveis refletem a fragilidade da atenção ao doente, mostrando a importância do tratamento diretamente observado, assim como das culturas e testes de sensibilidade ...

OBJECTIVE: To determine the drug resistance profile of Mycobacterium tuberculosis in the state of Mato Grosso do Sul, Brazil, between 2000 and 2006. METHODS: Descriptive study of reported tuberculosis cases in the Brazilian Case Registry Database. We included only those cases in which M. tuberculosis culture was positive and sensitivity to drugs (rifampicin, isoniazid, streptomycin and ethambutol) was tested. Lõwenstein-Jensen and Ogawa-Kudoh solid media were used for cultures, as was an automated liquid medium system. Sensitivity tests were based on the proportion method. RESULTS: Among the 783 cases evaluated, males predominated (69.7 percent), as did patients in the 20-49 year age bracket (70 percent), a diagnosis of pulmonary tuberculosis (94.4 percent) and positive HIV serology (8.6 percent); 645 (82.4 percent) were new cases, and 138 (17.6 percent) had previously been treated. Resistance to at least one drug was found in 143 cases (18.3 percent). The primary resistance (PR) rate was, respectively, 8.1 percent, 1.6 percent, 2.8 percent and 12.4 percent, for monoresistance, multidrug resistance (MDR), other patterns of resistance and resistance to at least one drug, whereas the acquired resistance (AR) rate was 14.5 percent, 20.3 percent, 10.9 percent and 45.7 percent, respectively, and the combined resistance (CR) rate was 9.2 percent, 4.9 percent, 4.2 percent and 18.3 percent, respectively. In PR, streptomycin was the most common drug, whereas isoniazid was the most common in AR and CR (7.2 percent and 3.7 percent, respectively). CONCLUSIONS: These high levels of resistance undermine the efforts for tuberculosis control in Mato Grosso do Sul. Acquired MDR was 12.7 times more common than was primary MDR, demonstrating that the previous use of drug therapy is an indicator of resistance. These levels reflect the poor quality of the health care provided to these patients, showing the importance of using the directly observed treatment, ...

Prevalence of Shigella species and their antimicrobial resistance patterns in Eastern Nepal.

The study was conducted to determine the prevalence of Shigella species and their antimicrobial resistance patterns in eastern Nepal. Stool samples submitted to the diagnostic laboratory of B.P. Koirala Institute of Health Sciences, Nepal, during August 2000-July 2004, were cultured for Shigella species and were confirmed by biochemical and serological tests. Of 53 Shigella species isolated, Shigella dysenteriae type 1 was the most predominant isolate (73.7%), followed by S. flexneri (23%) and S. boydii (4%). The majority (79%) of Shigella species were isolated from children aged less than five years. An overall high resistance was observed for trimethoprim-sulphamethoxazole, ampicillin, nalidixic acid, mecillinam, and ciprofloxacin. There was a statistically significant (p < 0.001) increasing trend in the prevalence of ciprofloxacin resistance in S. dysenteriae type 1. The results suggest reconsideration of the empiric use of these antimicrobial agents for shigellosis. A further study is required to evaluate additional antimicrobial agents.

Correlation of mutations detected by INNO-LiPA with levels of rifampicin resistance in Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVES: Due to emergence of drug resistance in Mycobacterium tuberculosis, there is a need to have accurate and rapid methods for detection of drug resistance to important drugs like rifampicin. The present study was aimed at evaluation of a commercially available INNO-LiPA assay, for the detection of mutation in rpoB gene region of M. tuberculosis and correlate these mutations with levels of rifampicin resistance for assessing their clinical relevance. METHODS: Fifty five well-characterized isolates of M. tuberculosis deposited from various regions of India in Mycobacterial Repository Centre at the CJILOMD, Agra were subjected to susceptibility testing for rifampicin at various concentrations of drug viz., 10, 40, 64, 128 microg/ml on Lowenstein- Jensen (LJ) medium. rpoB gene fragment (260 bp) was amplified using Rif-TB amplification kit and after hybridization, detection was done by using INNO-LiPA Rif TB kit. RESULTS: The rpoB gene could be amplified from DNA extracted from all the 55 culture isolates and showed clear hybridization pattern with M. tuberculosis complex specific probes on LiPA strips. Mutations detected were correlated with degree of rifampicin resistance. All the sensitive isolates (identified by MIC) were identified as rifampicin sensitive (100%) by INNO-LiPA as they exhibit positive for wild type 'S' probes and negative for 'R' probes. Two of the 5 isolates, resistant at 10 microg/ml and 40 microg/ml had either D516V, H526Y mutations or unknown mutations. Thirty (85.71%) isolates resistant at clinically relevant levels (64,128microg/ml) exhibited double, triple or more 'R' type mutations (R(2(D516V)), R(4a(H526Y)), R(4b(H526D)), R(5(S531L))) as well as unknown mutations present at 'S' probes region whereas remaining isolates did not show any mutation by this method. This method could identify with definitiveness 60 per cent ( 21/35) isolates as rifampicin resistant as mutations observed in others were also present in isolates with low levels of resistance. INTERPRETATION & CONCLUSION: The results indicate that INNO-LiPA Rif TB test is a rapid and easy to use method for detection of mutations associated with rifampicin resistance in M. tuberculosis. However, as some of these mutations are also present in isolates with low degree of resistance which are still microbiologically sensitive to rifampicin, there is a need to improve this assay by exclusion of some of the current probes and inclusion of more probes.

Pattern of drug resistant Helicobacter pylori in dyspeptic patients in Thailand.

Emergence of drug resistant Helicobacter pylori (H. pylori) has occurred in various countries and could compromise the efficacy of current treatment regimens. The aim of the study was to identify the pattern of antibiotic resistant H. pylori in Thailand and evaluate various factors associated with drug resistance. Between June 2001 and December 2002, a total of 560 dyspeptic patients who underwent upper gastrointestinal endoscopy at King Chulalongkorn Memorial Hospital were included in this study. Antral gastric biopsies were obtained for H. pylori cultures and susceptibility tests using Epsilometer test (E-test). The value of antibiotic resistant breakpoints were amoxicillin 0.5 microg/ml, clarithromycin 1.0 microg/ml, metronidazole 8 microg/ml, and tetracycline 4 microg/ml, respectively. H. pylori were detected in 315 patients using the rapid urease test (56.25%). Cultures for H. pylori were positive in 172 patients. E-test for all four antibiotics was successfully placed in 79 isolations. The prevalence of antibiotic resistant H. pylori were amoxicillin 13.9 per cent (11/79), clarithromycin 19.0 per cent (15/79), metronidazole 30.4 per cent (24/79), tetracycline 5.1 per cent (4/79), and multi-drugs 16.5 per cent (13/79), respectively. However, age, sex, or endoscopic findings did not differ between the patients with H. pylori resistant strains and sensitive strains. The emergence of antibiotic and multi-drug resistant H. pylori in Thailand were relatively high and these could compromise the efficacy of current treatment regimens. The factors associated with drug resistant H. pylori could not be demonstrated in the present study. Further study in a larger number of patients might be necessary to identify factors associated with resistant H. pylori.

Aetiology of shigellosis in northern Pakistan.

People of northern Pakistan face health hazards because of poor sanitation practices. Bacterial gastrointestinal infections are very common, and sometimes outbreaks occur. The present study was aimed at evaluating and analyzing infestation of Shigella spp. in patients with suspected gastroenteritis and ascertaining the status of antibiotic therapy. Five hundred and eighty-five faecal samples of patients with suspected gastroenteritis, referred to the District Headquarter Hospital Gilgit, were investigated for common enteropathogenic bacteria from July 1997 to September 1999. Seventy-seven (13.2%) of the faecal specimens were infected with different strains of Shigella spp., 61% of which were Shigella dysenteriae, 15.6% were S. flexneri, and 23.4% were Shigella sp. All Shigella strains were sensitive to ceftriaxone, cefotaxime, ciprofloxacin, and enoxacin. Sixty-one percent of the strains were resistant to both ampicillin and chloramphenicol, and 3.9% to ampicillin and nalidixic acid, while 10.4% were resistant to ampicillin alone and 14.3% to chloramphenicol only. Only 10.4% of the strains were sensitive to all the antibiotics tested. Sixty strains of Shigella spp. were processed for isolation of plasmids, and 58 (97%) of these antibiotic-resistant bacteria harboured at least one plasmid. The number of plasmids varied from 1 to 9. Escherichia coli C600 were transformed with the isolated plasmids. Transformants, containing 23-kb plasmid, resisted growth in media containing antibiotics, thereby indicating that antibiotic resistance is plasmid-borne. Based on the findings of the study, it is concluded that the infestation of Shigella spp. is high in northern Pakistan, the aetiological agents are highly resistant to chloramphenicol and ampicillin, and the antibiotic resistance is mediated by the 23-kb plasmid.

In vitro susceptibility of new generation of antibacterial antibiotics against pathogenic bacteria.

Antibiotic susceptibility of ten bacteria i.e. Neisseria catarrhalis, Salmonella typhi, S. enteritidis, Haemophilus influenzae, Bacillus subtilis, Pseudomonas fluorescence, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus and E. coli to twenty antibiotics i.e. cefpirom (30 mcg), ceftriaxone (30 mcg), erythromycin (15 mcg), doxycycline (30 mcg) lomefloxacin (10 mcg), sisomicin (30 mcg), vancomycin (30 mcg), augmentin (30 mcg), ampicillin (30 mcg), cotrimoxazole (25 mcg), cefotaxime (30 mcg), Chloramphenicol (30 mcg), cephalexin (30 mcg), tetracycline (30 mcg), ciprofloxacin (5 mcg), nitrofurantoin (300 mcg), nalidixic acid (30 mcg), pefloxacin (10 mcg), norfloxacin and ofloxacin (5 mcg) was studied to evaluate the antimicrobial efficacy of recently introduced second and third generation antibiotics. All the test strains were sensitive to pefloxacin, erythromycin, augmentin and chloramphenicol. Maximum resistance to cefpirom excluding E. coli and S. typhi and co-trimoxazole except S. typhi, Pseudomonas aeruginosa was observed, occasional resistance was seen against ceftriaxone, vancomycin and cefotaxime.

Determinación cuantitativa de la actividad de los agentes antimicrobianos CIM / Quantitative determination of the activity of the antimicrobial agents CIM

El documento ofrece el III Curso Latinoamericano de Actualización en Antimicrobianos realizado en Managua del 13 al 15 de junio del 2000. En el que aborda el tema sobre la determinación cuantitativa de la actividad de los agentes antimicrobianos (CIM), así como la cinetica de la actividad antimicrobiana, los casos en que se debe realizar una prueba cuantitativa (CIM), ,los métodos para la determinación cuantitativa de la sensibilidad a los antimicrobianos, los factores que afectan los resultados de las pruebas de sensibilidad por dilución, los criterios para establecer puntos de corte farmacocinetica y farmacodinamica de las drogas, asi como los criterios para establecer puntos de corte y las recomendaciones para evaluar la sensibilidad de microorganismos inusuales, los mecanismos de resistrencia a antibióticos Betalactamicosn en Haemophilus SPP