Serogroups, K1 antigen, and antimicrobial resistance patterns of Aeromonas spp. strains isolated from different sources in Mexico

A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek��. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60% were identified as A. hydrophila, 20.4% as A. caviae, and 19.25% as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90% respectively) and environmental sources (45 and 10% respectively). Using "O" antisera, only 42.5% (94/221) of the strains were serologically identified, 55% (121/221) were non-typable, and 2.5% (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60% of the serotyped strains. More than 50% of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67% of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.

Actinobaculum schaalii: clinical observation of 20 cases.

Actinobaculum schaalii is a new species that has so far been isolated from human blood, urine and pus. Its importance has probably been underestimated and other Actinobaculum spp. may also have been underdiagnosed. This retrospective study comprises all known cases of A. schaalii infections identified since 2004 in the canton of Neuch��tel (170,000 inhabitants), Switzerland. Strains were cultivated and isolated in the bacteriology laboratory using its routine procedure. Identification included a Rapid ID 32 A strip (bioMérieux) and 16S rRNA gene sequencing. Twenty-one positive samples were found in 19 patients (11 male, 8 female) of all ages (range 16-91 years): 10 from urine (50%), six from blood (30%), one from both blood and urine (5%), and three from pus (15%). Thirteen out of 17 (76%) cases with either blood or urine specimens had underlying genitourinary tract pathologies. When urine cultures were positive for A. schaalii, leucocytes were found in all samples (10/10, 100%) but all nitrite tests were negative (10/10, 100%). The onset of appropriate treatment was delayed due to the diminished sensitivity of A. schaalii to the antibiotics commonly used for UTIs (i.e. ciprofloxacin and trimethoprim/sulfamethoxazole) and to the delay in microbiological diagnosis. A. schaalii should specifically be searched in all cases of leukocyturia with a negative nitrite test but with Gram-positive rods in the Gram stain, in patients with underlying genitourinary tract pathology, instead of dismissing these findings as clinically irrelevant colonization by coryneform bacteria. This infection may be much more common than previously thought.

Preval��ncia de coloniza����o por estreptococos do grupo B em gestantes atendidas em maternidade p��blica da regi��o Nordeste do Brasil

OBJETIVO: avaliar a preval��ncia da coloniza����o pelo estreptococo do grupo B (EGB) em gestantes em pr��dromos ou em trabalho de parto. M��TODOS: foram colhidas culturas vaginal e retal de 201 gestantes atendidas no setor de admiss��o de maternidade p��blica da regi��o Nordeste do Brasil (S��o Lu��s, Maranh��o). As amostras obtidas foram inoculadas em meio seletivo de Todd Hewith e, posteriormente, subcultivadas em placas de ��gar sangue. O teste de CAMP (Christie, Atkins, Munch-Petersen) foi utilizado para identifica����o do EGB, confirmado sorologicamente pelo sistema de microteste kit Api 20 Strep da BioMérieux. As amostras positivas para EGB foram submetidas ao teste de sensibilidade para antibi��ticos. Foram estudadas as vari��veis sociodemogr��ficas, antecedentes gineco-obst��tricos e desfechos perinatais. Na an��lise estat��stica foram utilizados os programas Epi-Info 3.3.2, da Organiza����o Mundial de Sa��de e o Statistical Package for Social Sciences, vers��o 14.0. A raz��o de preval��ncia foi utilizada como medida de risco, considerando como n��vel de signific��ncia p<0,05, aceitando-se poder de 80%. RESULTADOS: a preval��ncia da coloniza����o materna pelo EGB foi de 20,4%. N��o foi encontrada associa����o entre as vari��veis sociodemogr��ficas ou antecedentes gineco-obst��tricos, com a maior presen��a de coloniza����o pelo EGB. Houve dois desfechos infecciosos entre os rec��m-natos de m��es colonizadas, por��m as hemoculturas foram negativas. Foram encontradas taxas de resist��ncia elevadas para os seguintes antibi��ticos: clindamicina, 25,4%; eritromicina, 23,6% e ceftriaxona 12,7%. CONCLUS��ES: a preval��ncia da coloniza����o materna pelo EGB foi elevada, semelhante �� descrita em outros estudos. As taxas elevadas de resist��ncia aos antimicrobianos, especialmente ceftriaxona, indicam a necessidade de mais estudos para determinar a sorotipagem deste agente e os protocolos de orienta����o para uso racional de antimicrobianos.

Avalia����o, identifica����o e atividade enzim��tica de bact��rias psicotr��ficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4��C, 10oC and 7��C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Infec����o do trato urin��rio em crian��as de um hospital p��blico do Par��-Brasil: perfil cl��nico-epidemiol��gico e genotipagem dos uropat��genos

A infec����o do trato urin��rio (ITU) �� uma das doen��as mais comuns na inf��ncia e em 80 a 90% dos casos �� causada por bact��rias da fam��lia Enterobacteriaceae, especialmente Escherichia coli e Klebsiella pneumoniae, as quais no mundo inteiro t��m emergido como produtoras de ESBL, um dos principais mecanismos de resist��ncia bacteriana a cefalosporinas de espectro-estendido e monobactans. A preval��ncia da ITU em crian��as, bem como as vari��veis, sexo, idade, febre, bact��ria mais frequente, presen��a de refluxo vesico-ureteral (RVU), presen��a de cicatrizes renais foram avaliadas no per��odo de janeiro de 2006 a mar��o de 2009, em hospital p��blico de bel��m, regi��o norte do Brasil e no per��odo de abril a agosto de 2009, isolados de cepas de E. coli e K. pneumoniae foram obtidos de urina de crian��as menores de 16 anos e avaliados fenotipicamente atrav��s do m��todo automatizado de caracteriza����o de ESBL, Vitek2, juntamente com a PCR para determinar se os genes^blaTEM, ^blaSHV e ^blaCTX-M1 estavam presentes em cada organismo. Foram confirmados 199 casos de ITU no per��odo estudado, 54,2% eram do sexo feminino, 46,2% eram menores de 02 anos de idade, febre ocorreu em 37,3% dos casos, RVU foi identificado em 38,6% das crian��as com ITU e cicatriz renal em 38%, a bact��ria mais frequente foi a E. coli (60%). Foram isoladas 43 amostras ( E. coli e K. pneumoniae, 74,4% e 25,6%, respectivamente), 95% foi resistente a ampicilina e sulfametoxazol-trimetroprim; 23,2% apresentaram fen��tipo ESBL. O gene^blaCTX-M1 foi o mais prevalente, encontrado em 19 cepas, seguido do gene ^blaTEM (18 cepas) e ^blaSHV (8 cepas). Esse estudo mostrou que bact��rias com perfil de resist��ncia ESBLcirculam no ambiente hospitalar em Bel��m e que os genes ^blaCTX-M1 e ^blaTEM e ^blaSHV est��o presentes em cepas de E. coli e K. pneumoniae causadoras de ITU em crian��as na regi��o norte do Brasil.

Estomatite prot��tica em pacientes com Diabetes mellitus: preval��ncia de Candida spp. e efici��ncia dos tratamentos com nistatina e desinfec����o de pr��teses por micro-ondas

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Pesquisa de genes de resist��ncia do M. tuberculosis a isoniazida e rifampicina por sequenciamento e pela tecnologia de DNA-STRIP

Tuberculose (TB), causada por Mycobacterium tuberculosis, �� uma das doen��as infecciosas que mais causam mortes. Estima-se que mais de dois bilh��es de pessoas estejam infectadas no mundo. O tratamento da TB consite em associa����o de f��rmacos, isoniazida, rifampicina, pirazinamida e etambutol, nos primeiros 2 meses e 4 meses de isoniazida e de rifampicina. Internacionalmente, s��o consideradas cepas multi resistentes (MDR), as que apresentam resist��ncia simult��nea a isoniazida e a rifampicina. A r��pida detec����o de resist��ncia �� essencial para o controle e tratamento da TB, reduzindo, assim, o custo do tratamento e a transmiss��o da doen��a. Neste projeto, os isolados j�� identificados fenotipicamente como resistentes a isoniazida e/ou rifampicina, foram submetidos ao sequenciamento de Sanger para pesquisa de 3 genes relacionados a resist��ncia a isoniazida (katG, inhA e ahpC) e 1 gene de resist��ncia a rifampicina (rpoB). Foi realizada uma compara����o destes genes mutados para a resist��ncia utilizando o novo teste desenvolvido pela Biomérieux, denominado GenoType�� MTBDRplus, que se baseia na tecnologia DNA-STRIP. Atrav��s deste novo teste, foi observada muta����o em 22 isolados cl��nicos de M. tuberculosis para genes de resist��ncia a isoniazida e/ou rifampicina, sendo 4 provenientes do MS e 18 de SP. J�� pelo sequenciamento gen��tico foi observada muta����o em 24 isolados para genes de resist��ncia a isoniazida e/ou rifampicina, sendo 6 provenientes do MS e 18 de SP. Portanto, atrav��s do sequenciamento de Sanger, foi poss��vel detectar um n��mero maior de isolados mutados e mais muta����es quando comparado ao teste GenoType�� MTBDRplus. Isso acontece porque na t��cnica de sequenciamento, um fragmento do gene como um todo �� analisado e no caso do teste GenoType�� MTBDRplus, �� verificada apenas a aus��ncia ou presen��a das muta����es mais frequentes descritas na literatura, al��m de n��o ser analisado o gene ahpC. A grande ...

The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase

OBJECTIVE: Enterobacteriaceae bacteria harboring Klebsiella pneumoniae carbapenemase are a serious worldwide threat. The molecular identification of these pathogens is not routine in Brazilian hospitals, and a rapid phenotypic screening test is desirable. This study aims to evaluate the modified Hodge test as a phenotypic screening test for Klebsiella pneumoniae carbapenemase. METHOD: From April 2009 to July 2011, all Enterobacteriaceae bacteria that were not susceptible to ertapenem according to Vitek2 analysis were analyzed with the modified Hodge test. All positive isolates and a random subset of negative isolates were also assayed for the presence of blaKPC. Isolates that were positive in modified Hodge tests were sub-classified as true-positives (E. coli touched the ertapenem disk) or inconclusive (distortion of the inhibition zone of E. coli, but growth did not reach the ertapenem disk). Negative results were defined as samples with no distortion of the inhibition zone around the ertapenem disk. RESULTS: Among the 1521 isolates of Enterobacteriaceae bacteria that were not susceptible to ertapenem, 30% were positive for blaKPC, and 35% were positive according to the modified Hodge test (81% specificity). Under the proposed sub-classification, true positives showed a 98% agreement with the blaKPC results. The negative predictive value of the modified Hodge test for detection was 100%. KPC producers showed high antimicrobial resistance rates, but 90% and 77% of these isolates were susceptible to aminoglycoside and tigecycline, respectively. CONCLUSION: Standardizing the modified Hodge test interpretation may improve the specificity of KPC detection. In this study, negative test results ruled out 100% of the isolates harboring Klebsiella pneumoniae carbapenemase-2. The test may therefore be regarded as a good epidemiological tool.

The modified Hodge test is a useful tool for ruling out Klebsiella pneumoniae carbapenemase

OBJECTIVE: Enterobacteriaceae bacteria harboring Klebsiella pneumoniae carbapenemase are a serious worldwide threat. The molecular identification of these pathogens is not routine in Brazilian hospitals, and a rapid phenotypic screening test is desirable. This study aims to evaluate the modified Hodge test as a phenotypic screening test for Klebsiella pneumoniae carbapenemase. METHOD: From April 2009 to July 2011, all Enterobacteriaceae bacteria that were not susceptible to ertapenem according to Vitek2 analysis were analyzed with the modified Hodge test. All positive isolates and a random subset of negative isolates were also assayed for the presence of blaKPC. Isolates that were positive in modified Hodge tests were sub-classified as true-positives (E. coli touched the ertapenem disk) or inconclusive (distortion of the inhibition zone of E. coli, but growth did not reach the ertapenem disk). Negative results were defined as samples with no distortion of the inhibition zone around the ertapenem disk. RESULTS: Among the 1521 isolates of Enterobacteriaceae bacteria that were not susceptible to ertapenem, 30% were positive for blaKPC, and 35% were positive according to the modified Hodge test (81% specificity). Under the proposed sub-classification, true positives showed a 98% agreement with the blaKPC results. The negative predictive value of the modified Hodge test for detection was 100%. KPC producers showed high antimicrobial resistance rates, but 90% and 77% of these isolates were susceptible to aminoglycoside and tigecycline, respectively. CONCLUSION: Standardizing the modified Hodge test interpretation may improve the specificity of KPC detection. In this study, negative test results ruled out 100% of the isolates harboring Klebsiella pneumoniae carbapenemase 2. The test may therefore be regarded as a good epidemiological tool.

Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain

60 strains (belonging to the genera Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus) were tested for their capacity to inhibit the growth of 3 strains of Campylobacter jejuni: Lactobacilli and bifidobacteria were left to grow in MRS or TPY broth at 37��C overnight in anaerobic conditions; Campylobacter jejuni was inoculated in blood agar plates at 37��C for 24-48 hours in microaerophilic conditions. The inhibition experiments were carried out in vitro using ���Spot agar test��� and ���Well diffusion assay��� techniques testing both cellular activity and that of the surnatant. 11 strains proved to inhibit the growth of Campylobacter jejuni. These strains were subsequently analised analised in order to evaluate the resistance to particular situations of stress which are found in the gastrointestinal tract and during the industrial transformation processes (Starvation stress, osmotic stress, heat stress, resistance to pH and to bile salts). Resistance to starvation stress: all strains seemed to resist the stress (except one strain). Resistance to osmotic stress: all strains were relatively resistant to the concentrations of 6% w/v of NaCl (except one strain). Resistance to heat stress: only one strain showed little resistance to the 55��C temperature. Resistance to pH: In the presence of a low pH (2.5), many strains rapidly lost their viability after approximately 1 hour. Resistance to bile salts: Except for one strain, all strains seemed to be relatively resistant to the 2% w/v concentration of bile salts. Afterward, strains were identified by using phenotipic and molecular techniques. Phenotipic identification was carried out by using API 50 CHL (bioMérieux) and API 20 STREP identification system (bioMérieux); molecular identification with species-specific PCR: the molecular techniques confirmed the results by phenotipic identification. For testing the antibiotic resistance profile, bacterial strains were subcultured in MRS or TPY broth and incubated for 18 h at 37��C under anaerobic conditions. Antibiotics tested (Tetracycline, Trimethoprim, Cefuroxime, Kanamycin, Chloramphenicol, Vancomycin, Ampycillin, Sterptomycin, Erythromycin) were diluted to the final concentrations of: 2,4,8,16,32,64,128,256 mg/ml. Then, 20 ��l fresh bacterial culture (final concentration in the plates approximately 106 cfu/ml) were added to 160 ��l MRS or TPY broth and 20 ��l antibiotic solution. As positive control the bacterial culture (20 ul) was added to broth (160 ul) and water (20 ul). Test was performed on plates P96, that after the inoculum were incubated for 24 h at 37oC, then the antibiotic resistance was determined by measuring the Optical Density (OD) at 620 nm with Multiscan EX. All strains showed a similar behaviour: resistance to all antibiotic tested. Further studies are needed.

In vitro activity of clinically implemented ��-lactams against Aerococcus urinae: presence of non-susceptible isolates in Switzerland.

We analyzed the in vitro susceptibility to several ?-lactams and vancomycin of 80 Aerococcus urinae isolates col- lected during 2011-2012 in Switzerland. MICs were determined by Etest (bioMérieux) on M��ller-Hinton agar with 5% sheep blood and interpreted according to the CLSI and EUCAST criteria set for viridans streptococci. MIC50/90 for penicillin, amoxicillin, ceftriaxone and vancomycin were 0.016/0.064 mg/l, 0.032/0.064 mg/l, 0.125/0.5 mg/l and 0.38/0.5 mg/l, respectively. Three (3.8%) isolates were resistant to ceftriaxone regardless of the criteria used (MICs ?2 mg/l); one of them was also non-susceptible to penicillin (MIC of 0.25 mg/l) according to CLSI. ?-lactam resis- tance in A. urinae is a concern and suggests that more studies are needed to determine the molecular mechanisms of such resistance.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BACKGROUND Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(��), bioMérieux, Marcy-L'��toile, France. Alere Determine HBsAg���, Iverness Biomedical Innovations, K��ln, Germany. Quick Profile���, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(��) assay (Abbott Laboratories, Sligo, Ireland). RESULTS The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile��� assay with a virus harboring a D144A mutation. CONCLUSIONS The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Giza papilomabirusa eta umetoki lepoko minbizia: aldakortasun genetikoa, biomarkatzaileak eta epidemiologia

148 p.