Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

Abstract Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2 μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125 μg/mL for both antimicrobials. The MIC90 were 4 μg/mL and 2 μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

Prevalence and Clinical Impact of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolated From Hospitalized Patients

BACKGROUND: We estimated the prevalence and clinical impact of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). The concordance between macromethod and glycopeptide resistance detection (GRD) E tests was determined. In addition, predictors of clinical outcomes in hospitalized patients with S. aureus bacteremia (SAB) or pneumonia (SAP) were evaluated. METHODS: We obtained 229 consecutive S. aureus isolates from all hospitalized patients at two university hospitals located in Busan and Yangsan, Korea. Standard, macromethod, and GRD E tests were performed. Additionally, we reviewed the medical records of all patients. Among the 229 patients, predictors of clinical outcomes were analyzed for 107 patients with SAB and 39 with SAP. RESULTS: Among the 229 isolates, 34.5% of S. aureus isolates and 50.7% of methicillin-resistant S. aureus isolates exhibited the hVISA phenotype based on the macromethod E test. hVISA was nearly associated with treatment failure in patients with SAB (P=0.054) and was significantly associated with treatment failure in patients with SAP (P=0.014). However, hVISA was not associated with 30-day mortality in patients with SAB or SAP. The concordance between the macromethod and GRD E tests was 84.2%. CONCLUSIONS: hVISA is quite common in the southeastern part of Korea. hVISA is associated with treatment failure in patients with SAP.

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

In vitro activity of vancomycin and teicoplanin against coagulase negative staphylococci

With the increase in nosocomial infections caused by coagulase negative staphylococci [CNS], laboratory diagnosis of CNS with reduced susceptibility to glycopeptides [vancomycin and teicoplanin] has become important. This study was designed to determine the glycopeptide susceptibility of clinical isolates of methicillin resistant coagulase negative staphylococci [MRCNS] at the department of microbiology, government medical college and hospital, Amritsar, India. A total of 250 CNS isolated from various clinical specimens were speciated and their methicillin resistance was detected by studying the minimum inhibitory concentration [MIC] of oxacillin by macrobroth dilution method. Glycopeptide susceptibility of 130 methicillin resistant strains obtained was determined for vancomycin by vancomycin screen agar test, MIC of vancomycin by macrobroth dilution/and E test. Teicoplanin susceptibility was determined using teicoplanin disc diffusion test and MIC was determined by macrobroth dilution method. All the MRCNS isolates were found to be susceptible to vancomycin and teicoplanin. MIC of vancomycin ranged between

Linezolid vancomycin resistant Enterococcus isolated from clinical samples in Tehran hospitals.

Background: Vancomycin-resistant enterococci pose an emerging health risk. The limitation in therapeutic options has resulted in the development of new drugs such as quinupristin/ dalfopristin and linezolid. Aim, Setting and Design: This study investigated the species prevalence and antibacterial resistance among enterococci isolated in selected Tehran hospitals. Materials and Methods: Between March 2006 and August 2007, 200 enterococcal isolates from urine, blood, stool and wound were recovered in 2 teaching hospitals of Tehran province. Susceptibility of all isolates was tested against vancomycin, teicoplanin and linezolid antibiotics by disk diffusion and agar dilution method. Results and Conclusion: Seventeen (8.5%), 6 (3%) and 4 (2%) of the isolates were resistant to vancomycin, teicoplanin and linezolid, respectively. Within the vancomycin-resistant isolates, 6 (35.2%), 4 (25%) and 1 (5.88%) showed vanA, vanB and vanC genotype patterns, respectively. Four (23.5%) of VRE isolates were resistant to linezolid with minimum inhibitory concentrations between 16 and 32 µg/mL. Two linezolid vancomycin resistant enterococci were E. faecium.

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans.

Garlic (Allium sativum) extract has been known to have inhibitory activity on various pathogenic bacteria, viruses and fungi. The objective of present investigation was to study in vitro inhibitory activity of garlic extract on multidrug-resistant (MDR) strains of Streptococcus mutans isolated from human carious teeth. Filter sterilized aqueous extract of garlic was prepared and used in the present study. For isolation of S. mutans, extracted human carious teeth were cultured in Todd-Hewit broth and Mitis-Salivarius-Bacitracin agar. S. mutans was characterized by colony morphology, biochemical tests and other conventional bacteriological procedures. Disk sensitivity tests and broth dilution methods were used to determine antibiotic sensitivity profile and inhibitory activity of garlic extract on S. mutans isolated from carious teeth. Of 105 carious teeth tested, 92 (87.6%) isolates of S. mutans were recovered, among which 28 (30.4%) were MDR since they were resistant to four or more antibiotics. The highest rate of resistance was observed for tetracycline (30.4%) and least resistance (0%) to teichoplanin and vancomycin while 22.8% and 23.9% of the isolates were resistant to penicillin and amoxicillin, respectively. Chlorhexidine minimum inhibitory concentration (MIC) for MDR and non-MDR S. mutans varied from 2 to 16 microg ml(-1) and from 0.25 to 1 microg ml(-1), respectively (P<0.05). All isolates, MDR and non-MDR of S. mutans were sensitive to garlic extract with the MIC ranging from 4 to 32 microg ml(-1). Considering in vitro data obtained in the present study, mouthwashes or toothpaste containing optimum concentration of garlic extract could be used for prevention of dental caries.

Heterogeneous resistance to vancomycin and teicoplanin among Staphylococcus spp. isolated from bacteremia

This study evaluated the BHIA screening method with 4 or 6 mug/mL of vancomycin to detect glycopeptides heteroresistant staphylococci strains isolated from bacteremia. A total of 213 staphylococci strains were isolated from 106 patients between October/2001 and November/2002 in a tertiary hospital in Rio de Janeiro city. Fifty-seven (53.8 percent) patients presented Staphylococcus aureus, while coagulase-negative staphylococci (CNS) were isolated from 49 (46.2 percent). Resistance rates for oxacillin of 26.3 percent and 81.6 percent were found for the staphylococci isolates, respectively. Thirteen CNS isolated from nine (8.5 percent) patients grew on agar screening with 4 mug/mL of vancomycin and showed heterogeneous profiles of resistance for vancomycin and teicoplanin by the population analysis profile method. Only 30.8 percent of them grew at the concentration 6 mug/mL. Bacterial infection and use of antimicrobial therapy were common among these patients. Alert about the emergence of oxacillin-resistant staphylococci presenting heteroresistance to glycopeptides is important in order to achieve judicious use of antimicrobials. Vancomycin agar screening test could help to confirm the presence of these isolates in hospitals.

In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus.

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.

Respiratory tract infection caused by bacteria (non-Mycobacterium) and their antibiogram in HIV-positive patients.

Abstract. This study was undertaken from 1995-2000 to investigate the cause of respiratory tract infection among 481 patients with human immunodeficiency virus (HIV) at Siriraj Hospital, Bangkok, Thailand. The positive rate of bacterial pathogens was 38.46%. Pseudomonas aeruginosa appeared to be the most common pathogen (32.97%), followed by Staphylococcus aureus (18.92%), Klebsiella pneumoniae (10.81 %), Haemophilus influenzae (7.57%), and Acinetobacter baumannii (5.95%). P. aeruginosa was sensitive to netilmycin, amikacin, imipenem, meropenem, cefoperazone/sulbactam, piperacillin/tazobactam, and gentamicin (67-84%). S. aureus was sensitive to vancomycin and teicoplanin (100%).

Análisis clínico-epidemiológico de la portación intestinal de enterococos resistentes a vancomicina en una unidad de terapia intensiva / Clinical and epidemiologic analysis of intestinal tract colonization with vancomycin-resistant enterococci in an intensive care unit

En un período de cinco meses y 25 días se investigó la portación intestinal de enterococos resistentes a vancomicina (EVR). Se estudiaron 124 pacientes (73%) de 171 admitidos en la unidad de terapia intensiva (UTI), 35 de los cuales (28%) resultaron ser portadores. Los aislamientos de EVR (n=35) fueron identificados como Enterococcus faecium (n=18), Enterococcus gallinarum (n=16) y Enterococcus raffinosus (n=1). Todos los aislamientos estudiados fueron resistentes a vancomicina (VAN) (CIM90= 512 µg/ml) y teicoplanina (CIM90= 32 µg/ml) y portaban el gen vanA. Los estudios de tipificación molecular mostraron un alto grado de homología entre los aislamientos de E. faecium (un clon dominante) y E. gallinarum (dos tipos clonales), sugiriendo su diseminación a modo de brote. No se encontraron diferencias significativas con la edad y el sexo de los pacientes no portadores (p>0,05), pero si con el tiempo de hospitalización y el uso de esquemas antibióticos de amplio espectro (p<0,05), estando estos dos factores asociados al estado de portación. Se deduce de este estudio, la importancia de maximizar las medidas de prevención y control de las infecciones nosocomiales, analizar los esquemas empíricos empleados, tratar de disminuir el tiempo de hospitalización y continuar con los estudios de vigilancia para evaluar la eficacia de las acciones implementadas.

Intestinal tract colonization with vancomycin resistant enterococci (VRE) was studied during five months and 25 days. Out of 171 patients hospitalized in the intensive care unit, 124 (73%) were included in this study. Thirty five of them (28%) were recognized as colonized with VRE. VRE isolates (n = 35) were identified as Enterococcus faecium (n=18), Enterococcus gallinarum (n=16), and Enterococcus raffinosus (n=1). All of them were resistant to vancomycin (MIC90= 512 µg/ml) and to teicoplanin (MIC90= 32 µg/ml), having the vanA gene. By means of molecular methods a high homology was found among E. faecium and E. gallinarum isolates, respectively, suggesting their spread as a kind of outbreak. No significant differences in age or sex were found among colonized and non-colonized patients (p>0.05). On the other hand, the hospitalization time and the use of broad-spectrum antibiotics were associated with colonization. From this study we highlight the importance of enhancing all measures of control and prevention of hospital infections, carefully analyzing the empiric antimicrobial schemes, trying to reduce the hospital stage, and following the surveillance to evaluate the efficacy of such procedures.

Glicopéptidos en el tratamiento de infecciones por microorganismo gram positivos / Glycopeptides in the treatment of gram positive infections

Glycopeptides have become a very important class of antibiotics because its widespread usage, the emergence of resistance to this antibiotics and need for new antibiotics to treat serious resistant gram positive infections. We discuss the general characteristics of this class of antibiotics, resistance to glycopeptides in gram positive organisms, the recommendations for the appropriate usage of glycopeptides, and the development of new antibiotics as alternatives to treat serious gram positive infections. Because it is important to limit the indiscriminate usage of glycopeptides, in order to avoid the emergence of resistance, recommendations for the appropriate and inappropriate usage of vancomycin are highlighted

Antibacterial activity of teicoplanin against Clostridium difficile.

The in vitro inhibitory action of teicoplanin, vancomycin, metronidazole and clindamycin against clinical isolates of Clostridium difficile was investigated. Minimum inhibitory concentrations (MICs) were determined using E test. Teicoplanin (MIC range 0.023-0.75 microgram/ml), vancomycin (MIC range 0.5-3 micrograms/ml) and metronidazole (MIC range 0.19-1 microgram/ml) were all very active against the isolates examined. No resistant strains of C. difficile to those three antimicrobial agents were observed, whereas resistance to clindamycin was found in 39.5% of the tested strains. Teicoplanin was about 4-times more potent than vancomycin. It appears to be a more promising antimicrobial for treatment of C. difficile enteric disease.

Estudo da sensibilidade de Staphylococcus sp. e Enterococcus sp. à teicoplanina e à vancomicina / Study of the susceptibility of Staphylococcus sp. and Enterococcus sp. to teicoplanin and vancomycin

Objetivo. Avaliar a sensibilidade de Enterococcus e Staphylococcus à teicoplanina e à vancomicina. Métodos. Foram estudadas 150 cepas de Enterococcus e 450 de Staphylococcus (298 Staphylococcus aureus e 152 de coagulase negativas) isoladas de três hospitais brasileiros. As CIMs foram determinadas utilizando o E Test. A concentraçao de antimicrobiano em cada fita foi de 256mcg/mL a 0,016mcg/mL. Também foi feito o estudo da sensibilidade pelo método de difusao empregando discos impregnados com 10mcg de teicoplanina e 30 mcg de vancomicina, respectivamente. Resultados. Todas as 298 cepas de Staphylococcus aureus foram sensíveis a dois antimicrobianos. Três cepas coagulase negativas apresentaram sensibilidade intermediária à teicoplanina (CIMs entre 8 e 16mcg/mL). Quatro das 150 cepas de Enterococcus apresentaram sensibilidade intermediária à vancomicina, mas foram totalmente sensíveis à teicoplanina. Conclusao. De acordo a estes resultados, teicoplanina e vancomicina sao boas opçoes terapêuticas nas infecçoes provocadas por Enterococcus e Staphylococcus.

Eficacia y seguridad de teicoplanina en el tratamiento de infecciones severas causadas por Staphylococcus / Efficacy and security of teicoplasnin in the treatment of severe infections caused by staphylococcus

Se realizó un estudio abierto no comparativo para evaluar la eficacia y seguridad de la administración de teicoplanina en pacientes con infecciones bacterianas causadas por gérmenes grampositivos, principalmente Staphylococus y Enterococcus. Fueron tratados 20 enfermos con infecciones de tejidos blandos, osteoarticulares y del aparato respiratorio inferior, causado por Staphylococcus aureus. Se les administró teicoplanina por vía intravenosa en infusión durante 30 minutos o por inyección intramuscular; la dosis inicial fue de 6 mg/kg, seguida de 3 mg/kg administrada una vez al día. Los resultados clínicos obtenidos fueron 65 por ciento de curación y 35 por ciento mejoría, con una erradicación bacteriológica del 100 por ciento. La tolerancia fue buena; sólo un enfermo fue retirado del estudio debido a que presentó rash, probablemente relacionado al fármaco en estudio. Podemos concluir que la administración de teicoplanina en dosis de 400 mg iniciales y 200 mg subsecuentes, una sola vez al día, por vía intravenosa o intramuscular, fue eficaz y segura en este estudio

Teicoplanina: uma nova opçäo para os gram-positivos / Teicoplanin: a new option to the gram-positives

Os autores fazem uma revisao da literatura e analisam as atividades da Teicoplanina em comparaçao principalmente com a Vancomicina. Ficou evidente que o espectro de açao destes dois fármacos é semelhante. A Teicoplanina mostrou-se eficaz contra a grande maioria dos cocos Gram-positivos. Porém, devido ao seu elevado custo, seu uso fica restrito a infecçoes hospitalares graves por Staphylococcus resistentes à Vancomicina.