Evaluation of Mycotube, a modified version of Lowenstein-Jensen (LJ) medium, for efficient recovery of Mycobacterium tuberculosis (MTB).

Timely diagnosis of tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is only achieved for ~58% cases. An improved, accurate, time- and cost-effective method for bacteriological confirmation of MTB is necessary. We evaluated Mycotube, a new variant of Lowenstein-Jensen (LJ) culture medium, by comparing it with Mycobacterium Growth Indicator Tube (MGIT) 960 (gold standard), local LJ, and bioMérieux LJ-T in terms of isolation rate and time-to-growth. Pulmonary and extra-pulmonary samples from treatment-naïve suspects (n = 207) were decontaminated by the N-acetyl-L-cysteine-sodium hydroxide method and used to inoculate the four media. Subjective and objective parameters were used for evaluation. Mycotube yielded 140 positive results, compared to 162, 69, and 141 from MGIT, local LJ, and LJ-T, respectively. Of these, 139 (67%) were true-positive results and 1 (0.5%) was false-positive. The mean time-to-growth detection was 17.4 days for Mycotube, compared to 14.5, 28.1, and 16.5 days for MGIT, local LJ, and LJ-T, respectively. The mean time-to-growth for local LJ significantly differed from that for MGIT, but not those for LJ-T and Mycotube. No contamination was observed. Mycotube had a sensitivity of 85.8% and a specificity of 97.8% as compared to MGIT. Mycotube offers good results, comparable with those observed for conventional LJ. It requires only basic laboratory infrastructure. The overall cost of the test should be nearly three times lower than that of MGIT. Mycotube helps with TB diagnosis and generates pure isolates for drug susceptibility testing.

Identification of bacterial species by untargeted NMR spectroscopy of the exo-metabolome.

Identification of bacterial species is a crucial bottleneck for clinical diagnosis of infectious diseases. Quick and reliable identification is a key factor to provide suitable antibiotherapies and avoid the development of multiple-drug resistance. We propose a novel nuclear magnetic resonance (NMR)-based metabolomics strategy for rapid discrimination and identification of several bacterial species that relies on untargeted metabolic profiling of supernatants from bacterial culture media. We show that six bacterial species (Gram negative: Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis; Gram positive: Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus saprophyticus) can be well discriminated from multivariate statistical analysis, opening new prospects for NMR applications to microbial clinical diagnosis.

A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens.

The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 10(5), 10(3), and 10 CFU yielded colonies in 5.7 ± 1.5 days, 7.6 ± 0.8 days, and 10.8 ± 1.7 days versus 1.5 ± 0.4 days, 3.5 ± 0.6 days, and 4.9 ± 1 days in MOD9 (P < 0.05, Student's t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P = 0.11, Fisher's exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P = 0.5195, Fisher's exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ± 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ± 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Student's t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis.

Rapid detection of carbapenemase activity: benefits and weaknesses of MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an identification procedure for bacteria and fungi. The MALDI-TOF MS-based analysis of resistance to ß-lactam antibiotics has been applied to detect hydrolysis of carbapenems by different bacterial strains. However, the detection of enzymatic carbapenem degradation by MALDI-TOF MS lacks well-standardized protocols and several methods and models of interpretation using different calculations of ratio-of-peak intensities have been described in the literature. Here, we used faropenem and ertapenem hydrolysis as model compounds. In an attempt to propose a universal protocol, the hydrolysis was regularly monitored during 24 h using well-characterized bacterial strains producing different types of carbapenemases (KPC, IMP, NDM, VIM, and OXA-48). Variable responses and different timing for detectable hydrolysis, depending on the enzyme produced, were observed. KPC degrades its template antibiotics very quickly (15 min for some KPC producers) compared to other types of enzymes (more than 90 min for other enzymes). Prior bacterial lysis was shown to be of no interest in the modulation or optimization of the hydrolytic kinetics. The adequate detection of carbapenem hydrolysis would, therefore, require several MALDI-TOF MS readouts for the timely detection of rapid hydrolysis without missing slow hydrolysis. This enzymatic constraint limits the implementation of a standard protocol in routine microbiology laboratories.

Evaluation of an automated repetitive sequence-based PCR system for subtyping Enterobacter sakazakii.

Enterobacter sakazakii is regarded as a ubiquitous organism that can be isolated from a wide range of foods and environments. Infection in at-risk infants has been epidemiologically linked to the consumption of contaminated powdered infant formula. Preventing the dissemination of this pathogen in a powdered infant formula manufacturing facility is an important step in ensuring consumer confidence in a given brand together with the protection of the health status of a vulnerable population. In this study we report the application of a repetitive sequence-based PCR typing method to subtype a previously well-characterized collection of E. sakazakii isolates of diverse origin. While both methods successfully discriminated between the collection of isolates, repetitive sequence-based PCR identified 65 types, whereas pulsed-field gel electrophoresis identified 110 types showing > or =95% similarity. The method was quick and easy to perform, and our data demonstrated the utility and value of this approach to monitor in-process contamination, which could potentially contribute to a reduction in the transmission of E. sakazakii.

Does nitrate co-pollution affect biological responses of an aquatic plant to two common herbicides?

Aquatic systems in agricultural landscapes are subjected to multiple stressors, among them pesticide and nitrate run-off, but effects of both together have rarely been studied. We investigated possible stress-specific and interaction effects using the new OECD test organism, Myriophyllum spicatum, a widespread aquatic plant. In a fully factorial design, we used two widely applied herbicides, isoproturon and mesosulfuron-methyl, in concentration-response curves at two nitrate levels (219.63 and 878.52mg N-NO3). We applied different endpoints reflecting plant performance such as growth, pigment content, content in phenolic compounds, and plant stoichiometry. Relative growth rates based on length (RGR-L) were affected strongly by both herbicides, while effects on relative growth rate based on dry weight (RGR-DW) were apparent for isoproturon but hardly visible for mesosulfuron-methyl due to an increase in dry matter content. The higher nitrate level further reduced growth rates, specifically with mesosulfuron-methyl. Effects were visible between 50 and 500µgL(-1) for isoproturon and 0.5-5µgL(-1) for mesosulfuron-methyl, with some differences between endpoints. The two herbicides had opposite effects on chlorophyll, carotenoid and nitrogen contents in plants, with values increasing with increasing concentrations of isoproturon and decreasing for mesosulfuron-methyl. Herbicides and nitrate level exhibited distinct effects on the content in phenolic compounds, with higher nitrate levels reducing total phenolic compounds in controls and with isoproturon, but not with mesosulfuron-methyl. Increasing concentrations of mesosulfuron-methyl lead to a decline of total phenolic compounds, while isoproturon had little effect. Contents of carbon, nitrogen and phosphorus changed depending on the stressor combination. We observed higher phosphorus levels in plants exposed to certain concentrations of herbicides, potentially indicating a metabolic response. The C:N molar ratio decreased strongly with isoproturon and increased with mesosulfuron-methyl. The C:P and N:P ratios did not vary for most herbicide concentrations, indicating homeostasis. Nitrate level had no effect on the C:N ratio, but the N:P ratio increased in high nitrate level treatments, indicating that the former is more strictly regulated by the plant than the latter. We conclude that the multi-stress impacts caused to aquatic primary producers by herbicides and nitrate enrichment, as often observed in agricultural run-off, not only affected growth and pigment content, but also structural traits (dry matter content) and other physiological traits (elemental stoichiometry, phenolic compounds). Changes in those might have indirect effects on biotic interactions and elemental cycles. We suggest considering some of these endpoints in future studies in environmental risk assessment for agricultural run-off.

Use of a DNA typing method to investigate a fatal perinatal infection due to Staphylococcus aureus.

The aim of this study was to determine, by random amplified polymorphic DNA analysis, the origin of a fatal Staphylococcus aureus perinatal infection which occurred after multiple cervical examinations before induction of labor for patient convenience. This DNA typing method was able to demonstrate that this infection originated from S. aureus genital carriage and urinary tract infection of the mother, and was not acquired in the hospital. This observation argues for the systematic screening of genital flora for highly virulent strains before induction of labor for non medical reasons. In addition, the DNA typing method used demonstrates that one neonate was subsequently infected by the strain responsible for the chorioamnionitis. This shows that molecular typing methods can help determine hospital staff responsibility.

Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia.

Yeast identification--past, present, and future methods.

The focus of this review is the evolution of biochemical phenotypic yeast identification methods with emphasis on conventional approaches, rapid screening tests, chromogenic agars, comprehensive commercial methods, and the eventual migration to genotypic methods. As systemic yeast infections can be devastating and resistance is common in certain species, accurate identification to the species level is paramount for successful therapy and appropriate patient care.

Preferential stimulation of human lymphocytes by oligodeoxynucleotides that copy DNA CpG motifs present in virulent genes of group A streptococci.

Immunostimulatory oligodeoxynucleotides (ODN) containing CpG dinucleotides have been shown to stimulate murine and human lymphocytes. We investigated the presence of stimulatory DNA motifs in specific group A streptococcal (GAS) genes to elucidate the potential role of DNA in the immunopathogenesis of GAS infections. Despite low GC content in GAS DNA, the emm1 gene encoding the streptococcal M1 protein contained a relatively high frequency of TTCG(T/C), TCGTCG and (G/A)TCGT motifs that preferentially stimulated human lymphocytes. Interestingly, these motifs were also found in genes encoding M-like proteins of group C and G streptococci. ODN copying the emm1 gene sequences harboring these motifs induced the proliferation of human B cells and up-regulated the expression of CD25 on their surface. T cells were not required for the response to the ODN, indicating a direct effect of these motifs on B lymphocytes. Inter-individual variations in responsiveness to ODN were observed, suggesting that host factors potentiate these responses. The finding that GAS DNA contains stimulatory motifs that induce activation of human B cells in a T cell-independent manner suggests that this may be an important mechanism by which the bacteria can target the innate arm of the immune system in the early stages of invasive infections.

Host variation in cytokine responses to superantigens determine the severity of invasive group A streptococcal infection.

Cytokines elicited by superantigens have been suggested to play a central role in severe systemic clinical manifestations of gram-positive sepsis. Here we provide evidence for a potent inflammatory cytokine response in acute invasive group A streptococcal infections, and show a direct correlation between the magnitude of this response and the severity of systemic manifestations of the disease. Severe invasive cases suffering from toxic shock and/or necrotizing fasciitis had significantly higher frequencies of IL-2-, IL-6-, and TNF-alpha-producing cells in their circulation as compared to non-severe invasive cases (p=0.05-0.01). This difference was even more accentuated when severe and non-severe cases infected with a clonal M1T1 strain were compared (p=0.03-0. 004). To determine whether host factors were responsible for this difference in magnitude of cytokine responses, paired age- and gender-matched severe and non-severe M1T1 cases (n=8) were tested in vitro during their convalescent phase for immune response to superantigens produced by their infecting isolate. The results showed persistent and inherent differences in the magnitude of proliferative and cytokine responses of severe and non-severe patients to the streptococcal superantigens to which they had been exposed during infection. Thus, the study provides evidence that patients with a propensity to produce higher levels of inflammatory cytokines in response to streptococcal superantigens develop significantly more severe systemic manifestations than patients who have a propensity to produce lower levels of inflammatory cytokines to the same superantigens. We therefore conclude that host factors influence the magnitude of cytokine responses to superantigens and consequently the clinical outcome of the infection.

Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis.

The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95).

Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis.

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).

Relatedness of Streptococcus suis serotype 2 isolates from different geographic origins as evaluated by molecular fingerprinting and phenotyping.

The genetic diversity of 88 Streptococcus suis serotype 2 isolates which were recovered from various countries was examined by randomly amplified polymorphic DNA (RAPD) analysis with three primers. This bacterial collection included 80 isolates of porcine origin and 8 of human origin. This investigation allowed the identification of 23 RAPD types containing 1 to 30 isolates originating from one to six countries. Common RAPD patterns were found between human and pig isolates. The isolates were also tested for the production of virulent factors such as hemolysin, muramidase-released protein (MRP), and extracellular factor (EF). All isolates exhibiting the virulent phenotype hemolysin+ MRP+ EF+ clearly clustered on the basis of fingerprinting by RAPD analysis. In a similar way, most of isolates with the hemolysin- MRP- EF- phenotype were assigned to one RAPD cluster. Therefore, RAPD clusters are more related to the phenotype defined with hemolysin, MRP, and EF than to the geographic origin of the isolates. These data indicate that RAPD analysis used in conjunction with phenotypic methods provides a reliable method for the assessment of the clonal relationship between S. suis isolates responsible for infections in pigs or humans, especially for those exhibiting the classic "virulent" phenotype hemolysin+ MRP+ EF+.

Phylogenetic diversity of Streptococcus suis strains of various serotypes as revealed by 16S rRNA gene sequence comparison.

The 16S rRNA gene sequences of reference strains of Streptococcus suis serotypes 1-34 and 1/2 were determined. A comparative sequence analysis showed that the degree of sequence similarity between S. suis reference strains ranged from 93.94 to 100%. A dendrogram was constructed from the similarity matrix. Thirty-two strains representing 32 serotypes fell into a major group divided into three clusters. The other strains, S. suis serotypes 32, 33 and 34, were more distant. Biochemical characterization of the six more distant strains, including S. suis serotypes 20, 22, 26, 32, 33 and 34, revealed a profile similar to that of other S. suis serotypes. Comparison of the 16S rRNA gene sequences of S. suis reference strains with sequences of other members of the genus Streptococcus indicated that, with the exception of S. suis serotypes 32, 33 and 34, reference strains did not cluster with any other species in the genus. In conclusion, 16S rRNA gene sequence analysis defined a major group of S. suis reference strains which were very closely related and a higher divergence for S. suis serotypes 32, 33 and 34. However, to date, there is no strong evidence to reclassify strains of these serotypes in another species.

Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases.

The relatedness of group A streptococcal (GAS) strains isolated from 35 Canadian patients with invasive disease of different severity was investigated by a variety of molecular methods. All patients were infected with M1T1 strains and, based on clinical criteria, were classified as severe (n = 21) and nonsevere (n = 14) invasive GAS infection cases. All the M1 strains studied had the emm1.0 allele and the same streptococcal pyrogenic exotoxin (Spe) genotype, speA(+) speB(+) speC speF(+) speG(+) speH smeZ(+) ssa. All isolates had the same speA allotype, speA2. The randomly amplified polymorphic DNA banding pattern with two different primers was identical for all strains, and pulsed field gel electrophoresis analysis showed that 33 and 30 isolates had identical banding patterns after DNA digestion with SfiI or SmaI, respectively; the nonidentical isolates differed from the main pattern by only one band. A relatively high degree of polymorphism in specific regions of the sic gene was observed among isolates; however, this polymorphism was not associated with disease severity. Likewise, although the phenotypic expression of SpeA, SpeB, and SpeF proteins varied among the M1T1 isolates, there was no correlation between the amount of Spe expressed and disease severity. Importantly, mitogenic and cytokine responses induced by partially purified bacterial culture supernatants containing a mixture of expressed superantigens were very similar for isolates from severe and nonsevere cases (P > 0.1). Together, the data indicate that highly related invasive M1T1 isolates, some indistinguishable, can cause disease of varying severity in different individuals. These findings underscore the contribution of host factors to the outcome of invasive GAS infections.