[Criterium pencil mines as an alternative to platinum rods (Inoclic) in achievement of the antibiogram in agar medium for countries with limited resources?] / Les mines de crayon pour critérium comme alternative aux tiges de platine (Inoclic) dans la réalisation de l'antibiogramme en milieu gélosé pour les pays à ressources limitées ?

The realization of the antibiotic susceptibility test in agar is the routine bacteriological examination for the determination and monitoring of bacterial susceptibility to antibiotics. In this study, we report the comparative results between pencil leads for criterium, as an alternative to platinum rods in the realization of the antibiotic susceptibility test. METHODOLOGY: Experimental study evaluating the comparability of the results between Criterium and Inoclic mines (by counting bacterial cells on agar after 5 successive dilutions of reason 10 from a bacterial suspension obtained after piercing through a colony; by measuring the inhibition diameters of 4 ATCC reference bacterial strains on an antibiogram in an agar medium) and evaluating the sterility of the criterium mines by culturing them on enriched broth (heart - brain type). RESULTS: 42 bacterial strains were used for bacterial cell counting. The results were of the same order of magnitude (107 CFU/mL) between Inoclic and criterium mines, for all strains and at all dilutions. The antibiotic susceptibility tests performed for the 4 reference strains by the Inoclics and criterium mines all complied (100%) with the expected limits for determining their sensitivity profile to the antibiotics tested. Compared to the bacterial growth inhibition diameters on antibiotic susceptibility tests, no intra-operator variability was observed, while significant inter-operator variability (both with Inoclic and 0.5 mm criterium mines) was observed with some strains and for inhibition diameters greater than 10 mm. The enriched broth cultures (BCC) and their subculture carried out on 10 criterium mines from 5 different batches were negative. CONCLUSION: Criterium mines seem to be a serious and less expensive alternative to Inoclic for the realization of antibiotic susceptibility testing in our resource-limited countries.

Assessment of predatory bacteria and prey interactions using culture-based methods and EMA-qPCR.

Traditional culture-based enumeration methods were compared with the ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) technique to assess Bdellovibrio-and-like-organisms (BALOs) predator-prey interactions. Gram-negative [Pseudomonas spp. and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium)] organisms were employed as prey cells, while a Bdellovibrio bacteriovorus strain (PF13) was used as the predator. The co-culture experiments were also compared in diluted nutrient broth (DNB) and HEPES buffer. In both media, K. pneumoniae (maximum log reduction of 5.13) and Pseudomonas fluorescens (P. fluorescens) (maximum log reduction of 4.21) were sensitive to predation by B. bacteriovorus PF13 as their cell counts and gene copies were reduced during all the co-culture experiments, while the concentration of B. bacteriovorus PF13 increased. The concentration of B. bacteriovorus PF13 also increased in the presence of S. aureus (HEPES buffer) and E. faecium (DNB), indicating that the predator interacted with these Gram-positive prey in order to survive. Moreover, as no predator plaques were produced in the co-culture experiments with P. aeruginosa (DNB and HEPES buffer), S. aureus (DNB and HEPES buffer) and E. faecium (HEPES buffer), EMA-qPCR proved to be beneficial in monitoring the concentration of B. bacteriovorus. In conclusion, the cell counts and/or EMA-qPCR analysis for the HEPES buffer and DNB assays were successfully employed to monitor the predation of P. fluorescens and K. pneumoniae by B. bacteriovorus, while E. faecium was sensitive to predation in DNB and S. aureus was sensitive to predation in HEPES buffer.

Identification of a conserved chromosomal region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and assessment of the role of fimbriae in pathogenicity.

Type 3 fimbriae are expressed by most clinical Klebsiella pneumoniae isolates and mediate adhesion to host structures in vitro. However, the role of type 3 fimbriae in K. pneumoniae virulence has not been evaluated by use of in vivo infection models. In this study, the type 3 fimbrial gene cluster (mrk) of the clinical isolate C3091 is described in detail. The mrk gene cluster was revealed to be localized in close proximity to the type 1 fimbrial gene cluster. Thus, a 20.4-kb fimbria-encoding region was identified and found to be highly conserved among different K. pneumoniae isolates. Interestingly, a homologue to PecS, known as a global regulator of virulence in Erwinia chrysanthemi, was identified in the fimbria-encoding region. Comparison to the previously characterized plasmid encoded mrk gene cluster revealed significant differences, and it is established here that the putative regulatory gene mrkE is not a part of the chromosomally encoded type 3 fimbrial gene cluster. To evaluate the role of type 3 fimbriae in virulence, a type 3 fimbria mutant and a type 1 and type 3 fimbria double mutant was constructed. Type 3 fimbria expression was found to strongly promote biofilm formation. However, the fimbria mutants were as effective at colonizing the intestine as the wild type, and their virulence was not attenuated in a lung infection model. Also, in a urinary tract infection model, type 3 fimbriae did not influence the virulence, whereas type 1 fimbriae were verified as an essential virulence factor. Thus, type 3 fimbriae were established not to be a virulence factor in uncomplicated K. pneumoniae infections. However, since type 3 fimbriae promote biofilm formation, their role in development of infections in catheterized patients needs to be elucidated.

Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes.

Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.