Molecular bacteriology

Malgré les progrès réalisés en réanimation ces dernières années, la mortalité du choc septique demeure inchangée. La reconnaissance des signes suspects d'infection méningococcique, l'institution d'une antibiothérapie urgente et d'un support hémodynamique intensif constituent toujours le traitement incontournable du purpura fulminans chez l'enfant. Des travaux récents soulignent l'importance des polymorphismes génétiques au niveau de plusieurs systèmes pour rendre compte des formes les plus sévères : profil cytokinique anti-inflammatoire IL-10/TNF-α, taux élevés d'inhibiteur-1 de l'activateur du plasminogène, déficit d'activation complémentaire via la mannose binding lectin. Ces données permettent de mieux comprendre l'échec général de l'immunothérapie du choc septique, et d'expliquer les désillusions des réanimateurs pédiatres. Aussi, un espoir raisonnable d'éradiquer cette maladie réside-t-il toujours dans la mise au point de nouveaux vaccins anti-méningocoques, malgré la complexité de leur diversité génétique et les recombinaisons intra-espèces au sein du réservoir rhinopharyngé. © 2001 Éditions scientifiques et médicales Elsevier SAS purpura fulminans / choc septique / cytokine / coagulation intravasculaire disséminée / polymorphisme génétique Summary -Severe meningococcal shock with purpura fulminans: untoward result of genetic polymorphisms? Despite significant progress in intensive care medicine, the mortality of septic shock has not changed in recent years. Early recognition of subtle signs in favor of meningococcal sepsis, early antibiotic treatment, and aggressive hemodynamic support remains the cornerstone of therapy of severe meningococcal shock in children. Recent work has emphasized the role of genetic polymorphisms in various systems to explain the most severe cases: anti-inflammatory cytokine profile IL-10/TNF-α, elevated levels of plasminogen activator inhibitor type-1, variants of the gene for mannose-binding lectin complement pathway. This may explain the disillusionment of pediatric intensivists, and the general failure of immunotherapy for sepsis. Reasonable hope lies upon new meningococcal vaccines. © 2001 Éditions scientifiques et médicales Elsevier SAS shock, septic / purpura fulminans / meningococcal infections / cytokine / coagulation protein disorders / genetic predisposition to disease *Correspondance et tirés à part. Adresse

ABSTRACT
In the present study five bacterial strains were
used to study the antibacterial activity of soap and detergent by disc method and
turbidity method. It was found that Staphylococcus aureus was good bactericidal as it unable to grow
in any of the detergent
concentration and other species showed varied lev
el of minimum inhibition concentration. It is possible that antibacterial soaps and
detergents have the antibacterial agents that can either kill or inhibit the bacterial cells. It mig
ht be possible that some
bacterial
strains become resistant which leads
to their survival even at high concentrations of soaps. The resistant bacterium against soap in
the present study was Pseudomonas aeroginosa.

Though the promotion of resistant bacteria worldwide has mainly been attributed to the injudicious use of many antibiotics, the role of non-antimicrobial drugs used in the clinical management of pathological conditions of non-infectious diseases in the modulation of microbial drug resistance or susceptibility needs to be assessed. The effects of the antibiotic non-antibiotic drug-drug interactions against bacteria in SCD patients and in other chronic diseases cannot be overemphasized. The interactions of nine SCD management drugs and fifteen phenotype modulating compounds on the activities of 19 standard antimicrobial drugs
against clinical isolates of methicillin-resistant S. aureus (MRSA GGP E120) and E. coli (E. coli GGP E100) were investigated by pairwise drug combinations between SCD drugs/phenotypic compounds and antimicrobial agents using the agar plate disc diffusion method. The comparative effects of the SCD drugs/PMCs on ethidium bromide (EtBr) uptake and efflux in the bacterial cells relative to verapamil were detected by measuring fluorescence of intracellular EtBr. SCD drugs and PMCs biofilm inhibitory and disrupting abilities were analyzed by reading absorbencies of crystal violet stained biofilms. Bacterial strains were obtained from the bacteriology departments of the Noguchi Memorial Institute for Medical Research, University of Ghana, Accra.

In the preliminary pairwise combination screen by disc diffusion, SCD drugs produced mostly resistance-inducing interactions with standard antibiotics particularly the cell wall inhibitors, amoxicillin and ampicillin and some few weak resistance-breaking features mostly with gentamicin. The opioid medication, morphine emerged as a key resistance-breaking (antibiotic enhancing) SCD drug. Methotrexate, an anti-inflammatory and anti-folate
compound generated significant interactions with antibiotics against both organisms. Resistance breaking and resistance inducing effects were characterized as increase and decrease in zones of inhibition by four millimetres or more respectively, compared to zones produced in the absence of SCD drug.

In E. coli, no SCD drug exceeded the accumulated intracellular ethidium bromide (EtBr) level achieved by verapamil, the control for maximum accumulation. Hydroxyurea and paracetamol induced higher uptake of EtBr within MRSA relative to verapamil. Results were analyzed using the fluorescence value of verapamil control as standard to assess and compare the effects of the SCD drugs, taking into consideration the overlapping of error bars.

Generally, all SCD drugs showed higher rates of efflux inhibition (low efflux activity) relative to the no drug treatment control (bacterial cells and EtBr only) in both organisms, following the course of the line curves. Efflux activities of SCD drugs were not significantly different from verapamil with the exception of deferasirox and methotrexate in E. coli. In MRSA, efflux activities of methotrexate, morphine, ibuprofen and paracetamol treated cells were not significantly distinct relative to efflux activity of verapamil, the control for minimum rate of efflux.

Using a 50% value of biofilm formed in the compound free medium as a cut-off to analyze the effects of SCD drugs, the most effective biofilm inhibiting abilities (approximately greater than 60%) was observed for methotrexate and pethidine against E. coli and none (greater than 50%) against MRSA. Deferasirox showed above 50% biofilm disrupting activity against E. coli biofilm and again none against MRSA biofilm. Overlapping of error bars representing standard error of the mean was carefully considered in the analyses. Biofilm formation and integrity tended to be more robust in MRSA than in E. coli.

Extracts from four fungal isolates – SBF 052, SBF 062, TEF 246 and TEF 256 showed antibacterial activities against both E. coli and MRSA in a preliminary disc diffusion assay.

The resistance-inducing interactions and few resistance-breaking interactions generated by SCD drugs to the antibiotics against both bacterial strains could not be attributed to their effects on efflux or biofilm formation and thus, the need for further investigations to establish the molecular mechanisms causing such interactions.

Further investigations are also necessary to provide healthcare providers with information on what antibiotics to prescribe for certain chronic disease patients who are continually taking particular management medications. Analyses of microbial stress responses to the unique antibiotic-non-antibiotic interactions in further studies would reveal a number of endogenous resistance-promoting genes that represent possible therapeutic targets in countering antimicrobial resistance.

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven mi-crosatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more dis-criminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC phylotype III.

Communications 569 tive pneumonia study: implications for underdiagnosis. JAMA 1983;249:3184-92. 1 I. Dournon E, Bibb WF. Rajagopalan P, Desplaces N. McKinney RM. Monoclonal antibody reactivity as a virulence marker for Legione/la pneumophila serogroup I strains. J Infect Dis 1988;157:496-501. 12. World Health Organization. Epidemiology. prevention and control of legionellosis: memorandum from a WHO meeting. Bull World Health Organ 1990;68:155-64. 13. Alary M. Joly JR. Risk factors for the contamination of domestic hot water systems by Legionellaceae. Appl Environ Microbiol 1991 ;57:2360-7. 14. Ciesielski CA, Blaser MJ, Wang WL. Role of stagnation and obstruction of water flow in isolation of Legione/la pneumophila from hospital plumbing. Appl Environ Microbiol 1984;48:984-7. 15. Stout JE, Yu VL, Best MG. Ecology of Legione/la pneumophila within water distribution systems. Appl Environ MicrobioI1985;49:221-8.

Background: Most urinary tract infections (UTI) are caused by Escherichia coli. Integrons have an important role in distributing antibiotic resistance genes among bacteria. Objectives: The aim of this study was to investigate the presence of class 1, 2 and 3 integrons and their association with antibiotic resistance in E. coli isolated from patient with UTI in Yasuj, Iran. Patients and Methods: In this cross-sectional study a total of 200 E. coli were collected from 1820 patients diagnosed with UTI that had been referred to two clinical laboratories between February 2013 and November 2014 in Yasuj city, southwest of Iran. Susceptibility of isolates to 11 different antibiotics was determined by the disk agar diffusion method. multiplex-polymerase chain reaction (PCR) was used for detection of class 1, 2 and 3 integrons. The data were analyzed using the SPSS software (version 16) and the chi-square test. A P value of < 0.05 was considered statistically significant. Results: The highest rate of resistance was observed toward cephalothin (99%) and amoxicillin (76%) while only two (1%) isolates showed resistance to imipenem. Overall, 79% of isolates were multi drug resistant (MDR). Class 1 and 2 integrons were detected in 104 (52%) and 5 (2.5%) isolates respectively, while none of the isolates were positive for class 3 integrons. A significant association was observed between the presence of integrons and resistance to co-trimoxazole, nalidixic acid, ciprofloxacin, amoxicillin, ceftazidime and tetracycline (P < 0.05). Conclusions: High MDR isolates of E. coli were observed in this study. The significant association between class 1 integrons and resistance to ciprofloxacin, nalidixic acid, co-trimoxazole, amoxicillin, ceftazidime and tetracycline showed that class 1 integrons have an important role in resistance to these antibiotics in this region.

The aim of this study was to investigate the antimicrobial activities of calcium ions and other cross-linking agents of alginate dressings, as well as their compatibility with commonly used topical antimicrobials. The antimicrobial activities of cross-linking agents and antimicrobials (five antibiotics and four antiseptics) were evaluated by the broth dilution method. The interactions between individual cross-linking agents and antimicrobials were evaluated using the chequerboard test against common skin pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. From the MIC determined, antibiotics were the most active, followed by the antiseptics and cross-linking agents. Calcium ions, which are commonly used to cross-link alginate, exhibited very weak antimicrobial activity and higher fractional inhibitory concentration than the other cross-linking agents. The use of calcium and gentamicin resulted in antagonism against S. aureus. In contrast, aluminium, zinc and copper ions exh...

We analyzed the restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) to document the occurrence of endogenous, systemic bacteremia and meningitis due to Enterobacter cloacae in a newborn. Five strains of E. cloacae were isolated from this newborn. Three of these strains were recovered from stool at counts of 10 8 , 10 9 , and 10 9 organisms/g offeces, respectively; one strain was isolated from blood; and one strain was isolated from cerebrospinal fluid. In addition, five epidemiologically unrelated strains of E. cloacae were studied for comparison. Our study clearly shows the genetic relatedness of the strains isolated sequentially from cultures of stool, blood, and cerebrospinal fluid. RFLP analysis of total DNA and ribotyping seem particularly well suited to the study of the epidemiology of nosocomial E. cloacae strains.

Background.Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of theRalstonia solanacearumspecies complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III.Methods.We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Differ...

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. D...

During a systematic survey, maternal carriage of Proteus mirabilis was found over a 25-day period in 18 pregnant women admitted to the delivery ward of our hospital maternity. Five neonates born to these mothers were found to be colonized with P. mirabilis. We report here on the use of DNA fingerprinting by the arbitrarily primed polymerase chain reaction technique (AP-PCR) for the epidemiological investigation of this sudden outbreak. This approach was compared with the analysis of restriction fragment length polymorphisms of ribosomal DNA regions (ribotyping). Results of the AP-PCR and of ribotyping were in complete agreement in showing the genetic unrelatedness of the isolates obtained from each mother. Moreover, the results showed mother-to-infant vertical transmission of P. mirabilis in the neonates. AP-PCR is a rapid and discriminative method which seems particularly well suited to the epidemiological study of P. mirabilis.

The current study aimed to identify the selected number of Campylobacter strains of poultry origin in India that were isolated in the Laboratory of International Prevention of Epidemics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan to the species level with the aid of cdtA/B/C gene-based multiplex PCR assays as well as to investigate their antimicrobial resistance profiles. C. jejuni (4 strains) and C. coli (16 strains) were identified. The poultry isolates identified were subjected to susceptibility testing with the aid of disk diffusion method using 12 antimicrobial agents. Again, the resistant and intermediate isolates confirmed by the disk diffusion method were subjected to determination of minimum inhibitory concentration by agar dilution method. To elucidate the mechanism of quinolone resistance, a total number of 20 quinolone resistant strains were subjected to sequence determination and analysis of the gyrA gene in the quinolone-resistance determining region. The results of the disk diffusion method were consistent with the results of the agar dilution method with slight variation in case of ampicillin, levofloxacin, ofloxacin and fosfomycin. All C. jejuni and C. coli strains had the Thr 86 to Ile substitution in gyrA. Results of this study support the emergence of resistance of C. jejuni and C. coli strains of poultry origin in India to a variety of antimicrobials especially fluoroquinolones.

A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5-7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.

Ribotyping with a nonradioactive probing system was used for the epidemiological evaluation of 15 Serratia marcescens nosocomial strains isolated from the stools of 12 children with no apparent illness in five different hospital wards over a 20-day period. Our results indicate that the occurrence of S. marcescens colonization was the result of the spread of a single epidemiological strain in the hematology ward, the oncology ward, and the gastroenterology ward and in two neonates in the neonatology ward, suggesting cross-contamination between Escherichia coli (Boehringer) was used as a probe and labeled by random oligopriming by using a mixture of

Vancomycin-resistant enterococci are now found with increasing frequency. Up to now, epidemiological studies of enterococci have been limited by the lack of convenient and accessible methods for comparing strains. In this study, we report an epidemiological investigation on 16 nosocomial vancomycin-resistant Enterococcus faecium strains isolated from 15 patients in four different wards of a children's hospital over a period of 17 months. Analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) was used as a typing approach. Each strain produced a different total DNA RFLP pattern after HindIll and PvuII digestion, except for two strains that were isolated from a single patient and that gave indistinguishable patterns. In our system, ribotyping was less discriminative than RFLP of total DNA. This approach, therefore, shows the genetic unrelatedness of the nosocomial strains studied and excludes patient-to-patient strain transmission either in the same ward or between wards.

We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiological investigation of 23 Pseudomonas cepacia isolates obtained from 11 cystic fibrosis (CF) patients attending our CF center. This approach was compared with ribotyping, pulsed-field gel electrophoresis (PFGE), and conventional phenotypic typing. AP-PCR and ribotyping were identical in resolving power, since the two methods generated four different profiles and identified the same group of strains. Six patients on the one hand and four on the other harbored strains of the same genotype, thus raising the possibility of either patient-to-patient transmission or acquisition from a common hospital environmental source. PFGE results were in good agreement with those of the other two methods, but PFGE seems more discriminative since it