Distribution Of Bacterial Species Research Articles

Psychobiotics and the gut-brain axis: in the pursuit of happiness.

The human intestine houses an astounding number and species of microorganisms, estimated at more than 1014 gut microbiota and composed of over a thousand species. An individual’s profile of microbiota is continually influenced by a variety of factors including but not limited to genetics, age, sex, diet, and lifestyle. Although each person’s microbial profile is distinct, the relative abundance and distribution of bacterial species is similar among healthy individuals, aiding in the maintenance of one’s overall health. Consequently, the ability of gut microbiota to bidirectionally communicate with the brain, known as the gut–brain axis, in the modulation of human health is at the forefront of current research. At a basic level, the gut microbiota interacts with the human host in a mutualistic relationship – the host intestine provides the bacteria with an environment to grow and the bacterium aids in governing homeostasis within the host. Therefore, it is reasonable to think that the lack of healthy gut microbiota may also lead to a deterioration of these relationships and ultimately disease. Indeed, a dysfunction in the gut–brain axis has been elucidated by a multitude of studies linked to neuropsychological, metabolic, and gastrointestinal disorders. For instance, altered microbiota has been linked to neuropsychological disorders including depression and autism spectrum disorder, metabolic disorders such as obesity, and gastrointestinal disorders including inflammatory bowel disease and irritable bowel syndrome. Fortunately, studies have also indicated that gut microbiota may be modulated with the use of probiotics, antibiotics, and fecal microbiota transplants as a prospect for therapy in microbiota-associated diseases. This modulation of gut microbiota is currently a growing area of research as it just might hold the key to treatment.

Organic amendments as ecosystem engineers: Microbial, biochemical and genomic evidence of soil health improvement in a tropical arid zone field site

The ability of organic biodynamic fertilizers to improve soil quality was evaluated in arid loamy sand soils in farmers’ fields in Rajasthan, India in cowpea cropping and citrus orchards. Water holding capacity, organic carbon and ammoniacal nitrogen improved significantly in organic farming. Microbial community was evaluated using both a culture dependent and independent approach. Actinomycetes increased significantly in organic cropping and orchard by 92 and 100%, respectively, compared to conventional management. Bacterial populations increased significantly on nutritionally diverse media in organic farm soils over conventional, both copiotrophs (+52–119%) as well as oligotrophs (+25–79%). The arbuscular mycorrhizal protein, glomalin increased by 56–82% in organic farms. Nitrogen fixers, ammonifiers, nitrifiers and sulfur oxidizers did not show significant differences. There was a consistent increase in soil enzymatic activities in organic farms—acid phosphatase (1.5× in cropping; 3× in orchard), flourescein diacetate hydrolysis (1.8×; 3.3×), dehydrogenase (2.4×; 3.5×) and β-glucosidase (2.2×; 6.3×). Quantification of 16S rDNA abundances in soil using qPCR showed a clear 1.8 fold increase in both organic cropping and organic orchard soils. The abundance of amoA gene decreased by 22 and 11 folds in organic cropping and orchards. The culture independent analysis of eubacterial 16S rRNA gene showed that organically cropped farms and orchards had more diverse bacterial community compared to the conventional. The distribution of bacterial species observed in organic cropping is more even. Representation of Proteobacteria among the eubacterial species was 20% lesser in organic as compared to conventional cropping whereas Actinobacteria were higher by 10% in organic cropping. Overall, the results demonstrated unequivocally that organic amendments improved the biological quality through an alteration of the microbial community structure and function. We conclude that organic manures may thus be appropriately included in the group of ‘Ecosystem Engineers’ that selectively modify the environment and make soil ecosystems more sustainable.

Mathematical modeling of competition and coexistence of sulfate-reducing bacteria, acetogens, and methanogens in multispecies biofilms

This work presents an integrated mathematical model able to simulate the physical, chemical, and biological processes prevailing in a sulfate-reducing biofilm under dynamic conditions. The model includes sulfate reduction by complete and incomplete sulfate-reducing bacteria (SRB); lactate removal by sulfate reduction and by acetogenic bacteria and acetate consumption via methanogenesis. Numerical integration based on the method of characteristics has been developed. The major problem of sulfate-reducing fixed-growth reactors is the formation of undesired bacterial species, which compete for space and substrate within the biofilm with SRB. The effect of COD/SO42-ratio on the reactor performances in terms of bacterial species distribution and substrate diffusion trends in the biofilm has been assessed. The simulation results reveal a stratification of microbial activities in biofilm reflecting the different ecological niches created by substrate gradients.

Distribution of Bacterial Species and Evaluation of their Antimicrobial Susceptibility Support a General Approach that Urinary Tract İnfections are Considered Complicated in Men

Background: Urinary tract infections (UTIs) in men are considered complicated, however few studies support this view. We compared the differences in the causative microorganism and their antimicrobial resistance between male and female patients presenting with an UTI. Methods: We evaluated the all microorganism isolated from the urine samples of patients which were admitted to outpatient clinics of our hospital during 2006 – 2011, which diagnosed with uncomplicated community acquired UTI. Results: Totally 8730 bacterial strains were isolated. Of these strains 5168 (59.2%) were isolated in women, whereas 3562 (40.8%) were isolated in men. Escherichia coli was the most frequently isolated microorganism (64.4%), followed by other enterobacteriaceae (15.1%) and enterococci (17.8%). However, the frequency of E. coli in men was significantly lower than in women, and frequency of other enterobacteriaceae and frequency of enterococci were higher than in women (49.4% versus 74.7%, 21.2% versus 10.8%, 24.7% versus 12.9%, respectively, p=0.001). The distribution of microorganism was evaluated according to age groups, similar results were observed in all age groups. In addition, E. coli, the most common strains for both sex. E. coli strains isolated in men were significantly less sensitive to ampicillin, ampicillin/sulbactam, cefuroxime, ceftriaxone, trimethoprim-sulphamethoxazole, gentamicin, ciprofloxacin, nitrofurantoin and fosfomycin than in women (p<0.05). Conclusion: The distribution of bacterial species isolated from urine culture of UTI is different in men. Even though E. coli is the most common strains in men as in women. In addition E. coli strains isolated in men were more resistant to antibiotics.

Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1-1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

Bacterial colonization and gut development in preterm neonates

Necrotizing enterocolitis (NEC) develops in 5–10% of preterm infants in association with enteral feeding and bacterial colonization. It remains unclear how diet and bacteria interact to protect or provoke the immature gastrointestinal tract. Understanding the factors that control bacterial colonization may provide the clue to prevent NEC, and studies in infants must be combined with animal models to understand the mechanisms of the microbiota–epithelium interactions. Analyses of infant fecal samples show that the density and distribution of bacterial species are highly variable with no consistent effects of gestational age, delivery mode, diet or probiotic administration, while low bacterial diversity and bacterial overgrowth are commonly associated with NEC. A series of recent studies in preterm pigs show that the mucosa-associated microbiota is affected by delivery method, prematurity and NEC progression and that diet has limited effects. Overgrowth of specific groups (e.g. Clostridia) appears to be a consequence of NEC, rather than the cause of NEC. Administration of probiotics either decreases or increases NEC sensitivity in preterm pigs, while in preterm infants probiotics have generally decreased NEC incidence and overall mortality. The optimal nature and amount of probiotic bacteria are unknown and host defense factors appear more important for NEC sensitivity than the nature of the gut microbiota. Host defense is improved by feeding the optimal amount of enteral diets, such as mother's colostrum or milk, that help the immature intestinal immune system to respond appropriately to the highly variable bacterial colonization.

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.

Molecular identification of bacteria by total sequence screening: determining the cause of death in ancient human subjects.

Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17th to the 19th century, we propose a methodology that includes the: 1) amplification of all 16S rDNA gene sequences present in each sample; 2) identification of all bacterial DNA sequences with a degree of identity ≥95%, according to quality criteria; 3) identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4) establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1) the correct identification of bacterial molecular sequence signatures by quality criteria; 2) the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3) the distribution of bacterial species for each subject and for each burial; and 4) the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17th century to the early 18th century.

Effects of intramammary infections on somatic cell score and milk yield in Sarda sheep

AIM: To evaluate the effects of intramammary infections (IMI) on somatic cell score (SCS) and milk yield in dairy ewes. METHODS: Monthly milk samples were collected from a flock of 202 Sarda sheep, over a period of 7 months, for bacteriological culture and measurement of somatic cell counts (SCC). During the same period, milk yield was measured daily using electronic milk meters connected to each half-udder cluster of the milking machine. SCC was transformed to SCS using a base-2 log transformation. One SCS is equivalent to a SCC of 25,000 cells/ml, and each increase of 1 in SCS is associated with doubling of the SCC. IMI was defined by the presence of five or more colonies of similar morphology isolated from a milk sample (≥500 cfu/ml). The effect of IMI on SCS and milk yield was assessed using a generalised estimating equation (GEE). RESULTS: There were 1,186 udder halves with IMI from 2,828 milk samples, a prevalence of 41.9%. The distribution of bacterial species within the 1,186 culture-positive samples was comprised of 476 (40.1%) Staphylococcus epidermidis, 172 (14.5%) Staph. chromogenes, 38 (3.2%) Staph. caprae, 134 (11.3%) Staph. simulans, 114 (9.6%) Streptococcus uberis, 123 (10.4%) Strep. dysgalactiae, and 129 (10.9%) Strep. equi subsp. zooepidemicus. SCS was greater in udder halves with IMI (mean 7.71; SD 0.82) than in udder halves without IMI (mean 5.53; SD 1.02) (p<0.01). IMI due to streptococcal species were associated with greater SCS (mean 8.24; SD 0.62) than those due to staphylococcal species (mean 7.48; SD 0.79) (p<0.01). Milk yield from udder halves with IMI was lower (mean 439 (SD 162) ml/half udder/day) than from udder halves without IMI (mean 602 (SD 170) ml/half udder/day) (p<0.01). IMI due to staphylococcal species was associated with a lower milk yield (mean 399 (SD 167) ml/half udder/day) than IMI due to streptococcal species (mean 427 (SD 156) ml/half udder/day) (p<0.01). CONCLUSIONS: These findings provide sheep milk producers with information on the losses associated with subclinical mastitis, which can be used to evaluate the economics of prevention and treatment protocols concerning udder health in ovine dairy flocks.

Alteration of the murine gut microbiota during infection with the parasitic helminth Heligmosomoides polygyrus

In a murine model of inflammatory bowel disease (IBD), treatment of colitis in IL-10 gene-deficient mice with the parasitic helminth Heligmosomoides polygyrus ameliorates colonic inflammation. The cellular and molecular mechanisms driving this therapeutic host response are being studied vigorously. One proposed mechanism is that H. polygyrus infection favors the outgrowth or suppression of certain bacteria, which in turn help modulate host immunity. To quantify the effect of H. polygyrus infection on the composition of the gastrointestinal (GI) tract microbiota, we conducted two independent microbial ecology analyses of C57BL/6 mice. We obtained and analyzed 3,353 bacterial 16S rRNA encoding gene sequences from the ileum and cecum of infected and uninfected mice as well as incective H. polygyrus larvae at the outset of the second experiment and adult worms taken directly from the mouse duodenum at the end of the second experiment. We found that a significant shift in the abundance and relative distribution of bacterial species in the ileum of mice is associated with H. polygyrus infection. Members of the bacterial family Lactobacillaceae significantly increased in abundance in the ileum of infected mice reproducibly in two independent experiments despite having different microbiotas present at the outset of each experiment. These data support the concept that helminth infection shifts the composition of intestinal bacteria. The clinical consequences of these shifts in intestinal flora are yet to be explored.

Operation of a sequencing batch reactor for cultivating autotrophic nitrifying granules

The granulation of nitrifying sludge in a sequencing batch reactor (SBR) fed with NH 4 + -N-laden inorganic wastewater was investigated. After 120-day operation spherical and elliptical granules with an average diameter of 0.32 mm were observed. The hydrophobicity surface, settling velocity and specific gravity of the matured granules increased with the processing of sludge granulation. Spatial distribution of bacterial species within the autotrophic granules was analyzed with fluorescence in situ hybridization. Both ammonia- and nitrite-oxidizing bacteria were observed in the granular sludge. The Michaelis–Menten equation was used to describe their NH 4 + -N utilization rate, and the kinetic coefficients were calculated to be v m = 18.0 mg/g-VSS/h and K m = 36.7 mg/l. Taking into account the NH 4 + -N utilization rate and removal efficiency together, an NH 4 + -N concentration range of 100–250 mg/l was found to be favourable for the operation of the SBR to cultivate nitrifying granules.

Distribution of Bacterial Species in Soil with a Vegetational Change from Japanese Black Pine (Pinus thunbergii) to Black Locust (Robinia pseudoacacia)

The effects of a nitrogen-fixing tree, black locust (Robinia pseudoacacia), on the distribution of bacterial species were examined in a Japanese black pine (Pinus thunbergii) and black locust-dominated area. DNA was extracted from the soil at depths of 0-5 and 5-10 cm, collected at the border between a Japanese black pine-dominated forest and a black locust-dominated forest, and the distribution of bacterial species was investigated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities did not differ between the two forests. The distribution of some bacterial species correlated significantly with soil pH, soil carbon [C], soil nitrogen [N], and soil N/phosphate [P], but not with soil C/N or soil P. The distributional relationships between ectomycorrhizal (ECM) fungal species and bacterial species were also analyzed. A positive correlation was observed between the distribution of some ECM fungi and bacterial species. These bacteria may have some interactions with ECM fungi in the field.

Bacterial species distribution and drug sensitivity in children acute bacillary dysentery

目的 了解儿童细菌性痢疾的志贺菌菌群分布及耐药性,以指导临床应用抗生素.方法 收集98例急性细菌性痢疾患者的大便标本进行细菌培养,采用纸片扩散法(K-B法),结果判断参照2002年版美国国家临床实验室标准化委员会(NCCLS)标准进行.结果 98株志贺菌中,福氏志贺菌77株,宋内志贺菌21株.志贺菌对头孢哌酮、头孢他啶、头孢曲松、头孢哌酮/舒巴坦、呋喃唑酮敏感;对氨苄西林、复方磺胺甲恶唑等药物耐药率高.结论 福氏志贺菌是儿童细菌性痢疾的主要致病菌,治疗志贺菌感染首选三代头孢菌素。

Effects of long-term routine use of selective digestive decontamination on antimicrobial resistance

To assess the distribution of bacterial species and antimicrobial resistance in an ICU during long-term use of selective digestive decontamination (SDD) in the context of national reference data. Five-year prospective observational study in a 24-bed interdisciplinary surgical ICU of a university hospital (study ICU) participating in the project "Surveillance of Antimicrobial Use and Antimicrobial Resistance in German Intensive Care Units" (SARI; reference ICUs). Resistance data were obtained from all patients; patients intubated for at least 2 days received SDD (colistin, tobramycin, amphotericin B). SDD was performed in 1,913 of 7,270 patients. Antimicrobial resistance was examined in 4,597 (study ICU) and 46,346 (reference ICUs) isolates. Methicillin-resistant Staphylococcus aureus (MRSA) remained stable (2.76 and 2.58 isolates/1000 patient days) in the study ICU; this was below the German average (4.26 isolates/1000 patient days). Aminoglycoside- and betalactam-resistant Gram-negative rods did not increase during SDD use. Aminoglycoside resistance of Pseudomonas aeruginosa was 50% below the mean value of SARI (0.24 vs. 0.52 isolates/1,000 patient days). The relative frequency of enterococci and coagulase-negative staphylococci (CNS) was higher than in the SARI ICUs (23.2% vs. 17.3%, and 25.0% vs. 20.6%, respectively). Routine 5-year-use of SDD was not associated with increased antimicrobial resistance in our ICU with low baseline resistance rates. Vigorous surveillance and control measures to search and destroy MRSA were considered a mandatory component of the SDD program. The relative increase in enterococci and CNS is of concern requiring further investigation.

Species diversity of campylobacteria in a wild bird community in Sweden

To analyse the occurrence and host species distribution of campylobacteria species in shorebirds, geese and cattle on grazed coastal meadows in Sweden. Species identification was performed through a polyphasic approach, incorporating Amplified Fragment Length Polymorphism (AFLP) profiling, 16S RNA gene sequence analysis together with extensive phenotypic characterization. From 247 sampled birds and 71 cattle, we retrieved 113 urease positive thermophilic Campylobacter (UPTC) and 16 Campylobacter jejuni ssp. jejuni isolates. Furthermore, 18 isolates of Helicobacter canadensis, and five isolates that potentially represent a new genus of micro-aerophilic, spiral and Gram-negative bacteria were isolated. The distribution of bacterial species on hosts was uneven: all H. canadensis isolates were retrieved from geese, while all but one of the Campylobacter lari UPTC isolates were found in shorebirds. AFLP type distribution of Camp. lari UPTC isolates among individual, resampled and breeding-paired Redshank birds generally indicated a constant shift in strain populations over time and absence of geographical clustering. The large number of isolated campylobacteria, including species that are zoonotic enteropathogens, indicates that these wild birds potentially may serve as reservoirs of human infections. However, despite a common environment, the different host species largely carried their own campylobacteria populations, indicating that cross-species transmission is rare. Our study is one of few that provide data on the occurrence of campylobacteria in wild animals, adding information on the ecology and epidemiology of micro-organisms that are of public health concern.

Maintenance of soil functioning following erosion of microbial diversity

The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.

Transport of bacteria across and along the large intestinal lumen of guinea pigs

Flow cytometry was used to observe the transport of fluorescently labelled viable bacteria in the large intestinal lumen of guinea pigs after the injection of the bacteria into the proximal colon. Bacteria were transported along the radial and longitudinal axes of the intestine and were separated from dietary residue, accumulated, and then transported back to the caecum. These observations, together with the heterogeneous distribution of bacterial species and chemical composition across and along the large intestine, suggest that there are several different microenvironments within the intestinal lumen between which bacteria and/or dietary residues move. The existence of different microenvironments within the intestinal lumen is consistent with poor mixing of the digesta within the large intestine of pigs and chickens.

Bacterial Population Analysis of Human Colon and Terminal Ileum Biopsies with 16S rRNA-based Fluorescent Probes: Commensal Bacteria Live in Suspension and Have No Direct Contact with Epithelial Cells

The commensal intestinal microflora has important metabolic and perhaps also immune modulatory functions. Evidence has accumulated that the microflora plays a role in the pathogenesis of inflammatory bowel disease. Therefore, there is a growing interest in the intestinal microflora and its interaction with the host. Presumably, this interaction takes place at the mucus layer. In this study, we investigated the microflora that is present at the mucus layer and addressed the following questions. Does a specific mucus-adherent microflora exist? Is there direct contact between commensal bacteria and epithelial cells? Snap-frozen biopsies were taken of 5 colon regions and of the terminal ileum in 9 subjects with a normal colon. Fecal samples were also collected. Bacteria were detected in cryosections with fluorescent in situ hybridization (FISH) with 16S ribosomal (r)RNA-targeted probes for all bacteria and specific probes for the major representatives of anaerobic microflora (bifidobacteria, Bacteroides, clostridia, atopobia) and aerobic microflora (Enterobacteriaceae, enterococci, streptococci, lactobacilli). With this sensitive technique, bacteria were only observed at the luminal side of the intestinal mucus layer. Very few microcolonies were present at the mucus layer, and the composition of the bacterial microflora present in the feces was similar to that at the mucus layer of the terminal ileum and colon regions. We did not observe direct contact between bacteria and epithelial cells. The equal distribution of bacterial species suggests that intestinal commensal bacteria live in suspension in the lumen and that there is no specific mucus-adherent microflora.

Ecology of mixed biofilms subjected daily to a chlorinated alkaline solution: spatial distribution of bacterial species suggests a protective effect of one species to another

Three bacterial strains (Kocuria sp. C714.1, Brevibacterium linens B337.1 and Staphylococcus sciuri CCL101) were grown together on stainless steel and were subjected daily to a commercial alkaline chlorine solution (22 mg l-1 of free chlorine, pH 11) over a period of 4 weeks. After the daily chemical shock, culture madia [1:20 dilution of tryptic soy broth (TSB-YE/20) or diluted whey] was deposited on the biofilms. The chemical shocks led first to a drop in the culturable population, followed by an increase and finally stabilization at around 106-107 CFU cm-2 by day 11 of the experiment. These changes in the microbial population can be attributed to a decreasing susceptibility to the antimicrobial agent with biofilm age, and to the consumption of free chlorine by biofilm exoproteins. The microbial composition appeared to be linked to the free chlorine concentration that depended on exoprotein production. At the end of the experiment, exoprotein production was greater for biofilms grown in TSBYE/20 than in whey. As a consequence, biofilms grown in whey did not neutralize the chlorine and the dominant strain was the one having the highest resistance to chlorine: K. varians. When biofilm were grown in TSBYE/20, chlorine was neutralized and the dominant strain was the one having the highest growth rate: S. sciuri. The presence of chlorine may also explain the distribution of S. sciuri cells as a ring around Kocuria sp. microcolonies. When chlorine was totally consumed by the biofilm during the chemical shock, S. sciuri was no longer grouped around Kocuria sp. microcolonies but was evenly scattered over the substratum as single cells or in small clusters, as it was before any chemical treatment. These findings strongly suggest protection of S. sciuri by Kocuria sp. microcolonies against the chlorinated solution. This phenomenon, added to the low susceptibility phenotype of the biofilm cells, could at least partly explain the survival of microbial cells in an adverse environment.

DISTRIBUTION AND PREVALENCE OF ANTIMICROBIAL RESISTANCE AMONG GRAM-NEGATIVE ISOLATES IN INTENSIVE CARE UNITS (ICU) IN BELGIAN HOSPITALS BETWEEN 1996 AND 1999

Objective : to assess the distribution and prevalence of resistance rates among Gram-negative isolates in Belgian intensive care units (ICUs) between 1996 and 1999.Methods : During 1996-1997 and 1998-1999, over a total period of 10 and 9 months respectively, members of the NPRS Belgian Study group collected, on clinical indications, 3029 consecutive initial isolates of Gramnegative bacteria from patients admitted to 26 Belgian hospitals and performed minimal inhibitory concentration (MIC) determinations by means of the E-test. Breakpoints were defined according to the criteria of the NCCLS.Results : The overall distribution of bacterial species was, in decreasing order of frequency : Pseudomonas aeruginosa >E. coli >E. aerogenes >K. pneumoniae >P. mirabilis >S. marcescens >E. cloacae >K. oxytoca >M. morganii >Stenotrophomonas maltophilia >Acinetobacter spp. All together these species and genera constituted about 90% of all isolates. The frequency of resistance for all the initial Gram-negative isolates in 1998-9 were : amoxicillin-clavulanic acid 60%, piperacillin 31%, piperacillin-tazobactam 20%, cefuroxime 58%, ceftriaxone 31%, ceftazidime 17%, aztreonam 23%, cefepime 10%, imipenem 13%, gentamicin 12%, amikacin 12% and ciprofloxacin 21%. Apart for an increase in multiple drug resistance among P. aeruginosa isolates, no significant trends were observed neither in species distribution nor in the overall prevalence of antimicrobial resistance among Gramnegative isolates from Belgian ICUs between 1996-7 and 1998-9.Conclusions : Among Gram-negative isolates in Belgian ICUs, a very high frequency of resistance was seen to amoxicillin-clavulanic acid and cefuroxime, and rather high frequencies of resistance to piperacillin, ceftriaxone and aztreonam. Taking into account the species distribution and the prevalence of resistance, cefepime, imipenem, amikacin and gentamicin appeared generally suitable for empirical therapeutic use in severe ICU-acquired Gram-negative infections in Belgium. However, the therapeutic strategy should be adapted according to the local ecology of resistance.