Opportunistic Enterobacteriaceae Research Articles

Клинические особенности затяжного течения кишечной инфекции, ассоциированной с Klebsiella pneumoniae, у ребенка грудного возраста

The development of complex criteria for the diagnosis, differential diagnosis, and optimization of treatment of infectious diarrhea associated with opportunistic Enterobacteriaceae is a pressing issue of pediatric research and practice. The paper reports a clinical case of protracted intestinal infection associated with Klebsiella pneumoniae in the form of moderate hemorrhagic enterocolitis in an infant, which is explained by the decrease in specific resistance due to unfavorable maternal obstetric and gynecological history, perinatal CNS injury, iron deficiency anemia, protein-energy malnutrition. The disease relapse associated with secondary norovirus infection was reported after the first hospitalization. Three courses of intestinal antiseptics and probiotics were required to achieve a beneficial treatment outcome, although usually in such a situation one course of such drugs is enough. The recovery process was accompanied by the nutritional status improvement, hemorrhagic colitis relief, normalization of gut microbiota.

Prevalence and antimicrobial resistance of Enterobacteriaceae in the north of Kazakhstan.

An increasing number of drugs are used each year in the treatment of small pets (cats and dogs), including medicines (cephalosporins and fluoroquinolones) used in human therapy. The purpose of this study was to isolate and explore the antibiotic resistance of opportunistic Enterobacteriaceae (Escherichia coli, Klebsiella, Proteus, Ci trobacter, Enterobacter) from cats and dogs, and to isolate resistance genes in the microorganisms. In 2021, 808 samples of biological material from small domestic animals were collected in veterinary clinics in Kostanay. From these, 210 microorganisms were isolated and identified. A large majority of the strains sampled belonged to E. coli-149 (70.9%), Enterobacter-11 (5.2%), Klebsiella-28 (13.3%), Proteus-12 (5.7%) and 10 Citrobacter isolates (4.8%). In all isolates identified, antibiotic resistance/sensitivity was determined by disc-diffusion method to ampicillin, cefoxitin, gentamicin, levomycetin, tetracycline, ciprofloxacin, norfloxacin, ofloxacin, cefoperazone, cefpodoxime, streptomycin, kanamycin, doxycycline, gemifloxacin, nalidixic acid, furazolidone, furadonine, amoxicillin, and enrofloxacin. The study has demonstrated that the greatest number of Enterobacteriaceae were sensitive to the action of meropenem, which belongs to the group of beta-lactam antibiotics; resistance was demonstrated against tetracycline, doxycycline, ampicillin, amoxicillin, ofloxacin, and cefpodoxime. The most common genes encoding antimicrobial resistance were as follows: BlaTEM and OXA in 41 and 28 isolates, respectively, encoding resistance to beta-lactams; StrA and StrB in 45 and 48 isolates encoding aminoglycosides; and tetA and tetB in 43 and 28 isolates encoding tetracyclines. Obtained data demonstrate that uncontrolled and frequent use of beta-lactam and tetracycline antibacterials, in cats and dogs, results in the spread of genotypic resistance among micro-organisms of the family Enterobacteriaceae.

Антибиотикорезистентность Enterobacteriaceae в микробиомах цыплят-бройлеров

Antibiotics have long been overused for non-therapeutic purposes. As a result, Escherichia, Enterobacter, Klebsiella, and Proteus in avian microbiomes have become reservoirs for genetic determinants of resistance, thus spreading resistance to antibiotics and contaminating raw materials and finished products. The food industry is looking for alternative means to preserve health and maintain high productivity of commercial poultry, e.g., probiotics, phytobiotics, organic acids, etc. The research featured the effect of antibiotics and phytobiotics on enteropathogenic bacteria in the microbiomes of broiler chicken.
 Escherichia coli bacteria were cultivated in vitro in subthreshold concentrations on nutrient media with antibiotics for 37 days to study the effect of low doses of antibiotics on the sensitivity of isolates. The study involved microbiocenoses of broiler chicken that received avilamycin A and a phytobiotic based on Brassica juncea, Linum usitatissimum, and Nigella sativa L. A set of experiments covered the species composition of opportunistic Enterobacteriaceae, the phenotypic sensitivity to antibiotics, and the genetic determinants of resistance, as well as the antimicrobial potential of phytobiotics.
 E. coli developed no resistance for 37 days when the antibiotic dose remained below minimal inhibitory. Opportunistic gram-negative Enterobacterales predominated in all litter samples. E. coli, Klebsiella pneumoniae, and Proteus mirabilis accounted for more than 30% of all isolated strains. Avilamycin A and the phytobiotic affected the coccal microflora but had no effect on the genus-species composition of Enterobacteriaceae. Litter samples from both experimental and control groups demonstrated K. pneumoniae with severe phenotypic resistance to ciprofloxacin, as well as blaDHA genes. In broiler farming, maintenance and circulation of resistance agents depends on litter microbiota. In this research, the chicken that received the phytobiotic showed the lowest level of resistance to ciprofloxacin while the groups that received avilamycin A had the highest resistance results.
 During a broiler’s life span, bacteria with no active resistance determinants in their genome remained sensitive to antibiotics, even though the contact with the latter was constant. Phytobiotics showed good prospects for broiler farming as food additive that could reduce and eventually eliminate the intake of antibiotics.

Sequencing and Characterization of M. morganii Strain UM869: A Comprehensive Comparative Genomic Analysis of Virulence, Antibiotic Resistance, and Functional Pathways.

Morganella morganii is a Gram-negative opportunistic Enterobacteriaceae pathogen inherently resistant to colistin. This species causes various clinical and community-acquired infections. This study investigated the virulence factors, resistance mechanisms, functional pathways, and comparative genomic analysis of M. morganii strain UM869 with 79 publicly available genomes. The multidrug resistance strain UM869 harbored 65 genes associated with 30 virulence factors, including efflux pump, hemolysin, urease, adherence, toxin, and endotoxin. Additionally, this strain contained 11 genes related to target alteration, antibiotic inactivation, and efflux resistance mechanisms. Further, the comparative genomic study revealed a high genetic relatedness (98.37%) among the genomes, possibly due to the dissemination of genes between adjoining countries. The core proteome of 79 genomes contains the 2692 core, including 2447 single-copy orthologues. Among them, six were associated with resistance to major antibiotic classes manifested through antibiotic target alteration (PBP3, gyrB) and antibiotic efflux (kpnH, rsmA, qacG; rsmA; CRP). Similarly, 47 core orthologues were annotated to 27 virulence factors. Moreover, mostly core orthologues were mapped to transporters (n = 576), two-component systems (n = 148), transcription factors (n = 117), ribosomes (n = 114), and quorum sensing (n = 77). The presence of diversity in serotypes (type 2, 3, 6, 8, and 11) and variation in gene content adds to the pathogenicity, making them more difficult to treat. This study highlights the genetic similarity among the genomes of M. morganii and their restricted emergence, mostly in Asian countries, in addition to their growing pathogenicity and resistance. However, steps must be taken to undertake large-scale molecular surveillance and to direct suitable therapeutic interventions.

Prevalence, antibiotic resistance patterns, and biofilm formation ability of Enterobacterales recovered from food of animal origin in Egypt.

The majority of animal-derived food safety studies have focused on foodborne zoonotic agents; however, members of the opportunistic Enterobacteriaceae (Ops) family are increasingly implicated in foodborne and public health crises due to their robust evolution of acquiring antimicrobial resistance and biofilms, consequently require thorough characterization, particularly in the Egyptian food sector. Therefore, this study aimed to determine the distribution and prevalence of Enterobacteriaceae family members in animal-derived foods, as well as their resistance to important antimicrobials and biofilm-forming potential. A total of 274 beef, rabbit meat, chicken meat, egg, butter, and milk samples were investigated for the presence of Enterobacteriaceae. All isolated strains were first recognized using traditional microbiological techniques. Following that, matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to validate the Enterobacteriaceae's identity. The isolated enterobacteria strains were tested on disk diffusion and crystal violet quantitative microtiter plates to determine their antibiotic resistance and capacity to form biofilms. There have been thirty isolates of Enterobacteriaceae from seven different species and four genera. Out of the three food types, Pseudomonas aeruginosa had the highest prevalence rate (4.1%). With three species, Enterobacter genera had the second-highest prevalence (3.28%) across five different food categories. In four different food types, the Klebsiella genera had the second-highest distribution and third-highest incidence (2.55%). Almost all isolates, except three Proteus mirabilis, showed prominent levels of resistance, particularly to beta-lactam antibiotics. Except for two Enterobacter cloacae and three P. mirabilis isolates, all isolates were classified as multidrug-resistant (MDR) orextensively multidrug-resistant (XDR). The multiple antibiotic resistance index (MARI) of the majority of isolates dropped between 0.273 and 0.727. The highest MARI was conferred by Klebsiella pneumoniae, at 0.727. Overall, 83.33% of the isolates had strong biofilm capacity, while only 16.67% exhibited moderate capacity. The MDR, XDR, and strong biofilm indicators confirmed in 83.33% of the currently tested Enterobacteriaceae from animal-derived foods suggest that, if not addressed, there may be rising risks to Egypt's economy and public health.

Effect of probiotics on the intestinal microbiota of patients with giardiasis and ascariasis.

Recently, many cases of mixed invasion by Giardia and ascarids have been registered. Gastrointestinal lesions in patients are often accompanied by dysbiotic changes. The aim was to study the effect of probiotics containing Saccharomyces bouvardias CNCM I-745 in the complex therapy of patients with giardiasis, ascariasis, and mixed invasion. 90 patients with giardiasis, ascariasis and mixed invasion were divided into 3 groups, depending on the type of invasion. Each group was divided into two subgroups, depending on the treatment (basic treatment and treatment with probiotics). When studying the state of the intestinal microbiota, the following changes were detected in all patients before treatment. The content of Bifidobacterium spp., Lactobacillus spp., Enterococcus spp. and the total content of E. coli was reduced. At the same time, there was an increase in the content of Peptostreptococcus spp., Bacteroides spp., E. coli with low enzymatic properties, hemolytic E. coli, opportunistic Enterobacteriaceae spp., Candida spp. and Staphylococcus spp. Under the influence of treatment, the subgroup with probiotics addition to the basic treatment, was more effective for all types of invasions. The use of probiotics containing Saccharomyces bouvardias CNCM I-745 in the complex therapy of patients with mixed invasion of giardiasis and ascariasis increased treatment efficiency following a significant improvement in intestinal microbiota.

Associations of clinical features of functional bowel disorders with gut microbiota characteristics in adolescents: A pilot study

Background. Obesity is a multifactorial disease that is one of the important public health problems in the 21st century. Functional bowel disorders in obese children and adolescents are one of the most common pathologies of the digestive tract. The aim: to characterize the bacteriological composition of the colon microbiota in obese adolescents and identify its associations with the clinical manifestations of functional bowel disorders.Materials and methods. The study included 20 adolescents aged 11–17 years. Stool consistency was assessed using the Bristol Stool Scale. The study of biological material was carried out using standard bacteriological methods. To assess the strength of the relationship between the nominal variables, Cramer’s V test was used.Results. It has been shown that in adolescents with obesity and functional bowel disorders, there is a decrease in the number of representatives of bifidoflora, an increase in the microbial density of Escherichia coli with altered enzymatic properties and an excessive growth of potentially pathogenic enterobacteria. In obese adolescents with functional bowel disorders, abdominal pain associated with the act of defecation is associated with the presence of enterobacteria and E. coli with altered enzymatic properties. Rare bowel movements are associated with an increased abundance of Enterococcus spp. and the presence of opportunistic Enterobacteriaceae microorganisms. Frequent bowel movements are associated with the presence of E. coli with altered enzymatic properties and Clostridium spp. In the presence of loose stool, Clostridium spp. is present; unstable stool (alternation of hard and liquid stools in one patient) is associated with a decrease in the number of lactobacilli and E. coli with normal enzymatic properties, the appearance of E. coli with altered enzymatic properties, opportunistic microorganisms of Enterobacteriaceae family and Clostridium spp. Conclusion. The relationship between the clinical symptoms of functional bowel disorders and changes in the composition of the gut microbiota in obese adolescents may be a justification for conducting in-depth studies aimed at finding early predictors of functional bowel diseases associated with obesity in the adult cohort.

Phage resistance of conditionally pathogenic intestinal microbiota bacteria in children with microbiocenosis disorders

Objective: to study the circulation and prevalence of phage-resistant strains among opportunistic enterobacteria, isolated from children with disorders of intestinal microflora, in relation to domestic preparations of bacteriophages.Materials and methods. A bacteriological study of the fecal microflora of the colon in 970 young children for dysbiosis was carried out. The sensitivity of 720 antibiotic-resistant strains of opportunistic enterobacteriaceae (S. aureus, K. pneumoniae, P. mirabilis and P. vulgaris, P. aeruginosa, E. coli, coagulase-negative staphylococci) isolated from children to domestic mono- and polyvalent drugs was studied (coliprotein, staphylococcal, Pseudomonas aeruginosa, purified Klebsiella pneumonia bacteriophage, “Sextafag”, Intesta bacteriophage). Determination of sensitivity to bacteriophages was carried out by the “sterile spot” method. Antibiotic sensitivity was determined by the disk diffusion method. Statistical processing of the results was carried out using the Microsoft Office Excel 2007 software package.Results. A large percentage of phage-resistant strains with low sensitivity to bacteriophages was revealed among opportunistic bacteria, amounting to 54,2%, with the highest circulation among coagulase-negative staphylococci, Proteus, Klebsiella (more than 50%). A significant spread of intestinal dysbiosis was established in young children (87,5%), characterized by a low content of bifidobacteria and a high level of allocation of opportunistic enterobacteria, including in associations.Conclusion. As a result of the studies, a significant prevalence of phage-resistant strains in children with dysbiosis was revealed, which indicates the need for preliminary determination of their sensitivity to bacteriophages in order to resolve the issue of their possible inclusion in the intestinal microflora correction system.

S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants

After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. We collected stool samples (n= 517) from full-term (n= 72) and preterm infants (n= 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

Microbiological monitoring of opportunistic Enterobacteriaceae of the Lena river

The aim is the study of the circulation, biological activity, persistent potential and genotypic characteristics of opportunistic Enterobacteriaceae of microbial community of the Lena River which is the one of the largest sources of water in Russia and Siberia. Gram-negative component of microbial community in the Lena River was mainly represented by Enterobacteriaceae (80%), including dominated Escherichia (dominant class) and Enterobacter and Klebsiella (subdominant class). In conditions of anthropogenic pollution there is a reorganization of microbial community of the Lena River in the direction of the increase in the proportion of opportunistic pathogens, their species diversity; there is a change of the biological activity of microorganisms, gains of share of strains characterized by antilysozyme activity, hemolytic activity, production DNase, phosphatase. The parameters of system “lysozyme-antilysozyme” are changing, the frequency of the occurrence of bacteria with antilysozyme activity is increasing. This shows the restructurization of the microbial community of water objects. Microorganisms of water objects in territories of anthropogenic pollution are characterized by multiple antibiotic resistance. The proportion of gram-negative opportunistic Enterobacteriaceae in microbial communities of water ecosystems determines the potential danger of water objects and the impact on the level of antibiotic resistance of bacteria. Opportunistic Enterobacteriaceae of microbial community of the Lena River near the city of Yakutsk are characterized by the presence of genetic determinants of pathogenicity (hlyA and sfaG). This is indicative of their potential epidemiological relevance. Microorganisms with high biological activity are markers of their epidemiological danger.

Isolation of Antibiotic-Resistant Micrococcus aloeverae from Milk Powder

The emergence of antibiotic resistance in humans via the food chain has become an area of great concern. Antibiotic resistance gene transfer from a food-borne bacteria or probiotic agent to a human gut microbe can lead to a serious food safety issue. Such seemingly harmless bacteria or probiotics can potentially become a threat to human health if they carry mobile genes conferring antibiotic resistance. The recent trend of probiotic application in powdered infant formula and milk products and the presently known contamination of these products by certain opportunistic Enterobacteriaceae bacteria serves as the rationale for evaluating their safety. In this study, 25 milk powder samples and 25 powdered infant formula samples were analyzed for the presence of antibiotic resistant bacteria of family Enterobacteriaceae using an isolation technique. Out of the 50 samples screened, an isolate was detected in one milk powder sample. Its biochemical characteristics, Gram character, antibiotic susceptibility, and motility were analyzed. It was found to be a Gram-positive, non-motile, catalase-positive and oxidase-negative microbe. The isolate was resistant to Rifampicin and Vancomycin, but susceptible towards Streptomycin, Ciprofloxacin, Ceftriaxone and Gentamycin. Molecular characterization by 16S rDNA sequencing identified the isolate as Micrococcus aloeverae. The unique occurrence of Micrococcus aloeverae in milk powder elicits an area of food safety concern for such samples. To the best of our knowledge, this study is the first to report antibiotic resistance by Micrococcus aloeverae. Although it may be a non-pathogenic microbe, its resistance towards antibiotics cannot be ignored. The possibility of Micrococcus aloeverae serving as a potential vehicle for transfer of antibiotic resistance genes to human gut microbiota represents a prospective food safety concern.

Potency and selectivity indices of acetone leaf extracts of nine selected South African trees against six opportunistic Enterobacteriaceae isolates from commercial chicken eggs

BackgroundThe rise in antimicrobial resistance in a plethora of nosocomial and opportunistic bacterial pathogens often isolated from commercial eggs, poses a serious public health concern. The existence of these contaminants may also serve as a drawback in the current efforts of improving the well-being of immunocompromised patients. The aim of this study was to determine the efficacy of plant extracts that had good activity on Escherichia coli in previous word on pathogens isolated from eggs for possible use in combating pathogens from eggs.MethodsAcetone leaf extracts of nine trees with good activities against Escherichia coli were tested for their in vitro antibacterial activity against six opportunistic bacterial isolates from commercial eggs (Stenotrophomonas maltophilia, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Proteus mirabilis, Enterobacter cloacae and Escherichia coli) using a serial microdilution method with tetrazolium violet as indicator of growth. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against Vero kidney cells, and selectivity index calculated.ResultsThe MIC values range of the different extracts against Stenotrophomonas maltophilia was 0.08-0.31 mg/ml, Klebsiella pneumonia 0.08-0.63 mg/ml, Salmonella ser. Typhimurium 0.08-0.63 mg/ml, Proteus mirabilis 0.02-1.25 mg/ml, Enterobacter cloacae 0.08-0.31 mg/ml and Escherichia coli 0.08-0.16 mg/ml respectively. Escherichia coli was the most sensitive while Proteus mirabilis was most resistant pathogen to the different test extracts, with mean MIC values of 0.08 mg/ml and 0.46 mg/ml respectively. Cremaspora triflora extracts had good activity against all the pathogenic egg isolates, with the exception of Proteus mirabilis. Maesa lanceolata and Elaeodendron croceum had the best total antibacterial activity (TAA), while generally the selectivity index of the extract was low (SI < 1).ConclusionThe exceptional activity of C. triflora extracts suggests that the plant has potential as a therapeutic agent against some members of the Enterobacteriaceae. Further pharmacological investigations may be interesting in the search for new antimicrobial leads.

EFFECT BY INTESTINAL MICROBIOM ON THE PROGRESSION OF VIRAL HEPATITIS

According to the modern concepts an intestinal microbiome has a significant effect on the functioning of the whole body including the immune system, digestive tract and liver in particular. This review displays current understanding of the intestinal microbiome impacting on the progression of chronic viral hepatitis caused by HCV- and HBV-infection, as well as changes in bowel microbiocenosis features depending on the duration of chronic process in the liver. It is indicated that chronic hepatitis to cirrhosis progression is accompanied by Bifidobacterium and strains of lactic acid (Lactobacillus, Pediococcus, Leuconostoc, Weissella) number decreasing and overgrowth of opportunistic species such as Enterococcaceae, Veillonellaceae, Enterobactericeae, Candida spp., Clostridia spp. This phenomena caused by PAMPs entry into the bloodstream including various types of toxins playing a role in liver immune inflammation processes progression. Thus patients with HBV and HCV infection are increased the number of CD4+, CD25+ in the blood and liver significantly, FOXP3+ Treg cell providing an immunosuppressive effect, and the function of specific CD8 lymphocytes is reduced and insufficient leveling virus significantly. Microbial imbalance has a negative effect on the biosynthesis of bile acids and sterolbiom functioning of our body as a result of changes in the balance between Bacteroides/Firmicutes, overgrowth of pathogenic and opportunistic Enterobacteriaceae, Veillonellaceae, Alcaligeneaceae and Porphyromonadaceae, Clostridium cluster XIVa, Helicobacter spp. and Clostridium difficile. These toxins formation and various carcinogenic metabolites from these strains leads to the inflammation development in the intestines and as a consequence to the progression of the inflammatory process in the liver. In turn, the reduction in the bacteria number producing short-chain fatty acid contributes to intestinal colonization by pathogenic representatives Gracilicutes (Salmonella, Shigella, enterohaemorrhagic E. coli) and Firmicutes (Clostridia), the toxins having a direct toxic effect on the liver. It is examined a little-studied question about a possible cofactor effect of enteric viruses (rotavirus, adenovirus, poliovirus type 1, Coxsackie virus, ECHO) and bacteria (Shigella, Salmonella, diarrheagenic E. coli, C. jejuni) on the progression of chronic hepatitis. In view of the above, it is necessary to study in further detailed the influence of the intestinal microbiome on the progression of chronic hepatitis HBV/HCV etiology in order to develop a comprehensive approach to treatment and reduce the risk of adverse outcomes in patients.

Increased resistance to quinolone in Catalonia, Spain

From 1989 to 1991, the level of resistance to ciprofloxacin in our hospital increased from 0.47% to 6.7% in opportunistic Enterobacteriaceae, from 9.9% to 16% in Pseudomonas aeruginosa and from 8.27% to 31.8% in Campylobacter jejuni-coli . We also observed an increase in quinolone consumption from 1.1 in 1989 to 1.5 defined daily doses per 1000 inhabitants per day in 1991.