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PubMed2006

Enterobacter sakazakii: an emerging pathogen in powdered infant formula.

Enterobacter sakazakii represents a significant risk to the health of neonates. This bacterium is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. Infants aged <28 days are considered to be most at risk. Feeding with powdered infant formula (PIF) has been epidemiologically implicated in several clinical cases. Infants should be exclusively breast-fed for the first 6 months of life, and those who are not should be provided with a suitable breast-milk substitute. PIF is not a sterile product; to reduce the risk of infection, the reconstitution of powdered formula should be undertaken by caregivers using good hygienic measures and in accordance with the product manufacturer's food safety guidelines.

Cronobacter sakazakii↗
PubMed1989

Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula.

We report an outbreak of Enterobacter sakazakii infection and colonization in neonates related to an infant formula contaminated during the manufacturing process. The outbreak occurred in a 20-bed neonatal intensive care unit during a six-week period in 1988, and involved a total of four infants. Three infants had sepsis and three had bloody diarrhea; all patients responded to intravenous antibiotics and recovered without complications. The E sakazakii isolated from the formula had the same plasmid and multilocus enzyme profile as those isolated from patients. This outbreak demonstrates the significance of commercially contaminated formulas and emphasizes the need to limit contamination and multiplication of bacteria in enteral formulas.

Cross Infection↗
PubMed2003

Enterobacter cloacae bloodstream infections in pediatric patients traced to a hospital pharmacy.

The sources of an outbreak of Enterobacter cloacae bloodstream infections in a pediatric hospital were investigated, as were the risk factors for acquiring the infection: Two retrospective case-control studies were conducted. The study sample included all patients admitted to the general pediatric wards from February 5 through March 30, 2001, who had a positive blood culture for E. cloacae. Pediatric ward and pharmacy infection-control practices were reviewed, personnel and environmental cultures were obtained, and pulsed-field gel electrophoresis (PFGE) molecular typing of the bloodstream isolates was conducted. Four subjects were identified. These infants were more likely than control patients to receive i.v. ranitidine (p < 0.01). Among patients receiving i.v. ranitidine, subjects were more likely than controls to receive i.v. ranitidine prepared by a pharmacist. No environmental or personnel cultures yielded E. cloacae. Patients' E. cloacae isolates had four different PFGE patterns, suggesting environmental rather than point-source contamination. Ranitidine multidose vials were kept connected to an automatic compounding machine for up to 48 hours at room temperature after the first dose was drawn, contrary to manufacturer recommendations. Further, preparation of ranitidine infusions was not conducted in accordance with recommendations for risk level 2 sterile i.v. products. The use of contaminated ranitidine multidose vials was the most likely cause of an outbreak of E. cloacae. However, a combination of other factors such as inadequate hand-washing techniques, presence of E. cloacae in the environment, noncompliance with guidelines for the preparation of sterile infusions and medications, and a susceptible population may have contributed to the infections.

Case-Control Studies↗
PubMed1995

Molecular epidemiology of infections due to Enterobacter aerogenes: identification of hospital outbreak-associated strains by molecular techniques.

Molecular techniques were used to study the epidemiology of infections due to Enterobacter aerogenes in a tertiary-care hospital. Sixty-two clinical isolates were collected from 43 patients over 3 months. Restriction endonuclease analysis (REA) of chromosomal DNA and repetitive-element polymerase chain reaction (rep-PCR) with primers based on repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) bacterial DNA sequences were used for genomic fingerprinting. REA with HindIII or EcoRI yielded complex banding patterns that differentiated community-acquired from some hospital-acquired organisms. Less complex fingerprints were obtained with rep-PCR, which also distinguished between epidemiologically unrelated strains. More discriminatory DNA fingerprints were provided by rep-PCR when REP primers rather than ERIC primers were used. Two clusters of genomically distinct isolates from patients with recent or current exposure to the hospital environment were identified by REA and rep-PCR. Most isolates within each cluster contained characteristic plasmids, and some isolates contained additional plasmids. These results suggest colonization and infection with genotypically related strains of E. aerogenes in a nosocomial setting. Although REA and plasmid profiling are useful techniques for the epidemiological typing of E. aerogenes, genomic fingerprinting with rep-PCR may offer the advantages of ease, speed, and broad species applicability over existing molecular-typing techniques.

Adult↗
PubMed1989

Neurotoxicity of benzylpenicillin in experimental renal failure and Enterobacter cloacae meningitis.

The neurotoxic potential of benzylpenicillin administered as a continuous intravenous infusion was studied in rabbits with intact blood-CNS barriers, experimentally established Enterobacter cloacae meningitis and experimental renal failure, secondary to cephaloridine-induced acute tubular necrosis after iv administration. The concentrations of benzylpenicillin in serum, CSF and brain tissue fluid were assayed at the onset of epileptogenic electroencephalographic activity. The brain tissue concentrations of benzylpenicillin were consistently higher than those in CSF in both infected and uninfected animals. The highest brain tissue fluid concentrations of benzylpenicillin were found in rabbits with renal failure after cephaloridine pretreatment. The brain tissue fluid concentrations of benzylpenicillin rather than the CSF concentrations were decisive for neurotoxicity. Cephaloridine-induced uraemia, but not the combination of uraemia and meningitis, resulted in a significantly increased tolerance of high intracerebral concentrations of benzylpenicillin before EEG-changes were precipitated.

Acute Kidney Injury↗
PubMed1991

Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes.

An imipenem-resistant strain, Enterobacter aerogenes EA-Z, was isolated from a blood culture. Outer membrane protein (OMP) profiles indicated the loss of a 40 kDa OMP, decreased expression of 42 and 44 kDa OMPs, and increased expression of a 50 kDa OMP in strain EA-Z when compared with imipenem-susceptible clinical isolates of E. aerogenes. The OMP profile of EA-Z was similar to that of strain EA-SI16, an imipenem-resistant E. aerogenes second-step mutant selected on imipenem-containing media. A single-step imipenem-resistant mutant, EA-SI8, had lost expression of only the 40 kDa OMP. No new beta-lactamase could be detected by isoelectric focusing, and no increase in imipenem hydrolysis was seen when EA-Z was compared with imipenem-sensitive controls, even in the presence of added zinc. These data suggest that the 40 kDa OMP of E. aerogenes might be required for the normal diffusion of imipenem across the outer membrane.

Bacteremia↗
PubMed1996

Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans.

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Journal Article↗
PubMed1976

Characterization and replication control of plasmids from Enterobacter cloacae DF 13.

It has been shown previously that Enterobacter clocacae DF13 harbours at least five different size classes of plasmids. A 45 x 10(6)-Mr self-transmissible R factor determining resistance against tetracyclin, sulfanilamide, streptomycin and chloramphenicol, a 6.0 x 10(6)-Mr bacteriocinogenic factor without sex factors activity and cryptic plasmids in the size classes of Mr 1.3 x10(6), 2.8 x 10(6) and 8.0 x 10(6) respectively. The present work deals with the determination of the homogeneity and molecular relationship of 1.3 x 10(6)-Mr (mini) and 2.8 x 10(6)-Mr (midi) cryptic plasmids and the 6.0 x 10(6)-Mr (maxi) bacteriocinogenic factor, their kinetics of replication and their replication control in response to inhibition of protein synthesis.

Base Sequence↗
PubMed1977

Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobacter cloacae (Clo DF13). Dissociation and reconstitution of the complex.

Cell of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmid-specific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.

Amino Acids↗
PubMed1998

In vitro selective concentrations of cefepime and ceftazidime for AmpC beta-lactamase hyperproducer Enterobacter cloacae variants.

METHODS: A mixed culture of a wild-type ceftazidime/cefepime-susceptible (4x107 CFU/mL) strain and an ampC derepressed Enterobacter cloacae (105 CFU/mL) strain (relative proportions 99.75% and 0.25%) was challenged for 4 h with different antibiotic concentrations of ceftazidime and cefepime (0.03-4096 mg/L), and then transferred to drug-free medium. The proportion of wild-type versus derepressed population was evaluated after 24 h. RESULTS: Ceftazidime and cefepime selected the derepressed variant at concentrations ranging from 1 to 4096 and from 0.12 to 16 mg/L respectively. CONCLUSIONS: These results suggest that serum concentrations attainable with a 2 g/8 h cefepime dosage may be able to suppress the emergence of derepressed ampC mutants.

Journal Article↗
PubMed1977

Characterization of Enterobacter agglomerans (Erwinia spp.) from clinical specimens.

The biochemical reactions of 18 anaerogenic and 45 aerogenic isolates of Enterobacter agglomerans are described and used for subdivision in the biogroups suggested by Ewing & Fife. The presence of special cultural characteristics (symplasmata and biconvex bodies) was also recorded and was found to be unrelated to formation of gas from glucose. Previous authors have reported such structures in anaerogenic cultures only. The amount of gas produced varied to a great extent between strains and also proved to be dependent on incubation temperature. The results do not support the division of E. agglomerans into one anaerogenic and one aerogenic major subgroup. The value of the gelatin liquefaction test for characterization of the species is emphasized.

Bacteriological Techniques↗
PubMed2005

Enterobacter sakazakii and other bacteria in powdered infant milk formula.

Recently there has been considerable concern related to the presence of bacteria, in particular Enterobacter sakazakii, in powdered infant formula milk. This paper considers the bacteria in these products at point of sale, with reference to current microbiological testing and the need for good hygienic practice in their subsequent preparation before feeding. The ingestion of raised numbers of E. sakazakii resulting from temperature abuse after reconstitution is highlighted as well as the uncertain routes of E. sakazakii product contamination.

Colony Count, Microbial↗
PubMed1973

Drug-resistant Escherichia coli and Klebsiella-Enterobacter in healthy adults in Thailand and the effect of antibiotic administration.

Antibiotic-resistant strains of Escherichia coli and, to a lesser extent, Klebsiella-Enterobacter were common in serial samples of the normal intestinal flora of healthy young adults in Thailand, and the resistance often was to multiple drugs and was transferable. Antibiotic administration promoted dominance of the flora by drug-resistant organisms regardless of the drug regimen and the predrug susceptibility pattern of the flora. The normal pattern was regained slowly after the cessation of drug administration.

Adult↗
PubMed1996

Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes.

We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.

Anti-Bacterial Agents↗
PubMed2006

Attachment of and biofilm formation by Enterobacter sakazakii on stainless steel and enteral feeding tubes.

Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25 degrees C were examined for the extent to which they attach to these materials. Higher populations attached at 25 degrees C than at 12 degrees C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4 degrees C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm(2), respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25 degrees C for up to 10 days. Biofilms were not produced at 12 degrees C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm(2) and 1.16 to 1.31 log CFU/cm(2) in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25 degrees C; biofilms were not formed on TSB and LJB at 25 degrees C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.

Bacterial Adhesion↗
PubMed2006

Comparison of media for the isolation of Enterobacter sakazakii.

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.

Agar↗
PubMed2006

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm.

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.

Biofilms↗
PubMed2006

Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture.

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.

Bacteriological Techniques↗