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PubMed2001

Spatial and Temporal Variation of Enterobacter Genotypes in Sediments and the Underlying Hyporheic Zone of an Agricultural Stream.

Population studies of enteric bacteria in an agriculturally impacted stream (Ledbetter Creek, Murray, Kentucky, USA) were conducted over a period of 2 years. Total number of bacteria, cultivated heterotrophic aerobic bacteria, and enteric bacteria showed significant differences between winter and summer. The cultivated numbers of heterotrophic aerobic bacteria and enteric bacteria were significantly more abundant in summer than in winter. The abundance of enteric bacteria was 12.9% in an upwelling zone and 9.8% in a downwelling zone in summer. Most of the enteric bacterial strains isolated on MacConkey agar were assigned to Enterobacter cloacae and E. agglomerans by API 20E and an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (ARDRA) with the enzyme Hpa II. E. cloacae and E. agglomerans genotypes isolated from three hyporheic and gravel bar depth intervals (0-10 cm, 15-25 cm, and 30-40 cm) in summer and fall showed significant spatial variation and were heterogeneously distributed along the stream. Temperature, inorganic nutrients, and occurrence of anoxic zones affected the distribution of enteric bacteria. These techniques can be used as a model to monitor shifts among different species in the stream ecosystem.

Journal Article↗
PubMed1976

Peptides isolated from Enterobacter nuclease as potential polyamine binding sites.

The Enterobacter nuclease, which cleaves RNA between the 3'-phosphate group of cytidylic acid and the 5'-hydroxyl group of adenylic acid, has been shown to be affected by the polyamines, spermidine, spermine and putrescine. These substances enhance the hydrolytic activity of the enzyme against both poly(C) and yeast RNA. Sperimidine and spermine also reverse the inhibition of the enzyme by the ordered polynucleotides, apparently by removing them from the surface of the enzyme. Treatment of poly(G)-bound peptides (obtained from tryptic digests of poly(G)-bound nuclease) with an excess of spermidine resulted in the isolation of spermidine-bound peptides. Purification of these peptides through ion-exchange chromatography resulted in the isolation of three spermidine-bound peptides which consisted of 17 residues (6 amino acids), 19 residues (10 amino acids), and 12 residues (9 amino acids). The binding ratio of spermidine to peptides varied from 1:1 to 3:1.

Amino Acids↗
PubMed2006

Enterobacter sakazakii in the mouths of stroke patients and its association with aspiration pneumonia.

BACKGROUND: Previous studies by our group have identified a high prevalence of Gram-negative bacilli in the mouths of stroke patients. Eradication of such organisms by selective decontamination of the digestive tract (SDD) has not previously been studied. Enterobacter sakazakii is a rarely found organism that has previously been identified as a cause of neonatal meningitis and sepsis but that has rarely been reported as a pathogen in older people. The purpose of this study was to look specifically for E. sakazakii in the mouths of stroke patients and to correlate its isolation with response to SDD gel and the presence of clinical complications such as pneumonia. METHODS: Following an acute stroke, 203 patients were recruited for the study and received placebo or SDD in a 50:50 ratio. Oral swabs were obtained on nine separate occasions and E. sakazakii was cultured from them. Clinical data were prospectively collected and analysed. RESULTS: E. sakazakii was identified in 7 of 203 patients. The organism was almost exclusively removed by SDD gel, but in those patients in whom an abnormal swallow was present, pneumonia was found in two of the four cases. CONCLUSION: E. sakazakii may colonise the mouths of acute stroke patients. Since SDD gel is not routinely administered to such patients, this organism may be implicated in patients who subsequently develop aspiration pneumonia.

Journal Article↗
PubMed2006

The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat.

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin using the kinetic colorimetric Limulus amoebocyte lysate (LAL) assay. The endotoxin levels ranged from 40 to 5.5 x 10(4) endotoxin units (EU) per gram and did not correlate with the number of viable bacteria. The neonate rat model was used to address the risk of endotoxin-induced bacterial translocation from the gut. Purified Escherichia coli LPS was administered to rat pups followed by inoculation with Enterobacter sakazakii ATCC 12868. Bacteria were isolated from the mesentery, spleen, blood and cerebral spinal fluid (CSF) of endotoxin-treated rats due to enhanced gut and blood brain barrier penetration. Histological analysis of the colon showed marked distension of the mucosal and muscular layers. It is plausible that the risk of neonatal bacteraemia and endotoxemia, especially in neonates with immature innate immune systems, may be raised due to ingestion of IFM with high endotoxin levels.

Animals↗
PubMed2004

A selective differential medium for Enterobacter sakazakii, a preliminary study.

Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen. The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc). Ent. sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium. DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E. Ninety-five clinical and food strains of Ent. sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method. The characteristics of 148 strains representing 17 genera of non-Ent. sakazakii Enterobacteriaceae were compared using the two methods. Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp. and 1/8 Citrobacter koseri strains gave false positive results on DFI agar. Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent. sakazakii using ID32E. Therefore the DFI medium enables the detection of Ent. sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium. In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent. sakazakii.

Colony Count, Microbial↗
PubMed2005

Enterobacter sakazakii: a coliform of increased concern to infant health.

The first cases of neonatal meningitis believed to have been caused by Enterobacter sakazakii were reported in 1961. Prompted by several subsequent outbreaks of E. sakazakii infections in neonates and an increasing number of neonates in intensive care units being fed rehydrated powdered infant formula, considered to be a source of the pathogen, public health authorities and researchers are exploring ways to eliminate the bacterium or control its growth in dry infant formula, processing environments and formula preparation areas in hospitals. Reviewed here are advances in taxonomy and classification of E. sakazakii, methods of detecting, isolating and typing the bacterium, antibiotic resistance, clinical etiology and pathogenicity. Outbreaks of E. sakazakii infections in neonates and adults are summarized. Reports on the presence of E. sakazakii in clinical settings, the environment and foods and food processing facilities are reviewed. Tolerance of the pathogen to environmental stresses, its behavior in powdered and rehydrated infant formulae and hazard analysis and risk management are discussed. Research needs are presented.

Cronobacter sakazakii↗
PubMed2006

Survival of Enterobacter sakazakii on fresh produce as affected by temperature, and effectiveness of sanitizers for its elimination.

A study was done to determine the survival characteristics of Enterobacter sakazakii on the surface of apples, cantaloupes, strawberries, lettuce, and tomatoes stored at 4, 12, and 25 degrees C for 8-28 days. Populations significantly decreased (p or=50 microg/ml, were equivalent in killing E. sakazakii on apples. Populations of E. sakazakii on apples treated with 10 microg/ml chlorine dioxide for 1 or 5 min were significantly reduced (p or=4.00 log CFU/apple. Reductions of >or=3.70 log CFU/tomato were achieved by treatment with 10 microg/ml chlorine or chlorine dioxide or 40 microg/ml Tsunami 200 for 5 min. Reductions in populations of E. sakazakii on lettuce treated with chlorine at 10, 50, and 100 microg/ml for 1 min ranged from 1.61 to 2.50 log CFU/sample (26+/-4 g), compared to populations remaining on lettuce washed with water. Chlorine was less effective in killing E. sakazakii on lettuce than on apples or tomatoes. Treatment of lettuce with Tsunami 200 (40 and 80 microg/ml) for 5 min caused a reduction of >or=5.31 log CFU/sample. Results provide insights to predicting survival characteristics of E. sakazakii on produce and the efficacy of sanitizers in killing the bacterium.

Chlorine↗
PubMed2006

A study of the Gamma hypothesis: predictive modelling of the growth and inhibition of Enterobacter sakazakii.

Although the temperature growth profile of the opportunistic pathogen Enterobacter sakazakii is known, few other environmental factors affecting growth have been analysed. Using a model based on the Gamma hypothesis--that antimicrobial factors in mixtures exert independent effects--a range of weak acids (lactic, acetic, propionic, citric, sorbic and benzoic), pH, salt and temperature and some of their combinations were examined. The weak acids examined inhibited principally with the acid-form of the weak acid, however, benzoic, sorbic and propionic acids also displayed an inhibitory contribution from their respective anionic forms. In all cases pH could be considered an independent inhibiting factor. The minimum pH and maximum salt concentration for growth were calculated to be 3.89 and 9.1% respectively. In combination, there was no suggestion of any interactive effect between them. Studies performed on combinations of Na acetate/pH between 25 and 41 degrees C showed that temperature did not affect the relative inhibitory effects of the weak acid/pH mixtures. The results of this study support the Gamma hypothesis suggesting that there are no synergistic interactions between inhibitory factors and that growth can be predicted from a library of known effects. More importantly to the food industry, the results can be used to design good quality shelf-life challenge tests by reducing the number of studies required.

Acids↗
PubMed2006

Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula.

Reconstituted infant formula has been implicated in outbreaks of Enterobacter sakazakii infections, causing high mortality and serious sequelae. Current prevention methods appear to be insufficient to ensure that such foods are free of E. sakazakii. In this study, the usefulness of bacteriophages for biocontrol of E. sakazakii was investigated. Of a total of six new E. sakazakii phages isolated from sewage and UV irradiated cultures, two were selected for further study by electron microscopy, DNA restriction analysis and SDS-PAGE of structural proteins. Purified phages were used to control bacterial growth in broth medium and reconstituted infant formula. Both phages effectively prevented development of E. sakazakii in formula at various temperatures (12, 24 and 37 degrees C), the efficiency of which was dependent upon intrinsic lysis properties and the applied phage concentration. We conclude that application of specific bacteriophages may provide a means for efficient prevention of E. sakazakii infection through reconstituted infant formula.

Bacteria↗
PubMed2004

Enterobacter sakazakii is a rare cause of neonatal septicemia or meningitis in VLBW infants.

To determine the rates of Enterobacter sakazakii (ES) infections among very low birth weight infants, culture data from the National Institute of Child Health and Human Development Neonatal Research Network were reviewed. Only one case of ES sepsis was identified among 10660 neonates. These data suggest that outside of the epidemic situation, ES is very rare in very low birth weight infants.

Bacteremia↗
PubMed2005

PCR and oligonucleotide array for detection of Enterobacter sakazakii in infant formula.

Enterobacter sakazakii has been implicated in a several form of neonatal meningitis with a high mortality rate. In the present study, the species-specific PCR and oligonucleotide array assays were developed to detect the 16S-23S rDNA internal transcribed spacer (ITS) of E. sakazakii. Two pairs of specific PCR primers and 10 oligonucleotide probes were designed by sequencing the ITS of six strains of E. sakazakii and BLAST of GenBank. The specificity and efficiency of the PCR and oligonucleotide array methods were tested against a panel of numerous strains from 88 different bacterial strains. All of the E. sakazakii strains generated positive signal, and no cross-reaction was observed with non-E. sakazakii strains in the PCR and oligonucleotide array detections based on ITS sequences. Sensitivity of the detections is 1.3 CFU/100 g infant formula with the selective enrichment. Both of the PCR and oligonucleotide array procedures take only 48 h including the enrichment culture, whereas the conventional methods required at least 5 days. This study demonstrated that both of the pathogenic detections are time-saved and reliable.

Cronobacter sakazakii↗
PubMed2005

Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula.

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E. sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.

Animals↗
PubMed2006

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism.

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.

Bacterial Proteins↗
PubMed2004

Occurrence of Enterobacter sakazakii in food production environments and households.

Enterobacter sakazakii occasionally causes illness in premature babies and neonates. Contamination of infant formulae during factory production or bottle preparation is implicated. Advice to health-care professionals focuses on bottle preparation, but the effectiveness of prevention depends on the degree of contamination and contamination sites, which are generally unknown. To keep contamination to a minimum in the finished product depends on knowledge of the occurrence of E sakazakii. We used a refined isolation and detection method to investigate the presence of this micro-organism in various food factories and households. Environmental samples from eight of nine food factories and from five of 16 households contained E sakazakii. The widespread nature of this micro-organism needs to be taken into account when designing preventive control measures.

Cronobacter sakazakii↗
PubMed2004

High-yield production of hydrogen by Enterobacter aerogenes mutants with decreased alpha-acetolactate synthase activity.

To enhance hydrogen (H2) production from glucose by Enterobacter aerogenes HU-101, two mutants, strains VP-1 and VP-2, with decreased alpha-acetolactate synthase activity, were isolated using the Voges-Proskauer (VP) test. In pH-uncontrolled batch culture, both mutants showed a lower 2,3-butanediol yield for the glucose consumed than that shown by the wild-type strain, although glucose remained in the medium after 12 h of culture. In the same cultures, compared to the H2 yield of 0.80 mol/mol-glucose of the wild-type strain, strain VP-1 showed a high H2 yield of 1.8 mol/mol-glucose with decreased lactate and increased succinate yields, while strain VP-2 showed an H2 yield of 1.0 mol/mol-glucose with an increased lactate yield. Increasing the phosphate buffer concentration, which contributes to maintaining the pH in the medium, increased the glucose consumption by both strains. However, in a pH-controlled batch culture at neutral pH, the H2 yield of strain VP-1 was decreased to 1.2 mol/mol-glucose due to the accumulation of formate, an intermediate of the H2-producing pathway, with the yield of H2 plus formate being 1.7 mol/mol-glucose.

Journal Article↗
PubMed2003

Treatment of winery effluent with upflow anaerobic sludge blanket (UASB)--granular sludges enriched with Enterobacter sakazakii.

Three upflow anaerobic sludge blankets (UASBs) were evaluated for the treatment of winery wastewater: the first was seeded with granular sludge enriched with Enterobacter sakazakii and reached a 90% COD removal within 17 d at hydraulic retention time of 24 h; the second was seeded with brewery granules and achieved 85% COD removal within 50 d, the third was seeded with just sludge and showed the typical problems encountered with conventional sludge seeding and had continuously to be re-seeded. A PCR-based technique was developed for the rapid detection of E. sakazakii in the granular sludge.

Bacteria, Anaerobic↗
PubMed2003

Desiccation and heat tolerance of Enterobacter sakazakii.

AIMS: Enterobacter sakazakii is an opportunistic pathogen which has been isolated at low levels from powdered infant formulas. This study was performed to demonstrate that Ent. sakazakii is not particularly thermotolerant, but can adapt to osmotic and dry stress. METHODS AND RESULTS: We determined the heat, osmotic and dry stress resistance of Ent. sakazakii. The D-value at 58 degrees C ranged from 0.39 to 0.60 min, which is comparable with that of other Enterobacteriaceae, but much lower than reported previously (Nazarowec-White and Farber 1997, Letters in Applied Microbiology 24: 9-13). However, stationary phase Ent. sakazakii cells were found to be more resistant to osmotic and dry stress than Escherichia coli, Salmonella and other strains of Enterobacteriaceae tested. Further analysis indicated that the dry resistance is most likely linked to accumulation of trehalose in the cells. CONCLUSIONS: The high tolerance to desiccation provides a competitive advantage for Ent. sakazakii in dry environments, as found in milk powder factories, and thereby increases the risk of postpasteurization contamination of the finished product. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding of the physiology and survival strategies of Ent. sakazakii is an important step in the efforts to eliminate this bacterium from the critical food production environments.

Colony Count, Microbial↗
PubMed2003

Nosocomial outbreak of Enterobacter gergoviae bacteraemia in a neonatal intensive care unit.

A nosocomial outbreak of bacteraemia, caused by Enterobacter gergoviae infected 11 babies, nine of whom were premature, and was investigated in the neonatal intensive care unit (NICU) of a general hospital in Johor Bahru, Malaysia. The strain that was isolated from the babies was also isolated from the dextrose saline used for the dilution of parenteral antibiotics and from the hands of a healthcare worker on duty in the nursery. Pulsed-field gel electrophoresis (PFGE) of Xba I-digested chromosomal DNA confirmed a possible cross-contamination of parenteral dextrose saline and the healthcare worker. Prompt and effective control measures were initiated within NICU and the nosocomial infection of E. gergoviae was brought to an abrupt end. To the best of our knowledge, this is the first documented outbreak of E. gergoviae in the NICU in a hospital in the state of Johor, Malaysia.

Bacteremia↗