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The taxonomic position of Enterobacter dissolvens was re-evaluated based on the analysis of the type strain ATCC 23373T and three clinical isolates. The strains were assigned to the genetic cluster of the species by phylogenetic sequence analysis in the frame of a recent population genetic study. The relatedness of E. dissolves to the other species of the E. cloacae complex was analyzed by DNA-DNA hybridization studies based on melting profiles in microplates. The genetic cluster of E. dissolvens fell into the same DNA-relatedness group like E. cloacae with mean deltaTm-values of 3.9 degrees C confirming the hybridization results of three former studies. Phenotypic analysis of the E. cloacae and E. dissolvens strains, respectively, based on 115 biochemical reactions yielded the esculin test as the only one differentiating between them by being positive for E. dissolvens and negative for E. cloacae strains. The name E. cloacae subsp. dissolvens comb. nov. is proposed for the group of organisms formerly referred to as E. dissolvens, and the name E. cloacae subsp. cloacae comb. nov. for the group of organisms formerly referred to as E. cloacae. The species descriptions of Enterobacter kobei and Enterobacter asburiae were emended based on the data collected on 17 and 15 strains, respectively. The strains were assigned to the respective species by a combination of phylogenetic sequence analyzes and DNA-DNA hybridizations. Phenotypic analyzes of 115 reactions gave detailed species profiles with new differentiating phenotypic properties.
A phenanthrene-degrading bacterium, strain TKP(T), was isolated from a fly ash dumping site of the thermal power plant in Panki, Kanpur, India, by an enrichment culture method using phenanthrene as the sole source of carbon and energy. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belonged to the genus Sphingobium, as it showed highest sequence similarity to Sphingobium herbicidovorans DSM 11019(T) (97.3 %) and Sphingomonas cloacae JCM 10874(T) (96.5 %), compared with only 91-93 % similarity to members of other genera such as Sphingomonas sensu stricto, Novosphingobium, Sphingopyxis and Sphingosinicella. In DNA-DNA hybridization experiments with strains that were closely related phylogenetically and in terms of 16S rRNA gene sequences, i.e. Sphingobium herbicidovorans DSM 11019(T) and Sphingomonas cloacae JCM 10874(T), strain TKP(T) showed less than 70 % relatedness. Strain TKP(T) contained sphingoglycolipids SGL-1 and SGL-2 and 18 : 1omega7c as the predominant fatty acid, with 16 : 0 as a minor component and 14 : 0 2-OH as the major 2-hydroxy fatty acid. Thus, phylogenetic analysis, DNA-DNA hybridization, fatty acid and polar lipid profiles and differences in physiological and morphological features from the most closely related members of the Sphingobium group showed that strain TKP(T) represents a distinct species of Sphingobium. The name Sphingobium fuliginis sp. nov. is proposed, with the type strain TKP(T) (=MTCC 7295(T)=CCM 7327(T)). Sphingomonas cloacae JCM 10874(T) formed a coherent cluster with members of Sphingobium, did not reduce nitrate to nitrite and had a fatty acid profile similar to those of Sphingobium species; hence Sphingomonas cloacae should be transferred to the genus Sphingobium as Sphingobium cloacae comb. nov., with the type strain JCM 10874(T) (=DSM 14926(T)).
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Mutants of Enterobacter cloacae 55 were studied to delineate more completely the genetics of inducible expression of AmpC beta-lactamase. E. cloacae 55M-L, derived by mutagenesis from a mutant with high-level cefotaxime resistance (MIC, greater than 64 micrograms/ml), E. cloacae 55M, demonstrated a novel phenotype by producing only low levels of AmpC constitutively. Neither the parental phenotype of E. cloacae 55M nor the wild-type phenotype of E. cloacae 55 could be restored in E. cloacae 55M-L by the introduction of functional ampR, ampC, or ampD genes. Cloning each of these genes from E. cloacae 55M-L confirmed the same genotype for this mutant as for its parental strain. Mutation of E. cloacae 55M-L to the E. cloacae 55M phenotype was found to occur spontaneously at a frequency of 10(-8). All such revertants demonstrated an inducible wild-type phenotype after introduction of a functional ampD. These results suggested that the E. cloacae 55M-L phenotype was due to a mutation in an as yet unrecognized gene, designated ampG. Verification of this gene was obtained by the restoration of the E. cloacae 55M phenotype in E. cloacae 55M-L by introduction of a cloned 2.9-kilobase BamHI fragment from the E. cloacae 55 chromosome. Transformation of both ampG and ampD into E. cloacae 55M-L reconstituted the inducible wild-type phenotype. These results indicate that ampG is required for the activation of ampC by AmpR. Without ampG, neither induction nor high-level expression of AmpC is possible. It is likely that the ampG gene product and AmpD together modulate the ability of AmpR to activate ampC expression.
To further elucidate the induction process of the carbapenem-hydrolyzing beta-lactamase of Ambler class A, NmcA, ampD genes of the wild-type (WT) strain and of ceftazidime-resistant mutants of Enterobacter cloacae NOR-1 were cloned and tested in transcomplementation experiments. Ceftazidime-resistant E. cloacae NOR-1 mutants exhibited derepressed expression of the AmpC-type cephalosporinase and of the carbapenem-hydrolyzing beta-lactamase NmcA. The ampD genes of Escherichia coli and E. cloacae WT NOR-1 transcomplemented the ceftazidime-resistant E. cloacae NOR-1 mutants to the WT level of beta-lactamase expression, while the mutated ampD alleles of E. cloacae NOR-1 failed to do so. The deduced E. cloacae NOR-1 WT AmpD protein exhibited 95 and 91% amino acid identity with the E. cloacae O29 and E. cloacae 14 WT AmpD proteins, respectively. Of the 12 ceftazidime-resistant E. cloacae NOR-1 strains, 3 had AmpD proteins with amino acid changes, while the others had truncated AmpD proteins. Most of these mutations were located outside the conserved regions that link the AmpD proteins to the cell wall hydrolases. AmpD from E. cloacae NOR-1 is involved in the regulation of expression of both beta-lactamases (NmcA and AmpC), suggesting that structurally unrelated genes may be under the control of an identical genetic system.
The ampR gene and its regulation of AmpC beta-lactamase synthesis were investigated for Enterobacter cloacae 1194E, a wild-type strain producing a group A (pI 8.7) enzyme. Expression of the cloned E. cloacae 1194E ampR-ampC region was examined initially in Escherichia coli HB101. However, transformants showed only constitutive beta-lactamase expression. For study of enzyme expression in a more closely related host, the cloned E. cloacae 1194E ampR-ampC region was transformed into E. cloacae 55, a wild-type strain producing a group B (pI 7.8) enzyme. Results indicated a functional E. cloacae 1194E ampR gene that could not be transcomplemented by E. cloacae 55. A comparative analysis of ampR nucleotide and amino acid-sequence data from E. cloacae 1194E and E. cloacae MHN1 revealed related but divergent genes. Thermal induction studies of AmpC beta-lactamase also indicated a difference between E. cloacae 1194E and E. cloacae 55 in ampR-ampC interaction. Thus, it appears that, in at least some strains of Enterobacter, significant intraspecies divergence of ampR has occurred. This heterogeneity in ampR would not have been detected with beta-lactamase expression studies conducted exclusively in E. coli.
The motility of the avian cloaca is under neural control, but little is known about the neural network that accomplishes this function. This present study was designed to determine the distribution of nitric oxide-synthesising neurons in the pigeon cloaca by enzyme histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). NADPH-d-positive staining was seen in the neurons and fibres in the cloaca. The highest density of nerve fibres was noted in the coprodeum and the lowest in the proctodeum. In the coprodeum, NADPH-d neurons were found singly, formed small groups of 2-10 neurons, or were seen in plexuses in the muscle layer, lamina propria, or around the arterioles. Several NADPH-d-positive neurons were also observed in the ganglia of the cloaca. NADPH-d fibres ran in the muscle layer, lamina muscularis mucosae and lamina propria, or surrounded blood vessels. The distribution pattern of acetylcholinesterase (AChE)-stained neurons and fibres in the cloaca was similar to that of NADPH-d. Double staining for NADPH-d and AChE showed colocalisation of the 2 enzymes in many neurons of the cloaca. Tyrosine hydroxylase (TH)-immunoreactive nerve fibres originating outside the cloaca were also noted. In the urodeum and proctodeum, neurons or fibres positive for NADPH-d, AChE or TH were scattered in the lamina propria. Nerve fibres immunoreactive for calcitonin-gene related peptide, galanin, methionine-enkephalin, substance P, and vasoactive intestinal peptide were found sparsely in the cloaca. Our results demonstrate that nitrergic neurons constitute a subpopulation which is closely associated with the cholinergic system in the pigeon cloaca.
The antibacterial activities of imipenem-cilastatin, meropenem-cilastatin, cefepime and ceftazidime against Enterobacter cloacae NOR-1, which produces the carbapenem-hydrolyzing beta-lactamase NmcA and a cephalosporinase, and against one of its in vitro-obtained ceftazidime-resistant mutant were compared by using an experimental model of pneumonia with immunocompetent rats. The MICs of the beta-lactams with an inoculum of 5 log(10) CFU/ml were as follows for E. cloacae NOR-1 and its ceftazidime-resistant mutant, respectively: imipenem, 16 and 128 microg/ml, meropenem, 4 and 32 microg/ml, cefepime, <0.03 and 1 microg/ml, and ceftazidime, 1 and 512 microg/ml. The chromosomally located cephalosporinase and carbapenem-hydrolyzing beta-lactamase NmcA were inducible by cefoxitin and meropenem in E. cloacae NOR-1, and both were stably overproduced in the ceftazidime-resistant mutant. Renal impairment was induced (uranyl nitrate, 1 mg/kg of body weight) in rats to simulate the human pharmacokinetic parameters for the beta-lactams studied. Animals were intratracheally inoculated with 8.5 log(10) CFU of E. cloacae, and therapy was initiated 3 h later. At that time, animal lungs showed bilateral pneumonia containing more than 6 log(10) CFU of E. cloacae per g of tissue. Despite the relative low MIC of meropenem for E. cloacae NOR-1, the carbapenem-treated rats had no decrease in bacterial counts in their lungs 60 h after therapy onset compared to the counts for the controls, regardless of whether E. cloacae NOR-1 or its ceftazidime-resistant mutant was inoculated. A significant decrease in bacterial titers was observed for the ceftazidime-treated rats infected with E. cloacae NOR-1 only. Cefepime was the only beta-lactam tested effective as treatment against infections due to E. cloacae NOR-1 or its ceftazidime-resistant mutant.
The transport of sperm in the cloaca and adjacent regions of the female red-spotted newt was examined. It was found that within 1 min after sperm were introduced into the vent, they progressed in a random pattern past the apertures of the spermatheca (the glandular, sperm storage organ that opens from the anterior roof of the cloaca) forward to the anterior end of the cloaca and on into the posterior regions of the hindgut and bladder. Sperm did not enter the dorsal recess of the cloaca into which the oviducts and ureters open. After 1 day, few sperm remained within the cloaca lumen. Sperm were not transported into the cloacae of artificially inseminated, anesthetized females without prior administration of norepinephrine to their cloacal mounds. Treatment of the cloacal mounds of naturally inseminated females with an antagonist of neuromuscular transmission (lidocaine) decreased the numbers of sperm in the anterior cloaca relative to those of saline-injected control specimens. Neither dead newt sperm nor live rabbit sperm entered the spermatheca. Rabbit sperm, however, entered the oviduct. It is argued that passive and active mechanisms of sperm transport work in concert. Contractions of smooth muscle, which may be initiated during courtship, probably serve to draw sperm passively into the cloaca and up to and beyond the apertures of spermathecal tubules, but sperm, once in the vicinity of those apertures, probably swim actively into them.
The cloaca, the caudal limit of the avian gastrointestinal tract, acts as a collecting chamber into which the gastrointestinal, urinary, and genital tracts discharge. It is intrinsically innervated by the enteric nervous system, which is derived from neural crest emigres that migrate from the vagal and sacral regions of the neural tube. Abnormal cloacal development can cause a number of anorectal anomalies, including persistent cloaca. Ablation of the vagal neural crest has previously been shown to result in an aganglionic hindgut to the extent of the colorectum. The aim of our study was to investigate the effect of vagal neural crest ablation on the cloaca, the limit of the hindgut in the developing chick embryo. Chick embryos were incubated until the 10-12 somite stage. The vagal neural tube corresponding to the level of somites 3-6 was then ablated, and eggs were incubated until harvested on embryonic day 11 (E11). Whole chick embryos were fixed, embedded in paraffin, and sectioned. Immunohistochemistry was then carried out using the HNK-1 monoclonal antibody to label neural crest cells, and results were assessed by light microscopy. Vagal neural crest ablation resulted in a dramatic decrease in the number of neural crest cells colonizing the chick embryo cloaca compared with control embryos. Ablated embryos contained only a small number of HNK-1-positive neural crest cells, which were scattered within the myenteric plexus in a disorganised pattern. Hypoganglionosis was also evident in other regions of the hindgut in ablated embryos. Ablation of the vagal neural crest results in a hypoganglionic cloaca in addition to hypoganglionosis of the hindgut. These results suggest that the cloaca is largely innervated by vagal neural crest emigres. Further studies involving quail-chick chimeras to investigate the exact contribution provided by both vagal and sacral neural crest cells to the cloaca should increase our understanding of the pathophysiology of conditions like persistent cloaca.
The intrinsic innervation of the developing gut has long been a subject of investigation, but little is known regarding that of the embryonic cloaca. The cloaca, like the rest of the gastrointestinal tract, is intrinsically innervated by the enteric nervous system. Nitrergic neurons and fibres make up a large part of this system, thus, their distribution provides us with a useful insight into its development. Cloacal and colorectal tissue specimens were removed from chick embryos at embryonic days 11 (E11), E15 and E19. NADPH-diaphorase (NADPH-d) histochemistry was carried out using whole mount tissue preparations. Ganglia density, the number of NADPH-d-positive cells per ganglia in the myenteric plexus and cell size were calculated and statistical analysis was performed to compare both regions of the gut (P<0.001). There were significant differences in the ganglia density in the cloaca compared to the colorectum at E11 (P<0.05) and E15 (P<0.01), with the colorectum having a much denser network. In both the cloaca and the colorectum, ganglia density significantly decreased with age (P<0.001), while significant differences were observed in the number of NADPH-d-positive cells per ganglia in both regions through development. Total cell size was similar in both the cloaca and colorectum at each stage and increased in both regions through development, predominantly due to an increase in the cytoplasm. Results reveal striking differences in innervation between the chick embryo cloaca and colorectum. The sparse network of innervation evident within the cloaca in contrast to the dense network within the colorectum emphasizes the individuality of both regions. These results highlight the need for a further in-depth analysis of the enteric nervous system's development within the embryonic cloaca.
Mixed cultures of Lactobacillus curvatus and Enterobacter cloacae were chosen as a model system to quantitatively study microbial interactions involved in food spoilage and food preservation. In this paper models were developed to predict the individual behaviour of L. curvatus and E. cloacae in pure suspension cultures as a function of the glucose and lactate concentration and the pH. In a second paper these models will be used to predict the moment of interaction in mixed suspension cultures and the cell concentrations at this moment. The effect of pH on the maximum specific growth rate could be described by a parabolic equation for L. curvatus and E. cloacae. E. cloacae is clearly more sensitive to a pH decrease than L. curvatus, as may be concluded from the theoretical minimum pH for growth of 5.6 for E. cloacae and 4.3 for L. curvatus. For both organisms no effect of glucose on the maximum specific growth rate could be detected and Monod kinetics with a low Monod constant was assumed. For L. curvatus the effect of lactate on the maximum specific growth rate at different initial pH values could be described by a linear relationship. For E. cloacae a slight effect of lactate on the maximum specific growth rate could only be detected at pH 7. However, this effect was negligible compared to the effect of the pH. For both organisms the lag time was modelled by the inverse of the specific growth rate. It can be concluded that, with regard to interactions between L. curvatus and E. cloacae, the pH is likely to be the most important factor in the case where L. curvatus is the dominant organism. Substrate limitation is likely to become important in the case where E. cloacae is the dominant organism or for media with a high buffer capacity.
Enterobacter cloacae is becoming an increasingly important nosocomial pathogen. Outbreaks of E. cloacae in intensive care units and burns units have been described frequently. In December 1999, a neonate with line sepsis was transferred from a university hospital to the neonatal unit of the Diakonessen Hospital. Blood culture yielded E. cloacae. An outbreak of E. cloacae was occurring in the university hospital at that time. In February 2000, a second neonate in our hospital developed line sepsis caused by E. cloacae. Direct measures taken included cohorting of infected children, disinfection of incubators, thermometers and wards, and screening patients. Of nine neonates, seven were colonized with E. cloacae. Despite these measures, the outbreak continued. Forty-one patients were screened; 15 were colonized. Environmental searches yielded E. cloacae in a sink and on two thermometers. Sixteen isolates were typed by arbitrarily primed PCR using four primers. All the patient isolates and the two isolates from thermometers were identical. The strain isolated from the sink was unrelated. Amplified fragment length polymorphism typing showed that the outbreak clone was identical to that in the university hospital. After the introduction of disposable thermometer covers, E. cloacae colonization slowly decreased.
OBJECTIVE: From October 1996 to March 1997, a cluster of 11 cases of neonatal sepsis caused by Enterobacter cloacae with similar antimicrobial susceptibility patterns occurred in a neonatal intensive care unit. This outbreak prompted an investigation. METHOD: Twelve isolates obtained from 6 neonatal patients who developed E cloacae sepsis during the outbreak were analyzed. Four E cloacae isolates from 2 preterm neonates without E cloacae infection on the same ward, and 1 isolate from the hands of a nurse, were also examined. No E cloacae were isolated from the environment. Bacterial DNA digested with XbaI or NotI was analyzed with pulsed-field gel electrophoresis. RESULT: Three distinct banding patterns were identified by pulsed-field gel electrophoresis. Of the 6 preterm infants with sepsis, strain I was identified in 1, strain II in 2, a mixed infection of strains I and II in 2, and strain III was found in only 1 infant. An isolate from the hands of a nurse was identified as strain II, as were the 4 isolates from the 2 preterm neonates without E cloacae infection. Thus, this outbreak of sepsis was caused by 2 genotypes of E cloacae. CONCLUSION: This study demonstrates that pulsed-field gel electrophoresis with restriction enzyme digestion is a valuable tool for genetic characterization of multidrug-resistant E cloacae strains during outbreaks.
OBJECTIVE: To investigate an outbreak in neonates of Enterobacter cloacae infection resistant to third-generation cephalosporins. DESIGN: A retrospective study of an outbreak in the neonatal intensive care unit (NICU) and review of E cloacae isolates in pediatric wards and other intensive care units from June 1992 through March 1993. SETTING: An academic tertiary care hospital. PATIENTS: Six patients admitted to the NICU were colonized or infected with E cloacae resistant to third-generation cephalosporins. In the period preceding the outbreak, four E cloacae isolates were available from four patients in the pediatric surgical ward. Nine isolates from four patients in two other intensive care units (ICUs) also were collected during the outbreak. Isolates were biotyped by the API 50CH system and genotyped by polymerase chain reaction (PCR) fingerprinting. RESULTS: Typing by interrepeat PCR showed that 21 isolates, which were obtained from five neonates, were identical. One neonate was colonized with a different strain. Some neonates were colonized with a single type of E cloacae for a relatively long period of time. Isolates of patients who were cared for in the pediatric surgical ward and the two other intensive care units (ICUs) showed different genotypes. One patient in an ICU was colonized with an E cloacae strain genetically identical to the outbreak strain. No predominant biotype could be established. CONCLUSIONS: E cloacae can colonize neonates for a long period of time and although colonization with E cloacae initially may arise endogenously, we were able to show further transmission by cross-contamination in a neonatal intensive care unit.
The effects of ceftizoxime (CZX), piperacillin (PIP), and PIP-tazobactam (PT) concentrations on the antibacterial activity and selection of resistant mutants of Bacteroides fragilis and Enterobacter cloacae were investigated in vitro in a mixed-culture anaerobic time-kill study and in vivo in a mixed-infection abscess model. Mixed cultures were incubated for 24 h with 0.125 to 512 micro g of CZX per ml or 0.125 to 2,048 micro g of PIP or PT per ml. Mice were treated every 2 h for 24 h with CZX at 6 to 1,536 mg/kg/day or with PIP or PT at 24 to 6,144 mg/kg/day starting 30 min before inoculation with different B. fragilis-E. cloacae combinations. There was a good correlation between the in vitro and in vivo activities of the antibiotics and their MICs obtained with high inocula (10(8) CFU/ml). The respective 50% effective doses (milligrams per kilogram per day) with B. fragilis and E. cloacae 22491 were 771 and 521 for CZX, 416 and 643 for PIP, and 85 and 554 for PT, and with the B. fragilis-E. cloacae 032349 combination, they were 81 and 21 for CZX and 77 and 766 for PT. Resistant mutants of E. cloacae 22491 were preferentially selected in vitro with 2 to 64 micro g of CZX per ml and in vivo with CZX at 12 to 384 mg/kg/day. There was no preferential selection of CZX-resistant B. fragilis or E. cloacae 032349. For CZX-resistant E. cloacae 22491, we found a 16- to 512-fold increase in the MIC of CZX and increased MICs of other expanded-spectrum cephalosporins, owing in part to the production of a stably derepressed cephalosporinase. In vitro and in vivo, PT did not select resistant mutants of E. cloacae and B. fragilis. Results demonstrate the adverse microbiological outcome of choosing an expanded-spectrum cephalosporin like CZX for empirical treatment of mixed infections involving a susceptible Enterobacter strain.