Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.
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We have previously demonstrated the existence of Klebsiella pneumoniae clinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates. (+info)
Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L.
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The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions. (+info)
Low resolution structure of the sigma54 transcription factor revealed by X-ray solution scattering.
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The sigma54 RNA polymerase holoenzyme functions in enhancer-dependent transcription. The structural organization of the sigma54 subunit of bacterial RNA polymerase in solution is analyzed by synchrotron x-ray scattering. Scattering patterns are collected from the full-length protein and from a large fragment able to bind the core RNA polymerase, and their low resolution shapes are restored using two ab initio shape determination techniques. The sigma54 subunit is a highly elongated particle, and the core binding fragment can be unambiguously positioned inside the full-length protein. The boomerang-like shape of the core binding fragment is similar to that of the atomic model of a fragment of the Escherichia coli sigma70 protein, indicating that, although the sigma54 and sigma70 factors are unrelated by primary sequence, they may share some structural similarity. Potential DNA binding surfaces of sigma54 are also predicted by comparison with the sigma54 core binding fragment. (+info)
Production of an extracellular polysaccharide bioflocculant by Klebsiella pneumoniae.
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Klebsiella pneumoniae H12 produced a newly identified extracellular polysaccharide in an ethanol medium with a yield of 3.0 g/l. The molar composition of the polysaccharide was 56.04% galactose, 25.92% glucose, 10.92% galacturonic acid, 3.71% mannose, and 3.37% glucuronic acid. The addition of 0.5%-1.5% NaCl increased production. The polysaccharide flocculated with kaolin clay in suspension at the concentration of 1 ppm in a 300-ppm solution of CaCl2. Almost all bacterial species cells aggregated in the polysaccharide solution. The ability to flocculate with kaolin clay changed with the pH and with the concentrations of coexisting cation and anion species. The polysaccharide flocculant may participate in in vivo bacterial aggregation or adherence to host organisms. (+info)
Febrile core temperature is essential for optimal host defense in bacterial peritonitis.
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Fever, a nonspecific acute-phase response, has been associated with improved survival and shortened disease duration in infections, but the mechanisms of these beneficial responses are poorly understood. We previously reported that increasing core temperature of bacterial endotoxin (LPS)-challenged mice to the normal febrile range modified expression of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-6, three cytokines critical to mounting an initial defense against microbial pathogens, but survival was not improved in the warmer animals. We speculated that our inability to show a survival benefit of optimized cytokine expression in the warmer animals reflected our use of LPS, a nonreplicating agonist, rather than an infection with viable pathogens. The objective of this study was to determine if increasing murine core temperature altered cytokine expression and improved survival in an experimental bacterial peritonitis model. We showed that housing mice at 35.5 degrees C rather than 23 degrees C increased core temperature from 36.5 to 37.5 degrees C to 39.2 to 39.7 degrees C, suppressed plasma TNF-alpha expression for the initial 48 h, delayed gamma interferon expression, improved survival, and reduced the bacterial load in mice infected with Klebsiella pneumoniae peritonitis. We showed that the reduced bacterial load was not caused by a direct effect on bacterial proliferation and probably reflected enhanced host defense. These data suggest that the increase in core temperature that occurs during bacterial infections is essential for optimal antimicrobial host defense. (+info)
Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae.
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Klebsiella pneumoniae ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was cloned, and its protein was expressed in Escherichia coli DH10B, where this pI-5. 8 beta-lactamase of a ca. 31-kDa molecular mass conferred resistance to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamases (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome-borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1. Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was characterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P(1) and P(2), for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3' conserved segment consisting of qacEDelta1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since they lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid and integron, thus providing it additional means for its spread and its expression. (+info)
Failure of an automated blood culture system to detect nonfermentative gram-negative bacteria.
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During a 1-year study we observed that both aerobic and anaerobic blood culture bottles from patients were negative by the BacT/Alert system during a 7-day incubation period. However, upon subcultivation of negative bottles, growth of Pseudomonas aeruginosa was detectable. In an attempt to explain this observation, aerobic BacT/Alert Fan bottles were seeded with a defined inoculum (0.5 McFarland standard; 1 ml) of Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, P. aeruginosa, Stenotrophomonas maltophilia, or Acinetobacter baumannii. Half of the inoculated bottles were loaded into the BacT/Alert system immediately, and the remainder were preincubated for 4, 8, 16, and 24 h at 36 degrees C. With preincubation all bottles seeded with the Enterobacteriaceae signaled positive during the next 1.5 h. Organisms in bottles seeded with the nonfermentative species P. aeruginosa and A. baumannii remained undetected by the BacT/Alert system for 7 days. S. maltophilia was detected if the preincubation time was equal or less than 8 h. Without preincubation all bottles seeded with the Enterobacteriaceae or nonfermentative species signaled positive. Since nonfermentative species seem to enter a state of bacteriostasis within the preincubation period, we reasoned that an unknown factor is consumed. Accordingly, a smaller inoculum should allow the detection of nonfermentative species, even after preincubation, and serial dilutions of P. aeruginosa were detected in preincubated bottles. In this case preincubated bottles signaled positive faster than bottles without preincubation. We conclude that all bottles from clinical settings should be subcultured prior to loading to avoid false negatives. An alternative may be preincubation at room temperature. (+info)
First identification of an SHV-12 extended-spectrum beta-lactamase in Klebsiella pneumoniae isolated in Italy.
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A clinical isolate of Klebsiella pneumoniae highly resistant to third- and fourth-generation cephalosporins, cephamycins and aminoglycosides, was isolated in 1991 from urine. Analysis of a crude extract showed the presence of three beta-lactamases with isoelectric points of 6.6, 7.5 and 8.2. The enzyme with pI 8.2 was transferred by conjugation into Escherichia coli K-12 J53 and was responsible for the resistance to third-generation cephalosporins and monobactams, but not to other antibiotics. Kinetic studies of partially purified beta-lactamase from the transconjugant strain confirmed that the enzyme was able to hydrolyse ceftazidime, cefotaxime and aztreonam but not cephamycins. Analysis of the transconjugant showed the presence of two small non-conjugative plasmids of 14 and 6 kb. A polymerase chain reaction was performed using primers specific for the bla(SHV) gene and a fragment of the expected size (about 961 bp) was obtained with both the K. pneumoniae clinical isolate and the transconjugant. Nucleotide sequence analysis of the fragment showed that it encoded the enzyme SHV-12, derived from SHV-5 (with Gln-35 to Leu). This is the first report of an SHV-12-like enzyme isolated in Italy. (+info)