Identification of two additives, locust bean gum (E-410) and guar gum (E-412), in food products by DNA-based methods.

Locust bean gum (E-410) and guar gum (E-412) are high molecular weight galactomannans used by the food industry as versatile food additives. The compounds, although chemically closely related, do not have the same functional properties when used in foods, and the substitution or unadvertised addition of either could change the desired qualities of the product. Analytical discrimination between E-410 and E-412 is technically difficult since they only differ in their galactose: mannose ratios, being 1 : 4 and 1 : 2 for locust bean gum and guar gum, respectively. A qualitative DNA-based method is reported for the authentication of additives E-410 and E-412 in finished food products (ice cream, dehydrated desserts, milk derivatives, dehydrated soups, salad dressing, marmalade and meat) from small quantities of food. DNA sequences from the nuclear ribosomal spacers of Ceratonia siliqua and Cyamopsis tetragonoloba, the plant sources of E-410 and E-412, respectively, were used to design polymerase chain reaction primers specific for each additive (PA23/PA21 and PG22/PG21). Twenty-two foods were analysed for the presence of E-410 and E-412 additives by this single-step polymerase chain reaction-based method. Positive DNA amplifications with the E-410 and/or E-412 primers were obtained in all 19 samples reported to contain either additive.

Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases.

The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.

Impairment of respiratory burst in polymorphonuclear leukocytes by extended-spectrum beta-lactamase-producing strains of Klebsiella pneumoniae.

The ability of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Klebsiella pneumoniae strains to induce a respiratory burst in polymorphonuclear leukocytes (PMNLs) was investigated. Ninety ESBL-producing and 178 non-ESBL-producing Klebsiella pneumoniae isolates were serotyped and their ability to induce a respiratory burst in PMNLs tested by monitoring the cells' chemiluminescence (CL) response. The percentage of isolates inducing high levels of CL response (CL>75%) was significantly higher among non-ESBL producers (52%) than among ESBL producers (32.2%) ( P<0.0001; OR=3.396; 95%CI=2.036-5.664). The median CL response was significantly higher among the non-ESBL producers (76.9%) than among the ESBL producers (52.6%) ( P=0.034). The two groups did not differ in their ability to resist intracellular killing by PMNLs ( P>0.05), with strains inducing high levels of CL response having significantly lower survival rates (31.8% vs. 42.4%) than strains inducing low levels of CL response (164% vs. 200%) ( P<0.01). The frequencies of the K2 and the K25 serotypes were significantly higher among ESBL-producing strains (17.8% and 22.2%, respectively) than among the non-ESBL producers (6.2% and 1.7%, respectively) ( P=0.0057 and P<0.0001). Of the 77 Klebsiella K serotypes, 71 were detectable among the non-ESBL producers, but only 24 were detectable among the ESBL producers. ESBL-producing Klebsiella pneumoniae strains might have a greater pathogenic potential by virtue of their ability to escape the phagocytic activity of PMNLs.

Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study.

Since 1992, there has been an increase in the incidence of Enterobacter sepsis in the neonatal intensive care unit (NICU) of the authors' hospital. From 1995 to 1997, a prospective molecular epidemiological survey of the colonizing and infecting strains isolated from neonates was conducted. Enterobacter cloacae was the most frequent cause of neonatal sepsis, accounting for 19.2% of all neonatal infections, reaching a peak incidence of 2.2/1000 during 1996. Fifty isolates from the NICU and four epidemiologically unrelated strains were characterized by pulse-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC)-PCR and plasmid profiling. PFGE was the most discriminatory technique and identified 13 types (two of them classified into two and three subtypes) compared with ERIC-PCR, plasmid profiling and ribotyping that identified 11, 11 and seven types, respectively. A good correlation was found between all techniques. Five different clones caused 15 cases of sepsis. Clones A and B were prevalent in 1995 and 1996, but they were not isolated in 1997. An outbreak caused by clone G in 1997 was controlled by cohort nursing and hygienic measures, without changing the antibiotic policy. Strains were characterized by their antibiotic resistance pattern and divided into three groups. Group I correlated with PFGE types A, B1 and B2, which hyperproduced Bush type 1 chromosomal beta-lactamase and expressed extended-spectrum ?-lactamases (ESBLs). Group II only hyperproduced Bush type 1 chromosomal beta-lactamase and correlated with PFGE-types D1, D2, D3 and I. Finally, Group III, with inducible beta-lactamases, correlated with the rest of PFGE types. The sudden disappearance of E. cloacae after reinforcement of hygienic measures confirms the importance of patient-to-patient transmission.

Increase of Enterobacter in neonatal sepsis: a twenty-two-year study.

BACKGROUND: Data on the incidence of Enterobacter infections in neonates over prolonged periods of time are scant. We determined the epidemiology of Enterobacter sepsis and/or meningitis and the trends of infection in a neonatal unit. METHODS: Retrospective review of sepsis and/or meningitis in inborn neonates admitted to Son Dureta University Hospital during a 22-year period. Molecular study by ribotyping of the Enterobacter strains isolated from 1995 to 1997. RESULTS: There were 513 cases of culture-proved sepsis and/or meningitis in neonates. In late onset infections Klebsiella pneumoniae and Staphylococcus epidermidis were the most frequent isolates in the period 1977 through 1991. Enterobacter was the most common isolate in the period 1992 through 1998. During this latter period Candida infections also increased, and the resistance rate of Enterobacter to cefotaxime was higher (59.2%). Decrease in early onset infections and increase in late onsets (4.6/1,000 live births) were observed in the second period. From 1977 to 1998, 45 episodes of sepsis and/or meningitis by Enterobacter species were identified in 44 patients (8.7% of all neonatal bacteremias). Three patients with Enterobacter bacteremia died (6.6%, 0.03/1,000 live births). During 1995 through 1997 5 different clones causing sepsis were identified and 3 were predominant. In 1997 there was an outbreak of Enterobacter disease. After cleaning, cohort nursing and hygiene reinforcement, Enterobacter was not isolated in the next 2 years. No change in the antibiotic policy was made. CONCLUSIONS: We observed a resurgence of Enterobacter infections in our neonatal intensive care unit. The sudden disappearance of this microorganism after reinforcement of hygienic measures, without withdrawing cefotaxime, confirms the importance of patient-to-patient transmission of this nosocomial infection. Further studies are needed to establish the role of antibiotics in the emergence of microorganisms in neonatal intensive care units.

Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal beta-lactamase and showing altered porin profiles.

Forty clonally related clinical isolates of Escherichia coli from hospitalized patients were resistant to cefoxitin (MICs, >256 microg/ml) and ceftazidime (MICs, 32 to 256 microg/ml) and were intermediate or resistant to cefotaxime (MICs, 16 to 128 microg/ml) but susceptible to both cefepime (MICs, 0.5 to 2 microg/ml) and imipenem (MICs, 0.125 to 0.25 microg/ml). Resistance to beta-lactams was related to high-level production of AmpC beta-lactamase and loss of OmpF porin.

Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae.

The activities of beta-lactams, chloramphenicol, tetracycline, fluoroquinolones and aminoglycosides against Klebsiella pneumoniae C3 (O1:K66, producing porins OmpK35 and OmpK36) and a set of isogenic mutants derived from it lacking the O antigen of lipopolysaccharide (LPS), capsular K antigen, or one or both porins were determined. MICs remained within one dilution step in mutants deficient in antigen O, in capsule or in one of the two porins. No increases in the MICs of aminoglycosides, fluoroquinolones, tetracycline and chloramphenicol were observed for strains deficient in the two porins, but the MICs of ampicillin, cephalothin, cefoxitin, cefotaxime and ceftazidime for this type of mutant increased four- to >256-fold. The highest MICs of beta-lactams were obtained in a porin-deficient mutant expressing increased beta-lactamase activity. It is concluded that isolated outer membrane alterations in K. pneumoniae are not decisive factors in increasing resistance to antimicrobial agents, but porin loss co-operates with beta-actamase production to increase resistance to beta-lactams.

Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila.

We cloned and sequenced the structural gene for Aeromonas hydrophila porin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that of ompF in Escherichia coli, is adjacent to aspC and transcribed in the same direction. However, upstream of the porin II gene no similarities with E. coli were found. We obtained defined insertion mutants in porin II gene either in A. hydrophila (O:34) or A. veronii sobria (serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.

Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.

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.

Roles of beta-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae.

Two clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae were noted to be less susceptible than expected to imipenem. Both were missing outer membrane proteins that serve as channels for antibiotic entry. The role of beta-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC beta-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with beta-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional ompK36 gene on a plasmid. Plasmids encoding AmpC-type beta-lactamases provided resistance to imipenem (up to 64 microg/ml) and meropenem (up to 16 microg/ml) in strains deficient in porins. Carbapenem resistance showed little inoculum effect, was not affected by clavulanate but was blocked by BRL 42715, and was diminished if OmpK36 porin was restored. Plasmids encoding TEM- and SHV-type ESBLs conferred resistance to cefepime and cefpirome, as well as to earlier oxyimino-beta-lactams. This resistance was magnified with an increased inoculum, was blocked by clavulanate, and was also lowered by OmpK36 porin restoration. In addition, SHV-2 beta-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 microg/ml, increasing to 16 microg/ml with a higher inoculum) when porins were absent. In K. pneumoniae porin loss can thus augment resistance provided either by TEM- or SHV-type ESBLs or by plasmid-mediated AmpC enzymes to include the latest oxyimino-beta-lactams and carbapenems.

Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance.

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes.

We have demonstrated by using an in vitro approach that interruption of the OmpK36 porin gene by insertion sequences (ISs) is a common type of mutation that causes loss of porin expression and increased resistance to cefoxitin in Klebsiella pneumoniae. This mechanism also operates in vivo: of 13 porin-deficient cefoxitin-resistant clinical isolates of K. pneumoniae, 4 presented ISs in their ompK36 gene.

Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniae clinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O- strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.

Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein.

The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355-3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups.

Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems.

Fifteen isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) isolated during a nosocomial outbreak were studied. The strains belonged to the same clonal type, as shown by pulsed-field gel electrophoretic analysis of chromosomal DNA. All the isolates were resistant to extended-spectrum cephalosporins, aztreonam, gentamicin, and fluoroquinolones and were susceptible to carbapenems, tobramycin, netilmicin, and amikacin. None of the isolates expressed the OmpK36 porin. Eight isolates, for which the MICs of cefoxitin were > or = 64 micrograms/ml, showed a diminished level or no expression of a 35-kDa porin. The MICs of meropenem, cefotaxime, and cefpirome were three to eight times higher for porin-deficient isolates than for isolates expressing the 35-kDa porin, but the MICs of imipenem increased two times for porin-deficient isolates compared to those for isolates expressing the porin. This MIC increase reverted to a level similar to that for the parental strain when porin-deficient isolates were transformed with the gene coding for the K. pneumoniae porin OmpK36. It is concluded that the high level of resistance to cefoxitin and the increase in the MICs of meropenem, cefotaxime, and cefpirome for the ESBL-producing K. pneumoniae isolates studied are associated with porin deficiency.

Energy-dependent accumulation of fluoroquinolones in quinolone-resistant Klebsiella pneumoniae strains.

The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed.

Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36.

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks.

Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae.

The majority of Klebsiella pneumoniae serum-resistant strains activate complement and bind C3b, the opsonic fragment of C3, without C5b-9 formation and bacterial killing. The mechanisms leading to C3b deposition without cell death were studied, and the results indicate that serum-resistant strains activate principally the alternative pathway and that serum-sensitive strains activate both the alternative and classical pathways. Bacterial molecules implicated in C3b deposition are the outer membrane porin proteins and smooth and rough lipopolysaccharides. Porins activate both complement pathways, and the rough lipopolysaccharide activates the classical pathway, causing deposition of C3b in serum-sensitive strains. The smooth lipopolysaccharide of serum-resistant strains activates only the alternative pathway, impeding the binding of C1q to porins (S. Albertí, G. Marqués, S. Camprubí, S. Merino, J. M. Tomás, F. Vivanco, and V. J. Benedí, Infect. Immun. 61:852-860, 1993; S. Albertí, F. Rodríguez-Quinónes, T. Schirmer, G. Rummel, J. M. Tomás, J. P. Rosenbusch, and V. J. Benedí, Infect. Immun. 63:903-910, 1995) and rough lipopolysaccharide molecules and thereby preventing activation of the classical pathway. After its deposition, C3b is quickly degraded to iC3b on both types of strains, but the higher-level deposition of C3b on serum-sensitive strains, resulting from activation of both the alternative and classical complement pathways, supports further complement activation and killing of serum-sensitive strains.