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Differences in clinical manifestations are due to virulence factors expressed by the organism., We studied 455 consecutive episodes of Klebsiella pneumoniae bacteremia occurring in 7 countries. Community-acquired pneumonia and an invasive syndrome of liver abscess, meningitis, or endophthalmitis occurred only in Taiwan and South Africa. Infections by K1 and K2 capsular serotype, the mucoid phenotype, and aerobactin production were important determinants of virulence. The mucoid phenotype was seen in 94% of isolates in patients with community-acquired pneumonia and in 100% of isolates that caused the invasive syndrome in Taiwan and South Africa, compared with only 2% of isolates elsewhere. Mortality of mice injected with mucoid strains (69%) was strikingly higher than that occurring in mice injected with nonmucoid strains (3%, p

Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase ( uge ) renders a strain with the O − :K − phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics and this resistance is steadily rising. Quinolones are included in the group of antimicrobial agents to which this microorganism is developing resistance. Therefore, the aim of this study was to analyze the epidemiological relationship among 22 clinical isolates of S. maltophilia as well as the molecular mechanisms responsible for the acquisition of quinolone-resistance in these strains. The results of the pulsed-field gel electrophoresis (PFGE) showed an heterogenicity of 82% among the strains used in the study. On the other hand, no amino acid changes were found in the quinolone resistance-determining region (QRDR) of either gyrA and parC genes among quinolone-susceptible and -resistant S. maltophilia strains. Besides, the amino acid of the GyrA found in the position equivalent to Ser-83 of E. coli was Gln instead of a Ser or Thr, the amino acids usually encountered in this position among Gram-negative bacteria. The results suggest that there is not a relationship between the presence of this Gln and the resistance to quinolones in S. maltophilia. We can conclude that, contrary to what has been described in other microorganisms, in these S. maltophilia isolates, the development of resistance to quinolones was not related to mutations in the QRDR of gyrA and parC genes. Thus, to our knowledge, this is the first report describing this phenomenon.

A Klebsiella pneumoniae clinical isolate was resistant to cefoxitin, cefotaxime, ceftazidime, ceftazidime-clavulanate, piperacillin-tazobactam (MICs, >256 μg/ml in all cases), and meropenem (MIC, 16 μg/ml) and was intermediate to imipenem (MIC, 8 μg/ml). Decreased expression of the OmpK36 porin and expression of an SHV-2 β-lactamase contributed to the observed resistance to these β-lactam-containing agents.

We recently described the use of mini-Tn 5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae , we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50°C) and oxidative stress (H 2 O 2 ) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae .

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.

The activities of β-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 β-lactams were obtained in a porin-deficient mutant expressing increased β-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 cooperates with β-lactamase production to increase resistance to β-lactams., This work was funded by grants from Ministerio de Educación y Cultura and Junta de Andalucía, Spain.

Fifteen isolates of Klebsiella pneumoniae producing extended-spectrum β-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 ≥64 μg/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.

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.

The capsular polysaccharide (CPS) ofKlebsiella pneumoniae is an important virulence factor. Salicylate, which inhibits CPS production, was used to expose subcapsular antigens and components that may play an important role in host defense. Salicylate treatment greatly increased phagocytosis of five O1 serotypes by human polymorphonuclear leukocytes with normal rabbit serum and rabbit antisera against purified O1 lipopolysaccharide (O1LPS) as opsonins (p

FC3-11, a bacteriophage specific for the Klebsiella pneumoniae porin OmpK36, was isolated by its ability to infect Escherichia coli strains expressing the cloned OmpK36 porin. Porin OmpK36 was shown to be the receptor for phage FC3-11 by the observations that K. pneumoniae and E. coli strains that do not express OmpK36 were resistant to phage FC3-11, the purified porin inactivated the phage, and mutants selected for FC3-11 resistance had lost OmpK36. The outer membrane protein OmpK35 was isolated from a K. pneumoniae phage-resistant mutant by using porin isolation methods and was shown to contain an N-terminal sequence typical of enterobacterial porins. Bacteriophage FC3-11, alone or in combination with previously described lipopolysaccharide-specific phages, is a valuable tool to obtain OmpK36-porinless mutants.Key words: Klebsiella pneumoniae, porins, bacteriophage.

In a retrospective study at Son Dureta Hospital (Palma de Mallorca, Mallorca, Spain) of the period 1977-1991, 334 cases of culture-proven sepsis and/or meningitis in neonates born at the facility were identified. Overall, there was an incidence rate of 4.9 cases per 1,000 live births. The case-fatality rate was 7.5%. Infection was more frequent in infants of low birth weight, with the exception of infants with meningitis and infections due to group B Streptococcus and Listeria species. The patterns of predominance among bacterial pathogens that were isolated changed during the period studied. From 1977 to 1984, Klebsiella pneumoniae was the most frequent such isolate, but this frequency declined in the following years. Group B Streptococcus organisms and Staphylococcus epidermidis replaced K. pneumoniae as the predominant pathogens in early- and Staphylococcus epidermidis replaced K. pneumoniae as the predominant pathogens in early- and late-onset infections, respectively. The frequency with which other pathogens were isolated did not vary significantly during the study period. Invasive infection caused by Candida organisms was found in two patients. The incidence of infection due to group B streptococci has increased in the last few years (to 2.4 cases per 1,000 live births in 1991) and has become a significant problem that requires a thorough epidemiological evaluation.

Sir, The most relevant single mechanism of resistance of Enterobacteriaceae to cephalosporins is the production of -lactamases. For Klebsiella pneumoniae, these enzymes are usually plasmid-mediated extended-spectrum -lactamases (ESBLs) or, less commonly, plasmid-mediated AmpC-type enzymes. K. pneumoniae produces two major porins, outer membrane proteins (Omps) K35 and K36, which are homologous to Escherichia coli porins OmpF and OmpC, respectively. In klebsiellae, simultaneous loss of both OmpK35 and OmpK36 causes increased resistance to cephalosporins and other -lactams when ESBL or AmpCtype -lactamase is also produced. ESBLs do not confer resistance to cephamycins and carbapenems. Cephamycins, however, are able to select for mutants that are deficient in porins, resulting in subsequent resistance to these agents. Cefepime and cefpirome are poorly hydrolysed by AmpCtype -lactamases but have less activity against ESBLproducing strains, particularly in porin-deficient organisms. ESBL-producing K. pneumoniae are more frequently resistant to aminoglycosides and fluoroquinolones than K. pneumoniae strains not producing these -lactamases. The purpose of this study was to evaluate the activity of cefepime, cefpirome, third-generation cephalosporins, carbapenems, amikacin and ciprofloxacin against clinical isolates of ESBL-producing K. pneumoniae with different porin profiles. Sixty-five non-consecutive clinical isolates of ceftazidime-resistant K. pneumoniae isolated at the Microbiology Laboratory of the University Hospital V. Macarena (Seville, Spain) in the period 1994–1997 were studied. Strains were selected to include 15 isolates deficient in porins (see below). E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and K. pneumoniae LB4 (producing SHV-5) were also included as controls for antimicrobial susceptibility testing. K. pneumoniae C3 (producing OmpK35 and OmpK36) and E. coli K-12 mutants deficient in OmpA, OmpF or OmpC were used as reference strains in the determination of porin profiles. The activities of cefepime, cefpirome, cefotaxime, ceftazidime, cefoxitin, imipenem, meropenem, ciprofloxacin and amikacin were evaluated by a microdilution assay according to NCCLS guidelines. A 8-fold decrease in the activities of either ceftazidime or cefotaxime with clavulanic acid (2 mg/L) compared with ceftazidime or cefotaxime alone was taken as an indication of ESBL production. Omps were obtained after sonication of bacteria and sarkosyl treatment of cell membranes. Omps were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using 11% polyacrylamide in the running gel. Bacteria were categorized as porin producers (P ) if at least one protein band corresponding to either OmpK35 or OmpK36 was detected, and as porin-deficient (P ) if no major bands in the region of porins were observed. Fifty of the 65 clinical isolates of ESBL-producing K. pneumoniae produced at least one porin (mostly OmpK36). The remaining 15 strains were deficient in both OmpK35 and OmpK36. The proportion of isolates expressing or not expressing porins does not reflect their actual frequency of isolation among clinical isolates, as K. pneumoniae strains deficient in both porins are much less common than would be predicted from these figures. The activities of nine antimicrobial agents against P and P strains are presented in the Table. The activity of cefepime and cefpirome against both P and P strains is higher than that of cefotaxime and ceftazidime. Nevertheless, and according to the NCCLS recommendations, ESBL-producing K. pneumoniae are considered resistant to all cephalosporins, independently of the in vitro activity of the different compounds within this group. The MIC50s and MIC90s of cefoxitin against P and P strains indicate that loss of porins is critical for resistance to this cephamycin in ESBL-producing K. pneumoniae. Porin loss contributes to increasing the level of resistance to the other cephalosporins in ESBL-producing strains. Both imipenem and meropenem were the most active

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., This work was supported by grants from the Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT) and project FEDER 2FD97- 0287. D.A. was supported by a predoctoral fellowship from CICYT.

We have studied the usefulness of commercially available silica gel-containing tubes (serum separator tubes) for lipopolysaccharide extraction. Bacterial lipopolysaccharides from different bacterial species have been extracted in regular test tubes and tubes containing silica gel. They were characterized by chemical analysts, gel electrophoresis and enzyme linked immunosorbent assay, and one of their biological activities has been assayed. They were found to be indistinguishable, except that higher lipopolysaccharide yields were obtained when the extraction was carried out in silica gel-containing tubes. The use of these tubes provides a safe and convenient alternative to standard lipopolysaccharide extraction.

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.

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.

OmpK35 from Klebsiella pneumoniae is the homologue of Escherichia coli OmpF porin. Expression of OmpK35 in K. pneumoniae strain CSUB10R (lacking both OmpK35 and OmpK36) decreased the MICs of cephalosporins and meropenem ≥128-fold and decreased the MICs of imipenem, ciprofloxacin, and chloramphenicol ≥8-fold. MIC reductions by OmpK35 were 4 times (cefepime), 8 times (cefotetan, cefotaxime, and cefpirome), or 128 times (ceftazidime) higher than those caused by OmpK36, but the MICs were similar or 1 dilution lower for other evaluated agents.

The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum β-lactamase (ESBL) production were determined for 53 clinical isolates of Klebsiella pneumoniae. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a ≥50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both gyrA and parC from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (P < 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (P < 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (P > 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (P < 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to fluoroquinolone resistance in K. pneumoniae was negligible. It is concluded that among K. pneumoniae strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications., This work was supported by the Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo of Spain, grants 00/0242 (to L.M.-M.) and 01/0034 (to V.J.B.). A.D.-S. was supported by a predoctoral fellowship from the Ministerio de Educación y Cultura of the Spanish government

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 β-lactamase and expressed extended-spectrum β-lactamases (ESBLs). Group II only hyperproduced Bush type 1 chromosomal β-lactamase and correlated with PFGE-types D1, D2, D3 and I. Finally, Group III, with inducible β-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. © 2001 The Hospital Infection Society., This work was supported by grants from Fondo de Investigaciones Sanitarias (FIS) and Comisión Interministerial de Ciencia y Tecnología (CICYT).

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

We cloned and sequenced the structural gene forAeromonas hydrophilaporin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that ofompFinEscherichia coli, is adjacent toaspCand transcribed in the same direction. However, upstream of the porin II gene no similarities withE. coliwere found. We obtained defined insertion mutants in porin II gene either inA. hydrophila(O:34) orA. 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.

Two clinical isolates of extended-spectrum β-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 β-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC β-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with β-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional ompK36 gene on a plasmid. Plasmids encoding AmpC-type β-lactamases provided resistance to imipenem (up to 64 μg/ml) and meropenem (up to 16 μg/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-β-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 β-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 μg/ml, increasing to 16 μg/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-β-lactams and carbapenems.

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many β-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 β-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 β-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to β-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

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.

We have developed a series of vectors for easy construction of translational fusions with the phoA gene (encoding the periplasmic alkaline phosphatase, PhoA) in the three reading frames. One plasmid series carries a multiple cloning site (MCS) followed by a promoterless and leaderless 5'-truncated phoA ('phoA), which in turn is followed by a kanamycin-resistance-encoding gene (kan). Another plasmid series contains two identical inverted MCS flanking the phoA-kan cluster. These latter vectors are devised as phoA-kan cassette delivery vectors. In-frame cloning results in the production of hybrid PhoA proteins which display PhoA activity if successfully exported beyond the cytoplasmic membrane. In order to test these vectors, we have constructed hybrid PhoE::PhoA proteins, which were used to analyze the activity of the phoE promoter and identify the hybrid gene products.

Early onset infection in neonates caused by group B Streptococcus and the prevalence of colonization by this microorganism in a group of 1,003 pregnant women and their neonates were studied. The capsular serotypes of the colonizing and infecting bacterial isolates and the components of C protein expressed were determined. Except for serotype IV, all the currently recognized capsular serotypes (including serotype V) were found. Among the colonizing strains, serotypes III, Ia, and Ib were almost equally represented and accounted for 82% of the isolates. Serotype III was predominantly found in cases of infection (55%), and serotypes Ia and V were also found in such cases. C protein was not detected in serotype III isolates, but alpha and/or beta components of this protein were found in isolates of all other serotypes. Neonatal infection and colonization due to this microorganism were observed in 1.2 cases per 1,000 live births and in 7.1% of pregnant women. Host factors for infection, such as low birth weight, premature delivery, and prolonged rupture of membranes, were uncommon. The level of specific antibodies to serotype III was also quantitated; lower levels were found in infected children and their mothers than in healthy infants and pregnant women. Further similar studies in Spain are necessary so that preventive measures can be planned.

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage. Key words: Klebsiella pneumoniae, bacteriophages, isolation, environmental conditions.

FC3-2, FC3-3 and FC3-6 are different Klebsiella-specific bacteriophages isolated on Klebsiella pneumoniae strain C3. The common bacteriophage receptor for these phages was shown to be the lipopolysaccharide (LPS), specifically the high-molecular-weight polysaccharide fraction (O-antigen). Mutants resistant to these phages were isolated and found to be devoid of lipopolysaccharide O-antigen by several criteria. We concluded that no other outer membrane (OM) molecules were involved in phage binding. At least 2 different types of lipopolysaccharide-core mutant were obtained.

High-molecular weight lipopolysaccharide (O antigen enriched fraction) from Klebsiella pneumoniae was determined to be the receptor for bacteriophage FC3-1. A methodology for the identification of the lipopolysaccharide component involved in FC3-1 bacteriophage reception was used that is suitable for other phages and host bacteria.

Bacteriophage FC3-9 is one of the several bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Mutants resistant to this bacteriophage were isolated and found to be devoid of lipopolysaccharide O antigen, modified in outer-membrane protein composition and sensitive to complement killing (serum-sensitive), unlike the wild-type strain. Serum-resistant mutants were isolated from these strains. They regained the lipopolysaccharide O antigen and the wild-type outer membrane protein composition and became sensitive to bacteriophage FC3-9.

SUMMARY: The role of lipopolysaccharide (LPS) in the susceptibility of Escherichia coli and Salmonella typhimurium to non-immune human serum was investigated using serum-sensitive strains of both enterobacteria. LPS from serum-resistant strains of E. coli and S. typhimurium could activate and completely remove the serum bactericidal activity, and also showed dose-dependent anti-complement activity. These properties were mainly due to the high-molecular-mass LPS: the low-molecular-mass LPS from serum-resistant strains of E. coli and S. typhimurium had only a slight effect on the serum bactericidal activity, and showed only low anti-complement activity, even at high concentration. The results suggest that LPS composition, especially the O-antigen polysaccharide chains, contributes to the susceptibility of E. coli and S. typhimurium strains to complement-mediated serum bactericidal activity.

The different mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes K1, K10, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed.

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.

Two porins, OmpK36 and OmpK35, have been described previously in Klebsiella pneumoniae, and they are homologous to the Escherichia coli porins OmpC and OmpF, respectively, at both the DNA and amino acid levels. Optimal resolution of the two K. pneumoniae porins by electrophoresis on polyacrylamide gels is not achieved using gel systems already described for E. coli and requires modifications of the bisacrylamide content of the resolving gels. Once resolved, identification of porins OmpK36 and OmpK35 cannot be based solely on their apparent molecular masses since in some strains the OmpK36 porin migrates faster than the OmpK35 porin, whilst in other strains OmpK35 is the faster-migrating porin. Expression of OmpK35 porin is increased in low-osmolarity medium and, combined with Western blot analysis, this allows for the identification of both porins. Application of this identification system showed that most isolates lacking expression of extended-spectrum /?-lactamases express the two porins, whereas most isolates producing these /?-lactamases express only porin OmpK36, and the OmpK35 porin is either very low or not expressed, This wo rk was supported by grants from Comision Inter- ministerial de Ciencia y Tecnologi'a (CICYT). S.H. -A., D.A., and S.A. were supported by fellowships from CICYT, a n d A.D.-S. was supported by a predoctoral fellowship from CSIC-CAROB SA.

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. pneumoniaeclinical 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., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnologı́a (CICYT) of the Spanish Government. S.H.A., S.A., and D.A. were supported by predoctoral fellowships/postdoctoral contracts from CICYT.