bluuebunny/crossref_metadata_2025 huggingface dataset:
Virulence Factors of
Escherichia coli
Isolates That Produce CTX-M-Type Extended-Spectrum β-Lactamases
Authors: Pitout Johann D. D. , Laupland Kevin B. , Church Deirdre L. , Menard Megan L. , Johnson James R.
Published: October 2005

This study determined the phylogenetic groups and virulence factors of 37
Escherichia coli
isolates producing types of CTX-M compared with those of 19 isolates producing different types of extended-spectrum β-lactamases (ESBLs) in a well-defined North American population. Most CTX-M-14 producers (97%) were from phylogenic group D; 67% of the CTX-M-15 producers were from group B2. A single CTX-M-14-producing strain belonged to clonal group A. There were significant prevalence differences for individual virulence factors among CTX-M producers and nonproducers; however, aggregate virulence factor scores were similar. CTX-M producers more commonly caused repeat urinary tract infections. Our results indicate that CTX-M type predicts phylogenetic background, and the virulence potential of ESBL-producing
E. coli
isolates is a complex issue, requiring further study and ongoing surveillance.
Organotypic Epithelial Raft Cultures as a Model for Evaluating Compounds against Alphaherpesviruses
Authors: Andrei Graciela , van den Oord Joost , Fiten Pierre , Opdenakker Ghislain , De Wolf-Peeters Chris , De Clercq Erik , Snoeck Robert
Published: October 2005

The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK
+
and TK
−
strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.
DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic
Escherichia coli
Transmissible R Plasmid
Authors: Johnson Timothy J. , Siek Kylie E. , Johnson Sara J. , Nolan Lisa K.
Published: October 2005

In this study, a 101-kb IncF plasmid from an avian pathogenic
Escherichia coli
(APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the
E. coli
F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal
E. coli
strain from an apparently healthy bird, a
Salmonella enterica
serovar Typhimurium strain, and a uropathogenic
E. coli
strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.
Mouse Adenovirus Type 1 Infection in SCID Mice: an Experimental Model for Antiviral Therapy of Systemic Adenovirus Infections
Authors: Lenaerts L. , Verbeken E. , De Clercq E. , Naesens L.
Published: October 2005

The importance of human adenovirus infections in immunocompromised patients urges for new and adequate antiadenovirus compounds. Since human adenoviruses are species specific, animal models for systemic adenovirus infections rely on a nonhuman adenovirus. We established mouse adenovirus type 1 (MAV-1) infection of BALB/c SCID mice as a model for the evaluation of antiadenovirus therapy. In vitro studies with mouse embryonic fibroblasts pointed to the acyclic nucleoside phosphonate cidofovir and the N-7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242) as markedly active compounds against MAV-1. SCID mice, infected intranasally with MAV-1, developed a fatal disseminated infection after approximately 19 days, characterized by hemorrhagic enteritis. Several techniques were optimized to monitor viral, immunological, and pathological aspects of MAV-1 infection. Real-time PCR quantification of viral DNA revealed that after replication in the lungs, virus disseminated to several organs, including the brain, liver, spleen, intestine, heart, and kidneys (resulting in viruria). Immunohistochemical staining showed that MAV-1 was localized in the endothelial cells of the affected organs. Using reverse transcription-PCR, tissue levels of proinflammatory cytokines (i.e., interleukin-1β and tumor necrosis factor alpha) were found to be markedly increased. The MAV-1/SCID model appears to be an appropriate model for in vivo evaluation of antiadenovirus agents. Treatment with cidofovir or S-2242 at a dose of 100 mg per kg of body weight resulted in a significant delay in MAV-1-related death, although these antivirals were unable to completely suppress virus replication despite continued drug treatment. These findings suggest that complete virus clearance during antiviral therapy for disseminated adenovirus infection may require an efficient adaptive immune response from the host.
Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency
Authors: Mejías Asunción , Chávez-Bueno Susana , Ríos Ana María , Aten Mónica Fonseca , Raynor Brett , Peromingo Estrella , Soni Perla , Olsen Kurt D. , Kiener Peter A. , Gómez Ana María , Jafri Hasan S. , Ramilo Octavio
Published: October 2005

Respiratory syncytial virus (RSV) is the leading viral pathogen responsible for bronchiolitis and pneumonia in infants and young children worldwide. We have previously shown in the mouse model that treatment with an anti-RSV neutralizing monoclonal antibody (MAb) against the F glycoprotein of RSV, palivizumab, decreased lung inflammation, airway obstruction, and postmethacholine airway hyperresponsiveness. MEDI-524, or Numax, is a new MAb derived from palivizumab with enhanced neutralizing activity against RSV. We compared the effects of these two MAbs on different markers of disease severity using the murine model of RSV infection. BALB/c mice were intranasally inoculated with RSV A2. Palivizumab or MEDI-524 was administered once at either 24 h before or 48 h after RSV inoculation. Regardless of the time of administration, all treated mice showed significantly decreased RSV loads in bronchoalveolar lavage samples measured by plaque assay. Only MEDI-524 given at −24 h significantly decreased lung RSV RNA loads on days 5 and 28 after RSV inoculation. Pulmonary histopathologic scores, airway obstruction, and postmethacholine airway hyperresponsiveness were significantly reduced in mice treated with MEDI-524 at 24 h before inoculation, compared with untreated controls and the other regimens evaluated. MEDI-524 was superior to palivizumab on several outcome variables of RSV disease assessed in the mouse model: viral replication, inflammatory and clinical markers of acute disease severity, and long-term pulmonary abnormalities.
Molecular Analysis of the Simultaneous Production of Two SHV-Type Extended-Spectrum Beta-Lactamases in a Clinical Isolate of
Enterobacter cloacae
by Using Single-Nucleotide Polymorphism Genotyping
Authors: Szabó Dóra , Melan Melissa A. , Hujer Andrea M. , Bonomo Robert A. , Hujer Kristine M. , Bethel Christopher R. , Kristóf Katalin , Paterson David L.
Published: October 2005

Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an
Enterobacter cloacae
isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of
bla
SHV
gene products gave a “double peak” at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different
bla
SHV
genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.
Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity
Authors: Dorr Patrick , Westby Mike , Dobbs Susan , Griffin Paul , Irvine Becky , Macartney Malcolm , Mori Julie , Rickett Graham , Smith-Burchnell Caroline , Napier Carolyn , Webster Rob , Armour Duncan , Price David , Stammen Blanda , Wood Anthony , Perros Manos
Published: October 2005

Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.
Molecular Mechanisms of β-Lactam Resistance Mediated by AmpC Hyperproduction inPseudomonas aeruginosaClinical Strains
Authors: Juan Carlos , Maciá María D. , Gutiérrez Olivia , Vidal Carmen , Pérez José L. , Oliver Antonio
Published: October 2005

The molecular mechanisms of β-lactam resistance mediated by AmpC hyperproduction in natural strains ofPseudomonas aeruginosawere investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with β-lactams. All 10 ceftazidime-resistant mutants hyperproduced AmpC (β-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them harbored mutations inampRor theampC-ampRintergenic region. On the other hand, six of them harbored inactivating mutations inampD: four contained frameshift mutations, one had a C→T mutation, creating a premature stop codon, and finally, one had a large deletion, including the completeampDEregion. Complementation studies revealed that only three of the sixampDmutants could be fully transcomplemented with eitherampD- orampDE-harboring plasmids, whereas one of them could be transcomplemented only withampDEand two of them (including the mutant with the deletion of theampDEregion and one with anampDframeshift mutation leading to anampDE-fused open reading frame) could not be fully transcomplemented with any of the plasmids. Finally, one of the four mutants with no mutations inampDcould be transcomplemented, but only withampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to theampDEoperon.
Spontaneous Conversion to Quinolone and Fluoroquinolone Resistance among Wild-Type
Escherichia coli
Isolates in Relation to Phylogenetic Background and Virulence Genotype
Authors: Johnson James R. , Johnston Brian , Kuskowski Michael A. , Colodner Raul , Raz Raul
Published: October 2005

Human clinical isolates of
Escherichia coli
that are resistant to quinolone or fluoroquinolone agents typically exhibit fewer extraintestinal virulence factors (VFs) than susceptible isolates, along with a different phylogenetic background. To experimentally assess the basis for this as-yet-unexplained phenomenon, 40
E. coli
strains (20
E. coli
Reference collection members and 20 Israeli cystitis isolates) were subjected to serial selective passaging to obtain derivatives resistant to nalidixic acid (NA) and ciprofloxacin (C). PCR-based VF profiling and phylotyping were performed on the parents and their respective resistant derivatives. All 40 susceptible parent strains yielded NA- and C-resistant derivatives after a median of 6 (range, 4 to 12) serial selective passages on agar plates containing increasing concentrations of NA and C. The numbers of passages required for resistance did not differ by collection origin, phylogenetic group, basal VF profile, source (urine versus fecal), or host group (human versus animal). With the development of C resistance, only one VF was lost in a single strain. Resistant derivatives exhibited the same phylotype as their susceptible parents. These findings suggest that the sparse VF profiles and the low-virulence phylogenetic background of NA- and C-resistant
E. coli
clinical isolates probably are not attributable to the loss of VFs from intrinsically high-virulence strains during conversion to resistance or to enhanced emergence of drug resistance among intrinsically low-virulence strains. A more likely explanation is the importation of resistant strains from an as-yet-undefined low-virulence external selection reservoir.
CTX-M-2 and a New CTX-M-39 Enzyme Are the Major Extended-Spectrum Beta-Lactamases in Multiple
Escherichia coli
Clones Isolated in Tel Aviv, Israel
Authors: Chmelnitsky Inna , Carmeli Yehuda , Leavitt Azita , Schwaber Mitchell J. , Navon-Venezia Shiri
Published: October 2005

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among
Escherichia coli
isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of
E. coli
ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing
E. coli
clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was
bla
CTX-M
; 16 isolates carried
bla
CTX-M-2
and three carried a new ESBL gene designated
bla
CTX-M-39
. Three strains carried
bla
SHV
(two
bla
SHV-12
and one
bla
SHV-5
), and two strains carried inhibitor-resistant ESBL genes,
bla
TEM-33
and
bla
TEM-30
; 18 strains carried
bla
TEM-1
and eight strains carried
bla
OXA-2
. Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that
bla
CTX-M-2
is plasmid borne. The wide dissemination of ESBLs among
E. coli
isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated
bla
CTX-M-39
, which revealed 99% homology with
bla
CTX-M-26
, with a substitution of arginine for glutamine at position 225.
Increased Mutation Frequencies in
Escherichia coli
Isolates Harboring Extended-Spectrum β-Lactamases
Authors: Baquero María-Rosario , Galán Juan Carlos , del Carmen Turrientes María , Cantón Rafael , Coque Teresa M. , Martínez José Luis , Baquero Fernando
Published: October 2005

Hypermutable (mutation frequency [
f
], ≥4 × 10
−8
)
Escherichia coli
strains were more frequently found (43%) in a collection of 89 extended-spectrum β-lactamase (ESBL)-producing isolates from different patients (77 pulsed-field gel electrophoresis clones, 12 ESBL types) than in non-ESBL
E. coli
(26%) strains (
P
= 0.03). Among urinary tract isolates, the frequency of hypermutation was 40% in ESBL versus 26% in non-ESBL isolates (
P
= 0.03).
Kinetics Study of KPC-3, a Plasmid-Encoded Class A Carbapenem-Hydrolyzing β-Lactamase
Authors: Alba Jimena , Ishii Yoshikazu , Thomson Kenneth , Moland Ellen Smith , Yamaguchi Keizo
Published: October 2005

The kinetic activity of KPC-3, a plasmid-encoded class A carbapenemase, was studied. It hydrolyzed penicillins, cephalosporins, carbapenems, and even sulbactam. The best substrate was cephalothin (
k

cat
/K

m
= 3.48 μM
−1
s
−1
). The efficiency of the enzyme was similar for imipenem and meropenem (
k
cat
/

K
m

, 1.4 and 1.94 μM
−1
s
−1
, respectively).
Recombination between the
dfrA12
-orfF-
aadA2
Cassette Array and an
aadA1
Gene Cassette Creates a Hybrid Cassette,
aadA8b
Authors: Gestal Alicia M. , Stokes H. W. , Partridge Sally R. , Hall Ruth M.
Published: October 2005

Homologous recombination between closely related gene cassettes, such as
aadA1
and
aadA2
, which are 89% identical, can create hybrid cassettes and hybrids of existing cassette arrays. A new cassette array,
dfrA12
-orfF-
aadA8b
, which was created by such a recombination event occurring within the
aadA2
cassette in the
dfrA12
-orfF-
aadA2
array, has been identified.
Use of an Efflux-Deficient
Streptococcus pneumoniae
Strain Panel To Identify ABC-Class Multidrug Transporters Involved in Intrinsic Resistance to Antimicrobial Agents
Authors: Robertson Gregory T. , Doyle Timothy B. , Lynch A. Simon
Published: October 2005

Thirteen derivatives of the
Streptococcus pneumoniae
TIGR4 strain in which putative drug efflux pumps were genetically inactivated were constructed and characterized. The results indicate that two linked genes encoding the ABC-type transporters SP2073 and SP2075 function together to confer intrinsic resistance to a series of structurally unrelated compounds, including certain fluoroquinolones.
High Prevalence of Glycopeptide Resistance Genes
vanB
,
vanD
, and
vanG
Not Associated with Enterococci in Human Fecal Flora
Authors: Domingo M.-C. , Huletsky A. , Giroux R. , Boissinot K. , Picard F. J. , Lebel P. , Ferraro M. J. , Bergeron M. G.
Published: October 2005

The presence of
Enterococcus
-associated vancomycin resistance genes
vanA
,
vanB
,
vanD
,
vanE
, and
vanG
in rectal swabs was investigated in two hospitals using PCR. All
vanA
genes detected were associated with vancomycin-resistant enterococci (VRE), whereas VRE-associated
vanB
genes were detected in only one hospital (4.7%). However, in both hospitals, high prevalences of
vanB
(6.2 and 2.3%),
vanD
(43.8 and 26.7%), and
vanG
(10.5 and 6.9%) genes not associated with enterococci were found.
Efficacy of Penicillin versus Cefdinir in Eradication of Group A Streptococci and Tonsillar Flora
Authors: Brook Itzhak , Foote Perry A.
Published: October 2005

Core tonsillar cultures were obtained from 40 children with recurrent tonsillitis treated with either penicillin or cefdinir. Group A beta-hemolytic streptococci were isolated from 11 penicillin- and 3 cefdinir-treated (
P
< 0.001) patients. β-Lactamase producers were recovered from 17 penicillin- and 3 cefdinir-treated (
P
< 0.01) patients. Inhibiting alpha-hemolytic streptococci were isolated less often from penicillin-treated patients than from cefdinir-treated patients.
Therapeutic Effects of Bacteriophage Cpl-1 Lysin against
Streptococcus pneumoniae
Endocarditis in Rats
Authors: Entenza J. M. , Loeffler J. M. , Grandgirard D. , Fischetti V. A. , Moreillon P.
Published: October 2005

Cpl-1, a pneumococcal phage lytic enzyme, was tested in rats with experimental endocarditis due to
Streptococcus pneumoniae
WB4. High-dose regimen Cpl-1 eliminated pneumococci from blood within 30 min and decreased bacterial titers in vegetations (>4 log
10
CFU/g) within 2 h. Rapid bacterial lysis induced by Cpl-1 treatment increased cytokine secretion noticeably.
Recovery of Interfering Bacteria in the Nasopharynx following Antimicrobial Therapy of Acute Maxillary Sinusitis with Telithromycin or Amoxicillin-Clavulanate
Authors: Brook Itzhak , Hausfeld Jeffrey N.
Published: October 2005

The effect on the nasopharyngeal flora of 7 days of amoxicillin-clavulanate or 5 days of 800 mg once a day was studied in 50 adults with acute sinusitis. The numbers of potential pathogens and interfering alpha-hemolytic streptococci were equally reduced after both therapies. However, the number of interfering
Prevotella
spp. declined more significantly after amoxicillin-clavulanate treatment.
Effectiveness of Anidulafungin in Eradicating
Candida
Species in Invasive Candidiasis
Authors: Pfaller M. A. , Diekema D. J. , Boyken L. , Messer S. A. , Tendolkar S. , Hollis R. J. , Goldstein B. P.
Published: October 2005

In a phase 2 open-label, dose-ranging study in patients with candidemia, anidulafungin was effective in eradicating
Candida albicans
and other species of
Candida
. The anidulafungin MIC distribution showed that
Candida albicans
and
C. glabrata
were the most susceptible species and
C. parapsilosis
was the least susceptible species.
Multiplex PCR for Simultaneous Detection of Macrolide and Tetracycline Resistance Determinants in Streptococci
Authors: Malhotra-Kumar Surbhi , Lammens Christine , Piessens Jasper , Goossens Herman
Published: October 2005

Resistance to macrolides and tetracyclines is increasing among streptococci and co-occurs as their resistance determinants are carried on the same mobile element. We developed a multiplex PCR to facilitate simultaneous and specific detection of resistance determinants for both macrolides [
erm
(A),
erm
(B), and
mef
(A/E)] and tetracyclines [
tet
(M),
tet
(O),
tet
(K), and
tet
(L)] in streptococci.
New
Mycobacterium avium
Antifolate Shows Synergistic Effect when Used in Combination with Dihydropteroate Synthase Inhibitors
Authors: Suling W. J. , Seitz L. E. , Reynolds R. C. , Barrow W. W.
Published: October 2005

Mycobacterium avium
complex (MAC) is resistant to trimethoprim, an inhibitor of bacterial dihydrofolate reductase (DHFR). A previously identified selective inhibitor of MAC DHFR, SRI-8858, was shown to have synergistic activity in combination with dapsone and sulfamethoxazole, two drugs that inhibit bacterial dihydropteroate synthase.
Shiga Toxin 1 Causes Direct Renal Injury in Rats
Authors: Yamamoto Elise T. , Mizuno Masashi , Nishikawa Kiyotaka , Miyazawa Shinobu , Zhang Lianshan , Matsuo Seiichi , Natori Yasuhiro
Published: October 2005

Infection with Shiga toxin (Stx)-producing
Escherichia coli
has been implicated to cause hemolytic uremic syndrome, which is characterized by histological abnormalities such as microvascular thrombi and tubular cell damage in the kidney. Although Stx is known to be the major virulence factor of the pathogen, it is still unclear whether Stx directly impairs renal cells in vivo to cause such histological changes and deterioration of renal function. To assess the consequence of the direct action of Stx on renal cells, left kidneys of rats were perfused with Stx1 from the renal artery through the renal vein and then revascularized. Kidneys of control animals were perfused with the vehicle alone. On day 1, apoptosis and induction of tumor necrosis factor alpha gene expression were noticed to occur in the medulla of the Stx1-perfused kidneys. On day 3, extensive tubular injuries were observed by light microscopy: aggregated platelets and monocytic infiltrates in both glomeruli and the medullary interstitium were detected by immunostaining. Tubular changes were more extensive on day 9, with areas of infarction seen in the cortex and medulla. These changes were not found to occur in the sham-operated kidneys. No obvious glomerular changes were detected by light microscopy at any time point. When nonperfused right kidneys were removed after the Stx1 perfusion of the left kidneys, the serum creatinine and blood urea nitrogen levels were increased from day 2, and acute renal failure followed on day 3. These results indicate that Stx1 caused glomerular platelet aggregation, tubular damage, and acute deterioration of renal function by acting directly on renal cells.
Disruption of Tight Junctions during Traversal of the Respiratory Epithelium by
Burkholderia cenocepacia
Authors: Kim Jason Y. , Sajjan Umadevi S. , Krasan Graham P. , LiPuma John J.
Published: October 2005

Burkholderia cenocepacia
is an opportunistic bacterial species capable of causing life-threatening respiratory tract infection in persons with cystic fibrosis (CF). Unlike most other pathogens in CF, which typically remain confined to the endobronchial spaces,
B. cenocepacia
can traverse airway epithelium to cause bacteremia and sepsis. The mechanisms by which this occurs, however, are unknown. We examined the transmigration of
B. cenocepacia
through polarized respiratory epithelium. Representatives of three “epidemic” lineages common among CF patients in North America were able to traverse polarized 16HBE14o
−
cells in vitro. Transmigration of bacteria was associated with significant perturbations in epithelial permeability, as measured by a loss of transepithelial electrical resistance and increased flux of bovine serum albumin across the cell layer. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and trypan blue exclusion assays, as well as lactate dehydrogenase levels, did not indicate excessive cytotoxicity or cell death in infected cell layers. Rather, confocal fluorescence microscopy demonstrated the loss of occludin from tight junctions. In contrast, zonula occludens 1 was well preserved along intercellular borders. Western blot analysis showed a shift in the major occludin isoforms from high- to low-phosphorylation states during infection. These observations suggest that
B. cenocepacia
traverses polarized respiratory epithelium by the dephosphorylation and dissociation of occludin from the tight-junction complex.
Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing
Escherichia coli
Authors: Riff Jason D. , Callahan John W. , Sherman Philip M.
Published: October 2005

The diarrheal pathogens enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 strain CL56 and enteropathogenic
Escherichia coli
(EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced α-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing
Escherichia coli
. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.
Demonstration of Factor H-Like Protein 1 Binding toTreponema denticola, a Pathogen Associated with Periodontal Disease in Humans
Authors: McDowell John V. , Lankford Justin , Stamm Lola , Sadlon Tania , Gordon David L. , Marconi Richard T.
Published: October 2005

Treponema denticolais an important contributor to periodontal disease. In this study we investigated the ability ofT. denticolato bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate thatT. denticolaspecifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface ofT. denticolato serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding byT. denticolamight be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.
Pathogenesis ofYersinia pestisInfection in BALB/c Mice: Effects on Host Macrophages and Neutrophils
Authors: Lukaszewski Roman A. , Kenny Dermot J. , Taylor Rosa , Rees D. G. Cerys , Hartley M. Gill , Oyston Petra C. F.
Published: October 2005

The pathogenesis of infection withYersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+macrophages and Ly-6G+neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103to 104CFU per spleen. Flow cytometric analysis revealed that there was even distribution ofY. pestiswithin both CD11b+macrophage and Ly-6G+neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages ofY. pestisinfection, splenic neutrophils are responsible for limiting the growth ofY. pestisand that splenic macrophages provide safe intracellular shelters within whichY. pestisis able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.
Human Airway Epithelial Cells SensePseudomonas aeruginosaInfection via Recognition of Flagellin by Toll-Like Receptor 5
Authors: Zhang Zhe , Louboutin Jean-Pierre , Weiner Daniel J. , Goldberg Joanna B. , Wilson James M.
Published: October 2005

Pseudomonas aeruginosa, an opportunistic respiratory pathogen that infects the majority of patients with cystic fibrosis, initiates host inflammatory responses through interaction with airway epithelial cells. The Toll-like receptors (TLRs) are a family of pathogen pattern recognition receptors that play key roles in host innate immunity. In this study we aimed to determine whether TLRs mediate the interaction betweenP. aeruginosaand airway epithelial cells. Individual murine TLRs (TLR1 to TLR9) and dual combinations of these TLRs that activate an NF-κB-driven luciferase reporter in response to PAO1 were screened in HEK 293 cells. TLR5, TLR2, a combination of TLR1 and TLR2, or a combination of TLR2 and TLR6 responded to PAO1. AnotherP. aeruginosastrain, strain PAK, activated TLR5 similarly, while the isogenic flagellin-deficient strain PAK/fliCand the flagellum-free bacteriumHaemophilus influenzaefailed to activate TLR5. Reverse transcription-PCR was used to probe the presence of multiple TLRs (including TLR5) in primary human airway epithelial cells (HAECs). Immunostaining with TLR5 antibodies showed that TLR5 was expressed in HAECs and on the apical surface of the human trachea epithelium. In HAECs, PAO1, PAK, andBurkholderia cepacia, but not flagellin-deficient strain PAK/fliCor aB. cepacia fliCmutant, activated the NF-κB reporter. Dominant negative TLR5 specifically blocked the response toP. aeruginosabut not to the response to lipoteichoic acid, a specific ligand of TLR2. We also determined that MyD88, IRAK, TRAF6, and Toll-interacting protein (Tollip), but not TIRAP, were involved in the TLR-mediated response toP. aeruginosain HAECs. These findings demonstrate that the airway epithelial receptor TLR5 sensesP. aeruginosathrough its flagellin protein, which may have an important role in the initiation of the host inflammatory reaction to clear the invading pathogen.
The Role of
Candida albicans NOT5
in Virulence Depends upon Diverse Host Factors In Vivo
Authors: Cheng Shaoji , Clancy Cornelius J. , Checkley Mary Ann , Zhang Zongde , Wozniak Karen L. , Seshan Kalpathi R. , Jia Hong Yan , Fidel Paul , Cole Garry , Nguyen M. Hong
Published: October 2005

We previously identified
Candida albicans
Not5p as an immunogenic protein expressed during oropharyngeal candidiasis (OPC). In this study, we demonstrate that
C. albicans NOT5
reverses the growth defects of a
Saccharomyces cerevisiae not5
mutant strain at 37°C, suggesting that the genes share at least some functional equivalence. We implicate
C. albicans NOT5
in the pathogenesis of disseminated candidiasis (DC) induced by intravenous infection among neutropenic and nonimmunosuppressed mice, as well as in that of OPC in mice immunosuppressed with corticosteroids. We find no role in virulence, however, among neutropenic and corticosteroid-suppressed mice with DC resulting from gastrointestinal translocation, nor do we implicate the gene in vulvovaginal candidiasis among mice in pseudoestrus. These findings suggest that the role of
NOT5
in virulence depends on the specific in vivo environment and is influenced by diverse factors such as tissue site, portal of entry, and the status of host defenses.
NOT5
is necessary for normal adherence to colonic and cervical epithelial cells in vitro, demonstrating that such assays cannot fully replicate disease processes in vivo. Lastly, antibody responses against Not5p do not differ in the sera of patients with OPC, patients with DC, and healthy controls, suggesting that the protein is associated with both commensalism and the pathogenesis of disease.
Immunosuppressive Effects of CCL17 on Pulmonary Antifungal Responses during Pulmonary Invasive Aspergillosis
Authors: Carpenter Kristin J. , Hogaboam Cory M.
Published: October 2005

Aspergillus fumigatus-sensitized CCR4-deficient (CCR4−/−) mice exhibit an accelerated clearance of conidia during fungal asthma. In the present study, we examined the roles of CCL17 and CCL22, two CCR4 ligands, during pulmonary invasive aspergillosis in neutropenic mice. Kaplan-Meier survival curve analysis revealed that wild-type C57BL/6 (CCR4+/+) mice were significantly protected from the lethal effects ofAspergilluscompared with their wild-type controls following systemic neutralization with anti-CCL17 but not anti-CCL22 antibodies. Systemic neutralization of CCL17 significantly increased whole-lung CCL2 levels. Mouse survival and histological analysis revealed that the receptor mediating the deleterious effects of CCL17 was CCR4 since mice genetically deficit in CCR4 (CCR4−/−) did not develop invasive aspergillosis. Enzyme-linked immunosorbent assay analysis of whole-lung samples at day 2 after conidial challenge in neutrophil-depleted CCR4−/−and CCR4+/+mice revealed that whole-lung IL-12 levels were significantly increased in the CCR4−/−group compared with the wild-type group. Also at day 2 after conidial challenge, significantly greater numbers of CD11c+F4/80+and CD11c+/CD86+but fewer CD3/NK1.1+cells were present in the lungs of CCR4−/−mice compared with their wild-type counterparts. Thus, CCL17-CCR4 interactions dramatically impair the pulmonary antifungal response againstA. fumigatusin neutropenic mice.
Association of Linear Plasmid 28-1 with an Arthritic Phenotype ofBorrelia burgdorferi
Authors: Xu Qilong , Seemanapalli Sunita V. , Lomax Larry , McShan Kristy , Li Xin , Fikrig Erol , Liang Fang Ting
Published: October 2005

Borrelia burgdorferi, the Lyme disease spirochete, has a genome comprised of a linear chromosome and up to 21 plasmids. Loss of plasmids is associated with decreased infectivity and pathogenicity. Sixteen transformants were generated by transforming the noninfectious clone 5A13 with the recombinant plasmid pBBE22. The transformants were classified into nine groups based on plasmid content analysis. An infectivity study revealed that all nine transformants examined, each of which represented one of the plasmid patterns, were infectious in mice with severe combined immunodeficiency (SCID) regardless of their genomic compositions. Tissue bacterial quantification revealed that the loss of plasmids significantly reduced the spirochete burden in the heart and joint tissues, not in the skin, suggesting virulence factors may be tissue specific. Four transformants containing lp28-1 induced severe arthritis in SCID mice, in contrast to the five transformants lacking lp28-1. These pathogenicity studies associated lp28-1 with an arthritic phenotype and further studies may identify factors that contribute to arthritic pathology.
Peptidoglycan Recognition Protein 2 (N-Acetylmuramoyl-l-Ala Amidase) Is Induced in Keratinocytes by Bacteria through the p38 Kinase Pathway
Authors: Wang Haitao , Gupta Dipika , Li Xinna , Dziarski Roman
Published: October 2005

Human peptidoglycan recognition protein 2 (PGLYRP2) is anN-acetylmuramoyl-l-alanine amidase that hydrolyzes bacterial peptidoglycan and is constitutively produced in the liver and secreted into the blood. Here we demonstrate that PGLYRP2 was not expressed in healthy human skin and had low expression in the eye. However, upon exposure to gram-positive and gram-negative bacteria or cytokines, PGLYRP2 expression was highly induced in keratinocytes and to a lower level in corneal epithelial cells. Expression of PGLYRP2 was not induced in nonepithelial cells. Exposure of keratinocytes to bacteria induced keratinocyte differentiation and stress response and inhibited activation of signal transduction molecules involved in cell proliferation. Induction of PGLYRP2 expression correlated with expression of differentiation markers (cytokeratins and transglutaminase). Bacteria induced activation of p38 mitogen-activated protein kinase (MAPK) in keratinocytes, which was required for the induction of PGLYRP2 expression, because induction of PGLYRP2 transcription by bacteria was inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by a dominant-negative p38 construct. Induction of PGLYRP2 expression by bacteria (in contrast to expression of human β-defensin-2) was not mediated by Toll-like receptor 2 or 4. PGLYRP2 may function in the skin and the eyes as an inducible scavenger of proinflammatory peptidoglycan.
CD4+-T-Cell Responses Generated during MurineSalmonella entericaSerovar Typhimurium Infection Are Directed towards Multiple Epitopes within the Natural Antigen FliC
Authors: Bergman Molly A. , Cummings Lisa A. , Alaniz Robert C. , Mayeda Laura , Fellnerova Ivana , Cookson Brad T.
Published: October 2005

The flagellar filament protein FliC is a natural antigen recognized by memory CD4+T cells recovered fromSalmonella entericaserovar Typhimurium-infected humans and mice. To further investigate T-cell responses to FliC, we derived FliC-specific CD4+-T-cell clones from mice of two different haplotypes following oralS. entericaserovar Typhimurium infection. Using C-terminal truncations of MalE-FliC recombinant fusion proteins, we mapped antigenic activity to four different regions of FliC; three of the four epitope-containing regions were present in both FliC and the alternate flagellin subunit FljB. We determined that two novel FliC epitopes were also present in flagellins from several gram-negative enteric bacterial species: Ek-restricted FliC 80-94 (amino acids 80 to 94) and Ab-restricted FliC 455-469. Further mapping confirmed the presence of two previously identified FliC epitopes: Ak-restricted FliC 339-350 and Ab-restricted FliC 428-442. Therefore, like the recognition site of the innate immune receptor Toll-like receptor 5, three of four FliC epitopes recognized by CD4+T cells colocalize in the D0/D1 domains of FliC.Salmonella-infected macrophages and dendritic cells stimulated epitope-specific CD4+-T-cell proliferation; infected dendritic cells also activated T cells to produce gamma interferon. These data demonstrate thatSalmonellainfection generates murine CD4+-T-cell responses to multiple epitopes in the natural antigen FliC and that recognition of infected phagocytes by FliC-specific CD4+T cells triggers effector functions known to be essential for protective immunity. Together, these data suggest that FliC-specific CD4+T cells may contribute to cell-mediated host defenses againstSalmonella.
The Nickel-Responsive Regulator NikR Controls Activation and Repression of Gene Transcription inHelicobacter pylori
Authors: Ernst Florian D. , Kuipers Ernst J. , Heijens Angela , Sarwari Roya , Stoof Jeroen , Penn Charles W. , Kusters Johannes G. , van Vliet Arnoud H. M.
Published: October 2005

The NikR protein is a nickel-dependent regulatory protein which is a member of the ribbon-helix-helix family of transcriptional regulators. The gastric pathogenHelicobacter pyloriexpresses a NikR ortholog, which was previously shown to mediate regulation of metal metabolism and urease expression, but the mechanism governing the diverse regulatory effects had not been described until now. In this study it is demonstrated that NikR can regulateH. pylorinickel metabolism by directly controlling transcriptional repression of NixA-mediated nickel uptake and transcriptional induction of urease expression. Mutation of the nickel uptake genenixAin anH. pylori26695nikRmutant restored the ability to grow in Brucella media supplemented with 200 μM NiCl2but did not restore nickel-dependent induction of urease expression. Nickel-dependent binding of NikR to the promoter of thenixAgene resulted in nickel-repressed transcription, whereas nickel-dependent binding of NikR to the promoter of theureAgene resulted in nickel-induced transcription. Subsequent analysis of NikR binding to thenixAandureApromoters showed that the regulatory effect was dependent on the location of the NikR-recognized binding sequence. NikR recognized the region from −13 to +21 of thenixApromoter, encompassing the +1 and −10 region, and this binding resulted in repression ofnixAtranscription. In contrast, NikR bound to the region from −56 to −91 upstream of theureApromoter, resulting in induction of urease transcription. In conclusion, the NikR protein is able to function both as a repressor and as an activator of gene transcription, depending on the position of the binding site.
Partially Assembled K99 Fimbriae Are Required for Protection
Authors: Ascón Miguel A. , Ochoa-Repáraz Javier , Walters Nancy , Pascual David W.
Published: October 2005

Antibodies to K99 fimbriae afford protection to F5+bovine enterotoxigenicEscherichia coli(ETEC). Previous studies show that murine dams immunized withSalmonellavaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion offanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion offanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion offanGHis sufficient to confer protection.