O-antigen serotyping and MALDI-TOF, potentially useful tools for optimizing semi-empiric antipseudomonal treatments through the early detection of high-risk clones.

The increasing prevalence of extensively drug-resistant (XDR) Pseudomonas aeruginosa infections is due to the global spread of defined high-risk clones (HRC). Among them, ST175 is particularly frequent in Spain and France. Here, we evaluated O-antigen serotyping and MALDI-TOF as typing methods for the early identification of ST175. O-antigen (O4) serotyping and MALDI-TOF biomarker peak-based recognition models were tested in several strain collections, including 206 non-duplicated P. aeruginosa clinical isolates collected in 2016. Resistance profiles were determined by broth microdilution and clonal epidemiology by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Up to 24.3% of the isolates were XDR and 28.2% non-susceptible to meropenem, while resistance to ceftolozane/tazobactam (2.9%) and colistin (0.5%) was infrequent. Half of all XDR isolates belonged to ST175 and most of them were only susceptible to ceftolozane/tazobactam and colistin. A model based on the detection of one MALDI-TOF biomarker peak yielded negative and positive predicted values (NPV/PPV) for the detection of ST175 of 100%/51.9%, whereas NPV/PPV for a model based on two biomarker peaks were 99.4%/87.1% and for O4 serotyping, 99.4%/84.1%. Both, O4 serotyping and MALDI-TOF biomarker peak analysis, proved to be sensitive and specific methods that could be easily incorporated in the routine workflow for the early detection of ST175 HCR. Since ST175 is associated with defined XDR profiles, with most isolates only being susceptible to colistin and ceftolozane/tazobactam, these simple techniques could be useful for optimizing semi-empiric antipseudomonal treatments in areas where this HRC is prevalent.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

Background: Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods: We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results: Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. Conclusions: This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

Host and Pathogen Biomarkers for Severe Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is among the leading causes of severe nosocomial infections, particularly affecting critically ill and immunocompromised patients. Here we review the current knowledge on the factors underlying the outcome of P. aeruginosa nosocomial infections, including aspects related to the pathogen, the host, and treatment. Intestinal colonization and previous use of antibiotics are key risk factors for P. aeruginosa infections, whereas underlying disease, source of infection, and severity of acute presentation are key host factors modulating outcome; delayed adequate antimicrobial therapy is also independently associated with increased mortality. Among pathogen-related factors influencing the outcome of P. aeruginosa infections, antibiotic resistance, and particularly multidrug-resistant profiles, is certainly of paramount relevance, given its obvious effect on the chances of appropriate empirical therapy. However, the direct impact of antibiotic resistance in the severity and outcomes of P. aeruginosa infections is not yet well established. The interplay between antibiotic resistance, virulence, and the concerning international high-risk clones (such as ST111, ST175, and ST235) still needs to be further analyzed. On the other hand, differential presence or expression of virulence factors has been shown to significantly impact disease severity and mortality. The likely more deeply studied P. aeruginosa virulence determinant is the type III secretion system (T3SS); the production of T3SS cytotoxins, and particularly ExoU, has been well established to determine a worse outcome both in respiratory and bloodstream infections. Other relevant pathogen-related biomarkers of severe infections include the involvement of specific clones or O-antigen serotypes, the presence of certain horizontally acquired genomic islands, or the expression of other virulence traits, such as the elastase. Finally, recent data suggest that host genetic factors may also modulate the severity of P. aeruginosa infections.

[Comparison of enteropathogenic Escherichia coli (EPEC) diagnosis by serology and by polymerase chain reaction (PCR)]. / Comparación entre el diagnóstico serológico y el diagnóstico por reación en cadena de la polimerasa (PCR) para Escherichia coli enteropatogénica (EPEC).

INTRODUCTION: The identification of EPEC in clinical laboratories is based on the determination of the serotypes by agglutination with O and H antiserum. Currently the proper diagnosis of EPEC should be done by the identification of the intimin gen (eaeA) by PCR. OBJECTIVES: To compare the diagnosis of EPEC by serotyping and by PCR. MATERIALS AND METHODS: We collected EPEC strains, identify by their O antigen, from 4 clinical laboratories in Lima from diarrheal samples in children less than 5 years of age. In those strains we have searched for virulence genes by a real time multiplex PCR for the diarrheagenic E. coli. RESULTS: We collected 113 strains; 82% from children less than 2 years of age. Only 15 strains (13.3%) had the intimin gene and therefore a confirmatory diagnosis of EPEC. In addition we found 3 enterotoxigenic (ETEC), 3 shiga toxin-producing (STEC), 1 enteroagreggative (EAEC) and 1 enteroinvasive (EIEC) strains. CONCLUSIONS: PCR should be use for the proper identification of EPEC. However, molecular methods are still not easily available in clinical laboratories worldwide.

Bivalent oral cholera vaccination induces a memory B cell response to the V. cholerae O1-polysaccharide antigen in Haitian adults.

The bivalent killed whole-cell oral cholera vaccine (BivWC) is being increasingly used to prevent cholera. The presence of O-antigen-specific memory B cells (MBC) has been associated with protective immunity against cholera, yet MBC responses have not been evaluated after BivWC vaccination. To address this knowledge gap, we measured V. cholerae O1-antigen MBC responses following BivWC vaccination. Adults in St. Marc, Haiti, received 2 doses of the BivWC vaccine, Shanchol, two weeks apart. Participants were invited to return at days 7, 21, 44, 90, 180 and 360 after the initial vaccination. Serum antibody and MBC responses were assessed at each time-point before and following vaccination. We observed that vaccination with BivWC resulted in significant O-antigen specific MBC responses to both Ogawa and Inaba serotypes that were detected by day 21 and remained significantly elevated over baseline for up to 12 months following vaccination. The BivWC oral cholera vaccine induces durable MBC responses to the V. cholerae O1-antigen. This suggests that long-term protection observed following vaccination with BivWC could be mediated or maintained by MBC responses.

The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.

Baseline Widal Titer Among Healthy Adult Males from the Greater Mymensingh Division of Bangladesh.

BACKGROUND: Widal test is the most widely used laboratory investigation for diagnosis of typhoid. However, the test interpretation remains controversial in the context of endemic regions such as Bangladesh, as agglutination occurs at varied titrations among a large percentage of healthy population. Paired Widal tests are often not feasible; hence single unpaired test has to be used for screening, diagnosis and treatment. OBJECTIVE: We aimed to assess the normal range of baseline titre for Anti TO, TH, AO, AH, BO agglutinins among healthy population in an endemic country with a view to guide the researchers and the clinicians, facilitating further investigation on updating cut off points of single Widal test for screening and diagnosis of typhoid fever in the context of Bangladesh. METHODS: A cross-sectional study was carried out in Mymensingh Medical College, Bangladesh on 2925 male immigration applicants. A single blood sample was collected for Widal test and interpreted using standard guidelines. RESULTS: The highest baseline titer for Anti TO, TH, AO, AH, BO agglutinins among 95% of the healthy participants was found to be 1:80 for each respectively. A titre of 1: 40 was observed for BH antigen. CONCLUSION: In case of singular Widal test, baseline values for the normal range was found to be 1:20 - 1:80 for all the antigens (TO, TH, AO, AH, BO, BH), except BH, for which it was 1:20-1:40. Further studies, inclusive of other sociodemographic groups and positive controls are required to determine the updated cut off values.

Endotoxemia-menace, marker, or mistake?

Endotoxemia is in its scientific ascendancy. Never has blood-borne, Gram-negative bacterial endotoxin (LPS) been invoked in the pathogenesis of so many diseases-not only as a trigger for septic shock, once its most cited role, but also as a contributor to atherosclerosis, obesity, chronic fatigue, metabolic syndrome, and many other conditions. Finding elevated plasma endotoxin levels has been essential supporting evidence for each of these links, yet the assays used to detect and quantitate endotoxin have important limitations. This article describes several assays for endotoxin in plasma, reviews what they do and do not measure, and discusses why LPS heterogeneity, LPS trafficking pathways, and host LPS inactivation mechanisms should be considered when interpreting endotoxin assay results.

Antibiotic resistance and virulence properties of Pseudomonas aeruginosa strains from mechanically ventilated patients with pneumonia in intensive care units: comparison with imipenem-resistant extra-respiratory tract isolates from uninfected patients.

We investigated the epidemiology of antibiotic resistance and virulence properties among Pseudomonas aeruginosa clinical isolates collected in 1999 from patients hospitalized in the intensive care units of the centre hospitalier d'Orléans, in France. We compared the totality of the strains from mechanically ventilated patients with pneumonia (33 non-duplicate isolates, group 1) to 15 randomly chosen, imipenem-resistant, extra-respiratory tract isolates, collected from non-infected patients hospitalized in the same units (group 2). The isolates were serotyped, typed by random amplified polymorphic DNA (RAPD), and screened for their pneumocyte cell adherence, cytotoxicity, and antibiotic resistance. A total of 35 RAPD profiles were found, and only two profiles were encountered in both groups, demonstrating a high genetic diversity. 84.8% of the group 1 and 93.3% of the group 2 isolates adhered to A549 cells. Three non-exclusive adhesive patterns were observed: a diffuse adhesion in 38 isolates, a localized adhesion in 14 isolates, and an aggregative adhesion in seven isolates. 78.8% of the group 1 and 93.3% of the group 2 isolates were cytotoxic. Considering all 48 isolates, there was a strong and statistically significant correlation between cytotoxicity and adherence. Among the three dominant serotypes, O:12 isolates were in majority avirulent, but the great majority of O:1 and all the O:11 isolates were found adherent and cytotoxic. Gentamicin was the least active antibiotic for both groups, and ceftazidime was the most active antibiotic for group 1 and amikacin for group 2. The penicillinase production phenotype was significantly correlated with a decrease in P. aeruginosa virulence.

Campylobacter jejuni O:19 serotype-associated Guillain-Barré syndrome in a child: the first case reported from Greece.

We present a case of Guillain-Barré syndrome (GBS) following Campylobacter jejuni HS serotype O:19 infection in a child. Antibodies against C. jejuni and autoantibodies to the peripheral nerve gangliosides GM1 were positive, a pattern correlating well with the existence of an inflammatory neuropathy like GBS. The patient shared the HLA-B35 and HLA-DR8 antigens, which have been found to be increased in GBS patients with previous C. jejuni infection. As this is the first diagnosed C. jejuni-associated GBS case reported from Greece, further clinical and epidemiologic investigations are warranted.

Escherichia coli strains from edema disease: O serogroups, and genes for Shiga toxin, enterotoxins, and F18 fimbriae.

The objectives of the research were to determine the presence of the gene sequences for Shiga Toxin 2e (Stx2e), enterotoxins (ST-I, ST-II and LT-I), and F18 fimbriae in 144 Escherichia coli strains isolated from pigs with edema disease; to assess the ability of stx2e(+) strains to produce Stx2e; and to determine the O serogroups of the E. coli strains. Presence of the genes was determined by polymerase chain reaction (PCR), production of Stx2e was assessed by cytotoxicity for Vero and Hela cells, O serogroups were identified by agglutination with specific antisera. Of the 144 strains tested, 99 were stx2e(+) by PCR, but only 45 of these were Stx2e(+) in the cell culture assays. Among the 99 stx2e(+) strains, PCR detected the genes for F18ab, ST-I, ST-II, LT-I in 76, 40, 31 and 16 strains, respectively. Forty-one of the 99 sxt2e(+) strains belonged to O group 139; the rest did not belong to the classical edema disease O serogroups. It is likely that the enterotoxins, whose genes were detected at high frequency, are responsible for diarrhea seem in pigs with edema disease in Brazil.

Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina. Evaluation of two procedures.

Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.

Additional O antigens of Vibrio fluvialis and Vibrio furnissii.

A total of 297 strains of Vibrio fluvialis and Vibrio furrnissii, which were collected from various countries for the past 15-year period of 1984-1998, were serogrouped. Of those examined, 239 strains of V. fluvialis and V. furnissii were classified into 29 known O serogroups; 9 strains were found to belong to R-form cultures, and the rest of the 49 strains could not be serogrouped. Of those serologically untypable strains, 26 novel O serogroups (O36 to O61) were established and added to our reference of the V. fluvialis and V. furnissii antigenic scheme. As all antisera against the O reference strains of the organisms contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera were absorbed with the reference rough strain, V. fluvialis GF25.

Isolation of Pasteurella multocida during an outbreak of infectious septicemia in Japanese quail (Coturnix coturnix japonica).

In May 1994, about fifty Japanese quails out of ninety being bred for experimental purposes at Miyazaki University died of acute septicemia within a few days. At autopsy, there were no gross pathological lesions, however, severe bacteremia was observed in all cases. Bacterial examination revealed the presence of Pasteurella multocida in blood and several organs in pure culture and they were of Carter's capsular type A, Heddleston's type 3-4 and Namioka's type O-8-9. The LD50 of bacteria in quails and mice were 4.3 x 10(4) cfu and 3.9 x 10(2) cfu, respectively. All of the three chickens experimentally infected with 4 x 10(4) of the isolate died within 20 hr after the infection and several bacteria were recovered from their blood and organs. This, to our knowledge, is the first report on an outbreak of fowl cholera in Japanese quails in Japan.

Enzyme-linked immunosorbent assay for screening of milk samples for Salmonella typhimurium in dairy herds.

We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive herd specificity, 0.9 and herd sensitivity, 1.0). A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S typhimurium infections, but further modifications are needed to test bulk tank milk samples.

Structural and serological studies on the O-antigen show that Citrobacter youngae PCM1505 must be classified to a new Citrobacter O-serogroup.

The O-polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter youngae PCM1505 was studied by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [Formula: see text]. Structural and serological data obtained earlier and in this work show that the strain studied is a candidate to a new Citrobacter O-serogroup.

Structure of the O-polysaccharide of Citrobacter youngae PCM 1503.

The O-polysaccharide (O-antigen) was obtained from the lipopolysaccharide of Citrobacter youngae PCM 1503, which is currently classified to the O7 serogroup. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy: Structural and serological data of the O-antigen suggest that strain PCM 1503 should be reclassified to a new Citrobacter serogroup.

Salmonella enterica Serotype Urbana Interference with Brucellosis Serology.

Sheep were immunized with killed Salmonella enterica serotype Urbana cells and their sera were tested in various serological tests for antibody to Brucella sp., Yersinia enterocolitica O:9 and Escherichia coli O:157 H:7. Of the eight sheep, all gave a positive agglutination reaction in the brucellosis buffered antigen plate agglutination test (BPAT), seven gave positive brucellosis standard tube agglutination test (TAT) and complement fixation test (CFT) results and four gave slightly positive reactions in a competitive enzyme immunoassay (CELISA). Seven sera were negative in an indirect enzyme immunoassay (IELISA-SLPS) using B. abortus smooth lipopolysaccharide (SLPS) antigen and all were negative in a fluorescence polarization assay (FPA-OPS) using B. abortus O-polysaccharide antigen. Two sheep gave a slight positive reaction in an IELISA using Brucella rough lipopolysaccharide antigen (IELISA-RLPS) and four sheep were slightly positive in an FPA using Brucella LPS core antigen (FPA-CORE). All sheep had high antibody responses to S. enterica serotype Urbana, Y. and E. coli O:157 and 7 were positive for antibody to Y. enterocolitica O:9 when tested by IELISA. The sheep were negative when tested in the FPA using OPS from Y. enterocolitica O:9 but all were strongly positive in the FPA using OPS from E. coli O:157 while seven sheep had titers to S. enterica serotype Urbana. The impact on diagnostic serology for brucellosis is discussed.