Capsular genotype and lipooligosaccharide locus class distribution in Campylobacter jejuni from young children with diarrhea and asymptomatic carriers in Bangladesh.

Campylobacter jejuni-related diarrheal diseases is one of the major health issues among young children (0-59 months old) in low-income countries. Monitoring of the capsular (capsule polysaccharide, CPS) types of virulent C. jejuni strains in regions where the disease is endemic is of great importance for the development of a customized capsule-based multivalent vaccine. Therefore, we aimed to determine the prevalence of CPS genotypes among C. jejuni strains isolated from young children with enteritis (n = 152) and asymptomatic carriers matched by age, sex, and residence defined as the control group (n = 215) in Bangladesh. CPS genotyping was performed using a newly established multiplex polymerase chain reaction (PCR) method and lipooligosaccharide (LOS) locus classes (A-E) were characterized using PCR as well. We identified 24 different CPS genotypes among the 367 isolates. Four prevalent capsular types, HS5/31 complex (n = 27, 18%), HS3 (n = 26, 17%), HS4A (n = 10, 7%), and HS8/17 (n = 10, 7%) covered almost 50% of the strains from enteritis patients and 43% of the isolates from controls. In combination, the CPS genotype and LOS class was not discriminative between cases and controls. Dominant capsular types previously identified in C. jejuni strains isolated from patients with Guillain-Barré syndrome in Bangladesh were rarely detected in strains isolated from the young children. A similar distribution was evident among enteritis- and control-related strains when comparison was done between CPS types and LOS classes. This is the first systematic study presenting the distribution of CPS genotypes of C. jejuni strains isolated in Bangladesh from children with diarrhea and controls, with capsular genotypes HS5/31 complex, HS3, HS4A, and HS8/17 being prevalent in both. In conclusion, systematic studies are required to develop a multivalent capsule-based vaccine for children in low-income countries.

Bacterial proteases: targets for diagnostics and therapy.

Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

Comparative population structure analysis of Campylobacter jejuni from human and poultry origin in Bangladesh.

Campylobacter jejuni is the most important cause of antecedent infections leading to Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The objective of the present study was to define the genetic diversity, population structure, and potential role of poultry in the transmission of Campylobacter to humans in Bangladesh. We determined the population structure of C. jejuni isolated from poultry (n = 66) and patients with enteritis (n = 39) or GBS (n = 10). Lipooligosaccharide (LOS) typing showed that 50/66 (76 %) C. jejuni strains isolated from poultry could be assigned to one of five LOS locus classes (A-E). The distribution of neuropathy-associated LOS locus classes A, B, and C were 30/50 (60 %) among the typable strains isolated from poultry. The LOS locus classes A, B, and C were significantly associated with GBS and enteritis-related C. jejuni strains more than for the poultry strains [(31/38 (82 %) vs. 30/50 (60 %), p < 0.05]. Multilocus sequence typing (MLST) defined 15 sequence types (STs) and six clonal complexes (CCs) among poultry isolates, including one ST-3740 not previously documented. The most commonly identified type, ST-5 (13/66), in chicken was seen only once among human isolates (1/49) (p < 0.001). Amplified fragment length polymorphism (AFLP) revealed three major clusters (A, B, and C) among C. jejuni isolated from humans and poultry. There seems to be a lack of overlap between the major human and chicken clones, which suggests that there may be additional sources for campylobacteriosis other than poultry in Bangladesh.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Emergence of multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh.

The main objective of this study was to investigate the prevalence of bla (NDM-1) in Gram-negative bacteria in Bangladesh. In October 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratories, 1,816 consecutive clinical samples were tested for imipenem-resistant Gram-negative organisms. Imipenem-resistant isolates were tested for the bla (NDM-1) gene. Among 403 isolates, 14 (3.5 %) were positive for bla (NDM-1), and the predominant species were Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. All bla (NDM-1)-positive isolates were resistant to multiple antibiotics. Among ß-lactamase genes, bla (CTX-M-1-group) was detected in ten isolates (eight bla (CTX-M-15)), bla (OXA-1-group) in six, bla (TEM) in nine, bla (SHV) in seven, and bla (VIM) and bla (CMY) in two isolates each. The 16S rRNA methylase gene, armA, was detected in five K. pneumoniae isolates and in one E. coli isolate. rmtB and rmtC were detected in a Citrobacter freundii and two K. pneumoniae isolates, respectively. qnr genes were detected in two K. pneumoniae isolates (one qnrB and one qnrS) and in an E. coli isolate (qnrA). Transferable plasmids (60-100 MDa) carrying bla (NDM-1) were detected in 7 of the 11 plasmid-containing isolates. Pulsed-field gel electrophoresis (PFGE) analysis grouped K. pneumoniae isolates into three clusters, while E. coli isolates differed significantly from each other. This study reports that approximately 3.5 % of Gram-negative clinical isolates in Bangladesh are NDM-1-producing.

Bacterial aetiology of diarrhoeal diseases and antimicrobial resistance in Dhaka, Bangladesh, 2005-2008.

Infectious diarrhoea caused by bacterial pathogens contributes to the high level of mortality in developing countries like Bangladesh. Following standard bacteriological procedures, a total of 14 428 bacterial pathogens were isolated from 56 132 stool samples and rectal swabs collected from diarrhoeal patients between 2005 and 2008. The rate of isolation and antimicrobial susceptibility data were retrospectively analysed for these isolates and among them Vibrio spp. (42·9%) were the most predominant, followed by Shigella spp. (20·3%), Aeromonas spp. (12·8%) and Salmonella spp. (6·4%). A decreasing trend in isolation of Vibrio spp. (P<0·001) and Salmonella spp. (P<0·001) was observed. While Vibrio cholerae isolates remained susceptible to ciprofloxacin, an increase in resistance was observed in Campylobacter spp. and Shigella flexneri. Variations in susceptibility to other tested antibiotics were observed among the isolated pathogens. Access to this current data will help in understanding the local burden of diarrhoeal disease and contribute to better design of prevention programmes.

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant.

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.

Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner.

Evidence of intra-familial transmission of Helicobacter pylori by PCR-based RAPD fingerprinting in Bangladesh.

Helicobacter pylori is a genetically diverse bacterial species, which has facilitated adaptation to new hosts and persists worldwide. The main objective of this study was to explore intra-familial transmission of H. pylori in Bangladesh. We characterized H. pylori in 35 families including 138 family members using random amplified polymorphic DNA (RAPD) fingerprinting. Forty-six percent of H. pylori isolated from the mother shared a related genotype with strains isolated from their children. Twenty-nine percent of H. pylori isolates of the mother are related to the youngest children. Only 6% of the parents shared related genotype of H. pylori. These findings suggest that mother-to-child transmission occurs in early childhood and is the most probable route of transmission of H. pylori in Bangladesh.

Rapid identification of mycobacteria by Raman spectroscopy.

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

ZikaPLAN: Zika Preparedness Latin American Network.

The ongoing Zika virus (ZIKV) outbreak in Latin America, the Caribbean, and the Pacific Islands has underlined the need for a coordinated research network across the whole region that can respond rapidly to address the current knowledge gaps in Zika and enhance research preparedness beyond Zika. The European Union under its Horizon 2020 Research and Innovation Programme awarded three research consortia to respond to this need. Here we present the ZikaPLAN (Zika Preparedness Latin American Network) consortium. ZikaPLAN combines the strengths of 25 partners in Latin America, North America, Africa, Asia, and various centers in Europe. We will conduct clinical studies to estimate the risk and further define the full spectrum and risk factors of congenital Zika virus syndrome (including neurodevelopmental milestones in the first 3 years of life), delineate neurological complications associated with ZIKV due to direct neuroinvasion and immune-mediated responses in older children and adults, and strengthen surveillance for birth defects and Guillain-Barré Syndrome. Laboratory-based research to unravel neurotropism and investigate the role of sexual transmission, determinants of severe disease, and viral fitness will underpin the clinical studies. Social messaging and engagement with affected communities, as well as development of wearable repellent technologies against Aedes mosquitoes will enhance the impact. Burden of disease studies, data-driven vector control, and vaccine modeling as well as risk assessments on geographic spread of ZIKV will form the foundation for evidence-informed policies. While addressing the research gaps around ZIKV, we will engage in capacity building in laboratory and clinical research, collaborate with existing and new networks to share knowledge, and work with international organizations to tackle regulatory and other bottlenecks and refine research priorities. In this way, we can leverage the ZIKV response toward building a long-term emerging infectious diseases response capacity in the region to address future challenges.

Effect of hospitalization on the antibiotic resistance of fecal Enterococcus faecalis of surgical patients over time.

The prevalence of antibiotic resistant Enterococcus faecalis was determined in fecal samples of 263 patients admitted to the surgical wards of three university-affiliated hospitals on admission, at discharge, and at 1 and 6 months after discharge. A slight increase in the prevalence of antibiotic resistance of E. faecalis was found at discharge for the antibiotics tested compared to those on admission, vancomycin excepted. At 6 months after discharge, the prevalence of resistance for amoxicillin (0%), ciprofloxacin (3%), erythromycin (47%), and oxytetracycline (60%) decreased to the level on admission (respectively 0%, 8%, 45%, and 64%). Gentamicin resistance was the same at discharge (10%) as 1 month later (12%), but decreased 6 months after discharge (8%) to the level on admission (7%). In conclusion, hospitalization resulted in the study population in a slight increase in the prevalence of resistant fecal E. faecalis isolates at discharge, which decreased again (slowly) to the level on admission 6 months after discharge. Thus, the influence of hospitalization on the prevalence of antibiotic resistance in the extramural situation disappears between 1 and 6 months after discharge in this population.

Rapid identification of Candida spp. in peritonitis patients by Raman spectroscopy.

This prospective study evaluated Raman spectroscopy for the identification of clinically relevant Candida spp. in peritonitis patients. A Raman database was developed by measuring spectra from 93 reference strains belonging to ten different Candida spp. Clinical samples were obtained from the surgical department and intensive care unit of a tertiary university hospital. In total, 88 peritoneal specimens from 45 patients with primary, secondary or tertiary peritonitis were included. Specimens were cultured initially on a selective Sabouraud medium that contained gentamicin to suppress bacterial growth. For conventional identification, a chromogenic medium was used for presumptive identification, followed by use of the Vitek 2 system for definitive identification (requiring a total time of 48-96 h). Raman measurements were taken on overnight cultures from Sabouraud-gentamicin medium. Thirty-one samples were positive for Candida by culture. Using multivariate statistical analyses, a prediction accuracy of 90% was obtained for Raman spectroscopy, which appears to offer an accurate and rapid (12-24 h) alternative for the identification of Candida spp. in peritonitis patients. The reduced turn-around time is of great clinical importance for the treatment of critically ill patients with invasive candidiasis in intensive care units.

Campylobacter jejuni capsular genotypes are related to Guillain-Barré syndrome.

In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.

Gastroenteritis-associated Guillain-Barré syndrome on the Caribbean island Curaçao.

BACKGROUND: The number of patients with Guillain-Barré syndrome (GBS) who have been observed in Curaçao, the Netherlands Antilles, may be increasing. METHODS: Clinical and serologic data were obtained from records of patients admitted between 1987 and 1999 and fulfilling National Institute of Neurological and Communicative Disorders and Stroke criteria for GBS. When possible, serum and stool samples were collected. The results were compared with a large Dutch epidemiologic study. RESULTS: The authors identified 49 patients, an overall crude incidence rate (IR) in Curaçao of 2.53/100,000 inhabitants (95% CI 1.87 to 3.35) (Dutch study 1.18, rate ratio (RR) of 2.14, p < 0.001). The IR in Curaçao increased from 1.62 in 1987 to 1991 to 3.10 in 1992 to 1999, RR 5.22 (95% CI 2.48 to 10.2, p = 0.02). The IR showed a curvilinear shape within a year. In comparison with the Dutch group, patients from Curaçao had a more severe course of the disease, with a mortality rate of 23% (3.4% in the Dutch group, p < 0.001), a higher percentage of preceding gastroenteritis (p < 0.001), and less sensory involvement (p < 0.001). In 8 of 10 serum samples, evidence was found for a recent infection with Campylobacter jejuni. CONCLUSIONS: The authors found a steady increase in incidence of GBS over the years in association with a more pronounced seasonal preponderance and a more severe course. The clinical characteristics suggest a role for C jejuni.

Campylobacter jejuni lipopolysaccharides from Guillain-Barré syndrome patients induce IgG anti-GM1 antibodies in rabbits.

Lipopolysaccharides (LPS) from Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome (GBS) display molecular mimicry with GM1. We immunized rabbits with C. jejuni LPS from GBS-associated strains containing a GM1-like epitope. All animals produced high titre anti-LPS antibodies that were cross-reactive with GM1. We conclude that C. jejuni strains from GBS patients are able to induce antibodies that cross-react with gangliosides and LPS. This study further confirms the role of molecular mimicry in the induction of anti-ganglioside antibodies in GBS patients.

Humoral immune response against Campylobacter jejuni lipopolysaccharides in Guillain-Barré and Miller Fisher syndrome.

In this study we characterized the IgG antibodies against lipopolysaccharides (LPS) of Campylobacter jejuni in serum from patients with Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), C. jejuni enteritis and normal controls. In patients with GBS and MFS long-lasting titers of IgG1 and IgG3 antibodies against LPS from GBS and MFS associated C. jejuni were found. The subclass and course of these antibodies were highly associated with those of antibodies against GM1 and GQ1b in GBS and MFS patients. However, in C. jejuni enteritis and normal controls anti-LPS antibodies were predominantly IgG2. Antibody binding with LPS was reduced after treatment with choleratoxin and sialidases, suggesting that the ganglioside-like epitopes in LPS are immunodominant. These results further indicate that antecedent C. jejuni infections determine the specificity and isotype of anti-ganglioside antibodies in GBS and MFS patients.

A case of Guillain-Barré syndrome following a family outbreak of Campylobacter jejuni enteritis.

We describe an outbreak of Campylobacter jejuni enteritis involving three family members of whom one developed Guillain-Barré syndrome (GBS). The patients' serum reacted strongly with several gangliosides and with the lipopolysaccharide (LPS) fractions from the C. jejuni strains isolated from his family members. Only low titer anti-ganglioside antibodies were found in his siblings. HLA-typing did not indicate a locus associated with auto-antibody production. Comparing the immune response in GBS patients and C. jejuni enteritis patients can be of great value in determining the additional factors that lead to post-Campylobacter GBS. Ganglioside mimicry alone is necessary but not sufficient for the induction of anti-ganglioside antibodies. Other susceptibility factors are required to induce an anti-neural immune response.

PCR-mediated DNA typing of Campylobacter jejuni isolated from patients with recurrent infections.

The polymerase chain reaction (PCR) and two primers aiming at the enterobacterial repetitive intergenic consensus (ERIC) and arbitrary DNA sequences, respectively, were used to fingerprint the genomic DNA of 24 Campylobacter jejuni strains isolated from five patients with recurrent C. jejuni infections. Results were compared with biotyping and serotyping. The latter two methods, when combined, distinguished 9 different types, whereas PCR-mediated DNA analysis discriminated 14 different patterns. For six strains, the results of PCR-mediated typing led to different interpretations. This method is proposed as an additional tool to further discriminate between C. jejuni strains that appear related by conventional typing methods. In view of its rapidity and simplicity, this method is a potential candidate to replace the relatively slow and laborious conventional methods. However, further study is needed to assess the sensitivity and specificity of PCR-mediated DNA analysis and to investigate the usefulness of this method as an epidemiological tool in outbreaks of Campylobacter infections.