Prevalence of extended spectrum β-lactamase, AmpC β-lactamase and metallo β-lactamase mediated resistance in Escherichia coli from diagnostic and tertiary healthcare centers in south Bangalore, India

Background: The increasing reports on multidrug resistant Escherichia coli has become a potential threat to global health. Here, we present a cross-sectional study to characterize extended spectrum β-lactamase, AmpC β-lactamase and metallo β-lactamase producing E. coli isolated from different human clinical samples.Methods: A total of 300 clinical Gram negative bacterial isolates were collected and re-characterized for the identification of E. coli following standard microbiological techniques. The antimicrobial susceptibility of E. coli isolates was initially screened by Kirby-Bauer disk diffusion and MIC methods. The resistant isolates were confirmed to be ESBL, AmpC and MBL producers by their respective phenotypic confirmatory tests of combined disc method.Results: We identified 203 (68%) E. coli and 97 (32%) Non-E. coli isolates. The highest recovery of E. coli was from urine samples 72 (35%). Combined disc method using ceftazidime/ceftazidime+clavulanic acid and cefotaxime/cefotaxime+clavulanic acid confirmed 156 (79%) and 144 (73%) E. coli as ESBL producers, respectively. Thirty-four (34%) and 16 (27%) resistant E. coli isolates were confirmed to be AmpC and MBL producers, likewise.Conclusions: Increased prevalence of ESBL, AmpC and MBL producing E. coli were observed. Beta-lactamase mediated resistance appears to be prime mechanism in the multidrug resistant E. coli. Thus, early detection of beta lactamase producing E. coli is necessary to avoid treatment failure and prevent the spread of MDR

Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.</p

Development of PCRSeqTyping—a novel molecular assay for typing of Streptococcus pneumoniae

Abstract Background Precise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need. Methods Polymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark). Results A 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step. Conclusion This study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies

The effectiveness of image-guided percutaneous catheter drainage in the management of acute pancreatitis-associated pancreatic collections

Purpose: Acute pancreatitis is commonly complicated by the development of pancreatic collections (PCs). Symptomatic PCs warrant drainage, and the available options include percutaneous, endoscopic, and open surgical approaches. The study aimed to assess the therapeutic effectiveness and safety of image guided percutaneous catheter drainage (PCD) in the management of acute pancreatitis related PCs. Material and methods: This was a single-centre prospective study covering a 4-year study period. Acute pancreatitisrelated PCs complicated by secondary infection or those producing symptoms due to pressure effect on surrounding structures were enrolled and underwent ultrasound or computed tomography (CT)-guided PCD. The patients were followed to assess the success of PCD (defined as clinical, radiological improvement, and the avoidance of surgery) and any PCD-related complications. Results: The study included 60 patients (60% males) with a mean age of 43.1 ± 21.2 years. PCD recorded a success rate of 80% (16/20) for acute peripancreatic fluid collections (APFC) and pancreatic pseudocysts (PPs), 75% (12/16) for walled-off necrosis (WON), and 50% (12/24) for acute necrotic collections (ANCs). Post-PCD surgery (necrosectomy ± distal pancreatectomy) was needed in 50% of ANC and 25% of WON. Only 20% of APFCs/PPs patients required surgical/endoscopic treatment post-PCD. Minor procedure-related complications were seen in 4 (6.6%) patients. Conclusion: PCD is an effective, safe, and minimally invasive therapeutic modality with a good success rate in the management of infected/symptomatic PCs

Structural, genetic, and serological elucidation of Streptococcus pneumoniae serogroup 24 serotypes: Discovery of a new serotype, 24C, with a variable capsule structure

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [β-Ribf-(1→4)-α-Rhap-(1→3)-β-GlcpNAc-(1→4)-β-Rhap-(1→4)-β-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without β-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-β-D-Galf2Ac-(1→3)-β-D-Galp-(1→3)-α-D-Galp-(1→3)-β-D-Galf-(1→3)-β-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F

A New Pneumococcal Capsule Type, 10D, is the 100th Serotype and Has a Large cps Fragment from an Oral Streptococcus

The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (cps) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has three large regions syntenic to and highly homologous with cps loci from serotype 6C, serotype 39, and an oral streptococcus strain (S. mitis SK145). The 10D cps region syntenic to SK145 is about 6 kb and has a short gene fragment of wciNα at the 5′ end. The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of cps loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination