Niyaz Ahmed

Mycobacterial Dormancy Systems and Host Responses in Tuberculosis

Tuberculosis (TB) caused by the intracellular pathogen, Mycobacterium tuberculosis (Mtb), claims more than 1.5 million lives worldwide annually. Despite promulgation of multipronged strategies to prevent and control TB, there is no significant downfall occurring in the number of new cases, and adding to this is the relapse of the disease due to the emergence of antibiotic resistance and the ability of Mtb to remain dormant after primary infection. The pathology of Mtb is complex and largely attributed to immune-evading strategies that this pathogen adopts to establish primary infection, its persistence in the host, and reactivation of pathogenicity under favorable conditions. In this review, we present various biochemical, immunological, and genetic strategies unleashed by Mtb inside the host for its survival. The bacterium enables itself to establish a niche by evading immune recognition via resorting to masking, establishment of dormancy by manipulating immune receptor responses, altering innate immune cell fate, enhancing granuloma formation, and developing antibiotic tolerance. Besides these, the regulatory entities, such as DosR and its regulon, encompassing various putative effector proteins play a vital role in maintaining the dormant nature of this pathogen. Further, reactivation of Mtb allows relapse of the disease and is favored by the genes of the Rtf family and the conditions that suppress the immune system of the host. Identification of target genes and characterizing the function of their respective antigens involved in primary infection, dormancy, and reactivation would likely provide vital clues to design novel drugs and/or vaccines for the control of dormant TB.

Risk of Transmission of Antimicrobial Resistant Escherichia coli from Commercial Broiler and Free-Range Retail Chicken in India

Multidrug-resistant Escherichia coli infections are a growing public health concern. This study analyzed the possibility of contamination of commercial poultry meat (broiler and free-range) with pathogenic and or multi-resistant E. coli in retail chain poultry meat markets in India. We analyzed 168 E. coli isolates from broiler and free-range retail poultry (meat/ceca) sampled over a wide geographical area, for their antimicrobial sensitivity, phylogenetic groupings, virulence determinants, extended-spectrum-β-lactamase (ESBL) genotypes, fingerprinting by Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and genetic relatedness to human pathogenic E. coli using whole genome sequencing (WGS). The prevalence rates of ESBL producing E. coli among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%. E. coli from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively) and extraintestinal pathogenic E. coli (ExPEC) contamination (5 and 0%, respectively). WGS analysis confirmed two globally emergent human pathogenic lineages of E. coli, namely the ST131 (H30-Rx subclone) and ST117 among our poultry E. coli isolates. These results suggest that commercial poultry meat is not only an indirect public health risk by being a possible carrier of non-pathogenic multi-drug resistant (MDR)-E. coli, but could as well be the carrier of human E. coli pathotypes. Further, the free-range chicken appears to carry low risk of contamination with antimicrobial resistant and extraintestinal pathogenic E. coli (ExPEC). Overall, these observations reinforce the understanding that poultry meat in the retail chain could possibly be contaminated by MDR and/or pathogenic E. coli.

Comparative Genomics of Escherichia coli Isolated from Skin and Soft Tissue and Other Extraintestinal Infections

ABSTRACT Escherichia coli, an intestinal Gram-negative bacterium, has been shown to be associated with a variety of diseases in addition to intestinal infections, such as urinary tract infections (UTIs), meningitis in neonates, septicemia, skin and soft tissue infections (SSTIs), and colisepticemia. Thus, for nonintestinal infections, it is categorized as extraintestinal pathogenic E. coli (ExPEC). It is also an opportunistic pathogen, causing cross infections, notably as an agent of zoonotic diseases. However, comparative genomic data providing functional and genetic coordinates for ExPEC strains associated with these different types of infections have not proven conclusive. In the study reported here, ExPEC E. coli isolated from SSTIs was characterized, including virulence and drug resistance profiles, and compared with isolates from patients suffering either pyelonephritis or septicemia. Results revealed that the majority of the isolates belonged to two pathogenic phylogroups, B2 and D. Approximately 67% of the isolates were multidrug resistant (MDR), with 85% producing extended-spectrum beta-lactamase (ESBL) and 6% producing metallo-beta-lactamase (MBL). The blaCTX-M-15 genotype was observed in at least 70% of the E. coli isolates in each category, conferring resistance to an extended range of beta-lactam antibiotics. Whole-genome sequencing and comparative genomics of the ExPEC isolates revealed that two of the four isolates from SSTIs, NA633 and NA643, belong to pandemic sequence type ST131, whereas functional characteristics of three of the ExPEC pathotypes revealed that they had equal capabilities to form biofilm and were resistant to human serum. Overall, the isolates from a variety of ExPEC infections demonstrated similar resistomes and virulomes and did not display any disease-specific functional or genetic coordinates. IMPORTANCE Infections caused by extraintestinal pathogenic E. coli (ExPEC) are of global concern as they result in significant costs to health care facilities management. The recent emergence of a multidrug-resistant pandemic clone, Escherichia coli ST131, is of primary concern as a global threat. In developing countries, such as India, skin and soft tissue infections (SSTIs) associated with E. coli are marginally addressed. In this study, we employed both genomic analysis and phenotypic assays to determine relationships, if any, among the ExPEC pathotypes. Similarity between antibiotic resistance and virulence profiles was observed, ST131 isolates from SSTIs were reported, and genomic similarities among strains isolated from different disease conditions were detected. This study provides functional molecular infection epidemiology insight into SSTI-associated E. coli compared with ExPEC pathotypes. Infections caused by extraintestinal pathogenic E. coli (ExPEC) are of global concern as they result in significant costs to health care facilities management. The recent emergence of a multidrug-resistant pandemic clone, Escherichia coli ST131, is of primary concern as a global threat. In developing countries, such as India, skin and soft tissue infections (SSTIs) associated with E. coli are marginally addressed. In this study, we employed both genomic analysis and phenotypic assays to determine relationships, if any, among the ExPEC pathotypes. Similarity between antibiotic resistance and virulence profiles was observed, ST131 isolates from SSTIs were reported, and genomic similarities among strains isolated from different disease conditions were detected. This study provides functional molecular infection epidemiology insight into SSTI-associated E. coli compared with ExPEC pathotypes.

Microbiological quality assessment of milk at different stages of the dairy value chain in a developing country setting

The main objective of the study was to assess the microbiological quality of milk at different stages of the dairy value chain from farm to the factory in Bangladesh. A total of 438 raw milk samples (387 from primary producers, 32 from collectors, 15 from chilling plants, 4 from local restaurants) and 95 commercially processed milk samples were collected from northern part of Bangladesh. Almost 72% (n = 280) of samples at producer level and 100% from both collectors (n = 32) and chilling plants (n = 15) were contaminated with coliforms while 57% (n = 220) of samples from producers, 91% (n = 29) of samples from collectors and 100% (n = 15) from chilling plants were contaminated with fecal coliforms. Around 31% (n = 119) of samples from producers were positive for E. coli whereas >60% (n = 20) and 100% (n = 15) samples from collectors and chilling plants, respectively were positive for E. coli. One quarter of samples from collectors were positive for B. cereus and coagulase positive staphylococci and 33% (n = 5) of samples from chilling plants were positive for both of these microorganisms. In case of commercially processed milk, 77% (n = 46) and 37% (n = 22) of pasteurized milk samples had a high aerobic plate count (APC) (104 CFU/ml) and coliform count (>10 CFU/ml), respectively. None of the samples was positive for Shigella spp., Salmonella spp., and Campylobacter spp. Among 158 E. coli positive raw milk samples, 9% (n = 14) contained pathogenic E. coli, and enteroaggregative E. coli (EAEC) and Shiga-toxin producing E. coli (STEC) were found to be the predominant pathotypes. Of the 23 pathogenic E. coli identified from 14 samples based on their gene contents, >95% (n = 22) were resistant to at least one antibiotic and 13% (n = 3) of isolates were resistant to ≥3 classes of antibiotics. Several factors including the time of milking, hygiene practices of the producers, cow breed and amount of milk produced by the cow were found to be significantly associated with high APC of milk samples. In conclusion, both raw and commercially pasteurized milk are highly contaminated with fecal organisms. For intervention, more emphasis should be given at producers level as microorganisms introduced to milk at this stage get the longest time for survival and multiplication.

Analysis of mutations in pncA reveals non-overlapping patterns among various lineages of Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug, resistance to which occurs primarily due to mutations in pncA (Rv2043c) that encodes the pyrazinamidase enzyme responsible for conversion of pro-drug PZA into its active form. Previous studies have reported numerous resistance-conferring mutations distributed across the entire length of pncA without any hotspot regions. As different lineages of Mycobacterium tuberculosis display a strong geographic association, we sought to understand whether the genetic background influenced the distribution of mutations in pncA. We analyzed the whole genome sequence data of 1,480 clinical isolates representing four major M. tuberculosis lineages to identify the distribution of mutations in the complete operon (Rv2044c-pncA-Rv2042c) and its upstream promoter region. We observed a non-overlapping pattern of mutations among various lineages and identified a lineage 3-specific frame-shift deletion in gene Rv2044c upstream of pncA that disrupted the stop codon and led to its fusion with pncA. This resulted in the addition of a novel domain of unknown function (DUF2784) to the pyrazinamidase enzyme. The variant molecule was computationally modelled and physico-chemical parameters determined to ascertain stability. Although the functional impact of this mutation remains unknown, its lineage specific nature highlights the importance of genetic background and warrants further study.

Colistin resistant Escherichia coli carrying mcr - 1 in urban sludge samples: Dhaka, Bangladesh

Of 48 bacteria belonging to the family Enterobacteriaceae tested from urban sludge samples, one Escherichia coli isolate was resistant to colistin and possessed the resistance marker gene mcr-1 found for the first time from Bangladesh. The colistin resistant E. coli was multidrug resistant showing resistance to 11 different antibiotics tested.

Mycobacterium tuberculosis DosR Regulon Gene Rv2004c Encodes a Novel Antigen with Pro-inflammatory Functions and Potential Diagnostic Application for Detection of Latent Tuberculosis

Approximately 1.7 billion people in the world harbor latent Mycobacterium tuberculosis (Mtb) with a substantial risk of progression to clinical outcome. Containment of these seed beds of Mtb is essential to eliminate tuberculosis completely in high burden settings such as India. Hence, there is an urgent need for the identification of new serological markers for detection or vaccine candidates to prevent latent tuberculosis infection (LTBI). DosR regulon antigens of Mtb might serve as attractive targets for LTBI diagnosis or vaccine development as they are specifically expressed and are upregulated during latent phase. In this study, we investigated the role of Rv2004c, a member of DosR regulon (exclusive to Mtb complex), in host–pathogen interaction and its immunogenic potential in LTBI, active TB, and healthy control cohorts. Rv2004c elicited strong antibody response in individuals with LTBI compared to active TB patients and healthy cohorts. Recombinant Rv2004c induced pro-inflammatory cytokine response in human peripheral blood mononuclear cells and THP-1 cells via NF-κB phosphorylation. Interaction of Rv2004c with toll-like receptor (TLR)-2 was confirmed using HEK-Blue hTLR-2 and pull-down assays. Rv2004c enhanced the surface expression of TLR-2 at mRNA and protein levels in THP-1 cells. Our findings revealed that Rv2004c induces strong humoral and cell mediated immune responses. Given these observations, we propose Rv2004c to be a potential diagnostic marker or an attractive vaccine candidate that can be useful against LTBI.

Author Correction: Analysis of mutations in pncA reveals non-overlapping patterns among various lineages of Mycobacterium tuberculosis.

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ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost

BackgroundExtended spectrum beta lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli infections are of global interest because of their clinical and economic impact. The ESBL resistance genes disseminate through plasmids, and are found in successful global lineages such as ST131 and ST648. The carriage of plasmids has been suggested to result in a fitness burden, but recently it was shown that ESBL-plasmids enhanced virulence in pandemic ST131 and ST648 lineages without affecting their fitness. Herein, we investigated the influence of ESBL-plasmids on bacterial competition and serum resistance, both of which are essential characteristics of ExPEC during infections.MethodsTriplets of ESBL-plasmid-carrying wildtype (WT), plasmid-cured variant (PCV) and transformant (T) of five ExPEC strains of ST131 and ST648 were used for bacterial competition experiments with colicin-producing commensal E. coli, competitive adhesion experiments and serum survival. In addition, resilience after SDS, acid, osmotic challenges and RNA sequence data were analyzed.ResultsIn all five strains tested, ESBL-plasmid carriage did not negatively influence E. coli fitness in direct bacterial competition with commensal E. coli in vitro. That is, WTs did not show any disadvantages when compared to their isogenic plasmid-free PCV. For one strain we even found the opposite as PCV17433 was out-competed by a commensal strain, which suggests an even protective role of the ESBL-plasmid carried by the WT17433. Similarly, in the serum-resistance experiments, the PCVs of two strains (PCV17433 and PCV17887) were more sensitive to serum, unlike WTs and Ts. The observed inter-strain differences could be explained by the different genetic content of plasmids carried in those strains.ConclusionsOverall, we found no compelling evidence for an increased burden resulting from the carriage of ESBL-plasmids in the absence of antimicrobial selection pressure in the strains of pandemic ST131 and ST648; rather, the possession of certain ESBL-plasmids was beneficial for some strains in regarding competition fitness and serum survival.

Multiresistant Uropathogenic Escherichia coli from a Region in India Where Urinary Tract Infections Are Endemic

ABSTRACT Escherichia coli sequence type 131 (O25b:H4), associated with the CTX-M-15 extended-spectrum beta-lactamases (ESBLs) and linked predominantly to the community-onset antimicrobial-resistant infections, has globally emerged as a public health concern. However, scant attention is given to the understanding of the molecular epidemiology of these strains in high-burden countries such as India. Of the 100 clinical E. coli isolates obtained by us from a setting where urinary tract infections are endemic, 16 ST131 E. coli isolates were identified by multilocus sequence typing (MLST). Further, genotyping and phenotyping methods were employed to characterize their virulence and drug resistance patterns. All the 16 ST131 isolates harbored the CTX-M-15 gene, and half of them also carried TEM-1; 11 of these were positive for blaOXA groups 1 and 12 for aac(6′)-Ib-cr. At least 12 isolates were refractory to four non-beta-lactam antibiotics: ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, and tetracycline. Nine isolates carried the class 1 integron. Plasmid analysis indicated a large pool of up to six plasmids per strain with a mean of approximately three plasmids. Conjugation and PCR-based replicon typing (PBRT) revealed that the spread of resistance was associated with the FIA incompatibility group of plasmids. Pulsed-field gel electrophoresis (PFGE) and genotyping of the virulence genes showed a low level of diversity among these strains. The association of ESBL-encoding plasmid with virulence was demonstrated in transconjugants by serum assay. None of the 16 ST131 ESBL-producing E. coli strains were known to synthesize carbapenemase enzymes. In conclusion, our study reports a snapshot of the highly virulent/multiresistant clone ST131 of uropathogenic E. coli from India. This study suggests that the ST131 genotypes from this region are clonally evolved and are strongly associated with the CTX-M-15 enzyme, carry a high antibiotic resistance background, and have emerged as an important cause of community-acquired urinary tract infections.