Molecular investigation of antibiotic resistant bacterial strains isolated from wastewater streams in Pakistan.
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ABSTRACT:
SUBMITTER: Saima S
PROVIDER: S-EPMC7410903 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The presented data provide new information on antibiotic resistance and virulence genes in the genomes of Aeromonas caviae strains TW-2 and TW-6, isolated from treated wastewater. The results confirm the presence of multi-antibiotic-resistant Aeromonas caviae strains with virulence properties as "high-risk isolates" in treated wastewater.
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Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and the community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan are limited. We performed a whole-genome-based comparative analysis of two (P10 and R46) MRSA strains isolated from two provinces of Pakistan to understand the genetic diversity, sequence type (ST), and distribution of virulence and antibiotic-resistance genes. The strains belong to ST113 and harbor the SCCmec type IV encoding mecA gene. Both the strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes, aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, a point mutation in fusA conferring resistance to fusidic acid, and in strain R46 a specific plasmid associated gene ant(4')-Ib. The strains harbor many virulence factors common to MRSA. However, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) or toxic shock syndrome toxin (tsst) genes were detected in any of the genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. These findings provide important information concerning the emerging MRSA clone ST113 in Pakistan and the sequenced strains can be used as reference strains for the comparative genomic analysis of other MRSA strains in Pakistan and ST113 strains globally.
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Project description:Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for Escherichia coli, Enterococcus spp., and other members of the family Enterobacteriaceae, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.
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Project description:INTRODUCTION:Pakistan has one of the highest burdens of pneumococcal diseases in the world, but unfortunately studies in this demanding research area are limited in the region. Pneumococcal surface protein A (PspA) is the next generation pneumococcal vaccine candidate as the protein locates on the Streptococcus pneumoniae surface. Its gene, pspA, might be encoded by all pneumococci, and the protein has proven immunogenicity. The molecular characterization of PspA, pneumococcal serotype distribution and antibiotic susceptibility are important for regional diversity studies. METHODS:In this study, we examined 38 pneumococcal isolates from pneumococcal diseased (pneumonia/meningitis) patients blood or cerebrospinal fluid. There were no specific inclusion or exclusion criteria, but all the individuals [ages 1 month to 12 years (male/female)] had undergone no antibiotic treatment in at least the past 3 months and had no vaccination history. We investigated the serotype distribution, antibiotic susceptibility, prevalence of the PspA family and its active domain's fusion, expression and antigenicity. RESULTS:Our finding shows that serotype 19F is the most prevalent (23.6%) followed by 18B (15.78%) (non-vaccine type) in all isolated pneumococcal strains. All strains were susceptible to chloramphenicol and linezolid, while 80% were resistant to gentamycin. Genotyping revealed that ~ 80% (N = 31/38) of pneumococcal strains produce PspA belonging to family 2 and clade 3. We further selected three active domains of PspA (family 2 and clade 3) by in silico analysis, merged together into a fusion gene for expression study, and its antigenicity was analyzed by Western blotting. CONCLUSION:Serotypes 19F and 18B (non-vaccine type) are the most prevalent in the Pakistani pneumococcal isolates. The PspA family 2 proteins produced by Pakistani pneumococcal isolates have high sequence homologies with each other and differ from those produced by strains isolated in the rest of the world. The PspA fusion peptide had a proven antigenic response in western blotting, with no considerable correlation among pneumococcal serotypes, antibiotic susceptibility and PspA family/clade distribution.
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Project description:A Kashmir Himalayan (India) soil isolate, Streptomyces sp. SM01 was subjected to small scale fermentation for the production of novel antimicrobials, picolinamycin (SM1). The production has been optimized which found to be maximum while incubated in AIA medium (pH 7) for 7 days at 30 °C. Seven days grew crude cell-free culture media (50 µL) showed a larger zone of inhibition against Staphylococcus aureus compared to streptomycin (5 µg) and ampicillin (5 µg). Extraction, purification, and chemical analysis of the antimicrobial component has been proved to be a new class of antibiotic with 1013 dalton molecular weight. We have named this new antibiotic as picolinamycin for consisting picolinamide moiety in the center of the molecule and produced by a Streptomyces sp. In general, the antimicrobial potency of this newly characterized antibiotic found to be higher against Gram-positive organisms than the tested Gram-negative organisms. The MIC of this antimicrobial compound was found to be 0.01 µg/ml for tested Gram-positive organisms and 0.02 to 5.12 µg/ml for Gram-negative organisms. Furthermore, it showed strong growth impairments of several multidrug resistance (MDR) strains, including methicillin-resistant strains of Staphylococci and Enterococci with the MIC value of 0.04 to 5.12 µg/ml and MDR (but methicillin-sensitive) strains of S. aureus with the MIC value of 0.084 µg/ml. It also showed anti-mycobacterial potential in higher concentrations (MIC is 10.24 µg/ml). Picolinamycin however did not show toxicity against tested A549 human cell line indicating that the spectrum of its activity limited within bacteria only.
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Project description:The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.
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Project description:BackgroundWe evaluated the distribution of carbapenem and colistin resistance mechanisms of clinical E. coli and K. pneumoniae isolates from Iran.Methods165 non-duplicate non-consecutive isolates of K. pneumoniae and E. coli were collected from hospitalized patients admitted to Iran's tertiary care hospitals from September 2016 to August 2018. The isolates were cultured from different clinical specimens, including wound, urine, blood, and tracheal aspirates. Antibiotic susceptibility testing was performed by disc diffusion and microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The presence of extended spectrum β-lactamases (ESBLs) genes, carbapenemase genes, as well as fosfomycin resistance genes, and colistin resistance genes was also examined by PCR-sequencing. The ability of biofilm formation was assessed with crystal violet staining method. The expression of colistin resistance genes were measured by quantitative reverse transcription-PCR (RT-qPCR) analysis to evaluate the association between gene upregulation and colistin resistance. Genotyping was performed using the multi-locus sequencing typing (MLST).ResultsColistin and tigecycline were the most effective antimicrobial agents with 90.3% and 82.4% susceptibility. Notably, 16 (9.7%) isolates showed resistance to colistin. Overall, 33 (20%), 31 (18.8%), and 95 (57.6%) isolates were categorized as strong, moderate, and weak biofilm-producer, respectively. Additionally, blaTEM, blaSHV, blaCTX-M, blaNDM-1, blaOXA-48-like and blaNDM-6 resistance genes were detected in 98 (59.4%), 54 (32.7%), 77 (46.7%), 3 (1.8%), 17 (10.30%) and 3 (1.8%) isolates, respectively. Inactivation of mgrB gene due to nonsense mutations and insertion of IS elements was observed in 6 colistin resistant isolates. Colistin resistance was found to be linked to upregulation of pmrA-C, pmrK, phoP, and phoQ genes. Three of blaNDM-1 and 3 of blaNDM-6 variants were found to be carried by IncL/M and IncF plasmid, respectively. MLST revealed that blaNDM positive isolates were clonally related and belonged to three distinct clonal complexes, including ST147, ST15 and ST3299.ConclusionsThe large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of diverse carbapenem- and colistin-resistant isolates in the future.
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Project description:Poultry husbandry is important for the economic health of Pakistan, but the Pakistani poultry industry is negatively impacted by infections from Escherichia coli. We performed Illumina whole genome sequencing on 92 E. coli isolates obtained from the livers of deceased chickens originating in five Pakistani geographical regions. Our analysis indicates that the isolates are predominantly from the B1 and A clade and harbor a diverse number of antibiotic resistance and virulence genes, with no linkage between phylogeny and antibiotic resistance gene presence but some association between phylogeny and virulence gene and SNP presence for the B1 and E phylogroups. The colistin resistance gene mcr-1 and the quinolone resistance gene qnrS1 were both found in 13/92 isolates. Alarmingly, 82/92 of the E. coli strains characterized in this study are multidrug resistant with 100% (92/92) resistance to lincomycin, 81.5% (75/92) to streptomycin, 79.3% (73/92) to ampicillin and 66.3% (61/92) to ciprofloxacin. These results provide a high-resolution analysis of poultry-associated E. coli isolates in an area with a high endemic burden of antibiotic resistance. Surveillance of antibiotic resistance in poultry associated E. coli isolates is an important pillar of the One Health concept to integrate analysis of potential pathogens in human, animal, and environmental niches.
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