Antimicrobial Resistance Studies Using Raman Spectroscopy on Clinically Relevant Bacterial Strains.

There has been a steep rise in the emergence of antibiotic-resistant bacteria in the past few years. A timely diagnosis can help in initiating appropriate antibiotic therapy. However, conventional techniques for diagnosing antibiotic resistance are time-consuming and labor-intensive. Therefore, we investigated the potential of Raman spectroscopy as a rapid surveillance technology for tracking the emergence of antibiotic resistance. In this study, we used Raman spectroscopy to differentiate clinical isolates of antibiotic-resistant and -sensitive bacteria of Escherichia coli, Acinetobacter baumannii, and Enterobacter species. The spectra were collected with or without exposure to various antibiotics (ciprofloxacin, gentamicin, meropenem, and nitrofurantoin), each having a distinct mechanism of action. Ciprofloxacin- and meropenem-treated sensitive strains showed a decrease in the intensity of Raman bands associated with DNA (667, 724, 785, 1378, 1480, and 1575 cm-1) and proteins (640 and 1662 cm-1), coupled with an increase in the intensity of lipid bands (891, 960, and 1445 cm-1). Gentamicin- and nitrofurantoin-treated sensitive strains showed an increase in the intensity of nucleic acid bands (668, 724, 780, 810, 1378, 1480, and 1575 cm-1) while a decrease in the intensity of protein bands (640, 1003, 1606, and 1662 cm-1) and the lipid band (1445 cm-1). The Raman spectral changes observed in the antibiotic-resistant strains were opposite to that of antibiotic-sensitive strains. The Raman spectral data correlated well with the antimicrobial susceptibility test results. The Raman spectral dataset was used for partial least-squares (PLS) analysis to validate the biomarkers obtained from the univariate analysis. Overall, this study showcases the potential of Raman spectroscopy for detecting antibiotic-resistant and -sensitive bacteria.

Localized outbreaks of Pseudomonas aeruginosa belonging to international high-risk clones in a south Indian hospital.

Introduction. Pseudomonas aeruginosa is now considered as a major bacterial pathogen associated with hospital infections. Frequently, multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa are being encountered. Unusual increase in the P. aeruginosa infections led to the suspicion of outbreaks in the urology ward and cardiothoracic and vascular surgery intensive care unit (CTVS-ICU).Hypothesis. We hypothesize that the localized outbreaks may have originated from environmental sources within the hospital premises. An alternative possibility is the transmission from a previously infected patient or hospital attendant. Understanding the drug-resistance profile and genome characteristics of these clinical samples would determine the likely source of infection and spread.Aim. To perform epidemiological and molecular investigations on the suspected outbreaks of P. aeruginosa in the study centre and identify potential sources of infection.Methodology. Fourteen drug-resistant P. aeruginosa isolated from patients of the urology ward, CTVS-ICU and tap waters collected during the suspected outbreaks were subjected to microbiological and genomic analysis. Comparative genome (CG) analysis of these 14 study genomes with 284 complete P. aeruginosa genomes was performed.Results. Multilocus sequence typing analysis revealed that the isolates belonged to five different sequence types (ST235, ST357, ST639, ST654 and ST1203) and clustered into three distinct groups while two CTVS-ICU isolates remained as singletons. Genome analysis distinguished that the outbreaks in the urology ward and CTVS-ICU are independent, epidemiologically unrelated to each other and with the tap-water isolates.Conclusion. This study highlights the presence of distinct, clonally unrelated, drug-resistant P. aeruginosa within a hospital setting. The genome analysis of the two localized outbreaks revealed their distinct genetic background and phylogenetically unrelated origin. Vigilant screening and effective implementation of infection control measures led to the successful containment of potential environmental reservoirs of P. aeruginosa within the premises.

Prediction of nitrofurantoin resistance among Enterobacteriaceae and mutational landscape of in vitro selected resistant Escherichia coli.

Nitrofurantoin (NIT) has long been a drug of choice in the treatment of lower urinary tract infections. Recent emergence of NIT resistant Enterobacteriaceae is a global concern. An ordinal logistic regression model based on PCR amplification patterns of five genes associated with NIT resistance (nfsA, nfsB, ribE, oqxA, and oqxB) among 100 clinical Enterobacteriaceae suggested that a combination of oqxB, nfsB, ribE, and oqxA is ideal for NIT resistance prediction. In addition, four Escherichia coli NIT-resistant mutants were in vitro generated by exposing an NIT-susceptible E. coli to varying concentrations of NIT. The in vitro selected NIT resistant mutants (NI2, NI3, NI4 and NI5) were found to have mutations resulting in frameshifts, premature/lost stop codons or failed amplification of nfsA and/or nfsB genes. The in vitro selected NI5 and the transductant colonies with reconstructed NI5 genotype exhibited reduced fitness compared to their parent strain NS30, while growth of a resistant clinical isolate (NR42) was found to be unaffected in the absence of NIT. These results emphasize the importance of strict adherence to prescribed antibiotic treatment regimens and dosage duration. If left unchecked, these resistant bacteria may thrive at sub-therapeutic concentrations of NIT and spread in the community.

Complete genome sequence of an extensively drug resistant (XDR) M. morganii SMM01 isolated from a patient with urinary and fecal incontinence.

OBJECTIVE: M. morganii is a gram-negative, non-lactose fermenting and an opportunistic pathogen frequently associated with nosocomial infections. Although first isolated in 1906 from a pediatric fecal sample, not many M. morganii isolates have been sequenced. The objective of this work is to determine the complete genome sequence of an XDR M. morganii strain (SMM01) isolated from the urine of a patient with urinary and fecal incontinence and to characterize its antimicrobial resistance profile. DATA DESCRIPTION: Here, we report the complete genome sequence of M. morganii SMM01 generated from the hybrid assembly of Illumina HiSeq X and Nanopore MinION reads. The assembly is 100% complete with genome size of 39,30,130 bp and GC content of 51%. Genomic features include 3617 CDS, 18 rRNAs, 78 tRNAs, 4 ncRNAs and 60 pseudogenes. Antimicrobial resistance profile was characterized by the presence of genes conferring resistance to aminoglycosides, ß-lactams, fluoroquinolones, chloramphenicol, and tetracyclines. Secondary metabolite biosynthetic gene clusters like NRPS, T1PKS, thiopeptide, beta-lactone, and bacteriocin were identified. The genome data described here would be the first complete genome of an Indian M. morganii isolate providing crucial information on antimicrobial resistance patterns, paving the way for further comparative genome analyses.

Genomic analysis of a multidrug-resistant Brucella anthropi strain isolated from a 4-day-old neonatal sepsis patient.

OBJECTIVES: Brucella anthropi is a Gram-negative, aerobic, motile, oxidase-positive, non-fermentative Alphaproteobacteria belonging to the family Brucellaceae. It is most commonly found in soil but is an emerging, opportunistic, nosocomial human pathogen. The objective of this study was to understand the genome features of a drug-resistant B. anthropi (SOA01) isolated from a blood culture of a 4-day-old neonate and to determine its antimicrobial resistance and pathogenic potential. METHODS: Hybrid genome assembly of B. anthropi strain SOA01 was generated using quality-trimmed short Illumina and long MinION reads. Identification and antimicrobial susceptibility profile were determined by MALDI-TOF, in silico ribosomal multilocus sequence typing (rMLST) and VITEK®2, respectively. PATRIC webserver and VFDB were used to identify antimicrobial resistance (AMR), virulence factor (VF) and transporter genes. RESULTS: Multidrug-resistant B. anthropi strain SOA01 has a genome of 4 975 830 bp with a G+C content of 56.29%. Several AMR, VF and transporter genes were identified in the genome. Antimicrobial susceptibility testing revealed resistance to different classes of antibiotics in strain SOA01. CONCLUSION: Brucella anthropi SOA01 is a multidrug-resistant strain. Several AMR and VF genes were identified in the genome, revealing the potential threat posed by this pathogen. The genome data generated in this study are likely to be useful in better understanding its AMR mechanisms, pathogenic potential and successful adaptation from its primary habitat of soil to the human system. Since it is often misidentified as Brucella melitensis or Brucella suis, genome characterisation and detailed understanding of its biology are crucial.

Draft Genome Sequence of Pandrug-Resistant Pseudomonas aeruginosa SPA03, Isolated from a Patient with Benign Prostatic Hyperplasia.

The draft genome of pandrug-resistant Pseudomonas aeruginosa strain SPA03, which belongs to global high-risk sequence type 357 (ST357) and was isolated from a patient with benign prostatic hyperplasia, is presented in this report. The genome assembly was generated by combining short-read Illumina HiSeq-X Ten and long-read Oxford Nanopore Technologies MinION sequence data using the Unicycler assembler.

Genomic and phylogenetic analysis of a multidrug-resistant Burkholderia contaminans strain isolated from a patient with ocular infection.

OBJECTIVES: The genus Burkholderia comprises rod-shaped, non-spore-forming, obligately aerobic Gram-negative bacteria that is found across diverse ecological niches. Burkholderia contaminans, an emerging pathogen associated with cystic fibrosis, is frequently isolated from contaminated medical devices in hospital settings. The aim of this study was to understand the genomic characteristics, antimicrobial resistance profile and virulence determinants of B. contaminans strain SBC01 isolated from the eye of a patient hit by a cow's tail. METHODS: A hybrid sequence of isolate SBC01 was generated using Illumina HiSeq and Oxford Nanopore Technology platforms. Unicycler was used to assemble the hybrid genomic sequence. The draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline. Antimicrobial susceptibility testing was performed by VITEK®2. Antimicrobial resistance and virulence genes were identified using validated bioinformatics tools. RESULTS: The assembled genome size is 8 841 722 bp with a G+C content of 66.33% distributed in 19 contigs. Strain SBC01 was found to possess several antimicrobial resistance and efflux pump genes. The isolate was susceptible to tetracyclines, meropenem and ceftazidime. Many genes encoding potential virulence factors were identified. CONCLUSION: Burkholderia contaminans SBC01 belonging to sequence type 482 (ST482) is a multidrug-resistant strain containing diverse antimicrobial resistance genes, revealing the risks associated with infections by new Burkholderia spp. The large G+C-rich genome has a myriad of virulence factors, highlighting its pathogenic potential. Thus, while providing insights into the antimicrobial resistance and virulence potential of this uncommon species, the present analysis will aid in understanding the evolution and speciation in the Burkholderia genus.

Resistance to nitrofurantoin is an indicator of extensive drug-resistant (XDR) Enterobacteriaceae.

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options.Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited.Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae.Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR.Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR (P=0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (ß-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1, bla NDM-1, bla OXA-48, ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible.Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae, harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.

Molecular Analyses of Biofilm-Producing Clinical Acinetobacter baumannii Isolates from a South Indian Tertiary Care Hospital.

OBJECTIVES: The aim of the study was to determine the presence of antimicrobial-resistance (AMR) genes, virulence genes, and mobile genetic elements (MGEs) in 14 biofilm-producing clinical isolates of Acinetobacter baumannii. MATERIALS AND METHODS: PCR amplification was performed to analyse the prevalence of genes associated with antibiotic resistance (extended-spectrum ß-lactamases [ESBLs] and metallo-ß-lactamases [MBLs]), virulence factors, MGEs (class 1 integron, Tn1213, and A. baumannii antibiotic resistance [AbaR]), and comM among the study isolates. Random amplified polymorphic DNA (RAPD) PCR was then deployed to understand their phylogenetic relationship. All the isolates were investigated for biofilm production. RESULTS: Two isolates were antibiotic-sensitive (AS), 3 were multi-drug-resistant (MDR), and the remaining 9 were extensively drug-resistant (XDR). The majority of the isolates were found to be positive for biofilm production and were sensitive against tetracycline and colistin only. Ab14 and Ab11 were found to be resistant to minocycline and colistin, respectively. blaTEM, blaOXA, blaNDM, blaVIM, blaSIM, and blaPER-1; class 1 integron; composite transposon Tn1213; AbaR island, and virulence factor genes were detected among the isolates. These pathogens were found to have originated from multiple clonal lineages. CONCLUSION: Biofilm-producing A. baumannii with multiple virulence and AMR genes pose serious clinical challenges. The presence of MGEs further compounds the situation as these isolates serve as potential reservoirs of AMR and virulence genes. Together with their capacity for natural competence, A. baumannii, if left unchecked, will lead to the spread of resistance determinants to previously sensitive bacteria and may aid in the emergence of untreatable pan-drug-resistant phenotypes.

Comparative genomic analysis of a naturally competent Elizabethkingia anophelis isolated from an eye infection.

Elizabethkingia anophelis has now emerged as an opportunistic human pathogen. However, its mechanisms of transmission remain unexplained. Comparative genomic (CG) analysis of E. anopheles endophthalmitis strain surprisingly found from an eye infection patient with twenty-five other E. anophelis genomes revealed its potential to participate in horizontal gene transfer. CG analysis revealed that the study isolate has an open pan genome and has undergone extensive gene rearrangements. We demonstrate that the strain is naturally competent, hitherto not reported in any members of Elizabethkingia. Presence of competence related genes, mobile genetic elements, Type IV, VI secretory systems and a unique virulence factor arylsulfatase suggests a different lineage of the strain. Deciphering the genome of E. anophelis having a reservoir of antibiotic resistance genes and virulence factors associated with diverse human infections may open up avenues to deal with the myriad of its human infections and devise strategies to combat the pathogen.

Detection of Antibiotic Resistance Determinants and Their Transmissibility among Clinically Isolated Carbapenem-Resistant Escherichia coli from South India.

OBJECTIVES: The aim of this study was to analyze the prevalence of the CTX-M, TEM, SHV, VIM, NDM, and OXA genes in carbapenemase-producing Escherichia coli and their transmissibility at a tertiary care hospital in south India. MATERIALS AND METHODS: Twenty-one carbapenem-resistant E. coli (carbapenem-resistant Enterobacteriaceae; CRE) were collected from the Sri Sathya Sai Institute of Higher Medical Sciences (Puttaparthi India). Resistance to antibiotics was analyzed by Vitek-2, and the identity of the isolates was confirmed by 16S rDNA sequencing. RAPD and enterobacterial repetitive intergenic consensus (ERIC)-PCR were performed for molecular typing. Metallo-ß-lactamase production was confirmed by a double disc synergy test. The presence of the extended-spectrum ß-lactamases CTX-M, TEM, and SHV and of the carbapenemases NDM, VIM, and OXA was determined by PCR. Carbapenemase variants were further confirmed by sequencing. The transmissibility of the genes was tested by conjugation. RESULTS: Twelve of the 21 (57%) carbapenem-resistant E. coli isolates were community acquired, indicating the spread of CRE in environmental samples. TEM and NDM-5 were found to be the major ß-lactamases produced by the pathogens. OXA-181 was found in 5 of the isolates. All 21 isolates were found to harbor more than one of the tested ß-lactamases, and all of the isolates were found to have the capacity to participate in conjugation; 15 of the transconjugants were found to have acquired the tested ß-lactamases, substantiating their ability to be transferred to other strains of bacteria. CONCLUSION: Monitoring of community-acquired carbapenem-resistant bacteria is very important as the association of resistance determinants with mobile genetic elements would present a serious clinical challenge.