Changes in antimicrobial resistance of Escherichia coli isolated from community-associated urinary tract infection before and during the COVID-19 pandemic in India.

PURPOSE: The impact of the COVID-19 pandemic on antimicrobial resistance (AMR) is largely studied in healthcare settings. There is a need to understand the fluctuations in AMR during pandemic at the community level. With urinary tract infection (UTI) being one of the most common infections in the community, the AMR profile of community-acquired UTI (CA-UTI) is considered representative AMR at the community level. METHODS: The study was taken in a cohort of patients with a clinical diagnosis of CA-UTI. The four study sites represented different community health centres in India. Escherichia coli isolates were analysed phenotypically and genotypically for AMR pre-COVID (October 2019-February 2020) and in the first (March 2020-February 2021) and second waves of COVID-19 (March 2021-December 2021). RESULTS: E. coli was the predominant uropathogen (229, 82%). Increased susceptibility to nitrofurantoin was observed during the pandemic. Reduced susceptibility to first-line oral antibiotics and carbapenems was seen during the second wave, and an increased minimum inhibitory concentration (MIC50) to beta-lactams and fluoroquinolones was seen during the pandemic. Genomic analysis of E. coli isolates showed some AMR genes (aacC1, aacC4, SHV, QepA) only during the second wave. CONCLUSION: One good outcome of the pandemic was increased susceptibility to nitrofurantoin, while drawback was a significant decrease in susceptibility to oral antibiotics during the second wave and increased MIC50 of some antibiotics. Decreased susceptibility to last-resort carbapenems and the occurrence of various AMR genes during the second wave of the pandemic are of great concern.

Systems-level profiling of early peripheral host-response landscape variations across COVID-19 severity states in an Indian cohort.

Host immune response to COVID-19 plays a significant role in regulating disease severity. Although big data analysis has provided significant insights into the host biology of COVID-19 across the world, very few such studies have been performed in the Indian population. This study utilizes a transcriptome-integrated network analysis approach to compare the immune responses between asymptomatic or mild and moderate-severe COVID-19 patients in an Indian cohort. An immune suppression phenotype is observed in the early stages of moderate-severe COVID-19 manifestation. A number of pathways are identified that play crucial roles in the host control of the disease such as the type I interferon response and classical complement pathway which show different activity levels across the severity spectrum. This study also identifies two transcription factors, IRF7 and ESR1, to be important in regulating the severity of COVID-19. Overall this study provides a deep understanding of the peripheral immune landscape in the COVID-19 severity spectrum in the Indian genetic background and opens up future research avenues to compare immune responses across global populations.

Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity.

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. Identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

Genome profiling of uropathogenic E. coli from strictly defined community-acquired UTI in paediatric patients: a multicentric study.

BACKGROUND: Urinary tract infection (UTI) in children is a common bacterial infection. The emergence of extended-spectrum beta-lactamases (ESBLs) poses a major challenge against the treatment of uropathogens. We aimed to characterize the E. coli isolates recovered from children with UTI for their resistance profile and circulating sequence types (ST). METHODS: Children (> 1.5-18 years of age) from different community health centres of India with symptoms of UTI were enrolled. Isolates causing significant bacteriuria were identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and tested for antimicrobial susceptibility by the automated system, VITEK-2 (Biomeriux, Durhum, US). Nineteen E. coli isolates (15 ESBL positive and 4 ESBL negative) were sequenced in Oxford Nanopore platform followed by core-genome phylogeny, accessory genome cluster analysis, identification of sequence types, mobile genetic elements, genetic antimicrobial resistance markers. The correlation between detection of antimicrobial resistance genes with phenotypic resistance profiles was also investigated. RESULTS: Eleven percent of children had significant bacteriuria [male:female-1:1, > 50% were 11-18 years of age group]. E. coli was predominant (86%) followed by K. pneumoniae (11%). Susceptibility of E. coli was highest against fosfomycin (100%) followed by carbapenems (90.7%) and nitrofurantoin (88.8%). ST131 (15.8%) and ST167 (10.5%) found as high-risk clones with the presence of plasmid [IncFIB (63.1%), IncFIA (52.6%)], and composite transposon [Tn2680 (46.6%)] in many isolates. Few isolates coharboured multiple beta-lactamases including blaNDM-5 (33.3%), blaOXA-1 (53.3%), blaCTX-M-15 (60%) and blaTEM-4 (60%). CONCLUSIONS: This study highlights horizontal transmission of resistance genes and plasmids in paediatric patients at community centers across the nation harbouring multidrug-resistant genes such as blaNDM-5 and blaCTX-M-15 associated with high-risk clones ST131 and ST167. The data is alarming and emphasizes the need for rapid identification of resistance markers to reduce the spread in community. To our knowledge, this is the first multicentric study targeting paediatric UTI patients from the community setting of India.

Antibiotic resistance of uropathogens among the community-dwelling pregnant and nonpregnant female: a step towards antibiotic stewardship.

BACKGROUND: Indiscriminate and widespread use of antibiotics has resulted in emergence of many antibiotic-resistant organisms. Antibiotic administration during pregnancy is mostly avoided, unless there is compelling medical condition. We hypothesized that the uropathogens isolated from pregnant women would be more susceptible to antibiotics compared to those isolated from nonpregnant women, thus will be helpful in formulating separate empiric guideline for pregnant women based on the resistance pattern. METHODS: This was a prospective cross-sectional study conducted over a period of 2 years in which females with the clinical diagnosis of either cystitis or asymptomatic bacteriuria during pregnancy were included from the community settings. Uropathogen species and their antimicrobial resistance pattern were compared between the pregnant and nonpregnant groups. After accounting for centre-to-centre variation and adjusting for age and socio-economic status, the adjusted odds ratio for antibiotic resistance was calculated and compared between pregnant and nonpregnant women using logistic regression analysis. RESULTS: A total of 1758 women (pregnant: 43.3%; nonpregnant: 56.6%) were screened in the study over a period of 2 years, out of which 9.3% (163/1758) were having significant bacteriuria. Escherichia coli and Klebsiella pneumoniae were the two commonest uropathogen in both the groups; their prevalence being 83.6% in pregnant women and 85.2% in nonpregnant women, respectively. Resistance against ampicillin, cefixime, cefoxitin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid were found significantly lower in the pregnant women compared to nonpregnant. After adjusting the age and socio-economic status accounting for centre-to-centre variation, the odds of resistance for cefixime, amoxicillin-clavulanic acid and co-trimoxazole were found lower and statistically significant among the pregnant women group. CONCLUSIONS: The antimicrobial resistance was significantly higher among the community-dwelling nonpregnant women compared to pregnant women in case of few antibiotics. The study highlighted the need of building local antibiogram that could help to initiate the empirical treatment and thus prevent emergence of antimicrobial resistance.

Culture-Independent Raman Spectroscopic Identification of Bacterial Pathogens from Clinical Samples Using Deep Transfer Learning.

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Simple and low-cost nucleic acid extraction methods for detection of SARS-CoV2 in self-collected saliva and dry oral swabs.

Background: Ongoing need of alternative strategies for SARS-CoV-2 detection is undeniable. Self-collected samples without viral transport media (VTM), coupled with simple nucleic acid extraction methods for SARS-CoV-2 PCR are beneficial. Objectives: To evaluate results of SARS-CoV-2 PCR using simple nucleic acid extraction methods from self -collected saliva and oral swabs without VTM. Methods: A cross-sectional single-centre study was conducted on 125 participants (101 SARS-CoV-2 positive cases and 24 controls). PCR was performed following five simple nucleic acid extraction methods on self -collect saliva and oral swabs without VTM and results were compared with gold standard PCR. For saliva, kit-based extraction (SKE), Proteinase K and Heat extraction (SPHE), only Heat extraction (SHE) methods and for dry oral swabs, Proteinase K and Heat extraction (DPHE) and only Heat extraction (DHE) was performed. Results: SARS-CoV-2 was detected in self-collected saliva and oral swabs. 93.07% were correctly classified as positive by SKE, 69.31% by SHE, 67.33% by SPHE, 67.33% by DPHE and 55.45% by DHE. Discriminant power of SKE was significantly higher than other methods (p-value < 0.001) with good- fair agreement of alternate extraction methods against gold standard. Conclusion: Combination of self-collected saliva/ oral-swab without VTM and alternative RNA extraction methods offer a simplified, economical substitute strategy for SARS-CoV-2 detection.

A 9-gene biomarker panel identifies bacterial coinfections in culture-negative COVID-19 cases.

Confirmatory diagnosis of bacterial coinfections with COVID-19 is challenging due to the limited specificity of the widely used gold-standard culture sensitivity test despite clinical presentations. A misdiagnosis can either lead to increased health complications or overuse of antibiotics in COVID-19 patients. With a multi-step systems biology pipeline, we have identified a 9-gene biomarker panel from host blood that can identify bacterial coinfection in COVID-19 patients, even in culture-negative cases. We have also formulated a qPCR-based score that diagnoses bacterial coinfection with COVID-19 with the accuracy, specificity, and sensitivity of 0.93, 0.96, and 0.89, respectively. This gene signature and score can assist in the clinical decision-making process of necessary and timely prescription of antibiotics in suspected bacterial coinfection cases with COVID-19 and thereby help to reduce the associated morbidity and mortality.

Virulence gene mutations as a differentiator of clinical phenotypes: insights from community-acquired uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) remains an important cause of urinary tract infection during pregnancy. Multiple molecular virulence determinants and antibiotic resistant genes facilitate its pathogenesis and virulence phenotype. Hence it is hypothesized that there will be considerable variation in genes among the isolates from symptomatic as well as asymptomatic bacteriuria (ABU) during pregnancy. The aim of this study was to decipher the genetic variation among the two phenotypes. Six different UPEC isolates collected from urine specimens of consecutive pregnant females (five, symptomatic bacteriuria and one, ABU) were tested for their growth kinetics, and biofilm formation. A total of 87 virulence determinants and 56 antibiotic resistance genes were investigated using whole-genome sequencing, to identify putative drives of virulence phenotype. In this analysis, we identified eight different types of fully functional toxin antitoxin (TA) systems [HipAB, YefM-YoeB, YeeU-YeeV (CbtA), YhaV-PrlF, ChpBS, HigAB, YgiUT and HicAB] in the isolates from symptomatic bacteriuria; whereas partially functional TA system with mutations were observed in the asymptomatic one. Isolates of both the groups showed equivalent growth characteristics and biofilm-formation ability. Genes for an iron transport system (Efe UOB system, Fhu system except FhuA) were observed functional among all symptomatic and asymptomatic isolates, however functional mutations were observed in the latter group. Gene YidE was observed predominantly associated with the biofilm formation along with few other genes (BssR, BssS, YjgK, etc.). This study outlines putative critical relevance of specific variations in the genes for the TA system, biofilm formation, cell adhesion and colonization among UPEC isolates from symptomatic and asymptomatic bacteriuria among pregnant women. Further functional genomic study in the same cohort is warranted to establish the pathogenic role of these genes.

Mycobacterium Species on the Cutaneous Microbiome of Very Preterm Neonates.

The neonatal skin microbiome consists of all the genomes and genetic products of microorganisms harboring on an infant's skin. Host and the microbiota develop a harmonious environment resulting in symbiosis. Any disruption of this environment could lead to pathological disease. This study was conducted to understand the neonatal skin microbiome of very preterm neonates (under 32 weeks) admitted to the Neonatal Intensive Care Unit(NICU) at a tertiary healthcare setting before and after kangaroo mother care (KMC), using next-generation sequencing (NGS). Skin swabs were collected on two different occasions and analyzed using the NGS technique after amplification via polymerase chain reaction. The results showed relative abundance for Mycobacterium tuberculosis in 83.33% and 66.67% (p = 0.29) and Mycobacteroides abscessus in 100% and 93.33% (p = 0.30) of the very preterm neonates on the skin microbiome before and after KMC, respectively as an incidental finding. The mere presence of these bacilli as commensals or as potential pathogens is alarming due to the risk of early exposure and incidence of tuberculosis from birth. These findings, in our view, are the first findings to be established in such a setting.

Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India.

Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.

Outbreak of Pichia kudriavzevii fungaemia in a neonatal intensive care unit.

PURPOSE: Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, Pichia kudriavzevii (a teleomorph of Candida krusei). To the best of our knowledge, this is the first report of neonatal sepsis due to P. kudriavzevii. METHODOLOGY: Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed. RESULTS: Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as P. kudriavzevii and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices. CONCLUSION: This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.

Detection and antifungal susceptibility testing of oral Candida dubliniensis from human immunodeficiency virus-infected patients.

CONTEXT: Candida dubliniensis, an opportunistic yeast that has been implicated in oropharyngeal candidiasis (OPC) in patients infected with Human Immunodeficiency Virus (HIV) may be under-reported due to its similarity with Candida albicans. Resistance to Fluconazole is often seen in C. dubliniensis isolates from clinical specimens. AIMS: To know the prevalence of C. dubliniensis in OPC in patients infected with HIV and their antifungal susceptibility pattern. SETTINGS AND DESIGN: One hundred and thirty-two HIV seropositive individuals and 50 healthy controls were included in the study. MATERIALS AND METHODS: Two oral swabs were collected from the site of the lesion from 132 HIV-infected patients. Oral rinse was obtained from 50 healthy controls. Samples were inoculated on Sabouraud's dextrose agar (SDA) medium and on HiCrome Candida Differential Agar (CHROM agar) medium. Isolates were speciated by standard tests. Dark green-colored, germ tube positive isolates, which failed to grow at 420C and negative for xylose assimilation were identified as C. dubliniensis. Antifungal susceptibility test was performed by Macro broth dilution technique (National Committee for Clinical Laboratory Standards guidelines). RESULTS AND CONCLUSIONS: From 132 patients, 22 (16.3%) C. dubliniensis were isolated; samples from healthy controls did not reveal their presence. Antifungal susceptibility test showed higher resistance among C. dubliniensis isolates to azoles compared to C. albicans. Five (22.7%) isolates of C. dubliniensis were resistant to Fluconazole followed by four (18.2%) to Ketoconazole. This study emphasizes the importance of identification and antifungal susceptibility testing of C. dubliniensis in HIV-infected patients.

Significance of isolation and drug susceptibility testing of non-Candida albicans species causing oropharyngeal candidiasis in HIV patients.

Oropharyngeal candidiasis (OPC) continues to be a common opportunistic infection in patients infected with Human Immunodeficiency Virus (HIV) and is predictive of increasing immunosuppression. Though Candida albicans remains the predominant isolate, a rise in the frequency of isolation of non-albicans Candida (NAC) species is being observed. The levels of virulence and the sensitivities to available antifungal drugs vary among these species. Of 340 HIV seropositive patients in this study, 132 (38.8%) had oral lesions suggestive of candidiasis. Samples were collected from the lesion using sterile cotton swabs. Isolation and speciation were done by standard techniques. Antifungal drug susceptibility testing was done by macro broth dilution. The total number of Candida isolates was 135, of which, 45 (33.3%) were NAC species and 90 were C.albicans (66.6%). Of the NAC species, C. dubliniensis was the predominant pathogen (22,48.9%). Antifungal susceptibility testing showed that 14 (31.1%) of the NAC species and 11 (12.2%) of C. albicans were resistant to fluconazole (MIC > 8 microg/ml). A very high MIC of > 32 microg/ml was noted among the NAC species resistant to fluconazole.