Spontaneous Emergence of Azithromycin Resistance in Independent Lineages of Salmonella Typhi in Northern India.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) pose a major threat to the effective treatment and control of typhoid fever. The ongoing outbreak of extensively drug-resistant Salmonella Typhi (S. Typhi) in Pakistan has left azithromycin as the only remaining broadly efficacious oral antimicrobial for typhoid in South Asia. Ominously, azithromycin-resistant S. Typhi organisms have been subsequently reported in Bangladesh, Pakistan, and Nepal. METHODS: Here, we aimed to understand the molecular basis of AMR in 66 S. Typhi organisms isolated in a cross-sectional study performed in a suburb of Chandigarh in Northern India using whole-genome sequencing and phylogenetic analysis. RESULTS: We identified 7 S. Typhi organisms with the R717Q mutation in the acrB gene that was recently found to confer resistance to azithromycin in Bangladesh. Six out of the seven azithromycin-resistant S. Typhi isolates also exhibited triple mutations in gyrA (S83F and D87N) and parC (S80I) genes and were resistant to ciprofloxacin. These contemporary ciprofloxacin/azithromycin-resistant isolates were phylogenetically distinct from each other and from those reported from Bangladesh, Pakistan, and Nepal. CONCLUSIONS: The independent emergence of azithromycin-resistant typhoid in Northern India reflects an emerging broader problem across South Asia and illustrates the urgent need for the introduction of typhoid conjugate vaccines in the region.

Prevalence, Virulence Gene Profiling, and Characterization of Enteroaggregative Escherichia coli from Children with Acute Diarrhea, Asymptomatic Nourished, and Malnourished Children Younger Than 5 Years of Age in India.

OBJECTIVE: To study the significance of enteroaggregative Escherichia coli (EAEC) as a pathogen causing acute diarrhea and a commensal in healthy nourished and malnourished children younger than five years of age in the Chandigarh region and to address possible traits of EAEC virulence genes, biofilm formation, phylogroups, and antibiotic resistance that would be correlated with diarrhea or carriage. STUDY DESIGN: Stool samples were obtained from children with acute diarrhea (n = 548), as well as nourished (n = 550), and malnourished controls without diarrhea (n = 110). E coli isolates were confirmed as EAEC by pCVD432 polymerase chain reaction. Multiplex polymerase chain reactions were used to identify 22 virulence-related genes and phylogeny. Antibiotic susceptibility, adherence, and biofilm-forming potential also were studied. RESULTS: Overall, 16.6% of children were malnourished. EAEC detection was greater among children with acute diarrhea (16%) than nourished (6%) and malnourished nondiarrheal controls (2.7%). We found an association of EAEC infections with age <2 years (P = .0001) in the diarrheal group. Adhesive variants adhesion fimbriae IV and adhesion fimbriae II were significantly associated with diarrhea. The aggR and aar genes showed a positive and negative association with the severity of disease (P = .0004 and P = .0003). A high degree of multidrug resistance was found (73.8%) in the diarrheal group. Most EAEC strains from the diarrheal group belonged to B2 and D phylogroups, whereas strains from non-diarrheal groups, which belonged to phylogroup B1. CONCLUSIONS: EAEC is a significant contributor to childhood diarrhea, its presence as a commensal, and the significance of the association of various virulence factors among the EAEC isolated from diarrheal and non-diarrheal stools. These data reinforce the importance of aggR and aar as positive and negative regulators and the contribution of AAF/II and AAF/IV fimbria for the pathobiology of EAEC.

Extensively Drug-Resistant Klebsiella pneumoniae Associated with Complicated Urinary Tract Infection in Northern India.

Klebsiella pneumoniae (Kp), which is associated with hospital-acquired infections, is extensively drug-resistant (XDR), making treatment difficult. Understanding the genetic epidemiology of XDR-Kp can help determine its potential to be hypervirulent (hv) through the presence of siderophores. We characterized the genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated tract infection at an Indian healthcare facility. The 18 organisms comprised the following sequence types (STs): ST14 (n = 9), ST147 (n = 5), ST231 (n = 2), ST2096 (n = 1), and ST25 (n = 1). Many patients in each ward were infected with the same ST, suggesting a common source of infection. Some patients had recurrent infections with multiple STs circulating in the ward, providing evidence of hospital transmission. ß-lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in 15 isolates, blaOXA-1 in 16 isolates, blaTEM-1D in 13 isolates, and blaOXA-48 in 3 isolates. Disruption of mgrB by various insertion sequences was responsible for colistin resistance in 6 isolates. The most common K-type among isolates was K2 (n = 10). One XDR convergent hvKp ST2096 mutation (iuc+ybt+blaOXA-1+blaOXA-48) was associated with prolonged hospitalization. Convergent XDR-hvKp has outbreak potential, warranting effective antimicrobial stewardship and infection control.

Are non-lactose-fermenting Escherichia coli important diarrhoeal pathogens in children and adults?

Introduction: Diarrhoeagenic Escherichia coli (DEC) remains one of the major causes of acute diarrhoea episodes in developing countries. The percentage of acute diarrhoea cases caused by DEC is 30-40 % in these countries. Approximately 10% of E. coli isolates obtained from stool specimens have been reported to be non-lactose-fermenting (NLF). The available literature is sparse regarding the pathogenicity of NLF E. coli causing infectious diarrhoea. Aim: We aimed to elucidate the importance of NLF E. coli in causing diarrhoea in both adults and children by detecting various DEC pathotypes among NLF E. coli in stool samples taken from gastroenteritis cases. Material and Methods: A total of 376 NLF E. coli isolates from 3110 stool samples from diarrhoea/gastroenteritis patients were included in the study. Up to three NLF colonies that were not confirmed as Vibrio cholerae , Aeromonas spp., Salmonella spp. or Shigella spp., but were identified as E. coli using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), were carefully picked up from each MacConkey agar plate and then meticulously streaked onto freshly prepared, sterilized nutrient agar plates, and biochemical reactions were conducted. Multiplex PCR was conducted for the EAEC, EPEC, ETEC and EHEC pathotypes and PCR for the ipaH gene was conducted for EIEC. The disc diffusion method was used for antibiotic sensitivity testing. Results: Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, with EAEC being the most predominant (n=31) followed by EIEC (n=22), EPEC (n=8) and ETEC (n=2). To further differentiate EIEC from Shigella , additional biochemical tests were performed, including acetate utilization, mucate and salicin fermentation, and aesculin hydrolysis. Antimicrobial susceptibility testing (AST) showed that maximum resistance was seen against ciprofloxacin (82.5 %) followed by ampicillin (77.8 %) and cotrimoxazole (68.2 %), and minimum resistance was seen against ertapenem (4.8 %). Conclusion: In our study two pathotypes (EAEC, EIEC) were predominant among NLF E. coli and these were not only important aetiological agents in children, but also in adults. Our study also sheds light on the epidemiology of EIEC, which is one of the most neglected DEC pathotypes, as hardly any microbiological laboratories process NLF E. coli for EIEC.

Exploring Synergistic Combinations in Extended and Pan-Drug Resistant (XDR and PDR) Whole Genome Sequenced Acinetobacter baumannii.

Background: The diminishing antimicrobial options for the treatment of XDR and PDR Acinetobacter baumannii is an increasing concern. In this study, we assessed the in vitro synergy of the fosfomycin (FOS) with meropenem (MEM), amikacin (AK), tigecycline (TGC), and colistin (CL) in whole genome sequenced isolates. Methods: Non-replicate whole genome sequenced (illumina next-generation sequencing platform, Clevergene, India), A. baumanii (7 XDR, 1PDR) were subjected to in vitro synergy testing by checkerboard (CB) and time kill assay (TKA) after MIC determination, with glucose-6-phosphate being incorporated in all runs. FOS was used as a cornerstone drug in four combinations and colistin in one. ResFinder, MLST, PlasmidFinder, and CSIPhylogeny tools were used. Results: Mortality occurred in three patients. Diverse MLST were observed, ST-1962 (3 isolates) and one each of ST2062, ST2063, ST1816, ST1806, ST234. FOS MICs ranged from 32 to 128 mg/L, MEM MIC: 16-64 mg/L, TGC MIC: ≤2-≤4 mg/L and AK MIC: >512 mg/L. CL: MIC range, 0.25-≤2 mg/L, PDR MIC > 16 mg/L. Synergy results by CB: FOS-MEM: synergy in ⅞ (90%) isolates. Synergy lowered MEM MICs to susceptibility breakpoints in 6/8 cases. CL-MEM: Excellent synergy (3/3) isolates. FOS-AK: Indifference in ⅞, antagonism ⅛ (AK-susceptible isolate). FOS-TGC: Partial synergy (PS) in 8/8 (TGC MIC dropped to ≤0.25 mg/L in 3/8). In the PDR isolate, synergy was seen in FOS-MEM, CL-MEM, PS in FOS-CL, FOS-TGC, indifference in FOS-AK. TKA: Excellent synergy was observed with FOS-MEM from 4 h, while FOS-AK and FOS-TGC demonstrated synergy at 24 h. Synergy was achieved despite presence of widespread resistance markers against aminoglycosides (AacAad, AadA, AadB, Aph3″Ia, ArmA, Arr, StrA, StrB), beta-lactams (ADC, BlaA1, BlaA2, Zn-dependent_hydrolase, OXA-23, OXA-51, PER-1,TEM-1D, CARB-5, Mbl), sulphonamides (SulII, SulI), phenicols (CatBx, CmlA), macrolides (MphE, MsrE) and tetracycline (TetB) were widespread. Carbapenemase, CARB-5 was present in one isolate. Beta-lactamase genes OXA-23, OXA-51, BlaA2, Zn-dependent_hydrolase, ADC, Mbl and macrolide resistance genes MphE, MsrE were present in all 8 isolates. Conclusions: FOS-MEM and CL-MEM are promising combinations against A. baumannii. Synergy of FOS-MEM in intrinsically resistant A. baumannii shows that this antibiotic combination might be useful in treating such XDR and PDR pathogens.

The genomic characterization of Salmonella Paratyphi A from an outbreak of enteric fever in Vadodara, India.

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines.

Phenotypic and genotypic characterization of antimicrobial resistance in clinical isolates of Vibrio cholerae over a decade (2002-2016).

PURPOSE: Emergence and spread of resistance among Vibrio cholerae have become a global public health problem. In India, no consolidated data is available on antimicrobial susceptibility patterns and antibiotic resistance genes. METHODS: A total of 110 representative isolates obtained over a period of 14 years were included. Antimicrobial susceptibility was tested by disc diffusion and micro broth dilution. Presence of 13 antimicrobial resistance genes was ascertained by using PCR. RESULTS: Antimicrobial resistance fluctuated for most of the antibiotics. Resistance to cotrimoxazole in our study was 92.72% and the SXT element was present in all isolates. Resistance to nalidixic acid, tetracycline, and cefotaxime was found to be 98.18%, 7.27%, and 10.9% respectively. Resistance to ampicillin saw a fluctuating trend with a recent fall. Resistance to ciprofloxacin and azithromycin was 12.72% and 29% by MIC. blaTEM was the most common ESBL gene (94.5%). Other were blaCMY (26.36%) and blaNDM (2.7%). We report blaCTX-M-15 and blaOXA-48 and ermB for the first time in the world. Newer antimicrobials like prulifloxacin and rifaximin were tested for the first time from India. CONCLUSIONS: Our study has shown very high levels of resistance to older antibiotics and the emergence of resistance to some of the newer classes of antibiotics. There is an urgent need for increased surveillance studies, rational use of the antimicrobials and preventive measures to control the disease.

Inland cholera in freshwater environs of north India.

In the freshwater environment of north India, cholera appears seasonally in form of clusters as well as sporadically, accounting for a significant piece of the puzzle of cholera epidemiology. We describe a number of cholera outbreaks with an average attack rate of 96.5/1000 but an overall low case fatality (0.17). Clinical cholera cases coincided with high rainfall and elevated temperatures, whereas isolation of V. cholerae non-O1 non-O139 from water was dependent on temperature (p < 0.05) but was independent of rainfall and pH (p > 0.05). However, isolation from plankton samples correlated with increased temperature and pH (p < 0.05). A lag period of almost a month was observed between rising temperature and increased isolation of V. cholerae from the environment, which in succession was followed by an appearance of cholera cases in the community a month later. Our results suggested that the aquatic environment can harbor highly divergent V. cholerae strains and serve as a reservoir for multiple V. cholera virulence-associated genes that may be exchanged via mobile genetic elements. In agreement with PFGE, AFLP data also proved that the V. cholerae O1 population was not clonal but was closely related. Our investigation did not support the concept that seasonal cholera outbreaks occur by movement of a single clonal strain across the region, as the clinical isolates from the same years were clearly different, implying that continuous evolution of V. cholerae O1 strains occurs in the cholera endemic area. Interestingly, the viable but non-culturable (VBNC) V. cholerae O1 cells were demonstrated in 2.21% samples from natural water bodies in addition to 40.69% samples from cholera-affected areas respectively. This suggests that aquatic environs do harbor the pathogenic O1 strain, though the isolation of culturable V. cholerae O1 is a rare event in the presence of relatively abundant non-O1 non-O139 isolates.

Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E. coli in India.

Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that causes acute and chronic diarrhea in developed and industrialized nations in children. EAEC epidemiology and the importance of atypical EAEC (aEAEC) isolation in childhood diarrhea are not well documented in the Indian setting. A comparative analysis was undertaken to evaluate virulence, phylogeny, and antibiotic sensitivity among typical tEAEC versus aEAEC. A total of 171 EAEC isolates were extracted from a broad surveillance sample of diarrheal (N = 1210) and healthy children (N = 550) across North India. Polymerase chain reaction (PCR) for the aggR gene (master regulator gene) was conducted to differentiate tEAEC and aEAEC. For 21 virulence genes, we used multiplex PCR to classify possible virulence factors among these strains. Phylogenetic classes were identified by a multiplex PCR for chuA, yjaA, and a cryptic DNA fragment, TspE4C2. Antibiotic susceptibility was conducted by the disc diffusion method as per CLSI guidelines. EAEC was associated with moderate to severe diarrhea in children. The prevalence of EAEC infection (11.4%) was higher than any other DEC group (p = 0.002). tEAEC occurrence in the diarrheal group was higher than in the control group (p = 0.0001). tEAEC strain harbored more virulence genes than aEAEC. astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination was present in more than 50% of strains. Also, 75.8% of EAEC strains were multidrug-resistant (MDR). Phylogroup D (43.9%) and B1 (39.4%) were most prevalent in the diarrheal and control group, respectively. Genetic analysis revealed EAEC variability; the comparison of tEAEC and aEAEC allowed us to better understand the EAEC virulence repertoire. Further microbiological and epidemiological research is required to examine the pathogenicity of not only typical but also atypical EAEC.

Phenotypic and molecular characterization of antimicrobial resistant Escherichia coli from urinary tract infections in Port-Harcourt, Nigeria.

INTRODUCTION: Multidrug resistance among Escherichia coli causing Urinary Tract Infections (UTIs) is a major public health problem, threatening the effective treatment of UTIs. This study investigated the phenotypic and molecular characteristics of E. coli associated with UTIs in Port-Harcourt, Nigeria. METHODS: Twenty-five non-duplicate isolates of E. coli from UTIs patients at the University of Port-Harcourt Teaching Hospital, Nigeria were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. The antimicrobial susceptibility patterns were determined using Kirby-Bauer disc diffusion technique. Phenotypic expression of Extended Spectrum Beta Lactamases (ESBLs) and AmpC beta-lactamase were determined using standard laboratory methods and polymerase chain reaction (PCR) was used to detect ESBLs, AmpC, Quinolones and Aminoglycosides resistance genes. RESULTS: The isolates exhibited high rates of resistance to co-trimoxazole (76%), nalidixic acid (68%), ciprofloxacin (60%), gentamicin (44%) and low resistance to cefotaxime (20%) but were fully susceptible to cefoperazone/sulbactam, amikacin, nitrofurantoin, colistin and carbapenems. Phenotypic expression of ESBLs was recorded in 6(24%) isolates while genotypic detection revealed the highest prevalence of blaTEM 22(88%), followed by blaCTX-M-15 16(64%), blaSHV 7(28%) and blaOXA-1 6(24%) while AmpC (blaCMY-2) gene was detected in 8(32%) isolates. Amongst the quinolone resistant isolates, qnr variants (qnrB, qnrD and qnrS) and aac(6')-Ib genes were detected in 7(28%) and 3(12%) isolates respectively while all gentamicin resistant isolates possessed the aacC2 gene. The co-expression of blaCTX-M-15 with quinolones and aminoglycoside genes were 20% and 40% respectively. The prevalence of multiple drug resistance was 52%. CONCLUSION: A high proportion of the studied E. coli isolates co-expressed ESBLs, quinolones and aminoglycosides resistance genes which call for prompt antibiotic stewardship and preventive strategies to limit the spread of these genes.

Evaluation of risk factors for colistin resistance among uropathogenic isolates of Escherichia coli and Klebsiella pneumoniae: a case-control study.

INTRODUCTION: The last few years have seen the emergence of multi-drug resistant (MDR) Gram-negative infections, which are associated with high morbidity and mortality. The indiscriminate use of colistin has led to the development of resistance, which can be diagnosed effectively by broth microdilution. Studies from India are limited, and this study was conducted in order to determine the prevalence and risk factors associated with colistin resistance. METHODS: Urine samples from patients admitted with urinary tract infection (UTI), growing MDR Escherichia coli and Klebsiella pneumoniae, were tested for the minimum inhibitory concentration (MIC) of colistin by broth microdilution. Isolates with an MIC >2 µg ml-1 (resistant) were subjected to polymerase chain reaction (PCR) for the mcr1, mcr2 and mgrB genes. A case-control study with 21 cases (resistant) and 42 matched controls (sensitive) was designed to evaluate risk factors and outcomes (recurrent UTI, readmission and hospital stay >2 weeks). RESULTS: Two hundred and fifty MDR isolates (E. coli=142/2319 and K.pneumoniae=108/775) from 216 patients were selected from the 25 046 isolates screened. Twenty-five isolates (20 K.pneumoniae and 5 E. coli) were resistant to colistin, with a prevalence of 3.52  % in E. coli and 18.5  % in K. pneumoniae among the MDR isolates. PCR for the mcr1 and mcr2 genes was negative. Multivariate regression showed that multiple episodes of hospitalization, hospital stay >2 weeks, exposure to >three antibiotic classes and abnormality/surgery of the lower urinary tract were the significant risk factors for colistin resistance. Previous use of colistin and colistin resistance had a significant effect on all outcomes. CONCLUSIONS: K. pneumoniae show six times higher prevalence of colistin resistance than E. coli, and the emergence of resistant organisms has led to an increase in morbidity in infected patients.

Endemic fluoroquinolone-resistant Salmonellaenterica serovar Kentucky ST198 in northern India.

Salmonellaenterica serovar Kentucky is an emergent human pathogen. Human infection with ciprofloxacin-resistant S. enterica Kentucky ST198 has been reported in Europe and North America as a consequence of travel to Asia/the Middle East. This is, to the best of our knowledge, the first study reporting the identification of this epidemic clone in India and South Asia.