Hypervirulent Capsular Serotypes K1 and K2 Klebsiella pneumoniae Strains Demonstrate Resistance to Serum Bactericidal Activity and Galleria mellonella Lethality.

Hypervirulent Klebsiella pneumoniae (hvKp) is a variant that has been increasingly linked to severe, life-threatening infections including pyogenic liver abscess and bloodstream infections. HvKps belonging to the capsular serotypes K1 and K2 have been reported worldwide, however, very scarce studies are available on their genomics and virulence. In the current study, we report four hypermucoviscous extended-spectrum ß-lactamase-producing hvKp clinical strains of capsular serotype K1 and K2 isolated from pus and urine of critically ill patients in tertiary care hospitals in Oman. These strains belong to diverse sequence types (STs), namely ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2). To study their virulence, a Galleria mellonella model and resistance to human serum killing were used. The G. mellonella model revealed that the K1/ST-23 isolate was the most virulent, as 50% of the larvae died in the first day, followed by isolate K2/ST-231 and K2/ST-14, for which 75% and 50% of the larvae died in the second day, respectively. Resistance to human serum killing showed there was complete inhibition of bacterial growth of all four isolates by the end of the first hour and up to the third hour. Whole genome sequencing (WGS) revealed that hvKp strains display a unique genetic arrangement of k-loci. Whole-genome single-nucleotide polymorphism-based phylogenetic analysis revealed that these hvKp isolates were phylogenetically distinct, belonging to diverse clades, and belonged to different STs in comparison to global isolates. For ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2), there was a gradual decrease in the number of colonies up to the second to third hour, which indicates neutralization of bacterial cells by the serum components. However, this was followed by a sudden increase of bacterial growth, indicating possible resistance of bacteria against human serum bactericidal activity. This is the first report from Oman detailing the WGS of hvKp clinical isolates and assessing their resistance and virulence genomics, which reinforce our understanding of their epidemiology and dissemination in clinical settings.

Presence of metallo-beta-lactamases (MBL), extended-spectrum beta-lactamase (ESBL) & AmpC positive non-fermenting Gram-negative bacilli among Intensive Care Unit patients with special reference to molecular detection of blaCTX-M & blaAmpC genes.

BACKGROUND & OBJECTIVES: Non-fermenting Gram-negative bacilli (NFGNB) including Pseudomonas aeruginosa and Acinetobacter baumannii have been implicated in a variety of infections, particularly in the Intensive Care Units (ICUs). This study was aimed to overview the burden of multidrug-resistant NFGNB causing infections in ICU and also to assess the occurrence of extended-spectrum beta-lactamases (ESBLs), AmpC and metallo-beta-lactamases (MBLs) among these isolates. METHODS: Bacterial culture, identification and antibiotic susceptibility were carried out. ESBLs and AmpC were detected both phenotypically and genotypically. MBL was detected by modified Hodge and imipenem-ethylenediaminetetraacetic acid double-disc synergy test. RESULTS: NFGNB represented 45 (37%) of total 121 Gram negative isolates. Multidrug resistance was observed in 66.9 per cent and 72.5 per cent isolates of P. aeruginosa and A. baumannii, respectively. Detection by phenotypic methods showed presence of ESBL, AmpC and MBL in 21.4, 51.1 and 21.4 per cent isolates, respectively. When detected genotypically by polymerase chain reaction, ESBL and AmpC were detected in 21.4 and 41.4 per cent of NFGNB isolates, respectively. BlaCTX-M (21.4%) was the most prevalent gene responsible for ESBL production. INTERPRETATION & CONCLUSIONS: Most of the NFGNB isolated from ICU patients were multidrug-resistant and producers of ESBL, AmpC and MBL. A regular surveillance is required to detect ESBL, AmpC and MBL producers, especially in ICU patients.

Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

Regional variations in antimicrobial susceptibility of community-acquired uropathogenic Escherichia coli in India: Findings of a multicentric study highlighting the importance of local antibiograms.

Objectives: Evidence-based prescribing is essential to optimize patient outcomes in cystitis. This requires knowledge of local antibiotic resistance rates. Diagnostic and Antimicrobial Stewardship (DASH) to Protect Antibiotics (https://dashuti.com/) is a multicentric mentorship program guiding centers in preparing, analyzing and disseminating local antibiograms to promote antimicrobial stewardship in community urinary tract infection. Here, we mapped the susceptibility profile of Escherichia coli from 22 Indian centers. Methods: These centers spanned 10 Indian states and three union territories. Antibiograms for urinary E. coli from the outpatient departments were collated. Standardization was achieved by regional online training; anomalies were resolved via consultation with study experts. Data were collated and analyzed. Results: Nationally, fosfomycin, with 94% susceptibility (inter-center range 83-97%), and nitrofurantoin, with 85% susceptibility (61-97%), retained the widest activity. The susceptibility rates were lower for co-trimoxazole (49%), fluoroquinolones (31%), and oral cephalosporins (26%). The rates for third- and fourth-generation cephalosporins were 46% and 52%, respectively, with 54% (33-58%) extended-spectrum ß-lactamase prevalence. Piperacillin-tazobactam (81%), amikacin (88%), and meropenem (88%) retained better activity; however, one center in Delhi recorded only 42% meropenem susceptibility. Susceptibility rates were mostly higher in South, West, and Northeast India; centers in the heavily populated Gangetic plains, across north and northwest India, had greater resistance. These findings highlight the importance of local antibiograms in guiding appropriate antimicrobial choices. Conclusions: Fosfomycin and nitrofurantoin are the preferred oral empirical choices for uncomplicated E. coli cystitis in India, although elevated resistance in some areas is concerning. Empiric use of fluoroquinolones and third-generation cephalosporins is discouraged, whereas piperacillin/tazobactam and aminoglycosides remain carbapenem-sparing parenteral agents.

Emergence of drug resistance-associated mutations in HIV-1 subtype C protease gene in north India.

A major cause of anti-retroviral therapy (ART) failure is the drug resistance-associated mutations in polymerase gene of HIV-1. Paucity of data regarding potential drug resistance to protease inhibitors (PIs) prompted us to carry out this study. Drug resistance (DR) genotyping of the entire protease gene was performed in 104 HIV-1 ART-naive and first-line ART-experienced patients. The DR pattern was analyzed using the Stanford HIV-DR database, International AIDS Society-USA mutation list and REGA algorithm version 8.0. Subtyping was done using Mega 4 and REGA HIV-1 subtyping tool-v2.01. Majority of our sequences 98 (96%) were subtype C and remaining four (3.92%) were subtype A1. In three (2.9%) DE patients, major DR-associated mutation at D30 N and M46I positions were detected. Approximately 70% polymorphisms as minor mutations were observed in protease gene, of which 14 distinct amino acids changes were linked to partial DR such as G16E, K20R, M36I, D60E, I62V, L63P, I64M, H69K, T74A/S, V77I, V82I, I85V, L89M, and I93L. The two major and several minor mutations detected in this study confer low/intermediate levels of resistance to most PIs independently or together. Our results conclude that resistance testing in HIV-1-infected patients should be performed before the initiation of PI therapy for better therapeutic outcome in this region. This information not only will shed light on the extent of current DR in HIV strains but also will aid in patient treatment and drug designing.

Prevalence and assessment of role of SEN virus in acute and chronic hepatitis in India.

BACKGROUND AND AIM: SEN virus (SENV), is a recently discovered single-stranded DNA virus of Annelloviridae family and is believed may play a role in non A-E hepatitis. We conducted this study to identify the prevalence and clinical association of SENV with acute and chronic hepatitis. METHODS: 135 liver disease patients were studied. Extent of liver damage was assessed using the Model for End Stage Liver Disease (MELD) score. A-E viruses and HIV were detected by enzyme immunoassay. Nested PCR was performed for detection of SENV and its genotypes D and H. RESULTS: 34 cases (25.18%) were positive for SEN virus DNA, a statistically significant finding (p < 0.01) of which 22 (64%) had acute viral hepatitis, 4 (11.76%) had chronic viral hepatitis, 3 (8.82%) fulminant hepatic failure and 5 (14.70%) cirrhosis. Mean AST was 47.85 IU/L, ALT 51.2 IU/L and INR 1.73, mean MELD score was 18.38 (11 to 24). 17.64% had severely deranged MELD score. SENV-D genotype was detected in 13 (38%) and SENV-H in 19 (58%) cases. SENV-H occurred in both acute (53%) and chronic hepatitis (47%). SENV-D was strongly associated with acute hepatitis (85%). Cirrhotic and FHF cases were SENV-H positive. 12 (44.11%) were co-infected with HBV, 5 (14.7%) with TTV, 4(11.76%) with HEV, 2 (5.88%) with HCV and 5 (14.4%) with HIV. CONCLUSION: Significant prevalence of SENV in hepatitis patients was observed. On the basis of clinical findings and abnormal liver function tests, we conclude that SENV appears to be not only hepatotropic but also capable of liver damage. Higher prevalence of SENV-H in cirrhotics may point to its possible role in the development of cirrhosis.

Comparative Genomic Analysis Reveals the Emergence of ST-231 and ST-395 Klebsiella pneumoniae Strains Associated with the High Transmissibility of blaKPC Plasmids.

Conjugative transposons in Gram-negative bacteria have a significant role in the dissemination of antibiotic-resistance-conferring genes between bacteria. This study aims to genomically characterize plasmids and conjugative transposons carrying integrons in clinical isolates of Klebsiella pneumoniae. The genetic composition of conjugative transposons and phenotypic assessment of 50 multidrug-resistant K. pneumoniae isolates from a tertiary-care hospital (SQUH), Muscat, Oman, were investigated. Horizontal transferability was investigated by filter mating conjugation experiments. Whole-genome sequencing (WGS) was performed to determine the sequence type (ST), acquired resistome, and plasmidome of integron-carrying strains. Class 1 integrons were detected in 96% of isolates and, among integron-positive isolates, 18 stains contained variable regions. Horizontal transferability by conjugation confirmed the successful transfer of integrons between cells and WGS confirmed their presence in conjugative plasmids. Dihydrofolate reductase (dfrA14) was the most prevalent (34.8%) gene cassette in class 1 integrons. MLST analysis detected predominantly ST-231 and ST-395. BlaOXA-232 and blaCTX-M-15 were the most frequently detected carbapenemases and beta-lactamases in the sequenced isolates. This study highlighted the high transmissibility of MDR-conferring conjugative plasmids in clinical isolates of K. pneumoniae. Therefore, the wise use of antibiotics and the adherence to effective infection control measures are necessary to limit the further dissemination of multidrug-resistant bacteria.

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.

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC ß-lactamases: is routine detection possible?

Background: Phenotypic characterization of the prevalent AmpC ß-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. Methods: A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. Results: The prevalence of AmpC ß-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. Conclusions: We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Trends of antimicrobial resistance in patients with complicated urinary tract infection: Suggested empirical therapy and lessons learned from a retrospective observational study in Oman.

Background: Complicated urinary tract infection (cUTI) is defined as an infection associated with structural, functional, or metabolic abnormalities of the genitourinary tract. These infections are caused frequently by multidrug-resistant Gram-negative bacilli. The rapid emergence of extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase (CR) producers has made the treatment of such infections increasingly more challenging. Objectives: The aims of the present study were threefold: to assess the clinical profile, trends in etiology, and antimicrobial susceptibility profile in cUTI over the past 10 years at a tertiary care center in Oman as an interrupted time series on the one hand and to develop guidelines for empirical management of such cases on the other. Materials and Methods: We conducted a retrospective analysis of cUTI in patients presenting at Sultan Qaboos University Hospital over 3 years (2008, 2013, and 2018) covering a span of 10 years. Data were obtained from the patient's electronic records in the hospital information system. Analysis was done using the Statistical Package for Social Sciences program (SPSS), version 23. Results: Among the 650 cases of cUTI, 284 (44%) were males and 366 (56%) were females, with dysuria being the most common symptom (34%). The biggest risk factor for developing cUTI was diabetes (35%). The predominant pathogen was Escherichia coli (53%), followed by Klebsiella spp. (16%), Enterococcus faecalis (7%), Pseudomonas aeruginosa (7%), Candida spp. (2%), and Enterobacter cloacae (2%). Over the years, E. coli emerged as the predominant ESBL and AmpC producer, Acinetobacter baumannii as the multidrug-resistant bug, and Klebsiella pneumoniae as the major carbapenem-resistant Enterobacterales (CRE) producer. Nitrofurantoin emerged as the most effective drug for cystitis. Aminoglycosides, piperacillin-tazobactam, and carbapenems demonstrated the highest activity with an overall resistance of less than 10%. Higher resistance (30%) was observed against cephalosporins, fluoroquinolones, and trimethoprim/sulfamethoxazole. Analysis of the 10-year trend threw up some unexpected results. As expected, resistance increased from 2008 to 2013. Surprisingly, however, antimicrobial resistance in 2018 was lower against majority of the antimicrobials compared to 2013. Conclusion: There is a paucity of data for developing evidence-based guidelines management of cUTI. Targeted antibiograms and not cumulative antibiograms are essential for promoting appropriate prescribing and optimizing patient care. The welcome decline in resistance may be attributed cascade reporting, introduction of more ID physicians. Another possibility is increased utilization of fluoroquinolones which spared the other groups of antimicrobials. Judicious heterogeneous mixing of antimicrobials should be spearheaded in both cystitis and pyelonephritis so that there is no undue pressure on one drug. We strongly recommend carbapenem-sparing protocols in treatment of cUTI when anticipating augmented resistance due to AmpC production. Synergistic combinations such as piperacillin-tazobactam plus aminoglycosides/fluoroquinolones may be prescribed. In sepsis, however, carbapenems are the drugs of choice.

Assessment of In-Vitro Synergy of Fosfomycin with Meropenem, Amikacin and Tigecycline in Whole Genome Sequenced Extended and Pan Drug Resistant Klebsiella Pneumoniae: Exploring A Colistin Sparing Protocol.

Fosfomycin has emerged as a very useful antimicrobial in management of extremely drug resistant (XDR) and pan drug resistant (PDR) Klebsiella pneumoniae. In this study, we assessed in-vitro synergy of colistin sparing combinations of fosfomycin (FOS) with meropenem (MEM), tigecycline (TGC) and amikacin (AK) against XDR and PDR Klebsiella pneumoniae. METHOD: Non-replicate fully characterised 18 clinical isolates of K. pneumoniae (15 XDR and 3 PDR strains) were subjected to in-vitro synergy testing by checkerboard and time kill assay. Combinations tested were FOS-MEM, FOS-TGC and FOS-AK with glucose-6-phosphate being incorporated in all runs.WGS was carried out on the Illumina next-generation sequencing platform. RESULTS: FOS-MEM and FOS-AK both demonstrated excellent synergy against all PDRs and all but one XDR. Synergy led to lowering of MICs to susceptible breakpoints. FOS-TGC demonstrated antagonism. MLST-231 K. pneumoniae predominated (14), followed by ST-395 (3) and ST147 (1). Majority harboured OXA-232 (n = 15), while n = 2 carried NDM-1 type and n = 1 co-carried NDM-5 + OXA-232. Mortality was high in both ST-231 (57.1%) and ST-395 (66.6%). Synergy was observed despite widespread presence of resistance markers against aminoglycosides [aph(3')-Ic, aacA4, and rmtf], beta-lactams [blaSHV-11, blaTEM-1b, blaCTX-M-15, and blaOXA-232], fosfomycin [fosA6 and fosA5] and presence of porin proteins OmpK37, OmpA and K. pneumoniae antibiotic efflux pumps Kpn F, H, G, and E. CONCLUSION: FOS + MEM and FOS + AK are excellent colistin sparing combinations against ST 231, ST-395 and ST-147 XDR and PDR K. pneumoniae. FOS with fewer side effects than colistin, excellent tissue distribution and minimal side effects may be recommended in combination with meropenem.

Whole Genome Sequencing of Methicillin-Resistant Staphylococcus epidermidis Clinical Isolates Reveals Variable Composite SCCmec ACME among Different STs in a Tertiary Care Hospital in Oman.

Staphylococcus epidermidis has been recently recognized as an emerging nosocomial pathogen. There are concerns over the increasing virulence potential of this commensal due to the capabilities of transferring mobile genetic elements to Staphylococcus aureus through staphylococcal chromosomal cassette (SCCmec) and the closely related arginine catabolic mobile element (ACME) and the copper and mercury resistance island (COMER). The potential pathogenicity of S. epidermidis, particularly from blood stream infections, has been poorly investigated. In this study, 24 S. epidermidis isolated from blood stream infections from Oman were investigated using whole genome sequence analysis. Core genome phylogenetic trees revealed one third of the isolates belong to the multidrug resistance ST-2. Genomic analysis unraveled a common occurrence of SCCmec type IV and ACME element predominantly type I arranged in a composite island. The genetic composition of ACME was highly variable among isolates of same or different STs. The COMER-like island was absent in all of our isolates. Reduced copper susceptibility was observed among isolates of ST-2 and ACME type I, followed by ACME type V. In conclusion, in this work, we identify a prevalent occurrence of highly variable ACME elements in different hospital STs of S. epidermidis in Oman, thus strongly suggesting the hypothesis that ACME types evolved from closely related STs.

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1.

PURPOSE: Carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India. METHODS: Fifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis. RESULTS: Isolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 â€‹% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 â€‹h detected 92.3 â€‹% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 â€‹h at cut off 17 â€‹mm while for NDM-1 CIM was the test of choice at 16 â€‹mm. CONCLUSION: CIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 â€‹h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 â€‹mm was considered adequate for detection of OXA-48-like and 16 â€‹mm of NDM-1 by mCIM at 4 â€‹h and CIM respectively.

Acute lower respiratory tract infections in children.

Acute lower respiratory tract infection (ALRTI) is a common illness, but there have been relatively few studies of the bacterial etiology in developing countries. Nasopharyngeal aspirates of 70 children under 10 years of age with ALRTI were cultured for aerobic bacterial pathogens. Klebsiella pneumoniae was the commonest organism (32.2%) isolated followed by S. pneumoniae (10%), E. coli (10%), P. aeruginosa (5.7%), S. aureus (2.8%) and H. influenzae (1.4%). There were significantly more bacterial pathogens isolated in children <1 year of age (73.7%) than in those > 1 year of age (56.2%) (P=0.03). A shift in spectrum from Gram-positive cocci to Gram negative bacilli in ALRTI was observed in our study.

Molecular characterization and phylogenetic analysis of human pathogenic Leptospira species circulating in a tertiary care hospital of Western Uttar Pradesh in India.

Leptospirosis is identified as an important reemerging zoonotic disease distributed worldwide, caused by Leptospira. This study was carried out to explore the genetic characterization and its phylogenetic analysis of circulating Leptospira species, among the Aligarh region of western Uttar Pradesh in India, utilizing secY gene-based nucleotide sequence. A total of 190 human samples were included in the study. Positive samples were identified by ELISA, MAT and PCR. MAT was carried out utilizing local circulating Leptospira serovars. Four positive samples including two MAT positive samples were subjected to DNA sequencing for further confirmation and phylogenetic tree was constructed. Out of the total of 190 samples, 24 patients were found positive by ELISA and 29 by PCR. Two samples were found reactive in MAT with L. interrogans serovars like hebdomadis and copenhageni. Phylogenetic analysis of four isolates based on partial secY gene nucleotide sequences revealed that species obtained from the Aligarh region clustered with the several published pathogenic Leptospira interrogans, while some of our isolates nucleotide sequences also clustered with the published sequence of intermediate and saprophytic Leptospira serovars like Leptospira inadai and Leptospira meyeri. This pilot study will help us to decipher the present scenario of circulating serovars of leptospira as well as to identify the nucleotide changes in secY gene, in this region.

ROS mediated antibacterial activity of photoilluminated riboflavin: A photodynamic mechanism against nosocomial infections.

Nosocomial infections are a major threat to modern therapeutics. The major causative agent of these infections is multidrug-resistant gram-negative bacteria, which impart high morbidity and mortality rate. This has led to an urge for the development of new antibiotics. Antimicrobial photodynamic therapy is a promising strategy to which till date no resistant strain has been reported. Since the efficacy of photodynamic therapy largely depends on the selection and administration of an appropriate photosensitizer, therefore, the realization of clinically active photosensitizers is an immediate need. Here, by using E. coli as a study model we have demonstrated the antimicrobial photodynamic potential of riboflavin. Intracellular ROS formation by DCFH-DA assay, lipid peroxidation, protein carbonylation, LDH activity was measured in treated bacterial samples. Enzymatic (SOD, CAT, GSH) antioxidants and non-enzymatic (GSH) was further evaluated. Bacterial death was confirmed by colony forming assay, optical microscopy and scanning electron microscopy. The treated bacterial cells exhibited abundant ROS generation and marked increment in the level of oxidative stress markers as well as significant reduction in LDH activity. Marked reduction in colony forming units was also observed. Optical microscopic and SEM images further confirmed the bacterial death. Thus, we can say that photoilluminated riboflavin renders the redox status of bacterial cells into a compromised state leading to significant membrane damage ultimately causing bacterial death. This study aims to add one more therapeutic dimension to photoilluminated riboflavin as it can be effectively employed in targeting bacterial biofilms occurring on hospital wares causing several serious medical conditions.

Rising prevalence of enteric fever due to multidrug-resistant Salmonella: an epidemiological study.

A prospective study of the prevalent aetiology of enteric fever was undertaken at a tertiary care hospital in North India at intervals of every 3 years. Salmonella spp. were isolated from 174 (7%) patients. Amongst these, 140 (80%) patients were infected by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and 16 (9%) by S. enterica serovar Paratyphi A; the remaining 11% were infected by other S. enterica serogroups, Typhimurium, Paratyphi C and Senftenberg, and other group E salmonella. A significantly greater number of S. Typhi were isolated in the summer and monsoon months. Multidrug resistance (resistance to chloramphenicol, ampicillin and co-trimoxazole) sequentially increased from 34% in 1999 to 66% in 2005. Increasing resistance was also noticed to the other antibiotics, especially to the cephalosporins. Moreover 8% of the S. Typhi isolates were found to be presumptive extended spectrum beta-lactamase producers. There was a gradual development of resistance to fluoroquinolones over the 7 years. No resistance was observed to fluoroquinolones in 1999, while in 2005 4.4% resistance was observed to sparfloxacin, 8.8% resistance to ofloxacin and a high resistance, 13%, to ciprofloxacin. This is an alarming development and it is of paramount importance to limit unnecessary use of fluoroquinolones and third generation cephalosporins so that their efficacy against salmonella is not jeopardized further.

Stomatococcus mucilaginosus meningitis in a healthy 2-month-old child.

Stomatococcus mucilaginosus is a Gram-positive, catalase-variable organism considered part of the normal human oral and upper respiratory tract flora. Although traditionally believed to be an organism of low virulence, Stomatococcus mucilaginosus has been reported to be an opportunistic pathogen in immunocompromised patients. We describe what we believe is the first reported case of Stomatococcus mucilaginosus meningitis in a healthy child. The isolate was multidrug-resistant, susceptible only to vancomycin. The patient was treated successfully with vancomycin after initial trials with amikacin and cefotaxime.

Alarming resistance of Neisseria gonorrhoeae in a tertiary care hospital of North India.

Background: This study attempted to elucidate the spectrum of sexually transmitted infections in a tertiary care centre in North India and to assess the antimicrobial resistance in Neisseria gonorrhoeae. Materials and Methods: Antimicrobial resistance pattern of N. gonorrhoeae was determined by the standard techniques. Genotypic detection of gyrA, parC and blaTEM genes was also carried out. The results of gyrA gene by polymerase chain reaction were confirmed by DNA sequencing. Results: N. gonorrhoea was identified in 10 (4.98%) patients, and antimicrobial sensitivity was performed in seven patients. All the seven patients tested were quinolone-resistant N. gonorrhoeae (QRNG), 5/7 were penicillinase-producing N. gonorrhoeae, 1/7 was chromosomally mediated penicillin-resistant N. gonorrhoeae and 3/7 were tetracycline-resistant N. gonorrhoeae. Minimal inhibitory concentration (MIC) by E-test was performed in five strains, and we observed that MIC90 for ciprofloxacin was ≥4 µg/ml, for penicillin was ≥6 µg/ml and for tetracycline was 12 µg/ml, which clearly brackets them as resistant isolates. The presence of TEM gene was confirmed genotypically in six out of seven cases. In all seven cases, gyrA and parC were observed, thus confirming their QRNG status. Conclusion: Alarming increase in the resistance to commonly used antimicrobials for gonorrhoea in our study, especially of fluoroquinolones, is a clarion call for the urgent need for prudence in prescribing them. Observing the rampant resistance exhibited by N. gonorrhoeae, it is clear that the day is not far when it will acquire a superbug status and become intractable to treatment by the available antibiotics.