Evaluation of Minimum Inhibitory Concentration of Heavy Metals Contained in Packaging Material Digest on Prominent Gut Microbiota.

Several scientific investigations have revealed that the leaching of metals from packaging material into the packed food is an unavoidable process. Hence, this study is aimed at investigating the effect of leached heavy metals from food packing materials on normal human gut flora. We analysed the effect of vanadium, arsenic, cadmium, and mercury present in digested packaging materials (DPM) on standard strains of Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70063, and Enterococcus faecalis ATCC 29212. The minimum inhibitory concentration (MIC) of laboratory-grade heavy metal salts and heavy metals present in DPM was determined by the agar dilution method. For all four bacteria, the MIC of cadmium and arsenic in the DPM was 7 µg/ml and 1.6 µg/ml, respectively. The MIC of mercury in DPM was 1.6 µg/ml for E. coli, K. pneumoniae, and E. faecalis and 1.4 µg/ml for P. aeruginosa. MIC of vanadium for E. coli, P. aeruginosa, and E. faecalis was 2.2 µg/ml, and for K. pneumoniae was 2.0 µg/ml. The difference in MICs of heavy metals in DPMs and heavy metal salts was not statistically significant. MICs were within CODEX-specified permissible levels. Though heavy metals in packaging material have not shown a deleterious effect on representative human gut flora, there is scope to study their effect on the gut microbiome. Thus, understanding the risk of heavy metal ingestion through unknown sources and avoiding any possible ingestion is crucial to preventing chronic diseases.

Molecular diagnosis of bacterial and viral diarrhoea using multiplex-PCR assays: An observational prospective study among paediatric patients from India.

PURPOSE: In developing countries, the aetiology of diarrhoea goes undiagnosed as only microscopy, stool culture or enzyme immunoassay are done to find the causative agent. The present study aims to detect common paediatric viral and bacterial diarrhoea pathogens by microscopy, stool culture for bacteria, and multiplex polymerase chain reaction (mPCR) for bacteria and virus detections. MATERIALS AND METHODS: Diarrheal stool samples (n â€‹= â€‹109) received at the laboratory from paediatric patients aged one month to 18 years were included in the study. They were cultured for common bacterial pathogens and simultaneously subjected to two multiplex PCRs one for the detection of Salmonella spp., Shigella spp., Enteroinvasive E.coli and Enteropathogenic E.coli, another for the detection of adenovirus, astrovirus, rotavirus and norovirus. RESULTS: Of the 109 samples cultured for bacterial aetiology, 0.9% (1/109) grew Salmonella enterica ser.Typhi and 2% (2/109) Shigella flexneri. By mPCR, 16% of samples (17/109) were positive for Shigella spp., 0.9% (1/109) for Salmonella spp., and 21% (23/109) for rotavirus. One sample (0.9%) had rotavirus and Shigella spp., which indicates mixed aetiology. CONCLUSIONS: Shigella spp. and rotavirus are the prime causative agents of childhood diarrhoea in our region. The rate of detection of bacterial aetiology by culture was poor. Isolation of pathogens by conventional culture helps to know the species, serotypes and antibiotic susceptibility of the pathogens. Virus isolation is cumbersome, time-consuming, and not available for routine diagnostic use. Therefore, real-time mPCR would be a better choice for early detection of pathogens, thereby ensuring timely diagnosis, treatment, and a reduction in mortality.

Phenotypic and genotypic characterization of clinical Pseudomonas aeruginosa.

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen that can cause many nosocomial infections. Biofilm formation, drug resistance, and motility contribute to virulence in P. aeruginosa. This study assessed the colistin minimum inhibitory concentration (MIC), biofilm formation, presence of mod A and psl A genes, and types of motilities in multidrug-resistant (MDR) and multidrug-susceptible (MDS) P. aeruginosa. Methods: Sixty-two P. aeruginosa from pus and 18 from urine samples were studied for their susceptibility to commonly used antibiotics, colistin MIC by agar dilution, and biofilm-forming ability by the microtiter plate method. All MDR and MDS P. aeruginosa isolates were tested for the presence of mod A and psl A genes by PCR, and different types of motilities using specific media. Results: Among the 40 MDR and 40 MDS isolates, 17 each were colistin-resistant and 23 each were colistin-intermediate. Nine MDR pus isolates and three MDR urine isolates showed all three types of motilities. Thirteen MDS pus isolates and four MDS urine isolates showed both swimming and swarming motility. MDS isolates did not show twitching motility. A higher number of MDR strains were strong biofilm producers (n = 19), whereas a higher number of MDS strains (n = 24) were moderate biofilm producers (p = 0.023). Twenty-seven MDR and twenty-eight MDS isolates were positive for both mod A and pslA genes. Among the strong biofilm-forming pus isolates, a greater number of MDR isolates (n = 13 each) had modA and pslA genes compared to MDS isolates (modA p = 0.017; pslA p = 0.014). Conclusions: Our findings clearly showed a statistically significant association among strong biofilm formation, modA, pslA genes, and drug resistance in P. aeruginosa isolated from clinical samples. Additional studies are needed to explore other genes and factors responsible for weak and moderate biofilm formation and drug resistance.

Heterogeneous Vancomycin Intermediate Staphylococcus aureus Infections in Diabetic and Non-Diabetic Patients - A Hospital-Based Comparative Study.

Purpose: To study the infections caused by methicillin resistant Staphylococcus aureus (MRSA) with emphasis on heterogeneous vancomycin intermediate S. aureus (hVISA) in diabetic and non-diabetic patients and their comparison. Patients and Methods: S. aureus strains isolated from diabetic and non-diabetic patients admitted in four tertiary care hospitals in Coastal Karnataka, South India, were tested for methicillin resistance and included in the present study. Demographic and clinical data of the patients were collected using structured proforma. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and MLSB phenotypes were identified using the D-test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar dilution. MRSA isolates were tested for hVISA using vancomycin screen agar and population analysis profile - area under the curve (PAP-AUC) test. Statistical analysis of the results was done using the chi-square test. SPSS version 29.0 was used for this purpose. Results: Out of 665 strains of S. aureus isolated, 220 (33.1%) were MRSA. Of these 220 MRSA strains, 122 (55.5%) and 98 (44.5%) were isolated from diabetic and non-diabetic patients, respectively. There was no significant difference in the antimicrobial resistance patterns of MRSA strains isolated from diabetic and non-diabetic patients. Foot infections and osteomyelitis caused by MRSA were significantly more among diabetic patients. Out of 220 strains of MRSA, 14 (6.4%) were hVISA. The rates of hVISA among MRSA isolated from diabetic and non-diabetic were 9.0% and 3.1%, respectively. This difference was statistically not significant. Conclusion: The rate of hVISA among all MRSA isolates was 6.4%. The risk of hVISA infection was three times more in diabetic patients. The results emphasize the importance of the detection of hVISA among MRSA isolates especially from diabetic patients.

Seropositivity for dengue and Leptospira IgM among patients with acute febrile illness: an indicator of co-infection?

INTRODUCTION: Presentation of febrile illness with nonspecific features, overlapping manifestations of dengue and leptospirosis, limited laboratory diagnostic tests, make the clinical diagnosis of pyrexia a challenge. The present study aimed to determine the prevalence of Leptospira and dengue IgM co-infection among acute febrile illness patients. METHODS: This is a retrospective hospital-based study which included patient data collected from June 2016 to May 2017. Inpatients' samples (n=2139) were tested for dengue and/or Leptospira IgM at the Microbiology Laboratory. Data like duration of fever, platelet count, hemoglobin, white blood cell count, erythrocyte sedimentation rate, results of liver and renal function tests, mode of treatment, were collected from medical records of laboratory-confirmed co-infection cases. RESULTS: Among 1612 serum samples tested for dengue IgM by ELISA, 382 (23.7%) were positive, 17 equivocal and 1213 were negative. Of the 811 Leptospira IgM ELISA done, 119 (14.7%) were positive, 17 equivocal and 675 negative. Two hundred eighty-four samples were tested for both infections and nine (3.2%) were positive for both and 275 were negative. These nine patients positive for dual infections showed elevated transaminases, alkaline phosphatase, serum bilirubin, creatinine, and blood urea, thrombocytopenia and leukocytosis. They received effective antibiotics along with supportive treatment and were cured of the infection. CONCLUSIONS: The study emphasizes the possibility of leptospirosis and dengue co-infection (3.2%) and need for confirmation by a highly specific test like PCR. If co-infection is suspected, treatment with specific antibiotics for leptospirosis and supportive treatment for dengue is mandatory, with due attention to complexity of organ involvement.

Antibiogram of Urinary Enterococcus Isolates from a Tertiary Care Hospital.

PURPOSE: Urinary tract infection (UTI) is one of the serious infections caused by the bacteria Enterococci. Vancomycin-Resistant Enterococci (VRE) is a persevering clinical problem globally. This study aims to detect high-level aminoglycoside and vancomycin resistance in uropathogenic Enterococcus spp. METHODOLOGY: A total of 75 clinically relevant Enterococcus spp. grown from urine samples, were collected following convenience non-random sampling method. Identified by standard biochemical tests and susceptibility to antibiotics was studied by Kirby Bauer's disc diffusion method. The MIC of vancomycin was detected by agar dilution test. Van A, and Van B genes in VREs were detected by PCR. RESULTS: Among 75 Enterococcal isolates, 43 (57.3%) were E. faecalis, 12 (16%) were E. faecium, 6 (8%) of each were E. pseudoavium and E. casseliflavus, 5(6.66%) were E. dispar and 3 (4%) were E. durans. E. faecalis (n=19) and E. faecium (n=3) were resistant to High Level Streptomycin (HLS). E. faecalis (n=21) and E. faecium (n=6) were resistant to High Level Gentamicin (HLG). 4 (9.3%) E. faecalis were vancomycin-resistant, out of which 3 were of Van A, and one was both Van A and Van B genotype. CONCLUSION: Isolation of high level aminoglycoside resistant (HLAR) Enterococci is a challenge for the treating physician because aminoglycoside cannot be used in combination with glycopeptide or ampicillin for such isolates. The occurrence of HLAR, Van A, and Van B VRE genotypes is a cause of concern as they may transfer drug resistance genes to other bacterial isolates, thus leading to limited therapeutic options.

Antibiogram, Virulence Genes, and Biofilm-Forming Ability of Clinical Salmonella enterica Serovars: An In Vitro Study.

Salmonella enterica serovar Typhi and Salmonella Paratyphi are causative agents of enteric fever. Salmonella Typhi persists as a biofilm on gallstones. Hence, we studied the biofilm formation, antibiogram, and virulence genes of S. enterica serovars. Antibiogram of S. enterica serovars from human blood and stool samples were studied by Kirby-Bauer disk diffusion method and biofilm by microtiter plate method. We studied the minimum inhibitory concentration of the isolates by Vitek-2 semiautomated system. Polymerase chain reaction was done to detect invA and spvC genes. Of the 55 isolates studied, 36 (65.45%) were Salmonella Typhi, 13 (23.63%) were Salmonella Paratyphi A, 2 (3.64%) were Salmonella Typhimurium, and 4 (7.28%) were Salmonella spp. Resistance to ciprofloxacin and nalidixic acid were found to be 81.8% and 92.7%, respectively. Chloramphenicol and cotrimoxazole-susceptible strains were 98.18%. One each of Salmonella Typhi, Salmonella Paratyphi A, and S. enterica isolates formed weak biofilm at 28°C. However, at 37°C eight Salmonella Typhi produced weak biofilm in the presence of bile. One Salmonella Paratyphi A and two Salmonella spp. formed weak biofilm in the absence of bile. All the isolates had the invA gene. Salmonella Typhimurium had invA and spvC genes. Bile may contribute to biofilm formation and persistence of the Salmonella Typhi on gallstones, which may lead to carrier state. Changing antibiotic susceptibility pattern of Salmonella serovars is observed in our geographic area. The presence of invA and spvC genes indicate the ability of invasiveness and intracellular survival.

Utility of smear microscopy and GeneXpert for the detection of Mycobacterium tuberculosis in clinical samples.

INTRODUCTION: Rapid identification of Mycobacterium tuberculosis (MTB), its resistance to rifampicin and differentiation of MTB from nontuberculous mycobacteria (NTM) is necessary in the management of mycobacterial diseases. Culture, the "gold standard" for the detection of MTB, is time consuming. In spite of its rapidity and low cost, smear microscopy has poor sensitivity for the detection of acid-fast bacilli (AFB). A real-time PCR based rapid diagnostic method like GeneXpert MTB/RIF assay can simultaneously detect M. tuberculosis and rifampicin (RIF) resistance. Hence, we aim to compare the performance of GeneXpert MTB/RIF assay with smear microscopy and culture. METHODS: In this descriptive cross-sectional study, we compared the performance of GeneXpert in pulmonary (N=127) and extrapulmonary (N=48) clinical specimens with other diagnostic methods like culture, Auramine O (AO), and Ziehl Neelsen (ZN) staining. Rifampicin resistance was detected only by GeneXpert. Demographic data and clinical history of the subjects were collected from the patient's hospital records. RESULTS: AO and ZN staining when compared with mycobacterial growth indicator (MGIT) culture showed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of 68.6, 95.7, 80, 92.4, 90.3% and 65.7, 95.7, 79.3, 91.8, 89.7%, respectively. The sensitivity, specificity, PPV, NPV and accuracy of GeneXpert was 88.6, 93.6, 77.5, 97.0 and 92.6%, respectively. CONCLUSIONS: GeneXpert is the best available rapid diagnostic method as it can detect MTB and rifampicin resistance gene simultaneously. Accuracy and negative predictive value of GeneXpert was found to be better than AFB staining. Thus, a negative GeneXpert test can rule out TB. Further, a negative GeneXpert and a positive smear microscopy results indicate the presence of NTM. However, GeneXpert is expensive and needs sophisticated instrument when compared to smear microscopy.

Antibiotic Susceptibility Pattern and Biofilm Formation in Clinical Isolates of Enterococcus spp.

Enterococcus is a commensal in the intestine and is now emerging as a drug-resistant pathogen. It produces different virulence factors. Enterococcus surface protein (esp) is a virulence factor that helps in the adhesion, but its role in biofilm formation is still contradictory. Moreover, in many bacterial species, strong biofilm producer exhibits multidrug resistance. Hence, this study is done to know the antimicrobial susceptibility pattern of Enterococcus spp. and to correlate the drug resistance with biofilm production and esp gene. Enterococcal isolates were collected from various clinical specimens. Antibiotic susceptibility testing was done by disc diffusion, and biofilm production was performed by microtiter plate method. PCR was performed for detection of esp gene. Two E. faecium strains resistant to vancomycin and high-level aminoglycoside (HLAR) were non-biofilm-producers and did not harbor esp gene. However, other biofilm-producing E. faecium harbored esp gene, and this association was found to be statistically significant (p=0.024). It was observed that there was no significant association between biofilm formation and presence of esp gene in E. faecalis. Moreover, a significant correlation was not found between drug resistance and biofilm production in both Enterococcus species. Thus, biofilm formation is not always associated with the presence or absence of esp gene and or drug resistance in Enterococcus spp.

Antifungal Susceptibility and Biofilm Production of Candida spp. Isolated from Clinical Samples.

OBJECTIVE: The study aims to speciate clinical Candida isolates and detect their biofilm-forming ability and antifungal resistance. METHODS: All the Candida spp. isolated from different clinical samples like pus, urine, blood, and body fluid were included in the study. Biofilm production was tested by the microtiter plate method. Antifungal susceptibility was studied by the disk diffusion method. Patient's demographic details such as age, sex, and clinical information were collected. Presence of other risk factors such as diabetes mellitus, history of antibiotic use, and any urinary tract instrumentations was also recorded. RESULTS: Among 90 Candida species isolated, most predominant species was found to be C. albicans (45.5%) followed by C. tropicalis (28.88%), C. krusei (20%), C. glabrata (3.33%), and C. parapsilosis (2.22%). Candida spp. were isolated from urine (43%), BAL/sputum (18.88%), high vaginal swab (8.88%), suction tips (7.77%), blood and wound swabs (6.66%), pus (3.33%), bile aspirate (2.22%), and deep tissue (1.11%). A larger number of females were affected than males, and the age group of 51 to 60 years was more susceptible to candidiasis. A higher number of C. albicans isolates produced biofilm followed by C. parapsilosis, C. tropicalis, and C. krusei. However, C. glabrata showed no biofilm production in our study. All Candida isolates were 100% sensitive to amphotericin B. Voriconazole was the next effective drug with 81.11% susceptibility. 24.44% of strains were resistant to fluconazole. CONCLUSION: Speciation of Candida isolates, detection of ability to form the biofilm, and monitoring of antifungal susceptibility testing are necessary for appropriate treatment.