Spatiotemporal genome diversity of SARS-CoV-2 in wastewater: a two-year global epidemiological study.

Wastewater surveillance locally and globally is important for the investigation of the molecular epidemiological features of SARS-CoV-2 in the environment. The current study investigated the genomic diversity and mutation profile of SARS-CoV-2 variants in wastewater for the period spanning COVID-19 pandemic up to December, 2022. A total of 3618 complete SARS-CoV-2 genome sequences from waste water samples submitted to the GISAID database were retrieved. The SARS-CoV-2 sequences were subjected to pairwise alignment against reference, followed by clade and lineage assignment (based on Nextstrain, GISAID and Pango), distance metric phylogenetic analysis, and detection of substitution mutations. Following GISAID, Nextstrain, and Pango nomenclatures, an overall agreement in clade and lineage determination in wastewater samples was observed. There was successive appearance, dissemination, and disappearance of SARS-CoV-2 lineages along time in wastewater. The SARS-CoV-2 genomes from wastewater were clustered into the variants of concern (VOC) as Alpha GRY (B.1.1.7 + Q.7), Delta GK (B.1.617.2 + AY.*), and Omicron GRA (BA.1*, BA.2* + B.1.1.529, BA.5*). The evolutionary rate was 9.63e-04 substitutions/site/year for SARS-CoV-2 in wastewater. B.1.1.7 was less prevalent than B.1.617.2 in 2021, appeared in succession, and BA.1, BA.2, BA.5 were serially detected in 2022, the latter strain continued to persist in wastewater. The N501Y, E484K/Q, K417N/T, L452R, T478K spike substitutions remained dominant attribute of SARS-CoV-2 VOCs. The study underlines the importance of wastewater surveillance for enumerating spatiotemporal diversity of SARS-CoV-2 variants and mutations, which might pave the way for novel antiviral and vaccine designing towards management and prevention of SARS-CoV-2 infection.

Principal component analysis exploring the association between antibiotic resistance and heavy metal tolerance of plasmid-bearing sewage wastewater bacteria of clinical relevance.

This paper unravels the occurrence of plasmid-mediated antibiotic resistance in association with tolerance to heavy metals among clinically relevant bacteria isolated from sewage wastewater. The bacteria isolated were identified following conventional phenotypic and/or molecular methods, and were subjected to multiple-antibiotic resistance (MAR) profiling. The isolates were tested against the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. SDS-PAGE and agarose gel electrophoretic analyses were performed, respectively, for the characterization of heavy metal stress protein and R-plasmid among the isolated bacteria. Principal component analysis was applied in determining bacterial resistance to antibiotics and heavy metals. Both lactose-fermenting ( Escherichia coli ) and non-fermenting ( Acinetobacter baumannii and Pseudomonas putida ) Gram-negative bacterial strains were procured, and showed MAR phenotypes with respect to three or more antibiotics, along with resistance to the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. The Gram-positive bacteria, Enterococcus faecalis , isolated had 'ampicillin-kanamycin-nalidixic acid' resistance. The bacterial isolates had MAR indices of 0.3-0.9, indicating their ( E. faecalis , E. coli , A. baumannii and P. putida ) origin from niches with high antibiotic pollution and human faecal contamination. The Gram-negative bacteria isolated contained a single plasmid (≈54 kb) conferring multiple antibiotic resistance, which was linked to heavy metal tolerance; the SDS-PAGE analysis demonstrated the expression of heavy metal stress proteins (≈59 and ≈10 kDa) in wastewater bacteria with a Cd2+ stressor. The study results grant an insight into the co-occurrence of antibiotic resistance and heavy metal tolerance among clinically relevant bacteria in sewage wastewater, prompting an intense health impact over antibiotic usage.

A rare case of childhood Hepatitis A infection with pleural effusion, acalculous cholecystitis, and ascites.

Hepatitis A is common among children of developing nations. Young children with Hepatitis A infection usually have a mild form of the disease. Serious manifestations like pleural effusion and acalculous cholecystitis are very rare in Hepatitis A infection in young children. There have been some reports of these manifestations of childhood Hepatitis A occurring in isolation but for these to co-exist, is extremely rare. In this article a young child with Hepatitis A infection who had all these three manifestations of pleural effusion, acalculous cholecystitis and ascites together, is reported.

Indigenous Probiotic Lactobacillus Isolates Presenting Antibiotic like Activity against Human Pathogenic Bacteria.

BACKGROUND: Indigenous lactic acid bacteria are well known probiotics having antibacterial activity against potentially pathogenic bacteria. This study aims to characterize the curd lactobacilli for their probiotic potentiality and antagonistic activity against clinical bacteria. METHODS: Four curd samples were processed microbiologically for the isolation of lactic acid bacteria (LAB). The LAB strains obtained were identified by conventional methods: cultural aspect, gram-staining, biochemical and sugar fermentation tests. The probiotic properties were justified with tolerance to low-pH, bile salt and sodium chloride, and the antagonistic activity of the lactobacilli against human pathogenic bacteria (Escherichia coli, Proteus vulgaris, Acinetobacter baumannii and Salmonella enterica serovar Typhi) was assessed. Hemolytic activity and antibiotic susceptibility were determined for the lactobacilli isolates, and the cumulative probiotic potential (CPP) values were recorded. RESULT: Four lactobacilli isolates, L. animalis LMEM6, L. plantarum LMEM7, L. acidophilus LMEM8 and L. rhamnosus LMEM9, procured from the curd samples, survived in low-pH and high bile salt conditions, and showed growth inhibitory activity against the indicator bacteria by agar-well (zone diameter of inhibition; ZDIs: 13.67 ± 0.58-29.50 ± 2.10 mm) and agar overlay (ZDIs: 11.33 ± 0.58-35.67 ± 2.52 mm) methods; the average growth inhibitory activity of lactobacilli ranged 233.34 ± 45.54-280.56 ± 83.67 AU/mL, against the test bacterial pathogens. All the lactobacilli were non-hemolytic and sensitive to most of the test antibiotics. The CPP values of the isolated LAB were recorded as 80-100%. CONCLUSION: The curd lactobacilli procured might be used as the valid candidates of probiotics, and bio-therapeutics against bacterial infection to humans.

Human cystic echinococcosis: epidemiologic, zoonotic, clinical, diagnostic and therapeutic aspects.

This review represents an updated scenario on the transmission cycle, epidemiology, clinical features and pathogenicity, diagnosis and treatment, and prevention and control measures of a cestode parasite Echincoccus granulosus (E. granulosus) infection causing cystic echinococcosis (CE) in humans. Human CE is a serious life-threatening neglected zoonotic disease that occurs in both developing and developed countries, and is recognized as a major public health problem. The life cycle of E. granulosus involves a definitive host (dogs and other canids) for the adult E. granulosus that resides in the intestine, and an intermediate host (sheep and other herbivores) for the tissue-invading metacestode (larval) stage. Humans are only incidentally infected; since the completion of the life cycle of E. granulosus depends on carnivores feeding on herbivores bearing hydatid cysts with viable protoscoleces, humans represent usually the dead end for the parasite. On ingestion of E. granulosus eggs, hydatid cysts are formed mostly in liver and lungs, and occasionally in other organs of human body, which are considered as uncommon sites of localization of hydatid cysts. The diagnosis of extrahepatic echinococcal disease is more accurate today because of the availability of new imaging techniques, and the current treatments include surgery and percutaneous drainage, and chemotherapy (albendazole and mebendazole). But, the wild animals that involve in sylvatic cycle may overlap and interact with the domestic sheep-dog cycle, and thus complicating the control efforts. The updated facts and phenomena regarding human and animal CE presented herein are due to the web search of SCI and non-SCI journals.

Enhancing chloramphenicol and trimethoprim in vitro activity by Ocimum sanctum Linn. (Lamiaceae) leaf extract against Salmonella enterica serovar Typhi.

OBJECTIVE: To evaluate the antibacterial activity of Ocimum sanctum (O. sanctum) leaf extract, alone, and in combination with chloramphenicol (C) and trimethoprim (Tm) against Salmonella enterica serovar Typhi (S. typhi). METHODS: The antibacterial activity of ethanolic extract of tulsi, O. sanctum, leaf (TLE; 500 µg) for 23 S. typhi isolates was determined following agar diffusion. The C (30 µg) and Tm (5 µg) activity alone and in combination with TLE (250 µg) was determined by disk diffusion. The zone diameter of inhibition (ZDI) for the agents was recorded, and growth inhibitory indices (GIIs) were calculated. RESULTS: The S. typhi isolates (n=23), which were resistant to both C (ZDI 6 mm) and Tm (ZDI 6 mm), had TLE (500 µg) ZDIs 16-24 mm. The ZDIs of C and Tm were increased up to 15-21 mm and 17-23 mm, respectively, when TLE (250 µg) was added to the C and Tm discs. The GIIs ranged 0.789-1.235 and 0.894-1.352, due to combined activity against S. typhi isolates, of C and TLE and Tm and TLE, respectively. CONCLUSIONS: The data suggest that TLE, in combination with C and Tm, had synergistic activity for S. typhi isolates, and hence O. sanctum is potential in combating S. typhi drug resistance, as well promising in the development of non-antibiotic drug for S. typhi infection.

Cholera: a great global concern.

Cholera, caused by the infection of toxigenic Vibrio cholerae (V. cholerae) to humans, is a life threatening diarrheal disease with epidemic and pandemic potential. The V. cholerae, both O1 and O139 serogroups, produce a potent enterotoxin (cholera toxin) responsible for the lethal symptoms of the disease. The O1 serogroup has two biotypes (phenotypes), classical and El Tor; each of which has two major serotypes (based on antigenic responses), Ogawa and Inaba and the extremely rare Hikojima. V. cholerae O1 strains interconvert and switch between the Ogawa and Inaba serotypes. Fluid and electrolyte replacement is the mainstay of treatment of cholera patients; the severe cases require antibiotic treatment to reduce the duration of illness and replacement of fluid intake. The antibiotic therapy currently has faced difficulties due to the rapid emergence and spread of multidrug resistant V. cholerae causing several outbreaks in the globe. Currently, cholera has been becoming endemic in an increasing number of geographical areas, reflecting a failure in implementation of control measures. However, the current safe oral vaccines lower the number of resistant infections and could thus represent an effective intervention measure to control antibiotic resistance in cholera. Overall, the priorities for cholera control remain public health interventions through improved drinking water, sanitation, surveillance and access to health care facilities, and further development of safe, effective and appropriate vaccines. Thus, this review describes the facts and phenomena related to the disease cholera, which is still a great threat mainly to the developing countries, and hence a grave global concern too.

Honey: its medicinal property and antibacterial activity.

Indeed, medicinal importance of honey has been documented in the world's oldest medical literatures, and since the ancient times, it has been known to possess antimicrobial property as well as wound-healing activity. The healing property of honey is due to the fact that it offers antibacterial activity, maintains a moist wound condition, and its high viscosity helps to provide a protective barrier to prevent infection. Its immunomodulatory property is relevant to wound repair too. The antimicrobial activity in most honeys is due to the enzymatic production of hydrogen peroxide. However, another kind of honey, called non-peroxide honey (viz., manuka honey), displays significant antibacterial effects even when the hydrogen peroxide activity is blocked. Its mechanism may be related to the low pH level of honey and its high sugar content (high osmolarity) that is enough to hinder the growth of microbes. The medical grade honeys have potent in vitro bactericidal activity against antibiotic-resistant bacteria causing several life-threatening infections to humans. But, there is a large variation in the antimicrobial activity of some natural honeys, which is due to spatial and temporal variation in sources of nectar. Thus, identification and characterization of the active principle(s) may provide valuable information on the quality and possible therapeutic potential of honeys (against several health disorders of humans), and hence we discussed the medicinal property of honeys with emphasis on their antibacterial activities.

Plasmid-mediated dimethoate degradation by Bacillus licheniformis isolated from a fresh water fish Labeo rohita.

The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F-) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid.

Scattergram analysis to explore the emerging problem related to in vitro susceptibility test for Salmonella enterica serovar Typhi to ciprofloxacin.

Ciprofloxacin susceptibility using a disk diffusion method and minimum inhibitory concentration (MIC) value determination for 421 S. Typhi isolates showed comparable results for 296 (70%) isolates with an MIC /=0.1 mg/L, signifying resistance, 123 (98.4%) showed discrepant results with the disk diffusion test (66 sensitive, 57 intermediately sensitive). This raises questions about National Committee for Clinical Laboratory Standards guidelines regarding the interpretative zone size for ciprofloxacin.

Synergism of ciprofloxacin and trimethoprim against Salmonella enterica serovar typhi isolates showing reduced susceptibility to ciprofloxacin.

The activity of the combination of ciprofloxacin and trimethoprim against 16 Salmonella enterica serovar typhi isolates from blood cultures were tested by agar dilution checkerboard technique. When used in combination, minimum inhibitory concentrations (MICs) of ciprofloxacin and trimethoprim ranged from 0.5 to 1.25 and from 10 to 125 microg/ml, respectively, and fractional inhibitory concentrations (FICs) from 0.025 to 0.125 and from 2.5 to 10 microg/ml, respectively. The FIC index was 0.140-0.483, indicating a marked synergy between ciprofloxacin and trimethoprim against trimethoprim-resistant S. enterica serovar typhi isolates (100%) with high MICs for ciprofloxacin.

Ofloxacin minimum inhibitory concentration versus disk diffusion zone diameter for Salmonella enterica serovar Typhi isolates: problems in the detection of ofloxacin resistance.

Using the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS), a total of 421 blood culture isolates of Salmonella enterica serovar Typhi obtained during 1991-2001 were tested for susceptibility to ofloxacin (OFX) by the disc diffusion method, and for the determination of minimum inhibitory concentration (MIC) values of OFX by the agar dilution method. Among 421 isolates, 248 were fully OFX-sensitive showing MICs of 0.0125-0.075 microg/ml and inhibitory zone diameters of > or =24 mm. The remaining 173 isolates (MICs of 0.5-1.5 microg/ml) that were treated with OFX did not respond to the therapy. However, 169 (97.69%) of the 173 isolates were determined to be susceptible (zone diameter > or =16 mm) by the disc diffusion method, whereas only 3 were intermediately susceptible (zone diameter 13-15 mm) and the final isolate showed OFX-resistance (zone diameter 12 mm). Thus, following the NCCLS guidelines, OFX-resistance in S. enterica serovar Typhi was not detected by the disc diffusion test. The present data suggest a revision of the NCCLS breakpoints in selecting OFX as the preferred treatment regimen for S. enterica serovar Typhi

Combination effect of ciprofloxacin and gentamicin against clinical isolates of Salmonella enterica serovar typhi with reduced susceptibility to ciprofloxacin.

The present study evaluated the in vitro efficacy of ciprofloxacin (CPFX) in combination with gentamicin (GM) using agar dilution checkerboard method against six blood culture isolates of Salmonella enterica serovar Typhi with CPFX minimum inhibitory concentration (MIC) values of 0.75 - 1.25 microg/ml and GM MIC values of 0.75 - 2 microg/ml. When used in combination, the fractional inhibitory concentration (FIC) values of CPFX and GM for the isolates ranged from 0.008 - 0.032 microg/ml and 0.1 - 0.2 microg/ml, respectively. The range of the FIC index from 0.121 - 0.216 indicated the synergistic effect between CPFX and GM for all the isolates. The time-kill method, which showed a 2.64 log(10) decrease in CFU/ml between the combination and its more active compound, also established synergism between CPFX and GM against one isolate employed in the method. These results may be helpful in making clinical decisions in the treatment of enteric fever due to the infection of multidrug resistant S. enterica serovar Typhi.

R-Factor in Salmonella enterica serovar typhi: transfer to and acquisition from Escherichia coli.

Blood culture isolates of Salmonella enterica serovar Typhi showing high degrees of resistance to ampicillin, chloramphenicol, cotrimoxazole and tetracycline (ACCoT-resistance) transferred their full resistance phenotype to antibiotic-sensitive S. enterica serovar Typhi strains through the primary recipient Escherichia coli C600. Transfer frequencies were 0.80 x 10(-5) and 0.80 x 10(-6), respectively, in the primary and secondary transfer experiments. The Escherichia coli isolates from urinary tract infection cases showing high minimum inhibitory concentration values (microg/ml) to A (2,000-5,000), C (2,000-5,000), Co (250-1,200), and T (500-2,000) also transferred ACCoT-resistance to the antibiotic-sensitive S. enterica serovar Typhi and then to E. coli C600 with transfer frequencies 0.61 x 10(-6) and 0.98 x 10(-5), respectively. Curing experiments revealed the loss of ACCoT-resistance from the original and the transconjugant S. enterica serovar Typhi strains. Results suggest that R-factor from other enteric bacteria is acquired by S. enterica serovar Typhi, and that it (R-factor) is unstable in nature.

In vitro efficacy of ciprofloxacin alone and in combination with amoxycillin against Salmonella typhi isolates.

Combined effect of ciprofloxacin (Ci) and amoxycillin (Ax) has been studied in vitro against 12 clinical isolates of S. typhi that showed Ci minimum inhibitory concentration (MIC) of > or =1 microg/ml. By agar dilution method, MIC values of Ax were 10-16 microg/ml for 11 isolates and 0.5 microg/ml for the remaining one isolate. The isolates, when treated with Ci and Ax in combination, showed fractional inhibitory concentration (FIC) of 0.004-0.256 microg/ml for Ci. FIC of Ax ranged from 6-10 microg/ml, except for a single isolate that showed Ax FIC of 0.25 microg/ml. Thus Ci was more efficacious in combination with Ax against S. typhi than Ci alone. The antibiotic combination exhibited an additive effect for all the isolates showing FIC index 0.504-0.832.