Synergy of Xpert (MTB/RIF) and Line probe assay for detection of rifampicin resistant strains of Mycobacterium tuberculosis.

INTRODUCTION: Early diagnosis and successful treatment of drug-resistant tuberculosis (TB) demands rapid, precise, and consistent diagnostic methods to minimise the development of resistance. Therefore, this comparative study was designed to evaluate the diagnostic performance of Xpert (MTB/RIF) and Line probe assay (LPA) for detecting drug-resistant TB. METHODOLOGY: This study comprised 389 (279 pulmonary and 110 extrapulmonary) samples from patients suspected of having TB. All samples were subjected to Xpert (MTB/RIF), LPA, solid culture, and drug-susceptibility testing. Out of 320 samples, only 180 culture (gold standard) positive were included in the final evaluation. The diagnostic characteristics for methods used were determined by calculating diagnostic sensitivity, specificity, and predictive values. The agreement between all methods was determined by calculating the kappa coefficient. RESULTS: The sensitivity and specificity for Xpert (MTB/RIF) for detecting TB were 88.5% and 96.4%, respectively, against the solid culture. On the other hand, LPA showed sensitivity and specificity at 94.3% and 100%, respectively. Xpert (MTB/RIF) showed moderate agreement (kappa 0.65, p < 0.01) - (73.3% sensitivity; 97.6% specificity) for the detection of rifampicin resistance. However, LPA achieved better diagnostic accuracy (kappa 0.80, p < 0.01) - (84.6% sensitivity; 98.4% specificity) against drug-resistant TB. CONCLUSIONS: Xpert (MTB/RIF) and LPA have outstanding diagnostic sensitivity and specificity against RIF resistance with a shorter turnaround time, which could result in a substantial therapeutic outcome. Our findings showed LPA superiority over Xpert (MTB/RIF) for drug resistance. However, due to operational challenges, the requirement of technical expertise and infrastructure issues, LPA cannot be used as point-of-care testing in resource-limited countries.

Coexistence of ß-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

INTRODUCTION: Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. METHODOLOGY: Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. RESULTS: 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced ß-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CONCLUSIONS: CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

New Delhi metallo-ß-lactamases among extensively drug-resistant clinical isolates from Lahore, Pakistan.

Aim: The study determines rates of carbapenem resistance (CR) and frequency of blaNDM in multidrug-resistance (MDR) or extensive drug resistance (XDR), and evaluates the potential of phenotypic tests for detecting NDM production. Materials & methods: Singleplex PCR was used to detect blaNDM. Phenotypic tests, including combination disc test (CDST) and modified Hodge test (MHT), were evaluated for NDM production. Results: Among 338 CR isolates, 47.63% were MDR, whereas 52.36% were XDR with 53.25% carrying blaNDM. MHT was found to be discriminative for detecting NDM production, whereas no significant association was observed for CDST. Conclusion: The high incidence of CR and MDR and XDR isolates possessing blaNDM presents an impending threat in therapeutics. Limitations of phenotypic tests suggest better testing, including molecular detection of the enzyme.

[Box: see text].

Bacterial ventriculoperitoneal shunt infections: changing trends in antimicrobial susceptibility, a 7-year retrospective study from Pakistan.

BACKGROUND: Ventriculoperitoneal (VP) shunt infections in adults represent a severe complication and make treatment more challenging. Therefore, drug susceptibility patterns are crucial for therapeutic decisions and infection control in neurosurgical centers. This 7-year retrospective study aimed to identify the bacteria responsible for adult VP shunt infections and determine their drug susceptibility patterns. METHODS: This single-center study was performed from 2015 to 2021 in Lahore, Pakistan, and included CSF cultures from VP shunt infections. Demographic data, causative organisms, and antimicrobial susceptibility testing results were collected. Multivariate analysis of variance (MANOVA) and two-sample t-tests were used to analyze and compare the antibiotic sensitivity trends over the study period. RESULTS: 14,473 isolates recovered from 13,937 CSF samples of VP shunt infections were identified and analyzed for their susceptibility patterns to antimicrobials. The proportion of Gram-negative and Gram-positive bacteria were 11,030 (76%) and 3443 (24)%, respectively. The predominant bacteria were Acinetobacter species (n = 5898, 41%), followed by Pseudomonas species (n = 2368, 16%) and coagulase-negative Staphylococcus (CoNS) (n = 1880, 13%). 100% of Staphylococcus aureus (S.aureus) and CoNS were sensitive to vancomycin and linezolid (n = 2580). However, 52% of S. aureus (719/1,343) were methicillin-resistant Staphylococcus aureus (MRSA). Acinetobacter showed maximum sensitivity to meropenem at 69% (2759/4768). Pseudomonas was 80% (1385/1863 sensitive to piperacillin-tazobactam, Escherichia coli (E. coli) showed 72% to amikacin (748/1055), while Klebsiella spp. was 57% (574/1170) sensitive to piperacillin-tazobactam. The sensitivity of piperacillin-tazobactam and meropenem for Gram-negative bacteria decreased significantly (p < 0.05) over 7 years, with 92.2% and 88.91% sensitive in 2015 and 66.7% and 62.8% sensitive in 2021, respectively. CONCLUSION: The significant decrease in the effectiveness of carbapenem and beta-lactam/beta-lactamase inhibitor combination drugs for the common Gram-negative causative agents of VP shunt infections suggests that alternative antibiotics such as colistin, fosfomycin, ceftazidime/avibactam, ceftolozane/tazobactam, and tigecycline should be considered and in consequence included in testing panels. Additionally, it is recommended to adopt care bundles for the prevention of VP shunt infection.

Corrigendum to "Plants as Useful Vectors to Reduce Environmental Toxic Arsenic Content".

[This corrects the article DOI: 10.1155/2014/921581.].

Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement

ABSTRACT Background: Since 1960s, the organophosphate pesticide chlorpyrifos has been widely used for the purpose of pest control. However, given its persistence and toxicity towards life forms, the elimination of chlorpyrifos from contaminated sites has become an urgent issue. For this process bioremediation is the method of choice. Results: Two bacterial strains, JCp4 and FCp1, exhibiting chlorpyrifos-degradation potential were isolated from pesticide contaminated agricultural fields. These isolates were able to degrade 84.4% and 78.6% of the initial concentration of chlorpyrifos (100 mg L-1) within a period of only 10 days. Based on 16S rRNA sequence analysis, these strains were identified as Achromobacter xylosoxidans (JCp4) and Ochrobactrum sp. (FCp1). These strains exhibited the ability to degrade chlorpyrifos in sterilized as well as non-sterilized soils, and were able to degrade 93-100% of the input concentration (200 mg kg-1) within 42 days. The rate of degradation in inoculated soils ranged from 4.40 to 4.76 mg-1 kg-1 d-1 with rate constants varying between 0.047 and 0.069 d-1. These strains also displayed substantial plant growth promoting traits such as phosphate solubilization, indole acetic acid production and ammonia production both in absence as well as in the presence of chlorpyrifos. However, presence of chlorpyrifos (100 and 200 mg L-1) was found to have a negative effect on indole acetic acid production and phosphate solubilization with percentage reduction values ranging between 2.65-10.6% and 4.5-17.6%, respectively. Plant growth experiment demonstrated that chlorpyrifos has a negative effect on plant growth and causes a decrease in parameters such as percentage germination, plant height and biomass. Inoculation of soil with chlorpyrifos-degrading strains was found to enhance plant growth significantly in terms of plant length and weight. Moreover, it was noted that these strains degraded chlorpyrifos at an increased rate (5.69 mg-1 kg-1 d-1) in planted soil. Conclusion The results of this study clearly demonstrate that the chlorpyrifos-degrading strains have the potential to develop into promising candidates for raising the productivity of crops in pesticide contaminated soils.

Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement.

BACKGROUND: Since 1960s, the organophosphate pesticide chlorpyrifos has been widely used for the purpose of pest control. However, given its persistence and toxicity towards life forms, the elimination of chlorpyrifos from contaminated sites has become an urgent issue. For this process bioremediation is the method of choice. RESULTS: Two bacterial strains, JCp4 and FCp1, exhibiting chlorpyrifos-degradation potential were isolated from pesticide contaminated agricultural fields. These isolates were able to degrade 84.4% and 78.6% of the initial concentration of chlorpyrifos (100mgL(-1)) within a period of only 10 days. Based on 16S rRNA sequence analysis, these strains were identified as Achromobacter xylosoxidans (JCp4) and Ochrobactrum sp. (FCp1). These strains exhibited the ability to degrade chlorpyrifos in sterilized as well as non-sterilized soils, and were able to degrade 93-100% of the input concentration (200mgkg(-1)) within 42 days. The rate of degradation in inoculated soils ranged from 4.40 to 4.76mg(-1)kg(-1)d(-1) with rate constants varying between 0.047 and 0.069d(-1). These strains also displayed substantial plant growth promoting traits such as phosphate solubilization, indole acetic acid production and ammonia production both in absence as well as in the presence of chlorpyrifos. However, presence of chlorpyrifos (100 and 200mgL(-1)) was found to have a negative effect on indole acetic acid production and phosphate solubilization with percentage reduction values ranging between 2.65-10.6% and 4.5-17.6%, respectively. Plant growth experiment demonstrated that chlorpyrifos has a negative effect on plant growth and causes a decrease in parameters such as percentage germination, plant height and biomass. Inoculation of soil with chlorpyrifos-degrading strains was found to enhance plant growth significantly in terms of plant length and weight. Moreover, it was noted that these strains degraded chlorpyrifos at an increased rate (5.69mg(-1)kg(-1)d(-1)) in planted soil. CONCLUSION: The results of this study clearly demonstrate that the chlorpyrifos-degrading strains have the potential to develop into promising candidates for raising the productivity of crops in pesticide contaminated soils.

Bacterial community analysis of cypermethrin enrichment cultures and bioremediation of cypermethrin contaminated soils.

Cypermethrin is widely used for insect control; however, its toxicity toward aquatic life requires its complete removal from contaminated areas where the natural degradation ability of microbes can be utilized. Agricultural soil with extensive history of CM application was used to prepare enrichment cultures using cypermethrin as sole carbon source for isolation of cypermethrin degrading bacteria and bacterial community analysis using PCR-DGGE of 16 S rRNA gene. DGGE analysis revealed that dominant members of CM enrichment culture were associated with α-proteobacteria followed by γ-proteobacteria, Firmicutes, and Actinobacteria. Three potential CM-degrading isolates identified as Ochrobactrum anthropi JCm1, Bacillus megaterium JCm2, and Rhodococcus sp. JCm5 degraded 86-100% of CM (100 mg L(-1) ) within 10 days. These isolates were also able to degrade other pyrethroids, carbofuran, and cypermethrin degradation products. Enzyme activity assays revealed that enzymes involved in CM-degradation were inducible and showed activity when strains were grown on cypermethrin. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates JCm1, JCm2, and JCm5 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0516, 0.0425, and 0.0807 d(-1), respectively, following first order rate kinetics. The isolated bacterial strains were among dominant genera selected under CM enriched conditions and represent valuable candidates for in situ bioremediation of contaminated soils and waters.

Determination of cypermethrin degradation potential of soil bacteria along with plant growth-promoting characteristics.

The pyrethroid insecticide cypermethrin is in extensive use since 1980s for insect control. However, its toxicity toward aquatic animals and humans requires its complete removal from contaminated areas that can be done using indigenous microbes through bioremediation. In this study, three bacterial strains isolated from agricultural soil and identified as Acinetobacter calcoaceticus MCm5, Brevibacillus parabrevis FCm9, and Sphingomonas sp. RCm6 were found highly efficient in degrading cypermethrin and other pyrethroids. These bacterial strains were able to degrade more than 85 % of cypermethrin (100 mg L(-1)) within 10 days. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates MCm5, FCm9, and RCm6 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0406, 0.0722, and 0.0483 d(-1) following first-order rate kinetics. Enzyme assays for Carboxylesterase, 3-PBA dioxygenase, Phenol hydroxylase, and Catechol-1,2 dioxygenase showed higher activities with cypermethrin treated cell-free extracts compared to non-treated cell-free extracts. Meanwhile, SDS-PAGE analysis showed upregulation of some bands in cypermethrin-treated cells. This might suggest that cypermethrin degradation in these strains involves inducible enzymes. Besides, the isolates displayed substantial plant growth-promoting traits such as phosphate solubilization, Indole acetic acid production, and ammonia production. Implying the efficient biodegradation potential along with multiple biological properties, these isolates can be valuable candidates for the development of bioremediation strategies.

Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation.

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas ß-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6-7.4 mg L(-1) day(-1) of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L(-1) day(-1) of CP (100 mg L(-1)). Addition of glucose as an additional C source increased the degradation capacity by 8-14 %. After inoculation of contaminated soil with CP (200 mg kg(-1)) disappearance rates were 3.83-4.30 mg kg(-1) day(-1) for individual strains and 4.76 mg kg(-1) day(-1) for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.

Plants as useful vectors to reduce environmental toxic arsenic content.

Arsenic (As) toxicity in soil and water is an increasing menace around the globe. Its concentration both in soil and environment is due to natural and anthropogenic activities. Rising arsenic concentrations in groundwater is alarming due to the health risks to plants, animals, and human beings. Anthropogenic As contamination of soil may result from mining, milling, and smelting of copper, lead, zinc sulfide ores, hide tanning waste, dyes, chemical weapons, electroplating, gas exhaust, application of municipal sludge on land, combustion of fossil fuels, As additives to livestock feed, coal fly ash, and use of arsenical pesticides in agricultural sector. Phytoremediation can be viewed as biological, solar-driven, pump-and-treat system with an extensive, self-extending uptake network (the root system) that enhances the natural ecosystems for subsequent productive use. The present review presents recent scientific developments regarding phytoremediation of arsenic contaminated environments and its possible detoxification mechanisms in plants.

Plant growth promotion traits and Cr (VI) reduction potentials of Cr (VI) resistant Streptomyces strains.

The most toxic form of chromium [Cr (VI)] can be converted to less toxic Cr (III) by reduction with the help of microbes. A total of 6 Streptomyces strains (S. matansis BG5, Streptomyces sp. RSF17, S. vinaceus CRF2, Streptomyces sp. CRF14, S. pulcher CRF17, S. griseoincarnatus SCF18) which were previously isolated from saline farmlands of Punjab, Pakistan, were used in this work. These strains showed varying levels of resistance to Cr (VI) from 800 µg ml(-1) to 1000 mg l(-1) . Their plant growth promoting and Cr (VI) reduction potentials were assessed. Two strains showed positive phosphate solubilization activity. All the strains had ability to produce indole acetic acid (IAA) except one strain (CRF17). The maximum production of IAA was observed by strain BG5 that was 16 mg l(-1) in the presence of 50 mg l(-1) of Cr (VI). All the strains were able to produce ammonia both in the absence and presence of Cr (VI). The highest Cr (VI) reduction in majority of the strains was observed at 28 °C and pH 9. The complete reduction of 150 mg l(-1) of Cr (VI) occurred after 168 hrs. The chromium (VI) concentration of 200 mg l(-1) could be reduced above 90% by most of these strains. The presence of other metals also enhanced Cr (VI) reduction by most of the strains. The results indicate the potential capacity of Streptomyces strains as tool for plant growth promotion and Cr (VI) bioremediation and also is the first report about plant growth promoting traits of Cr (VI) resistant Streptomyces strains.

Bioaerosols in residential micro-environments in low income countries: a case study from Pakistan.

Our knowledge of the concentrations of bioaerosols in residential micro-environments in low income countries is scanty. The present investigation was conducted to assess the culturable concentration and size distribution of bacteria, gram negative bacteria and fungi in two rural and an urban site in Pakistan. The highest indoor culturable bacteria concentration was found at Rural Site II (14,650 CFU/m3) while the outdoor maximum occurred at the urban site (16,416 CFU/m3). With reference to fungi, both indoor and outdoor concentrations were considerably higher at Rural Site I than the other sites. The size distribution of culturable bacteria at all sites showed greater variability than that of culturable fungi. At all sites more than the half (55-93%) of the culturable bacterial and fungal counts were observed in the respirable fraction (<4.7 µm) and so had the potential to penetrate into lower respiratory system.

Chromium (VI) reduction by cell free extract of Ochrobactrum anthropi isolated from tannery effluent.

Chromium-resistant bacteria isolated from industrial wastes can be used to detoxify toxic chromium from contaminated sources. From effluent of Shafiq Tannery, Kasur, Pakistan, bacterial strain STCr-1 that could endure 40 mg mL(-1) of potassium chromate in nutrient agar medium was isolated. STCr-1, identified as Ochrobactrum anthropi by 16S rRNA gene sequence homology, demonstrated substantial Cr(VI) reduction at pH 7 and temperature 37°C. It completely reduced 250 µg mL(-1) of Cr(VI) and showed 71.2 % Cr(VI) reduction at Cr(VI) concentrations of 550 µg mL(-1). Rate of Cr(VI) reduction increased with increase in cell and Cr(VI) concentration. The presence of Cu(2+), Co(2+) and Mn(2+) significantly stimulated Cr(VI) reduction. Assay with cell free extracts clearly indicated that Cr(VI) reduction was solely associated with the soluble fraction of the cell.

Reduction of toxic hexavalent chromium by Ochrobactrum intermedium strain SDCr-5 stimulated by heavy metals.

A Cr(VI) resistant bacterial strain SDCr-5, identified as Ochrobactrum intermedium on the basis of 16S rRNA gene sequencing, was tolerant to high concentrations of Cr(VI) up to 15 mg ml(-1) in acetate minimal medium. O. intermedium SDCr-5 reduced Cr(VI) under a wide range of concentrations from 100 to 1500 microg ml(-1) and reduction was optimum at 37 degrees C and pH 7. It reduced 200 and 721 microg ml(-1) Cr(VI) within 72 and 96 h, respectively. The rate of Cr(VI) reduction increased with concentration from 100 to 1500 microg ml(-1). The presence of heavy metal cations such as Cu(2+), Co(2+), Mn(2+) and Ni(2+) stimulated Cr(VI) reduction. Strain SDCr-5 might be useful for Cr(VI) detoxification under a wide range of environmental conditions.