Screening, biochemical characterization and antibiotics resistance/susceptibility of bacteria isolated from native soil and water samples / Triagem, caracterização bioquímica e resistência/suscetibilidade a antibióticos de bactérias isoladas de amostras nativas de solo e água

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.

O presente estudo foi realizado para isolar e caracterizar bactérias de amostras de água e solo retiradas do Canal Lahore, em Lahore, em diferentes locais, ou seja, Mall Road, Mohlanwal e Khera. As cepas bacterianas isoladas foram identificadas com base em testes morfológicos e bioquímicos. A identificação foi confirmada por cultura de bactérias em testes de meios seletivos. O teste de resistência aos antibióticos também foi realizado para observar a resistência das bactérias a diferentes antibióticos. Foi realizado o teste de ágar sangue para identificar diferentes bactérias patogênicas. O resultado revelou que amostras de água e solo do Canal Lahore, Lahore, de diferentes localidades estavam contaminadas com Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. e Staphylococcus spp. Por causa da presença desses patógenos, essa água não é adequada para qualquer uso doméstico e de irrigação. O estudo revelou que a água do Canal Lahore é prejudicial à saúde humana, pois está contaminada com bactérias que podem causar doenças graves, por exemplo: Escherichia coli pode ocasionar gastroenterite; Bacillus spp. pode causar náuseas e vômitos; Enterococcus sp. pode infectar o trato urinário; Salmonella sp. é responsável pela bacteremia; Staphylococcus spp. pode causar febre leve; e Vibrio sp. pode ser a razão da cólera. Assim, torna-se imprópria para uso humano, como natação, banho, lavagem etc., até que algumas medidas corretivas sejam empregadas para erradicar microrganismos patogênicos por WASA e LWMS de acordo com os padrões da OMS. Da mesma forma, é bastante prejudicial, quando usada para irrigação sem tratamento adequado.

Screening, biochemical characterization and antibiotics resistance/susceptibility of bacteria isolated from native soil and water samples.

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.

Utility of the microcolony method for evaluation of multidrug-resistant tuberculosis patients in Karachi, Pakistan.

OBJECTIVE: To compare the microcolony method (MCM) with the reference culture method to evaluate culture conversion in multidrug-resistant tuberculosis (MDR-TB) patients.MATERIAL AND METHODS: Adult patients with Mycobacterium tuberculosis culture-positive MDR-TB undergoing second-line anti-tuberculosis treatment were recruited from two tertiary care chest clinics from January 2013 to October 2014. The MCM was performed in addition to MGIT™ and Löwenstein-Jensen medium (reference method) on sputum samples submitted on a monthly basis.RESULTS: Of 140 patients, culture conversion could be evaluated in 95 patients. The MCM showed 100% agreement with the reference M. tuberculosis culture in 83 of 95 patients who achieved culture conversion. In smear-positive and smear-negative cases, the mean time to positivity was 9.1 and 11.4 days for the MCM and 16.1 and 23.2 days for the reference M. tuberculosis culture respectively. The contamination rate for the MCM was 4.5% in comparison with 6.1% for the reference M. tuberculosis culture. The cost of MCM was estimated to be 30% that of the reference method.CONCLUSION: The MCM can be used in non-urban laboratories as a safe, rapid and cost-effective substitute for the reference M. tuberculosis culture to assess culture conversion in MDR-TB patients.Note: Abstract has been published in International Journal of Mycobacteriology 2015; 4: 159-160.

Serious fungal infections in Pakistan.

The true burden of fungal infection in Pakistan is unknown. High-risk populations for fungal infections [tuberculosis (TB), diabetes, chronic respiratory diseases, asthma, cancer, transplant and human immunodeficiency virus (HIV) infection] are numerous. Here, we estimate the burden of fungal infections to highlight their public health significance. Whole and at-risk population estimates were obtained from the WHO (TB), BREATHE study (COPD), UNAIDS (HIV), GLOBOCAN (cancer) and Heartfile (diabetes). Published data from Pakistan reporting fungal infections rates in general and specific populations were reviewed and used when applicable. Estimates were made for the whole population or specific populations at risk, as previously described in the LIFE methodology. Of the 184,500,000 people in Pakistan, an estimated 3,280,549 (1.78%) are affected by a serious fungal infection, omitting all cutaneous infection, oral candidiasis and allergic fungal sinusitis, which we could not estimate. Compared with other countries, the rates of candidaemia (21/100,000) and mucormycosis (14/100,000) are estimated to be very high, and are based on data from India. Chronic pulmonary aspergillosis rates are estimated to be high (39/100,000) because of the high TB burden. Invasive aspergillosis was estimated to be around 5.9/100,000. Fungal keratitis is also problematic in Pakistan, with an estimated rate of 44/100,000. Pakistan probably has a high rate of certain life- or sight-threatening fungal infections.

Fluoroquinolone consumption and -resistance trends in Mycobacterium tuberculosis and other respiratory pathogens: Ecological antibiotic pressure and consequences in Pakistan, 2009-2015.

OBJECTIVE/BACKGROUND: Fluoroquinolones (FQs) are important anti-tuberculous drugs for the treatment of multidrug-resistant (MDR) tuberculosis. Resistance to FQs leads to fewer options for treatment of tuberculosis (TB), and infection with such strains may also require longer treatment duration. Trends of resistance in Mycobacterium tuberculosis (MTB) are indicators of MTB-resistance evolution. Drivers of such resistance need to be understood and studied to inform preventive strategies. METHODS: Here, we present FQ-resistance rates and trends in Pakistan from 2010 to 2015 and compare rates with FQ-consumption data and rates in other community pathogens. RESULTS: Our results reveal a recent decrease in FQ-resistance rates in MTB, but an increase in resistance for Haemophilus influenzae and Shigella spp. Correlation of FQ resistance with FQ consumption at the population level was weak for MTB, although strong associations were noted for H. influenzae and Shigella spp. CONCLUSION: We discuss the possible reasons for the decrease in resistance rates in TB, putative drivers of resistance other than volume of FQ consumption, and the possible impact of the National Tuberculosis Programme and drug regulatory activities.

Invasive candidiasis in Pakistan: clinical characteristics, species distribution and antifungal susceptibility.

This study reports for the first time, to our knowledge, descriptive epidemiological data for 188 invasive Candida isolates from Pakistan, including species identification and antifungal susceptibility against fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin and amphotericin. Risk factors for invasive candidiasis (IC) were determined for 96 patients from Karachi, Pakistan. In adults and neonates, Candida tropicalis (38 and 36 %, respectively) was the most common species, followed in adults by Candida parapsilosis (17.8 %), Candida glabrata (15.9 %) and Candida albicans (12.3 %). C. albicans (21 %) was the second most common in neonates. In children, C. albicans (31.9 %), C. tropicalis (26.4 %) and C. parapsilosis (19.4 %) were the most common. C. albicans IC was significantly associated with paediatric age [crude odds ratio (COR) 3.46, 95 % confidence interval (CI) 1.63-7.32]. Rare species made up 17.5 % of the total isolates studied. Resistance to fluconazole was seen in C. glabrata (15 .0%) and Candida krusei (100 .0%). Only one isolate (C. glabrata) was resistant to all three echinocandins. Low MICs of fluconazole for 98 % (184/188) of isolates tested support its continued use as an empiric therapy for IC. Non-C. albicans IC was associated with the use of ß-lactam inhibitor combinations (COR 3.16, 95 % CI 1.05-9.57). Use of healthcare devices was documented in 85.4 % of IC patients, whilst 75 .0% had been admitted to special care units. Surprisingly, 66.7 % of patients with IC were not obviously immunosuppressed. The high frequency of modifiable risk factors in this population indicates that candidaemia can be reduced with stringent antibiotic and infection control measures. These data will be useful for empiric selection of antifungals in Karachi, and contribute to global assessments of antifungal resistance.

Use of p-nitrobenzoic acid in 7H10 agar for identification of Mycobacterium tuberculosis complex: a field study.

We report the efficiency and cost-effectiveness of p-nitrobenzoic acid (PNB) testing in Middlebrook 7H10 agar medium for the identification of Mycobacterium tuberculosis complex (MTC). PNB-7H10 was compared with PNB-MGIT and BACTEC-NAP using 200 clinical mycobacterial isolates. PNB-7H10 showed 100% agreement with PNB-MGIT and BACTEC-NAP tests, and reduced the cost of PNB-MGIT test by 80%. PNB-7H10 agar is therefore an effective alternative to the costly PNB-MGIT and BACTEC-NAP tests, especially in resource-poor settings.

Increased isolation of Vibrio cholerae O1 serotype Inaba over serotype Ogawa in Pakistan.

Although the predominant Vibrio cholerae serotype in Pakistan is Ogawa and serotype Inaba is rare, there has been a significant increase in the isolation of Inaba in our referral laboratory in Karachi. This paper reports this observation and further analysis of previous cholera data from 1993 to 2005 to assess the trend of occurrence and resistance pattern of V. cholerae strains. From January to September 2005, 245/3292 (7.4%) specimens yielded growth of V. cholerae. Of these, 243 were serotype Inaba, outnumbering serotype Ogawa. This recent Inaba strain is 100% resistant to cotrimoxazole, 3% resistant to chloramphenicol and not resistant to ampicillin, tetracycline and ofloxacin. This sensitivity pattern is almost similar to that of the previous predominant serotype Ogawa.

Increased isolation of Vibrio cholerae O1 serotype Inaba over serotype Ogawa in Pakistan

Although the predominant Vibrio cholerae serotype in Pakistan is Ogawa and serotype Inaba is rare, there has been a significant increase in the isolation of Inaba in our referral laboratory in Karachi. This paper reports this observation and further analysis of previous cholera data from 1993 to 2005 to assess the trend of occurrence and resistance pattern of V. cholerae strains. From January to September 2005, 245/3292 [7.4%] specimens yielded growth of V. cholerae. Of these, 243 were serotype Inaba, outnumbering serotype Ogawa. This recent Inaba strain is 100% resistant to cotrimoxazole, 3% resistant to chloramphenicol and not resistant to ampicillin, tetracycline and ofloxacin. This sensitivity pattern is almost similar to that of the previous predominant serotype Ogawa

Re-emergence of Vibrio cholerae O139 in Pakistan: report from a tertiary care hospital.

OBJECTIVE: This study reports re-emergence of Vibrio cholerae O139 in Pakistan in 2000-2001 from a tertiary care hospital in Karachi, Pakistan. METHODS: This descriptive study was conducted from 2000-2001. Stool samples were taken from inpatients or those referred to the laboratory from other hospitals, clinics and general practitioners. Samples were processed and Vibrio cholerae isolates were identified according to standard protocols. Tellurite Taurocholate Gelatin agar was used as a selective medium for Vibrio cholerae. Serogroups were identified by slide agglutination with polyvalent antisera. Antimicrobial sensitivities were performed by Kirby Bauer technique. Data was entered and analyzed using SPSS, p values were calculated using t test and two independent samples test. RESULTS: During the study period, 144 samples were found to be infected with Vibrio cholerae O139 in comparison with 545 Vibrio cholerae O1. Infection with O139 was characteristically observed in the older population (mean age = 40 years) in contrast with Vibrio cholerae O1 strains (mean age = 23 years) (p. value = <0.001). Sensitivity pattern of 2000-2001 Vibrio cholerae isolates was markedly different to that of 1993-1994. The earlier isolates were resistant to Cotrimoxazole (99%) and Chloramphenicol (35%) whereas the recent isolates are almost 100% sensitive. CONCLUSION: In conclusion this re-emergent strain seen 6 years after previous episode infected large number of people especially older population suggesting that prior infection with O1 does not provide immunity against O139 and therefore Vibrio cholerae O139 has a potential to cause a major epidemic in an immunologically naïve population.

Comparison of double disc and combined disc method for the detection of extended spectrum beta lactamases in enterobacteriaceae.

OBJECTIVE: To compare double disc approximation and combined disc method for their ability to detect extended spectrum b lactamase (ESBL) production in enterobacteriaceae and determine the percentage of isolates which are falsely reported as sensitive in absence of ESBL detection, in a clinical microbiology laboratory of a tertiary care hospital between September-October 2002. METHODS: Selected isolates were identified according to standard biochemical tests. Disc susceptibility tests were performed according to NCCLS. ESBL detection by combined disc [cefotaxime (30 ug) versus cefotaxime plus clavulanate (30+10 ug)] was compared with detection using double discs [amoxy-clavulanic acid (20+10 ug) and aztreonam (30 ug) applied 10 mm apart]. Results were interpreted according to NCCLS and analysed on SPSS version 10. RESULTS: ESBL production was detected in 140 (30%) isolates by combined disc method and 139 (29.5%) by double disc method. There was no significant difference between two methods. Of the ESBL positive isolates 41 (29%) gave zone diameters that were within the sensitivity range cutoff and would have been falsely reported as being beta lactam sensitive in absence of ESBL detection. CONCLUSION: ESBL detection should be routinely performed in clinical laboratories as false reporting would result in treatment failure despite in vitro sensitivity. No difference was found between the combined disc and double disc methods hence either of two could be used.

Haemotoxicity to chicken (Gallus gallus domesticus) by technical and formulation grades of some phosphoric and synthetic pyrethroid esters.

Acute toxicity was studied by administering an encapsulated single dose to chickens (G.g. domesticus) and observing them for 21 days. Azodrin-71 (Tech.) was found to be extremely toxic, whereas Cypermethrin-92 (Tech.), Cypermethrin-25 EC and Permasect-25 EC (Form.) were practically non-toxic based on LD50 value determinations. Sub-acute oral haemotoxicity of technical and formulation grades of these insecticides was also studied by administering encapsulated low, medium and high daily doses for 21 days to chickens and recording clinical symptoms, mortality and haematological parameters pre and post-dosing. Clinically high doses of Azodrin-71 (Tech.) caused tremors and ataxia among chicks on the 10th day after dosing. Synthetic pyrethroids caused slight tremours in the whole body accompanied by salivation. In general, hyperactivity to external stimuli and loss of appetite and body weight were also observed. With sub-acute oral doses, Permasect-25 EC (Form.) more potently affected haemoglobin (Hb), red cell (RBC) counts and chloride level. Cypermethrin-92 (Tech.) was most potent towards thrombocytes and clotting time. Azodrin-71 (Tech.) was more potent to white cell (WBC) counts and serum protein level. The present Haematological studies have disclosed the possible reaction of blood and blood forming organs to these insecticides.

Effect of dermal application of phosphamidon-92 (technical) on different tissues and hematobiochemical parameters in albino rat.

The histological disturbances occurring from the dermal application of low, medium, and high doses of phosphamidon-92 (technical) to rats as observed by light microscope and analysis of hematobiochemical parameters of blood are presented. Groups of 10 male and 10 female albino rats (Wistar strain) were treated with the test material at dose levels of 0.48 (low), 2.2 (medium), and 3.98 (high) mg/kg X d for 3 wk, followed by a 2-wk observation period. During application, a reduction in food intake and in body weight was recorded with all three treatments. However, gain in body weight and food intake resumed during the observation period and was marked with the high-dose treatment only. Symptoms like hypersalivation and frothing were noticed in both the sexes, as well as a relative decrease in liver weight and gross pathological alterations on microscopical examination of skin, lung, kidney, and testis; significant alterations in some hematobiochemical parameters of blood were observed.