Positive Isolates Research Articles

Frequency of Clostridioides difficile Infection Among Hospitalized Patients in Kerman City, Iran

Background: Clostridioides difficile is one of the major causes of nosocomial infections, being responsible for 15 to 25% of antibiotic-associated diarrhea. It is important to determine the epidemiology and prevalence of this bacterium at hospitals and healthcare centers. Objectives: This study aims to investigate the prevalence of C. difficile infection (CDI) by identifying toxigenic isolates of C. difficile in different wards of the hospital. Methods: A total of 417 diarrheal stool samples were taken from hospitalized patients in different wards of three educational hospitals in Kerman City, Iran from 2018 to 2020. The samples were cultured on cycloserine-cefoxitin fructose agar and C. difficile suspected colonies were isolated. Identification of the cdd-3 gene for definitive diagnosis of C. difficile and identification of toxin genes in the positive isolates was performed using the PCR method. Results: A total of 68 isolates (16.3%) of C. difficile were isolated from the specimens. Besides, 8.6% (36/417) and 7.6% (32/417) of the isolates were toxigenic and nontoxigenic, respectively; thus, the prevalence of CDI was 8.6%. Most of the toxigenic isolates had the A+B+CDT- toxin phenotype. The highest prevalence of CDI was observed in males, ICU ward, and age group of 41 - 60. Conclusions: A total of 8.6% of hospitalized patients with diarrhea were infected with C. difficile. The prevalence of CDI in Kerman City is lower than that in Europe, East Asia, and other parts of Iran, but it is almost the same as that in the Middle East.

Fluoroquinolone resistance among fecal extended spectrum βeta lactamases positive Enterobacterales isolates from children in Dar es Salaam, Tanzania

BackgroundFluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria.MethodsA cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing.ResultsA total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacterspp. (100%, 5/5), followed by Klebsiella.pneumoniae (76.1%; 35/46), Escherichiacoli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6’)-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx,qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E.coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E.coli isolates, while ST607 was more common out of 12 sequence types detected among the K.pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids.ConclusionThe ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.

Genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021

Objective: To characterize the prevalence and genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021. Methods: Based on the Shenzhen Infectious Diarrhea Surveillance System, acute diarrheal patients were actively monitored in sentinel hospitals from 2013 to 2021. Whole-genome sequencing (WGS) of Vibrio parahaemolyticus isolates was performed, and the genomic population structure, serotypes, virulence genes and multilocus sequence typing were analyzed. Outbreak clusters from 2019 to 2021 were explored based on single-nucleotide polymorphism analysis. Results: A total of 48 623 acute diarrhea cases were monitored in 15 sentinel hospitals from 2013 to 2021, and 1 135 Vibrio parahaemolyticus strains were isolated, with a positive isolation rate of 2.3%. Qualified whole-genome sequencing data of 852 isolates were obtained. Eighty-nine serotypes, 21 known ST types and 5 new ST types were identified by sequence analysis, and 93.2% of strains were detected with toxin profile of tdh+trh-. 8 clonal groups (CGs) were captured, with CG3 as the absolute predominance, followed by CG189. The CG3 group was dominated by O3:K6 serotype and ST3 sequence type, while CG189 group was mainly O4:KUT, O4:K8 serotypes and ST189a and ST189 type. A total of 13 clusters were identified, containing 154 cases. About 30 outbreak clusters with 29 outbreak clusters caused by CG3 strains from 2019 to 2021. Conclusion: Vibrio parahaemolyticus is a major pathogen of acute infectious diarrhea in Shenzhen City, with diverse population structures. CG3 and CG189 have been prevalent and predominant in Shenzhen City for a long time. Scattered outbreaks and persistent sources of contamination ignored by traditional methods could be captured by WGS analysis. Tracing the source of epidemic clone groups and taking precise prevention and control measures are expected to significantly reduce the burden of diarrhea diseases caused by Vibrio parahaemolyticus infection in Shenzhen City.

Prevalence, multiple antibiotic resistance and virulence profile of methicillin-resistant Staphylococcus aureus (MRSA) in retail poultry meat from Edo, Nigeria.

Staphylococcus aureus causes staphylococcal food poisoning and several difficult-to-treat infections. The occurrence and dissemination of methicillin-resistance S. aureus (MRSA) in Nigeria is crucial and well documented in hospitals. However, findings on MRSA from meat in the country are yet to be adequately reported. The current study determined the prevalence, virulence profile and antibiogram characteristics of MRSA from a raw chicken product from retail outlets within Edo. A total of 368 poultry meat samples were assessed for MRSA using a standard culture-based approach and characterized further using a molecular method. The antimicrobial susceptibility profile of the isolates was determined using the disc diffusion method. The biofilm profile of the isolates was assayed via the crystal violet microtitre-plate method. Virulence and antimicrobial resistance genes were screened using polymerase chain reaction via specific primers. Of the samples tested, 110 (29.9%) were positive for MRSA. All the isolates were positive for deoxyribonuclease (DNase), coagulase and beta-hemolysis production. Biofilm profile revealed 27 (24.55%) weak biofilm formers, 18 (16.36%) moderate biofilm formers, and 39 (35.45%) strong biofilm formers. The isolates harboured 2 and ≤17 virulence genes. Enterotoxin gene profiling revealed that 100 (90.9%) isolates harboured one or more genes. Resistance against the tested antibiotics followed the order: tetracycline 64(58.2%), ciprofloxacin 71(64.6%), trimethoprim 71(64.6%) and rifampin 103(93.6%). A total of 89 isolates were multidrug-resistant, while 3 isolates were resistant to all 22 antibiotics tested. The isolates harboured antimicrobial-resistant determinants such as methicillin-resistant gene (mecA), tetracycline resistance genes (tetK, tetL), erythromycin resistance genes (ermA, ermC), trimethoprim resistance gene (dfrK). All the staphylococcal cassette chromosome mec (SCCmec) IVa and SCCmec V positive isolates harboured the Panton-Valentine Leukocidin Gene (PVL). In conclusion, S. aureus was resistant to commonly used antibiotics; a concern to public health concerning the transmission of these pathogens after consuming these highlight the significance of antimicrobial and enterotoxigenic monitoring of S. aureus in food chains.

Status of MDR and Plasmid Profiling of ESBL producing E. coli and Klebsiella spp. isolated from milk of Bovine in Gangetic Plain Zone of Uttar Pradesh

Nowadays resistance to antibiotics of β-lactam groups is expanding rapidly and is one of the latest challenges faced by scientific community due to therapeutic failure. In the present study total 180 milk samples were collected from two district of Gangetic Plain Zone of Uttar Pradesh (India). Total 65 (36.11%) isolates were confirmed comprising 44 (24.4%) E. coli and 21 (11.67%) Klebsiella spp. by PCR analysis using uidA gene and 16S rRNA gene respectively, 35/180 (19.44%) isolates were confirmed as ESBL producers, constituting 12 (6.67%) E. coli and 23 (12.8%) Klebsiella spp. All ESBL positive isolates were subjected to antimicrobial resistance pattern using 17 antibiotics of 10 different classes. In this study all the isolates of E. coli and Klebsiella spp. were found 100% resistant to cefotaxime, cefpodoxime, ceftriazone and ampicillin. Both the isolates of E. coli and Klebsiella spp. revealed different susceptibility pattern to different non-β-lactam antibiotics except aminoglycosides class of antibiotics for which both the isolates were found 100% sensitive. The multi-drug resistance pattern was assessed by using MAR index. Out of total ESBL producers, 80.0% isolates were found MDR with 78.0% E. coli and 83.0% Klebsiella spp. Overall 12 resistance pattern were observed for E. coli ranging from 5 to 12 antibiotics and 09 for Klebsiella spp. ranging from 5 to 14 antibiotics. Plasmid profiling revealed total 71.42% isolates carrying plasmids with average molecular weight ranged between 1.0 Kb to > 10 Kb and number1-4.

Bacterial Co-infections and Susceptibility patterns among admitted COVID 19 patients during 3rd wave of pandemic, in a Tertiary Care Hospital. Lahore.

Objective: To check the Bacterial Co-infections and Susceptibility patterns among admitted COVID-19 patients during 3rd wave of pandemic. Study Design: Descriptive Cross Sectional study. Setting: Department of Microbiology, Combined Military Hospital Lahore Pakistan. Period: May 2021 to August 2021. Material & Methods: Six hundred and twelve COVID-19 positive patients having positive bacterial cultures were processed, Antibiotic susceptibility testing was done by Kirby-Bauer diffusion technique, all antibiotics were reported using breakpoints recommended in clinical and laboratory standards institute (CLSI 2021). Results: Out of 612 patients, 348 (56.9%) were male and 264 (43.2%) were female. Mean age of the patients was 57.2 ± 14.4 years with a range of 22 to 89 years. Bacterial coinfection was present in 70.4% of the patients. Gram negative bacteria (94.4%) were more prevalent in COVID-19 patients as compared to gram positive isolates (5.6%). Antibiotic sensitivity pattern of Staphylococcus aureus showed a high resistance against penicillin, ampicillin, tetracycline and doxycycline. Conclusion: Our study reported a high prevalence of bacterial coinfections in COVID-19 patients infected during the third wave of pandemic. A high percentage of gram negative species were identified in our study population, this could be due to the suppression in the immunity of individuals due to severity of COVID-19 infection and already present Antimicrobial resistance.

Molecular Determination of Some Important Viruses Causing Infection in Potato Fields in Turkey

Potato is one of the most important agricultural crops worldwide and known to be susceptible to more than 40 viruses in nature. In this research, 298 leaf samples collected from potato fields in Afyon, Nevşehir and Bolu provinces in the previous study were used to determine viruses affecting potato production in the region. The leaves of potato plants showing virus symptoms were subjected to RT-PCR using virus-specific primers, in order to detect the presence of Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus X (PVX) and Potato virus M (PVM). As a result of the study,one or more viruses were detected in 46 (15.43%) of the 298 leaf samples tested. A total of 43 samples infected with PLRV (14.42%) and 3 sample infected with PVX (1.%). It was determined that the most common virus in plant samples collected from Afyon, Nevşehir and Bolu Central regions was PLRV followed by PVX. PVA and PVM were not detected in the samples. The sequences of some positive isolates of PLRV were obtained and used along with the isolates registered in the GenBank to reveal phylogenetic relationships among them. PLRV isolates from Turkey were classified in Group 2 along with isolates from Serbia, Canada, and Pakistan based on phylogenetic analyses.

Occurrence and Diversity of Biofilm Producing Multidrug Resistant Escherichia coli in Dug Wells Used for Domestic Purposes in Ile-Ife, Southwest Nigeria

Antibiotic resistance in Escherichia coli has emerged as a serious and growing threat to public and environmental health and domestic wells have been fingered as a potential reservoir of resistant E. coli due to faecal contamination, and are ideal for microbial biofilm formation. Therefore, this study was carried out to investigate biofilm production among 60 multidrug-resistant (MDR) E. coli recovered from domestic wells in Ile-Ife, Nigeria. All biofilm positive isolates were screened for seven associated (papC, iroN, biofilm, fimH, cna, fbna and bcsa) genes by multiplex polymerase chain reaction (PCR). The diversity of the isolates was investigated using (GTG)5-PCR, and a dendrogram was drawn with interactive-tree-of-life software. Overall, 45 of 60 (75%) isolates produced biofilms, comprising strong (n=15; 25%), moderate (n=16; 26.7%), and weak (n=14; 23.3%) producers. Moreover, 35 (77.8%) isolates had at least one biofilm gene, 30 (66.7%) had multiple genes, and 5 (11.1%) had one. The biofilm gene (biofilm, fimH, bcsa, iroN) combination was identified in 10 (28.7%) of the 60 isolates. The isolates (n=14; 40%) with moderate biofilm production had the highest number of genes dominated by the biofilm gene (biofilm, fimH, bcsa) combination, while isolates (n=8; 22.9%) with weak production had the least number of genes and comprised mainly the fimH and cna gene combination. The biofilm formers showed 32 distinct fingerprints and were grouped into nine clades, with clade 6 having the most strains (31.6%). The occurrence of MDR biofilm-producing organisms in domestic wells constitutes a public health concern. There is a need to develop strategies to curb faecal contamination of wells to prevent outbreaks of untreatable diseases.

Molecular characterization of class 1 integrons in carbapenem-resistant Enterobacterales isolates

ObjectiveCarbapenem-resistant Enterobacterales (CRE) infections result in higher treatment costs and mortality rates. Integrons play important roles in emergence and spread of antibiotic resistant genes. To get a better understand on the effects of integron on CRE resistance, distribution of common carbapenemase genes and class 1 integron in clinical CRE isolates were investigated. MethodCarbapenemase genes, including blaKPC, blaVIM, blaIMP, blaNDM, blaGES, blaVEB and blaOXA-23, were screened in 161 CRE isolates and subtypes of these genes were confirmed through sequence analysis. Class 1 integron was screened and common promoter and gene cassette arrays were determined by sequencing. The resistant rates to clinical commonly used antibiotics between integron positive and integron negative CRE isolates were compared. ResultsOf 161 CRE isolates, the most prevalent carbapenemase gene was blaKPC-2, which was detected in 139 isolates, including 99 Klebsiella pneumoniae. Class 1 integron was detected in 78 isolates. Twenty different gene cassettes, including two carbapenemase genes blaVEB-1 and blaIMP-4, and nine different gene cassette arrays, including blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1, aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3, were detected. Five types of common promoters were identified. Relative weak promoter PcH1 was the dominant type. Resistant rates of CRE isolates containing class 1 integrons to ceftazidime, amikacin, trimethoprim/sulfamethoxazole and gentamicin were higher than those without class 1 integrons (P < 0.05). ConclusionClass 1 integrons play important roles in the emergence and spread of CRE resistance. To the best of our knowledge, this is the first report of aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3 in the same Providencia rettgeri isolate and blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1 in P. rettgeri.

Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA.

As safe agents of last resort, carbapenems are reserved for the treatment of infections caused by multidrug-resistant organisms. The impact of β-lactam antibiotics, cefotaxime, and meropenem on the frequency and diversity of carbapenemase-producing organisms recovered from environmental samples has not been fully established. Therefore, this methodological study aimed at determining β-lactam drugs used in selective enrichment and their impact on the recovery of carbapenemase-producing Enterobacterales (CPE) from untreated wastewater. We used a longitudinal study design where 1L wastewater samples were collected weekly from wastewater treatment plant (WWTP) influent and quarterly from contributing sanitary sewers in Columbus, Ohio USA with 52 total samples collected. Aliquots of 500 mL were passed through membrane filters of decreasing pore sizes to enable all the water to pass through and capture bacteria. For each sample, the resulting filters were placed into two modified MacConkey (MAC) broths, one supplemented with 0.5 μg/mL of meropenem and 70 μg/mL of ZnSO4 and the other supplemented with 2 μg/mL cefotaxime. The inoculated broth was then incubated at 37° C overnight, after which they were streaked onto two types of correspondingly-modified MAC agar plates supplemented with 0.5 μg/mL and 1.0 μg/mL of meropenem and 70 μg/mL of ZnSO4 and incubated at 37°C overnight. The isolates were identified based on morphological and biochemical characteristics. Then, up to four distinct colonies of each isolate's pure culture per sample were tested for carbapenemase production using the Carba-NP test. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) MALDI-TOF MS was used to identify carbapenemase-producing organisms. In total 391 Carba-NP positive isolates were recovered from the 52 wastewater samples: 305 (78%) isolates had blaKPC, 73 (19%) carried blaNDM, and 14 (4%) harbored both blaKPC and blaNDM resistance genes. CPE genes of both blaKPC and blaNDM were recovered in both types of modified MAC broths, with 84 (21%) having a blaKPC gene, 22 (6%) carrying blaNDM and 9 (2%) harbored both a blaKPC and blaNDM of isolates recovered from MAC medium incorporated with 0.5ug/mL meropenem and 70ug/mL ZnSO4. The most prevalent isolates were Klebsiella pneumoniae, Escherichia coli, and Citrobacter spp.

Screening and Biochemical Characterization of Indigenous Polyhydroxyalkanoates Producing Bacteria

Exploration for promising polyhydroxyalkanoates (PHA) producers is a continuous process as effort to mitigate the accumulation of synthetic plastic is on the rise. The present study focuses on isolation of indigenous polyhydroxyalkanoates producing bacteria from soil, waste water effluent and organic waste sample of Obio-Akpor, LGA. Screening of the selected bacteria isolates for polyhydroxyalkanoates production was based on viable plate method followed by microscopic visualization using Sudan black B. The PHA producing abilities of the selected isolates were estimated by gravimetric method and the positive isolates were detected for the presence of PHA synthase gene and identified up to genus level. Results revealed that out of 106 isolates, 55 were suspected to accumulate PHA after screening and produced PHA in the range of 0.2-1.1 g/L. Six potential isolates produced highest PHA yield and their phaC genes were successfully amplified from genomic DNA. Based on morphological, cultural and biochemical characteristics, the six isolates were tentatively identified to the genus Bacillus sp, Lysinibacillus sp and Pseudomonas sp. The result of the present study indicated that these bacterial isolates have the potential of producing PHA and their potential harnessed for future purposes.

Assessment of artemisinin tolerance in Plasmodium falciparum clinical isolates in children with uncomplicated malaria in Ghana

BackgroundArtemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana.MethodsSix months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana’s Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing.ResultsOf the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates.ConclusionsThe observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Exploration of Endophytes from Azadirachta indica for Antifungal Activity

Endophytes are the plant associated microbes that live within the living tissues of their host plants without causing any harm to them. Bioprospecting of endophytes from understudied ecosystems is a promising source for extracting novel antifungal metabolites useful for pharmaceutical/cosmetic purposes. The aim of present study was to explore endophytes for antifungal activity against Aspergillus species and Candida species. To achieve this, endophytes were isolated from Azadirachta indica leaves. Cross streak and agar overlay methods were followed for screening for antifungal activity. Secondary antifungal activity was carried out for positive antifungal isolates using agar well diffusion method. In present study, twenty four fungal endophytes and fourteen bacterial endophytes were isolated from leaves of Azadirachta indica by optimizing suitable surface sterilization protocol. Out of these six bacterial isolates were found to produce antifungal compound against Candida albicans and two isolates were found to be inhibitory towards Aspergillus species. In secondary antifungal assay, one bacterial isolate showed anticandida activity. The results of this study suggest that these endophytes producing antifungal compounds may have medicinal value. Further studies related to characterization and identification of these compounds may lead to the development of novel products beneficial to human kind.

First report of root rot caused by Fusarium solani on roselle in Nanning, Guangxi, China.

Roselle (Hibiscus sabdariffa L.) is an annual herbaceous plant in the Malvaceae family with high anthocyanin and is widely cultivated in Nanning, Guangxi of China due to its economic and nutritional importance. In August 2021, a severe root rot disease with incidence of 42.4% (860 plants in the field) was observed in roselle plants in an open-field crop in Nanning (108°33"E, 22°84"N), Guangxi, China. The roots of the diseased plants were discolored and rotten, and the xylem became black, extending along the main root to the junction of the rhizomes. The above-ground symptoms were leaf yellowing, vascular tissue browning, wilting and death. Three diseased samples were rinsed thoroughly with sterile distilled water, cut with a sterile scalpel into approximately 0.5-cm pieces, surface disinfested with 75% ethanol for one minute, rinsed 3-4 times with sterile water, and finally incubated on potato dextrose agar (PDA) at 28 °C in the dark for 3 days. Emerging colonies were transferred to new PDA two-three times until a single colony was obtained. The aerial mycelium was initially white, turning pale yellow after 5 days of growth on PDA. Microscopic observations revealed that microconidia were hyaline and ovoid with sizes of 5.13 to 15.12 and 2.50 to 4.20 μm (average 9.02, 3.32 μm, n=30). Macroconidia were falciform with 3- to 4-septate, with sizes of 19.08 to 24.35 and 4.5 to 8.00 μm (average 24.35 and 5.23 μm, n=30). The morphological characteristics of the microscopic images were identical to those described for Fusarium solani (Leslie and Summerell 2006). A representative isolate (GXRST29) was selected for DNA extraction for further characterization. The internal transcribed spacer rRNA regions (ITSs), beta tubulin gene sequence and a fragment of the translation elongation factor 1-alpha (EF 1-α) gene sequence were amplified using the primer pairs ITS1/ITS4 (Chehri 2014), Bt-1/Bt-2 (Wang et al. 2014) and EF1-F/EF2-R (O'Donnell et al. 2010), respectively. PCR products were sequenced and deposited in GenBank (accession Nos. OL314654, ON157430 and ON157431, respectively). BLASTn analysis showed that the ITS sequence had 96.99% homology with sequence of F. solani (NR 163531), and 99.26% for Fusarium cf. solani (MG775565) obtained from Homo sapiens. The beta tubulin sequence had 97.96% similarity with BLAST sequence of F. solani (MN295052.1) and EF 1-α gene had 100% identity to published F. solani (MN977912.1). The fungus was identified as F. solani. Five roselle plants at the 5-leaf stage were artificially inoculated by root dipping into a 106-107-mL-1 spore suspension of the isolated GXRST29 for pathogenicity testing. The experiment was conducted three times, and the negative controls were replaced with sterile water. Compared to the control, the growth of plants was significantly inhibited, leaves turned yellow, plants dwarfed and wilted, and roots decayed three days post-inoculation. One week post-inoculation, all plants exhibited symptoms similar to those observed in the field, and F. solani was steadily reisolated from those diseased plants, while no positive isolations were obtained in the controls. F. solani has been reported to cause root rot on roselle in Upper Egypt (Hassan et al. 2014) and lisianthus in China (Xiao et al. 2018). To our knowledge, however, this is the first report of Fusarium wilt caused by F. solani in roselle plants in Nanning, Guangxi, China, and could result in severe crop losses.

Bacteriological and Genetic study of some species of Bacteria that isolated from patients and healthy of Diabetes

The study was conducted in the Laboratories of Teaching Tikrit Hospital and the laboratories of the Biology Department - College of Science –Tikrit University from January 2010 to the January 2011. In this study five hundreds thirty four samples of urine and wounds from patients with diabetes and healthy were collected from both sexes and all ages to isolation and identification of pathogenic bacteria by morphological, cultural and biochemical characteristics then determination of virulence factors and genetic variation between dominant type depending on the isolating source and the type of infection in diabetic patients. The counts of urinary tract infections and wound infection in non _diabetic patients were 118 and 52 respectively.The percentage of positive isolation of bacteria for both of them were 44, and 38.4% respectively and from the same patients with urinary tract Infections and wounds and Insulin Depended Diabetes (IDD) patients were 158 and 68 samples respectively and the percentage of bacterial isolation were at 78.5 and 67.6% respectively. The patients with Insulin Non-dependent Diabetes (INDD) were 69 and 42 samples, where positive isolates from bacteria were 75 and 71.4% respectively. The infections females from the Healthy and Diabetes Patients that (IDD) or (INDD) were larger than that of the males patients and with the same state with the wounds infections state for (IDD) patients, whereas the rate was smaller than of males for wounds infections to patients with the other diabetes infections type. The age group between 41-60 years was the larger percentage with all infections, except with wounds infections to (INDD) patients, while the age group between 16-40 years was the larger, and the infections were the largest means in the Winter and Autumn compare the other seasons. The higher rate of bacteria that isolated from patients with Urinary Tract Infections was Escherichia coli then other types like Citrobacter diversus , Proteus mirabilis, Morganella morganii, and Enterobacter aerogenes. The larger rate of bacteria in patients with diabetes and wounds Infections was Escherichia coli then Citrobacter diversus, Proteus mirabilis, Enterobacter aerogenes, Morganella morganii and Staphylococcus aureus. The rate of diabetes infections with urinary tract infections and wounds increased in winter and autumn seasons more than of summer and spring seasons, Most bacterial isolates where sensitive for chloramphenicol. Other antibiotics were highly variable in their ability to inhibit bacterial isolates. the bacterial isolates were different in their ability to produce virulence factors, the diabetes infections was the reason of increase the variation in their ability to produce that virulence factor and the bacteria that isolated from diabetes patients produced haemolysine factor and capsule .PCR technique was used to show the genetic variations for the more repeats bacterial isolates isolated from all sources infections and used the Specific Primers (KPSMT II) group II capsule, (CNF1) Cytotoxic Necrotizing factor, (CNFs) and (HLY A) haemolysine, the bands appeared after electrophoresis to represent the used Primers, one band was appeared in the sample of diabetes Patients with Urinary Tract Infections (KPSMT II) at molecular weight 270 bp ,and one band appeared in the sample of diabetes with urinary tract infections for Primer (HLY A) and it is molecular weight was 177 bp. One band appeared in the sample of diabetes patients for the primer (CNF1) hg with Wound Infections and it is molecular weight 450bp, and there is not any band in the Primer (CNFs).

Genotype MTBDRsl version 2 and phenotypic drug resistance detection of Mycobacterium tuberculosis for fluoroquinolones and aminoglycosides.

Information on genotypic with comparison of phenotypic drug sensitivity test of anti-tuberculosis (TB) has been reported in several studies, which have variable results. The present study aimed to assess the Genotype MTBDRsl version 2.0/Line probe assay (LPA) for the detection of fluoroquinolones (FQ) and aminoglycosides (AMGs) resistance mutations among drug-resistant Mycobacterium TB (MTB) strains and also to compare the patterns of genotypic mutations of gyrA/B, rrs, and eis with mycobacteria growth indicator tube (MGIT 960). A total of 1416 samples were subjected to Genotype MTBDRsl version 2.0 assay. One hundred and twenty sputum smear positive MTB isolates and 37 sputum smear negative MTB isolates confirmed multiple drug resistance resistant to FQ and AMG by the Genotype MTBDRsl version 2.0 were subjected to phenotypic drug susceptibility testing (DST) were analyzed. The association sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the resistance detection between MGIT (DST) and the Genotype MTBDRsl version 2.0 assay was significant (P < 0.01) of moxifloxacin (MFX) concentration. Sensitivity and specificity value for kanamycin (KAN) resistance was 76% and 89%; 47% and 94% for capreomycin (CAP); and 60% and 76% for low-level KAN, respectively. Our results indicate that MFX (0.25and 1 μg/mL), KAN (2.5 μg/mL), and CAP (2.5 μg/mL) significantly (P < 0.01) and support the World Health Organization guidance to test FQ and AMG by genotypic test.

Detection of extended spectrum beta-lactamase producing organisms and its antibiotic resistance pattern among Enterobacteriaceae isolates in a teaching hospital of Eastern India

Introduction: ESBL-producing Enterobacteriaceae have been responsible for innumerable outbreaks of infection throughout the world and are becoming a real challenge to control the spread of infection. Aim To determine the prevalence of ESBL producing organisms among Enterobacteriaceae isolated from different clinical samples and to detect their antibiotic susceptibility patterns. Material and Methods The study was conducted in the Department of Microbiology, College of Medicine and JNM hospital. A total of 241 samples showing pure growth of Enterobacteriaceae were included in the study. Identification followed by antibiotic susceptibility tests were performed according to CLSI guidelines. Organisms showing reduced susceptibility to third generation cephalosporins were further tested by double disc synergy test to detect ESBL production and those ESBL screening positive isolates were further tested by Phenotypic confirmatory test according to CLSI 2019 guidelines. Results: Prevalence of ESBL producing Enterobacteriaceae was found to be 31.04% with the highest rate among E.coli (34.5%) followed by K. pneumoniae (27.6%). and P. mirabilis (24.2%) .All the ESBL isolates were sensitive to meropenem and imipenem. Nitrofurantoin (84.2%) was found to be most effective drug for ESBL isolates from urine samples. Among β-Lactam/ β-Lactam inhibitor drugs, amoxicillin/clavulanic acid was found to be less sensitive compared to cefoperazone/sulbactam (67%) . Most of the ESBL isolates were found to be multidrug resistant. Conclusion: Screening of all the gram-negative organisms for ESBL detection and judicial use of antibiotics as per sensitivity report is utmost important to prevent the spread of resistance strains in the community as well as in hospital. Formulation of infection control committee in every hospital may help to combat the present situation.

Susceptibility of Bacterial Isolates From Wound Swabs in Bangladesh: Laboratory – Based Surveillance Study

Background: Wound infection is one of the major health problems that occur frequently. Infections of the wound result from entry of the organisms through breached skin. It plays an important role in the development of chronicity, delaying wound healing. It is evident that wound infection is a challenging situation for the physicians. Multiple bacteria can cause wound infection. Both broad spectrum and narrow spectrum antibiotics are available for the treatment. It is ideal to give proper antibiotic after culture and sensitivity of the wound swab. Improper and irrational use of antibiotics can lead to drug resistance. Objective: To isolate and identify the bacteria causing wound infection and to determine the antimicrobial susceptibility pattern. Materials and method: This descriptive cross-sectional study was carried out at the Microbiology Laboratory, Popular Diagnostic Centre Ltd, Dhanmondi, Dhaka, Bangladesh, from November 2019 to January 2020. A total of 62 wound swabs were collected and analyzed for culture and antibiotic sensitivity. Results: From total wound swab samples, 49 (79%) were culture positive. The most common isolated pathogen was Acinetobacter spp. 20 (40.81%). Other isolates were Staphylococcus aureus 10 (20.41%); Escherichia coli 07 (14.29%); Pseudomonas aeruginosa 07 (14.29%); Klebsiella 04 (08.16%) and Proteus 01 (02.04%). Among the Gram negative isolates, Acinetobacter spp showed 100% sensitivity to colistin, 85% to imipenem, 30% to amikacin and 25% to piperacillin + tazobactum. E. coli showed 100% sensitivity to imipenem and colistin and least sensitivity to third generation cephalosporin. Pseudomonas showed 100% sensitivity to piperacillin+tazobactum, 85.71% to imipenem and aztreonam. Among the Gram positive isolates, Staphylococcus aureus showed 100% sensitivity to linezolid, vancomycin, imipenem, cloxacillin, amikacin and least sensitivity to azithromycin. Conclusion: Acinetobacter spp. was the most frequently isolated pathogen from wound swab and the antibiotic sensitivity pattern of various isolates will help the clinician in appropriate selection of antibiotics against wound infection. J Dhaka Med Coll. 2021; 30(2) : 180-188

Evaluation of a MALDI-TOF MS cephalosporinase assay for blood culture isolates

Objectives: Rapid detection of beta-lactamases has the potential to improve antimicrobial stewardship and sepsis outcomes. The aim of this study was to evaluate a recently TGA-approved cephalosporinase detection method utilising matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) on blood culture isolates. This was compared to standard antimicrobial susceptibility testing and phenotypic cephalosporinase detection. Methods: A total of 114 gram negative blood culture isolates were retrospectively analysed. Isolates were selected to either be ceftriaxone non-susceptible by Vitek 2 semi-automated system (version 08.01; bioMerieux, USA, AST-N246 card) (n=31) and/or demonstrate an inducible cephalosporinase or ESBL on disc approximation testing (n=32). This cohort was compared to an equal number of ceftriaxone-susceptible isolates by Vitek 2 (n=57). The range of organisms included Escherichia coli (n=62), followed by Serratia marcescens (n=12), Pseudomonas aeruginosa (n=11), Enterobacter cloacae complex (n=10), Klebsiella aerogenes (n=5), Klebsiella pneumoniae (n=3), Klebsiella oxytoca (n=3), Morganella morganii (n=3), Acinetobacter calcoaceticus-A. baumannii complex (n=3), and Citrobacter freundii (n=2). Sub-cultured colonies were prepared with the MBT STAR-Cepha IVD Kit and third-generation cephalosporin hydrolysis was detected using the MBT STAR-BL IVD Module (Bruker Daltronics) on MALDI-TOF MS after 30 min incubation time. Results: The MALDI-TOF MS assay sensitivity and specificity for third-generation cephalosporinase activity was 61.3% and 77.1% respectively. The assay detected 11/11 (100%) ESBL producers and 5/6 (83.3%) AmpC type third-generation cephalosporinase producers. Additionally, third generation cephalosporinase activity was detected by the MALDI-TOF MS assay in 17 of 26 inducible cephalosporinase positive isolates (65.4%) that tested susceptible to ceftriaxone by Vitek 2. Of all species included, Pseudomonas aeruginosa had the lowest concordance of MALDI-TOF MS and Vitek 2 results, with only 1/11 isolates (9%) positive by MALDI-TOF MS. Conclusions: Overall the MALDI-TOF MS cephalosporinase assay lacked sensitivity in detection of third generation cephalosporinases compared to antimicrobial susceptibility testing by Vitek 2. The risk for false negative results in screening for resistance in blood culture isolates is too high to consider its implementation in the standard workflow, although there may be possible benefit in detecting ESBL and ampC type cephalosporinase activity following species identification as an alternative to standard disc approximation testing.

Utilization of industrial waste - Liquid cheese whey for the batch fermentation of lovastatin using Fusarium nectrioides (MH173849) an endophytic fungus: Screening, production and characterization

In this current research, a novel way of utilizing the plant weed and dairy industrial waste for the cost-effective production of Lovastatin by the novel fungus Fusarium nectrioides (MH173849) under controlled conditions was reported for the first time with scientific evidence. A total of 25 endophytic fungi were isolated from the 90 tissue fragments of Euphorbia hirta (L) and identified based on morphological and microscopical characteristics. All the fungal isolates were screened for Lovastatin production using Neurospora crassa bioassay. Among the 25 fungal isolates, Fusarium sp2, Nigrospora sphaerica, and Fusarium sp 4 showed maximum zone of inhibition and they were further verified by Thin Layer Chromatography. Since the Rf values of Fusarium sp 4 and standard Lovastatin were the same, further characterization was preceded only with Fusarium sp 4. An evolutionary relationship of two positive isolates, Fusarium sp 2 and Fusarium sp 4 was studied with other Lovastatin-producing fungi. Gene sequencing and BLAST revealed that a novel fungus, Fusarium sp 4 was found to be Fusarium nectrioides (MH173849) and it was further used for batch fermentation of Lovastatin in the modified media using liquid cheese whey under controlled conditions, which enhanced the productivity up to 43.40 μg/mL with the minimum purification steps. LC-MS-MS and NMR studies confirmed the production of Lovastatin by F. nectrioides (MH173849) due to the presence of Pyran molecule hydrogen, Hydrogen fusing two molecules as intermediate with triplet signal groups, methylbutanoic acid, and hexahydro naphthalene. Therefore, this fungus may be utilized by industries for the cost-effective production of Lovastatin.