Laboratory Identification of Haemophilus influenzae A Practical Laboratory Study

To assess the in vitro efficacy of doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii using Epsilometer strips. Cross-sectional study. Department of Microbiology, Army Medical College, Rawalpindi and National University of Sciences and Technology, Islamabad, from May 2014 to September 2014. A total of 60 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa collected from various clinical samples received from Military Hospital were included in the study. The specimens were inoculated onto blood, MacConkey and chocolate agars. The isolates were identified using Gram staining, motility, catalase test, oxidase test and API 20NE (Biomeriux, France). Organisms identified as Acinetobacter baumannii and Pseudomonas aeruginosa were included in the study. Bacterial suspensions equivalent to 0.5 McFarland turbidity standard of the isolates were prepared and applied on Mueller Hinton agar. Epsilometer strip was placed in the center of the plate and incubated for 18-24 hours. Minimum Inhibitory Concentration (MIC) was taken to be the point where the epsilon intersected the E-strip. MIC of all the isolates was noted. For Pseudomonas aeruginosa isolates, MIC(50) was 12 µg/mL and MIC(90) was 32 µg/mL. For Acinetobacter baumannii MIC(50) and MIC(90) was 32 µg/mL. Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting.

Diabetic foot is a major problem lead to a serious complications. Immune impairment in this group of patients predispose to bacterial infections. Different bacterial species were identified from samples taken from diabetic foot including gram positive and gram negative species. Antimicrobial considered important part of treatment regime and selection of effective antimicrobial will lead to better recovery results. This study was aimed to identify bacterial species infected diabetic foot and their antimicrobial patterns. Sterile cotton swab applied on the lesions and transferred to the laboratory immediately. Standard microbiology techniques were used to identify bacterial species whereas different media were used; MacConkey agar, Chocolate agar and blood agar. Multiple biochemical API 20 E test was used to identify gram negative bacteria. Catalase and coagulase test also used for gram positive collection identification. Disc diffusion method used to evaluate the antimicrobial susceptibility of collected identified isolates. Polymicrobial infections were 47.5% (19/40). Dominant combination of identified bacterial species was gram positive and gram negative group 15 (78.9). The highest prevalent bacterial species among gram positive isolates was S. aureus 31.7% (20/63) followed by coagulase negative staphylococci (CNS)15.9% (10/63) while Pseudomonas aeruginosa 17.5% (11/63) was the most prevalent among gram negative bacterial species. Methicillin resistant isolates was detected in 55% (n=11/20) of identified S. aureus collection. Antimicrobial resistance, Amikacin showed the highest effect against gram negative bacteria and ciprofloxacin against gram positive bacteria. High prevalence of polymicrobial infection in diabetic foot increase the concern of serious complications. S. aureus and CNS infection occurrence was high and their resistance to methicillin was a major concern. Most combination of polymicrobial infection consisted from gram positive and negative bacterial species which require antimicrobials effective against both groups.

Besides the gastric bacterium Helicobacter pylori, strains of Pseudomonas, Campylobacter,Bacteroides, Bifidobacterium Eubacterium, andStaphylococcus-like bacteria can be found in the human stomach in a state of disease (1, 2). Pseudomonas aeruginosa infection can lead to false-positive results of immunological tests to detect H. pylori (3) and may inhibit its in vitro growth (4). Contaminants or gastric non-H. pylori bacteria can grow in cultures of gastric specimens. We use brucella agar plates containing 5% calf serum, vancomycin (10 mg/ml), polymyxin B (10 mg/ml), and nalidixic acid (20 mg/ml) for culturing H. pylori from gastric biopsy specimens. We have found pure cultures of gram-negative bacilli that were positive for rapid urease, catalase, and oxidase tests but with colony characteristics (white small colonies rather than the transparent ones typical of H. pylori) that made us suspect that the strains were not H. pylori. We decided to further investigate three of these strains (isolated from three different patients) that could have lead to false identification of H. pylori, if only cell morphology and biochemical tests would have been considered. Unlike H. pylori, our three isolates could grow aerobically. Electron microscopy observations showed gram-negative bacilli with multiple unipolar flagella, also found in H. pylori. Two isolates had a unipolar tuft of two or more flagella, and the other had one polar flagellum. Analysis of the cell membrane fatty acids of the three strains (Midi System; Hewlett Packard) indicated a high index of identity with Pseudomonas aeruginosaand the organisms formerly known as Pseudomonas cepaciaand Pseudomonas diminuta (currently known asBurkholderia cepacia and Brevundimonas diminuta, respectively). All three species have been previously encountered in clinical specimens. Pseudomonas species may have a polar monotrichous flagellum (P. diminuta) or fewer than three flagella (P. aeruginosa [96%]) or have a tuft of three to eight flagella (P. cepacia [99%]). They are catalase-positive and can also be positive for urease (P. aeruginosa [72%]), P. cepacia [46%]), andP. diminuta [<10%]), oxidase, and resistant to polymyxin (P. aeruginosa [3%]), P. cepacia[>90%]), and P. diminuta [10%]).

Background Pneumonia is a condition, where bacterial infections are implicated as the most common causes of morbidity and mortality in humans. The actual burden of HIV-infected patients with pneumonia is not well documented in Mekelle region of Ethiopia. This study estimated the prevalence of bacterial pneumonia in HIV patients, antimicrobial susceptibility patterns of pathogens implicated in pneumonia, and associated risk factors in Mekelle zone, Tigray, Northern Ethiopia, during August-December 2016. Methods Sputum specimens were collected from 252 HIV seropositive individuals with suspected pneumonia. Data on sociodemographics and risk factors were also collected using a structured questionnaire. Blood, Chocolate, and Mac Conkey agar plates (Oxoid, Hampshire, UK) were used to grow the isolates. The isolated colonies were identified based on Gram stain, colony morphology, pigmentation, hemolysis, and biochemical tests. The antimicrobial susceptibility test was performed using the modified Kirby-Bauer disc diffusion method. The analysis was performed using SPSS version 22 and p-value < 0.05 with corresponding 95% confidence interval (CI) was considered statistically significant. Results Out of the 252 samples, 110 (43.7%) were positive for various bacterial species. The predominant bacterial species were Klebsiella pneumoniae (n=26, 23.6 %) followed by Streptococcus pneumoniae (n=17, 15.5 %), Escherichia coli (n=16, 14.5%), Klebsiella spp. (n=15, 13.6%), Staphylococcus aureus (n=9, 8.2%), Enterobacter spp. (n=7, 6.3%), Pseudomonas aeruginosa (4, n=3.6%), Proteus spp. (n=4, 3.6%), Citrobacter freundii (n=7, 6.3%), Streptococcus pyogenes (3, 2.7%), and Haemophilus influenzae (n=2, 1.8%). Young age (18-29), recent CD4+ count less than 350 cells/mL, alcohol consumption, and HIV WHO stage II showed significant association with the occurrence of bacterial pneumonia. Resistance to penicillin, co-trimoxazole, and tetracycline was observed in 81.8%, 39.8%, and 24.5% of the isolates, respectively. Conclusions The problem of pneumonia among HIV patients was significant in the study area. The high prevalence of drug-resistant bacteria isolated from the patient's samples possesses a health risk in immunocompromised HIV patients. There is a need to strengthen and expand culture and susceptibility procedures for the administration of appropriate therapy to improve patients management and care which may aid in decreasing the mortality.

Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Månsson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MS(n)). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MS(n) provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection, MEF samples were analyzed at 2, 5, and 9 days postinfection by CE-ESI-MS following minimal passage on chocolate agar. As previously observed, sialic acid-containing glycoforms were detected during the early stages of infection, but a trend toward more-truncated and less-complex LPS glycoforms that lacked sialic acid was found as disease progressed.

Legionella species, a Gram negative bacterium is the causative agent of Legionnaires’ disease, a potentially fatal pneumonic syndrome of widely recognized public health importance. The aim of the study was to determine the presence of Legionella pneumophila in domestic water (borehole) in Benin metropolis by cultural method. One hundred and ninety-eight (198) water samples from the eight-two facilities (grouped into three: public apartments/hotels, private apartments and eateries) were cultured on BCYE made selective with the addition of legionella supplement IV and growth supplement after concentration and heat treatment at 50 0 C for 30 minutes and incubated at 37 0 C. Isolates were identified by doing gram stain, oxidase, catalase and hippurate test, final identification was done by using PCR, sequence and blast search using National Center for Biotechnology Information (NCBI). The results obtained showed that Legionella pneumophila or Legionella species was not isolated though other bacteria (such as Burkholderia bacterium MSMB7 (32%), Pseudomonas antarctica (28%), Cupriavidus gilaridii (14%), Microbacterium paraoxydans (14%), Bacillus thuringensis (4%), Bacillus cereus (2%), Acinetobacter johnsonii (6%)) were detected. In conclusion zero percent (0%) prevalence of Legionella species in the water systems investigated though other bacteria with pathogenic potential were recovered. This finding suggests that water systems in Benin metropolis may not present vehicles for the transmission of diseases associated with Legionella species. Keywords: Legionella pneumophila , domestic water, search, BCYE, Benin metropolis

Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non-typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of licl and lic2, which contain translational switches based on intragenic tandem repeats of 5'-CAAT-3'. Genetic analysis showed that opaque organisms have an out-of-frame number of repeats in both licl, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galalpha1-4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C-reactive protein (CRP). The addition of ChoP by licl was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galalpha1-4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host-like antigen blocks antibody-mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galalpha1-4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.

Introduction and Objective: Sheep blood is the recommended type of blood for supplementation of agar media. In Sri Lanka, due to lack of availability of sheep blood, expired citrated human blood is used which gives poor haemolysis and causes difficulties in identification of some organisms. In addition, human blood contains antibodies and other antibacterial factors including antibiotics which may inhibit bacterial growth. Human blood may also contain blood borne pathogens which could be a risk for laboratory staff. The objective of this study was to explore available alternatives in the Sri Lankan setting. Methods: Clinical isolates and standard strains of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae and Haemophilus influenzae were tested for growth, identification characteristics and antibiotic susceptibility on human, cattle, goat and sheep blood agars. The performances were compared. Identification of Listeria monocytogenes as well as Streptococcus agalactiae was carried out using the CAMP test. Results: All tested organisms gave similar isolation rates at the tested dilutions in the four tested agar plates. Human blood gave noticeably smaller colonies. S. pyogenes and S. agalactiae gave equally large zones of beta-haemolysis and S. pneumoniae gave obvious alpha-haemolysis on all animal blood agar plates. Both types of haemolysis were faint on human blood. Typical arrow head shape haemolysis for S. agalactiae and match-head shape haemolysis for Listeria were seen in the CAMP test on the three animal blood agar plates whereas human blood gave negative results. All blood agar plates gave comparable positive results in the satellitism test for H. influenzae. There was no difference in bacitracin and optochin sensitivity tests for identification of S. pyogenes and S. pneumoniae respectively. Inhibitory zones were unreadable when antibiotic susceptibility was done for H. influenzae on goat and cattle chocolate agar. Sheep and human chocolate agar were inferior to Haemophilus test medium. ABST results were equivalent but goat blood gave hazy, irregular margins for other organisms. Conclusions: Cattle and goat blood show similar performance to sheep blood in growth and identification tests for common fastidious pathogens. Cattle blood is equivalent to sheep blood for ABST, but goat blood is inferior.

This is summarized in Table 8.1. a) Microscopy— A cell count is performed on sterile fluids and CSF samples using the Neubauer chamber or a similar device. The number of white blood cells (WBC) and red blood cells seen under the microscope are reported as well as the differential WBC count (i.e. the number or percentage of lymphocytes and neutrophils in the sample). A Gram stain is then done and the presence of any organism reported. b) Culture samples are plated onto the appropriate media and streaked out for single colonies as shown. Blood agar is normally used; however, other media are used depending on the site of the specimen, e.g. chocolate agar is used if a fastidious organism is a potential pathogen such as Haemophilus sp.; anaerobic agar for anaerobes; selective agar such as MacConkey can be used on non-sterile specimens to differentiate between the colony types. Plates are incubated for eighteen to forty-eight hours at the correct conditions; most plates being CO2, others at O2 and anaerobically. c) Identification plates are examined for growth. Potentially significant isolates are identified either by MALDI-TOF MS, by API, or other biochemical tests. d) Sensitivities are performed on significant organisms by manual and automated methods. This is summarized in Table 8.2. Selective agar is necessary when isolating pathogens from faeces, although further confirmatory tests are needed. ● Black or colourless colonies on xylose lysine deoxycholate (XLD) or other chromogenic agar plates are tested with oxidase reagent. ● Oxidase negative isolates are identified by MALDI-TOF, API and or biochemically using triple-sugar iron (TSI) tubes. ● Serology is then performed on suspicious isolates and sent to a reference laboratory for confirmation. ● Campylobacter is confirmed by testing grey flat colonies on campylobacter agar with oxidase reagent. Oxidase positive samples are Gram stained and if ‘seagull’-shaped gram-negative bacteria are observed under the microscope, campylobacter is confirmed. The catalase test is a simple biochemical test to differentiate between Staphylococcus species and Streptococcus species, with the use of hydrogen peroxide (H2O2). It tests for the presence of the enzyme catalase which is found in Staphylococcus species.

Community-acquired pneumonia (CAP) is mostly caused by bacteria and viruses. Identifying pathogenic bacteria and viruses using traditional culture techniques is challenging. Therefore, multiplex real-time PCR (MPL-rPCR) has the capacity not only to concurrently identify the causative bacteria, atypical bacteria, and viruses but also to quantify their load and detect co-infections. This study was carried out on patients with CAP who were admitte to the Respiratory departments of Nguyen Tri Phuong Hospital, Nhan Dan Gia Dinh Hospital and University Medical Center, from April 2021 to March 2023, using a cross-sectional descriptive design in prospect. Sputum samples, evaluated by the Barlett scale, were collected and processed using the MPL-rPCR technique at Nam Khoa Company’s laboratory. Bacterial pathogens and viruses were detected at rates of 67.7% and 57.5% (p&lt;0.05). Gram – negative bacteria included Klebsiella pneumoniae at 18.5%, Acinetobacter baumannii at 17.3%, and Haemophilus influenzae at 14.1%. Among Gram-positive bacteria, Streptococcus pneumoniae was found at 16.4%. The Epstein–Barr virus was the most frequently identified virus at 34.9%, followed by Cytomegalovirus at 16.7%, and Influenza virus type A at 10.3%. One sputum sample showed the presence of more than one bacterium or virus, with high rates observed for Epstein–Barr virus and Cytomegalovirus. Gram – negative bacteria are found in high proportions, and viruses were predominant, particularly Epstein–Barr virus, Cytomegalovirus, Influenza virus types A and B. Almost all viruses were co-infected with pathogenic bacteria, and multiple bacteria or viruses were identified in one sputum sample.

Moxifloxacin (Bay 12-8039) is a new 8 methoxy quinolone antibacterial. The MIC90 values are < 0.25 mg/l for Streptococcus pneumoniae (irrespective of penicillin susceptibility), Haemophilus influenzae (β-lactamase positive or negative), Morexella catarrhalis, Bordetella pertussis, Legionella sp., Mycoplasma pneumoniae, Clamydia pneumoniae, Mycobacterium tuberculosis, methicillin-sensitive Staphylococcus aureus, β-haemolytic streptococci (macrolide-sensitive or -resistant), Listeria sp., most Enterobacteriaceae, Salmonella sp., Shigella sp., Neisseria gonorrhoeae, N. menigitidis, Pasteurella spp., spp. andYersinia enterocolitica. For Mycobacterium intracellularae, methicillin-resistant S. aureus (MRSA), ciprofloxacin-resistant S. aureus, Citrobacter freundii, Providencia sp., Serratia sp., P. aeruginosa and other non-fermentive Gram-negative rods, MIC90s are in the range 0.5 - 4 mg/l. For anaerobic bacteria species, MIC90s are also in the range 0.25 - 4 mg/l. Moxifloxacin is bactericidal at concentrations 2- to 4-fold higher than the MIC and is rapidly bactericidal against most common pathogen groups at concentrations achieved in serum with a 400 mg dose that is between 0.5 - 4 mg/l. There is a post-antibiotic effect against Gram-positive and -negative bacteria. Resistant mutants are at present difficult to select in the laboratory but in general, moxifloxacin has poorer activity against strains resistant to ciprofloxacin compared to those which are susceptible. Animal and laboratory pharmacodynamic models indicate that the MIC and area under the serum concentration time curve predict outcome. Various animal models mainly of respiratory tract infection indicate equivalent or superior results compared to existing or other developmental agents. Human pharmacokinetics in healthy volunteers indicate linear pharmacokinetics over the dose range 50 - 800 mg/day. A single dose of 400 mg produces a maximum serum concentration of 2.5 - 4.5 mg/l, half-life of 11 - 15 h, AUC of 25 - 40 mg x h/l and volume of distribution of 2.5 - 3.5 L/kg. Protein binding is about 50% and two metabolites have been identified (M-1 and M-2). Bioavailability is >> 85% and a minority of clearance is via the kidneys. No dose modification is required in renal impairment. Extra vascular penetration, where studied, is comparable to that of other quinolones. At present undergoing clinical trials, with a focus on respiratory tract infection, it is likely that moxifloxacin will provide effective therapy for pathogens with MICs of < 0.25 - 0.5 mg/l. The safety profile in a large number of human subjects is awaited.

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria are well characterized and may be useful to infer a solution to better understand the translocation process in Brucella.

Children are more susceptible to ear infection than adults worldwide, especially in developing countries. In Bangladesh like other developing countries antibiotics is a common choice of treatment in an ear infection. This study was sought to determine causative agents both in right and left ear infection and their antibacterial susceptibility pattern from patients with an ear infection. Specimens of ear discharge were collected aseptically using a sterile swab and cultured on MacConkey’s agar, blood agar, and chocolate agar. After inoculating on appropriate culture media bacterial isolates were identified by their colony morphology, culture characteristics, Gram reaction, and biochemical tests. In this study, a total of 70 positive cases of patients were included. Among these 27 (38.6%) were male, and 43(61.4%) were female. The prevalence of ear infection was significantly high in females (61.4%) than males (38.6%). The predominant bacterial isolates from positive culture were Staphylococcus aureus 43(61.4%), followed by Pseudomonas spp., 13 (18.6%), Streptococcus spp., 8(11.4%), Proteus spp., 5(7.2%), and Klebsiella spp., 1(1.4%). The antibacterial agent like Amikacin and Gentamicin showed a high level of antibacterial effect on all identified bacterial isolates. On the other hand, 98.6% of isolates were found highly resistant to Co-Trimoxazole and Flucloxacillin. Moreover, Streptococcus spp., Proteus spp., Klebsiella spp., and Pseudomonas spp., were highly resistant to multiple drugs (more than 4). A high degree of antibiotic resistance was observed among most of the drugs used in this study. However, Amikacin and Gentamicin were highly effective against the isolated bacterial species. Therefore, culture and susceptibility tests are vital for the appropriate treatment of ear infection.

Objective:Pneumonia is an opportunistic infection and it is a major cause of mortalityand morbidity among human immunodeficiency virus/acquired immune deficiencysyndrome-positive patients. Previous studies have shown the dominantpathogens bacterial isolates were K. pneumoniae 27.0%,S. aureus 20.8%, S. pneumoniae 18.8%and E. coli 8.3%. This study aimed to determinebacteriology of community-acquired pneumonia, antimicrobial susceptibilitypattern and associated risk factors among human immunodeficiency viruspatients in the Northeast Ethiopia: cross-sectional study.Methods:A health facility-based cross-sectional study was conducted from January toApril 2021 at six health facilities in Dessie Town. A total of 378community-acquired pneumonia patients suspected to be human immunodeficiencyvirus-positive were recruited using a consecutive sampling technique.Sociodemographic and clinical data were collected using a structuredquestionnaire. A two-milliliter sputum specimen was collected asepticallyfrom each study participant. Samples were cultivated on blood agar,chocolate agar and MacConkey agar to isolate bacterial pathogens. Toidentify bacteria pathogens Gram stain, colony morphology and biochemicaltests were performed. The Kirby-Bauer Disc Diffusion method was used toperform the antimicrobial susceptibility test. Descriptive statistics,logistic regression analysis was carried out using Statistical package forsocial science version 25 software. p-value < 0.05 witha corresponding 95% confidence interval (CI) was considered for statisticalsignificance.Result:The overall prevalence of bacterial pneumonia was 175 (46.3%). Gram-negativebacteria accounted for 119 (68%) and the predominant isolates identifiedwere Streptococcus pneumoniae 49 (28%) followed byKlebsiella pneumoniae 46 (26.3%), Pseudomonasaeruginosa 34 (19.4%). There were 148 (84.6%)multidrug-resistant bacteria overall. Statistically significant factorsincluded viral load, cigarette smoking, cluster of differentiation 4 count,alcohol use, World Health Organization clinical stages III and IV and lowwhite blood cell count.Conclusion:The study found that both multidrug resistance and bacterial pneumonia werehigh. Thus, bacterial culture and antimicrobial susceptibility tests shouldbe routinely performed in health facilities in order to prevent and controlthe spread of bacterial infection and concurrent drug resistance.

Agriculture is an important part of the economy of the undivided Midnapore district. Agricultural land is its asset and most importantly its means of sustenance as well as survival. Earthworms are invertebrates that play a key role in recycling organic matters in soils. Since the intestines of earthworms harbor wide ranges of microorganisms, enzymes, hormones etc., these half digested materials decompose rapidly and are transformed into a stabilized material called vermicompost which is very useful for increasing the soil fertility. One has to look for these characters before recommending any species for vermiculture. In the present study, Perionyx excavatus specimens were collected from the undivided Midnapore district and from the Earthworms gut, bacteria, fungus, actinobacteria, and yeast were isolated and identified using various morphological and biochemical tests. All the bacterial isolates were identified using morphological study, staining techniques, and different biochemical tests such as catalase test, KOH test, H2 SO4 test, Starch hydrolysis test, oxidase test, and sucrose hydrolysis test. All the fungal, actinobacteria, and yeast isolates were subjected to staining and morphological characterization (color and texture of fungal colony). Bacterial isolates of genus Bacillus sp., Staphylococcus sp., Enterococci, Micrococcus sp., Enterobacter sp., and Citrobacter sp. were identified. Among the fungal isolates Aspergilus sp., and P. boydii were identified. Streptomyces sp., Nocardia sp. among the actinobacteria and Candida sp. among yeast were also found to be present in earthworm gut and these might play an important role along with the earthworm to increase the quality and fertility of soil.