Routine Bacteriological Media Research Articles

Isolation and molecular diagnosis of Citrobacter freundii in raw meat (beef, mutton and fish) in AL-Rusafa district of Baghdad city

Citrobacte freundii one of the important foodborne contaminated bacteria in meat and fish, while in Iraq there are limited researches about this bacterium, for that, the objectives of this study include isolate, diagnosed and molecular characterization of Citrobacter freundii in raw meat (beef, mutton) and carp fish samples collected from local markets and different butcher’s shop in a AL-Rusafa district of Baghdad city. One hundred and fifty samples were collected aseptically as 50 samples from each of meats type, there were submitted to laboratory examinations, to culture on routine bacteriological media as a first step for bacterial diagnosis. Bacterial identification done by biochemical test and confirmed by Vitek-2 and Molecular assays (polymerase chain reaction (PCR)). Result showed there are 23 from 150 samples having Citrobacter freundii. Highest C. freundii percentage found in carp samples at (36%), flowed by mutton and beef (6%, 4%) respectively. We conclude that carp fish represent one of the important sources for bacterial foodborne disease where it used as a maine source of proteins and minerals in Iraq.

Bacterial Co-infections and Antibacterial therapy in retrospective COVID-19 patients in AL Fallujah General Teaching Hospital

In order to determine a retrospective study data were collected from all 2031 COVID-19 patients detected by PCR (nasal swap) from January/ 2020- January /2021 for Fallujah General Teaching Hospital ,which total number of Positive covid19 were 210 in isolation ward and Negative number of COVID-19 were 1821.The study condense on bacterial co-infections and antibacterial therapy for this bacteria. Clinical information were : the genera females were 60 ( 28.5%) and were males 150 (71.4%). microbiological detected isolation from blood sample through 48hour of 210 COVID-19 patients in isolation ward detected by BACT/ALERT® Culture Media(1)then cultured on routine bacteriological media according to(2)for positive growth isolated bacteria then identification were done by biochemical test. The result of bacterial isolation from blood of patients assumptive co-infections among 210 samples, positive growth isolated of bacteria which were 121 (57,6%) and included S. pneumoniae , S. aureus , S. epidermidis, K. pneumoniae, p. aeroginosa and E. coli and total negative isolated which indeed enclose 89 (42.3%). Depending on ABS achieve guidelines, the most commonly used antibiotic way was Meropenem, Azithromycin and Cefixime with a median duration of 6 (range 1–13) days in isolation ward and when using Antibiotic Susceptibility test expressed each resistant and sensitive bacteria to antibiotic discs for all positive isolate and detected by sensitivity patterns of VITEK2 system to some species as ensuring method, but; the study focus on co-infection had the highest frequency of Staphylococcus aureus 57 (47.1%) isolates which were resistant to Ciprofloxacin, Chloramphenicol, Gentamycin, Amikacin, azithromycin, Cefotaxime, tetracycline ,Trimethoprim/ Sulphamethoxazole and sensitive to Levofloxacin and imipenem.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

In cholera-endemic areas, toxigenic Vibrio cholerae persist in the aquatic ecosystem mostly in a biofilm-associated state in which the bacteria remain embedded in an exopolysaccharide matrix. The biofilm-associated cells often enter into a dormant form referred to as conditionally viable environmental cells (CVEC), which resist cultivation on routine bacteriological media. However, these cells can naturally resuscitate into the active planktonic form through various mechanisms, multiply, and cause epidemics of cholera. This study was conducted to study possible effects of environmental bacteriophages on the prevalence and distribution of the pathogen between the biofilm associated state, and the planktonic form.MethodsPhages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored.ResultsA phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms.SignificanceBesides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.

Association between pyuria and uropathogen in suspected urinary tract infection

Background: Urinary tract infections (UTI) are one of the most common infectious diseases diagnosed in outpatients as well as in hospitalized patients, and can lead to significant mortality. Several rapid methods have been developed in diagnosing urinary tract infections such as microscopic examination, chemical tests, and automated systems. Thus, in this study we aimed at determining pyuria in suspected UTI patients and their association with uropathogen. Materials and Methods: A total of 379 clean catch mid-stream urine samples were collected in sterile containers. Manual microscopy is used to determine pyuria. All the urine samples inoculated on routine bacteriological media. Results: Out of 379 urine samples, 181 yielded significant bacterial growth. Patients belonged to age group 21-30 were more commonly had significant growth followed by 11-20 years age group. Escherichia coli was the most common organism isolated. Majority of urine specimens which showed plenty of pus cells per high power field yielded significant growth. Escherichia coli was most commonly associated with pyuria. Conclusion: As the number of pus cells in urine increases, the chance of yielding significant bacterial growth is also high. Overall, significant pyuria was observed among specimens yielded Gram negative bacilli than Gram positive cocci. Among Gram negative bacilli, Escherichia coli was most commonly accompanied with pyuria.A

Edwardsiella tarda의 비배양성 생존상태(VBNC) 유도 및 소생 특성

Viable but nonculturable (VBNC) 상태에 들어간 세균은 일반적인 증균 배지에서는 집락을 형성하지 않지만, 죽은 것이 아니라 낮은 대사활성상태로 유지되고 있다. 본 연구에서는 <TEX>$10^{\circ}C$</TEX>의 저온 빈영양 해수에서 Edwardsiella tarda를 VBNC 상태로 유도한 후, 해수 온도를 10에서 <TEX>$25^{\circ}C$</TEX>로 상승시킬 때 첨가된 유기물의 종류에 따라 VBNC 상태인 균의 소생 가능성을 알아보고자 하였다. E. tarda가 접종된 빈영양 해수 microcosm을 <TEX>$10^{\circ}C$</TEX>에 유지하였을 때 VBNC 유도 기간은 42-84일까지 다양하였다. 유도 기간 동안 acridine orange direct counting법으로 계수한 총 균수는 초기 접종 농도인 약 <TEX>$10^8cells/ml$</TEX>로 일정하였으며, direct viable counting법으로 계수한 생존 균수는 약 <TEX>$10^4cells/ml$</TEX>로 감소되었다. VBNC E. tarda에 효모추출물, 넙치근육추출물 그리고 혈청을 첨가하여 <TEX>$25^{\circ}C$</TEX>에서 소생을 유도한 결과 전체 시료 개수의 37%, 23%, 37%에서 각각 소생이 확인되었으며 소생된 E. tarda의 특성은 VBNC 유도 전 원래의 세균과 일치하였다. 소생된 E. tarda를 넙치(Paralichthys olivaceus)에 복강 주사 하였을 때 접종 후 5일 이내에 시험어가 모두 사망함으로써 VBNC 상태의 E. tarda가 독력을 유지하고 있었음을 시사하였다. 그러므로 E. tarda는 우리나라 남해 연안 겨울의 저온 빈영양 해수에서 VBNC 상태로 유도되었다가 여름과 가을 시기에 수온 상승과 더불어 소생되어 양식 넙치에 지속적인 발병 요인이 되고 있는 것으로 생각된다. Bacteria in the viable but nonculturable (VBNC) state fail to produce colonies on routine bacteriological media, but are still alive in the state of very low metabolic activity. The aim of the present study was to induce the VBNC state of the Edwardsiella tarda using sea water microcosm under starvation conditions at <TEX>$10^{\circ}C$</TEX> and to investigate resuscitation of the VBNC cells in temperatures changed from 10 to <TEX>$25^{\circ}C$</TEX>, with and without additives. E. tarda entered into the VBNC state within about 42-84 days of incubation in the microcosm. Throughout this period, the total cell counts as determined using acridine orange direct counting remained near the original inoculum level of <TEX>${\sim}10^8cells/ml$</TEX>. The live cell counts measured with direct viable counting, on the other hands, declined to <TEX>${\sim}10^4cells/ml$</TEX>. When the VBNC cells were incubated with addition of yeast extract, fish muscle extract or serum at <TEX>$25^{\circ}C$</TEX>, the ratios of resuscitated samples were 37%, 23%, and 37%, respectively. The characteristics of resuscitated E. tarda were consistent with those of the original E. tarda. When the resuscitated E. tarda were intraperitoneally injected into olive flounders, all fishes died within 5 days, indicating that the VBNC E. tarda might retain its pathogenic potential. Therefore, E. tarda under starvation conditions in the winter enter into the VBNC state and the VBNC E. tarda cells resuscitated at summer and autumn seawater temperature are considered to be pathogen continuously to olive flounder on the southern coast of Korea.

Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples.

Cholera epidemics have long been known to spread through water contaminated with human fecal material containing the toxigenic bacterium Vibrio cholerae. However, detection of V. cholerae in water is complicated by the existence of a dormant state in which the organism remains viable, but resists cultivation on routine bacteriological media. Growth in the mammalian intestine has been reported to trigger "resuscitation" of such dormant cells, and these studies have prompted the search for resuscitation factors. Although some positive reports have emerged from these investigations, the precise molecular signals that activate dormant V. cholerae have remained elusive. Quorum-sensing autoinducers are small molecules that ordinarily regulate bacterial gene expression in response to cell density or interspecies bacterial interactions. We have found that isolation of pathogenic clones of V. cholerae from surface waters in Bangladesh is dramatically improved by using enrichment media containing autoinducers either expressed from cloned synthase genes or prepared by chemical synthesis. These results may contribute to averting future disasters by providing a strategy for early detection of V. cholerae in surface waters that have been contaminated with the stools of cholera patients or asymptomatic infected human carriers.

A lysozyme and magnetic bead based method of separating intact bacteria

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Laboratory Aspects in the Diagnosis of Coccidioidomycosis

Coccidioides immitis and Coccidioides posadasii, the two recognized causes of coccidioidomycosis, may be detected by direct microscopy, culture, and serologic documentation. Two useful stains include the Grocott methenamine silver (GMS) and the calcofluor white (CFW). Other useful stains used in histopathologic studies include hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). Nucleic acid amplification tests (NAATs) have been introduced for detection of Coccidioides spp. in specimens, but are not yet commercially available. Isolation of Coccidioides spp. by culture is not difficult as many fungal as well as routine bacteriologic media are available. For the safe isolation of Coccidioides spp., the laboratory should maintain a biological safety level 2 or 3. Identification of Coccidioides spp. uses the organisms' phenotypic or genotypic characteristics. Phenotypic identification to genus level may be achieved by visualization of spherules in specimens and/or by the presence of arthroconidia in culture. Isolates may be confirmed as Coccidioides spp. by molecular probes. Separation of species into C. immitis and C. posadasii is best achieved by specialized molecular techniques which are not normally available in routine clinical laboratories. Humoral antibodies can be used for the diagnosis and prognosis of coccidioidomycosis. Although positive serologic results may be helpful in the diagnosis of coccidioidomycosis, negative serologic results cannot be used to rule out the disease. Enzyme immunoassays (EIA) and immunodiffusion methods are commonly used for detection of both IgM and IgG antibody groups. Sequential complement fixation (CF) studies for IgG class of antibody are useful for the prognosis of coccidioidomycosis.

Pneumonia of Turkey Breeder Hens Associated with Mycoplasma Synoviae

Turkey breeder hens showed an increase in mortality beginning at 38 wk of age with no other clinical signs or changes in egg production. While no respiratory signs were observed in live turkeys, those that died consistently had gross lesions of pneumonia. Histopathology of lungs revealed serofibrinous bronchopneumonia, lymphofollicular reaction, and other features suggesting a bacterial etiology. However, except for incidental findings, bacteria were not visualized in the sections examined, and none were isolated in meaningful numbers on routine bacteriologic media. At 42 wk of age the flock showed serologic evidence of infection with Mycoplasma synoviae (MS), and MS was identified by both mycoplasma culture and polymerase chain reaction (PCR) procedures in samples from choanal clefts and tracheas. Results of lung histopathology and PCR tests were consistent with a diagnosis of pneumonia caused by MS.

Mucoid Encapsulated Enterococcus faecalis: An Emerging Morphotype Isolated from Patients with Urinary Tract Infections

Three strains of encapsulated Enterococcus faecalis,which produced highly mucoid coalescing colonies on routine bacteriologic media, were isolated from urine specimens of patients with urinary tract infection. Encapsulation could be demonstrated through India ink preparations. The occurrence of this unusual enterococcal colony morphotype, which resembles that of a Gram-negative bacterium, may delay true identification.

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a serious human pathogen, accounting for 95% of all seafood-related deaths in the United States. During the winter months, when coastal water temperatures drop below 10 °C, investigators have repeatedly reported their inability to isolate this estuarine bacterium from the environment. We now realize that this apparent ‘die-off’ is actually due to entry of the cells into a ‘viable but non-culturable’ state, a survival response to the low temperature stress. Cells in this state appear dormant, and cannot be cultured in or on routine bacteriological media, but are capable of returning to the actively metabolizing state when the environmental stress is removed. This review describes this non-culturable state in V. vulnificus, and its role in the ecology, physiology, and epidemiology of this pathogen.

The viable but non-culturable state in the human pathogen Vibrio vulnificus.

Vibrio vulnificus is a serious human pathogen, accounting for 95% of all seafood-related deaths in the United States. During the winter months, when coastal water temperatures drop below 10 degrees C, investigators have repeatedly reported their inability to isolate this estuarine bacterium from the environment. We now realize that this apparent 'die-off' is actually due to entry of the cells into a 'viable but non-culturable' state, a survival response to the low temperature stress. Cells in this state appear dormant, and cannot be cultured in or on routine bacteriological media, but are capable of returning to the actively metabolizing state when the environmental stress is removed. This review describes this non-culturable state in V. vulnificus, and its role in the ecology, physiology, and epidemiology of this pathogen.

Mycobacterium chelonei infection of a Broviac catheter insertion site

Mycobacterium chelonei infection developed at the insertion site of an indwelling Broviac catheter in a child with erythroleukemia. Direct adherence to and colonization of the intra- and extra-luminal surfaces of the catheter, with extension to the adjacent subcutaneous tissue, by this rapidly growing mycobacterium may have been the primary factor underscoring the infection. Nontuberculous mycobacteria such as Mycobacterium chelonei grow readily on routine bacteriologic media and resemble Corynebacterium spp. (diphtheroids) in their Gram staining and microscopic characteristics. The persistence of the infectious process and a diphtheroid-like microorganism despite antimicrobial therapy should raise the suspicion for a mycobacterial species.

ASSOCIATION OF UREAPLASMA UREALYTICUM INFECTION OF THE LOWER RESPIRATORY TRACT WITH CHRONIC LUNG DISEASE AND DEATH IN VERY-LOW-BIRTH-WEIGHT INFANTS

Endotracheal aspirates from 200 infants who weighed ≤ 2500 g and who had evidence of respiratory disease were cultured within 24 h of birth for mycoplasmas, chlamydiae, viruses, and bacteria to evaluate the relation between lower respiratory tract infection and development of chronic lung disease and/or death. Ureaplasma urealyticum, an organism not visible on gram stain, not recovered on routine bacteriological media, and not susceptible to antibiotics commonly used to treat neonatal infections, was the single most common organism isolated. 14% of isolates were from infants born by caesarean section with intact membranes, which indicated that infection had occurred in utero. The findings probably represented true infection of the lower respiratory tract because the organism was recovered in pure culture in numbers > 10 3 from 85% of the infants, and also from the blood in 26% of infants. Those infants ≤ 1000 g with Ureaplasma urealyticum infection of the lower respiratory tract were twice more likely to have chronic lung disease or to die than were infants of similar birth-weight but who were uninfected, or infants > 1000 g. Very-low-birth-weight infants infected with ureaplasmas did not differ from those uninfected, either in demographic features or in potential risk factors for chronic lung disease.

CHRONIC UREAPLASMA UREALYTICUM AND MYCOPLASMA HOMINIS INFECTIONS OF CENTRAL NERVOUS SYSTEM IN PRETERM INFANTS

Summa ry In a prospective study of meningitis in 100 predominantly preterm infants, Ureaplasma urealyticum was isolated from the cerebrospinal fluid (CSF) of 8 and Mycoplasma hominis from the CSF of 5 babies undergoing investigation of suspected sepsis or treatment of hydrocephalus. U urealyticum was isolated from 6 infants with severe intraventricular haemorrhage and from 3 with hydrocephalus. In 4 babies multiple isolations were made over several weeks. There were clinical features of congenital infection with major neurological impairment in 1 infant infected with M hominis. Diagnosis is difficult because these organisms cannot be seen on gram stain and cannot readily be cultivated on routine bacteriological media, and CSF pleocytosis may be absent. This study, which used appropriate mycoplasmal media, shows that U urealyticum and M hominis are the most common microorganisms isolated from the CSF of newborn infants in a high-risk population.

TATLOCK bacterium (Pittsburgh pneumonia agent) presumptively identified in five cases of pneumonia.

Excerpt Recently Rogers and co-workers (1) described five cases of pneumonia caused by a bacterium that could not be isolated on routine bacteriologic media. They suggested that the organism might ...

Vitamin B6-dependent Streptococcus mitior (mitis) isolated from patients with systemic infections.

Strains of nutritional-variant viridans Streptococcus were isolated from two patients with bacterial endocarditis and from one patient with a pancreatic abscess. All three strains grew as satellite colonies of other bacteria but did not grow as pure cultures in media lacking sufficient thiol compounds. For one-half maximal growth these organisms required the addition of one of the active forms of the B6 vitamin, pyridoxal with HCl (0.16-1.45 mug/ml) or pyridoxamine dihydrochloride (0.67-2.0 mug/ml), or addition of l-cysteine (0.235-0.425 mg/ml) to routine bacteriological media. The active forms of vitamin B6 are essential coenzymes in the synthesis of l-cysteine and of other thiol compounds. Use of media supplemented with 0.001 percent pyridoxal with HCL led to identification of the strains as Streptococcus mitior (mitis). Incorporation of pyridoxal with HCl, pyridoxamine dihydrochloride, or l-cysteine into media is recommended for the isolation and identification of vitamin B6-dependent viridans streptococci.

Laboratory identification of Torulopsis glabrata: typical appearance on routine bacteriological media.

The colonial morphology of the yeast Torulopsis glabrata on sheep blood-agar is characteristic and was useful in the identification of 24% of clinical isolates.

Laboratory Identification of Torulopsis glabrata: Typical Appearance on Routine Bacteriological Media

The colonial morphology of the yeast Torulopsis glabrata on sheep blood-agar is characteristic and was useful in the identification of 24% of clinical isolates.