Primary Subcultures Research Articles

Rapid Detection of PBP2a in Staphylococci from Shortly Incubated Subcultures of Positive Blood Cultures by an Immunochromatographic Assay.

ABSTRACTStaphylococcus aureus, as well as coagulase-negative staphylococci (CoNS), can cause a wide range of human infections both in nosocomial and community settings. Βeta-lactams are the antibiotics of choice for the treatment of bloodstream infections (BSI) caused by these microorganisms. Resistance to virtually all β-lactams (also referred to as methicillin resistance) primarily results from the production of an alternative penicillin-binding protein (PBP2a) encoded by the mecA gene. While β-lactams are still used as first-line therapy against BSI caused by S. aureus, BSI with CoNS are usually treated with vancomycin due to the high prevalence of methicillin resistance. Rapid detection of methicillin resistance is thus critical for continuation or adjustment of the empirical therapy and therewith to improve the clinical outcome of the patients. The revised version of the immunochromatographic assay PBP2a SA culture colony test (SACCT) is a rapid, inexpensive, and easy method that enables reliable detection of PBP2a in mecA-positive staphylococcal isolates after18 to 24 h of incubation. Here, we evaluated the diagnostic performance of the SACCT using primary subcultures of spiked blood cultures after short incubation (4 to 6 h) and established a modified procedure with an equal analytical performance to that of longer-grown cultures. With the proposed method the SACCT can be employed for PBP2a detection from shortly incubated subcultures of clinically relevant staphylococcal isolates, thereby allowing more rapid and effective management of BSI caused by these organisms.IMPORTANCE Antibiotic resistance poses a major threat to health and incurs high economic costs worldwide. Rapid detection of resistance mechanisms can contribute to improving patient care and preventing the dissemination of antimicrobial resistance. Here, we describe a rapid method to detect the most important beta-lactam resistance mechanism (the plasmid-encoded alternative transpeptidase PBP2a) in staphylococcal isolates causing BSI. We show that, using a modified procedure, PBP2a can be reliably detected from primary subcultures of spiked blood cultures after short incubation (4 to 6 h) with a rapid, inexpensive, and simple immunochromatographic test (SACCT). We provide an accurate, inexpensive, and rapid method to facilitate appropriate management and control of infections in patients suffering from invasive staphylococcal infections.

Evaluation of standardized automated rapid antimicrobial susceptibility testing of Enterobacterales-containing blood cultures: a proof-of-principle study.

Rapid antimicrobial susceptibility testing (RAST) of bacteria causing bloodstream infections is critical for implementation of appropriate antibiotic regimens. We have established a procedure to prepare standardized bacterial inocula for Enterobacterales-containing clinical blood cultures and assessed antimicrobial susceptibility testing (AST) data generated with the WASPLabTM automated reading system. A total of 258 blood cultures containing Enterobacterales were examined. Bacteria were enumerated by flow cytometry using the UF-4000 system and adjusted to an inoculum of 106 cfu/mL. Disc diffusion plates were automatically streaked, incubated for 6, 8 and 18 h and imaged using the fully automated WASPLabTM system. Growth inhibition zones were compared with those obtained with inocula prepared from primary subcultures following the EUCAST standard method. Due to time-dependent variations of the inhibition zone diameters, early AST readings were interpreted using time-adjusted tentative breakpoints and areas of technical uncertainty. Inhibition zones obtained after 18 h incubation using an inoculum of 106 cfu/mL prepared directly from blood cultures were highly concordant with those of the EUCAST standard method based on primary subcultures, with categorical agreement (CA) of 95.8%. After 6 and 8 h incubation, 89.5% and 93.0% of the isolates produced interpretable results, respectively, with CA of >98.5% and very low numbers of clinical categorization errors for both the 6 h and 8 h readings. Overall, with the standardized and automated RAST method, consistent AST data from blood cultures containing Enterobacterales can be generated after 6-8 h of incubation and subsequently confirmed by standard reading of the same plate after 18 h.

In vitro resistance acquisition in Mycoplasma gallisepticum against ofloxacin, tylosin and spectinomycin

To clarify a mechanism of acquired resistance of two Mycoplasma gallisepticum (MG) strains against ofloxacin (OFLX) along with tylosin (TS) and spectinomycin (SPCM) as the controls, in vitro resistance acquisition test was carried out for 10 subcultures of each strain with increasing the amount of antimicrobials, and maximum growth allowance concentrations (MAC) in 10th and primary subcultures were compared. Acquisition of resistance in the strains against OFLX was moderate and MAC of the 10th subculture increased twice for one strain and 16 times for another one as compared to that of their primary subcultures. Acquisition of resistance in the strains against TS was also moderate and MAC of the 10th subculture increased 8 times for both strains as compared to that of the primary subcultures. Acquisition of resistance against SPCM was quite different between the strains. Namely the MAC of one strain increased 512 times, while another one did not acquire any resistance.

Distribution of phenotypically disparate myocyte subpopulations in airway smooth muscle

Phenotype and functional heterogeneity of airway smooth muscle (ASM) cells in vitro is well known, but there is limited understanding of these features in vivo. We tested whether ASM is composed of myocyte subsets differing in contractile phenotype marker expression. We used flow cytometry to compare smooth muscle myosin heavy chain (smMHC) and smooth muscle-alpha-actin (sm-alpha-actin) abundance in myocytes dispersed from canine trachealis. Based on immunofluorescent intensity and light scatter characteristics (forward and 90 degrees side scatter), 2 subgroups were identified and isolated. Immunoblotting confirmed smMHC and sm-alpha-actin were 10- and 5-fold greater, respectively, in large, elongate myocytes that comprised approximately 60% of total cells. Immunohistochemistry revealed similar phenotype heterogeneity in human bronchial smooth muscle. Canine tracheal myocyte subpopulations isolated by flow cytometry were used to seed primary subcultures. Proliferation of subcultures established with myocytes exhibiting low levels of smMHC and sm-alpha-actin was approximately 2 x faster than subcultures established with ASM cells with a high marker protein content. These studies demonstrate broad phenotypic heterogeneity of myocytes in normal ASM tissue that is maintained in cell culture, as demonstrated by divergent proliferative capacity. The distinct roles of these subgroups could be a key determinant of normal and pathological lung development and biology.

Inhibition of cartilage development in organ cultures of chick somites by the thymidine analog, 5-bromo-2′-deoxyuridine

Organ cultures of stage 17–18 chick somites with or without notochord were exposed to 5-bromo-2′-deoxyuridine (BUdR) at 0–3, 1–4, 2–5, and 3–6 days, reincubated in BUdR-free medium, and assayed for the appearance of functional chondroblasts 10–20 days later. In all organ cultures, exposure to BUdR from day 0 to day 3 prevented the appearance of methylene blue-staining cartilage nodules. Delaying the BUdR exposure allowed increasing numbers of cartilagepositive cultures to appear. Exposure to BUdR on days 0 to 3 also inhibited the subsequent incorporation of glucosamine- 3H or sulfate- 35S into chondroitin 4-sulfate assayed by high-voltage electrophoresis. In these cultures synthesis of hyaluronic acid and a glycoprotein component was relatively unaffected. Attempts were made to recover functional chondrocytes from BUdR treated organ cultures by dissociating the cells and growing them as monolayers. No chondrogenic colonies appeared in primary subcultures of organ cultures treated with BUdR from 0 to 3 days, and few chondrogenic colonies emerged from secondary subcultures. Organ cultures treated with BUdR at later periods, when subcultured, produced increasing numbers of chondrogenic colonies. It is postulated that incorporation of BUdR from 0 to 3 days prevents the shift from the sclerotome phenotype to the chondrogenic phenotype. BUdR resistance in older cultures may reflect the gradual withdrawal of chondrogenic cells from the mitotic cycle.

Primary subcultures ofHistoplasma capsulatum

The colonial and micromorphology of 36 primary subcultures of H. capsulatum from various sources have been studied in depth. Most strains separate into 2 main types on the basis of colony appearance. The A or albino type with white aerial mycelium and smooth macroconidia and the B or brown type consisting almost solely of masses of tuberculate macroeonidia. The implications of this in identification and epidemiology are discussed. Several new or improved cytological observations are reported including racket hyphae, nuclei and nucleoli, gelatinous wall sculptures and perforate septa. Although much is being written (Ajello, 1967; Campbell, 1967; Nielsen, 1967) about the medical, serological, diagnostic and epidemiologic aspects of histoplasmosis and its etiological agent, Histoplasma capsulatum, little has been done systematically to characterize the morphology and cytology of the many strains which different investigators use. This often makes comparisons of results meaningless. It was therefore decided to study in depth as many primary isolates from as many sites and geographic areas as were available to me in a year period. Observations recorded here relate only to the macromorphology and cytology of the mycelial phase. Studies on the physiology, morphology and pathogenicity of the yeast phase are in progress and will be reported later. Only strains for which a complete history was available were used. They were received as isolated, in the plates or tubes of media planted usually with livers and spleens of mice inoculated with specimens from patients and nature, and only occasionally with specimens directly from the patient. As especially requested they were not subcultured before shipping. This seemed to be the only logical approach to the study of an organism as seemingly exasperating in its morphological and physiological variation as H. capsulatum if meaningful correlations were to be established among source, morphology, pathogenicity, antigenic properties and conversion ability.