Tiny Colonies Research Articles

Growth of Streptococcus pneumoniae on Macconkey Agar: Possibility of Impossible?

Dear Editor Streptococcus pneumoniae (S. pneumoniae) is Gram-positive lanceolate-shaped diplococci which is a significant human pathogenic bacterium causing pneumonia, meningitis and various other infections. It is a fastidious organism and grows well in 5% carbondioxide (CO2) and media containing blood (blood agar, chocolate gar) as growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide (H2O2) produced by the bacteria [1]. Hence, it does not grow on MacConkey agar (MA) due to the absence of blood as well as due to the presence of high concentration of bile in the medium which is inhibitory to the growth of S. pneumoniae. However, to the best of our knowledge, we are reporting the first and unusual case of S. pneumoniae growth on MA. We received cerebrospinal fluid from a 21-year-old male patient who was admitted with fever, headache, vomiting and clinically had signs of meningitis. Gram stain revealed few pus cells. Biochemical parameters showed decreased glucose, moderately increased protein. Culture revealed pure growth of tiny opaque lactose-fermenting colonies on MA and alpha-haemolytic colonies on blood agar (BA) and chocolate agar (CA). Colony smear from all the three plates showed lanceolate-shaped Gram positive cocci in pairs and short chains. Identification and antibiotic sensitivity testing was done by Vitek-2 compact (BioMerieux) using GPC identification card (GP 21 342) and Streptococcal AST card (AST-ST01) respectively. The organism was identified as Streptococcus pneumoniae which was sensitive to all the antibiotics tested except benzylpenicillin. However, the growth of the isolate on MA created a dilemma and hence the same was sent for identification by MALDI-TOF method. The later identified the isolate as Streptococcus pneumoniae with score value of 2.114. Several studies have highlighted evolving clinical problems with S. pneumoniae. Increasing resistance to antimicrobial agents, and failure of traditional optochin(ethylhydrocupreine) sensitivity had been a growing concern [2]. Dias CA et al., reported the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase [3]. Clinical isolate with intermediate sensitivity to penicillin was first reported in 1967. Since then, penicillin non-susceptible S. pneumoniae has been reported with increasing frequency in many countries.The resistance to penicillin arises from mosaic mutation of penicillin binding protein (PBP) genes due to interspecies recombination of homologous genes [4]. Growth of S. pneumoniae on MA may be a beginning of the changing trend of the organism’s fastidious growth requirements. One of the possible explanations for the same may be some mutation causing alteration in hydrogen peroxide producing capacity of S. pneumoniae and hence bypassing the need for catalase in the media used.Some strains of S. pneumoniae with such mutations have been used in experimental studies to create an environment with low concentration of H2O2 [5,6]. Pericone et al., used loss-of-function mutations of spxB gene in P62 and P878 strains of S. pneumoniae to study the effect of low H2O2 on rate of spontaneous gene mutation. The lower spxB expression resulting in under-expression of pyruvate oxidase results in the lower production of hydrogen peroxide. A spxB-defective pneumococcal mutant produces opaque colony and opaque variants show increased virulence for non-nasopharyngeal infections [5]. In our case also the colony morphology was opaque on MA, and the infection site was meninges. So, these findings provides an assumption that the spx-B mutation can be occurring in the clinical isolates like in our case thus leading to diminished production of hydrogen peroxide and hence not requiring catalase in the growth medium. Due to unavailability of molecular methods at our institution the genetic analysis could not be performed in this case. However, it is advisable that in future if any such scenario is encountered by any institution the genetic analysis could be of great help.

Reinterpretation of the Cambrian ‘bryozoan’ Pywackia as an octocoral

Pywackia baileyi Landing in Landing et al., 2010, from the upper Cambrian Yudachica Member of Oaxaca State, southern Mexico, consists of small, phosphatic, proximally tapering cylindrical rods covered by shallow polygonal calices. The bryozoan-like morphology of this fossil prompted its interpretation as the first bryozoan known from the Cambrian. However, restudy of some of the original material, employing scanning electron microscopy for the first time, questions the assignment of Pywackia to the Bryozoa. Striking similarities between Pywackia and the modern pennatulacean octocoral Lituaria lead to an alternative hypothesis interpreting Pywackia an early fossil octocoral. While Pywackia is probably not a true pennatulacean, a group with a definitive fossil record stretching back only to the Late Cretaceous, it can be envisaged as having had a similar skeletal structure and ecology to Lituaria, the rods representing mineralized axes of tiny colonies that lived with their proximal ends buried in the sediment and distal ends covered by feeding polyps. Landing et al. (2010) considered the phosphatic composition of Pywackia specimens to be the result of diagenetic replacement, but the evidence is equivocal. If Pywackia had a primary phosphatic skeleton, this would support the hypothesized existence of phosphatic biomineralization early in the evolutionary history of Cnidaria, as well as providing further evidence that Pywackia is not a bryozoan.

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as β-lactams.

NeonatalGranulicatella elegansBacteremia, London, UK

Neonatal<i>Granulicatella elegans</i>Bacteremia, London, UK

A Case ofHelicobacter cinaediBacteremia in an Asplenic Patient

Helicobacter cinaedi is an enterohepatic species. It can cause bacteremia, gastroenteritis, and cellulitis, particularly in immunocompromised individuals, such as those with acquired immunodeficiency syndrome, malignancy, or alcoholism. There are no previous reports of H. cinaedi infection in Korea. A 71-yr-old man was admitted to the emergency room because of dyspnea on November 9, 2011. He had undergone splenectomy 3 yr ago because of immune hemolytic anemia. Chest plain radiography revealed bilateral pleural effusion. He developed fever on hospital day (HD) 21. Three sets of blood cultures were taken, and gram-negative spiral bacilli were detected in all aerobic vials. The isolate grew in tiny colonies on chocolate agar after 3-day incubation under microaerophilic conditions. This organism tested positive for catalase and oxidase, and negative for urease. The 16S rRNA gene sequence of this isolate exhibited 99.8% homology with the published sequence of H. cinaedi CCUG 18818T (GenBank accession no. ABQT01000054) and 98.5% homology with the sequence of Helicobacter bilis Hb1T (GenBank accession no. U18766). The patient was empirically treated with piperacillin/tazobactam and levofloxacin, and discharged with improvement on HD 31. To our knowledge, this is the first report of H. cinaedi bacteremia in an asplenic patient. Asplenia appears to be a risk factor for H. cinaedi bacteremia.

Identify of Granulicatella adiacens from blood cultures of a patient bearer of prosthetic valve

The clinical case studied concerns a woman 81 years old, with a history of prosthetic valve and mitral insufficiency, admitted to internal medicine ward of NOCSAE hospital as a result of a recurrent fever. Due to the suspicion of endocarditis and with the aim to identify the presence of aerobic/anaerobic microorganisms, two set of blood cultures collected within 24 hours were sent to the Laboratory of microbiology. All the bottles were incubated into the Bact-Alert 3D System (bioMerieux). After an 19 hours incubation time, the samples were identified as positive by the automated system; consequently they cultured on a blood agar and selective media, according to our laboratory operational protocol. In the same time Gram stain of the cultural broth revealed the presence of Gram positive cocci arranged in chains different in length. Since there wasn’t an evident microbial growth on solid media after 24-48 hours of incubation, a new culture was carried out on blood and chocolate agar after the addition of Staphylococcus aureus ATCC 25923. After 24 hours of incubation it was possible appreciate the growth of tiny colonies around the S. aureus ones. These colonies were identified by Vitek2 and Api Rapid 32 Strep (bioMerieux) as Granulicatella adiacens. The results were confirmed by PCR and sequencing of the groESL gene. MIC values obtained by the means of E-test (bioMerieux) were: 0.016mg/L for penicillin, 0.125mg/L for cefotaxime, 1mg/L for both vancomicin and levofloxacin. Resistance was observed for cloramphenicol (MIC=16mg/L). The timely communication of these findings, supported by clinical data like the appearance of vegetation on mitral valve highlighted by trans-oesophageal echocardiography, allowed to establish an adequate antibiotic therapy, rapid resolution of fever and normalisation of inflammatory parameters.

Colony image acquisition system and segmentation algorithms

This paper presents a novel colony analysis system including an adjustable image acquisition subsystem and a wavelet-watershed-based image segmentation algorithm. An illumination box was constructed-both front lightning and back lightning illuminations can be chosen by users based on the properties of Petri dishes. In the illumination box, the lightning is uniform, which makes image processing easy. A digital camera at the top of the box is connected to a PC computer; all the camera functions are controlled by the developed computer software in this study. As usual, in the image processing part, the hardest task is image segmentation which is carried out by the four different algorithms: 1. recursive image segmentation on gray similarity; 2. canny edge detection-based segmentation; 3. the combination of 1 and 2, and 4. colony delineation on wavelet and watershed. The first three algorithms can obtain good results for ordinary colony images, and for the images including a lot of small (tiny) colonies and dark colonies and overlapping (or touching) colonies, the algorithm 4 can obtain better results than the others. The algorithms are tested by using a large number of different colony images, and the testing results are satisfactory.

Actinobaculum schaalii- invasive pathogen or innocent bystander? A retrospective observational study

BackgroundActinobaculum schaalii is a Gram-positive, facultative anaerobic coccoid rod, classified as a new genus in 1997. It grows slowly and therefore is easily overgrown by other pathogens, which are often found concomitantly. Since 1999, Actinobaculum schaalii is routinely investigated at our hospital, whenever its presence is suspected due to the detection of minute grey colonies on blood agar plates and negative reactions for catalase. The objective of this study was to determine the clinical significance of Actinobaculum schaalii, identified in our microbiology laboratory over the last 11 years.MethodsAll consecutive isolates with Actinobaculum schaalii were obtained from the computerized database of the clinical microbiology laboratory and patients whose cultures from any body site yielded this pathogen were analyzed. Observation of tiny colonies of Gram-positive, catalase-negative coccoid rods triggered molecular identification based on 16S rRNA gene sequencing.Results40 isolates were obtained from 27 patients during the last 11 years. The patient's median age was 81 (19-101) years, 25 (92.6%) had underlying diseases and 12 (44.4%) had a genitourinary tract pathology. Actinobaculum schaalii was isolated in 12 urine cultures, 21 blood cultures, and 7 deep tissue biopsies. Twenty-five (62.5%) specimens were monobacterial, the remaining 15 (37.5%) were polybacterial 7/7 deep tissue samples (three bloodcultures and five urine cultures). Recovery from urine was interpreted as colonization in 5 (18.6%) cases (41.6% of all urine samples). Six (22.2%) suffered from urinary tract infections, six (22.2%) from abscesses (skin, intraabdominal, genitourinary tract, and surgical site infections) and 10 (37.0%) from bacteremia.ConclusionsIn this largest case series so far, detection of Actinobaculum schaalii was associated with an infection - primarily sepsis and abscesses - in 81.5% of our patients. Since this pathogen is frequently part of polymicrobial cultures (42.5%) it is often overlooked or considered a contaminant. Detection of Actinobaculum schaalii in clinical isolates mainly reflects infection indicating that this Gram-positive rod is not an innocent bystander.

H 2O 2 metabolism during sweet orange ( Citrus sinensis L. Osb.) ‘Hamlin’ Xanthomonas axonopodis pv. citri interaction

Sweet orange ( Citrus sinensis L. Osb.) ‘Hamlin’ is a canker ( Xanthomonas axonopodis pv. citri: Xac) susceptible citrus genotype grown commercially worldwide. Canker causes severe economic losses and restricts the marketability of crop for export. Little is known about the role of oxidative stress in canker development. In the present investigation, sweet orange ‘Hamlin’ leaves were artificially inoculated with Xac to determine the impact of Xac infection on hydrogen peroxide (H 2O 2) metabolism. Characteristic symptoms following artificial inoculation were water soaking of the infiltrated zone between 2 and 8 days after inoculation (dai); raised epidermis accompanying tiny yellow colored bacterial colonies at 8 dai; and yellowing and necrosis of the infected zone by 12–16 dai. In planta Xac population increased 1000 fold by 14 dai from an initial population of 7.3 × 10 6 cfu cm −2 (0 dai). Peak concentrations of H 2O 2 were observed at 24 h and between 8 and 10 dai and coincided with higher activity of total superoxide dismutase (SOD). Lower levels of H 2O 2 in infected leaves were maintained by Xac induced higher activities of catalase (CAT), ascorbate peroxidase (APOD), and guaiacol peroxidase (POD). It appears Xac altered H 2O 2 metabolism in C. sinensis L. Osb. ‘Hamlin’ to enhance survival and growth.

Importance of Suspicion for the Identification ofMycoplasmain Wound Culture: A Case Report

Genital mycoplasmas are rare in extraintestinal specimens, but can cause disseminated infections in immunocompromised patients and wound infections after surgery or injury. We report two cases of Myoplasma hominis wound infections after lung lobectomy and kidney transplantation, and a case of M. salivarium wound infection after aortic graft replacement. Mycoplasmas grew in aerobic and anaerobic cultures as tiny colonies but were not observed by gram- or acid fast stain and were confirmed by MYCOFAST EvolutioN 2 kit or 16S rRNA sequencing. These cases indicated that mycoplasmas were probably underestimated in wound infections because they were not in suspicion. We suggest that Mycoplasma should be suspected when microorganisms are not readily observable in Gram stains but can be cultured. (Korean J Clin Microbiol 2010;13: 121-124)

Characterization of aStaphylococcus aureusSmall Colony Variant (SCV) Associated with Persistent Bovine Mastitis

Staphylococcus aureus is a common cause of bovine mastitis and foodborne and other diseases in humans. This study tested the hypothesis that small colony variants (SCVs) of S. aureus are implicated in chronic bovine mastitis. Six S. aureus isolates from foremilk samples from 11 chronically infected cows were investigated. Five isolates had typical morphology and were hemolytic and coagulase positive; one was a heterogeneous population of typical and SCV phenotype (tiny nonhemolytic colonies). In the presence of gentamicin, three of the previously typical S. aureus developed SCVs that were confirmed as S. aureus by biochemical and genetic analyses; these SCVs reverted to the typical form on antibiotic-free medium. The SCV isolate (Heba3231) from the heterogeneous population had a slow growth rate and prolonged lag phase and did not revert during 10 h of incubation. Transcriptional analysis showed that SCV Heba3231 had reduced expression of agr, hla, and coa and increased expression of indicators of fermentation pathways compared to the parent strain. Invasion of and persistence in a primary culture of bovine aortic endothelial cells (BAEC) showed that SCV Heba3231 had minimal deleterious effects, whereas the parent strain or the Newbould 305 strain caused severe damage. Recovery of the parent strain from BAEC yielded a mixture of the parent and SCV phenotypes. This study reports for the first time the isolation of S. aureus SCV from persistent bovine mastitis and suggests that SCV may be an important contributor to the prolonged survival of S. aureus in some cases of mastitis.

Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India

To investigate and characterize Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease, attendants of animals with suspected infection, and healthy humans, using multiple diagnostic tests. A total of 119 samples (35 stool, 76 serum, three blood clots, and five biopsies) were collected from five patients with Crohn's disease, eight attendants of animals with Johne's disease, and 93 apparently normal control subjects (Agra region) from North India. Samples were screened for the presence of MAP by smear examination, culture of stool, blood clot and biopsies, and ELISA. Colonies obtained by culture were further characterized using polymerase chain reaction (PCR) with IS900 MAP-specific primers. Using all diagnostic modalities, MAP and/or MAP antibodies were identified in 100% (5/5) of subjects with Crohn's disease; 75.0% (6/8) of attendants of MAP infected animals were positive and 38.0% (27/71) of apparently normal controls were also positive. Most sensitive test was ELISA (100%, 5/5), followed by culture (80.0%, 4/5), and acid-fast staining. Ziehl-Neelsen staining was positive in 37.5% (3/8) of subjects with active animal husbandry practices. In 71 serum samples from control subjects, seroprevalence of MAP was 38.0% using indigenous protoplasmic antigens (PPA) and 36.6% using commercial PPA. Of the serum samples from the Crohn's disease patients, 100% (5/5) were positive by ELISA using indigenous PPA and 40.0% (2/5) were positive by ELISA using commercial PPA. IS900 PCR was used to characterize tiny colonies of MAP that grew extremely slowly on Herrold's egg yolk medium, and of 15 (42.8%) cultures, 14 (93.3%) were typed as MAP. Paper documented the presence of MAP in all patients with Crohn's disease, in some animal attendants who had the history of working with goat herds infected with Johne's disease and in few normal healthy individuals. Presence of Ziehl Neelsen positive MAP. In the stool of attendants working with MAP-infected animals was unique to humans. ELISA based on antigens derived from indigenous MAP 'bison type' genotype of goat origin was most sensitive modality for screening Crohn's disease patients.

Protracted Cervical Lymphadenitis With Mycobacterium avium and Methylobacterium mesophilicum

To the Editors: Methylobacterium mesophilicum has rarely been isolated from specimens of febrile severely immunocompromised hosts1 and in catheter-related infections.2 Hitherto, 5 pediatric patients with bloodstream infection caused by M. mesophilicum have been reported who received intensive anticancer chemotherapy3,4 or who were immunocompromised because of other reasons.5 A 3-year-old white boy was referred to the oncology outpatient unit with an enlarged and obdurate nuchal tumor. The boy used to play and dig in the family’s vegetable garden. Ultrasound examination showed enlarged lymph nodes that measured up to 3 cm in diameter. Mendel-Mantoux and serologic testing for viral pathogens and for Bartonella hensellae yielded negative results. Histologic evaluation of the lymph node revealed epitheloid-cell granulomatous necrotizing lymphadenitis. Aerobic cultures of lymph node tissue on Columbia sheep blood (5%) and chocolate agar yielded a slow-growing pure culture of tiny oxidase-positive colonies; MacConkey agar remained negative. After an incubation time of 4 days at 35°C, 1-mm-diameter colonies were produced, which appeared pink pigmented in incandescent light. Bacteriologic standard procedures resulted in the identification of M. mesophilicum, susceptible in vitro to ceftazidime, ceftriaxone, meropenem, gentamicin, amikacin, and ciprofloxacin. In addition, Mycobacterium avium was cultured from the specimen. The boy was readmitted with high temperatures (39.7°C) and a left-sided tender and warm nuchal swelling with multiple enlarged lymph nodes, and received intravenous ceftazidime and gentamicin (according to the in vitro susceptibility data of M. mesophilicum). After 10 days, the patient was released from hospital in a good condition with oral ciprofloxacin for another week. Four follow-up examinations at 2-week intervals showed a healthy boy with a small residual nuchal induration. To our knowledge, this is the first report of a protracted lymphadenitis yielding growth of Mycobacterium avium and M. mesophilicum in an apparently immunocompetent child. Louria et al5 described an infant and an adolescent with systemic disease and Methylobacterium sp., isolated from blood, lymph node, and bone marrow; both children were severely immunocompromised. In addition, a 5-year-old boy, who received intensive chemotherapy for T-cell lymphoma, had a Hickman catheter-related M. mesophilicum bacteremia. Eventually, the catheter was removed after unsuccessful treatment with ticarcillin-clavulanate and gentamicin.4 Playing and digging in the family’s garden is one possible source of the infection with these 2 ubiquitous opportunistic environmental pathogens in our patient. Tap water used for oral irrigation in patients with mucositis after bone marrow transplantation has been related to an outbreak. Arne Simon, MD Sebastian Völz, MD Department of Pediatric Hematology and Oncology Children’s Hospital Medical Centre University of Bonn Bonn, Germany [email protected] Soo-Mi Reiffert, MD Institute for Medical Microbiology Immunology and Parasitology University of Bonn Bonn, Germany Hans Bölefahr, MD Department of Pediatric Surgery Marienhospital Bonn Bonn, Germany Günter Marklein, MD Institute for Medical Microbiology Immunology and Parasitology University of Bonn Bonn, Germany Reinhard Büttner, MD Institute for Pathology University of Bonn Bonn, Germany Nico Hepping, MD Udo Bode, MD Gudrun Fleischhack, MD Department of Pediatric Hematology and Oncology Children’s Hospital Medical Centre University of Bonn Bonn, Germany

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific beta-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.

Actinomycosis Of Hand And Wrist: A Case Report

Actinomycosis is a chronic granulomatous disease characterized by external sinuses through which tiny colonies of organisms called sulphur granules are discharged. We hereby report a case of actinomycosis of wrist and hand – a rare site, in a 33 years old male patient which resisted usual treatment of curettage and penicillin therapy and amputation was required to eradicate the disease.

Evaluation of isolation media for the detection of Bartonella henselae--isolation of Bartonella henselae from domestic cats

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

Genetic toxicology of remifentanil, an opiate analgesic.

Compounds that interact with opioid receptors are commonly used as analgesics. Opioid agonists vary in their potency and pharmacokinetic properties as well as in their affinity for distinct opioid receptors. The fentanyl opiate analogues are an important group of analgesics that interact with the mu opioid receptor. Remifentanil (GI87084) is a particularly interesting member of this group of opioids because its action is especially short in duration. This report examines the genetic toxicology of remifentanil. Remifentanil was not genotoxic in an Ames test, an in vitro chromosome aberration assay in Chinese hamster ovary cells, an in vivo micronucleus assay in rat erythrocytes, or an in vivo/in vitro unscheduled DNA synthesis assay in rat hepatocytes. In the in vitro L5178Y tk(+/-) mouse lymphoma assay, remifentanil produced a genotoxic response at dose levels >or=308 microg/mL only in the presence of rat liver S9 metabolic activation; primarily tiny and small mutant colonies were produced. This pattern of activity in a battery of genetic toxicology assays is not unique to remifentanil, but has also been observed for other pharmaceuticals, including the opioid fentanyl. A weight-of-evidence analysis, taking into consideration genotoxic mechanisms, in vivo results, and the conditions of clinical use, suggests remifentanil does not pose a genotoxic risk to patients.

Pleural Effusion Due toCorynebacterium Propinquumin a Patient with Squamous Cell Carcinoma

Pleural Effusion Due to<i>Corynebacterium Propinquum</i>in a Patient with Squamous Cell Carcinoma

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants.

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.

The atpIBEXF operon coding for the F0 sector of the ATP synthase from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus.

The atpIBEXF operon coding for the F0 sector of the ATP synthase from Rhodobacter capsulatus was cloned and sequenced. The genes for the five subunits were present in the order: atpI (subunit I), atpB (subunit a), atpE (subunit c), atpX (subunit b'), and atpF (subunit b). The transcription initiation site was defined by primer-extension analysis. A duplicated and divergent copy of the b subunit gene (subunit b') was present. This duplication is found only in photosynthetic prokaryotes and in plant chloroplasts. F0 deletion mutants formed tiny colonies during anaerobic growth in the dark but could not sustain continuous growth. Based on the results of the present work, we conclude that a functioning ATP synthase is essential for normal growth under all conditions tested.