Non-Fermentative Gram negative bacilli

Excellent data about NFGNB

1. Non-Fermentative Gram Negative Bacilli Presented By Nagaraj.S Medical Microbiologist

2. Introduction Non-fermentative gram-negative bacilli (NFGNB) are group of aerobic, non-spore forming bacilli that either do not use carbohydrate as source of energy or degrade them through metabolic pathways other than fermentation. They exhibit resistance to beta-lactams including carbapenems and also to other group of antibiotics . These organisms have been implemented as a cause of clinical diseases including meningitis, septicemia, wound infections and urinary tract infections. The non-fermentative gram-negative bacilli (NFGNB) are ill defined taxonomic group of bacteria that comprises more than species. Recent studies say that, these organisms have been increasingly isolated from hospitalized patients .

3. These are saprophytic in nature, have emerged as important health care associated pathogens. Data from the Surveillance and Control of pathogens of Epidemiological importance (SCOPE) study revealed that, approximately one fourth of gram negative bacteremia attributed to NFGNB. The proper identification of NFGNB up to the species level together with monitoring their susceptibility patterns are mandatory for proper management of infections caused by these pathogens. the antibiotic susceptibility pattern may change with time and may vary from hospital to hospital.

4. Classification of NFGNB There is 11 species of NFGNB, which results wide range of clinical manifestations in humans. Apart from that, other species has not much clinical importance. So, the classification of NFGNB is based on some characteristic features. Which is mentioned below:

5. Broadly NFGNB can be grouped as: Motile and oxidase + ve Non-motile and oxidase - ve Motile and oxidase - ve Non-motile and oxidase + ve Based on these differential characterization, along with lysine-decarboxylase test, OF glucose test, polymyxin B sensitivity, pigmentation of MacConkey, the NFGNB are classified into different species. They are: Acinetobacter ( A. baumannii and A.lwoffii ) Stenotrophomonas maltophilia Burkholderia spp. Sphingomonas spp . Chryseomonas spp. Moraxella spp . Flavobacterium Sphingobacterium Chryseobacterium Pseudomonas spp . Alcaligenes spp .

6. Scheme of differentiation of NFGNB

7. Pseudomonas The generic name  Pseudomonas  created for these organisms was defined in rather vague terms by  Walter Migula  in 1894 and 1900 as a genus of Gram-negative, rod-shaped and polar- flagellated  bacteria with some sporulating species, the latter statement was later proved incorrect and was due to refractive granules of reserve materials. Despite the vague description, the type species,  Pseudomonas pyocyanea ( basonym of  Pseudomonas aeruginosa ), proved the best descriptor. Morphology Pseudomonas is a straight or slightly curved, gram negative bacilli measuring 0.5-1.0 micrometers in size. Arranged in pairs and short chains. Motile by polar flagella, non-capsulated, non- sporing and fimbriated.

8. Cultural characteristics These are strictly aerobic bacteria. P. aeruginosa grows over a wide range of temperature (5-32 C), the optimum temperature being 37 C. On nutrient agar, it produces large, opaque, translucent and irregularly rounded colonies. On MacConkey, the organism produces Non-Lactose Fermenting colonies, on nutrient broth it produces a dense turbidity with surface pellicle. They are oxidase positive and OF test positive. Odor Colonies emit a characteristic musty to fruity odor due to production of aminoacetophenone from the amino acid tryptophan.

9. Pigmentation Pyocyanin (green)is a specifically produced by P. aeruginosa . The pigment is soluble in chloroform and water. Pyoverdin or fluorescein (yellow), is produced by P. aeruginosa as well as by many other Pseudomonas species. It is soluble in water but not in chloroform. Pyorubin is a red pigment, which is soluble in water but not in alcohol. Pyomelanin is a brown pigment. Virulence factors Exotoxin A is one of the most important virulence factor produced by P. aeruginosa . It is like a diphtheria toxin acts by preventing synthesis of protein in eukaryotic cells. It is responsible for tissue damage in chronic pulmonary infection, dermatonecrosis in burns wound, and destruction of cornea in ocular infections. Exotoxins S and T are the toxins produced by P. aeruginosa . these toxins show adenosine diphosphate ribosyl transferase activity. It causes damage in epithelial cells. The elastase enzyme causes damage in parenchymal tissues of the lung and produce haemorrhagic lesions associated with spreading of pseudomonas infections. Alkaline protease is also responsible for the destruction of tissue and dissemination of P. aeruginosa infection. Phospholipase C is a heat-labile haemolysin and Rhamnolipid is a heat stable haemolysin, contributes to breaking down of lecithin containing tissues. The antigenic structures of cell wall, which includes pili, loose slime layer, LPS, and pyocyanin.

10. Spectrum of disease NFGNB have been isolated from variety of clinical specimens, including blood, sputum, urine, body fluids, stool, CSF, throat swabs, pus, peritoneal fluids etc. Pseudomonas aeruginosa is an opportunistic pathogen that can cause community- or hospital- acquired infections. It is associated with community acquired infections : skin (folliculitis); external ear canal (otitis externa); eye, following trauma; bone (Osteomyelitis), following trauma; heart (endocarditis) in IV drug abusers; and respiratory tract (patients with cystic fibrosis). Hospital acquired infections : respiratory tract, urinary tract, wounds, blood stream (bacteremia) and central nervous system infections.

11. Antimicrobial therapy and susceptibility testing Pseudomonas spp. is susceptible to antipseudomonal beta-lactam with or without aminoglycosides; certain quinolones may be also used. Specific agents include piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin.

12.   Burkholderia The genus burkholderia was proposed by Yabuchi et. al. (1992) to encompass the species formerly grouped with in rRNA group II of the genus pseudomonas. This species complex owes its present responsible for its present epithet to the description by Burkholderia (1950) of the bacterium responsible for rot in onion bulbs. Morphology NFGNB organisms are gram negative, straight or slightly curved rods. some other are coccobacilli, plump coccoid rods, sea gull shaped, spirals and pleomorphic. the cells range from 1 to 5 micrometers in length and 0.5 to 1 micrometers in width.

13. Cultural characteristics Mostly NFGNB have no distinctive appearance on blood agar. All produce Non-lactose fermenting colonies on MacConkey agar. Colonies on blood agar may be smooth, slightly raised or mucoid to dry or wrinkled colonies or non-wrinkled, flat colonies or spreading with serrated edges or dry adherent or glistening entire colonies or opaque, circular colonies, translucent, punctate beta hemolytic or convex, semi opaque colonies or pitting sticky colonies. They are oxidase positive/negative and motile/ non motile and resistant to Polymixin /colistin.

14. Pigmentation Colonies may be chalky white, orange, lavender green, brown to tan, yellow, buff, grey, whitish or pink to coral coloured .   Odour They may produce different smells like grape, corn taco, dirt, popcorn, rose, apple or straw berry and bleach. virulence factors Mucin production may help in attachment in patients with cystic fibrosis – Burkholderiacepacia . Bacilli can survive within phagocytes – B. pseudomallei .

15. Spectrum of disease NFGNB have been isolated from variety of clinical specimens, including blood, sputum, urine, body fluids, stool, CSF, throat swabs, pus, peritoneal fluids etc. Burkholderiacepacia infections range from asymptomatic infection to melioidosis, including infections of the skin and respiratory tract, multi system abscess formation, bacteremia with septic shock, infections in patients with cystic fibrosis or chronic granulomatous disease; nonfatal infections of the urinary tract, respiratory tract and other sterile body sites. skin infections range from localized acute or chronic suppurative infections of skin at site of inoculation. Antimicrobial therapy and susceptibility testing Burkholderia spp. are susceptible to potentially active agents include piperacillin, ceftazidime, imipenem, ciprofloxacin,ciprofloxacin , chloramphenicol, Trimethoprim/ sulphamethaxozole , meropenem. MIC by broth micro dilution and E- test are preferred for this group of organisms.

16. Acinetobacter The current genus designation,  Acinetobacter  (from the Greek α κινɛτοσ [ akinetos ], i.e., nonmotile), was initially proposed by Brisou and Prévot in 1954 to separate the nonmotile from the motile microorganisms within the genus  Achromobacter . It was not until 1968 that this genus designation became more widely accepted. Baumann et al. published a comprehensive survey and concluded that the different species listed above belonged to a single genus, for which the name  Acinetobacter  was proposed, and that further subclassification into different species based on phenotypic characteristics was not possible. These findings resulted in the official acknowledgment of the genus  Acinetobacter  by the Subcommittee on the Taxonomy of  Moraxella  and Allied Bacteria in 1971. In the 1974 edition of  Bergey's Manual of Systematic Bacteriology , the genus  Acinetobacter  was listed, with the description of a single species,  Acinetobacter calcoaceticus  (the type strain for both the genus and the species is  A. calcoaceticus  ATCC 23055). In the “Approved List of Bacterial Names,” in contrast, two different species - A. calcoaceticus and  A. lwoffii , were included, based on the observation that some Acinetobacter were able to acidify glucose whereas others were not.

17. Morphology Species of the genus  Acinetobacter  are  strictly aerobic ,  non-fermentative ,  Gram-negative   bacilli . They show mostly a  coccobacillary morphology on nonselective agar. Rods predominate in fluid media, especially during early growth. Cultural characteristics The morphology of  Acinetobacter  species can be quite variable in Gram-stained human clinical specimens and cannot be used to differentiate  Acinetobacter  from other common causes of infection. Most strains of  Acinetobacter , except some of the  A.lwoffii  strain, grow well on  MacConkey agar  (without salt). Although officially classified as not lactose-fermenting, they are often partially lactose-fermenting when grown on MacConkey agar. They are  oxidase -negative, catalase-positive, indole-negative,  non motile , and usually  nitrate negative. Bacteria of the genus  Acinetobacter  are known to form intracellular inclusions of  polyhydroxy alkenoates  under certain environmental conditions (e.g. lack of elements such as phosphorus, nitrogen, or oxygen combined with an excessive supply of carbon sources).

18. Transmission Risk factors include long-term intubation and tracheal or lung aspiration. In most cases of ventilator-associated pneumonia, the equipment used for artificial ventilation such as endotracheal tubes or bronchoscopes serve as the source of infection and result in the colonization of the lower respiratory tract by  A. baumannii . In some cases, the bacteria can go on to enter the bloodstream, resulting in bacteremia with mortality rates ranging from 32% to 52%. UTIs caused by  A. baumannii  appear to be associated with continuous catheterization, as well as antibiotic therapy.   A. baumannii  has also been reported to infect skin and soft tissue in traumatic injuries and postsurgical wounds.  A. baumannii  commonly infects burns and may result in complications owing to difficulty in treatment and eradication.

19. Virulence factors one factor in particular, OmpA , a member of the Outer membrane proteins (OMPs), has been determined to contribute significantly to the disease-causing potential of the pathogen.   A. baumannii   OmpA bind to the host epithelia and mitochondria, once bound to the mitochondria, it induces mitochondrial dysfunction and causes the mitochondria to swell. This is followed by the release of cytochrome c, a heme protein, which leads to the formation of apoptosome . These reactions all contribute to apoptosis of the cell.  OmpA , being the most abundant surface protein on the pathogen, is also involved in resistance to complement and the formation of biofilms. The ability of  A. baumannii  to form biofilms allows it to grow persistently in unfavourable conditions and environments. Indeed,  A. baumannii  has been shown to form biofilms on abiotic surfaces, which can include glass and equipment used in intensive care units, and/or on biotic surfaces such as epithelial cells. The most common factors that control biofilm formation can include nutrient availability, the presence of pili and outer membrane proteins and macromolecular secretions. Pili assembly and production of biofilm-associated protein (BAP) both contribute to the initiation of biofilm production and maturation after  A. baumannii  attach to particular surfaces. When pili attach to abiotic surfaces, they initiate the formation of microcolonies, followed by the full development of biofilm structures. BAP is present on the surface of bacterial cells and they contribute to biofilm development and maturation by stabilizing the mature biofilm on abiotic or biotic surfaces.

20. Environmental signals, such as metal cations, also play a role in controlling the formation of biofilms, increasing the ability of  A. baumannii  to adhere to particular surfaces. Other key proteins that have been shown to contribute to  A. baumannii  virulence include phospholipase D and C. While phospholipase D is important for resistance to human serum, epithelial cell evasion and pathogenesis ,  phospholipase C enhances toxicity to epithelial cells.   Along with OmpA , fimbria, also expressed on the surface of the bacterial cell, contribute to the adhesion of the pathogen to host epithelia.

21. Spectrum of disease Hospital-acquired pneumonia Ventilator associated pneumonia (VAP) is commonly linked to infection. Longer periods of hospitalization, longer time on mechanical ventilation and prior use of antibiotics are the recognized factors increasing the risk of VAP due to Acinetobacter.Nosocomial outbreaks have also been described due to health care professionals with colonized hands and poor personal hygiene; Such individuals may act as opportunist carriers of an epidemic stain. Contaminated ventilators or respiratory care equipment as well as intra-hospital transmission may also contribute to the beginning of an outbreak. Community-acquired pneumonia Pneumonia acquired outside of the hospital setting and caused by Acinetobacter has been noted in Australia and Asia. The source of infection may be throat carriage, which occurs in up to 10% of community residents with excessive alcohol consumption.  It is characterized by a severe and sudden onset coupled with secondary bloodstream infection and has a mortality rate of between 40% and 60%.

22. Bloodstream infections In a seven-year review (1995–2002) of nosocomial bloodstream infections in the United States, Acinetobacter accounted for 1.3% of all monomicrobial bloodstream infections. Acinetobacter was a more common cause of ICU-acquired bloodstream infection than of non-ICU-ward infection (1.6% vs. 0.9% of bloodstream infections, respectively, in those locations). Crude mortality figures overall from Acinetobacter bloodstream infection was 34.0% to 43.4% in the ICU and 16.3% outside the ICU. Acinetobacter bloodstream infections had the third highest crude mortality rate in the ICU, exceeded only by  P. aeruginosa  and  Candida  spp. infections. Battlefield trauma and other wounds Acinetobacter is a well-documented pathogen of burns units and is difficult to treat in patients with severe burns. However, infection of the skin and soft tissue outside of a military environment is uncommon. Meningitis Nosocomial, Post-neurosurgical Acinetobacter meningitis is becoming increasingly more common with many other Gram-negative bacteria also becoming problematic in Post-operative care.   Installation of an external ventricular drain becomes a site for opportunistic infection. The mortality rate may be as high as 70%; however, it is not possible to discern the definitive cause of mortality.

23. Antimicrobial therapy and susceptibility testing Acinetobacter  species are innately resistant to many classes of antibiotics, including  penicillin , chloramphenicol and aminoglycosides. Resistance to  fluoroquinolones  has been reported during therapy, which has also resulted in increased resistance to other drug classes mediated through active drug  efflux . A dramatic increase in  antibiotic resistance  in  Acinetobacter  strains has been reported by the  Centers for Disease Control and Prevention  (CDC), and the carbapenems are recognised as the gold-standard and treatment of last resort. Acinetobacter  species are unusual in that they are sensitive to  sulbactam , which is commonly used to inhibit bacterial beta-lactamase, but this is an example of the antibacterial property of sulbactam itself.

24. Moraxella Moraxella  is a  genus  of  Gram-negative   bacteria  in the  Moraxellaceae  family. It Is named after the Swiss  ophthalmologist   Victor Morax . The organisms are short  rods ,  coccobacilli , or as in the case of  Moraxella catarrhalis ,  diplococci  in morphology, with asaccharolytic ,  oxidase -positive, and  catalase -positive properties.  M. catarrhalis  is the clinically most important species under this genus. Morphology It is a gram-negative coccus measuring about 0.8um in diameter. It is arranged in singly or in pairs with adjacent sides flattened. It is also found in groups and tetrads.

25. Cultural characteristics These are aerobes. They grow well at an optimum temperature of 36 c. Most strains are grown in nutrient agar, blood agar, and chocolate agar. On blood agar, after 24 hrs of incubation, they produce non-haemolytic, white or greyish, convex colonies with entire margins. On prolonged incubation at 4 hrs, the colonies become large with elevated margins and raised opaque centre. They are non motile, oxidase negative, OF test negative and sensitive to Penicillin.

26. Virulence factors Outer Membrane Proteins CopB is a surface protein expressed that is believed to be involved in acquisition of iron from host lactoferrin or transferrin molecules. uspA1 and uspA2 uspA1 and uspA2 is a dimeric protein associated with cell adherence to human epithelial cells. It has been shown that isolates of the protein have a high affinity to extracellular fibronectin, a component of integrands, and vitronectin. Pili The initial onset of symptoms from pili forming strains is much more rapid than non-pili strains. However, non-pili strands seem to be more delayed, drawn out response. The scores for non-pili were significantly higher than pili. Transferrin-Binding Proteins - Complement activation against the pathogen. Lactoferrin-Binding Proteins – Facilitates a strong immune response Lipo-oligosaccharides LOS is a prominent component on the surface of the pathogen. It consists of an A lipid chain linked to ketodeoxyoctulonic acid which is then linked to the inner core. The A lipid is very similar to the typical type of members of the Enterobacteriaceae family. There are also epitopes expressed which are homologous with other gram-negative bacteria. LOS serotyping is used to classify strains of  M. catarrhalis.

27. Spectrum of diseases It is an opportunistic pathogen. It is a common cause of acute otitis media (middle ear infections). And also, People with a lung disease who spend a lot of time in hospitals have the highest risk of developing pneumonia due to  M. catarrhalis . In adults with weakened immune systems or chronic lung conditions,  M. catarrhalis can cause bronchitis. M. catarrhalis  is the  second most common  bacterial cause of worsening COPD. It can increase mucus production, make mucus thicker, and make it even harder to breath. It causes pink eye in both children and new-born. Antimicrobial therapy and susceptibility testing Moraxella was resistant to penicillins due to production of beta-lactamases and susceptible to other antibiotics, such as trimethoprim-sulfamethoxazole, erythromycin, tetracyclines, and cephalosporins.

28. Alcaligenes Alcaligenes  is a genus of  Gram-negative ,  aerobic , rod-shaped bacteria. The species are motile with one or more peritrichous  flagella and rarely nonmotile. It is a genus of nonfermenting bacteria (in the family  Alcaligenaceae ). A. faecalis  was isolated in 1896 by Petruschky from stale beer. Morphology This species is motile, flagellated, slender, slightly curved, not spore-forming, slowly growing, Nonfermenting, Capsule forming , Gram-negative aerobe of the family Alcaligenaceae . Alcaligenes faecalis  occur in water and soil. The microbe has peritrichous flagellar arrangement which allows for motility. It is a gram-negative, rod-shaped organism observed at 0.5-1.0 µm x 0.5-2.6 μm in diameter. An aerobic microbe,  A. faecalis  is optimal at temperature between 20-37 °C.

29. Cultural characteristics These are aerobes of the family Alcaligenaceae . This species is most commonly found in the alimentary tract as a harmless saprophyte in 5-19% of the normal population. It causes a greenish discoloration of blood agar medium. A key biochemical feature of this species is its ability to reduce nitrate but not nitrite. They are motile, oxidase positive and OF test negative. Odor Some strains (A. odorans ) produce a characteristic fruity odor Virulence factors An extracellular serine protease ( Esp ) has potential to be a nematocidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita.

30. Spectrum of disease Alcaligenes  spp. have been isolated from various clinical sources such as blood, feces , sputum, urine, CSF, wounds, burns and swabs taken from throat, eyes and ear discharges. They are infrequent causes of hospital-acquired infections in patients who often have severe underlying disease. Rare cases of peritonitis, pneumonia, bacteraemia, meningitis and UTI have been reported. In many instances the organism is considered to be a colonizer. Nosocomial outbreaks are usually associated with an aqueous source.  Alcaligenes  spp. are predominantly isolated from respiratory tract specimens and recovery of these organisms from the sputum of CF patients has been associated with an exacerbation of pulmonary symptoms. Alcaligenes xylosoxidans  has been implicated in BSI (mostly catheter-related), pneumonia, endocarditis, meningitis, osteomyelitis, peritonitis and urinary tract infection, often in patients with underlying malignancy, HIV and CF. Antimicrobial therapy and susceptibility testing The bacterium is known to be resistant to all cephalosporins and often to aminoglycosides, aztreonam and cotrimoxazole.

31. Stenotrophomonas maltophila Stenotrophomonas maltophila is an aerobic, non-fermentative gram-negative bacterium. It is an uncommon bacterium and human infection is difficult to treat. Initially classified as Bacterium bookeri , then renamed Pseudomonas   maltophilia ,  S.maltophilia  was also grouped in the  genus   Xanthomonas  before eventually becoming the  type species  of the genus  Stenotrophomonas  in 1993. Morphology S. maltophilia  is slightly smaller (0.7–1.8 × 0.4–0.7  μm ) than other members of the genus. They are motile due to  polar   flagella , and grow well on  MacConkey agar  producing pigmented colonies.  S. maltophilia  is  catalase -positive,  oxidase -negative (which distinguishes it from most other members of the genus) and has a positive reaction for extracellular  DNase .

32. Cultural characteristics On  MacConkey agar  producing pigmented colonies. It is a motile rod, possesses polar multitrichous flagella, and can be easily distinguished from other pseudomonads by virtue of being lysine and DNase positive, but oxidase negative. Most strains of S. maltophila are susceptible to colistin and polymyxin B. Virulence factors   Type II protein secretion (T2S) is a major facilitator of virulence in lung pathogens, including Pseudomonas aeruginosa and Legionella pneumophila . Proteins secreted by T2S usually include toxins and tissue-degrading enzymes.  Type IV pili (T4P) promote lung infection . Spectrum of disease S. maltophila has been reported to cause a wide spectrum of disease, including pneumonia, bacteremia, endocarditis, catheter-related infections, cholangitis, urinary tract infections, meningitis and serious wound infections, particularly in patients with cancer.

33. Antimicrobial therapy and susceptibility testing S. maltophila is inherently resistant to most of the commonly used antipseudomonal drugs, including aminoglycosides and many beta-lactam agents, including those effective against P. aeruginosa. S. maltophila is inherently susceptible to trimethoprim- sulfamethoxazole, a drug that has no activity against P. aeruginosa or most other pseudomonas species.

34. Sphingomonas The genus sphingomonas was described by Yabuuchi et al. in 1990 and was embedded by Takeuchi et al. in 1993. Organisms in this genus are gram-negative, non-spore forming rods with that have a single polar flagellum when they are motile. By 2001, the genus included more than 20 species that were quite diverse in terms of their  phylogenetic , ecological, and physiological properties. As a result,  Sphingomonas  was subdivided into 4 genera: Sphingomonas ,  Sphingobium ,  Novosphingobium ,  Sphingosinicella , and  Sphingopyxis . Morphology Sphingomonas  was defined in 1990 as a group of  Gram-negative , rod-shaped,  chemoheterotrophic , strictly  aerobic  bacteria. They possess  ubiquinone  10 as their major  respiratoryquinone ,contain   glycosphingolipids  (GSLs),specifically  ceramide , instead of  lipopolysaccharide  (LPS) in their cell envelopes, and typically produce yellow-pigmented colonies. It is motile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium.

35. Cultural characteristics The colonies appear as smooth, shiny, convex, yellow colored colonies. It produces diffusible pigment on nutrient agar. the optimal growth temperature is about 30 C. It can grow on trypticase soya agar +/-5% sheep blood, nutrient agar, muller Hilton agar. Positive results for esculin &gelatin hydrolysis, beta- galactosidase(ONPG), utilization of gluconate, adipate, L-malate, L-arabinose, mannose, maltose, fructose, glucose, sucrose, trehalose , and xylose. They are oxidase negative and motile bacteria. Negative results for oxidase ( Bergey’s manual), denitrification, indole production, DNase, gelatin hydrolysis, lecithin, lysine & ornithine decarboxylase, urease, utilization of citrate, mannitol, sorbitol, L-tryptophan, histidine, mucate , D and L- arabitol . Variable result in starch hydrolysis.

36. Virulence factors There is a phylogenetic divergence between  Sphingomonas  and  Pseudomonas , they shared many major virulence factors, such as adherence, anti-phagocytosis, iron uptake, proteases, and quorum sensing.   Sphingomonas  spp. contains several major virulence factors resembling  Pseudomonas  sp.,  Legionella  sp.,  Brucella  sp., and  Bordetella  sp. Similarity of virulence factors did not match phylogenetic relationships.  Sphingomonas  spp. is potential virulent bacterial pathogen. A recent study has identified several virulence genes in the genome of  S. paucimobilis . That have similarity to those of Pseudomonas aeruginosa including Pyoverdine and Pyochelin ( sidephores -iron chelating agents), alginate genes (alginate, an anionic polysaccharide, can have antiphagocytosis activity), Rhamnolipid (a biosurfactant), Phospholipase C (a toxin), several Protease genes (Alkaline protease and LasA and LasB ) and several genes involved in adherence such as flagella genes and Type IV pili genes. The effects the protein products of these genes might have in vivo is unknown.

37. Spectrum of disease Initially  S. paucimobilis  was thought to be a non-pathogenic environmental isolate however it has since been reported to cause a variety of infections. Cases have included wound infections, meningitis, a case of septic shock in a burns patient, catheter associated bacteremia, ventilator associated pneumonia, splenic abscess, urinary tract infection, empyema and several cases of bacteremia and peritonitis. Antimicrobial therapy and susceptibility testing Most strains are susceptible to tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, and aminoglycosides. Their susceptibility to other anti-microbial agents, including fluoroquinolones.

38. Chryseobacterium C. indologenes  was first isolated from a clinical specimen and described by Yabuuchi et. al. in 1983. 42 It is most commonly isolated from human specimens but has rare clinical significance. There have been recent reports of bacteremia related to  C. indologenes  as it is often seen in indwelling devices, such as a catheter, but little is known of its significance in clinical disease. 43-46 Morphology C. indologenes  is a Gram-negative filamentous rod bacterium of approximately 0.5 µm in diameter and 1.0 – 3.0 µm in length. The rods have rounded ends and parallel sides.  Colonies are circular, convex, entire, smooth and up to 2 mm in diameter with an aromatic odor.  C.indologenes  colonies are a deep yellow and are thinner in the central portions than in the peripheral portions.

39. Cultural characteristics The chryseobacterium species generally grow poorly on MacConkey agar and are considered to be glucose oxidizers. Most of the strains will slowly ferment glucose after prolonged incubation. They are oxidase positive, non motile and indole positive. C. indologenes  is a Non-fastidious, chemo-organotrophic organism that can respire in anaerobic conditions with nitrate or fumarate used as the terminal electron acceptor. Acid is produced from D-fructose, D-glucose, glycerol, maltose, trehalose , glycogen and mannose, but not from lactose, L-arabinose, ethanol, sucrose, or D-xylose. Catalase, oxidase, phosphatase and strong proteolytic activities are observed in  C. indologenes  as well as oxidation of carbohydrates. Glycerol and trehalose are not oxidized.  C. indologenes  is also capable of degrading esculin, DNA, starch and Tween-80 and produces indole. It does not, however produce ß-galactosidase or L-phenylalanine deaminase.

40. Virulence factors The resistome of this multidrug resistant C. indologenes MARS 15 includes a total of nine antibiotic resistant genes including Ambler class A and B β-lactamases encoding BlaCIA and BlaIND2 genes, chloramphenicol acetyltransferase gene CAT and multidrug efflux pump AcrB . The specific features such as the presence of urease operon, iron acquisition mechanisms, pigmentation of bacterium, amino acid metabolism that may be involved to adaptation under selective pressures in order to enhance chance of survival under chronical infection diseases as fibrosis cystic. Spectrum of disease Chryseobacterium   indologenes  can cause serious infections in immunosuppressed patients. Intravascular devices such as venous catheters, intravascular implants, urinary catheters, intubation and mechanical ventilation are the risk factors for  C.   indologenes  infection. Most of the cases of  C.   indologenes  were reported from long-term hospitalized patients on mechanical support associated with the underlying disease. This case indicates that  C.   indologenes  might cause symptomatic disease in immunocompetent patients with otherwise no associated underlying risk factors.

41. Antimicrobial therapy and susceptibility testing The organism is found to be resistant to most antimicrobial agents commonly used to treat Gram-negative bacteria, however, susceptible to those used to treat Gram-positive bacteria. C. indologenes  is resistant to cephalothin, cefotaxime, ceftriaxone, aztreonam, aminoglycosides, erythromycin, clindamycin, vancomycin and teicoplanin, while susceptible to piperacillin, cefoperazone , ceftazidime, imipenem, quinolones, minocycline and trimethoprim-sulfamethoxazole.

42. Sphingobacterium Sphingobacterium  is a genus in the family  Sphingobacteriaceae . The genus Sphingobacterium is characterized by the high concentrations of  sphingophospholipids  as lipid components. 70 Morphology Laboratory tests conducted demonstrate  Spingobacterium multivorum  is a rod shaped, non-spore forming bacteria. It also has evidence towards aerobic metabolism, using a multitude of sugars and the ability to reduce nitrogen to nitrogen gas.  Spingobacterium multivorum  got its genus for the high composition of sphingolipid. Cells are Gram-stain-negative, non-motile, non-spore forming, short rods, 0.6–0.8 mm61.3–2.2 mm in size and have pili. Colonies on LB are yellow, circular, and convex, grow at 10–40°C (optimum, 25–30°C), pH 5.0– 10.0 (optimum, pH 6.0–8.0) and with 0–5 % NaCl (w/v) (optimum, 1 %).

43. Cultural characteristics Bacteria that belong to the genus  Sphingobacterium  are Gram-negative, non-lactose-fermentative microorganisms that are positive for catalase and oxidase tests and produce small, circular, convex, smooth and opaque colonies with light yellow pigment when incubated at 35°C in either carbon dioxide or ambient air for a minimum of 24 hours. Growth on MacConkey agar is usually detectable within 24 hours of inoculation.  71 An important feature of bacteria that belong to this genus is the presence of high concentrations of sphingophospholipids in their cell membrane. 72 It is oxidase positive, yellow pigmented. they are differentiated from chryseobacterium by their failure to produce indole from tryptophan. Cells are positive for catalase and oxidase activities and can hydrolases starch, DNA and Tween 20. Using API 20E and 20NE kits, strain N7T is positive for beta-galactosidase, beta- glucosidase,Voges –Proskauer reaction, nitrate reduction, fermentation of glucose and urease activity.

44. Negative for arginine dihydrolase , gelatinase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, and H2S and indole production. Acid production occurs on D-glucose and L-arabinose. D-Glucose, N-acetylglucosamine, salicoside , sucrose, D-mannose, mellibiose and maltose are used as growth substrates, while D-mannitol, valeric acid, capric acid, malate, sodium acetate, L-serine, potassium 5-ketogluconate, glycogen, citrate, adipic acid, L-fucose, D-sorbitol, propionic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3-hydroxybutyric acid, gluconate, L-histidine, amygdalin, potassium 2- ketogluconate, phenylacetic acid, L-proline, lactic acid, itaconic acid, suberic acid, sodium malonate, L-rhamnose, D-ribose, inositol and L-alanine are not used as sole carbon sources.

45. Virulence factors Three genes ( gldA , gldF , and gldG ) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility. Spectrum of disease S. multivorum  has caused blood stream infection, spontaneous bacterial peritonitis, meningitis, and lung infections.  Antimicrobial therapy and susceptibility testing Sphingobacterium species are generally resistant to aminoglycosides and polymyxin B, while they are susceptible to invitro to the quinolones and trimethoprim- sulphmethoxazole . Susceptible to beta-lactam antibiotics is variable, requiring testing of individual isolates.

46. Chryseomonas Morphology Colonies are R or S type, sometimes wrinkled, yellow colonies (water- insoluble pigment). Strictly aerobic, optimal growth temperature 30 c. can grow at 42 c but not at 5 c. Cultural characteristics Media: Nutrient, trypticase soy agar, MacConkey, CASO agar. Distinguished from phenotypically similar Flavimonas orhyzihabitans by positive test reactions for nitrate reduction, sorbitol utilization, hydrolysis of ONPG, esculin and arginine. Positive results for catalase, nitrate reduction, urease, DNase, gelatin hydrolysis, esculin hydrolysis, ONPG, acid and alkaline phosphatases, arginine dihydrolase , acid production from: L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, D-galactose, l-rhamnose, maltose, trehalose , mannitol, inositol, salicin . L-arabinose, D-xylose, d-ribose, D-glucose, D-fructose, D-mannose, D-galactose, maltose, trehalose , mannitol, glycerol, acetate, pyruvate, malonate, fumarate, 2-ketogluconate, gluconate, succinate, p-hydroxy benzoate, and glutamate are utilized.

47. Negative results for indole production, H 2 S production, starch hydrolysis, TWEEN 80, lysine decarboxylase, ornithine decarboxylase, phenyl alanine deaminase. Acid production from sucrose, lactose, cellobiose, adonitol, sorbitol and inulin. Sucrose, lactose, raffinose, inulin, starch, phenol, alpha-hydroxy benzoate and m-hydroxy benzoate are not utilized. Virulence factors The strain is also known to produce an  exopolysaccharide  (EPS) utilized in the adsorption of nickel and copper. In order to adsorb Ni and Cu at significant levels, the strain must be immobilized in a  calcium alginate  beads. With this enhancement, maximum adsorption capacities range from 45.87-50.81 mg g-1 and 52.91-61.73 mg g-1, respectively. Spectrum of disease The pathogenic form of  Pseudomonas luteola  is a  saprophyte . It is an  opportunistic pathogen  that can cause bacteraemia, meningitis, prosthetic valve  endocarditis , peritonitis in humans and animals. It also induces septicaemia , meningitis,endocarditis,or peritonitis.

48. Antimicrobial therapy and susceptibility testing Antimicrobial susceptibility by agar disk diffusion methods showed that the  C. luteola  culture was susceptible to imipenem, colistin, ofloxacin, ciprofloxacin, amikacin, netilmicin, and doxycycline and resistant to amoxicillin, ceftazidime, cefotaxime, ceftriaxone, gentamicin, and trimethoprim-sulfamethoxazole. Most strains are susceptible to broad-spectrum antibiotics, such as cephalosporins, aminoglycosides, and ciprofloxacin .  However, infections associated with foreign material are highly resistant, and infected prostheses have to be removed if possible.

49. Flavobacterium Flavobacterium psychrophilum  is a psychrophilic,  gram-negative  bacterial rod (3-5 µm in length), belonging to the  Bacteroidetes . It is the causative agent of  bacterial cold-water disease  (CWD) and was first isolated in 1948 during a die-off in the salmonid  Oncorhynchus kisutch . Morphology According to Bernardet et.al.  Flavobacteria  are gram-negative aerobic rods, 2-5 um long, 0.3-0.5 um wide, with rounded or tapered ends that are motile by gliding, yellow (cream to orange) colonies on agar, decompose several polysaccharides but not cellulose, G+C contents of 32 - 37 %, and are widely distributed in soil and freshwater habitats. The type species is  F. aquatile .

50. Cultural characteristics When grown on  Cytophaga Agar ,  F. psychrophilum  produces bright yellow colonies with thin spreading margins not greater than 3mm in diameter.  Motility is achieved by gliding, movement that does not involve the use of pili or flagella. The bacterium is positive for  gelatin hydrolysis ,  albumindigestion ,  tributyrin digestion ,  tributyrin hydrolysis ,  E.coli  cell autolysis, and  casein hydrolysis .  On its fish host, the pathogen can be found on external and internal sites such as skin/mucus, gills, brain, ascites, lesions, mucus, kidney, spleen, and reproductive excretions of spawning adults. Colonization is evident by faint, white areas on the host. Catalase-positive. Oxidase-positive. Phosphatase-positive. Indole-positive. Urease-negative. DNase-negative.

51. Virulence factors The genome encodes 13 assumed secreted proteases that are involved in virulence and destruction of the host’s tissues. Also, the genome encodes for bacterial hemolysins that cooperate with proteases for tissue destruction and thiol-activated cytolysins like proteins that are responsible for host tissue damage. Spectrum of disease Flavobacteria are generally commensal bacteria that live in soil and water and are opportunistic pathogens. The can be found in water and soil in many areas around the world. Found in humans and cats as pathogenic.  Flavobacterium meningosepticum   is well recognized as a cause of neonatal meningitis and such infections have a poor prognosis. Meningitis due to  F. meningosepticum   in adults is rare, as is septicaemia , and all the patients in whom it has been described had underlying diseases predisposing to Gram-negative infection. This species may also cause pneumonia in both infants and adults. Colonization of the respiratory tract in compromised hosts may occur under endemic situations. The pathogenicity for man of other  Flavobacterium  species is less well documented but cases of meningitis, bacteremia, and upper respiratory tract colonization of seriously ill patients have been attributed to groups

52. Antimicrobial therapy and susceptibility testing The organism was susceptible to amikacin, chloramphenicol and sulphonamides when tested by the disc diffusion method, therapy with these drugs was ineffective. Intravenous therapy with cotrimoxazole gave complete recovery. Various strains of flavobacteria including F. meningosepticum type B were isolated from the environment. Antimicrobial susceptibilities of patients and environmental strains against chloramphenicol, sulphonamides and rifampicin showed no correlation between disc diffusion results and minimum inhibitory concentration (MIC). On the other hand, all the strains were found by both methods to be susceptible to amikacin, erythromycin, clindamycin and trimethoprim. Disc diffusion method should be supplemented with determination of MIC when testing antimicrobial susceptibility of Flavobacterium spp.

53. Thank you….