1. The following presentation and its content is exclusive property of CPM SAS and is considered strictly confidential and reserved exclusively to the partecipants. It is prohibited the divulgation without the consent of CPM SAS

4.  Normally the urinary tract is sterile  The UTI is the most common bacterial in all age groups  Patients with UTI generally have bacteriuria equal to or greater than 10 5 colony forming units (CFU) / mL (significant bacteriuria), while a charge of less than 10 4 CFU / mL in asymptomatic patients involves a valutation (not significant/significant bacteriuria depending of the patient condition / contamination).

5. 1. Clinical situation of patient 2. Urine collection procedure, the container used for the collection, the time between sample collection and examination, the diuresis importance, urinary pH, the recent consumption of antibacterial drugs and high leucocyturia

6. Age Female sex Diabetes Pregnancy Vesicoureteral reflux Urinary stasis (malformations, IPB, neurogenic bladder, ureteral or urethral stenosis, calculi, etc.). Foreign bodies in the urinary tract (including calculi) Instrumentation in the urinary tract Sexual intercourse Intrauterine contraceptive, spermicide creams Incorrect voiding habits Irregularities of alvus

7. In the interpretation of a urine culture must considered all these aspects. This is the reason why this is usually one of the more complex diagnostic tests

8. Oyron WELL D-ONE® is a system designed for the presumptive identification of microorganisms most frequently responsible of urinary tract infections (UTI) and to test their susceptibility to antibiotics

9. Oyron WELL D-ONE® consists of a plate made of polypropylene in which there are 32 conical wells with flat well to allow better visualization of the colorimetric reactions that occur in each of them following the growth of a specific m.o. according to specific metabolic characteristics.

10. An essential characteristic of this system is that it can perform the presumptive identification of the pathogen directly from the sample, after dilution, without previous isolation and other manipulations that require time and more materials for execution of test A single plate of Oyron WELL D-ONE®, thanks to the presence of specific media, at the same time allows both the identification and the susceptibility test necessary to eradicate the infecting m.o.

12. Operative procedure 1. Collect 200 µL (4 drops) of urine directly from the sample 2. The 200 µL (4 drops) must diluted in 10 mL of sterile saline solution 3. From this dilution 150 µL (3 drops) is inoculated in each well of plate OYRON WELL D-ONE® 4. Incubate at 37°C for 24 hours (24 for Candida Albicans, 24/48 for Candida spp.) 5. Add 1 drop of Reagent A and B 6. Reading

13. OYRON WELL D-ONE NEGATIVE

14. The well 1 allows the investigation on the production of nitrite or not. The nitrates are converted to nitrites in the urine via an enzymatic reaction. The positivity is evidenced by a change of the color from yellow to red/dark orange after addition of a drop of reagent A (Naphthylamine) and a reagent B (sulfanilic acid).

15. The well 2 represents a growth control (GC). If negative there is a transparent white colour, in the case of bacterial presence well assumes a color white turbid well evident. The presence of turbidity indicates concentration ≥ 10⁵CFU.

16. The wells 3-4 allow a presumptive identification of Gram positive and Gram negative respectively. The positivity of the well 3 opaque white turns to deep yellow, while in the case of positivity of the well 4 to gram positive the well turns to turbid red The presence of turbidity indicates a concentration≥ 10⁵CFU. The well 3 can give an intense yellow color without turbidity. Well 3 positivity are caused only by Gram negative bacteria. Well 4 positivity are caused by Gram positive bacteria. Multi-resistance bacteria, such as., KPC, ESBL, MRSA and Proteus spp. cause a colorimetric reactions. The presence of Gram negative and Gram positive bacteria in the sample may cause positivity in well 3 and 4. NEGATIVE GRAM NEGATIVE GRAM POSITIVE

17. 5 6 Presumptive identification of Escherichia coli 75-95 % of urinary tract infections (UTI) are associated with Escherichia coli The wells 5 e 6 are formulated for identification with selective media this m.o., well 5, in case of positivity, change from yellow to aqua green, while the well 6 from pink to light blue. The presumptive identification is realized observing the resultswells 5 and 6. The combination of chromogenic media (well 5) and selective (well 6) provides presumptive identification especially for E. coli. Some INACTIVE E. coli strains do not produce color change in well 6, which remains turbid pink.

18. Positivity for E.Coli, wells 1,2,3,5 and 6 positive.

19. 7 8 Presumptive identification of beta-hemolytic Streptococcus group B, if the microorganism is present causes a color change from light yellow to green. The well 8 contains a selective culture medium for Streptococcus agalactiae, the growth of this m.o. in this well causes an increase in turbidity of the same. Some strains of non-hemolytic Streptococcus agalactiae do not produce a color change in the well. It is recommended to perform sodium hippurate test and serological test.

20. Therefore, the presumptive identification of Streptococcus agalactiae is given by the negativity of the well 1, positivity of well 2 (turbid), White turbid / opaque of the 3, turbid red of 4 and positivity in the well 7or 8 or negativity in 7 but with a hippurate test and a serological test positive by taking in a well 8 turbid.

21. Designed for the presumptive identification of Pseudomonas spp., The presence of this organism in the sample causes a color change from white to aqua green turbid. Some strains of P. Aeruginosa develop an aquamarine–green color at room temperature. It is recommended to leave the identification panel at room temperature from 1 to 3 hours, after having remove from the incubator. All strains of P. Aeruginosa cause a color change of the well to turbid aquamarine-green. Some bacteria, such as Enterococcus spp and some enterobacteria may cause a color change to light green without causing turbidity.

22. Commonly, the members of this group are considered opportunists, as pathogenic for individuals with compromised defenses, are also readily available in nosocomial environments, in respirators, in dialysis equipment etc.. If there is the presence of these microorganisms should be positive wells 2, 3 and 9.

23. The well 10 contains a selective culture medium with a chromogen for presumptive identification of Staphylococcus aureus. The growth of this m.o. in well causes a color change from light yellow to violet. The well 11 helps in the identification of this m.o. as it contains an indicator of growth for the presumptive identification of S. aureus, the growth causes a color change from light yellow to black. The presumptive identification of Staphylococcus aureus is evidenced by the results of both wells. Negativity of well 3 and positivity of well 4. Positive reactions may occur in the well 10 when in the sample are present strains of Enterococcus spp. at higher concentrations of 10^3 CFU. (Make observation of Enterococcus spp. well 17)

24. 12 13 Wells 12 and 13 allow the presumptive identification of Enterobacter spp. with a color change in well 12 from slight yellow to white turbid and well 13 to pink turbid Presumptive identification is made for the positivity of both wells. Some Gram-negative bacteria can give a slight pink color in well 13 but well 12 maintains a transparent color.

25. Enterobacter spp The presence of Enterobacter spp. is evidenced by a color change in wells 1, 2, 3, 12 and 13, and well 16 (dark blue)

26. For the presumtpive identification of Proteus / Providencia When there is a color change from slight yellow to dark brown in well 14 and a color change in well 15 from sligh yellow to white turbid is Proteus; if only well 14 is positive and not 15 is presumptively Providencia The color change of this well is evidenced of a strain presumably of Proteus /Providencia. Enterobacter spp. causes a strong turbid slight grey or white.

27. Proteus -> positivity of the wells 1, 2, 3, 14 and 15.

28. Klebsiella Enterobacter KES GROUP : Enterobacter spp, Klebsiella spp., Serratia spp. There is the distinction of colors from blue purple to green when we are in the presence of the genus Klebsiella spp, from blue purple to very dark blue when there is a m.o. as Enterobacter spp. and a color variability for Serratia.

29. So the presence of these m.o. presumptively must be identified by the positivity of the well 1, 2 and 3 and 16 blue (Enterobacter spp), 1,2,3 and 16 green (Klebsiella spp) and 1,2,3 and 16 variable (Serratia spp).

30. For the presumptive identification of Enterococcus spp, The well change from slight yellow to black

31. The presence of this kind of m.o. can be evidenced presumptively with the positivity in the wells 4 and 17. It is possible that when there is an increase of more than 1000 CFU / mL of this m.o. it can possible a change of color in the well 10

32. 18 19 AFTER 24 HRS, checked with conventional and molecular tests with 99 % of specificity and sensitivity. The presumptive identification of Candida albicans is evidenced by the positivity of well 18 (green) and 19 (turbid) -> AFTER 24 HRS, checked with conventional and molecular tests with 99 % of specificity and sensitivity. Other species of Candida spp. may cause other different colors in well 18 (Candida dubliniensis - Green, Candida tropicalis - Violet) and turbidity in the well 19( 24-48 hrs ). It is recommended to confirm by microscopic observation (40x) to observe the characteristic structures of chlamydospores and / or hyphae.

33. FROM WELL 20 TO WELL 32 IS EVIDENCED THE SUSCEPTIBILITY TO ANTIBIOTICS WITH RESISTANCE EVIDENCED BY A COLOR CHANGE TO TURBID RED WHILE THE SUSCEPTIBILITY FROM TURBID RED TO WHITE/PINKISH EUCAST RESISTANCE SUSCEPTIBILITY

34. It is important the evaluation of all plate, the sample history and the clinical situation of patient to allow a complete diagnosis.

35. ResultsOYRONConventional Positive*828399 % Negative*29429299 %* Contamination505198 % Total426 78 %** MicroorganismOYRONConventional E. coli495294 % Enterobacter spp.131185 % Kleibsella spp.99100 % Serratia spp.11100 % Proteus spp33100 % S. aureus11100 % Candida spp.33100 % Pseudomona spp.22100 % Enterococcus spp.91-

36. NEXT PHOTOS NEGATIVE LOW COUNT CONTAMINATION POSITIVE POSSIBLE RESULTS

37. NEGATIVE

38. LOW COUNT

40. CONTAMINATION

42. POSITIVE for E. coli and Enterococcus spp.

43. POSITIVE for Kleibsella spp.

44. POSITIVE for Proteus spp./Providencia spp.

45. CONVENTIONAL METHODSOYRON D WELL ONE ® MEDIAPRESUMPTIVE IDENTIFICATION By chromogenic and selective medium combinations. URISLIDE (COUNTING) Escherichia coli Streptococcus spp. Staphylococcus spp. Enterococcus spp. Pseudomonas spp. KES group Proteus/Providencia spp. Candida albicans Candida spp. Presumptive diagnosis requires confirmation by biochemistry or agglutination tests. Detection well for NO 3 Gram + and Gram – differentiation well Counting well Escherichia coli Streptococcus agalactiae Staphylococus aureus Pseudomonas spp Enterococus spp Klebsiella spp. Enterobacter spp. Proteus spp. Proteus/Providencia spp Candida albicans Candida spp. Presumptive diagnosis does not require confirmation by biochemical or agglutination tests. MAC CONKEY AGAR MUG COLUMBIA CNA BLOOD AGAR CETRIMIDE AGAR CLED AGAR SLANETZ BARTLEY AGAR ENTEROCOCCO AESCULIN AGAR (SELECTIVE ENTEROCOCCUS) CHROMOGENIC URINE AGAR SABOURAUD DEXTROSE AGAR +CAF+CYCLOHEXIMIDE CHROMOGENIC CANDIDA AGAR

46. Complementary methods Urea Test Oxidase Test Test Catalase Additional commercial testComplementary techniques can be realized from the identification wells. Only to confirm the diagnosis Agglutination test Latex tests, specific antisera. Antigen Detection Identification card. Coagulase Test Latex tests; Free coagulase and bound coagulase detection tests. Traditional antimicrobial susceptibility test. Additional media and antibiotics discs are required. Antimicrobial susceptibility MIC TEST manual/ Automatic method.Included in the kit Biochemical tests They should be performed from the culture. Additional media or automatic method are required. Included in the kit IDENTIFICATION TIME 2/4 days24 HOURS READING OF RESULTS Experience and/or equipment are required.VISUAL, EASY AND EVIDENT INTERPRETATION OF RESULTS Experience is required.EASY ADDITIONAL EQUIPMENT REQUIRED yesNO LABORATORY CONDITIONS MEDIUM/HIGHMINIMUM HANDLING SPECIFIC ABILITIES ARE REQUIRED INOCULATION AND READING OF DIFFERENT PLATES ARE REQUIRED THE PROCESSING AND THE ASSEMBLY REQUIRED LONG TIME. EASY TO PERFORM INOCULATION OF ONLY ONE PLATE IS REQUIRED THE PROCESSING AND THE ASSEMBLY DO NOT REQUIRE LONG TIME

47. CONCLUSION The greater danger for most of us lies not in setting our aim too high and falling short; but in setting our aim too low, and achieving our mark “The greater danger for most of us lies not in setting our aim too high and falling short; but in setting our aim too low, and achieving our mark.” (Michelangelo) THANKS! Prof. Isis Tamargo Martinez, PhD Dr.ssa Sabina Ripani Dr. Francesco Pacella La seguente presentazione ed il suo contenuto è di proprietà della CPM SAS ed è da ritenersi strettamente confidenziale e riservata esclusivamente ai destinatari. Se ne vieta la divulgazione senza il consenso della CPM SAS