Key findings:

Key findings include: among Klebsiella spp. cases, 12 exhibited resistance to Ampicillin, 10 to Ceftazidime, while 5 were susceptible to Amikacin, 4 to Imipenem, and 2 to Gentamicin; 12 cases of Staphylococcus spp. were identified, with 8 demonstrating resistance to Clindamycin, 5 to Oxacillin, while 9 were sensitive to Gentamicin, 8 to Nitrofurantoin, and 5 to Vancomycin.

What is known and what is new?

The known aspect is the general importance of bacterial identification and antimicrobial susceptibility testing in clinical microbiology. The new contribution is the specific study on the VITEK system's performance in identifying and testing anaerobic bacteria in clinical isolates from Wasit province, providing insights into antimicrobial resistance patterns and treatment options for UTI cases.

What is the implication, and what should change now?

The implication is that the VITEK system provides valuable insights for enhancing bacterial identification and susceptibility testing practices in Wasit province and its clinical laboratories. Changes needed include implementing effective antimicrobial therapy strategies based on the results, particularly using Amikacin and Gentamicin for UTI cases, to reduce and eradicate bacterial infections effectively.

The detection of bloodstream infections is one of the most important tasks performed by the clinical microbiology laboratory. Rapid bacterial identification and susceptibility testing not only improve patient therapy and outcomes but also reduce costs [1]. Both automated blood culture systems and automated systems for the identification and susceptibility testing of bacteria have been on the market for a number of years [2]. Clinical microbiology laboratories often play an important role in detecting the causative pathogens of bloodstream infections. However, the information cannot be obtained rapidly with conventional blood culture procedures because time is required to grow colonies of bacteria from positive BCs, and then identify the bacteria involved and determine their antimicrobial susceptibility. Therefore, the standard procedure does not allow the early development of precise therapeutic strategies for patients. Furthermore, the receipt of discordant empiric antibiotic therapy was associated with increased overall mortality [3]. The classical method of detecting drug resistance in a positive BC requires overnight subculture of the organism on an agar medium. Clinicians sometimes have to initiate empirical treatment before the results are available. This increases the likelihood of an unreasonable treatment. Furthermore, the increases in multidrug-resistant Gram-negative bacteria (MDR-GNB) have increased the failure of empirical treatments. This demonstrates the clinical importance of the early-stage diagnosis of drug resistance. Such early results can be obtained with various methods. PCR can be used to directly detect certain antibiotic resistance genes in positive BCs, but a major limitation is that negative amplification does not necessarily indicate susceptibility [4,5]. The VITEK 2 system (produced and developed by the French company BioMérieux) is a new automated bacterial identification and susceptibility testing system that uses fluorescence-based technology. This device is one of the best devices in the world for identifying all types of pathogenic bacteria and yeasts very accurately and within a short period, identifying their classification, and testing their degree of sensitivity to antibiotics. Previous studies showed that this system could give reliable identification and susceptibility results with pure bacterial cultures [6]. The current study will highlight the utilization of the Vitek 2 system in the identification and susceptibility of bacterial infections and measure its accuracy in this regard. It would compare the potential benefits of implementing this system in the clinical laboratories of Wasit Province.

2.1 Bacterial isolation and identification

A lab-based study on 65 different clinical samples collected from patients, attending different hospitals for laboratory bacterial investigations in Wasit Province for the period of January 10^th to February 27^th, 2024. All the samples were already conducted by cultivating them on selective media, such as MacConkey agar and blood agar media. The automated VITEK-2 compact system was used to recognize the isolated bacteria to the genus and species level. 

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2.2 The automated identification by VITEK-2 System

The VITEK-2 system uses a turbidimetric method for susceptibility testing and fluorogenic technology to identify organisms. It uses 64-well cards with a barcode containing the card type, expiration date, lot number, and unique identification number. Available test kits include ID-GN (identification of Gram-negative bacilli) and ID-GP (identification of Gram-positive susceptibility). Within 10 hours, the VITEK-2 ID-GN card can identify 154 Enterobacteriaceae and a limited number of glucose-non-fermenting Gram-negative bacteria. In up to eight hours, the VITEK-2 ID-GP card can detect 124 species of Staphylococcus, Streptococcus, Enterococcus, and some other Gram-positive organisms. (Figure 1)

In this analysis, the VITEK-2 method was used to confirm the identification of Enterobacteriaceae as follows: 

1. In a sterile test tube, a single isolated colony of pure bacteria is suspended in 3 mL of saline.
2. The bacterial suspension and the conventional turbid standard solutions were compared. The final concentration must be between 0.5 and 0.63.
3. According to the diagnostic Gram stain, the VITEK2 card or cassette was chosen.
4. The cassette and test tube racks are delivered to the system, and after being placed in the first filling area (filler), the boxes are automatically filled with bacterial suspension, and the device sends out an end signal.
5. The cassette is left on for 24 hours at 37°C. The results are read to diagnose bacteria. The manufacturer reported results as 96% to 100% excellent identification; 93% to 95% very good identification; 89% to 92% good identification; 85% to 88% acceptable identification, and conversely no identification in other isolates

Fig. 1: Vitek-2 system utilized for identification the bacterial infections and antibiotics.

1. Antimicrobial susceptibility testing

This test with the VITEK-2 compact system was performed by means of an AST N281 card according to the manufacturer’s directions. The VITEK-2 AST N281 vulnerability card is proposed for use with the VITEK-2 systems in clinical laboratories as an in-vitro test to decide the vulnerability of clinically significant aerobic gram-negative bacilli to antimicrobial agents [7]. Antibiotics tested in AST N281 card incorporated Cefepime, Levofloxacin, Mero-penem, ticarcillin/Clavulanic acid, Gentamicin, Imipenem, Ceftazidime, Doripenem, Cefoperazone/Sulbactam, Ciprofloxacin, Minocycline, Tigecycline, Amikacin, Trime-thoprim/Sulfamethoxazole (Cotrimoxazole), Colistin, Tobramycin, Piperacil-lin/Tazobactum, Cefuroxime, Ceftriaxone, cefotaxime, the cards were overflowing with inoculums (ready by transfer 200μL of culture suspension from the 0.5 McFar-land culture suspension used for satisfying the identification cards into a clean 3mL sterile saline solution obtain a concluding turbidity of 8x106 cfu/mL).In the satisfying hall, the VITEK-2 System automatically processes the antimicrobial susceptibility cards until MICs are obtained, the VITEK-2 compact system next corrects, where essential for MIC’s or clinical group in agreement with the interior folder of likely phenotypes for microorganism antimicrobial agent combination [8]. The VITEK system originated in the 1970s as an automated system for identification and AST and has evolved today into the VITEK 2 system, which automatically performs all of the steps required for identification and AST after a primary inoculum has been prepared and standardized [9].

2.4 Direct Vitek 

Direct Vitek 2 AST was carried out as described above. In brief, the cell density of the remaining bacterial suspension was adjusted to a density of 0.5 McFarland after dilution in 0.45% saline; 145 μL of the bacterial suspension was drawn into 3 mL of 0.45% saline solution to further adjust the bacterial cell density. Vitek cards were inoculated with the suspension vials and loaded into the Vitek 2 automated reader-incubator. Vitek cards AST-GN13, AST-Gp67, and AST-Gp68 were used for gram-negative bacteria, and Staphylococci/Enterococci/Streptococci [10].

4.1 Samples

A total of 65 were studied. They were clinical isolates consecutively collected in Al-Karama Teaching Hospital in Al-Kut, Wasit Province over 2 months (single-patient isolates).

4.2 Identification with the VITEK 2 system

After the overnight incubation of samples and the subsequent growth of colonies, each colony was suspended to achieve a standardized turbidity/spectrometry reading. Subsequently, the kit corresponding to each group (Gram-negative, Gram-positive, antibiotics) of the Vitek system was inoculated and introduced into the system. The reading of the Vitek 2 system is obtained as described in the following table 1.

Table 1 The performance of the VITEK 2 system for the identification of individual species or bacteria groups.

Out of the 22 cases of E.coli, 14 were resistant to Ampicillin, 9 were resistant to Ceftazidime, and 5 were resistant to Ceftriaxone; and 13 sensitive to Amikacin, 6 sensitive to Nitrofurantoin, and 5 sensitive to Gentamicin. (Figure 2)

Fig 2: Antibiotic resistance and sensitive patterns of E. coli.

Out of 12 cases of klebsiella spp. , 10 were resistant to Ampicillin, 6 were resistant to Ceftazidime; 5 were sensitive to Amikacin, 4 were sensitive to Imipenem, and 2 were sensitive to Gentamicin. (Figure 3)

Fig. 3 Antibiotics resistance and sensitive patterns of Klebsiella spp.

And out of the 12 cases of staphylococcus spp. , 8 were resistant to Clindamycin, 5 were resistant to Oxacillin;  9 were sensitive to Gentamicin, 8 were sensitive to Nitrofurantoin, and 5 were sensitive to Vancomycin. (Figure 4).

Fig. 4:  Antibiotic resistance and sensitive patterns of Staphylococcus spp.

Describing what has been mentioned above, the study can state that E. coli is most resistant to Ampicillin [1] and sensitive to Amikacin [2], Klebsiella spp. most resistant to Ampicillin [3] and sensitive to Amikacin [2] and staphylococcus spp. most resistant to Clindamycin [4] and sensitive to Gentamicin [5].

Bacteremia caused by non-fastidious aerobic gram-negative bacilli is common in Asia, and the antimicrobial susceptibility profiles of these bacteria vary widely [11]. The possibility of combining an automated blood culture system with an automated identification and susceptibility testing system by direct inoculation from positive blood cultures has been studied by several groups of investigators, but their results vary. The VITEK 2 direct identification testing of gram-negative bacilli had a correct identification rate of 82.2% for the positive aerobic blood cultures with a single type of organism; none of the results gave a wrong identification [12].

The ability of the VITEK 2 system and ID-GPC card to accurately give a rapid identification of clinically significant was first assessed by Bassel et al. (Abstr. 8th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P255, 1997), who showed, with isolates of staphylococci, streptococci, and enterococci, 98.0% overall agreement (86.8% agreement to species level without supplemental testing required and 11.2% agreement to species level after supplemental testing), whereas 1.7% of isolates were misidentified and 0.3% of isolates were unidentified. In particular, stock isolates and fresh isolates of S. aureus were identified with an accuracy of 98.8 and 96%, respectively. Stock and fresh isolates of the following organisms were identified with the indicated accuracies, respectively: S. epidermidis, 92.7 and 97%; Staphylococcus haemolyticus, 83.8 and 97%; E. faecalis, 97.7 and 97%; E. faecium 94.6 and 91%; S. agalactiae, 97.7 and 95%; and S. pneumoniae, 95.8 and 76%. With the VITEK 1 system and GPC cards, the range of correct identification among CNS was 67 to 83% and that among S. epidermidis isolates was 88 to 95%.

Antimicrobial susceptibility test.

The Food and Drug Administration has established minimal performance characteristics to assess antimicrobial susceptibility tests (7). These guidelines indicate that CA should be >90%, ME should be <3% and VME should be ≤1.5%. 

The detection of Ampicillin-resistant Amikacin-sensitive E. coli, Ampicillin-resistant Amikacin-sensitive Klebsiella spp., and Clindamycin-resistant Gentamicin-sensitive Staphylococcus spp. with the VITEK 2 system was very accurate, Our results agreed with those reported in the few studies which have assessed the performance of the VITEK 2 system (1,2,3,4, and 5).In a recent multi-site study, the performance of the VITEK 2 GN was evaluated using 562 isolates of both commonly and rarely observed species of Gram-negative bacilli, including 153 non-fermentative strains. The reference identification was determined with api® 20 E and API 20 NE identification kits. Overall, the VITEK 2 GN correctly identified 96.8% of the isolates, including 6.4% low discrimination with the correct species listed. Misidentifications occurred at 3.0% and no identifications occurred at 0.2%. 

The database performance of the VITEK 2 BCL was evaluated using 1436 isolates of both commonly and rarely observed species of Gram-positive aerobic spore-forming bacilli. The reference identification was determined with the API 50 CHB identification kit and other conventional test methods. 

In a recent multi-site study, the performance of the VITEK 2 GP was evaluated using 457 isolates of both commonly and rarely observed species of Gram-positive cocci. The reference identification was determined with API STAPH and API 20 STREP identification kits. Overall, the VITEK 2 GP correctly identified 96.5% of the isolates, including 2.2% low discrimination with the correct species listed. Misidentifications occurred at 3.3% and no identifications occurred at 0.2%.

Furthermore, other studies showed that the performance of the VITEK 2 YST was evaluated using 623 isolates of both commonly and rarely observed species of yeast and yeast-like organisms. The reference identification was determined with API 20C AUX identification kits. Overall, the VITEK 2 YST correctly identified 98.9% of the isolates, including 11.7% low discrimination with the correct species listed. Misidentifications occurred at 0.6% and no identifications occurred at 0.5%.

Overall, the VITEK 2 BCL correctly identified 96.1% of the isolates, including 9.3% low discrimination with the correct species listed. Misidentifications occurred at 2.6% and no identifications occurred at 1.3%

This study validates that:

1. The VITEK 2 system is an easy-to-use system that provides a rapid (4 to 15 h) and accurate means for identification and AST of the most commonly isolated species.
2. The VITEK 2 system allows the reliable identification of UTI pathogens directly from urine samples, especially in cases of Gram-negative bacterial infections. 
3. Using VITEK 2 is required to enhance the performance of the hospital’s microbiology laboratory which is essential for accurate diagnosis and prompt effective treatment of bloodstream infections.
4. Amikacin and Gentamicin reflect high efficiency in reducing and eliminating the bacterial infections of E. coli, Staphylococcus spp., Proteus, and Klebsiella of UTI cases. 

Funding: No funding sources.

Conflict of interest: None declared.

Ethical approval: The study was approved by the Institutional Ethics Committee of Kut University College.

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