1. IntroductionInfections due to resistant Gram-negative bacteria (GNB) have emerged as a global public-health problem [1,2]. The impact on lives lost and costs for the health systems have been extensively studied in the medical literature [3]. Up to 40% of patients diagnosed with sepsis and septic shock due to multi-drug resistant germs die due to these infections. Additionally, survivors have prolonged hospital stays, with annual costs for the health system estimated at USD 16.7 billion in the United States alone [4,5]. Early antibiotic therapy improves the survival of patients with sepsis and bloodstream infections caused by GNB [6,7,8,9,10,11,12]. Still, the emergence of antibiotic resistance poses a growing challenge to achieving this objective. Owing to their genetic plasticity, GNBs rely on various resistance mechanisms that allow them to respond to a wide range of environmental threats; of these mechanisms, the main and most versatile is the production of extended-spectrum β-lactamases (ESBL) [13]. The global expansion of ESBLs and carbapenemases is an unprecedented event favored by the high-mobility conditions of the population [14] and the non-prudent use of antibiotics in the food and healthcare industries, among other factors [15,16].This problem has significantly affected Latin America [17,18]. Irrespective of the type of β-lactamase involved, epidemiological surveillance studies have shown a growing prevalence of Enterobacterials resistance to oxyimino-cephalosporins in Latin-American hospitals, which has led to the massive use of carbapenems with the resulting emergence of isolates-resistant to these agents [19]. In Colombia, GNBs resistant to carbapenems are endemic. Colombia was the first Latin-American country to deliver isolate producers of Klebsiella pneumoniae carbapenemase (KPC) [20] and the first country in the world to identify isolates of KPC-producing Pseudomonas aeruginosa [21]. Equally, the national epidemiological surveillance has detected an endemic circulation of various carbapenemase types (i.e., OXA, NDM, and VIM) [22,23].The use of conventional methods, such as cultures in solid media and subsequent identification through biochemical tests by automated systems, allow the differentiation of ESBL- and carbapenemase-producing Gram-negative bacilli, but the time required can be up to 24 h after obtaining the isolates in culture and up to an additional 48 h for phenotypic confirmation tests, making them very time-consuming methods. However, rapid techniques have recently been evaluated to detect multi-drug resistant GNB in blood culture samples [24]. In addition to molecular methods [25,26], biophysical techniques such as mass spectrometry, flow cytometry, laser nephelometry, immunochromatography, chemiluminescence, and bioluminescence are currently available [27]. Implementing these techniques leads to decreased hospital stays, ICU stays, costs, and mortality rates [28]. However, there are limitations, such as their high cost, the use of physical space, and the need for trained staff. More importantly, these technologies are not available in every hospital worldwide.Unlike molecular methods, laser nephelometry (HB&L® Alifax, Italy) offers excellent diagnostic performance at a lower cost. They have various uses, including sifting strategies, quantitative cultures, and antimicrobial susceptibility determination. The most significant contribution in terms of antibiotic resistance has been implementing this technology as an active surveillance strategy for the early identification of multi-drug resistant GNB carriers in rectal swabs [29,30,31]. Its use in blood samples has been limited [32,33]. We hypothesize that this technique is efficient for rapidly detecting multi-drug-resistant GNB in patients with bacteremia. Therefore, the objective of this study is to evaluate the operational characteristics resulting from a new modified protocol designed to detect Gram-negative bacteria producing carbapenemases and extended-spectrum β-lactamases from positive blood cultures using HB&L® laser nephelometry, compared with the reference standard. 2. Materials and Methods

This is a prospective cohort observational study of diagnostic tests in which the operational characteristics are evaluated, in addition to the degree of concordance and the diagnostic time resulting from the combination of the Bact/Alert 3D®, HB&L® laser nephelometry, and Vitek2® techniques compared with the reference standard in the development of a new protocol for the direct assembly of positive blood cultures in a high-complexity hospital in the city of Bogota, Colombia. The research protocol was approved by the Shaio Clinic Foundation’s Ethics and Research Committee (Memorandum of Approval No. 273). Obtaining informed consent was unnecessary because of the absence of direct intervention in patients and the observational characteristics of the study.

The HB&L® equipment (Alifax, Polverara, Italy) is designed to perform quantitative kinetic counting in colony-forming units per milliliter (CFU/mL) in urine samples, biological fluids, and rectal swabs by laser nephelometry methodology. For the detection of carbapenemases and ESBL in rectal swabs, it has kits which contain a reagent composed of a mixture of antibiotics, including vancomycin; an antifungal; and carbapenems to eliminate the accompanying microbiota in the rectal samples, and allow the growth of GNB resistant to carbapenems. In our study, under our design, we apply this kit to detect carbapenemases and ESBL in positive-blood-culture bottles and evaluate their performance.

2.1. Sample Processing

All blood cultures sent to the microbiology laboratory from 1 July 2017 to 31 March 2019 were collected consecutively and prospectively. All positive blood cultures with GNB identified via direct Gram-stain microscopy were included. The only exclusion criterion in our study was blood cultures in which the presence of Gram-positive cocci, Gram-positive bacilli, and yeast was observed.

2.1.1. Setting up Blood Cultures for Laser Nephelometry Using HB&L-ESBL/AmpC® and HB&L-Carbapenemase® Kits (New Protocol)With the help of a syringe, a blood sample was taken from the positive-blood-culture bottle, and two drops of blood were released into a plastic tube with 2 mL of 0.9% saline solution and mixed using a vortex. Based on this prepared suspension, 200 μL was released into the green-lidded vial from the HB&L-ESBL/AmpC® kit, together with 200 μL of the reagent from the kit and another 200 μL into the red-lidded vial from the HB&L® Carbapenemase kit with 200 μL of the specific reagent (including vancomycin, an antifungal, and carbapenems); the vials were then deposited in the automated HB&L® system in the pre-established programs ESBL and carbapenemase, respectively. The results were subsequently read after 6 h (Figure 1). 2.1.2. Setting up the Tests through the Conventional Method

Culturing was performed through the conventional method in solid culture mediums. For this, a positive blood-culture sample was extracted, releasing a drop into blood agar and a drop into MacConkey agar. Subsequently, culturing was performed by dropping using a handle, and cultures were incubated at 37 °C for 24 h. The following day, colonies were subjected to biochemical identification and susceptibility testing using the Vitek2® automated system (Biomerieux®) with GNB and AST-272 cards.

2.1.3. Phenotypic Tests for Confirmation of ESBL, AmpC, and Carbapenemase-Producing GNBIsolates with antimicrobial susceptibility test with MICs greater than or equal to 1 ug/mL for ertapenem or greater than or equal to 2 ug/mL for imipenem or meropenem (according to CLSI 2020) [34], underwent confirmatory tests for the detection of carbapenemase production, such as the Hodge test, synergy tests with EDTA disks, and synergy tests with boronic acid disks.For the confirmation of ESBL and AmpC, the double-disc test with third-generation cephalosporin discs combined with clavulanic acid was used as the reference method, according to CLSI standards [34]. In the study period of this investigation, the Hodge test was the method used to confirm the presence or absence of carbapenemases, and synergy tests with EDTA discs and boronic-acid discs were used to differentiate the type of carbapenemase present (serine or metallobetalactamase). 2.1.4. Test Controls

The following control strains were used in setting up each test: K. pneumoniae ATCC BAA 1705 (blaKPC+), K. pneumoniae ATCC BAA 2146 (blaNDM+), ESBL-producing K. pneumoniae ATCC 700603, Escherichia coli ATCC 25922, and P. aeruginosa ATCC 27853.

2.2. Statistical Analysis

The operational characteristics were analyzed separately for each test (HB&L-ESBL/AmpC® and HB&L-Carbapenemase® kits, Alifax, Padua, Italy). Kappa coefficient, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios (LR) with 95% confidence intervals (CI) were calculated. In addition, the positivity time (identification of multidrug-resistant BGN) was evaluated using the proposed identification protocol and the diagnosis time used by the conventional method. Data were analyzed using SPSS 28, IBM, USA.

4. DiscussionOur new protocol for the rapid detection of ESBL and carbapenemases demonstrated high sensitivity, which is why it can be used in blood-culture samples with excellent results. A 42-h reduction was noted in identifying carbapenem and oxyimino-cephalosporin resistance in GNB from blood cultures (Figure 3). The reduction in time positively impacts two main objectives: (1) initiation of targeted antibiotic therapy according to the type of resistance identified and (2) reduction in direct hospitalization costs by having these results in less time. Additionally, it was found that compared with the conventional method, the combination of the Bact/Alert 3D®, HB&L®, and Vitek2® techniques resulted in a concordance of 100% for the early detection of carbapenemase and a concordance of 95% in the detection of phenotypic resistance to oxyimino-cephalosporins (Table 3). This time saved can positively impact the clinical outcomes of such patients.The early initiation of effective antibiotic therapy predicts the outcome of bacteremia due to GNB [6,8,9,10,11,12]. Tumbarello et al. [8] showed that the initiation of inappropriate antibiotic treatments was a strong predictor of mortality in patients with bacteremia due to ESBL-producing GNB (59.5% vs. 18.5%; OR: 2.28; 95% CI: 1.76–3.22; pp = 0.02) [12]. Kang et al. reported an 11% reduction in the total crude mortality when adequate empiric antibiotic treatment was administered [6]. Likewise, the INCREMENT cohort reported a 22% increase in mortality resulting from the initiation of inappropriate empiric therapy [11]. Therefore, it is evident that tests are necessary to detect multi-drug-resistant germs to ensure adequate antibiotic treatment rapidly.In various studies, rapid detection tests showed a reduction in hospital and ICU stays, mortality rate, and costs [25,26,27]. Perez et al. incorporated the rapid identification of pathogens through matrix-assisted laser desorption/ionization time (MALDI-TOF) and susceptibility through BD PhoenixTM, with a decrease by more than 50% in the microbiological identification times (40.6 vs. 14.5 h; ppp = 0.001) and ICU stay (16 vs. 10.7 days; p = 0.008), and 30-day mortality (21% vs. 8.9%; p = 0.01) [28]. Sakarikou et al. also used MALDI-TOF for identification purposes as well as VITEK-2® for susceptibility tests in blood-culture samples with GNB. In this case, the sample was taken directly from the blood culture without moving it to a solid medium, saving 8 h compared with the conventional method (5 vs. 11 h; p ≤ 0.001, without taking time for blood-culture positivity into account) and with a concordance of 98.5% [35]. This evidence confirms that identifying multi-drug resistant bacteria early makes it possible to provide a rapid, targeted therapy and improve clinical outcomes. Unfortunately, the MALDI-TOF availability is limited to a few hospitals owing to the high initial installation cost.PCR-based techniques conducted microbiological identification in 1–2 h. However, antibiotic susceptibility is limited to the genes included in each panel, such as the Film Array® platform (Biofire®, Salt Lake City, UT, USA), where the BCID 1 or 2 panel is still used in some Latin-American countries, detects the presence of different carbapenemase genes. Additionally, in some of these techniques, a decreased efficiency is observed in polymicrobial infections, thereby making the combination of additional techniques necessary [26,27,28]. On the other hand, the Bact/Alert 3D®, VITEK®, and HB&L® techniques are less costly and do not require specialized training, resulting in easy implementation in hospitals. Studies such as those conducted by Hogan and Höring [36,37] on GNB-positive blood culture involved direct inoculation in evaluating susceptibility through VITEK-2®; the concordance with the conventional method was over 95% in both studies. However, false susceptibilities to carbapenemases were documented, constituting a major error. Recently, Athamna et al. used Uro4 HB&LTM laser nephelometry, compared with VITEK-2®, in their study on ESBL/AmpC susceptibility in Enterobacterials, with 91.3% concordance. However, carbapenemase identification was not included, and for P. mirabilis, the concordance was only 58.3% with zero sensitivity [32]. Other studies were performed by rapid incubation, such as the EUCAST RAST test, which uses discs of different antibiotics in short incubation times (4 to 6 h) [38].Currently, there are new Rapid ESBL NP® colorimetric tests (Liofilchem, Italy) based on the hydrolysis of cefotaxime which detect the presence of ESBL very quickly, but to perform them directly from positive blood cultures requires additional kits which allow obtaining the bacterial sediment but increase the cost of testing and further steps in assembly [39]. Additionally, the immunochromatography tests for ESBL and carbapenemases, CARBA 5 and NG-Test® CTX-M (NG-Test®, Guipry, France) are performed from colonies of the isolates in solid culture [40] and have a higher cost than the laser nephelometry tests (USD 14 versus USD 6 respectively).In contrast, our study integrated the Bact/Alert 3D®, VITEK®, and HB&L® techniques in GNB-positive blood cultures, with direct inoculation using both kits (ESBL/AmpC® and Carbapenemase®) (Figure 1). Identification time and susceptibility decreased by 42 h compared with the conventional methodology, which involves a significant decrease in the identification times which could potentially improve the clinical outcomes of patients with bacteremia due to GNB. Occasionally, the efficiency was 100% in all operational characteristics using the HB&L Carbapenemase® kit (Table 2), including the identification of serine carbapenemases and Metallo-β-lactamases. Thus, this study opens the way for new low-cost strategies aimed at rapidly detecting these pathogens; however, it must be assessed whether the routine use of this protocol impacts the clinical outcomes of patients with bacteremia.The strengths and weaknesses of our study are noteworthy. Despite being a unicentric study, the incidence of antibiotic resistance resembles that at the international level; thus, similar endemic populations with multi-drug resistance could benefit from this methodology and the results presented in this manuscript. Two other important limitations are the small number of samples included in the study and the lack of genomic sequencing or PCR for the molecular identification of ESBL, AmpC, and carbapenemases. Concerning cost, the systematic use of HB&L® for all GNB-positive blood cultures could initially have a negative impact; however, it could be considered cheap when comparing it with alternative quick tests and evaluating the impact of the optimal initiation of therapy in terms of other cost-effective scenarios such as hospital stay, ICU stay, antibiotic savings, and mortality rate. However, the assessment of this test’s economic and clinical impacts was out of this study’s scope and needs further study in other research. The phenotypic diagnosis led to limitations owing to the presence of combinations in increasing resistance mechanisms; however, in these limited-resource scenarios, we believe this methodology is one of the best diagnostic options. Moreover, it provides another fast diagnostic opportunity using concentrated biomass in the HB&L® equipment to apply other methods, such as immunochromatography or lateral flow for detecting carbapenemases and ESBL, as demonstrated in recent studies of direct protocols with good sensitivity and specificity [40].