Characteristics of Trauma Patients with Multidrug-resistant Bacteria from an Epidemiological, Clinical, and Microbiological Perspective

Abstract Trauma, healthcare-associated infections, and antimicrobial resistance are three major public health issues worldwide due to the high probability of patient morbidity and, ultimately, a tragic outcome. In the current study included all patients admitted to The NRI Medical College Hospital, (Mangalagiri, Andhra Pradesh, India) intensive care units between October 20, 2022 and April 20, 2023. Patients with first bacteremia were found to have MRSA, VRE, and carbapenem-resistant Gram-negative bacteria. In terms of demographic and clinical data, patients with MR bacteremia in the general and trauma cohorts were compared to patients without MR bacteremia. PCR was used to detect carbapenemases and the SCCmec gene in the strains. We included 1528 patients, 302 of whom had trauma and 66 (4.3%) of whom had MR-agent-induced bacteremia. Mechanical ventilation, renal replacement therapy, and surgery were all independent risk factors for MR agent bacteremia. In the trauma cohort, haemodialysis alone was associated with an increased risk of MR agent bacteremia. S. aureus, MRSA (n=27), and K. pneumoniae (n=26) were the two most common isolating agents. The blaKPC gene was found in 83% of the K. pneumoniae strains (while the NDM gene was found in only one). Injuries have no effect on the development of bacteremia caused by MR agents. Certain risk factors appear to be associated with the severity of MR bacteremia cases.


Introduction
Trauma is defined medically as acute overexposure to any form of energy (mechanical, thermal, electrical, chemical, or radiation) in excess of the threshold of physiologic tolerance, resulting in severe organic level injury (Nauth et al., 2021).
Knowing that up to 25% of patients who suffer polytrauma from traffic accidents have a fatal outcome due to inadequate management, the health system and its members must be prepared to handle this type of situation, have knowledge, tools, process equipment for proper damage control when assisting a patient who has suffered a polytrauma and thus be able to offer them better chances of survival (Weihs et al., 2022). There are 13-15 accidental deaths in India per hour. The number of individuals killed in RTAs in India in 2011roughly 142,485-was the most it had ever been anywhere in the globe. Based on current trends, it is estimated that the number of lives lost on the world's roads would rise by almost 66% in the next two decades. The rate is only about 28% in high-income and industrialised countries, but it's risen by over 92% in China and 147% in India due to deaths caused by road traffic accidents. In 2018, there were a reported 4,67,044 traffic accidents in the 50 states and 7 union territories (UTs). By 2024, the RTA fatality rate could reach 2/10,000 in low-income nations, while it is expected to drop in high-income countries. By 2020, it is expected that road traffic injuries in India would account for 150% more deaths and hospitalisations than they do today. Although trauma injuries are widely established in terms of their nature, their aetiology remains a mystery to many. The evidence on the prevalence of trauma in underdeveloped nations is limited, as there are few studies on the topic. (Kashid et al., 2020) Microorganisms that are resistant to multiple kinds of antibiotics are said to be multiresistant (MMR) from an epidemiological standpoint. The idea of multiresistant bacteria may have different nuances depending on whether one takes a clinical, microbiological, or epidemiological stance. To be more precise, Gram-negative bacteria (GNB) are considered multiresistant if they show resistance to three or more classes of antibiotics to which they typically respond favourably. These classes include beta-lactams (penicillin and cephalosporins), carbapenems, aminoglycosides, and quinolones (Huang et al., 2019).
In 2019, the results of the SENTRY (Shortridge et al., 2019) antimicrobial surveillance program, were published. Over 20 years, 264,901 strains were isolated from more than 200 medical centers located in 45 countries. The frequency of Enterobacteriaceae classified as MDR increased from 6.2% between 1997 and 2000 to 15.8% between 2013 and 2016, mainly among hospital isolates. The frequency of these agents also varies by region, with the highest rates observed in Latin America (28.1%). Recently, there has been an increase in the number of publications of cohorts of patients transferred from wars in Syria, Lebanon, Afghanistan and Iraq to support hospitals located in European countries (notably, Germany). Colonization and infection rates by MDR agents, mainly carbapenem-resistant Enterobacteriaceae (ERC), were published, and their resistance mechanism was identified through molecular biology techniques and, in some cases, genetic sequencing. Through a prospective cohort of patients admitted to the ICU, trauma victims or not, with epidemiological and clinical data collection, it would be possible to identify risk factors for bacteremia by MDR agents. Unlike the other studies presented in the introduction, a cohort of trauma patients in an urban area would be formed. In addition, resistance mechanisms would be identified through molecular biology and genetic sequencing techniques in order to assess their clonality. We believe that the study could be justified by the scarcity of data on factors associated with bacteremia by MDR agents in a population of critically ill patients and trauma victims in Indian urban area. The current study aims to Identify risk factors for bacteremia by MDR agents (bloodstream infection by carbapenem-resistant Enterobacteriaceae, methicillin-resistant S. aureus, vancomycin-resistant Enterococcus sp; carbapenem-resistant P. aeruginosa, and carbapenem-resistant A. baumannii) in the population of patients hospitalized in the Intensive Care Unit for trauma and in the general cohort and to identify the epidemiological and clinical characteristics of the population hospitalized in the ICU regarding bacteremia caused by MDR agents.

Materials and Methods: 2.1 Location and Sampling
The NRI Medical College, Hospital has 1170 beds, 87 of which are adult intensive care units. There are two ICUs in the service, which are geographically separated and have different medical and nursing teams. Admission to the ICU is determined by availability (no distinction is made between clinical and surgical patients). The study will include all patients who are newly admitted to either of the two intensive care units at NRI Medical College, General Hospital in Guntur between October 20, 2022 and April 20, 2023. The exclusion criteria will be this patient's readmission to any ICU during this hospitalization; the patient being considered unknown (no date of birth recorded) until the moment of its outcome; and the patient having an ICU stay of less than 24 hours.

Collection of Microbiological Data
Clinical samples: The bacterial identification of the strains and their respective sensitivity tests were performed using an automated method (bioMeuriex Vitek 2, Hazelwood, MO, USA) and the cutoff points of the Clinical and Laboratory Standards Institute (2015) were used.
Any Enterobacteriaceae that showed resistance to at least one of the tested carbapenems would be considered: ertapenem, meropenem or imipenem. The isolates were considered resistant to meropenem or/and imipenem with MIC ≥ 4 µg/dl and/or to ertapenem with MIC ≥ 2 µg/dl. As for non-fermenting gram negative bacteria, isolates whose MIC for these drugs was > 4 µg/dl were considered resistant to meropenem or/and imipenem. As for the S. aureus isolates, they were considered resistant if the MIC for oxacillin was ≥ 4 µg/dl. Vancomycin resistance in enterococci was defined when the MIC for vancomycin was ISSN: 2583-7283 Volume 1 (2): 2023 https://tamjed.com equal to or greater than 32 µg/ml. All isolates that met the above criteria were sent to the Laboratory of Medical Investigation of Bacteriology, NRI General Hospital of NRI medical College, where tests were performed to detect the genes described below. The isolates were stored in BHI (brain heart infusion)glycerol at -70º. Variables collected from all patients included in the study: Using a standardized instrument, the following data were collected: age, gender, reason for admission to the ICU, previous diagnoses of hypertension (SAH), diabetes (DM), neoplasia, other comorbidity (smoking or alcohol consumption were not considered) , severity score Simplified Acute Physiology Score (SAPS III) calculated at admission to the ICU by a nurse hired for this purpose, presence of invasive devices (indwelling urinary catheter, mechanical ventilation, central venous access, catheter for haemodialysis), time of use of the devices, surgical intervention, length of stay in the ICU ((Falcone, 2016), antimicrobials prescribed and time of use, total length of stay in the institution and outcome.
Regarding trauma patients, the following data were collected: type of trauma (classified as ATT, physical aggression, unintentional event, occupational accident, attempted self-extermination, fall to the ground and fall from another height); trauma severity (measured by the Revised Trauma Score-RTS) (Dkhar et al., 2019); involvement of the segments (cranioencephalic/face injury; abdominal injury; thoracic injury; injury to the extremities and pelvis) and open fracture of the skull or long bones. As for ATT, victims were classified as occupants of cars, motorcycles, buses or trucks and cyclists or victims of being run over. As for physical aggression, whether there was beating, use of a firearm or bladed weapon. Unintentional events included accidents during leisure time.
Variables collected from patients diagnosed with MDR agent bacteraemia: MDR agents were defined as those resistant to at least one agent from three or more antimicrobial classes. Despite the definition, we chose to consider enterobacteria resistant to at least one of the carbapenems, P. aeruginosa and carbapenem-resistant A. baumannii, methicillinresistant S. aureus and vancomycin-resistant Enterococcus sp. Bacteremia was defined as growth in blood culture collected from a patient admitted to the ICU of one of the aforementioned organisms (only the first positive blood culture of the patient at that hospitalization was considered). MDR etiologic agents were defined as carbapenem-resistant enterobacteria, carbapenemresistant P. aeruginosa, Acinetobacter baumannii resistant to more than three classes of antimicrobials, MRSA, and vancomycin-resistant Enterococcus spp., and only the initial episode of bacteremia was considered. The presence of one of these organisms in a blood culture was considered bacteremia, and the first time a blood culture was taken was considered the commencement of bacteremia. The sites of infection were defined according to the criteria used for epidemiological surveillance of HAI recommended by the National Healthcare Safety Network (NHSN).

Detection of genes encoding KPC, OXA-48 and NDM through polymerase chain reaction (PCR):
In-house PCR was performed to identify KPC, OXA-48 and NDM genes in all enterobacterial isolates. First, the bacterial lysate was made by thermal lysis, 15 minutes at 95ºC and immediate centrifugation at 13,000 rpm for 15 minutes at 4ºC. The supernatant was separated and kept at -20°C until use. Multiplex PCR for blaKPC, blaNDM and blaOXA-48 was performed according to the technique already described by Poirel et al., (2011).
The primers used to detect the genes encoding KPC, OXA-48 and NDM are described below in Table 1: Detection of carbapenemase production through phenotypic methodology: Enterobacteriaceae isolates were also submitted to a colorimetric assay to detect carbapenemases. In this case, Carbapenembac® (Probac, India) was used.
Assays were performed according to the manufacturer's instructions. The isolates were placed in a Petri dish with the Carbapenembac tape together with 150 µl of a suspension with turbidity equal to 10 on the McFarland scale of the selective solution. The plate was incubated between 35º and 37º for 60 minutes. Subsequently, 200 µl of the special iodine solution was placed on the tape. Reading was performed after 30 minutes.
Detection of genes encoding SCCmec from strains of MRSA: SCCmec type characterization was performed on all samples identified as MRSA. For molecular characterization of SCCmec, the multiplex polymerase chain reaction (m-PCR) method was used, as described by Zhang et al., (2005). The primers used in these reactions are described in Table 2 below.

R ATGCAATATATAAGGATGCTGG
Analysis of the clonality of MRSA strains: For clonality analysis, the MRSA isolates were subjected to pulsed field gel electrophoresis (PFGE) as described above.

Statistical analysis
Bivariate analyses were performed to assess factors associated with MDR bacteremia, bacteremia in trauma patients, carbapenem-resistant K. pneumoniae bacteremia, and MRSA bacteremia. For dichotomous variables, relative risk was determined with a confidence interval of 95%. For continuous variables, t test was used. P values less than 0.05 were considered significant.

Clinical and epidemiological characteristics of the cohort:
From October 20, 2022 and April 20, 2023, 1621 patients were admitted to NRI General Hospital's two Intensive Care Units. 86 were omitted because they were admitted for the second time; 6 because they were unknown (no identification and no date of birth); and 3 because they stayed in this sector for less than 24 hours. As a result, 1528 patients were included in the research. Table 3 describes the clinical and epidemiological features of the patients who took part in the study. The diagnosis of MDR agent bacteremia was made in 63 patients and 69 strains were isolated. Two patients were diagnosed with co-infection: the first with bloodstream infection by S. aureus and A. baumannii and the second by M. morgannii, S. aureus and K. pneumoniae.
The third site of bacteremia was attributed to nosocomial pneumonia (n = 7), whose main agents were K. pneumoniae (n = 3) and P. aeruginosa (n = 2). Urinary tract infection was responsible for two cases of bacteremia and the etiologic agent responsible was K. pneumoniae.

Clinical and epidemiological characteristics of the trauma cohort
Among the patients diagnosed with trauma (n=302), there was a predominance of males (77.15%) and 88 patients were aged over 60 years (29.14%). The main causes of trauma are shown in Graph 1 and the classification of RTA in Graph 2. The outcomes of each type of injury are shown in table. Next, Table 4 shows the clinical characteristics of the trauma cohort.   In-hospital mortality of patients diagnosed with trauma is lower than that of patients without trauma (30% vs. 43%; RR 0.81; 95% CI 0.74 -0.88; p < 0.0001).

Results of bivariate analysis between patients with and without MDR agent bacteremia
The overall features of the group of patients admitted to the ICU with MDR etiologic agent infection and those without MDR infection are shown below (Table 6).  In the bivariate analysis, open fracture, use of mechanical ventilation, use of renal replacement therapy, use of antimicrobials and surgical procedure are associated with MDR bacteremia in trauma patients.

Results of the bivariate analysis of factors associated with MRSA bacteremia in the cohort of 1,528 patients
We chose to analyse the risk factors associated with MRSA bacteremia in the cohort of critically ill patients, since it was the most prevalent etiologic agent of bacteremia The results were as follows (Table 8). submission to renal replacement therapy, and use of antimicrobials were associated with MRSA bacteremia.

Results of the bivariate analysis of factors associated with K. pneumoniae bacteremia in the cohort of 1,528 patients
As it is the second most prevalent agent in our cohort, we chose to assess factors associated with carbapenem-resistant K. pneumoniae bacteremia The results were as follows (Table 9):  Mechanical ventilation is required when there is reduced level of consciousness (eg, TBI and/or hypovolemic shock).

MRSA cohort
In our cohort of 1528 patients, the frequency of bacteremia

Conclusion
In a sample of 1528 patients, the use of mechanical ventilation, renal replacement treatment, and surgical procedure were all independently associated with an increased risk of MDR bacteremia. Trauma did not appear to be a risk factor for MDR bacteremia. In this cohort, the prevalence of MDR bacteremia was 4.3% (n = 62), and the mortality rate was 75%. MRSA (28 strains) and K.
pneumoniae (26 strains) were the most common MDR agents in this cohort. All MRSA strains tested positive for the mecA