Hospital-acquired infections, also known as nosocomial infections (NIs), by gram-negative bacteria are a major threat to health worldwide ( Karaiskos & Giamarellou, 2014). These bacteria are consistently evolving and developing novel mechanisms of resistance to commonly used antibiotics, especially when these antibiotics are overused, which generates a selection pressure ( Peleg & Hooper, 2010). Increasingly, non-lactose-fermenting gram-negative bacilli (NLFGNB) have become significant pathogens in healthcare facilities ( Akbar et al., 2014; Hiltunen et al., 2017). The public health risk of NLFGNB is growing rapidly worldwide; consequently, warnings of the disturbing spread of anti-infection-resistant microorganisms causing NIs, which have cost lives and resources, have also risen ( Djordjevic et al., 2017; Leski et al., 2016; Ranjbar et al., 2018).

The over-prescription and use of antimicrobial agents may be playing a major role in the escalating number of NIs ( Mataseje et al., 2016; Prasert et al., 2020; Rabirad et al., 2014). Bacteria are identified phenotypically and characterized by biochemical tests; however, these tests are not as accurate as required ( Bush & Jacoby, 2010). Advanced techniques in molecular biology and sequencing approaches are needed for the discovery, identification, and characterization of emerging and re-emerging pathogens ( Wilson et al., 2014). 16s rRNA gene sequencing offers a practical and reliable molecular identification method ( Brown-Elliott et al., 2006; Clarridge, 2004). 16s rRNA gene sequence analysis can better identify poorly described, recognize inadequately depicted, infrequently isolated, or phenotypically deviant strains, can be routinely utilized for identification of bacteria, and can prompt the acknowledgment of novel pathogens and non-cultured bacteria ( Case et al., 2007; Crosby & Criddle, 2003; Dahllöf et al., 2000; Reller et al., 2007). A significant function for 16s rRNA gene sequencing is to identify precisely assembled living organisms for additional study ( Goldenberger et al., 1997; Jalava et al., 1995). Gram-negative organisms are involved in both network- and emergency clinic-procured contaminations ( Mataseje et al., 2016; Prasert et al., 2020). NLFGNB infections cause a significant public health problem in hospitals, especially when they develop multi-drug resistance ( Kaye & Pogue, 2015; Lochan et al., 2017). These microorganisms can develop several mechanisms of resistance, including β-lactamase creation, overexpression of multi-drug efflux pumps, target site mutations, and reducing the permeability of the external membrane ( Djordjevic et al., 2017).

The correct identification of these bacteria by conventional microbiology methods is often limited by phenotypic misidentification ( AbdulWahab et al., 2015; Alby et al., 2013; Plongla et al., 2016). The spread of extended-spectrum β-lactamase (ESBL)-making strains restricted the range of antimicrobial agents that could be used to treat patients effectively and raised concerns for control of infections caused by NFGNB and limitation of treatment to prohibitively expensive antibiotics ( Manyahi et al., 2017; Ranjbar et al., 2018; Zhang et al., 2009). The mutation of TEM-1, TEM-2, and SHV-1 β-lactamases produced the ESBLs, which were discovered over 1980–1990 and first detected in Western Europe ( Bubpamala et al., 2018; Bush et al., 1995). DNA sequencing technologies offered a sensitive diagnostic tool that improved the detection, identification, and characterization of drug-resistant bacteria ( Livermore, 1995; Ranjbar et al., 2018).

NLFGNB is commonly associated with hospital-acquired infections; therefore, identification of the species and the study of antimicrobial susceptibility patterns are imperative. ESBL genes are the most common resistance genes associated with gram-negative bacilli. Thus, it is essential to determine both the prevalence of ESBL among NLFGNB and the recognizable proof of microscopic organisms at the species level, which is essential to determine the most effective treatment and best guide case management ( Bush et al., 1995; Livermore, 1995).

In Sudan, recognizable proof of NLFGNB at the species level is difficult to achieve by conventional methods. Alternative strategies, (e.g., API or VITEK) are not affordable; therefore, the current study was undertaken to provide more sensitive and accessible methods that require 16s rRNA to detect and confirm the bacterial pathogens separated from patient samples in Soba University Hospital in Khartoum, Sudan.

NLFGNB were previously considered to be contaminants but have now emerged as significant healthcare-associated pathogens ( Malini et al., 2009). Pseudomonas and Acinetobacter species are known to be the regular nosocomial pathogens ( Gales et al., 2001; Tortoli et al., 2001). In this study, the most common NLFGNB isolated was P. aeruginosa, followed by A. baumannii, which is similar to the results obtained by Sarkar et al. (2018) and Malini et al. (2009), who reported P. aeruginosa as the most common isolate, followed by A. baumannii ( Malini et al., 2009; Wauters & Vaneechoutte, 2015). Other NLFGNB, such as B. cepacian, S. maltophilia, and Stenotrophomonas pavanii vary from study to study, both in terms of their genera and prevalence. However, their role as opportunistic pathogens in immunocompromised and debilitated individuals has been invariably established ( Chawla et al., 2013). Strains isolated by biochemical methods were recorded a Pseudomonas spp., Coccobacilli, and other GNB. The sequencing test identified the isolates to many strains, such as P. aeruginosa, A. baumannii, B. cepacian, S. maltophilia, and other GNB . The prevalence of ESBLs in this study showed SHV to be the highest level of the total sample, followed by CTX-M, with TEM being the lowest of the resistance isolates. ESBLs are enzymes that demonstrated resistance to the third generation of cephalosporin. As these enzymes are plasmid-encoded, the spread of bacterial resistance disseminates rapidly ( Jena et al., 2017). ESBLs are responsible for resistance against β-lactam antibiotics, such as penicillin, cephalosporin, monobactams, and sometimes also carbapenems ( Cantón et al., 2008). Resistance of NLFGNB and multidrug resistance have risen widely according to numerous studies. B. cepacia is another NLFGNB that colonizes and infects patients with chronic bacteremia and UTI. It is known to cause infections in hospitalized patients and, once infected, is hard to eliminate ( Akbar et al., 2014; Chawla et al., 2013; Plongla et al., 2016).

In this study, the isolate of B. cepacian recorded 6.8% of the non-fermenters isolated from blood and urine culture. B. cepacian is known for its inherent resistance to numerous beta-lactam drugs, including aminoglycosides, colistin and polymyxin B, first-line therapeutics against serious pseudomonas infections ( Rahbar et al., 2010). This isolate showed maximum (100%) susceptibility to imipenem, in accordance with Sidhu et al. who also reported 100% susceptibility to imipenem. Similarly, in a study done by Grewal et al., (2017) and Gautam et al., (2009). B. cepacia isolates showed excellent susceptibility to imipenem and Meropenem.

Three strains of S. maltophilia were isolated during the study; it is now considered a common non-fermenter that causes infection in hospital settings ( Verweij et al., 1998). Our study has shown the isolation of this bacterium in 6.4% of isolates. Earlier A’Court and Garrard reported S. maltophilia to account for 5% of nosocomial pneumonias ( A’Court & Garrard, 1992). It showed 100% susceptibility to some of the antibiotics, notably ciprofloxacin. Similar results were obtained by Malini et al. (2009).

The majority of our isolates were resistant to ampicillin (98%), followed by cefotaxime (94.5%) then amoxicillin/clavulanate acid (90.1%). Resistance rates also rise in ciprofloxacin (50.1%), gentamicin (52.5%), and amikacin (22.3%). Imipenem and meropenem were the most effective antibiotics tested ( Malini et al., 2009; Rahbar et al., 2010), with 100% sensitivity to ampicillin, cefotaxime, and ciprofloxacin.

A phylogenetic tree for the most common NLFGNB shows the relationships among various species, with their phylogeny based on similarities and differences in their genetic characteristics.

This study covered this gap by using advanced molecular techniques to identify, characterize, and determine the spread of hospital-associated bacterial infections to permit infection control personnel to recognize potential sources of pathogens more effectively, and encourage physicians, healthcare workers, and stakeholders to develop treatment and control strategies against these rapidly evolving organisms.

In conclusion ,NLFGNB in communities and hospitals can help inform antibiotic treatment. This study showed a significantly higher prevalence of P. aeruginosa and A. baumannii among NLFGNB associated with hospital infections. The isolation of MDR ( P. aeruginosa) and MDR ( A. baumannii) in the present study raises the concern of rapidly emerging antibiotic resistance in this group of bacteria in Sudan. Proper screening of non-fermenters in nosocomial settings and regular assessment of their antibiotic susceptibility profiles are suggested for effective management of infections and limitation of the emergence of multidrug resistance. Assessment of 16s rRNA gene sequences permits bacterial identification that is stronger, more reproducible and more exact than that obtained by phenotypic testing. Thus, when ordinary methods do not yield an excellent (or very good) species identification, non-fermenter GNB should be exposed to 16s rRNA gene sequencing if adequate species assignment is of concern. Since the spread of MDR and ESBL-producing NLFGNB isolates reduces treatment options and increases hospital cost, it is important to keep up to date on prevailing resistant patterns of any locality, which will help determine a suitable antimicrobial therapy.