Isolation of Carbapenem and Colistin Resistant Gram-Negative Bacteria Colonizing Immunocompromised SARS-CoV-2 Patients Admitted to Some Libyan Hospitals

ABSTRACT The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating effect, globally. We describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing SARS-CoV-2 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of SARS-CoV-2 in the eastern part of Libya. In total, at first, 109 samples were collected from 43 patients, with the samples being recovered from oral (n = 35), nasal (n = 45), and rectal (n = 29) cavities. Strain identification was performed via matrix assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic susceptibility testing was carried out on Mueller-Hinton agar, using the standard disk diffusion method. MIC determination was confirmed via E-TEST and microdilution standard methods. A molecular study was carried out to characterize the carbapenem and colistin resistance in Gram-negative bacterial strains. All of the positive results were confirmed via sequencing. Klebsiella pneumoniae (n = 32), Citrobacter freundii (n = 21), Escherichia coli (n = 7), and Acinetobacter baumannii (n = 21) were the predominant isolated bacteria. Gram-negative isolates were multidrug-resistant and carried different carbapenem resistance-associated genes, including NDM-1 (56/119; 47.05%), OXA-48 (15/119; 12.60%), OXA-23 (19/119; 15.96%), VIM (10/119; 8.40%), and the colistin resistance mobile gene mcr-1 (4/119; 3.36%). The overuse of antimicrobials, particularly carbapenem antibiotics, during the SARS-CoV-2 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae, A. baumannii, and colistin-resistant E. coli strains. Increased surveillance as well as the rational use of carbapenem antibiotics and, recently, colistin are required to reduce the propagation of multidrug-resistant strains and to optimally maintain the efficacy of these antibiotics. IMPORTANCE In this work, we describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing COVID-19 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of COVID-19 in the eastern part of Libya. Our results confirmed that the overuse of antimicrobials, such as carbapenem antibiotics, during the COVID-19 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae and A. baumannii, as well as colistin resistance.

In the current study, we focused on the strains identified on MacConkey agar with ertapenem and LBJMR media. Identification via MALDI-TOF showed that these strains were as follows: Enterobacterales ). The non-Enterobacterales (n = 46) are distributed as below: A. baumannii (n = 21), P. aeruginosa (n = 17), Stentrophomonas maltophilia (n = 5), P. guariconensis (n = 2), and P. entomophila (n = 1). The distribution of the strains is illustrated in Fig. 1, and the clinical and isolate information are summarized (data not shown).
For C. freundii, resistance to gentamicin and amikacin was reported to be 47.6% and 42.85%, respectively, whereas resistance to quinolones and sulfonamides was reported to be 66.66% and 38.09% of the strains. However, fosfomycin and nitrofurans were the most active antibiotics on the tested strains (4.76%). For other species, resistance was mainly assigned to quinolones, aminoglycosides, sulfamides, nitrofurans, and fosfomycin. Doxycyclin and colistin seem to be the most effective molecules against these pathogens.
The imipenem E-TEST showed that all K. pneumoniae were resistant to imipenem, with MICs ranging from 3 mg/L to .32 mg/L, and ertapenem, with MICs ranging from 1.5 mg/L to .32 mg/L. 13 strains had a MIC of .32 mg/L for imipenem, and 24 strains had a MIC of .32 mg/L for ertapenem. Concerning C. freundii, the imipenem E-TEST revealed that strains were highly resistant to ertapenem, with MICs ranging from 1 mg/L to .32 mg/L, and to imipenem, with MICs ranging from 2 mg/L to .32 mg/L.
The less spread strains (K. oxytoca [n = 5], E. coli [n = 8], and R. ornithinolytica [n = 3]) were mainly resistant to quinolones, aminoglycosides, sulfamides, and fosfomycin. The imipenem ETEST showed that these strains were resistant, with MICs ranging from 1.5 mg/L to .32 mg/L, and they all remained resistant to ertapenem, with MICs ranging from 0.5 mg/ L to .32 mg/L. In addition, the nonfermenting GNB isolates showed high resistance to Gram-Negative Bacteria Colonizing SARS COV-2 Patients Microbiology Spectrum beta-lactams, particularly A. baumannii. These strains also exhibited resistance to other antibiotic families, such as quinolone, sulfamides, and aminoglycosides, with 100% resistance to each. Colistin and fosfomycin were the most effective antibiotics. Moreover, all strains were resistant to carbapenem drugs, namely, imipenem and meropenem, and E-TEST showed that all strains were highly resistant, with MICs of .32 mg/L for both drugs. Furthermore, all strains of P. aeruginosa were sensitive to imipenem and meropenem, and only two strains of P. guariconensis were extremely resistant. The phenotypic test results are mentioned in Fig. 3. Molecular analyses. (i) Molecular mechanisms of carbapenem resistance in Enterobacterales. Of the 76 ertapenem-resistant Enterobacterales, 65 strains harbored at least one of the tested carbapenemase encoding genes. The NDM metalloenzyme was the most prevalent carbapenemase, and it was identified in 56 strains, mainly in K. pneumoniae (n = 29), which was followed by C. freundii (n = 21), R. ornithinolytica (n = 3), C. youngae (n = 2), and K. oxytoca (n = 1). OXA-48 was detected in 15 strains, mostly in K. pneumoniae (n = 11), K. oxytoca (n = 3), and C. freundii (n = 1). 4 OXA-48 positive strains remained susceptible to imipenem (MICs of #2 mg/L). The VIM enzyme was detected in 7 strains. None of the strains harbored the IMP and KPC genes. On the other hand, 11 strains harbored at least two carbapenemase enzymes with three combinations, as follows: bla NDM 1 bla OXA-48 , bla NDM 1 VIM , and bla NDM 1 bla OXA-48 1bla VIM . By sequencing the NDM positive strains, three different variants were identified, namely, NDM-1, NDM-2, and NDM-5. On the other hand, from the VIM positive strains, two different variants were identified, namely, VIM-1 and VIM-4 (Table 1).
(ii) Molecular mechanisms of carbapenem resistance in non-Enterobacterales. Among nonfermenting bacteria, A. baumannii was the most prevalent carbapenem-resistant strain. The OXA-23 enzyme was the most common carbapenemase gene that was detected   Table 2). Molecular mechanisms of colistin resistance. PCR screening for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-8) was performed on all of the strains. The results showed that four strains of E. coli harbored the mcr-1 encoding gene. For these strains, the MICs were 4 mg/L, and they were sensitive to carbapenem antibiotics (imipenem and ertapenem) and also remained resistant, mainly to quinolones and sulfamides (Table 3).
Colistin resistance, mediated by alterations of the mgrB gene. The entire mgrB gene of the 32 K. pneumoniae strains was amplified via standard PCR and sequenced. Among these 32 K. pneumoniae strains, three strains were resistant to colistin, and their MICs ranged from 16 to 32 mg/L. The results of the sequencing confirmed the resistance to colistin via alterations in the mgrB encoding gene (deleterious mutations and a truncated gene) ( Table 3).

DISCUSSION
A new virus has been described by the International Committee on Taxonomy of Viruses (ICTV) as severe acute respiratory syndrome coronavirus (SARS-CoV-2). However, the recent increase in carbapenemase-producing bacteria (CPB) can be attributed, in part, to the SARS-CoV-2 pandemic, long hospitalization, inappropriate use of antibiotics (as empirical therapies or as treatments), and noncompliance to infection prevention and control measures. The inappropriate use of antibiotics has contributed to the wide spread of multidrug-resistant bacteria (9,10), which are essentially resistant to "carbapenem" antibiotics, which are considered to be the last resorts with which to treat Gram-negative bacterial infections.
In addition, broad-spectrum antibiotics were administered in nearly 70% of SARS-CoV-2 related hospital admissions and in 80 to 100% of SARS-CoV-2 related intensive care unit admissions (5). The impact of the SARS-CoV-2 pandemic on the propagation of antibiotic resistance in health care settings has not yet been fully described.
In this context, we describe the spread of carbapenemase-producing, Gram-negative bacteria, mainly due to carbapenem-resistant Enterobacterales (CRE) and A. baumannii (CRAB).       A large study performed in Wuhan Union Hospital emphasized that of the bacteria isolated, Gram-negative bacteria represented 85.5% (11). These findings are in concordance with our results; the most frequently isolated bacteria in our study were K. pneumoniae, C. freundii, and A. baumannii, at 26.89% (32/119), 17.64% (21/119), and 17.64% (21/119), respectively. A study published by Li et al. in 2020 in Wuhan, China, found that the most prevalent isolated strains were A. baumannii (35.8%), K. pneumoniae (30.8%), and S. maltophilia (6.3%). Also, Cano-Martín et al. reported in a study that was published in 2021 that K. pneumoniae, K. oxytoca, C. freundii, E. coli, Enterobacter cloacae, A. baumannii, and P. aeruginosa were the Gram-negative bacteria that were responsible for coinfection in SARS-CoV-2 patients (12). Furthermore, several reports described the increases rate of multidrug-resistant Enterobacterales, particularly K. pneumoniae, suggesting the overuse of antimicrobials during the SARS-CoV-2 pandemic (13). The most prevalent enzyme was the metallob-lactamase NDM, occurring mainly by the variant NDM-5 and NDM-1 in K. pneumoniae, at 21.5% and 12.65%, respectively. Several findings have referred to the spread of NDM, especially the NDM-5-producing E. coli strains in the ICU dedicated to SARS-CoV-2 patients in France (14). Our results are in line with another report performed on SARS-CoV-2 patients in Italy, which found that 14/41 patients (34%) were infected with carbapenemase-producing K. pneumoniae (15).
Furthermore, this metallo-b-lactmase was found in Citrobacter spp., mainly C. freundii. They are recognized as opportunistic pathogens and cause many health care-associated infections, particularly respiratory infections and urinary tract infections. A case report that was conducted by Du et al. and was published in 2013 suggested the occurrence of NDM-1 via C. freundii isolated from a urine culture from a 63-year-old Chinese male who developed urethral stricture and suffered from dysuria (16). In China, another work highlighted the detection of NDM-1 from a 52-year-old Chinese person (17). On the other side of the world, a recent study conducted in Africa mentioned the isolation of NDM-1 producing C. freundii from a rectal swab of a patient (18). It is of major concern to note that all of the patients in the current study suffered from chronic diseases, including diabetes mellitus, hypertension, and cancer.
The emergence of carbapenem-resistant A. baumannii (CRAB) is a serious concern in clinical practice, as it limits treatment options for patients (23). In 2013, the Centers for Disease Control and prevention (CDC) listed the multidrug-resistant Acinetobacter as a "serious threat". However, in 2019, carbapenem-resistant A. baumannii (CRAB) has been reported as an "urgent threat pathogen". This organism causes untreatable infections due to its high antimicrobial resistance and its arsenal of virulence agents, which are responsible for its high case mortality rate (24). Several studies highlighted the major role of multidrug resistant (MDR) A. baumannii and its implication in coinfection in SARS-CoV-2 patients who were admitted into an intensive care unit (ICU) (25). The main concern is that CRAB can transmit resistance determinants through mobile genetic elements between strains in health care settings, thereby limiting the availability of antimicrobial therapeutic options (23).
The current study suggested that OXA-23 was linked to carbapenem resistance, and 19 isolates were observed to be carrying this gene. Worldwide, several findings showed that bla OXA-23 is the most prevalent gene conferring resistance to carbapenem antibiotics in A. baumannii (26,27).
Furthermore, bla NDM-1 was shown in nine strains, and similar findings were reported by Ramadan et al., suggesting the presence of resistance genes other than OXA genes, including NDM-1, TEM, and CTX-M in A. baumannii isolated from SARS-CoV-2 patients (28). Our findings are in concordance with another study conducted by Moubareck et al. in 2020, which found that CRAB harbored the gene encoding OXA-23 carbapenemase as well as an additional carbapenemase gene encoding NDM (29).
The global clinical problem is the increase of multidrug-resistant bacteria, particularly carbapenem-resistant bacteria. Colistin was initially used on humans in 1950 to treat Gram-negative bacterial infections. Later, it was gradually abandoned because of its presumed nephrotoxicity, and it was replaced by new antibiotics with similar a spectrum and effectiveness but without the toxicity, such as the second-generation and third-generation cephalosporins (30). In fact, this has contributed to the reintroduction of colistin into clinical therapy to treat infections caused by multidrug-resistant bacteria (31).
This part of the study describes the occurrence of colistin-resistant Enterobacterales (K. pneumoniae and E. coli) in SARS-CoV-2 patients. We found that K. pneumoniae strains carried carbapenem-resistant genes, but this is not the case for E. coli that carry only colistinresistant genes. It is worth noting that many different genetic modifications were observed in our three colistin-resistant K. pneumoniae mgrB negative regulator genes, which resulted from deleterious mutations in two strains and a missing part of the mgrB in one strain. Colistin-resistant clinical isolates have been previously reported in the Middle East region, including the Arabian Peninsula, Turkey, and Egypt (32), as well as in Africa (Tunisia and Algeria) (33,34).
Of note, mcr-1 has rarely been reported in K. pneumoniae, compared with E. coli. Here, we describe the occurrence of mcr-1 in E. coli. Initially, the plasmid-mediated colistin gene mcr-1 was documented in 2015 in China (35). Since then, it has dramatically diffused worldwide, and it has become the most reported pathway implicated in colistin resistance in both animals and humans. So far, 10 MCR variants have been defined, accounting for a varied geographic distribution (36). MCR-1 was widely detected in Europe, Asia, and Africa, mainly in animals and in the environment (wastewater and sewage water), with few findings documenting mcr-1 in clinical isolates (37). To the best of our knowledge, this is the first time that the mobile colistin resistant gene (mcr-1) has been detected in a hospital setting in Libya.
This is the first comprehensive study to investigate the carriage of mcr-1 by Enterobacterales among individuals with SARS-CoV-2 in clinical settings in Libya. NDM was the major carbapenemase, with NDM-1 and NDM-5 being the most dominant types. The strains that were positive for mcr-1 were all E. coli, and they remained sensitive to carbapenem antibiotics. For K. pneumoniae, resistance to colistin was enhanced by mgrB modification.
Increased surveillance, continuous epidemiological monitoring at the local and national scales, the application of hygiene practices, and, above all, the rational use of carbapenem antibiotics and, recently, colistin (according to the dosage) are required to reduce the propagation of MDR strains and to maintain the efficacy of these antibiotics.

MATERIALS AND METHODS
Data collection and patients. This study was performed in SARS-CoV-2 isolation centers in the eastern part of Libya (Benghazi and Shahat), in different hospital settings (Benghazi Medical Center, New Marwa Hospital, Hawari Hospital, and Mansoura Hospital), from May to June of 2021. Patients (.40 years) were admitted to these hospital settings. Moreover, laboratory confirmations of SARS-CoV-2 infections were performed, following the Gene Expert protocol of nasopharyngeal swabs. 109 samples were collected from 43 patients. The swabs were recovered from the oral cavity (n = 35), nasal cavity (n = 45), and rectal cavity (n = 29). Patients' clinical information underlying diseases, comorbidities, age, and gender are summarized (data not shown).
Swab collection, culture media, and bacterial isolation. The sampling was performed using sterile cotton swabs. The swabs were kept on transport media. These swabs were stored at 4°C and were transported to the IHU Mediterranean Infection in order to achieve the isolation steps in the laboratory. The collected samples were incubated in 10 mL of brain heart infusion broth medium (bioMérieux, Marcy l'Etoile, France) for 48 or 72 h at 37°C. The broth samples were then cultured on MacConkey agar medium (bioMérieux, Marcyl'Etoile, France) that was supplemented with cefotaxime (2 mg/L) and ertapenem (2 mg/L) (Bio-Rad, Marnes-la-Coquette, France) in order to isolate third-generation cephalosporins (3GC) and carbapenem-resistant Gramnegative bacteria. To detect colistin resistance, the swabs were cultured on Lucie Bardet-Jean-MarcRolain (LBJMR) medium. This medium is Purple Agar Base (31 g/L) that is supplemented with colistin (4 mg/L), vancomycin (50 mg/L), and glucose (7.5 g/L) as a fermentative substrate (38).
Following 24 h of incubation at 37°C, the cultures yielded morphologically different colonies (differing in color, size, and shape) on each plate. Well-isolated colonies that were growing on the selective media were taken separately and purified.