Multidrug-resistant Acinetobacter baumannii outbreaks: a global problem in healthcare settings

Abstract INTRODUCTION: The increase in the prevalence of multidrug-resistant Acinetobacter baumannii infections in hospital settings has rapidly emerged worldwide as a serious health problem. METHODS: This review synthetizes the epidemiology of multidrug-resistant A. baumannii, highlighting resistance mechanisms. CONCLUSIONS: Understanding the genetic mechanisms of resistance as well as the associated risk factors is critical to develop and implement adequate measures to control and prevent acquisition of nosocomial infections, especially in an intensive care unit setting.


OVERVIEW OF A. BAUMANNII ANTIBIOTIC RESISTANCE
The key resistance mechanisms of A. baumannii are the low permeability of the outer membrane, alteration in antibiotic binding sites, and mutations, which can cause upregulation or downregulation of efflux system activity 4,10 . Among these mechanisms, alteration of bacterial membrane permeability by the outer membrane proteins (OMPs) is associated with the loss or reduced expression of porins 8 . This group is represented by OmpA, OprD, and CarO proteins 14 . The OccD1 (OprD) channel of the Pseudomonas aeruginosa species plays an important role in the uptake of molecules such as imipenem and meropenem. This OM channel is closely related to the OM family in A. baumannii and is the largest pore described amongst Occ proteins with efficient in vitro uptake responsible for transporting small molecules, presenting a huge potential for future antibiotic design 15 .
The efflux system expels toxic compounds to the extracellular environment. Within it, five families of systems have been described in A. baumannii, such as the major facilitator super family (MFS), ATP binding cassette (ABC), resistance nodulation division (RND), small multidrug resistance family 1 (SMR), multidrug and toxic compound extrusion (MATE), and drug/metabolite transporter (DMT) 16 . The RND family is well characterized and is represented by the AdeABC, AdeIJK, and AdeFGH efflux system 17 . Mutations can influence the expression of the efflux system, resulting in increased cases of clinical infections. A study highlighted resistance to aminoglicosides, tetracyclines, chloramphenicol, fluoroquinolones, some beta-lactams, and tigecycline related to mutations on the chromosome or plasmids 18 . The efflux systems CraA, AmvA/AedF, Tet(A), and Tet(B) of the MFS system are known to have a drug-specific substrate profile, and are involved in chloramphenicol, erythromycin, chlorhexidine, and tetracycline resistance 19,20 . The expression of Acel protein is strictly related to chlorhexidine transportation and the AbeM gene (a member of the MATE family), which confers resistance to fluoroquinolones through the H + antiport 20,21 . Quinolone resistance can be related to the AbaQ gene, which belongs to the MFS transporter and has its N-and C-ends located in the cytoplasm, which confers its characteristic as a drug H + antiporter-1 (DHA1). AbaQ knockout in A. baumannii confirmed its involvement with quinolone susceptibility, resulting in decreased susceptibility caused by active efflux transportation 22 .
It is known that the fluoroquinolone resistance mechanism is mainly encoded by mutations in DNA gyrase (gyrA, gyrB genes) and topoisomerase IV (parC, parE ), with gyrB and parE mutated at a lower frequency. These mutations are sequential, as primary mutations in gyrA81 are followed by mutations in parC88 and parC84 in A. baumannii. However, a study described strains carrying mutations in only the parC gene, revealing the involvement of other resistance mechanisms for fluoroquinolone [23][24] .
One of the main mechanisms of resistance to beta-lactam antibiotics is associated with changes in the structure or expression profile of penicillin binding proteins (PBPs) 25 . PBPs are transglycosylases, transpeptidases, and carboxypeptidases, enzymes located in the plasma membrane, and are involved in the synthesis of peptidoglycan, an essential component of the bacterial cell wall.
Once a PBP is acylated by a beta-lactam antibiotic, it is unable to catalyze hydrolysis of the covalent acyl-enzyme intermediate and is inactivated. Peptidoglycan transpeptidation cannot occur; thus, the cell wall is weakened 25 .
PBPs are divided into high molecular mass (HMM) and low molecular mass (LMM). The first is responsible for insertion into the cell wall, which, depending on the structure and catalytic activity of the N-terminal domain, can be classified as class A or B 26 . Therefore, changes in PBP expression lead to decreased susceptibility to these antimicrobial agents, favoring the occurrence of beta-lactamresistant strains 27 . Due to the lack of interaction that occurs in the connection between beta-lactams and PBPs, the susceptibility of A. baumannii strains to beta-lactams has been observed [27][28][29] .
Mutations can occur and modify the binding of antibiotics, inactivating some lipids, such as lipid A 30 . Polymyxins interact with lipid A through the addition of phosphoethanolamine (PEtn), resulting in displacement of cations Mg 2+ and Ca 2+ , which destabilizes the membrane. These molecules are mediated by the pmrCAB operon [31][32][33] . Alterations in the pmrA-pmrB two-component system, which is also involved in lipid A biosynthesis, upregulate pmrC, influencing the synthesis of PEtn. It is known that LPS is synthesized through the lpx pathway; mutations in lpxA, lpxC, and lpxD genes lead to deficiency in LPS production and its complete loss, conferring the colistin resistance phenotype 34,35 . Colistin resistance can be chromosomal or plasmid-encoded, carrying the mcr gene (mcr-1 to mcr-5) 36,37 .
Oxacillinases belong to class D of Ambler (1980) and group 2 of Bush and Jacob (2010) and are encoded by the bla OXA genes. These proteins hydrolyze carbapenems and penicillins at a low level and has weak hydrolysis of second and third generation cephalosporins 44 . Oxacillinases have been reported in clinical isolates of A. baumannii associated with hospital outbreaks 46 . Six subgroups of Class D carbapenem-hydrolyzing enzymes (CHDLs), including OXA-23, OXA-24, OXA-51, OXA-58, OXA-143, and OXA-235, were identified 47 . These enzymatic groups hydrolyze penicillins at a high level and carbapenems at a low level. However, the presence of insertion sequence (IS) is considered a strong promoter for the increase of oxacillin expression and dissemination 48 . It was reported that the ISAba1/bla  or ISAba1/bla OXA-51 combination amplified resistance to carbapenems 49 .
Aminoglycosides bind to 16S rRNA in the 30S ribosomal subunits and inhibit protein synthesis. Resistance is mediated by aminoglycoside-modifying enzymes (AMEs), such as acetyltransferases (AAC), adenyltransferases (ANT), and phosphotransferases (APH), which are found on mobile elements such as transposons and plasmids. AAC enzymes are responsible for modifying amino groups, while the ANT and APH enzymes act on hydroxyl groups, breaking bonds and inactivating the antibiotic molecule 10 . Methylase production (armA, rmtA, rmtB, rmtC, rmtD) decreases the affinity of the aminoglycosides for 30S ribosomal subunits 50 . A study with carbapenem-resistant (CR) A. baumannii identified 97.2% of the isolates carrying the aph(3´)-VI gene, with the majority found in 4 different clusters (A, B, C, and E), conferring resistance to amikacin, and group D, harboring AME genes (aac(6´)-Ib, aac(3)-Ia, and aph(3´)-Ia), responsible for gentamicin resistance and intermediate resistance to amikacin 51,52 . The presence of methylase armA coexisting with bla OXA-23 in MDR A. baumannii has been previously described and identified in quinolone-resistant A. baumannii 53,54 .
In addition to the multiple mechanisms of resistance, A. baumannii can acquire resistance genes through mobile genetic elements. Mobile elements, such as IS, transposons, genomic islands, integrons, and plasmids, are related to variations in the insertion site and carry strong transcriptional promoters that are abundantly synthesized 55,56 . Multiple A. baumannii plasmids have been reported: pA297-1, carrying gentamicin, kanamycin, and tobramycin resistance genes; pA297-3, carrying sulfonamide and streptomycin resistance genes; and pAb-G7-2, carrying an amikacin resistance gene 57,58 .
Transposons, such as Tn2006, Tn2007, and Tn2008, increase the spread of resistance genes and may present integrons, which were captured and express exogenous resistance genes 40,48,59 . Thus, integrons are composed of gene cassettes, and classes 1 and 2 are commonly found in A. baumannii clinical isolates [60][61][62] . As previously stated, insertion sequences act as strong promoters that increase the resistance levels of OXA carbapenemases in A. baumannii isolates 47,59,63 . Insertion sequence Acinetobacter baumannii (ISAba) can be located upstream of the resistant gene, overexpressing genes such as AmpC and OXA-51, which increases cephalosporin resistance 64,65 . Resistance to colistin in A. baumannii clinical isolates was related to the presence of the ISAba125 at the 3' end of the hns gene, disrupting the normal expression of a transcriptional gene regulator 66 .

RISK FACTORS RELATED TO A. BAUMANNII
Risk factors are directly related to increased susceptibility in hospitalized patients who develop some type of infectious disease involving bacterial resistance, consequently resulting in mortality in nosocomial environments. Investigation of the risk factors associated with A. baumannii infection/colonization contributes to the prevention and control of bacterial resistance, reducing the impact of A. baumannii isolates 67 (Table 1 and Table 2). The prevalence of A. baumannii infection and colonization is higher in ICUs, since patients with severe clinical conditions are hospitalized in such wards. In addition, these patients have compromised immune systems due to the presence of comorbidities, altered nutritional status, prolonged hospitalization, invasive procedures, immunosuppressive drugs, and broad-spectrum antibiotics 67,68 .
Skin colonization, length of hospital stays > 7 days, use of corticosteroids, and invasive procedures such as central venous catheter or tracheostomy, were the main risk factors related to the development of pneumonia associated with mechanical ventilation by MDR A. baumannii in hospitalized patients ( Table 1) 69,70 . Risk factors such as use of urinary catheters for more than 6 days, ICU contact pressure > 4 days, presence of gastrectomy tubes, chemotherapy, organ transplantation, chronic diseases, invasive procedures, recent bacteremia, tumors, hematological diseases, recurrent hospitalizations, hospitalization time > 7 days, transfer from another hospital, and previous use of carbapenems or broadspectrum cephalosporins were related to acquisition of MDR A. baumannii infection in adult patients hospitalized in the ICU 69,71 . Isolation of MDR A. baumannii after medical ICU (MICU) admission was related to a greater likelihood of the patient being older 72 . Previous hospitalization was associated with the isolation of A. baumannii after admission to the surgical ICU (SICU). Positive colonization in SICU was strongly correlated with heart failure, paralysis, human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV-AIDS), and rheumatoid arthritis 73 .
Bloodstream infections by A. baumannii are frequent in ICUs and have been associated with central venous catheters, mechanical ventilation, pneumonia, drain use, and respiratory and cardiovascular failure 74 . The risk of bacteremia caused by A. baumannii was associated with respiratory failure, mechanical ventilation, endotracheal tubes, central venous catheters, surgical procedures, and previous use of antibiotics 75,76 .
Newborns are considered susceptible to A. baumannii colonization and infections, since they have immature immune systems. The risk is greater for newborns if they are also preterm (< 28 weeks) and underweight (< 2,500 g) 76,77 . Birth weight < 2500 grams, respiratory syndromes, parental feeding, re-intubation, carbapenem use, mechanical ventilation, hematologic diseases, neutropenia > 3 days, previous use of broad-spectrum antibiotics, use of invasive devices, immunosuppressants, corticosteroids, previous hospitalization, and ICU stay > 3 days were considered risk factors for the acquisition of A. baumannii infections in the neonatal ICU ( Table 2) [78][79][80] .
Bloodstream infections caused by A. baumannii in neonates were related to the use of mechanical ventilation, and additionally to the presence of traumatic brain injury, previous use of antibiotics, hospitalization > 7 days, and use of mechanical ventilation > 7 days [81][82][83] . The weight of newborns (1000-1499 g), previous use of cephalosporins, surfactant replacement therapy, re-intubation, and umbilical artery catheterization were also indicated as risk factors 4/13    for the development of neonatal pneumonia caused by carbapenemresistant A. baumannii 84 . Maternal infection, gestational age among 26 to 36 weeks, use of central venous catheters, surgical procedures, blood transfusions, prolonged intubation, use of mechanical ventilation, central peripheral venous catheters, umbilical catheters, total parental nutrition, ICU stay > 7 days, surgical procedures, and bronchopulmonary dysplasia were described as risk factors for sepsis by A. baumannii 77,85 . Cholestasis, gestational age < 29 weeks, prematurity, low birth weight (70% < 1500 g), prolonged intubation, central venous catheters, use of imipenem for up to 5 days, mechanical ventilation, and prior carbapenem exposure are related to A. baumannii bacteremia in neonates 10,86,87 . Similar results were reported for colonization in neonates 88 . These studies pinpoint persistent endemic isolates in hospitals, highlighting the need to implement efficient control measures and prevent outbreaks.

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Seasonality of A. baumannii infection is another risk factor that should be taken into consideration. A systematic review compiled studies showing 57.1% (12/21) of A. baumannii infections occurred in warmer seasons. The hypothesis for this was that it was due to enhanced lipid A moiety regulation, which was responsible for the virulence; it was also reported there was biofilm formation and a higher flow of people entering the hospital facility (carriers, patients, healthcare workers, and sanitation workers) in warmer months. This study highlights the importance of correlating different factors of A. baumannii adaptability in the ambient environment to implement preventive measures for seasonal peaks of infection 89 .
Information related to colonization pressure (CP) is important for mediating risk factors. CP is a tool to measure the proportion of A. baumannii reservoirs within a health care facility. For A. baumannii surveillance, CP can help enhance patient screening and determine infection control measures 90,91 .

MOLECULAR EPIDEMIOLOGY OF A. BAUMANNII IN BRAZIL
In Brazil, the first outbreak associated with OXA-23-producing A. baumannii isolates was in 1999 92 . Subsequently, different outbreaks were reported 93 . A. baumannii dissemination in different Brazilian hospitals was associated with bla OXA-51 and bla OXA-23 genes and highlighted the prevalence of ISAba1/OXA-23 and ISAba1/ OXA-51 genetic profiles 94 . Isolates carrying the bla OXA-51 , bla OXA-58 , and bla OXA-23 genes, and ISAba1 upstream of OXA-51 and OXA-23 were found in different ICUs, indicating an outbreak of crosscontamination among patients, equipment, or medical staff 94 . The bla OXA-58 and bla OXA-65 genes with the upstream ISAba1 sequence for both genes have been reported. The bla OXA-58 gene is prevalent in Argentina, indicating a possible spread from the border with Rio Grande do Sul 95 . In addition, two genotypes of OXA-23-producing A. baumannii were present at 8 hospitals in the same city, suggesting the spread of isolates in these environments 93 . The sequence type (ST) 156, ST25, and ST160 were identified in a Brazilian hospital 96 . Cephalosporin-resistant A. baumannii and producers of extendedspectrum beta-lactamases (ESBL) were identified in a neonatal intensive care unit (NICU), causing septicemia in hospitalized neonates ( Table 3) 5 . A study in neonates described most isolates as belonging to ST1 and had ISAba1 upstream of the bla OXA-51 and bla OXA-23 genes 88 .
A study in Recife, Brazil described isolates belonging to ST1, ST15, ST25, ST79, ST113, and ST881 (related to ST1). Among them, ST79 and ST113 were found to be more virulent and presented resistance genes. ST113 and ST15 were commonly found in all 5 hospitals of the study, while ST79 was found in 4 hospitals and ST1 in 3 hospitals. Among the CCs circulating between hospitals, Leal et al. described CC1, CC15, and CC113, which are globally 7/13 spread types, and CC79, which is found in South America, North America, and Europe 97 .
A study carried out in nine hospitals in South America identified A. baumannii clinical isolates presenting bla OXA-51 , bla OXA-23 , bla OXA-72 , bla OXA-132 , bla OXA-65 , bla OXA-69 , and bla OXA-64 genes. Multilocus sequence type (MLST) analysis identified ST79, ST25, and ST15 98 . The two major clonal complexes (CC) found in bla OXA-23 multidrug-resistant A. baumannii are CC15 and CC79, and CC15 has already been described in 9 Brazilian states 77 . In addition, ST15 was described in other countries, such as Argentina and Turkey, and ST79 was described in the United States, Canada, and Spain 99 . Of the clonal profiles identified, ST15 and ST79 were described in several countries, indicating their spread among hospitals around the world and high mortality rates 100 .
Molecular typing of A. baumannii provides a better understanding of the epidemiology of outbreaks and identification of crosstransmission, as well as assisting in the monitoring and control of nosocomial infections 47,153 . Thus, several methods have been used to study the molecular epidemiology of A. baumannii and analyze the mechanisms involved in the resistance of this microorganism.

CONCLUSION
The increase in healthcare-associated infection (HAI) rates connected to A. baumannii antimicrobial resistance has become a major public health challenge worldwide. A. baumannii possesses several resistance mechanisms. However, hydrolysis by OXA-type carbapenemases and metallo-β-lactamases are considered the most prevalent mechanisms conferring resistance to most beta-lactam antibiotics and reduce therapeutic options. This study highlights the occurrence of outbreaks in hospital settings, especially in ICUs, which are commonly related to prolonged hospital stays and invasive procedures. Thus, epidemiological studies are important for monitoring the occurrence of A. baumannii clinical isolates and may assist in the implementation of appropriate measures, contributing to the control of hospital infections.

ACKNOWLEDGMENTS
We are grateful to the Universidade Federal da Grande Dourados (UFGD) and the research group in Molecular Biology of Microorganisms of this institution for their support.