Associations between β-Lactamase Types of Acinetobacter baumannii and Antimicrobial Resistance

Background and objective: Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen that not only possesses intrinsic resistance to many classes of antibiotics, but is also capable of rapidly developing antimicrobial resistance during treatment. The aim of this study was to determine the characteristics of resistance of A. baumannii strains to β-lactams and other tested antibiotics, to evaluate the associations between the phenotypes of resistance to β-lactams and other tested antibiotics, and to evaluate the changes in antibiotic resistance of A. baumannii strains over 5 years by comparing the periods of 2016–2017 and 2020–2021. Materials and methods: A total of 233 A. baumannii strains were isolated from different clinical specimens of patients treated at the Hospital of Lithuanian University of Health Sciences in 2016–2017 (n = 130) and 2021–2022 (n = 103). All clinical cultures positive for A. baumannii were analyzed. The type of β-lactamase was detected by phenotypic methods using ESBL plus AmpC screen disk tests and the combination meropenem disk test. Results: In both periods, all A. baumannii strains were resistant to ciprofloxacin; resistance to carbapenems, piperacillin/tazobactam, gentamicin, and tobramycin was noted in more than 80% of strains. A comparison of two periods showed that the percentages of A. baumannii strains producing two or three types of β-lactamases were significantly greater in 2021–2022 than in 2016–2017 (94.2% and 5.8% vs. 17.7% and 2.3%, respectively, p < 0.001). Isolates producing two or three types of β-lactamases were more often resistant to tigecycline, tetracycline, and doxycycline than strains producing one type of β-lactamase (p < 0.001). Conclusions: The frequency of isolation of A. baumannii strains producing two different types of β-lactamases (AmpC plus KPC, AmpC plus ESBL, or ESBL plus KPC) or three types of β-lactamases (AmpC, KPC, and ESBL) and the resistance rates to ampicillin/sulbactam, tigecycline, tetracycline, and doxycycline were significantly greater in 2020–2021 as compared with 2016–2017. The production of two or three types of β-lactamases by A. baumannii strains was associated with higher resistance rates to tetracyclines.


Introduction
Acinetobacter baumannii (A.baumannii) is an obligate aerobic, nonfermenting, Gramnegative nonmotile bacterium that was discovered by Dutch microbiologist Martinus Willem Beigerinck in 1911 [1].The ability of the microorganism to survive in various environmental conditions and to last longer on wet or dry surfaces has turned it into an endemic, health care-associated pathogen and a common cause of infection outbreaks [2,3].People who have a weakened immune system, chronic lung disease, or diabetes are at a greater risk of being more susceptible to A. baumannii-caused infections, especially patients with a prolonged hospital stay, those with open wounds, or anyone with urinary or other catheters [4].
A. baumannii is an important nosocomial pathogen that not only possesses intrinsic resistance to many classes of antibiotics, but is also capable of rapidly developing antimicrobial resistance during treatment.This pathogen has become resistant to almost all antimicrobial agents currently available, including aminoglycosides, quinolones, and broad-spectrum β-lactam antibiotics [5].Nosocomial infections caused by A. baumannii are difficult to treat due to multidrug resistance (MDR), which severely limits the options for therapeutic treatment due to increased resistance to carbapenems, which are used as a last resort for antibiotic therapy to eliminate infections caused by multidrug-resistant Gram-negative bacteria [6].When bacteria are responsible for antibiotic resistance to at least one antimicrobial agent from three or more classes of antimicrobials (for example, penicillins, aminoglycosides, cephalosporins, fluoroquinolones, or tetracyclines), they are considered to be multidrug resistant [7].Antibiotic resistance in A. baumannii occurs due to the enzymatic degradation of antibiotics, mutations/modification of target sites, reduced expression of porins, and overexpression of multidrug efflux pumps [8].Resistance to carbapenems is often mediated by β-lactamases including extended-spectrum β-lactamases (ESBLs) and AmpC [9].ESBLs conferring resistance to broad-spectrum cephalosporins and carbapenemases conferring resistance to carbapenems are the greatest concern.An ESBL and an AmpC β-lactamase in a single isolate confer resistance to carbapenems that are usually the drug of choice in the treatment of A. baumannii infection [10].Due to resistance to multiple antibiotics, A. baumannii is associated with high mortality [11].In 2017, this pathogen was included in the World Health Organization (WHO) global priority list of drug-resistant bacteria in order to highlight the need for research development and the urgent need for new antibiotics [12].However, clinical data evaluating potentially effective treatment methods are currently insufficient, and randomized clinical trials have not shown that a single antimicrobial agent or combination therapy is more efficient [13].Therefore, it is very important to maintain the effectiveness of already available antibiotics and to pay considerable attention to the prevention of the spread of A. baumannii infection in healthcare facilities [13,14].
According to the European Antimicrobial Resistance Surveillance Network (EARS-Net) report, the resistance rate of Acinetobacter spp. to carbapenems in Lithuania in 2021 was 96.1% [15].The aim of this study was to determine the characteristics of resistance of A. baumannii strains to different antibiotics, to evaluate the associations between the phenotypes of resistance to β-lactams and other tested antibiotics, and to evaluate the changes in antibiotic resistance of A. baumannii strains after 5 years by comparing the periods of 2016-2017 and 2020-2021.

Bacterial Strains
A. baumannii strains were isolated from different clinical specimens of patients treated in the Hospital of Lithuanian University of Health Sciences.Isolates were defined as MDR if they were resistant to at least one antimicrobial agent from three or more classes of antimicrobials: penicillins (ampicillin/sulbactam, piperacillin/tazobactam) or cephalosporins (ceftazidime, cefepime), fluoroquinolones (ciprofloxacin), and aminoglycosides (amikacin, tobramycin, gentamicin).Specimens were obtained from wounds, biopsy, bronchial secretions, blood, sputum, pus, abdominal fluid, implants, pleural fluid, urine, and cerebrospinal fluid of hospitalized patients.All the isolates were cultivated on blood agar and MacConkey agar for 17-24 h, and pure cultures were identified using a MALDI-TOF MS mass spectrometer (Brucker Daltonics Gmbh, Bremen, Germany).

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed by a disk diffusion method on Müller-Hinton agar (MH II according to EUCAST, Graso Biotech Microbiology Systems, Owidz, Poland).All inoculated plates were incubated for a total of 16-20 h at 35 • C ± 1 • C in an ambient air incubator after inoculation with organisms and placement of disks.All the strains were tested for sensitivity to the following antibiotics: ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, ampicillin/sulbactam, piperacillin/tazobactam, imipenem, meropenem, doxycycline, tigecycline, tetracycline, and sulfamethoxazole/trimethoprim disks (Becton Dickinson Microbiology Systems) were used.The diameter of the inhibition zone was measured in millimeters using a ruler.Inhibition zone diameters were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [16].The results were interpreted according to the EUCAST breakpoints.All isolated and identified A. baumannii strains were frozen at −80 • C.

Phenotypic Methods for the Determination of ESBL Type
A total of 233 A. baumannii strains resistant to carbapenems (imipenem and/or meropenem) were tested using a four-combination disk test (CDT) to determine the production of ESBL and AmpC (Abtek Biologicals, Liverpool, UK, in 2016 and 2017; Liofilchem ® , Roseto degli Abruzzi, Italy, in 2021 and 2022).Bacterial suspensions (0.5 McFarland) of A. baumannii strains were thawed at room temperature for about 15 min and cultured on MH agar using different swabs.With all aseptic precautions, the following antibiotic disks were placed on inoculated MH agar plates: 30 µg cefotaxime (CTX 30), 30 µg cefotaxime plus 10 µg clavulanic acid (CTL 40), 30 µg cefotaxime plus 200 µg cloxacillin (CTC 230), 30 µg cefotaxime plus 200 µg cloxacillin plus 10 µg clavulanic acid (CTLC 240).The plates were incubated at 37 • C for 18-24 h in an ambient-air incubator.An increase of ≥5 mm in the zone diameter with the CTL disk as compared with the CTX disk alone indicated ESBL production.The isolate was defined as an AmpC producer when the following was observed: (a) there was an increase of ≥5 mm in the zone diameter with the CTC 230 disk as compared to the CTX 30 disk or (b) there was an increase of ≥5 mm in the zone diameter with the CTLC 240 disk as compared to the CTL 40 disk or (c) there was an increase in the diameter with the CTLC 240 disk measuring < 5 mm as compared to the CTC 230 disk.
A. baumannii strains were also considered ESBL and AmpC producers if the diameter of the CTL 40 disk was smaller than ≤5 mm as compared to the CTX 30 disk, but the diameter of the CTLC 240 disk was greater than ≥5 mm as compared to the CTL 40 disk and the diameter of the CTLC 240 disk was greater than ≥5 mm as compared to CTC 230 disk.If the diameters of the disks differed from each other by 2 mm or less, the strain being tested was neither an ESBL nor an AmpC producer.
All A. baumannii strains resistant to carbapenems (imipenem and/or meropenem) were tested using a combination meropenem disk test.The advantage of the test is that it discriminates between carbapenem-susceptible, KPC-producing, metallo β-lactamase (MBL)-producing, and double carbapenemase-producing bacteria.Bacterial suspensions (0.5 McFarland) of A. baumannii were made and cultured on Mueller-Hinton agar using different swabs, and then four meropenem disks were placed.EDTA (0.1 M, 10 µL) was added on the second disk; 20 µL of phenylboronic acid (20 g/L) on the third disk; and 20 µL of phenylboronic acid (20 g/L) plus 10 µL EDTA (0.1 M) on the fourth disk.The plates were incubated at 37 • C for 18-24 h in an ambient-air incubator.Interpretation of the results of the combination meropenem disk test was based on the comparison among the inhibition zones of four meropenem disks.If no carbapenemase was present, the diameter of the disk with added inhibitors was similar in size (≥5 mm) compared to the diameter of the meropenem disk alone.If an isolate was a KPC producer, there was an increase of ≥5 mm in the diameters of the disks supplemented with phenylboronic acid as compared to the disks without phenylboronic acid.MBL production was confirmed by an increase of ≥5 mm in the diameters of the disks that were supplemented with EDTA.In the case of a KPC plus MBL producer, the fourth disk had the largest zone diameter of all.EDTAand phenylboronic acid-supplemented disks were ≥5 mm larger in diameter than the meropenem disk alone.Figure 1 shows the workflow diagram.The Kaunas Regional Biomedical Research Ethics Committee approved this study (No.BE10-0016).

Statistical Analysis
The chi-square (χ 2 ) criterion was used for the comparison of categorical data, and continuous data were compared with Student's t test.The results were considered statistically significant at p < 0.05.Statistical package SPSS 27.0 for Windows was used for the data analysis.

Results
A total of 233 A. baumannii strains were isolated from different clinical specimens of patients treated in the Hospital of Lithuanian University of Health Sciences: 130 in 2016-2017 and 103 in 2021-2022.In 2016-2017, 44 (33.8%) isolates were collected from women and 86 (66.2%) from men, with a mean age of 62.9 (SD 17.5) years.In 2021-2022, 29 The Kaunas Regional Biomedical Research Ethics Committee approved this study (No.BE10-0016).

Statistical Analysis
The chi-square (χ 2 ) criterion was used for the comparison of categorical data, and continuous data were compared with Student's t test.The results were considered statistically significant at p < 0.05.Statistical package SPSS 27.0 for Windows was used for the data analysis.

Results
A total of 233 A. baumannii strains were isolated from different clinical specimens of patients treated in the Hospital of Lithuanian University of Health Sciences: 130 in 2016-2017 and 103 in 2021-2022.In 2016-2017, 44 (33.8%) isolates were collected from women and 86 (66.2%) from men, with a mean age of 62.9 (SD 17.5) years.In 2021-2022, 29 (28.2%)isolates were collected from women and 74 (71.8%) from men, with a mean age of 62.7 (SD 14.4) years.

Assessment of Antibiotic Resistance Based on β-Lactamase Type
Resistance rates of A. baumannii strains producing and not producing AmpC, ESBL, and KPC in response to all antibiotics investigated are shown in Table 3. AmpC-producing A. baumannii strains were more frequently resistant to tigecycline,

Assessment of Antibiotic Resistance Based on β-Lactamase Type
Resistance rates of A. baumannii strains producing and not producing AmpC, ESBL, and KPC in response to all antibiotics investigated are shown in Table 3. AmpC-producing A. baumannii strains were more frequently resistant to tigecycline, tetracycline, and doxycycline than those strains not producing AmpC (p < 0.001 each).However, AmpCproducing strains were less frequently resistant to gentamicin (p < 0.001), tobramycin (p = 0.031), amikacin (p < 0.001), and trimethoprim/sulfamethoxazole (p < 0.001) as compared with AmpC-non-producing strains.Significant differences in the resistance rates between ESBL-producing and ESBL-non-producing A. baumannii strains were noted only for two groups of antimicrobial drugs-ampicillin/sulbactam (p = 0.014) and tigecycline (p = 0.021)-with the resistance rates being greater in ESBL-non-producing strains.The percentages of KPC-producing strains resistant to piperacillin/tazobactam (p = 0.012), carbapenems (p = 0.040), tigecycline (p < 0.001), tetracycline (p < 0.001), and doxycycline (p < 0.001) were significantly greater than those not producing KPC.The resistance rate of KPC-producing strains to trimethoprim/sulfamethoxazole was significantly lower than that of KPC-non-producing strains (p = 0.038).
Antibiotic resistance rates of A. baumannii strains producing different types and numbers of β-lactamases are presented in Table 4. Isolates producing one and two types of β-lactamase were more often resistant to ampicillin/sulbactam as compared to all three types of β-lactamase-producing strains (87.7% and 85.0% vs. 55.6%,p = 0.031).A. baumannii strains producing one type of β-lactamase were more often resistant to trimethoprim/sulfamethoxazole as compared with strains producing two or three types of β-lactamases (97.5% vs. 70.0%and 77.8%, p < 0.001).A. baumannii strains producing two or three types of β-lactamases were more often resistant to tigecycline as compared with strains producing one type of β-lactamase (87.5% and 77.8% vs. 67.9%,p < 0.001), tetracycline (94.2% and 88.4% vs. 64.2%,p < 0.001), and doxycycline (88.3% and 88.9% vs. 25.9%,p < 0.001) as compared with strains producing one type of β-lactamase.Strains producing one type of β-lactamase were more often resistant to antibiotics indicated by letter a , Strains producing two, three types of β-lactamase were more often resistant to antibiotics indicated by letter b , Strains producing three types of β-lactamase were more often resistant to antibiotics indicated by letter c .

Discussion
The number of cases of bacterial nosocomial infections resistant to antimicrobial agents has increased worldwide over the last decade [4]. A. baumannii is considered one of the most important causes of antimicrobial resistance in health care facilities.A. baumannii strains cause outbreaks around the world due to their remarkable ability to adapt to the changes in the environment and to acquire different resistance mechanisms against multiple antimicrobials [4].This study aimed to determine the types of β-lactamases and their combinations produced by MDR A. baumannii strains.It also aimed to evaluate associations between antibiotic resistance and produced types of β-lactamases and to evaluate the changes in antibiotic resistance during the periods of 2016-2017 and 2020-2021.
According to the 2017 European Antimicrobial Resistance Surveillance Network (EARS-Net) report, the proportions of carbapenem-resistant Acinetobacter spp.collected from invasive infections were particularly high in many southern and eastern European countries, where resistance proportions often exceeded 50% (such as 95% in Greece, 79% in Italy, and 53% in Hungary).The EARS-Net analysis also showed that, on average, more than 70% of carbapenem-resistant Acinetobacter spp.were also resistant to ciprofloxacin or gentamicin in southern and eastern Europe [17].In our study, all A. baumannii strains were resistant to ciprofloxacin, and resistance rates of A. baumannii strains to carbapenems, piperacillin/tazobactam, gentamicin, and tobramycin were high (more than 80%), supporting the findings of the EARS-Net report.
In our study, only 11.6% (n = 27) of all A. baumannii isolates were ESBL producers.However, we did not investigate to which particular β-lactamase class the investigated ESBLs belonged.The study by Kaur et al., investigated the prevalence of ESBLs in A. baumannii isolates obtained from various clinical samples of one Indian institution and reported that of the 116 A. baumannii isolates, ESBL production was documented in 32 isolates (27.5%) [18].Another study carried out in one teaching and referral Ethiopian hospital showed that ESBL production was observed in 55.8% (n = 24) of the 38 A. baumannii isolates collected from different wards such as the ICU, delivery room, and operating room [19].In both studies, the ESBL production by A. baumannii strains was greater as compared with that in our study.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by Kaunas Regional Biomedical Research Ethics Committee (permission No. BE10-0016; dated 28 December 2021).
Informed Consent Statement: Patient informed consent was waived as this study analyzed bacterial strains and their resistance to antibiotics.

Figure 1 .
Figure 1.Diagram of the study performed.

Figure 1 .
Figure 1.Diagram of the study performed.

Figure 3 .
Figure 3. Percentage distribution of A. baumannii strains producing different types of β-lactamases or their combinations in 2016-2017 and 2021-2022.

Figure 3 .
Figure 3. Percentage distribution of A. baumannii strains producing different types of β-lactamases or their combinations in 2016-2017 and 2021-2022.

Table 1 .
Distribution of Acinetobacter baumannii strains by isolation sources and two different periods.

Table 3 .
Comparison of antimicrobial resistance between A. baumannii strains producing and not producing only one type of β-lactamase (AmpC, ESBL, and KPC).

Table 4 .
Comparison of antimicrobial resistance among A. baumannii strains producing different types and numbers of β-lactamases.