Carbapenemase-Encoding Genes in MDR Acinetobacter baumannii Isolated from ICUs and Surgical Wards in Minya, Egypt

are among the last resort classes of antibiotics having the greatest efficacy against severe infections caused by bacteria that are resistant to many other antibiotics. This research was done in order to detecte the frequency of carbapenememase genes in A. baumannii isolated from Surgical Wards and Intensive Care Units (ICUs) in Minia, Egypt by phenotypic and genotypic methods. Methods: 310 patients who were undergoing surgery and receiving care in ICUs participated in the current study between August 2017 and October 2020. The isolates were obtained from a variety of clinical samples, including sputum, bronchial lavage, Pus and wound fluid. All clinical samples have been collected under complete aseptic conditions. A. baumannii isolates were identified by standard microbiological tests. The resistance patterns of the obtained isolates were detected by the disc diffusion method. Production of carbapenem-resistant genes was determined by phenotypic tests the Modified Hodge Test (MHT) and confirmed by the modified carbapenem inactivation method (mCIM). Various carbapenem-resistant genes were detected by PCR. Results :Out of 310 samples, Twenty-Five 25 (8%) isolates of A. baumannii were obtained. Antibiotic and sensitivity tests detect that 16(64%) and 12(48%) of A.baumannii isolates were resistant to meropenem and imipenem respectively. Out of the Carbapenem-resistant A. baumannii (CRAB): 9(56%) were (MHT) positive, and 13 (81%) were (mCIM) positive. Regarding resistance genes, bla OXA-23like and bla OXA-51like were detected in 94% and 100% of CRAB isolates respectively, while bla OXA-24like , bla oxa-48like , bla oxa-58like and bla NDM were not detected in CRAB isolates.


INTRODUCTION
Gram-negative Acinetobacter baumannii (A.baumannii) is a major health hazard because it causes healthcare-associated infections among critically ill patients like patients with immunosuppression.They are a common reason for sepsis, ventilator-acquired pneumonia, wound infection, and (UTI) urinary tract infection, particularly in ICUs (Sookkhee et al., 2022).The emergence of extensively drug-resistant strains (XDR) or multidrug-resistant (MDR) A. baumannii became a complex challenge and put physicians in a complicated situation owing to the reduced number of antibiotic treatment options available leading to failure or delay of antimicrobial treatment, as well as the increase in mortality rate especially with the presence of CRAB (Mukherjee et al., 2023).
Carbapenems are efficient broadspectrum β-lactam antibiotics They are often regarded as the final line of treatment when using antibiotics to treat severe infections caused by A. baumannii.Other antimicrobials like colistin and tigecycline may be used in case of carbapenem resistance; however, they have poor efficacy and/or high toxicity (Azimi et al., 2020).Unfortunately, carbapenems are frequently misused and are becoming ineffective due to the development of many resistance mechanisms produced by gram-negative bacteria.
Carbapenemresistance mostly resulted from the upregulation in the chromosomally mediated blaOXA-51-like gene and may be attributed to the acquisition of some OXA variants especially the blaOXA-23-like or by acquiring Metallo betalactamases (Gupta et al., 2022).Carbapenemase genes detection in A. baumannii strains is extremly significante in evading hospital-acquired infections by resistant bacteria.The availability of accurate and affordable carbapenemase detection techniques may encourage laboratories to investigate this issue and contribute to the prevention of a serious threat of bacterial antibiotic resistance.

MATERIALS AND METHODS
In this study, 310 specimens were obtained from Patients admitted to ICUs, neurosurgery and surgery departments in the period between August 2017 and October 2020.Various clinical samples, i.e. sputum, bronchial lavage, wound fluid and Pus, were collected under complete aseptic conditions.A. baumannii isolates were identified by using standard microbiological tests.The Ethics Committee of the Beni-Suef University Faculty of Science authorised each experimental procedure.Prior to collecting data, patients provided written informed permission.The study was carried out as per the Helsinki declarations.

1-Bacterial Isolation And Identification:
The bacterial strains were isolated from sputum, wound fluid, pus and bronchial lavage.Each sample was placed in a sterile container, put in an ice pack box and transported within 2 hours to the laboratory for processing.The bacterial isolates were identified by traditional biochemical tests including Citrate, Triple sugar iron (TSI), Oxidase, Catalase, Indole, and Voges-Proskauer (VP) test.Finally, the isolates were stored at -20°C in brain heart infusion [BHI] media containing 20% glycerol (Howard., 1956)

Modified Hodge Test (MHT):
MHT was mainly used to detect carbapenemases production in A. baumannii isolates.Escherichia coli ATCC 25,922 was swabbed in three distinct directions on plates of Mueller-Hinton agar media, using a diluted culture (0.5 McFarland standard).Each plate had a 10 μg meropenem or imipenem disc (Oxoid) in the middle of it.Beginning at the disc's edge and terminating at the plate's edge, CRAB isolates were streaked as a thin line.At 37C°, bacterial growth was permitted for 18 hours.Escherichia coli 25922 developed an area resembling a clover leaf along the growing streak in the disc diffusion zone, according to an MHT Positive test result (Pasteran et al., 2016).

Modified Carbapenem Inactivation Method (mCIM):
This method was used to confirm (MHT) as it is more sensitive than (MHT) for detecting of carbapenemases (Jing et al., 2018); one µl of the test strain suspended in 2 ml of trypticase soy broth (TSB) was emulsified.For 10 to 15 seconds, the bacterial suspension was vortexed.The bacterial suspension was then aseptically infused with a 10-g imipenem disk.The tube was then kept at 37C for another four hours.By using the direct colony suspension method, a half MF suspension of the indicator strain E. coli ATCC25922 was created (it was created shortly before the 4-hour carbapenem inactivation stage was finished).Inoculations were made on Muller Hinton agar plates using the conventional disc diffusion susceptibility testing method.The imipenem or carbapenem disc was then transferred to the inoculated MHA plate and incubated for 18 to 24 hours at 37C o using an inverted position after being removed from the TSB bacterial suspension using a 10-l inoculating loop.The boiling method was used to prepare DNA, by centrifuging 1.5 ml of the bacterial broth for 5 min at 11.000 rpm.The pellet taken out, and the supernatant was suspended and mixed well with 200 microliters of molecular biology-grade water.Tubes were boiled at 100C ο in a water bath for 20 min.Then quickly cooled in ice to block the reaction.Centrifugation at 14.000 rpm for 2 minutes.The Supernatant was stored at -20 C ο and then used for DNA amplification (Chen et al., 2019).
DNA ampilification was carried out in 25 μl reactions, using My Taq TM red Master Mix "2 μl of DNA template; 12.5 μl master mix; 8.5 μl of sterile water, 1 μl of each forwared and reverse primers".The reaction conditions for each gene were listed (Table 2).

Agarose Gel Electrophoresis for detection of PCR Products
Products of PCR reaction were separated on 1 %-1.5 % agarose gel stained by 0.5 μg/ml ethidium bromide and bands were visualized under UV light.

5-StatisStatistical Analysis:
Descriptive statistics, data are presented as percentage and frequency.For analytical statistics, Using the Chi-square test as a significance test to numerical variables comparison, with less than 0.05 a P value indicating high significance.
RESULTS From a total of 310 Clinical samples, Twenty five (8%) isolates were identified as A. baumannii in accordance with cultural characteristics and biochemical reaction results.Out of 25, 10 (40%) and 15(60%) isolates of A. baumannii belonged to female and male patients, respectively with Mean age ± SD (50.9±12.25 years).The majority of isolates were obtained from patients admitted to the ICUs was (52%) and from surgical departments was (28%).The majority of isolates were obtained from patients.with severe illnesses such as diabetes (44%), Asthma (36%), Cancer, or renal failure (4%).Also, (44%) and (20%) were recovered from patients with mechanical ventilators and urinary catheters respectively.The distribution of collected isolates from the specimen sources was as follows: Pus and wounds (36%), Sputum and bronchial lavage (28%) for each and urine (8%) (Table 3).

Table 3:
The prevalence of A. baumannii isolates according to some demographic factors.
Out of 25 A. baumannii isolaes just (16) isolates were resistant to carbapenems, they were classified according to the impact of some demographic factors which may affect the resistance ( Table4).All CRAB isolates were tested for carbapenemases production by both MHT and mCIM.9 (56%) of CRAB isolates were positive for MHT and 13(81%) were Mcim positive (Fig. 1).All CRAB isolates harbored blaOXA-51like gene which confirmed A. baumannii species, out of them we found that 15 isolates were positive for blaOXA-23like.While blaOXA-24like, blaoxa-48like, blaoxa-58like and blaNDM were not detected in CRAB isolates of this study.

DISCUSSION
Resistance to Carbapenems is a global public health issue that is predominantly found in A. baumannii.Antibiotic resistance is rapidly spreading, particularly when it is spread through transferable carbapenemase-encoding genes like Metallo β lactamases, resulting in large outbreaks and limiting treatment options (Vamsi et al., 2022).By the analysis of this study results, we found that A. baumannii prevalence was (8%) and it is relatively agrees with that obtained by (Mohammed et al., 2022) who detected a (10%) prevalence of A. baumannii, and this indicates that that in Egypt, A. baumannii does not pose a serious health risk, this is the same opinion mentioned in a recent study done in El-Minia hospitals by (Abd El-Baky et al., 2020).Most CRAB isolates were obtained from ICUs patients and this was statistically significant and agreed with (Ibrahim et al., 2022).Because of their weak immune responses, most ICU patients have a higher risk of infection and the use of numerous procedures and intrusive instruments like Central venous catheterization (CVC), mechanical ventilation and urinary tract catheters; they also require a prolonged stays in ICUs, which increased the risk of infection, particularly when drugs with potent antimicrobial activity and a wide antibacterial spectrum were used.In addition, patient's medical records, having an acute disease like diabetes or cancer are at risk and this was agreed with results reported by (Pachori et al., 2019).In our investigation, we found that patients with mechanical devices or underlying disease were more vulnerable to CRAB infection and this was statistically significant but this was opposite to research by (Pachori et al., 2019) who mentioned That, the prevalence of carbapenemases was independently predicted by catheter devices use and the existence of underlying diseases.In this investigation, While 48% of A. baumannii isolates were resistant to imipenem, they were highly resistant to other antibiotics like, cortimoxazole and ceftazidime.Carbapenems were once thought to be the only option for treating infections brought on by multi-drug resistant bacteria.Due to the widespread use of these antibiotics, carbapenem-resistant strains (CRAB) first appeared a few years ago.Additionally, the spread of CRBA across hospitals is a result of insufficient infection control efforts.Resistance to other kinds of antibiotics as well as a high mortality rate was found to be related to carbapenem resistance.(Hu et al., 2023) reported high resistance of A.baumannii against meropenem, cefepime, ceftazidime, imipenem and ciprofloxacin.However, in our investigation low resistance rate for meropenem and imipenem, high resistance rate for ceftazidime and cefepime was observed.Furthermore, we reported that imipenem was the most effective antibiotic, but they demonstrated that tigecycline was just the most effective antibiotic against A. baumannii.(Dias et al., 2017).
Carbapenemases represent three classes of β-lactamases.lactamases the Ambler class A, class β carbapenemases (zinc dependant) They are referred to as (MBLs) "metallo β -lactamases" because metal chelators like EDTA inhibit these enzymes from working; and D-carbapenemases (serine carbapenemases).All β -lactam antibiotics can be hydrolyzed by metallo β-lactamases (MBLs), whereas monobactams cannot.These enzyme-encoding genes may be plasmid or chromosomally-mediated.Phenotypic tests revealed that out of CRAB (56%) were (MHT) positive and this result was lower than other studies like (Abouelfetouh et al., 2019) which recorded 78 % positive MHT.In our study, we found (81%) of CRAB were (mCIM) positive, and this is higher than (Neupane et al., 2023) who reported (69.7%) positive for (mCIM).Previous studies recorded a lower dominance of blaOXA-23like among CRAB in Egypt (Al-Hassan et al., 2019) reported (55.8%).Our results indicate an apredominant increase in the prevalence of class D Oxacillinase (blaOXA23-like) and this may be explained by the existence of several clusters and horizontal gene transfer across different strains of the species, both of which have been implicated in the steady rise in carbapenem resistance over time.blaOXA-51like is expressed naturally and it is used for confirming A. baumannii.None of the isolates in our investigation was harboured blaOXA 24-like and blaOXA 58-like genes which were in concordance with (Ibrahem et al., 2022).No strains under investigation had blaNDM found, but in a research by (Hosseinzadeh et al., 2018) who detect about 10% of the isolates carried the blaNDM gene.Also, none of the isolates had the blaOXA-48-like gene and this was reported by (Anggraini et al., 2022).

Conclusion
Our data revealed that A. baumannii had a great ability of resistance to many antibiotics as they were detected as MDR strains.Resistance to imipenem and meropenem was detected in the strains isolated from hospitals in EL-Minia, this was due to the presence of carbapenemases especially oxacillinases, which make serious problems in forcing these pathogens.Most CRAB strains were from ICUs which was considered the potential source of infection in our hospitals.The most accurate method for carbapenemase detection is PCR because of the low sensitivity of MHT and mCIM.Carbapenemases were extremely complex determinants of resistance as they spread rapidly across the bacterial community creating a threat to the treatment with carbapenems.We noticed that the main cause of resistance in A. baumannii was the expression of carbapenem hydrolyzing class D b-lactamase encoded by the acquired gene blaoxa-23 like.The intrinsic gene blaoxa-51like was a good indicator in the characterization of A. baumannii.Determinant factors like the site of admission, use of mechanical devices and the presence of underlying diseases had a great influence on the resistance of A. baumannii to carbapenems.

REFERENCES
The loop was used to remove any excess liquid during the transfer.. Zone diameter (≤ 15 mm) around the disk indicates positive results and explains the ability of the test organism to produce carbapenemases, (16-19 mm) indicates intermediate results and ( ≥ 20mm) indicates negative results (Pierce et al., 2017).

Table 1 :
Primers used for PCR reaction.

Table 2 :
Conditions for PCR reactions:

Table 4 :
Distribution of CRAB in relation to some demographic factors.