Rapid identification of carbapenemases by CarbAcineto NP test and the rate of beta-lactamases among Acinetobacter baumannii from a teaching hospital

Background and Objectives: Acinetobacter baumannii has emerged as a major organism accounting for hospital acquired infections particularly in intensive care units. Due to production of different kinds of beta lactamases these bacteria have developed drug resistance rendering the treatment of such infections very difficult and expensive. Rapid identification of A. baumannii producing such beta-lactamases is the need of the hour in reducing morbidity and mortality associated with A. baumannii infections. Materials and Methods: A. baumannii was isolated from clinical samples like endotracheal aspirates, sputum, urine, exudates using standard culture techniques. Identification and drug sensitivity was done using Vitek 2 system. All the isolates were subjected to detection of ESBLs using phenotypic confirmatory test, plasmid mediated AmpC beta-lactamase by AmpC disc test, Carbapenemase production by CarbAcineto NP Test and Modified hodge method. Results: 149 A. baumannii isolates were analysed for antimicrobial susceptibility and various beta-lactamase production. Results were evaluated for statistical significance using Chi-Square and P value. 81.8% of isolates were from male patients with majority of them above 50 years of age. 88.5% of samples were from ventilator associated pneumonia patients. 83.8% of isolates were sensitive to tigecycline. Only 10% to 12% of isolates were sensitive to carbapenems. 23.4% of isolates were ESBL producers and 46.9% of them were AmpC producers. Modified Hodge test method identified 63.7% of A. baumannii as carbapenemase producers where as CarbAcineto NP test identified 63% and exibiting 94.74% sensitivity, 93.22% specificity when compared to Modified Hodge test. Conclusion: Multidrug resistant Acinetobacter spp. is on the rise. Present study showed that high percentage of drug resistance in A. baumannii could be due to production of ESBLs, AmpC and carbapenemases. Among all beta lactamases carbapenemase producers are more and quickly raising in A. baumannii. Rapid, cost effective assay which can be adopted in all clinical laboratories is critical to prevent their further transmission particularly in hospital environment.

A. baumannii infections are frequently treated with β-lactam antibiotics like cephalosporins and carbapenems or with other group of antimicrobials like aminoglycosides, polymyxins and glycylcycline. Majority of A. baumannii strains have developed resistant to most of these antibacterial agents due to the presence of mobile genetic elements, such as insertion sequences (ISs), plasmids, integrons and resistance islands (2). The most important mechanism of β-lactam resistance in A. baumannii is production of various types of beta-lactamases like extended spectrum betalactamases (ESBLs) , AmpC and carbapenemases. The Ambler classification divides the β-lactamases into four major molecular classes (A-D). The extended-spectrum β-lactamases (ESBLs) belong to class A and can hydrolyze first, second and third-generation of Cephalosporins, Penicillins, and monobactams but are inhibited by clavulanate (3). AmpC β-lactamases production is mediated either by chromosomes or by plasmids and cannot be inhibited by clavulanic acid, but can be inhibited by cloxacillin. The enzymes belong to the molecular class C (4). Chromosomally encoded AmpC beta-lactamases in A. baumannii have a low level of expression that does not cause clinically appreciable resistance, however, the addition of a promoter insertion sequence ISA-ba1 next to the ampC gene increases beta-lactamase production, causing resistance to cephalosporins (5). Plasmid mediated AmpC and ESBLs can effortlessly move from one bacteria to another resulting in the faster spread of the resistance genes (2). Due to the emergence of ESBLs and AmpC beta-lactamase, Carbapenems like imipenem, meropenem are used extensively to treat infections in hospitalized patients. This resulted in unresponsiveness among bacteria to carbapenems. carbapenem resistance in A. baumannii is due to a range of mechanisms like decreased expression of outer membrane proteins and production of carbapenemases (6). Increasing proportion of A. baumannii producing carbapenemases has resulted in growing therapeutic concern worldwide due to very few treatment options available right now. The rapid identification of such isolates is the need of the hour to manage infections associated with such organisms and to prevent their spread (7). This study was carried out with an intention to quickly identify the alarmingly emerging carbapenemases and also to know the rate of ESBL and AmpC beta-lactamase from A. baumannii.

MATERIALS AND METHODS
The present study was carried out in a tertiary care teaching hospital for a period of one year. A total of 149 A. baumannii isolates were collected from clinical samples like endotracheal aspirates, sputum, urine, exudates etc from patients admitted during study period. Isolation of A. baumannii was done by standard culture techniques by inoculating clinical samples on to blood agar and MacConkey agar and incubating aerobically at 37°C.
Identification and drug sensitivity of A. baumannii was done using Vitek 2 system according to the manufacturer's guidelines. All the isolates were subjected to phenotypic detection of ESBL, AmpC and Carbapenemase.

ESBL detection.
A lawn culture of A. baumannii was done on quality control passed and sterility checked Mueller Hinton agar plate. A sterile disc of ceftazidime (30 μg) and ceftazidime and clavulanic acid (30 μg/10 μg) (Himedia) was applied at a distance of 20 mm from centre to centre on Mueller Hinton agar plate, and incubated at 37°C for 18 to 24 hours. A difference in the zone of more than or equal to 5 mm between the two discs was considered as ESBL producer (1).
AmpC disc test. Using 0.5 McFarland's matched broth of ATCC E. coli 252922 a lawn culture was done with sterile cotton swab. A sterile 30 µg cefoxitin disc (Himedia) was kept on agar surface and a blank sterile disc was moistened with sterile saline and was inoculated with few colonies of A. baumannii isolate and placed touching the cefoxitin disc. The plates were incubated at 37°C for 18 to 24 hours. The organism was considered as AmpC producer if flattening or indentation of inhibition zone of cefoxitin disc in the vicinity of the disc with A. baumannii (8).
Carbapenemase production: modified Hodge method. Using 0.5 McFarland's matched broth of ATCC E. coli 252922 a lawn culture was done with sterile cotton swab. 10μg meropenem disc (Himedia) was placed at the centre of the agar plate and A. baumannii isolate was streaked from the disc's edge to the periphery of the plate. The plate was then incubat-ed at 37°C for 18 to 24 hours. Appearance of a clover leaf shaped zone of inhibition along the growth of A. baumannii indicates carbapenemase production (9).
CarbAcineto NP. From overnight growth of A. baumannii on Mueller-Hinton agar (MHA) two to three colonies were placed in two 1.5-ml Eppendorf tubes (A and B) containing 100 µl of 5 M NaCl and mixed using a vortexer for 5 s. Both the tubes were mixed with 100 µl of a pH adjusted 0.05% phenol red containing 0.1 mmol/liter ZnSO4. Then to the tube B 12 mg/ml Imipenem-cilastatin injectable drug was added. Both the tubes were incubated at 37°C for 2 hours. The carbapenemase produced by the bacterial strains could hydrolyze the imipenem into a carboxylic derivative and result in decrease in pH. This reduction in pH is indicated by color change of the phenol red solution. Any change in color from red to orange/yellow in the antibiotic containing tube (tube B) indicates carbapenemase activity (9).

RESULTS
During study period, 149 A. baumannii non repetitive clinical isolates were analysed for antimicrobial susceptibility and various beta-lactamase production. Results were evaluated for statistical significance using Chi-Square and P value.
Out of 149 isolates, 122 (81.8%) were from male patients and 28 (18.7%) were from female patients. Maximum number of isolates studied were from patients in the age group of 41-50 (20%) and 51-60 years (18.6%). Age wise and gender wise distribution of patients from whom these clinical isolates grown is shown in Table 1. Table 1. Age and sex wise distribution of the cases Maximum number of clinical isolates were from endotracheal aspirates (88.5%) of patients with suspected ventilator associated pneumonia (VAP) followed by sputum from patients diagnosed with nosocomial pneumonia and exudates samples (4.6%) two each from diabetic wound and burns wound. Distribution of isolates according to sample source is shown in Table 2.
Out of 132 patients with suspected VAP 84 were admitted in neuro ICU, 37 were admitted in RICU and 11 patients were in MICU. Piperacillin-tazobactam, meropenem or imipenem were predominant antibiotics started in these patients. Later most of these patients were started with tigecycline or colistin based on antimicrobial susceptibility which was performed using N-281 card of Vitek 2 system. Majority of the isolates were resistance to commonly used cephalosporins like ceftazidime, cefipime. Only 10% to 12% of isolates were sensitive to carbapenems like imipenem, meropenem and doripenem. The most susceptible drug was found to be the tigecycline (83.8%) followed by minocycline (61.7%). Antimicrobial resistance pattern of these isolates is depicted in Table 3. Out of 149 A. baumannii only 23.4% of isolates were ESBL producers where as 46.9% of them were AmpC producers. Distribution of these beta-lactamases is shown in Table 4

DISCUSSION
A. baumannii belongs to the Moraxellaceae family commonly associated with infections like hospital-acquired and ventilator-associated pneumonia, urinary tract infections and pyogenic infections. A. baumannii is one of the ESKAPE organisms that pose a global threat to human health (5).
A. baumannii has developed resistance to many groups of antibiotics due to indiscriminate use of antimicrobials (10). One of the major concerns is development of resistance to beta-lactam group of antibiotics as this result in restricted treatment options for treating A. baumannii infections and can contribute to increased morbidity and mortality in ICU patients (11). The overall prevalence of multidrug-resistant strains in patients with A. baumannii hospital-acquired and ventilator-associated pneumonia is estimated to be 79.9%, ranging from 56.5% in Argentina and 61.8% in Taiwan to 100% in Central America, Pakistan, Lebanon, Qatar, and Croatia, while its overall mortality can be as high as 56.2%. Carbapenems were the treatment of choice for multidrug resistant A. baumannii infections. Their extensive use has led to increased incidence of carbapenem resistance in recent years (5). One of the frequent ways of developing resistance to carbapenems in A. baumannii is production of carbapenemase especial-ly Ambler class D β-lactamases and to a lesser extent class B β-lactamases [metallo-β-lactamases (MBLs)] which includes four types like IMP, VIM, NDM, and SIM. Most of the class D β-lactamases are not inhibited by β-lactamase inhibitors (12). Other types of beta-lactamases in A. baumannii includes Class A beta-lactamases like ESBLS, KPCs and Class C beta-lactamases, which are chromosomally encoded cephalosporinases (Acinetobacter derived cephalosporinase, ADC. Carbapenem resistant A. baumannii was ranked in 2018 by WHO as number one priority for antibiotic research and development (5). So need of the hour is rapid identification of A. baumannii producing such carbapenemase. In the present study around 149 A. baumannii were screened for rapid identification of carbapenemase and also other beta lactamases like ESBLs and AmpC.
In the present study 122 (81.8%) clinical isolates included were from male patients with highest number of isolates from patients above 50 years of age. In accordance to present study TrangDinhVan et al. also observed male predominance (72.7%) and aged above 50 years (13). These findings suggest that male elderly were more vulnerable for infections in both the studies. 88% of A. baumannii during study period were from endotracheal aspirates followed by exudate samples. In a study done by Uzma Rehman et al. also reported similar findings (8). These findings highlights that A. baumannii is the predominant organism causing ventilator associated pneumonia.
In this study 83.8% of A. baumannii were sensitive to tigecycline. Sensitivity to carbapenems ranged between 10% to 12%. Michelle Lowe et al. from South Africa also, reported carbapenem sensitivity in the range of 11% to 13% and 90% of the isolates were resistant to tigecycline (12). In a study from Lebanon, Micheline Hajjar Soudeiha et al. reported carbapenem sensitivity between 16% to 23% and only one isolate was reported as resistant to colistin (14). These findings suggest that antimicrobial susceptibility patterns vary from place to place. Since present study was done in a tertiary care hospital where the patients are referred who are terminally ill and treated with broad spectrum antibiotics elsewhere could be the reason behind increased resistance to higher antibiotics.
ESBL identification in Acinetobacter spp. is not easy as standard protocols are not available unlike in Enterobacteraceae (15). 23 (23). Phenotypic detection of carbapenemases has the advantages of low cost, ease of procedure and the absence of complicated or expensive equipment; however, it suffers from poor specificity and sensitivity unlike PCR. So it is better to use more than one method for not missing out carbapenemase producers however it may not be possible to adapt all the different methods in routine laboratory practice. It is better to use more rapid, reliable, cost effective methods like CarbAcineto NP or modified carbapenem inactivation method (mCIM) methods. One of the limitations of the present study is not evaluating sensitivity and specificity of phenotypic methods by genotypic detection methods due to cost constraints.

CONCLUSION
Acinetobacter are "superbugs" causing infections in the intensive care units. Multidrug resistant Acinetobacter spp. is on the rise making the treatment more difficult. The present study showed that high percentage of drug resistance in A. baumannii and also prevalence of ESBLs, AmpC and carbapenemase among these isolates is high. Rapid, cost effective, assay which can be adopted in all clinical laboratories is critical to prevent their further transmission particularly in hospital environment.