Detection and phenotypic characterization of carbapenem non susceptible gram-negative bacilli isolated from clinical specimens

Background Multi-drug resistant, extremely drug-resistant, pan-drug resistant, carbapenem-resistant, and carbapenemase-producing gram-negative bacteria are becoming more common in health care settings and are posing a growing threat to public health. Objective The study was aimed to detect and phenotypically characterize carbapenem non susceptible gram-negative bacilli at Ethiopian Public Health Institute. Materials and methods Prospective cross-sectional study was conducted from June 30, 2019, to May 30, 2020, at the national reference laboratory of the Ethiopian Public Health Institute. Clinical samples were collected, inoculated, and incubated in accordance to standard protocol for each sample. Antimicrobial susceptibility testing was done using Kirby Bauer disk diffusion. Identification was done using the traditional biochemical method. Multidrug-resistant and extensively drug-resistant were classified using a standardized definition established by European Centers for Disease prevention and control and the United States Centers for Disease prevention and control experts. Carbapenemase production was confirmed by modified carbapenem inactivation and a simplified carbapenem inactivation method. Meropenem with EDTA was used to differentiate serine carbapenemase and Metallo β-lactamase. Results A total of 1337 clinical specimens were analyzed, of which 429-gram negative bacilli isolates were recovered. Out of 429 isolates 319, 74, and 36 were Enterobacterales, Acinetobacter species, and P. aeruginosa respectively. In our study, the prevalence of Multidrug-resistant, extensively drug-resistant, Carbapenemase-producing, and carbapenem non-susceptible Gram-negative bacilli were, 45.2%, 7.7%, 5.4%, and 15.4% respectively. Out of 66 isolates screened for Carbapenemase, 34.8% (23/66) were Carbapenemase enzyme producers. Ten out of twenty-three Carbapenemase-positive organisms were Metallo-beta-lactamase producers. Thirteen out of twenty-three isolates were serine carbapenemase producers. Three out of 13 serine Carbapenemase positive organisms were Klebsiella pneumoniae Carbapenemase. Conclusion The finding from this study revealed a high prevalence of Multidrug-resistant, extremely drug-resistant, carbapenemase-producing gram-negative bacteria, particularly among Intensive care unit patients at the health facility level, this necessitates a robust laboratory-based antimicrobial resistance monitoring and infection prevention and control program.

Hinton agar to determine antimicrobial susceptibility patterns of the isolates and CLSI M100 2020 147 was used to interpret the results [12]. 148 The Antimicrobial discs used for Kirby Bauer disk diffusion method were in the following Carbapenemase detection, but this method can only be used for the accurate detection of KPC-type 157 Carbapenemase in Enterobacteriaceae [13]. CLSI (2012) recommended the Carba NP test method 158 for the detection of Carbapenemase in gram-negative bacilli; however, the preparation of the 159 reagents required for this test is complicated and the solutions cannot be stored for extended periods, 160 limiting its clinical application [14]. 161 In 2015 a new detection method, the carbapenem inactivation method (CIM), which is easy to 162 operate and highly sensitive in the detection of Carbapenemase was designed [15]. In 2017, based 163 on the CIM method, CLSI recommended the modified carbapenem inactivation method (mCIM),

164
However; it is a relatively complex method and can only be used to detect Carbapenemase in 165 Enterobacteriaceae and P. aeruginosa [16]. In 2018, based on the mCIM, a simplified carbapenem 166 inactivation method (sCIM) was designed for simple and accurate detection of Carbapenemase in 167 gram-negative bacilli [17].
Modified Carbapenem Inactivation method. 169 In the mCIM, 1 mL loop full of Enterobacteriaceae or 10 mL loop full of P. aeruginosa from blood 170 agar plates was emulsified in 2 mL trypticase soy broth (TSB mm was considered to be a negative result [12].

180
The sCIM is based on the mCIM with an improvement of experimental procedures. Instead of 181 incubating the antimicrobial disk in the organism culture media for 4 hours as in the mCIM, the 182 organism to be tested was smeared directly onto an antimicrobial disk in the sCIM. To perform 183 the sCIM, for Acinetobacter species, a 0.5 McFarland standard suspension (using direct colony 184 suspension method) of E. coli ATCC 25922 was diluted 1:10 in saline and inoculated onto the 185 MHA plate, following the routine disk diffusion procedure. Plates were allowed to dry for 3-10 186 min [17]. 187 Then, 1-3 overnight colonies of the test organisms grown on blood agar were smeared onto one 188 side of an imipenem disk (10µg); immediately afterward, the side of the disk having bacteria was 189 placed on the MHA plate previously inoculated with E. coli ATCC 25922. An imipenem disk placed 190 on an MHA plate was used as the control [17].

191
All plates were incubated at 35C for 16-18 h in ambient air. Bacterial strains that produced 192 Carbapenemase can hydrolyze imipenem; hence the susceptible indicator strain grew unchecked.

193
Zone of inhibition around the disk shows a diameter of 6-20 mm, or the satellite growth of colonies 194 of E. coli ATCC 25922 around the disk with a zone diameter ≤22 mm, was considered 195 carbapenemase positive; a zone of inhibition ≥26 mm was considered to be a negative result; a zone 196 of inhibition of 23-25 mm was considered to be a carbapenemase indeterminate result [17]. negative. [18]. 214 Well, isolated colonies from an overnight blood agar plate were suspended into saline to achieve a   The highest number of MDR, XDR, and carbapenemase-producing isolates were recovered from 261 specimens referred from Aabet hospital, Ras Desta hospital, and Saint Peter hospital respectively.  Out of four hundred twenty-nine (429) Table 2. Three KPC isolates 293 were, E. coli, K. Pneumoniae, and P. aeruginosa Table 2.  Out of 194 MDR GNB isolates, 45% were isolated from patients admitted to the intensive care unit, 83.4% were isolated from patients previously exposed to different antimicrobial agents, 28% were isolated from patients under mechanical ventilation and/or urinary catheterization, and 34.7% were isolated from patients with hospital-acquired pneumonia and hospital-acquired infection Table 3.  Table 3.  vulgaris were intrinsically resistant to ampicillin, cefazolin, and cefuroxime and were not reported in Table 5. M.morganii, P.mirabilis, and P.vulgaris intrinsically to Antimicrobials were summarized in Table 5

Discussion
The prevalence of XDR, MDR, Carbapenemase-producing, and Carbapenem-resistant GNB is increasing [5,6,19]. In our study, the prevalence of MDR, XDR, Carbapenemase-producing, and carbapenem non-susceptible GNB is high. The most frequently isolated XDR organisms were pneumoniae accounts for 100%, 40%, 50%,36%, and 75% of the gram-negative bacilli isolates respectively, the observed difference could be attributed to the types of gram-negative bacteria analyzed, as most of them only included Enterobacteriaceae, the techniques used, geographical location, and so on.
The total resistance profile of Enterobacterales to extended-spectrum cephalosporins ranges from 57.7% to 80.8%, which marginally agrees with results of study conducted by Beyene et al. 73.5% to 73.9% [19], but higher than the study findings of Teklu

Conclusion and Recommendations
The finding from this study revealed a high prevalence of Multidrug-resistant (MDR), Extremely drug-resistant (XDR), carbapenemase-producing gram-negative bacteria, particularly among Intensive care unit (ICU) patients at the health facility level This necessitates a robust laboratorybased antimicrobial resistance monitoring and infection prevention and control program.

Limitations of the study
The responsible genes for carbapenemase production were not genotypically assessed. Possible patient clinical impact was not assessed. Tigecycline, colistin, and Fosfomycin were not available and not used for AST.