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M,
JOURNAL OF CLINICAL MEDICINE OF KAZAKHSTAN
(E-ISSN 2313-1519)
Original Article
DOI: https://doi.org/10.23950/jcmk/12648
Molecular analysis of metallo-ß-lactamase genes in some gram-negative bacteria and examination of the phylogenetic relationships of isolates
Farzad Heydari1, Fatih Koksal1, Cansu Onlen Guneri2, Suna Kizilyildirim3
'Department of Medical Microbiology, Medical Faculty, Cukurova University, Adana, Turkey
department of Medical Laboratory Techniques, Gulhane Vocational School of Health Services, University of Health Sciences, Ankara, Turkey Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Suleyman Demirel University, Isparta, Turkey
Abstract
Aim: This study aimed to determine the susceptibility of carbapenem-resistant Gr (-) bacilli isolated from various clinical infections to various antibiotics and identify genes causing carbapenem resistance and their clonal relationships to elucidate the distribution of resistance in community and/or hospital-acquired strains.
Material and methods: In this study, antibiotic susceptibilities of 450 carbapenem-resistant Gr (-) bacilli isolated from clinical specimens at Cukurova University, Faculty of Medicine, Balcali Hospital, were investigated using phenotypic methods. The presence of carbapenems and D-lactamase genes were searched using polymerase chain reaction (PCR) and sequence analysis methods. Pulsed-field gel electrophoresis (PFGE) method was used to evaluate the phylogenetic relationship of the isolates.
Results: Based on the results, it was determined that 99.23% of the strains had gained resistance to meropenem, whereas 5.38% had developed resistance to colistin. The most dominant carbapenems genes in all isolates were OXA-51, OXA-23-like and OXA-24-like.
Conclusion: It was observed that the only antibiotic that could be used safely in carbapenem-resistant Gr (-) bacilli infections was colistin. In addition, when the clonal relationship of the strains was examined, it was found that the clones considered to be closely related persisted, and these clones settled in different clinics of our hospital.
Key words: Carbapenem, MBL, PFGE, CLSI
Received: 2022-08-26. Accepted: 2022-10-21
© ®
This work is licensed under a Creative Commons Attribution 4.0 International License
J Clin Med Kaz 2022; 19(6):18-26
Corresponding author: Cansu Onlen Guneri. E-mail: [email protected]; ORCID: 0000-0002-6112-0693
Introduction
Gr (-) bacteria are among the leading factors of hospital and community-acquired infections. Due to the outer membrane structure in the cell walls, it is resistant to many antibiotics compared to gram-positive bacteria, and it gains multiple resistance characteristics with the transfer of genetic material and/or the selective pressure of antibiotics in the hospital environment, creating problems for infections treatment caused by these bacteria. Resistant strains of these bacteria are more fatal when they cause infections, especially in patients who are observed in intensive care units. Enterobacteriaceae
species and non-fermentative bacteria, such as Acinetobacter and Pseudomonas, are very important because they cause infections transmitted during hospital service and can easily transfer genes encoding resistance enzymes [1-3].
Enterobacteriaceae species, which are commensally found in flora, cause many infections as a primary or secondary pathogen in extraintestinal colonization, especially in people with diabetes, immunodeficiency, using immunosuppressive drugs, susceptible to infections, such as cancer patients, invasive instrument users and the elders. Escherichia coli, Klebsiella spp.,
Enterobacter spp., Proteus spp., Serratia spp., and Salmonella species are frequently isolated, especially in infections acquired during hospital service [4-7].
Acinetobacter spp and P aeruginosa species are at the forefront among clinically important non-fermentative Gr (-) bacteria. Due to the increasing antibiotic resistance, there are challenges in the treatment of infections caused by these bacteria [8].
Many p-lactamase enzymes that can hydrolyze penicillins, cephalosporins, monobactams, and carbapenems have been found in the majority of Gr (-) bacteria. [9]. Metallo-p-lactamases (MBL) plays a critical role in developing resistance against carbapenem group antibiotics, which is a good option in treating severe infections caused by resistant bacteria against most antibiotic groups. Carbapenems are a type of p-lactamase that causes a broader spectrum of antibiotic resistance [10]. Carbapenems, also known as MBL, are quite common, especially among E. coli and Klebsiella strains. In hospitals, it is essential to know the type of p-lactamase in the causative pathogen to optimize the treatment protocols for the patients infected with Expanded Spectrum p-lactamase, induced p-lactamases and MBL-producing strains. This study aimed to enlighten the regional epidemiology of MBL positive isolates that cause major problems in treatment and to type the carbapenemase enzymes. Thus, the presence, types, and frequencies of MBLs in Gr (-) bacilli isolated from clinical samples of patients treated at Cukurova University, Balcali Hospital were investigated in this study. Phylogenetic analysis was performed to elucidate the distribution of the clonal association and the resistance of the community or the hospital-acquired strains.
resistance. Escherichia coli ATCC 25922 was selected as a reference strain. Imipenem-EDTA double-disk synergy test was used to detect MBL production in the isolates included in the study. To determine the susceptibility of the isolates included in the study against various antibiotics, the Kirby-Bauer Disk Diffusion test was performed according to CLSI recommendations [11]. Sequence analysis studies were conducted for the presence of carbapenemase and ß-lactamase genes in isolates, using the automated system of "ABI Prism 310 DNA sequencer (Applied Biosystems)" with PCR-multiplex-PCR [12-14]. The data obtained by sequence analysis were compared with the gene bank database using the BLAST program on the "National Center for Biotechnology Information" (NCBI) web page (htpp: //www.ncbi.nlm.nih.gov/BLAST/). The pulsed-field gel electrophoresis method, which is the gold standard, was used to evaluate the phylogenetic relationship between isolates [15, 16]. This study was approved by the Non-Interventional Clinical Research Ethics Committee of the Medical Faculty of Cukurova University (Date: 06.12.2013 and Decision No: 19).
Results
In the current study, 450 isolates of MBL-resistant Gr (-) bacilli (A. baumannii (n=290), P. aeruginosa (n=75), K. pneumoniae (n=40), E. coli (n=20), P. mirabilis (n=10), Enterobacter cloacae (n=13) and Chryseobacterium indologenes (n=2)) were subjected to double-disk synergy and MHT tests; 130 of these isolates were found to be MBL positive. A. baumannii (n=88), P. aeruginosa (n=26), K. pneumoniae (n=9), E. coli (n=3), P. mirabilis (n=1), E. cloacae (n=1), and C. indologenes (n=2) were among the isolates. (Table 1).
Material and methods
A total of 450 Gr (-) bacilli isolates were isolated from various clinical materials at Cukurova University, Faculty of Medicine, Balcali Hospital and identified with the VITEK-II device, and were discussed to shed light on the epidemiology of nosocomial infections. Isolates were verified with conventional culture methods, biochemical tests, and the BD-Crystal Enteric/ Nonfermented Identification kit. Phenotypic Carbapenems production was performed by Modified Hodge Test (MHT) according to Clinical Laboratory Standards Institute (CLSI) criteria. MHT is considered the gold standard for carbapenem
Species distribution of 130 isolates included in the study
Isolates No Percentage %
A. baumannii 88 67.69
P. aeruginosa 26 20
K. peumoniae 9 6.92
E. coli 3 2.30
C. indologenes 2 1.53
P. mirabilis 1 0.76
E. cloacae 1 0.76
Total 130
Antibiotic susceptibility test result of A. baumannii isolates
Table 1
ANTIBIOTIC RESISTANT INTERMEDIATE SUSCEPTIBLE
Number Percentage % Number Percentage % Number Percentage %
AMIKACIN 51 57.95 3 3.4 34 38.63
AMPICILLIN SULBACTAM 85 96.59 3 3.40 - -
CEFEPIME 13 97.72 1 2.27 - -
CEFTAZIDIME 87 98.86 1 1.13 - -
CIPROFLOXACIN 85 96.59 1 1.13 2 2.27
COLISTINE 1 1.13 - - 87 98.86
GENTAMICIN 79 89.77 - - 9 10.22
IMIPENEM 87 98.86 1 1.13 -
LEVOFLOXACIN 77 87.5 11 12.5 -
MEROPENEM 88 100 - - -
PIPERACILLIN 88 100 - - -
TETRACYCLINE 67 76.13 1 1.13 20 22.72
TIGECYCLINE 12 13.63 18 20.45 58 65.90
TRIMETOPRIM SULFAMETOXAZOL 82 93.18 - - 6 6.81
PIPERACILLIN/TAZOBACTAM 88 100 - - -
Kirby-Bauer Disk Diffusion test was performed according to CLSI recommendations to determine the sensitivity of 130 isolates included in the present study against various antibiotics. According to these results, more than 50% resistance was observed in A. baummanii isolates against other tested antibiotics except tigecycline and colistin antibiotics. Moreover, Pseudomonas isolates were more than 50% resistant to all tested antibiotics. In K. pneumoniae, high resistance rates were found
Table 3
against antibiotics other than colistin and amikacin. Also, in E. coli high resistance rates were found against antibiotics other than colistin and fosfomycin. The results of Kirby-Bauer Disk Diffusion antibiotic susceptibility tests are given in Tables 2-7. Considering all the isolates included in our study, the highest resistance developed against meropenem with 99.23%, and the lowest resistance against colistin with 5.38%.
Antibiotic susceptibility test result of P. aeruginosa isolates
ANTIBIOTIC RESISTANT INTERMEDIATE SUSCEPTIBLE
Number Percentage % Number Percentage % Number Percentage %
AMIKACIN 19 73.07 - - 7 26.92
AMPICILLIN SULBACTAM 26 100 - - - -
CEFEPIME 16 61.53 7 26.92 3 38.46
CEFOPERAZONE SULBACTAM 18 69.23 7 26.92 1 3.84
CEFTAZIDIME 16 61.53 6 23.07 4 15.38
CIPROFLOXACIN 18 69.23 - - 8 30.76
COLISTINE - - - 26 100
GENTAMICIN 17 65.38 - - 9 34.61
NETILMICIN 16 61.53 6 23.07 4 15.38
IMIPENEM 26 100 - - -
LEVOFLOXACIN 18 69.23 - - 8- 30.76
MEROPENEM 25 96.15 1 3.84 - -
PIPERACILLIN 2 76.92 6 23.07 - -
TETRACYCLINE 26 100 - - -
TIGECYCLINE 25 96.15 1 3.84 - -
TRIMETOPRIM SULFAMETOXAZOL 26 100 - - - -
PIPERACILLIN/TAZOBACTAM 26-100 - - - - -
Antibiotic susceptibility test result of K. peumoniae isolates
ANTIBIOTIC RESISTANT INTERMEDIATE SUSCEPTIBLE
Number Percentage % Number Percentage % Number Percentage %
AMIKACIN 4 44.44 5 55.55 - -
AMPICILLIN 9 100 - - - -
AMOXICILLIN-CLAVULANATE 9 100 - - - -
CEFEPIME 8 88.88 1 11.11 - -
CEFOXITINE 9 100 - - - -
CIPROFLOXACIN 9 100 - - - -
COLISTINE 3 33.33 - - 6 66.66
ERTAPENEME 9 100 - - -
GENTAMICIN 7 77.77 - - 2 22.22
IMIPENEM 8 88.88 1 11.11 - -
MEROPENEM 9 100 - - - -
TRIMETOPRIM SULFAMETOXAZOL 8 88.88 - - 1 11.11
PIPERACILLIN/TAZOBACTAM 9 100 - - - -
Antibiotic susceptibility test result of E. coli isolates
ANTIBIOTIC RESISTANT SUSCEPTIBLE
Number Percentage % Number Percentage %
AMIKACIN 2 66.67 1 33.33
AMOXICILLIN-CLAVULANATE 3 100 - -
AMPICILLIN 3 100 - -
CEFOXIDINE 3 100 - -
CEFTRIAXONE 3 100 - -
CEFUROXIM 3 100 - -
CYPROFLOXACIN 3 100 - -
ERTAPENEM 3 100 - -
FOSFOMYCIN - - 3 100
GENTAMICIN 2 66.67 1 33.33
IMIPENEM 3 100 - -
MEROPENEM 3 100 - -
NITROFURANTOIN 3 100 - -
PIPERACILLIN/TAZOBACTAM 3 100 - -
TRIMETHOPRIM/SULFAMETHOXAZOLE 3 100 - -
COLISTIN - - 3 100
Table 6
Antibiotic susceptibility test result of Chryseobacterium indologenes isolates
ANTIBIOTIC RESISTANT SUSCEPTIBLE
Number Percentage % Number Percentage %
AMIKACIN 2 100
TICARCILLIN/CLAVULANIC ACID 2 100
CEFTAZIDIME 2 100
CEFOPERAZONE/SULBACTAM 2 100
CIPROFLOXACIN - - 2 100
COLISTIN 2 100
CEFAZOLIN 2 100
ERTAPENEM 2 100
CEFEPIME 2 100
CEFOXITINE 2 100
GENTAMICIN 2 100
IMIPENEM 2 100
LEVOFLOXACIN - - 2 100
MEROPENEM 2 100
PIPERACILLIN-TAZOBACTAM 2 100
TRIMETHOPRIM/SULFAMETHOXAZOLE - - 2 100
AZTREONAM 2 100
Antibiotic susceptibility test result of Proteus mirabilis and Enterobacter cloacae isolate
ß-lactamase genes amplified by PCR in P.
ANTIBIOTIC Resistant (R)- Intermediate (I)- Susceptible (S)
Proteus mirabilis Enterobacter cloacae
AMIKACIN S R
AMPICILLIN SULBACTAM R R
CEFEPIME I R
CEFOPERAZONE/SULBACTAM R R
CEFTAZIDIME I R
CIPROFLOXACIN S S
COLISTIN R S
GENTAMICIN S R
IMIPENEM R R
LEVOFLOXACIN S S
MEROPENEM R R
NETILMICIN S R
PIPERACILLIN R R
PIPERACILLIN-TAZOBACTAM R R
TETRACYCLINE R R
TIGECYCLINE R R
TRIMETHOPRIM/SULFAMETHOXAZOLE R R
PCR result amplified ß-lactamase genes in A. baumanii (88) isolates
ß-lactamase genes Number Percentage%
oxa 23 like 4 1S.3S
oxa 48 like 4 1S.3S
GES 2 7.70
CTX-M 1 3.SS
VIM 1 1 3.SS
1 ß-lactamase genes amplified by PCR in K. ^■■■¡¡■^^I pneumonia (9) isolates.
ß-laktamaz genleri Number Percentage%
oxa 4Slike 4 44.44
oxa 24 like 2 22.22
CTX-M 4 44.44
CTX-M 9 4 44.44
ß-lactamase genes Number of isolates Percentage%
OXA 51 LIKE 88 100
OXA 23 LIKE 54 боб
OXA 24 LIKE 15 17.04
NDM-1 2 2.2S
TEM 20 22.73
OXA 48 LIKE 3 3.40
GES 5 S.69
CTX-M 6 6.S2
CTX-M 1 1 1.14
CTX-M 2 1 1.14
CTX-M 9 2 2.2S
In-house PCR and Multiplex-PCR techniques targeting frequently seen p-lactamase genes were amplified in order to characterize carbapenemase genes.The most dominant carbapenems genes in all isolates had been found as OXA-51, OXA-23-like and OXA-24-like in Tables 8-11.
Sequence analyzes were performed by amplifying the detected MBL genes with specific primers. Also, it was defined at the sub-type of level. PCR results defining p-lactamase gene profiles within the species and the profiles of the gene sequenced are given in Table 12. Carbapenemase genes determined by sequence analysis were OXA-48, GES-11, VIM-1, NDM-1.
Other PCR analyses also revealed the presence of NDM-1 in one of three E. coli isolates, OXA-48 like genes in two isolates, and NDM-1, OXA-48 like genes, and CTX-M genes in one E. cloacae isolate. OXA-48, GES, and NDM-1 genes are given in Figure 1.
When the phylogenetic relationships of MBL resistant A. baumannii isolates (n: 88) were examined, it was determined that they were distributed in 17 clusters in a close relationship.
Table 11
Distribution of ß-lactamase genes determined by multiplex-PCR in all isolates
Code Species ß-lactamases Code Species ß-lactamases
1 A. baumannii OXA-51-like + OXA-24-like 66 A. baumannii OXA-51-like+OXA-23-like
2 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 67 A. baumannii OXA-51-like+NDM-1
3 A. baumannii OXA-51-like+OXA-24-like 68 A. baumannii OXA-51-like+TEM+ OXA-23-like
4 A. baumannii OXA-51-like+ OXA-24-like 69 A. baumannii OXA-51-like+GES+ OXA-23-like
5 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 70 A. baumannii OXA-51-like+ OXA-23-like
6 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 71 A. baumannii OXA-51-like+ OXA-23-like
7 A. baumannii OXA-51-like+ OXA-23-like+OXA-24-like 72 A. baumannii OXA-51-like+CTX-M9
8 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 73 A. baumannii OXA-51-like+ OXA-23-like
9 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 74 A. baumannii OXA-51- like +TEM+ GES +OXA-23-like
10 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 75 A. baumannii OXA-51-like+CTX-M+ OXA-23-like
11 A. baumannii OXA-51-like+CTX-M2 76 A. baumannii OXA-51-like+TEM+OXA-23-like+ OXA-48-like
12 A. baumannii OXA-51-like+ OXA-24-like 77 A. baumannii OXA-51-like+ OXA-23-like
13 A. baumannii OXA-51-like 78 A. baumannii OXA-51-like+ OXA-23-like
14 A. baumannii OXA-51-like 79 A. baumannii OXA-51-like+OXA-23-like
15 A. baumannii OXA-51-like 80 A. baumannii OXA-51-like+ OXA-23-like +OXA-24-like
16 A. baumannii OXA-51-like+ OXA-23-like 81 A. baumannii OXA-51-like+OXA-23-like+ OXA-24-like
17 A. baumannii OXA-51-like 82 A. baumannii OXA-51-like+ OXA-24-like
18 A. baumannii OXA-51-like 83 A. baumannii OXA-51-like
19 A. baumannii OXA-51-like 84 A. baumannii OXA-51-like+ CTX-M9+ OXA-23-like+ OXA-24-like
20 A. baumannii OXA-51-like+ OXA-23-like+ OXA-48-like 85 A. baumannii OXA-51-like
21 A. baumannii OXA-51-like 86 A. baumannii OXA-51-like
22 A. baumannii OXA-51-like 87 A. baumannii OXA-51-like+GES+ OXA-23-like
23 A. baumannii OXA-51-like 88 A. baumannii OXA-51-like+CTX-M1
24 A. baumannii OXA-51-like 89 P. aeruginosa -
25 A. baumannii OXA-51-like+GES 90 P. aeruginosa -
26 A. baumannii OXA-51-like+TEM+OXA-23-like 91 P. aeruginosa OXA-23-like
27 A. baumannii OXA-51-like+ TEM 92 P. aeruginosa OXA-48-like
28 A. baumannii OXA-51-like+ TEM+ OXA-23-like 93 P. aeruginosa OXA-48-like
29 A. baumannii OXA-51-like+ OXA-23-like 94 P. aeruginosa -
30 A. baumannii OXA-51-like+ TEM+ OXA-23-like 95 P. aeruginosa VIM1
31 A. baumannii OXA-51-like+ OXA-23-like 96 P. aeruginosa -
32 A. baumannii OXA-51-like 97 P. aeruginosa GES
33 A. baumannii OXA-51-like+ OXA-23-like 98 P. aeruginosa -
34 A. baumannii OXA-51-like 99 P. aeruginosa -
35 A. baumannii OXA-51-like+ TEM 100 P. aeruginosa CTX-M
36 A. baumannii OXA-51-like+ TEM 101 P. aeruginosa OXA-48
37 A. baumannii OXA-51-like+ OXA-23-like 102 P. aeruginosa -
38 A. baumannii OXA-51-like+ OXA-23-like 103 P. aeruginosa OXA-48
39 A. baumannii OXA-51-like+ OXA-23-like 104 P. aeruginosa -
40 A. baumannii OXA-51-like+TEM+OXA-23-like 105 P. aeruginosa OXA-23
41 A. baumannii OXA-51-like+NDM-1+ OXA-23-like 106 P. aeruginosa -
42 A. baumannii OXA-51-like 107 P. aeruginosa -
43 A. baumannii OXA-51-like+TEM+OXA-23-like 108 P. aeruginosa OXA-23
44 A. baumannii OXA-51-like+TEM+ OXA-23-like 109 P. aeruginosa -
45 A. baumannii OXA-51-like+ OXA-23-like 110 P. aeruginosa -
46 A. baumannii OXA-51-like+ OXA-23-like 111 P. aeruginosa GES
47 A. baumannii OXA-51-like+- OXA-23-like 112 P. aeruginosa OXA-23
48 A. baumannii OXA-51-like+ TEM 113 P. aeruginosa -
49 A. baumannii OXA-51-like 114 P. aeruginosa -
50 A. baumannii OXA-51-like 115 K. pneumoniae OXA-48+ CTX-M9
51 A. baumannii OXA-51-like+ OXA-23-like+ OXA-24-like 116 K. pneumoniae OXA-48+ CTX-M9
52 A. baumannii OXA-51-like+ OXA-23-like 117 K pneumoniae -
53 A. baumannii OXA-51-like+OXA-23-like 118 K pneumoniae OXA-48
54 A. baumannii OXA-51-like 119 K pneumoniae IMP
55 A. baumannii OXA-51-like+ OXA-23-like 120 K pneumoniae -
56 A. baumannii OXA-51-like+ TEM 121 K pneumoniae OXA-24+-CTX-M9
57 A. baumannii OXA-51-like+ TEM+ OXA-23-like 122 K pneumoniae OXA-24+CTX-M9
58 A. baumannii OXA-51-like+TEM+OXA-23-like 123 K pneumoniae OXA-48
59 A. baumannii OXA-51-like+TEM+OXA-23-like 124 E.coli OXA-48
60 A. baumannii OXA-51-like+CTX-M+OXA-23-like 125 E.coli NDM-1
61 A. baumannii OXA-51-like+ OXA-23-like 126 E.coli OXA-48
62 A. baumannii OXA-51-like+TEM+OXA-23-like 127 Chryseobacterium indologenes
63 A. baumannii OXA-51-like+ TEM+ OXA-23-like 128 Chryseobacterium indologenes
64 A. baumannii OXA-51-like+GES+OXA-23-like 129 Enterobacter cloacae NDM-1+OXA-48+CTX-M1
65 A. baumannii OXA-51-like+ TEM+ OXA-23-like+ OXA-48-like 130 Proteus mirabilis
Table 12
Carbapenemase genes determined by sequence analysis
OXA-48 GES VIM NDM
A. baumannii 3* OXA-48 5*GES-11 - 2*NDM-1
P. aeruginosa 4* OXA-48 1*GES-11+1*GES-12 1*VIM-1 -
K pneumoniae 4* OXA-48 - - -
E.coli 2* OXA-48 - - 1*NDM-1
Chryseobacterium indologenes - - - -
Enterobacter cloacae 1* OXA-48 1*NDM-1
Moreover, the strains that form the A1, A2, C1, D, F, H3, L1, N, O1, O2, P1, P2 and R clusters among these clusters were 100% similar. The largest cluster was formed by the L cluster with four sub-members (L1- 100%, L1-L2 96.8%, L2-L3
Figure 1 - Gel image showing the band profile of the 281 bp OXA-48 gene, 399 bp GES gene, 129 bp NDM-1 gene.
Figure 2 - Phylogenetic relationships of A. baumannii isolates.
96%, L3-L-4 94%), and the second-largest cluster was the four sub-membered C cluster (C1-100%, C1-C2 95.8%, C2-C3 93.3%, C3-C4 87.9%). When we examined the distribution of p-lactamase genes in A. baumannii isolates, which we evaluated as closely related, OXA-51-OXA-24 genes in A cluster, OXA-51-OXA-23-OXA-24 genes in B, C, D clusters, OXA-51-TEM gene in cluster E, OXA-51-TEM-OXA23 genes in clusters F, G, H, I, J, OXA-51- OXA23 genes in clusters K, N, O, P, OXA-51-OXA-48- OXA-23 genes in cluster L, also OXA-51-GES-OXA-23 genes in cluster M. OXA-51 gene were detected in 88 A. baumannii isolates. OXA-23 gene was the second most common in closely related isolates. Later, TEM and OXA-24 genes were detected. In addition to this, A. baumannii isolates were persistent at Balcali Hospital and these isolates were settled in different clinics of the hospital (Figure 2).
When the phylogenetic relationships of P. aeruginosa isolates (n: 26) were evaluated, it was observed that they were distributed in six clusters (A-B-C-D-E-F) in close relation. It was analyzed that the C1 cluster was 100% similar and three sub-members (C1 100%, C1-C2 91.4%) constituted the largest cluster C. In addition, we determined that the members in the C cluster had the OXA-48 gene, the D cluster members had the GES gene, and the members in the E and F clusters had the OXA-23 p-lactamase gene (Figure 3).
As a result of the phylogenetic analysis of K. pneumoniae isolates (n: 9), we found that they were distributed into two closely related clusters (A-B). We determined that A1 and A2 clusters, which were sub-members of the four-member A cluster that made up the largest cluster, were 100% similar. We determined that the A1 cluster was closely related to the A2
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Figure 3 - Phylogenetic relationships of P. aeruginosa isolates
cluster at a rate of 94.1% and the B1-B2 members that formed the B cluster were closely related to each other at the rate of 92.1%. We determined that the OXA-48-CTXM-9 genes in the A1 cluster, the OXA-24-CTXM-9 genes in the A2 cluster and the OXA-48 gene in the B cluster (Figure 4).
Figure 4 - Phylogenetic relationships of K. pneumoniae
Discussion
Bacteria showing resistance to antibiotics have become a major problem in the world and our country. Incorrect and unconscious use of antibiotics has led to an increase in multi-resistant Gr (-) bacilli. Resistance genes are studied with considerable interest by researchers worldwide.
In our study, as a result ofphenotypic antibiotic susceptibility tests, we found that the isolates were 99.23% (129/130) resistant to meropenem, 98.46% (128/130) to imipenem, 88.46% (115/130) to ciprofloxacin, 86.92% (113/130) to cefepime and ampicillin- sulbactam, 70% (91/130) to tetracycline group antibiotics and 5.38% (7/130) to colistin. It is thought that there is a high degree of resistance against almost all antibiotics among non-fermenter bacillus and Enterobacteriaceae strains that have clinical importance in our region, and colistin can be used as the last option in untreatable infections.
After determining the antibiotic susceptibility of the isolates, carbapenemase genes were investigated genotypically. In A. baumannii isolates, OXA-51-like (100%), OXA-23-like (61.36%), TEM (22.73%), OXA-24-like (17.04%), CTX-M (6.82%), GES (5.69%), OXA-48-like (3.40%) and NDM-1 (2.28%) type p-lactamase genes were identified, respectively. In the study conducted by Ergin et al., they detected the genes OXA-23-like (31%), OXA-58-like (23%) and OXA-51-like [17]. When compared with our study, we found that OXA-51-like and OXA-23-like genes became more prevalent over the years. Another finding in our study was the detection of GES-11 type A group carbapenemase in five isolates (5.69%). The first isolate producing GES enzyme in our country was found by Bogaerts et al. in a patient transferred to Belgium in 2010 [18]. Later in 2013, it was reported that the GES enzyme was synthesized by A. baumannii isolates in two different studies conducted by the groups of Cicek and Zeka [19,20]. The high resistance rates in A. baumannii isolates in our study can be interpreted due to either carbapenemase genes or combined p-lactamases. However, future studies on porin protein permeability and efflux systems are important in terms of elucidating these mechanisms.
In our study, OXA-23-like, OXA-48, GES-11, GES-12, CTX-M and VIM-1 p-lactamase genes were detected in P. aeruginosa isolates, respectively. In their review of the literature, Breidenstein et al. attributed the reason why P aeruginosa is much more resistant to antimicrobials than other Gr (-) bacilli due to its reduced outer membrane permeability, improved efflux systems, different porin numbers and structures from other bacteria, and various resistance mechanisms, such as these [21]. Fernandez et al. reported that it could adapt better than other bacteria against various environmental stress factors, such as antibiotic pressure, nutrient deficiency, and insufficient breeding environment [22]. Such changes are controlled by genes, such as crc, lon, psrA, ampD, gyrA, nalA, nfxB, mexZ, phoQ in the genome [21]. In line with this information, the fact that p-lactamase genes were detected in P. aeruginosa isolates was because carbapenem resistance occurred with efflux systems or membrane permeability modifications.
We identified OXA-48 (44.44%), OXA-24 (22.22%), CTX-M (44.44%) and CTX-M9 genes in K. pneumoniae isolates included in this study, respectively. Guran et al., in a study they conducted in 2011, determined the frequency of CTX-M genes in community-acquired K. pneumoniae isolates as 88.8% [23]. However, in a similar study conducted in 2014, they found the total CTX-M frequency among Carbapenem-resistant K. pneumoniae isolates as 52% (13/25) [11]. In this
study, the frequency of CTX-M was 44.44%. The most common ß-lactamase genes in K. pneumoniae isolates in our study were OXA-48 and CTX-M genes, with a rate of 44.44%. Alp et al. reported in their epidemiological study that the prevalence of the OXA-48 gene in K. pneumoniae isolates was 91.5% in 2013 [24]. Nazik et al. reported that the prevalence of the OXA-48 gene in K. pneumoniae isolates was common in their study between 2011-2012 [25, 26]. In our study, we detected NDM-1 in one of the 3 E.coli isolates and OXA-48-like genes in 2. OXA-48 enzyme in K. pneumoniae isolates was identified for the first time in 2001 in Turkey. In later years, it was detected in various Enterobacteriaceae species and E. coli [27, 28]. Turkey is a country that is now considered to be endemic to the OXA-48 enzyme. It is stated that the strains carrying this enzyme are now circulating in society [29].
Carbapenemase enzyme types differ between regions or countries depending on the preferred frequency of antibiotic use. The movements of the strains producing carbapenemase enzyme should be limited with the help of molecular epidemiological methods in the community and hospital also the development of new resistance should be minimized. In molecular epidemiological studies conducted in many countries, especially in developing countries. It was observed that there was a large increase in the prevalence of strains producing carbapenemase enzyme in Gr (-) bacilli isolated from both hospital and community-acquired infections.
In the study conducted by Yang et al. in Korea, it was determined that all the carbapenem-resistantA. baumannii strains were clonally related to PFGE method [30]. In a study conducted in Greece, Pournaras et al. analyzed 17 carbapenem-resistant A. baumannii isolates using the PFGE method and identified six different clones [31]. In the 2009 SENRTY surveillance study covering ten countries (China, India, Indonesia, Thailand, Korea, Taiwan, Singapore, Australia, Hong Kong, and the Philippines), the results of the PFGE analysis of the Carbapenem-resistant A. baumannii isolates carrying the OXA-23 gene were determined and it was emphasized that it had an epidemic potential for these countries [32]. In a study conducted in the USA, it was stated that Carbapenem-resistant A. baumannii isolates carrying the OXA-23 gene were a significant threat for hospital infections [33]. Vahapoglu et al. stated in their study that the genes in carbapenem-resistant A. baumannii isolates were plasmid-derived and showed multiple clonalities with PFGE [34]. In a study conducted in South Korea, it was determined that there was 85% and more clonal association in 35 P. aeruginosa isolates with the IMP-6 gene using the PFGE method. It was observed that a single isolate spread throughout the country [35]. Between 2002 and 2006, Pitout et al. investigated MBL production and the presence of clonal association in 528 carpaneme-resistant P. aeruginosa isolates, 518 of which were isolated from four different hospitals and 10 from environmental samples. They detected VIM-2 in 178 isolates and IMP-7 in seven isolates. 178 VIM-positive isolates showed a binary closely related pattern. One hundred fifty-four of them formed a group, while 21 formed a different group. They found that 3-VIM-positive isolates were unrelated to these patterns. They reported that the IMP positive isolates were in a different group from these groups [36].
In our study, which was conducted to monitor the prevalence and movements of the strains producing hospital and community-acquired carbapenemase enzyme, it was observed that A. baumannii isolates (n: 88) were distributed in 17 clusters (A1, A2, C1, D, F, H3, L1, N, O1, O2, P1, P2, R) closely related, the strains forming these clusters were 100% similar. The most
distributed p-lactamase genes were OXA-51, OXA-23, TEM, OXA-24. It was detected that P. aeruginosa isolates (n: 26) were distributed in six clusters (A-B-C-D-E-F) closely related, C1 cluster was 100% similar, members in cluster C had OXA-48, E and F cluster members had OXA-23 p-lactamase genes. K. pneumoniae isolates (n: 9) were distributed in two closely related clusters (A-B). It was determined that the sub-member A1-A2 cluster of the four-member A cluster forming the largest cluster was 100% similar and the closely related K. pneumoniae isolates had OXA48-CTXM9 p-lactamase genes. In our study, similar to other studies, it was observed that some clones that we evaluated in close relation with the PFGE method persist in our hospital and these clones were located in different clinics. In our study, it was determined that there was a high degree of resistance against almost all antibiotics among non-fermenter bacillus and Enterobacteriaceae strains, which were clinically important in our region as in the whole world. An increase in the prevalence of strains with carbapenemase activity was found. In addition, it was observed that strains synthesizing NDM-1 type carbapenemase, which are very difficult to treat, became widespread in our country.
As a result, considering the data of epidemiological surveillance studies in our region, it is necessary to take urgent measures to develop new and more effective control measures, review and revise existing control measures, and develop and implement more rational, disciplined strategies. More regional epidemiological studies are needed in our country to restrict the
motility of strains carrying antibiotic resistance genes.
Disclosures: There is no conflict of interest for all authors.
Acknowledgments: We would like to thank Prof. Dr. Akgun Yaman for his contributions to our study, and we would like to thank Cukurova University for its support.
Funding: This study was supported by Cukurova University Research Projects as project number TF2014DI.
Ethics approval: This study was carried out with the approval of Cukurova University, Faculty of Medicine, Non-Interventional Clinical Research Ethics Committee (Date: 06.12.2013 and Decision No: 19).
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