Highest Resistance Rates in Hvkp Were Agains
Ann Clin Microbiol Antimicrob. 2020; 19: 12.
The emergence of the hypervirulent Klebsiella pneumoniae (hvKp) strains among circulating clonal complex 147 (CC147) harbouring bla NDM/OXA-48 carbapenemases in a 3rd care center of Iran
Omid Pajand
1Microbiology Section, Faculty of Medicine, Semnan Academy of Medical Sciences, Semnan, Islamic republic of iran
2Educatee Research Commission, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran
iiiSocial Determinants of Health Research Center, Semnan University of Medical Sciences, Semnan, Iran
Narges Darabi
oneMicrobiology Department, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran
Maedeh Arab
iiStudent Research Committee, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran
Raheb Ghorbani
3Social Determinants of Health Enquiry Middle, Semnan University of Medical Sciences, Semnan, Islamic republic of iran
Zakaria Bameri
ivInfectious disease and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran
Ali Ebrahimi
2Student Research Committee, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Islamic republic of iran
Zoya Hojabri
iMicrobiology Department, Kinesthesia of Medicine, Semnan University of Medical Sciences, Semnan, Islamic republic of iran
2Student Enquiry Committee, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Islamic republic of iran
Received 2022 October xiv; Accustomed 2022 Feb 12.
- Data Availability Argument
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The data can be accessible to the interested researchers by the corresponding writer on behalf of all authors on reasonable asking.
Abstract
Background
Klebsiella pneumoniae is a public health business organisation considering of its ability to develop multidrug resistance and hypervirulent genotypes, of those capsular types K1 and K2 cause community and nosocomial life-threatening infections. This study aimed to determine the antibiotic susceptibility patterns and genotypic traits of a collection of Klebsiella spp. isolates. Furthermore, the clonal relatedness of bla NDM producing strains was investigated.
Methods
During a 19-months surveillance study, 122 Klebsiella spp. isolates were cultured from extraintestinal specimens of patients admitted to the 3rd referral infirmary in Semnan, Iran. Isolates were identified using biochemical tests and subjected to determination of phylogroups, capsular types and virulence/resistance genes content. Hypervirulent K. pneumoniae (hvKp) strains were detected genotypically, and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting was used to determine the clonality of bla NDM producing strains.
Results
Multidrug resistant phenotype was detected in 75 (61.5%) isolates and amikacin was found equally the most strong antibiotic with the susceptibility rate of 85.two%. The carbapenemase genes were detected in 45 (36.8%) strains, including 21 (17.2%) bla OXA-48, 7 (5.vi%) bla NDM-i, 14 (eleven.4%) bla NDM-i/OXA-48 and 3 (two.4%) bla IMP- carrying strains, while 55 (45.08%) isolates showed carbapenem resistant phenotype. The firstbla NDM-ane carrying strain was cultured from a sputum specimen on March 2015, while the last positive i was recovered from blood culture on September 2016. Most of the isolates (lxxx.3%) belonged to phylogroup I, and bla NDM-i was identified among all three phylogroups. The ERIC-PCR clustered the 101 bla NDM negative and 21 bla NDM-1 positive isolates into 25 and v clusters, respectively, and the latter grouping belonged to clonal circuitous 147 (CC147). One K1 and 15 K2 bla NDM-1 negative isolates were detected, of those three strains were identified every bit hvKp. Five K2 positive strains, including four bla OXA-48 producer and ane hvKp sequence type 86 (ST86) were carbapenem resistant. Among carbapenem resistant isolates, CC147 strains harboured higher rates of siderophores iutA and ybtS.
Determination
The nowadays findings showed a hospital circulation of CC147 bla NDM-1 or bla NDM-1/OXA-48 producing strains, disseminated in different wards. The hvKp/ST86 strain expressing K2 capsular type and carbapenem resistant phenotype wasn't reported from Iran so far. And so, information technology seems that nosotros must be aware of the emergence and spread of new Thou. pneumoniae clones associated with resistant and hypermucoviscous phenotypes.
Keywords: hvKp, NDM, OXA-48, rmtC, Phylogroup, Siderophore, Capsular blazon
Background
Klebsiella pneumoniae, ane of the nigh of import members of the Enterobacteriaceae family, is the leading cause of both of the community and healthcare-associated infections. The sheathing, as a critical virulence factor in this organism, plays an important function in its pathogenesis and avoids phagocytosis [1]. Over the past decade, the emergence of hypervirulent variants of K. pneumoniae (hvKp) which characteristically limited hypermucoviscosity phenotype caused serious concerns [2]. These strains conduct virulence genes associated with invasive illness and may cause severe infections such every bit pyogenic liver abscesses and endophthalmitis, in immunocompetent, healthy individuals [2]. While the hvKp strains were rarely resistant to commonly used antibiotics when firstly described, the emergence of multidrug resistant (MDR) phenotypes among these strains are increasingly reported in recent years due to the dissemination of mobile genetic elements encoding drug resistance [2]. Of special business is the acquisition of carbapenem resistance genes and development of carbapenem resistant phenotype, since these agents are the last resort of antibiotics for treatment of infections caused by multidrug resistant organisms [ii].
Increasing recognition of carbapenemase producing K. pneumoniae, including grade A (KPC), course B (IMP, VIM and NDM) and class D (OXA-48-like enzymes) carbapenemases has led to international concern, as they are carried on transposable elements in association with other resistance determinants, such as Extended spectrum β-lactamases (ESBLs), ampC cephalosporinases and 16S rRNA methyltransferases [iii]. Indeed, the convergence of carbapenemase product and hypermucoviscosity phenotype in this organism poses an important threat to public health.
The bla NDM, a relatively newly described Metallo-β-lactamase (MBL), was first identified in K. pneumoniae and E. coli isolated from a Swedish patient who was hospitalized in India in 2008 [4]. Since and then, information technology has spread worldwide and now NDM producing Gram-negative bacilli have been reported from more than twoscore countries, and the Indian subcontinent and the Middle Eastward are considered equally the main reservoirs for bla NDM producing bacteria [5]. Iran, equally i of the Middle East countries, neighbor countries where the NDM and OXA-48 producing bacteria are endemic [6]. While bla NDM producing K. pneumoniaeastward isolates have been reported from unlike cities of Iran in recent years, sequence types of these isolates are determined and published from 3 cities located in the center, southward, and due south-eastward of this country [half-dozen–8]. In our previous study, we reported a relatively high prevalence of bla OXA-48/bla NDM-1 producing Enterobacteriaceae isolates collected from the large tertiary hospital of Semnan [9], an important city along the historical Silk Road. And so in this survey, our goal was first to decide the phylogenetic groups, capsular genotypes, hypermucoviscosity biomarkers, and resistance determinants of One thousand. pneumoniae isolates nerveless during 19-months surveillance study and 2d, to carry out sequence typing of representatives of NDM producing isolates based on the Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting.
Methods
Sample collection
A 19-months surveillance study was conducted in the main tertiary teaching hospital of Semnan, Iran (Kosar hospital). During March 2022 to September 2016, 122 non-indistinguishable K. pneumoniae isolates were recovered from clinical specimens of patients admitted to the infirmary. The isolates were cultured from different extraintestinal specimens including urine, wound, sputum, blood and tracheal aspirate. Specimens were collected as the routine diagnostic purposes. K. pneumoniae isolates were identified based on the biochemical reactions, including reaction on Triple Sugar Iron (TSI) agar, SH2/Indole/Motility (SIM) pattern, growth on Simmon-citrate agar medium, urease product on urea agar, Methyl Crimson/Vogues Proskauer (MR/VP), and Ornithine decarboxylase (OD) test. Isolates were confirmed by PCR in which both thou. pneumoniae subsp. Pneumoniae and subsp. ozaenae requite a 130 bp band, and subsp. rhinoscleromatis is negative [ten]. Grand. oxytoca species were identified based on the VP +/Indole +/OD negative, tests results [11].
Antimicrobial susceptibility testing
Antibiotic susceptibility patterns for 16 antibiotics were obtained using standard disc improvidence test. For carbapenem not-susceptible isolates (resistant to either of the imipenem, meropenem, and ertapenem) carbapenem MICs were determined using gradient East-exam strips (Liofilchem, Italian republic). Susceptibility testing results were interpreted according to the Clinical and Laboratory Science Institute (CLSI) recommendations. The halo zones of ≥ 23 mm and ≥ 22 mm were considered as susceptible breakpoints for imipenem/meropenem and ertapenem, respectively [12]. Multidrug-resistant (MDR) isolates were those resistant to at least one representative of ≥ 3 antimicrobial classes—that is, penicillins (ampicillin/sulbactam, piperacillin-tazobactam), Extended Spectrum Cephalosporins (ESCs)/monobactams (cefotaxime, ceftazidime, cefepime/aztreonam), aminoglycosides (gentamicin), fluoroquinolones (ciprofloxacin), and antifolate agents (trimethoprim-sulfamethoxazole) [13]. Extended spectrum β-lactamase (ESBL) producing strains were identified using phenotypic combined deejay test as recommended past CLSI [12].
Detection of resistancegenes and capsular typing
Genomic DNA of collected isolates was extracted using Cetyl trimethylammonium bromide (CTAB) method [14, fifteen]. The isolates conveying carbapenemases (bla NDM-, bla IMP-, bla VIM-, bla OXA-48, bla KPC) [xvi], extended spectrum β-lactamases (bla TEM-, bla SHV-, bla OXA-1, and CTX-M clusters including CTX-M-G1, G2, G8, G9 and G25) [sixteen], plasmid mediated quinolone resistance (PMQR) (qnrA, qnrB, qnrS, aac-6Ib-cr) [17], aminoglycoside resistance determinants (ARD) aac-6Ib, aac3IIa and 16S rRNA methyltrasfrases (armA, rmtB, rmtC) were detected as described previously [16]. The capsular genotypes, including K1 (using magA primers), K2, K5, K20, K54 and K57, which are strongly associated with invasive illness, were determined by multiplex PCR every bit described previously [10]. The presence of plasmid-encoding virulence genes, including iucA (aerobactin siderophore biosynthesis), the plasmid-borne rmpA gene (prmpA), prmpA2, (regulators of the mucoid phenotype via increased capsule production), and peg-344 (putative transporter) which have been shown experimentally to contribute to hypervirulence in in vivo infection models, was assessed equally described by Russo et al. [18]. Of the virulence genes which are associated epidemiologically with putative hvKp strains, carriage of the iroB (salmochelin siderophore biosynthesis), iutA (receptor for hydroxamate siderophore), allS (associated with allantoin metabolism), mrkD (type iii fimbrial adhesion) and ybtS (yersiniabactin) genes was investigated by multiplex PCR [xviii, xix]. Furthermore, the K2 positive strains were subjected to a multiplex PCR to identify the principal hvKp clones, including clonal group (CG) 380, sequence blazon (ST) ST86, ST65 and ST375, as described by Davenet et al. [twenty].
Phylogenetic determination and clonal relatedness
For phylogenetic analysis, gyrA PCR–RFLP using restriction enzymes TaqI and HaeIII was performed as described by Brisse et al. [21]. The clonality of strains was determined by ERIC-PCR fingerprinting and the obtained dendograms were analyzed with Bionumerics software, version 6.ane (Applied Maths, Sint-Martens-Laten, Belgium). The similarities in amplicon profiles were compared using a Die coefficient at 1% tolerance and 0.v% optimization, and a dendogram was constructed using the unweighted-pair grouping method, with a cutoff of 80% similarity [22]. Sequence type of isolates was adamant for representatives of NDM positive strains of each cluster of ERIC dendogram according to the K. pneumoniae MLST website (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html).
Statistical analysis
Dichotomous variables were described using frequencies and percentages, and they were compared using Chi square examination, as advisable. The criterion for statistical significance was P< 0.05. Data were analyzed with software SPSS version 16.
Results
Patients and isolates
In this written report, a total of 122 Klebsiella spp. isolates were recovered from all clinical specimens except for stool. Almost of the isolates were cultured from urine (80, 65%), followed past sputum (33, 26.eight%), wound (5, 4.1%), claret (2, 1.6%) and chest tube (1, 0.viii%). 4 strains, including two Klebsiella subsp. ozaenae (one cultured from urine, other ane from sputum), one Klebsiella subsp. rhinoscleromatis (cultured from urine) and one Klebsiella oxytoca (cultured from urine) were identified among collected isolates.
Antibody susceptibility patterns and frequency of resistance genes
The highest susceptibility rate was obtained confronting amikacin (104, 85.2%), followed by meropenem (81, 66.4%), imipenem (77, 63.1%) and gentamicin (74, sixty.7%). The presence of bla OXA-48, bla NDM-, both of bla OXA-48 and bla NDM- (bla OXA-48/NDM) and bla IMP- genes was detected amongst 21 (17%), vii (5.6%), 14 (xi.3%) and three (2.4%) of isolates, respectively. The NDM PCR products from all positive strains were subjected to sequencing and identified as NDM-1 variant [9]. The virtually active agents against bla NDM-1 producers were amikacin (13, 61.ix%) and trimethoprim/sulfamethoxazole (3, xiv.3%) (Tableane).
Table i
Antibiotic resistance rates and frequency of resistance genes among NDM positive and negative isolates
| NDM positive (due north = 21) | NDM negative (n = 101) | NDM positive vs. NDM negative P value | |
|---|---|---|---|
| Resistance rates n (%) | |||
| Imipenem | 21 (100) | 24 (23.8%) | 0.001 |
| Meropenem | 21 (100) | xx (19.eight%) | 0.001 |
| Ertapenem | 21 (100) | 30 (29.7%) | 0.001 |
| Cefepime | twenty (95.ii) | 39 (38.half-dozen%) | 0.001 |
| Ceftazidime | 21 (100) | 52 (51.5%) | 0.001 |
| Cefotaxime | 21 (100) | 66 (65.3%) | 0.001 |
| Aztreonam | 21 (100) | 51 (50.5%) | 0.001 |
| Ampicillin/sulbactam | 21 (100) | 54 (53.5%) | 0.001 |
| Piperacillin/tazobactam | 21 (100) | 36 (35.3%) | 0.001 |
| Amoxicillin/clavulanate | 21 (100) | 53 (52.v%) | 0.001 |
| Ciprofloxacin | xx (95.2) | 48 (47.5%) | 0.001 |
| Levofloxacin | xx (95.2) | 37 (36.6%) | 0.001 |
| Gentamicin | 20 (95.ii) | 28 (27.7%) | 0.001 |
| Tobramycin | twenty (95.2) | 33 (32.7%) | 0.001 |
| Amikacin | 8 (38.1) | 10 (9.9%) | 0.003 |
| Trimethoprim/sulphamethoxazole | 18 (85.7) | 46 (45.five%) | 0.001 |
| MDR | 21 (100) | 54 (53.5) | 0.001 |
| Presence of resistance and capsular markers n (%) | |||
| armA | 10 (47.half-dozen) | sixteen (15.8%) | 0.003 |
| rmtC | 3 (14.3) | 3 (3%) | 0.06* |
| aac6Ib | xiv (66.7) | 35 (34.7%) | 0.01 |
| aac3IIa | 19 (xc.5) | 28 (27.5%) | 0.001 |
| qnrB | ane (4.8) | 22 (21.8%) | 0.1 |
| qnrS | 18 (85.7) | 17 (16.8%) | 0.001 |
| aac6Ib-cr | 19 (90.v) | 47 (47%) | 0.001 |
| bla OXA-48 | 14 (66.vii) | 21 (20.eight%) | 0.001 |
| bla IMP- | 0 | 3 (3%) | 1 |
| CTX-M-G1 | 21 (100) | 53 (52.5%) | 0.001 |
| CTX-M-G2 | 0 | 3 (3%) | 0.five |
| CTX-One thousand-G8 | 0 | 2 (ii.0%) | 0.6 |
| CTX-M-G9 | 0 | 2 (2.0%) | 0.6 |
| CTX-M-G25 | 0 | vii (vii%) | 0.2 |
| bla TEM- | 12 (57.1) | 29 (28.vii%) | 0.02 |
| bla SHV- | nineteen (90.5) | 67 (66.3%) | 0.03 |
| bla OXA-1 | 15 (71.4) | 25 (24.viii) | 0.001 |
| K1 | 0 | 1 (1%) | i |
| K2 | 0 | xv (fourteen.viii) | 0.07 |
One of the 21 bla NDM-1 producing isolates was identified equally subsp. ozaenae (isolate no. 500A, cultured from sputum). The bla NDM-1 producers were mostly cultured from urine (10, 47.5%), followed by sputum (9, 42.9%), wound and blood (ane, 4.8% for each of them). Carbapenemase encoding gene, including either of the bla OXA-48, bla NDM-1, or bla IMP-was detected in 43 strains out of 55 carbapenem resistant (resistant to at least i of the three carbapenems) isolates (78.2%, P< 0.001), and the two bla IMP- carrying strains were found susceptible to carbapenems. All of the bla NDM-1 conveying strains were found non-susceptible to carbapenems, with the MICs ranging from 1.5 to > 32 µg/ml [ix]. Every bit compared to bla NDM negative isolates, bla NDM-1 carrying strains showed significantly higher carriage rates of any resistance determinants (PMQR, ARD or β-lactamases) and higher resistance rates confronting whatever studied antibiotics (P< 0.001) (Fig.i). Of the ARD studied, the presence of armA (P: 0.01), aac6Ib (P< 0.001), and aac3IIa (P< 0.001) was associated with resistant phenotype to any of the aminoglycoside antibiotics. Of the PMQR determinants, the wagon of qnrS (P< 0.001) and/or aac6-Ib-cr (P: 0.03) genes was associated with resistance to whatsoever of the fluoroquinolones. For β-lactamase genes, isolates harbouring the bla TEM-, bla SHV-, bla OXA-1 and/or CTX-M-G1 were resistant to any of the three studied cephalosporins (P< 0.001).
Frequency (pct) of any resistance determinants, and resistant phenotype to whatever carbapenems, whatsoever cephalosporins, fluoroquinolones and any aminoglycosides in two groups of NDM positive and negative isolates. Whatever carbapenemase: bla OXA-48, bla NDM-1, bla IMP-; any PMQR: qnrB, qnrS, aac6Ib-cr; any β-lactamase (BL): bla TEM-, bla SHV-, CTX-Grand, bla OXA-1; any ARD: aac3IIa, aac6Ib, armA, rmtC
The resistance determinants which detected significantly amid NDM producers were bla OXA-48, bla TEM-, bla SHV-, bla OXA-1, armA, aac3IIa, aac-6Ib, aac6-Ib-cr, qnrS, and CTX-M-G1. The co-carriage of rmtC and bla NDM-1 was not constitute statistically significant, all the same this association was on the border of significance (P: 0.06).
Genetic relatedness and sequence typing of bla NDM producers
Based on the ERIC-fingerprinting of bla NDM negative isolates, 25 clusters were detected, including of two to x isolates in each cluster. Twenty-half-dozen isolates were likewise constitute equally singletons (Fig.2). In contrast, NDM carrying strains were grouped into five clusters (Fig.3). Consequently, as the representatives of NDM producers, one strain was selected from each of the cluster and subjected to MLST assay. 2 sequence types, ST392 (due north = ix) and ST147 (north = 12) were identified among NDM positive isolates. Noted that the ST392 is the clonal complex (CC) of ST147. The kickoff bla NDM-1 strain was obtained on March 2022 from internal intensive intendance unit (ICU) followed by the couple of isolates retrieved on May from surgical ICU. On June, there was only one strain isolated from a patient hospitalized in internal ward. On July, we obtained one bla NDM-1 isolate from each of the internal ICU and surgical ICU sites. On August, there were three isolates; two from internal ICU and i from internal ward. On the following months from October 2022 to February 2016, ix strains were isolated from patients hospitalized at internal ICU. On March, we obtained 1 isolate from internal ward and afterwards nosotros received two other isolates from coronary care unit (CCU) and surgery wards on April. The final positive strain recovered from a dialysis patient whom was initially hospitalized in internal ICU on September 2016.
ERIC-PCR based dendogram of bla NDM negative isolates. Cluster assay of the Dice similarity indices based on the unweighted pair group method using average linkages (UPGMA) was done to generate a dendogram describing the human relationship amid the ERIC profiles. ESBL: indicate the phenotypic results, IMP: imipenemase
ERIC-PCR based dendogram of bla NDM producers. 5 representatives were subjected to MLST and two STs, including ST147 and ST392 were identified. ESBL: indicate the phenotypic results
Phylogroups, capsular genotypes and hypervirulent clones
Except for Klebsiella oxytoca strain (strain no. 511b) which showed a dissimilar RFLP-pattern, all the remaining 121 studied isolates were grouped in three phylogroups, including 98 (lxxx.iii%) in group I, xx (16.iv%) in group Two and three (ii.v%) in group 3. Nearly of the NDM-1 producing isolates, including 17 (81%) strains, belonged to the group I, and the remaining four strains belonged to group Ii and 3. Of the CTX-M genes, the CTX-1000-G2, G8 and G9 were detected exclusively amongst isolates of phylogroup I. Of the six capsular types studied; K1 and K2 genotypes were identified among i and xv isolates, respectively. The K1 positive strain was cultured from urine specimen, while 12 (80%) and iii (20%) of K2 strains were recovered from urine and sputum cultures, respectively. Carbapenem resistant and MDR phenotypes were detected amidst 5 (33.3%) and xi (73.iii%) K2 positive strains, respectively. The K1 strain detected in phylogroup I, while the K2 isolates belonged to both phylogroup I (xiv [93.three%]) and 2 (1 [6.vii%]) (P> 0.05).
Based on the carriage of peg344, iucA, iroB, rmpA and rmpA2 biomarkers, three strains, including K1 (strain 542, carbapenem susceptible) and two K2 (strain 290, carbapenem resistant and 533, carbapenem susceptible) isolates were identified as hvKp. Virulence genes iutA, mrkD and ybtS were detected in these three hvKp strains, except for K1 isolate which was negative for the latter gene. The ii K2+/hvkp strains (strains 533 and 290) were identified every bit ST86 using the hvKP-K2 multiplex PCR. As expected, this multiplex PCR was completely negative for the only K2+/phylogroup II strain (strain 608). The alls gene was not detected amongst report isolates. None of the bla NDM-1 producers were positive for K1/K2 capsular types and were non identified every bit hvkp.
We too studied the presence of virulence genes in all carbapenem resistant isolates and peg344, iucA, rmpA, rmpA2, iroB1, iroB2, iutA, mrkD and ybtS were detected in 1.8%, one.8%, ane.8%, i.8%, 7.3%, 12.vii%, 18.2%, 89.1% and 67.three%, of strains, respectively. Higher rates of iutA and ybtS carriage were detected amidst bla NDM-one positive (19% and 71.4%) strains as compared to negative (17.half dozen% and 64.7%) ones, while the differences were not statistically significant (P> 0.05). Of the resistance genes studied, the railroad vehicle of qnrB and qnrS had positive and negative association with K2 capsular type, respectively.
Discussion
The beginning study of NDM producing Gram–negative bacilli from Islamic republic of iran was described in 2013 in a Grand. pneumoniae strain, which was cultured from the urine specimen of a patient with kidney transplant rejection history [23]. In the current study, we investigated the molecular epidemiology of a group of bla NDM-i producing isolates cultured from various extraintestinal specimens collected from Kosar infirmary, Semnan, Iran. In our study, NDM-1 producing K. pneumoniae strains were mostly derived from urine specimens, followed by sputum of patients admitted to different hospital wards.
In our study and comparing to NDM negative isolates, bla NDM-1 carrying strains were much resistant to different classes of β-lactam and non β-lactam antibiotics by disk diffusion assay. It has been shown that tigecycline, fosfomycin and colistin are the nigh potent agents against NDM producing isolates [8]. While nosotros didn't determine the susceptibility patterns of NDM producing strains against these antibiotics, amikacin which found with the highest susceptibility rates may be considered as a treatment choice due to some limitation of the extensive clinical usage of the three aforementioned antibiotics.
Phylogrouping of study Klebsiella isolates identified three groups, with the phylogroup I as the dominant group, followed by group II and III. It has been shown that the level of resistance for most of the antibiotics is highest amongst phylogroup I, intermediate in grouping Ii and lowest in grouping III, with the highest number of normal microbiota amid the latter group [24]. Accordingly, well-nigh of the bla NDM-1 producing strains (81%) belonged to group I and co-harbored dissimilar resistance genes, including bla SHV-, CTX-G-G1, qnrS, aac6-Ib-cr and aac3IIa. While the resistance is a critical parameter for the transmission of phylogroup I M. pneumoniae strains, detection of NDM/OXA-48 carbapenemases and consequently carbapenem resistant phenotype among phylogroup Two (strains: 500A, 718, 293 M) and Three (isolate: 247) strains, ostend the function of this mainly nosocomial and opportunistic pathogen in the dissemination of resistance elements.
Among the bla NDM-1 producers, 11 (52.3%) strains co-harboured any β-lactamase gene, CTX-One thousand; any PMQR; and 16S rRNA methylase cistron armA or rmtC. PMQR circumstantial with any β-lactamase gene was likewise high (21 [100%]). A stiff association betwixt the carriage of NDM and 16S rRNA methylase encoding factor, specifically rmtC methylase has been shown [25]. While the prevalence of rmtC among bla NDM-1 producing strains was higher than the negative ones, this association was institute borderline. In dissimilarity, the co-occurrence of armA or the other aminoglycoside resistance genes with NDM was establish significant. Then our results indicate that NDM producing Klebsiella isolates, have acquired a broad spectrum of singular and different resistance determinants.
It has been reported that carbapenemase producing Chiliad. pneumoniae strains accept a different population and less genetic variety equally compared to carbapenem susceptible isolates [26]. In our report, ERIC-PCR showed five clusters and higher genetic homogeneity among bla NDM-1 producers as compared to negative ones. Appropriately, the MLST assay identified two STs among NDM producing isolates. ST392, which is an SLV of ST147, has been associated with the wagon of bla KPC, bla NDM and bla OXA-48 [27]. In the current study, ST392 has detected among NDM positive isolates with different ERIC patterns. Some other detected ST, ST147 which is an international clone, has been reported from centre, southward and south e of Iran [vi–8]. In our study, ST147 has been associated with CTX-1000-G1, bla SHV-, aac6Ib-cr and armA. This ST has been recognized as a pandemic clone and is considered as a threat to public wellness worldwide [28]. ST147 was firstly observed by Papagiannitsis et al., as hosting the bla VIM cistron, and so Samuelson et al. reported this clone among Thousand. pneumoniae isolates imported to Scandinavia, mostly from Greece [28]. So, these findings imply that ST147 has the potential to larn unlike resistance elements, of note, bla NDM/OXA-48 carbapenemases and facilitate their rapid spread into the other pandemic clones of M. pneumoniae.
String test, a phenotypic analysis that is commonly used to identify the hvKp strains, has been shown to performed suboptimally, particularly in low prevalence areas [18]. And so, the identification of some genetic markers including peg344, irob, rmpA, rmpA2 and iucA has been suggested for accurate differentiation between hvKp and classical K. pneumoniae strains [18]. Accordingly, one K1 and ii K2 ST86 strains were identified as hvKp, of those one ST86 strain was MDR and carbapenem resistant. While these capsular types have not generally been associated with acquired resistance genes at the time which were identified, in the terminal few years increasing reports of resistant strains were observed among these genotypes [29]. In agreement with these new reports, four out of five carbapenem resistant K2 strains harboured the bla OXA-48 gene. Information technology has been reported by Turton et al., that isolates of CC147 carrying bla OXA-48 or bla NDM which was resistant to colistin, harboured many antibody resistance determinants, and contained a quarter of the virulence genes which were found in the K1-ST23/OXA-48+ isolates [30]. While our CC147 strains (all conveying NDM) were K1/K2/hvkp negative, they harboured relatively higher rates of siderophores iutA and ybtS as compared to another carbapenem resistant NDM negative isolates. So, concerning this finding that the acquisition of whatever one of the siderophore clusters increases the chance of complicated infections [2, 30], the combination of virulence and antibiotic resistance in this pandemic clone is extremely worrying.
Our study had some limitations. The bla OXA-48 producers and K1/K2 strains were not subjected to sequence type determination. Furthermore, the TEM- and SHV- variants of positive isolates were non adamant. We investigated the carriage of some express virulence factors among carbapenem resistant strains, while the other of import virulence determinants remained to exist characterized among both of carbapenem susceptible and resistant isolates.
Conclusions
In summary, clonal dissemination of bla NDM-i carrying One thousand. pneumoniae that co-harbour different β-lactamases, aminoglycoside modifying enzymes, and PMQR determinants take been observed. Isolation of carbapenem resistant Chiliad. pneumoniaeastward strains from clinical sources has been reported from Iran, previously. Still, their clan with K1 or K2 hypervirulent capsular types has never been reported. The emergence of the CC147 carbapenem resistant K. pneumoniae strains warrants urgent surveillance because not only are they considered equally international clone, but likewise they simultaneously have higher rates of siderophores in a pandrug resistance profile.
Acknowledgements
The authors would like to appreciate Prof. Thomas A. Russo for his helpful comments.
Authors' contributions
OP designed the proposal of the project, ND, AB and MA carried out the laboratory work. RGH participated in statistical analysis. ZB and ZH participated in drafting the manuscript. All authors read and approved the concluding manuscript.
Funding
This work was supported fully by Semnan University of Medical Sciences (Grants No. 1362, 871, 898, 918).
Availability of data and materials
The information tin be accessible to the interested researchers by the respective writer on behalf of all authors on reasonable request.
Ideals approval and consent to participate
The ethical clearance and consent to participate were canonical past the Iranian Health Research Council.
Consent for publication
The clinical isolate samples used in this research were role of the routine hospital laboratory procedure. We do non use patients' names or personal information so no need to accept writing consent.
Competing interests
The authors declare that they have no competing interests.
Footnotes
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