Distribution of blaCTX − M, blaTEM, blaSHV and blaOXA genes in Extended-spectrum-β-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan

Background Frequency of extended-spectrum-β-lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-β-lactamase-producing Clinical isolates in Pakistan. Methods A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results A total of 497/995 (50%) isolates including Escherichia coli 65% (n = 321), Klebsiella spp. 25% (n = 124) and Pseudomonas. 5% (n = 24), Enterobacter spp. 4% (n = 20) and Acinetobacter spp. 2% (n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% (n = 278) followed by pus 20% (n = 99) and wound swab 6% (n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% (n = 392). PCR revealed blaCTX − M (76%) gene followed by blaOXA (52%), blaTEM (28%) and blaSHV (21%) were most prevalent among ESBL-producers detected by CDST. blaCTX − M − 1(65%), blaOXA (78%) and blaTEM (57%) genes were carried on plasmids. Amplicon sequencing revealed blaCTX − M − 15 (75%), blaOXA − 1 (49%) and blaTEM − 1B (34%) and 21 (n = 28) isolates carried three genes in them. Conclusion Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding blaCTXM − 15 gene and narrow spectrum blaOXA − 1 and blaTEM − 1B were also prevalent.


Background
Multidrug resistant clinical isolates have important clinical consequence in community and hospital settings [1]. They have evolved as a global concern, exacerbated by under reporting in some regions of the world [2]. The tendency of these isolates concurrently resistant to other groups of antibiotics significantly limits the selection of antibiotics for treatment of infections [3]. The development of resistance for third generation cephalosporin is attributed to production of β-lactamases including extended-spectrum-β-lactamases (ESBLs), AmpCs and carbapenemases [4]. The most significant β-lactamase genes are variants of CTX-M, SHV, TEM, VEB, GES, PER, TLA and OXA which have broadened the substrate specificity against ceftazidime, cefotaxime and ceftriaxone [4,5]. These genes have broad host range but predominantly found in Escherichia coli and Klebsiella spp. [6]. While, OXA genes are found predominantly in Pseudomonas spp. and Acinetobacter spp. [7]. Moreover, many clinical pathogens harbor more than one β-lactam genes [8]. Plasmid association of these genes makes them easily spreadable. Due to the diversity of these enzymes, multiplex-PCR based detection methods have become a widely used tool for epidemiological surveys [8][9][10].
Asian countries are highly affected by extended spectrum-β-lactamase-producers inducing multidrug-resistant phenotype [11][12][13][14]. Several studies have reported the community-association of ESBL-producers [11,14,15]. In Pakistan, an increase in the number of ESBL-associated infections has been observed in last few decades [16][17][18][19][20][21]. Lack of regular surveillance programs at national or international levels, inadequate infection control agencies, lack of facilities and inappropriate diagnostic approaches contribute to the emergence of the antibiotic resistance in bacteria [2,10,22]. Moreover, dissemination of these isolates in the community demands the urgent call for surveillance of resistance and molecular characterization for extended-spectrum-β-lactamase-producers [23]. This study was designed to check molecular epidemiology of bla CTX

Bacterial Isolates
A total of 2,372 samples were processed during study period; 77 % (n=1835) cultures were positive and 54 % (n=995) gram negative non-duplicate clinical isolates from various sources were collected by standard culturing methods. Antibiotic susceptibility testing (AST) was performed according to the guidelines provided by clinical laboratory standards institute [24] by using standard antibiotic discs as mentioned in our previous study [16]. Multiple-antibiotics resistance (MAR) value was calculated as reported before [25]. E. coli ATCC 25922 was used as positive control and K. pneumoniae ATCC 700603 was used as negative control [26]. Analytical profile index (API 20-E) was used for biochemical characterization of isolates resistant to third generation cephalosporins.

Molecular detection
The DNA used for multiplex-PCR was extracted by the heat lysis method [16]. In Multiplex-PCR, 2 μl whole cell lysate DNA for each isolate was used separately in 25 μl PCR-master mix and amplification primers as previously mentioned [16,28,29]. PCR amplification conditions were: Initial step of denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min then annealing at 56°C for 1.5 min, extension at 95°C for 1 min and the final extension was done at 95°C for 10 min (Table 1).
Amplicon sequencing and in-silico analysis PCR amplified products were sequenced by Advance Bioscience International, Pakistan in collaboration with 1st Base, Malaysia [30]. Nucleotide sequence similarity searches were performed using the services of National Centre for Biotechnology Information (NCBI) (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST, CLUSTALX, and MEGA 7.0 software were used for sequence alignment of amplicon sequenced obtained with already submitted sequences of bla CTX−M , bla TEM , bla SHV and bla OXA in GenBank.

Statistical Analysis
All statistical analyses were performed using IBM-SPSS statistics 23. Bivariate analyses were performed using chi-square test for categorical variables. All p-values were two sided. The percentage values included in this article are the "valid percentages," which exclude the missing data.

Molecular detection
After screening, ESBL-producing isolates (n = 392) as detected by combination disc test were processed for the OXA-R AAACCCTTCAAACCATCC   (Table 8).

Discussion
Extensive use of antibiotics has resulted in resistance against variety of antibiotics including cephalosporins. They affect countries all over the world but control and prevention of ESBL-producers is severely compromised in underdeveloped countries [31][32][33].
Here, high prevalence of third generation cephalosporin resistant isolates (50%) was observed which has subsequently increased by 1.13-fold from 2014 to 2017. This high resistance also indicates high selection pressure for third generation cephalosporin resistant isolates [34]. This increase of resistance is worrisome as we are left with few treatment options including cephalosporins. Widespread usage of antibiotics might be the  factor of such increase in resistance in our hospital settings [16,35]. E. coli had high 3GC-R burden compared to Klebsiella spp. and Enterobacter spp. Bari et al., reported similar findings in a study conducted in 2013 in Lady Reading Hospital Peshawar [36]. These results are comparable to findings in Tanzania where 45% ESBL-producers have been reported [37]. Similar findings from different regions of the world were observed as previously studied [38,39]. Nahid et al., reported very high prevalence of ESBL-producers (87.5%) but this is because she worked on Metallo-β-lactamase producers which are highly resistant organisms [40].
ESBL infectivity rate in males was moderately high as compared to females. This rate is quite similar to the rate reported by Afirdi et al. [41]. In our study ESBL infections were significantly higher in the mean age group of 41-60 years whereas, high infection rates have been reported in old age individuals who are immuno-compromised and hence, more prone to infections [42]. We have found isolates originating from females were more frequent ESBL-producers. According to many reports males have significantly higher rates of hospital-acquired infection and communityacquired infections are more prevalent in females [42][43][44][45][46]. These findings represent that males are more   Enterobacter spp. 20 (4) 14 (70) 6 (30) Acinetobacter spp. 8 (2) 7 (88) 1 (13) often exposed to the hospital settings compared to the females. Studies indicated prevalence of ESBL-producers is variable in different regions of world as detected by phenotypic detection tests [47][48][49][50]. DDST determined only 54 % strains as ESBL-producers while CDST determined 79 % as ESBL-producers. Ejaz et al., reported similar detection efficiency of CDST as we reported here [17]. Prevalence of ESBL-producing isolates is quite higher than from other parts of the world including India (42.3 %), Bangladesh (37.8 %). Dalela et al., reported 90 % sensitivity of CDST for the detection of ESBL-producers [51]. E-test revealed that 61 % strains were ESBL-producers while 39 % remained non-determined by this technique. Mohanty et al. also reported 61 % positivity rate for ESBL-producers by E-test technique [52]. Such discrepancies between susceptibility data and phenotypic test results have increased the demand for more sensitive methods of ESBL-producer detection for implementation into routine susceptibility testing procedures.Despite of high resistance burden of ESBL-producers, the usage of molecular detection methods is not very common. A recent meta-analysis describes only 11% studies that reported PCR-based detection methods for screening of ESBL-producers in Pakistan [20]. Lack of knowledge and technical staff triggers the use of PCR-based methods as it is the rapid and reliable method of ESBL-producer detection [8]. It seems that bla CTX − M is predominant genotype in this region of the world. Another study from Pakistan indicated 72% of isolates had bla CTXM − 15 gene which was lower than prevalence of bla CTX-M gene found in this study [16]. Few studies from other parts of world have shown different prevalence of bla CTX-M gene  [53][54][55]. We observed bla TEM and bla OXA genes were less common in our settings with 50% prevalence. Report from Hamad Medical Corporation, Qatar stated that CTX-M group has evolved through mutations in bla TEM and bla SHV genes and is recent endemic [56].
-Acinetobacter baumannii isolates had OXA variants (bla OXA − 23,58 and others) which are carbapenemaseencoding genes [57]. These variants have previously been isolated from France, Spain and Turkey which indicates the global spread [50]. bla OXA − 23 was amplified from pan-drug resistance A. baumannii only which is in accordance with our results [58]. But these Acinetobacter baumannii isolates did not carry any of the ESBL-encoding genes which terminate the co-existence of carbapenemase and ESBL-encoding genes. This is in accordance with already published article which states no significant relation between both groups [59]. Appearance of different variants might provide extra advantage for these isolates to spread them and complicate the therapeutics.
With the passage of time increase in co-resistance of different ESBL-producing genes is worrisome as co-existence of multiple genes hinders the detection of ESBL-producers and complicates the treatment strategy for clinicians. Moreover, high plasmid burden was found these plasmids are involved in gene-transfer and they also carry additional antibiotic resistance genes along with β-lactam antibiotics.  (21)

Conclusions and Recommendations
In conclusion, bla CTX−M -type ESBL-producing genes and bla OXA -type narrow spectrum-β-lactamases are prevalent among the isolates in our health care settings. Isolates had high resistance towards cephalosporins. Resistance towards cephalosporins and carbapenems has increased many folds during study period. Co-expression of multiple genes complicates the treatment strategy. bla CTXM−15 , a pandemic genotype is quite prevalent and their plasmid association is a big thread for the community. There is a dire need for efficient molecular diagnostic tools for the detection of bla genes at laboratory level.