Distribution of bla_{CTX − M}, bla_TEM, bla_SHV and bla_OXA genes in Extended-spectrum-β-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan

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 [8‐10].

Asian countries are highly affected by extended spectrum-β-lactamase-producers inducing multidrug-resistant phenotype [11‐14]. 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 [16‐21]. 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−M, bla_TEM, bla_SHV and bla_OXA genes among ESBL-producers in Pakistani population to have a generalized view about the situation in our region.

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 [31‐33].

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 community-acquired infections are more prevalent in females [42‐46]. These findings represent that males are more 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 [47‐50]. 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 among isolates including 84.7% (Chile), 98.8% (China) and 13.6% (Tanzania) [53‐55]. 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 carbapenemase-encoding 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.

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.