High frequency and molecular epidemiology of metallo-β-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan

Dissemination of life-threatening infections caused by β-lactamase-producing pathogens is a major setback to antimicrobial therapy. The widespread use of carbapenems has resulted in the emergence of carbapenemases, conferring resistance against carbapenem drugs [1‐3]. Resistance to carbapenems is worrisome because of the very limited therapeutic options available to treat resistant infections [4, 5]. The diverse mechanisms of resistance to imipenem include AmpC enzymes accompanied by membrane porin alterations and upregulation of efflux pumps [5, 6]. The second phenomenon is carbapenem hydrolysis by carbapenemases [7‐9]. The epicentre for the emergence of carbapenemases and that of extended spectrum beta lactamases (ESBLs) were different, but the association of their genes is apparent through some studies, where ESBL genes are found to exist in MBL-producing isolates [10].

Among carbapenemases, metallo-β-lactamases (MBLs) are of prime importance for the region under study because of the emergence of new variants of MBL, such as New Delhi metallo-β-lactamase (NDM) [11] and various IMP variants from the subcontinent. MBLs belong to class B carbapenemases according to the Ambler classification system [12]. bla_IMP, bla_NDM and bla_VIM are important MBL gene clusters that are carried by mobile plasmids compatible with a vast array of clinically important pathogens [13, 14]. In addition, oxacillinases belonging to class D include serine β-lactamases and are known to be associated primarily with Enterobacteriaceae carbapenem-resistant epidemics.

With more than 37 types of IMP carbapenemases known [15], IMP-1 was the first to be reported in Japan in 1991[16]. IMP-4-type enzymes, first discovered in Hong Kong during the 2000s [17], were later found to be responsible for an outbreak in 2005 in Melbourne, Australia [18]. The dissemination of resistance genes from Serratia spp. and Pseudomonas aeruginosa to other members of Enterobacteriaceae caused these genes to become endemic in Australia. Approximately 20 different subtypes of IMP enzymes have been described to be associated with Pseudomonas spp., Acinetobacter spp. and Enterobacteriaceae infections throughout the globe [19].

The first case of VIM-1 enzyme-conferred resistance was reported in 1999 in Verona, Italy [20]. With reports of VIM-2 being highly prevalent in Europe, Asia, America and Africa [21], a recent global surveillance study reported four new variants of VIM [22]. After their discovery in New Delhi in 2009 [23], NDM-1-producing Klebsiella pneumoniae and E. coli have been widely reported throughout the globe, including various European countries as well as China, Kenya, Japan, Algeria and Syria [24‐27]. A research study has indicated dissemination of the NDM-1 carbapenemase gene through horizontal gene transfer in Pakistan, India and the UK [28].

The rise in carbapenem resistance in Asian countries has been evident, as reported imipenem resistance rose from 20% in the Philippines [29] to 40% in Vietnam [30]. Recently, > 50% carbapenem-resistant Klebsiella pneumoniae isolates have been recorded in India [31]. With the first report of emergence among Acinetobacter baumannii clinical isolates in Scotland in 1985 [32], plasmid-borne OXA-48 carbapenemases were reported to be highly disseminated in isolates from 18 countries in Europe and Africa [33].

The resistance pattern of metallo-β-lactamases in Pakistan has not been widely studied. In Pakistan, imipenem was only rarely available prior to the year 2000 [34]. Later, carbapenem resistance was reported mainly in P. aeruginosa in Lahore and Karachi [35, 36]. A report from Rawalpindi confirmed that 78% of isolates were MBL producers, with major incidence of MBL production in Acinetobacter baumannii and Pseudomonas aeruginosa [37].

A very limited number of reports have been published from Pakistan that are based on molecular analysis of genes acquired by carbapenem-resistant isolates. This study aimed to determine the incidence of MBLs through phenotypic and genotypic analyses. Moreover, this study is based on the detection of the gene variants responsible for resistance to carbapenem drugs. To the best of our knowledge, this is the first study from Lahore on the molecular epidemiology and coexistence of bla_ESBLs and bla_MBLs.

Pakistan is a country where empirical drug therapy and misuse of antibiotics are common practice. Poor sanitation, filthy practices in clinical settings, and ill-informed health care workers are factors in the dissemination of nosocomial pathogensto the community. Routinely used second-generation drugs are quickly being replaced by drugs of last resort, and this situation is ultimately an enduring threat to mankind. According to one estimate, first-line antibiotic-resistant pathogens account for 25,692 neonatal deaths annually in Pakistan [45]. Resistance to carbapenems has been significantly observed in Asian countries, including Pakistan [30, 46, 47]. At present, numerous reports of MBL producers and ESBL producers from Pakistan present clinical catastrophes and alarming health issues [41, 48, 49]. The present study demonstrates the frequency of imipenem resistance among clinical isolates and the incidence of MBLS producers associated with imipenem resistance in connection with various demographic factors and types of infection. The frequency of imipenem resistance in our study was 56%, which is significantly higher than the data reported from Asian countries in the last decade (2002–2012), with one epidemiological study reporting 1.9% resistance to imipenem and 2.4% resistance to meropenem [4].

Comparison of the antibiotic resistance profiles of all the pathogens with those reported in other recent studies has revealed relatively similar patterns. The resistance pattern of Acinetobacter spp. to imipenem in our study (61.89%) is in conformity with reports by Anwar et al., but a lower rate (77.5%) of imipenem resistance in this region was reported by Shamim et al. [50]. Acinetobacter spp., the pathogen renowned for hospital-acquired infections, was predominantly found to be associated with wound infections and was the causative agent of 92% infections associated with imipenem resistance. Pseudomonas spp. was the second most prominent pathogen associated with the acquisition of imipenem resistance, with a frequency of 61.89%, and has been noted in other reports from Pakistan, presenting imipenem resistance rates of 13.42% in 2011 and of 28% and 49.5% in 2015, thus demonstrating a sharp rise in the frequency of imipenem resistance [49, 51, 52]. The victims of Pseudomonas spp. infections were found predominantly in the group of patients with post-burn infections. This finding is in conformity with studies on invasive burn wound infections that document P. aeruginosa as a leading pathogen among gram-negative organisms [53, 54]. Wound infections were particularly found to be associated with Acinetobacter spp. (OR = 1.79 [1.16–2.75]) and Pseudomonas spp. *OR = 1.29 [0.89–1.85]).

The incidence of MBL among imipenem-resistant Acinetobacter spp. was calculated to be 89%, which is comparable to that reported in a study by Anwar et al., presenting a frequency of 83.3% MBLs among carbapenem-resistant isolates [55]. A total of 78% of the imipenem-resistant Pseudomonas spp. isolates were detected to be MBL producers by the MHT test, representing a lower incidence compared to the study by Shan et al. that stated the incidence rate to be 87.5% [49].

Our study demonstrates the OXA-type variants to be predominantly associated with resistance to imipenem. All of the isolates harbouring bla_IMP were also found to harbour bla_TEM. Despite the uncommon origin of two major groups of β-lactamases, bla_ESBL and bla_MBL, their association is imminent according to recent research [10]. We analysed the isolates for the coexistence of MBL-type and ESBL-type variants. The bla_VIM gene variant for MBL production was also found to be in coexistence with bla_TEM and bla_SHV in Providencia stuartii and Enterobacter spp. A total of 12.5% isolates were found to coexhibit the bla_IMP-1 and bla_SHV genes, whereas 10% were positive for the bla_OXA gene along with the bla_IMP-1 gene.

The abovementioned mechanism of coexistence of genes has been reported in Klebsiella pneumoniae, Escherichia coli, Salmonella spp. and Enterobacter spp. [56]. Plasmids containing the bla_NDM-1 gene have been observed to coexhibit genes for CTX-M, TEM-1 and OXA-1 enzymes. Major ESBL and MBL genes, including bla_CTX-M, bla_SHV, bla_TEM, and bla_OXA-51, and genes for the VIM-family and IMP-family, have been reported to coexist in clinically resistant Acinetobacter baumannii in Iran [57]. However, one study concluded that there was no significant relationship between ESBL and MBL production genes [57]. Ertapenem-resistant, ESBL-producing Klebsiella pneumoniae isolates have been reported in Italy and shown to carry novel porin variants that contributed to the reduced susceptibility of isolates to meropenem and imipenem [58].

The coexistence of the genes for MBL and ESBL variants in our isolates indicates the simultaneity of the emergence of different variants of β-lactamases among pathogens in our clinical settings. This finding also suggests that the resistance against imipenem in our isolates is mediated by MBL-type enzymes along with the overproduction of ESBL-type enzymes, as suggested by other studies [5, 7]. To the best of our knowledge, this is the first study from Pakistan reporting the coexistence of bla_IMP with bla_TEM-type and SHV-type variants. None of the isolates was found to coexhibit all the tested genes (bla_OXA, bla_TEM, bla_SHV, bla_IMP-1 and bla_VIM).

In conclusion, ESBL- and MBL-producing bacterial isolates are emerging very rapidly in the region. A great number of carbapenem-resistant clinical bacterial species are resistant to most of the commonly used antibiotics, demonstrating the rise of super-bacteria and their pan-resistance to antimicrobial therapy. Determining the resistance mechanisms and the root cause for their elimination are of great importance. It is also important to implement the routine screening of ESBLs and MBLs in laboratory procedures before antibiotic therapy begins. Further studies are required to specify other types of gene variants prevalent among clinical isolates in our region for the implication of medication in clinical settings.