Diversity of Carbapenem Resistance Mechanisms in Clinical Gram-Negative Bacteria in Pakistan

Abstract

Antibiotic resistance is a health challenge worldwide. Carbapenem resistance in Gram-negative bacteria is a major problem since treatment options are very limited. Tigecycline and colistin are drugs of choice in this case, but resistance to these drugs is also high. The aim of this study was to describe the diversity of resistance mechanisms in carbapenem-resistant clinical Gram-negative bacteria from Pakistan. Carbapenem-hydrolyzing enzyme-encoding genes were detected using PCR and DNA sequencing and clonal types determined by multilocus sequence typing (MLST). Forty-four carbapenem-resistant isolates were collected from the microbiology laboratory of Fauji Foundation Hospital and Al-Syed Hospital, Rawalpindi, Pakistan, including Klebsiella spp., Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Achromobacter xylosoxidans. bla_NDM-1, bla_NDM-4, bla_NDM-5, bla_NDM-7, bla_OXA-48, and bla_OXA-181 were detected in Enterobacteriaceae; bla_OXA-23, bla_OXA-72, and bla_NDM-1 in A. baumannii, and bla_VIM-6 and bla_VIM-11 in P. aeruginosa. MLST analysis revealed several predominant clonal types: ST167 in E. coli, ST147 in Klebsiella pneumoniae, ST2 in Acinetobacter, and ST664 in P. aeruginosa. In Acinetobacter, a new clonal type was observed for the first time. To the best of our knowledge, this is the first study describing the clonality and resistance mechanisms of carbapenem-resistant Gram-negative bacteria in Pakistan.

Introduction

Dissemination of carbapenem resistance in Gram-negative bacterial pathogens is a major problem worldwide.¹ Due to their broad-spectrum activity and resistance to degradation by extended-spectrum β-lactamases (ESBL), carbapenems have been used for treating severe infections caused by ESBL-positive bacteria for decades? However, the long use of this antibiotic has also decreased the effectiveness of this class of antibiotics.² It is estimated that ESBL prevalence in Escherichia coli is more than 80% in Pakistan (REF). This overuse of carbapenems has caused an increase in the number of carbapenem-resistant bacteria. Over the past 15 years, infections caused by carbapenem-resistant Enterobacteriaceae (CRE), such as Klebsiella pneumoniae and E. coli have increased.^1,2 Carbapenem-resistant Acinetobacter baumannii (CRAB) have become endemic and have been associated with high morbidity and mortality rates.³ Similarly, the increase in carbapenem resistance in Pseudomonas aeruginosa has also become a serious challenge for clinicians.⁴

One of the most common mechanisms of carbapenem resistance is due to bacterial production of carbapenemases, carbapenem-hydrolyzing enzymes. Carbapenemase-encoding genes represent a global health threat because of their ability to be carried by mobile genetic elements.¹ Carbapenemases can be classified into various categories according to the Ambler classification scheme as A, B, and D.⁵

K. pneumoniae carbapenemases (KPC) are clinically the most common enzymes among the class A carbapenemases.^1,2 New Delhi metallo-β-lactamases (NDM), Verona integron-encoded metallo-β-lactamases (VIM), and active-on-imipenem (IMP) types belong to class B metallo-β-lactamases (MBLs),¹ whereas oxacillinases, including OXA-23, OXA-24, OXA-58, and OXA-48, are part of the class D enzymes.^1,2,5

Previous studies have demonstrated prevalence of diverse carbapenemases in Gram-negative pathogens. In A. baumannii, class D oxacillinases are predominant, while class B carbapenemases were mostly found in P. aeruginosa.⁵

Besides producing carbapenem-degrading enzymes, there are various other mechanisms for the development of resistance against carbapenems. For instance, it is believed that alteration in porin proteins plays a critical role. OprD is an outer membrane porin protein facilitating the excretion of carbapenems across the cell membrane. Alteration of the porin-coding oprD gene is also considered to be one of the main mechanisms conferring resistance to carbapenems in P. aeruginosa.⁶

In Pakistan, the high prevalence of ESBL-producing bacteria has led to the elevated use of carbapenems.^7,8 The most common carbapenemase-encoding gene was bla_NDM.⁷ Some other studies also focused on the diversity of the different gene variants responsible for carbapenem resistance or studied the genetic relationship among the clinical isolates.^7–11

The purpose of this study was to describe the diversity of carbapenem-resistant strains, their mechanisms of resistance to these antibiotics, as well as their clonal types in Gram-negative bacteria from clinical samples in Pakistan.

Materials and Methods

Bacterial isolates and species identification

Bacterial isolates were collected from patients attending Kidney Center (Al-Sayed Hospital) and Fauji Foundation Hospital, Rawalpindi, Pakistan, from May to December 2016. The samples were from urine, Foley catheter, pus, throat swab, endotracheal tube, bronchoalveolar lavage, wound swab, double-J stent, blood culture, central venous pressure tubes, and nasogastric tubes, and were cultured on blood agar and MacConkey's agar at 37°C for 24 hours.

Bacterial isolates were identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (Microflex; BrükerDaltonics, Bremen, Germany).¹²

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing (AST) was initially performed by conventional Kirby Bauer disc diffusion to screen carbapenem-resistant isolates. Afterward, all carbapenem-resistant isolates were tested to determine minimum inhibitory concentrations (MIC). The MIC of imipenem and meropenem was determined by broth microdilution and by using E-test strips (bioMérieux, Marcy-l'Etoile, France). Colistin resistance was also determined by UMIC (Biocentric, Bandol, France) according to EUCAST recommendations. UMIC colistin is a ready-to-use broth microdilution kit for determining the MIC of colistin.

For Enterobacteriaceae strains, a panel of 16 antibiotics was tested, including amoxicillin, amoxicillin-clavulanic acid, cefepime, ceftriaxone, cefalothin, piperacillin-tazobactam, ertapenem, imipenem, meropenem, nitrofurantoin, fosfomycin, doxycycline, trimethoprim-sulfamethoxazole, amikacin, gentamicin, and ciprofloxacin. For non-Enterobacteriaceae isolates, other panels of 16 antibiotics consisting of ticarcillin, ticarcillin-clavulanic acid, cefepime, ceftazidime, piperacillin-tazobactam, imipenem, meropenem, nitrofurantoin, fosfomycin, doxycycline, trimethoprim-sulfamethoxazole, amikacin, tobramycin, gentamicin, ciprofloxacin, and rifampicin were used. Interpretations were made according to EUCAST breakpoints.¹³

DNA extraction

Bacterial DNA was extracted using the automatic robot EZ1 tool (Qiagen, Hilden, Germany), following the manufacturer's instructions. The extracted DNA was eluted in 200 μL elution buffer.

Determination of molecular mechanisms of antibiotic resistance

Real-time PCR was performed on all strains to screen for the presence of ESBL genes (bla_CTX-M group A [CTX-M-1 and CTX-M-9 groups], bla_CTX-M group B [CTX-M-2, CTX-M-8, and CTX-M-25 groups], bla_TEM, and bla_SHV)¹⁴ and carbapenem-hydrolyzing enzyme-encoding genes [bla_NDM, bla_KPC, bla_OXA-48, bla_VIM, bla_OXA-23, bla_OXA-24, and bla_OXA-58].^14,15

Positive samples were further confirmed by standard PCR and the amplicons were sequenced using the BigDye terminator chemistry on an ABI 3500 automated sequencer (Thermo Fisher Scientific, Waltham, MA). The sequences of the antibiotic resistance genes obtained were interpreted using the ARG-ANNOT database.¹⁶

Carbapenem resistance due to mutations in the oprD gene was investigated in P. aeruginosa isolates.¹⁷ The sequences obtained were compared against PA01 oprD sequence AAG04347 using blastN and blastP.

Molecular typing by multilocus sequence typing

Molecular typing of E. coli, Enterobacter cloacae, K. pneumoniae, P. aeruginosa, and Acinetobacter spp. was performed to determine the genetic relationship among different clinical isolates using multilocus sequence typing (MLST).

Clonal types were determined for E. coli, E. cloacae, and P. aeruginosa in MLST Databases and according to the Pasteur schemes for A. baumannii and K. pneumoniae.

Results

Bacteria strain identification

A total of 44 isolates displayed carbapenem resistance during the study period, including K. pneumoniae (n = 13), E. coli (n = 9), A. baumannii (n = 10), P. aeruginosa (n = 6), E. cloacae (n = 4), Klebsiella oxytoca (n = 1), and Achromobacter xylosoxidans (n = 1) (Table 1).

Table 1.

Phenotypic and Genotypic Features of Enterobacteriaceae and Non-Enterobacteriaceae Clinical Isolates

Isolates	Susceptible phenotype	Resistance phenotype	IMP MIC (μg/mL)	β-lactamase genes	Carbapenems resistance genes	Sequence type
Enterobacteriaceae
Escherichia coli 6, 12a, 24 (n = 3)	FF, NF, DO, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, AK, GEN, CIP	>32	bla_CTX-M15 (n = 2), bla_TEM-206 (n = 1)	bla _NDM-1	167
E. coli 31a	FF, DO, SXT, CT	AMX, AMC, FEP, CRO, KF, TZP, NF, ERT, MEM, IMP, AK, GEN, CIP	>32	bla _TEM-206	bla _NDM-7	167
E. coli 26b, 27b (n = 2)	FF, NF, GEN, DO, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, AK, CIP	4–16	bla_CTX-M15, bla_TEM-169	bla _OXA-48	617
E. coli 29	FF, NF, GEN, AK, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, DO, CIP	>32	bla _TEM-206	bla _NDM-5	405
E. coli 32	FF, NF, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, DO, AK, GEN, CIP	>32	bla_CTX-M15, bla_TEM-206	bla_OXA-181, bla_NDM-4	410
E. coli 41b	FF, NF, IMP, SXT, GEN, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, AK, DO, CIP	1.5	bla _CTX-M15	bla _OXA-181	410
Klebsiella pneumoniae 35B	FF, SXT, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, NF, DO, AK, GEN, CIP	>32	bla_CTX-M15, bla_SHV-28, bla_TEM-104	bla _NDM-1	15
K. pneumoniae 3a, 11a, 13b, 14b, 15b, 16a, 21b, 34, 40a (n = 9)	CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, FF, NF, DO, AK, GEN, CIP	8–>32	bla_CTX-M15, bla_SHV-67, bla_TEM-206	bla _OXA-48	147
K. pneumoniae 33b, 43 (n = 2)	FF, NF, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, DO, AK, GEN, CIP	4	bla_CTX-M15, bla_SHV-67, bla_TEM-206	bla _NDM-1	273
K. pneumoniae 36	FF, SXT, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, NF, DO, AK, GEN, CIP	6	bla_CTX-M15, bla_SHV-28, bla_TEM-206	bla _NDM-1	101
Klebsiella oxytoca 28	FF, NF, GEN, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, AK, DO, CIP	>32	bla _CTX-M15	bla _NDM-1	ND
Enterobacter cloacae 8	FF, NF, AK, DO, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, GEN, CIP	>32	bla _TEM-206	bla _NDM-1	177
E. cloacae 10	DO, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, FF, NF, AK, GEN, CIP	>32	bla _TEM-206	bla _NDM-1	177
E. cloacae 35A	FF, NF, AK, CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, DO, GEN, CIP	16	bla _TEM-104	bla _NDM-1	177
E. cloacae 25	CT	AMX, AMC, FEP, CRO, KF, TZP, ERT, MEM, IMP, SXT, FF, NF, DO, AK, GEN, CIP	>32	bla_CTX-M15, bla_TEM-198	bla _NDM-1	182
Non-Enterobacteriaceae
Acinetobacter baumannii 2, 12b, 4a, 38b (n = 4)	DO, CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, FF, NF, SXT, TOB, AK, GEN, RA, CIP	>32	/	bla _OXA-23	2 (n = 2), 1,106 (n = 1), 1,209 (n = 1)
A. baumannii 3	DO, FF, TOB, CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, NF, SXT, AK, GEN, RA, CIP	>32	/	bla _OXA-23	2
A. baumannii 7	DO, TOB, CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, FF, NF, SXT, AK, GEN, RA, CIP	>32	/	bla _OXA-23	2
A. baumannii 37a	CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, DO, FF, NF, SXT, TOB, AK, GEN, RA, CIP	>32	/	bla_OXA-23; bla_OXA-72	2
A. baumannii 5a, 9b (n = 2)	DO, AK, CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, FF, NF, SXT, TOB, GEN, RA, CIP	>32	/	bla _OXA-23	1,106
A. baumannii 82	DO, TOB, CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, FF, NF, SXT, AK, GEN, RA, CIP	>32	/	bla_OXA-23, bla_NDM-1	1
Pseudomonas. aeruginosa 1, 19 (n = 2)	CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, DO, FF, NF, SXT, TOB, AK, GEN, RA, CIP	>32	/	bla_VIM-6, lost oprD	815
P. aeruginosa 15	CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, DO, FF, NF, SXT, TOB, AK, GEN, RA, CIP	>32	/	bla_VIM-11, lost oprD	316
P. aeruginosa 13a, 15a, 18 (n = 3)	CT	TIC, TIM, CAZ, FEP, TZP, MEM, IMP, DO, FF, NF, SXT, TOB, AK, GEN, RA, CIP	>32	/	stop oprD (n = 2), lost oprD (n = 1)	664
Achromobacter xylosoxidans 22	TIM, CAZ, TZP, SXT, CT	TIC, FEP, MEM, IMP, FF, NF, GEN, TOB, AK, DO, RA, CIP	8	/	/	ND

AK, amikacin; AMC, amoxicillin-clavulanic acid; AMX, amoxicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CT, colistin; DO, doxycycline; ERT, ertapenem; FEP, cefepime; FF, fosfomycin; GEN, gentamicin; IMP, imipenem; KF, cefalothin; MEM, meropenem; MIC, minimum inhibitory concentrations; ND, newly detected; NF, nitrofurantoin; RA, rifampicin; SXT, trimethoprim-sulfamethoxazole; TIC, ticarcillin; TIM, ticarcillin-clavulanic acid; TOB, tobramycin; TZP, piperacillin-tazobactam.

AST of isolated strains

Antibiotic susceptibility testing results revealed high levels of resistance in most of the isolates to tested antibiotics (Table 1). Enterobacteriaceae isolates were resistant to all β-lactams, including imipenem (MIC ranging from 1.5 to >32 μg/mL). In addition, resistance to ciprofloxacin and aminoglycosides (amikacin and gentamicin) was also observed in all K. pneumoniae strains. Resistance to gentamicin and trimethoprim-sulfamethoxazole was observed in E. cloacae isolates. However, all E. coli strains were sensitive to fosfomycin, while 35.7% of Klebsiella spp. and 50% of E. cloacae were also sensitive (Table 1).

For non-Enterobacteriaceae isolates, all A. baumannii isolates were resistant to all tested β-lactams and also to imipenem, nitrofurantoin, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin. A. xylosoxidans was susceptible to ceftazidime and piperacillin-tazobactam, but resistant to imipenem, fosfomycin, and aminoglycosides. Furthermore, all P. aeruginosa isolates were resistant to all antibiotics tested, except colistin. Colistin was found to be active against all isolates with MICs ranging from 0.125 to 1 μg/mL.

Molecular mechanisms of antibiotic resistance

The number of resistance genes in E. coli (n = 9) were as follows: ESBL genes bla_TEM-206 = 4, bla_TEM-169 = 2, and bla_CTX-M-15 = 7, and carbapenem-hydrolyzing enzyme-encoding genes bla_NDM-1 = 3, bla_NDM-4 = 1, bla_NDM-5 = 1_, bla_NDM-7 = 1, bla_OXA-48 = 2, and bla_OXA-181 = 2, whereas one strain #32 carried both bla_OXA-181 and bla_NDM-4 gene (Table 1).

Antibiotic resistance genes in the case of Klebsiella isolates (n = 14) were as follows: bla_TEM-206 = 12, bla_TEM-104 = 1, bla_CTX-M-15 = 14, bla_SHV-67 = 10, bla_SHV-28 = 2, bla_SHV-11 n = 1, bla_OXA-48 n = 9, and bla_NDM-1 = 5. Bla_TEM-206 = 2, bla_TEM-104 = 1, bla_TEM-198 = 1, bla_CTX-M-15 = 1, and bla_NDM-1 = 4 genes were found in the four E. cloacae isolates (Table 1).

All imipenem-resistant A. baumannii isolates harbored bla_OXA-23gene, except isolates 37a and 80 that carried thebla_OXA-72 and bla_NDM-1 genes, respectively. In the case of six P. aeruginosa isolates, mutations leading to a stop codon on oprD gene were found in two isolates and the loss of oprD gene was observed in the other four isolates. In addition, carbapenem-hydrolyzing enzyme-encoding gene bla_VIM-11 was found in isolate 15 and bla_VIM-6 in isolates 1 and 19 (Table 1).

In summary, the frequency of antibiotic resistance genes in Enterobacteriaceae was as follows: bla_NDM = 55.5%, bla_OXA-48-like = 48.1%, bla_TEM = 85.2%, bla_CTX-M-15 = 81.5%, and bla_SHV = 48.1%. In P. aeruginosa, carbapenem resistance is due to the presence of bla_VIM gene (50%) and oprD modifications (100%). A. baumannii isolates harbored bla_OXA-23 gene (100%), bla_OXA-72 (10%), and bla_NDM genes (10%).

Molecular typing by MLST

From the MLST analysis, four sequence types (STs) were identified in E. coli, including ST167 (n = 4), ST617 (n = 2), ST410 (n = 2), and ST405 (n = 1). For K. pneumoniae, majority of STs were ST147 (n = 9) followed by ST273 (n = 2), ST101 (n = 1), and ST15 (n = 1). All E. cloacae belonged to two sequence types, that is, ST 177 (n = 3) and ST182 (n = 1) (Table 1).

Five STs were detected for Acinetobacter isolates (ST2 [n = 5], ST1106 [n = 3], ST207 [n = 1], ST1 [n = 1], and ST1209 [n = 1]). ST1209 is a new ST type assigned by the Pasteur MLST Database. In P. aeruginosa, ST664 (n = 3), ST815 (n = 2), and ST316 (n = 1) were found (Table 1).

Discussion

The results of this study show a high level of antibiotic resistance in clinical Gram-negative isolates against commonly used antibiotics in general and carbapenems in particular. In recent years, the rise in levels of antibiotic resistance, including increasing prevalence of ESBL-producing strains, has been reported from different areas of Pakistan. Therefore, carbapenems have become the drugs of choice to treat such cases. Increased and injudicious use of carbapenems in Pakistan have resulted in rise of carbapenem resistance,^8,18 which is also a cause for concern as the number of available therapeutic options is now limited. Carbapenem resistance is due to diverse mechanisms, including the production of carbapenemases in Gram-negative bacteria. Bacteria producing these enzymes can hydrolyze not only carbapenems but also other antibiotics such as penicillins, cephalosporins, and monobactams as well.^1,5 In addition, these hydrolyzing enzymes are spreading globally because of their prime location on plasmids.^1,2 It is therefore necessary to monitor resistance to detect emergence of different and new resistance mechanisms to prevent the dissemination of novel resistance genes to the rest of the world.¹

As reported in previous studies, bla_NDM was also found to be the most prevalent carbapenemase-encoding gene in Enterobacteriaceae isolates.¹⁰ The second most prevalent genes were bla_OXA-48-like and bla_OXA-181.^9,10 bla_OXA-181 differs from bla_OXA-48 by only four amino acid sequences and has been reported from several countries, including the Indian subcontinent.¹¹ The concurrent presence of different combinations of bla_OXA-48 variants and bla_NDM genes has already been reported in America,¹⁹ Singapore,²⁰ Saudi Arabia,²¹ India,²² and Pakistan.⁹ It is detected primarily in K. pneumoniae strains, and in E. coli isolates. Interestingly, a diversity of carbapenemase-encoding genes has been observed, including the variants bla_NDM-1, bla_NDM-4, bla_NDM-5, bla_NDM-7, bla_OXA-48, and bla_OXA-181 in E. coli isolates. In the Indian subcontinent, the variants bla_NDM-5 and bla_NDM-7 have already been described.^23,24

In Pakistan, only a few studies have described the clonal type of carbapenem-producing Gram-negative bacteria, making it difficult to monitor the emergence or spread of certain specific clones.^9,11,25 Previous studies have reported K. pneumoniae belonging to ST147,¹¹ ST11, ST14, ST15, ST101, and ST307.⁹ In our study, multiple other clones like ST15, ST147, ST101, and ST273 were also detected. ST147, ST101, and ST15 are international clones responsible for the spread of ESBLs and also dissemination of carbapenemase-encoding genes.²⁶ ST273 is an emerging clone described in K. pneumoniae carbapenem-resistant strain.^27,28

In E. coli isolates, the majority of isolates belonged to common ST complexes, such as ST10 complex (ST167 and ST617) followed by ST23 and ST405 complexes. In Pakistan, ST10 and ST405 have been described in NDM-producing E. coli and ST617 in ESBL-producing E. coli from migratory avian species.^25,28–30 Carbapenem-resistant E. coli strains belonging to ST167 and ST405 are present in the Indian subcontinent, notably in Nepal and India.^23,24 In Pakistan, the epidemiology of CRE seems similar to regional epidemiology in the Indian subcontinent.

A. baumannii is considered a leading cause of nosocomial infections throughout the world. Carbapenems are commonly used to treat these infections that result in the emergence and spread of carbapenem-resistant A. baumannii strains. In Pakistan, the frequency of CRAB is very high ranging from 62% to 100% in some hospitals.¹⁸ This study confirms the predominant presence of CRAB carrying bla_OXA-23 and the emergence of strains harboring bla_NDM or bla_OXA-72.^10,31 MLST typing revealed that most carbapenem-resistant A. baumannii isolates belonged to the ST2 (CC2 complex) and some isolates were ST1 (CC1 complex), ST1106, and ST1209. A. baumannii infections worldwide belong to international complexes CC1, CC2, and CC3 and these CC are frequently linked to the production of OXA-23, OXA-24, or OXA-58 enzymes.^15,32 Alternatively, the rare ST1106 clone seems to be emerging in Pakistan where it has already been reported in clinical strains.³³ In addition, the detection of a new ST1209 clone in carbapenem-resistant A. baumannii isolates should be monitored.

Carbapenem-resistant P. aeruginosa are frequent in Pakistan. A previous study reported that 61.89% of Pseudomonas spp. were carbapenem-resistant P. aeruginosa isolates, including 78% of MBL producers in a Lahore hospital.⁸ Genes encoding the VIM enzyme have also been detected in Pakistan, notably bla_VIM-1, bla_VIM-2, bla_VIM-4, and bla_VIM-5, but not bla_VIM-6 and bla_VIM-11, suggesting a change in the epidemiology of these genes.¹⁰ These variants of bla_VIM are endemic in India, highlighting an epidemiology common to the Indian subcontinent.³⁴

Carbapenem resistance due to the alteration of the porin-coding oprD gene has not been investigated in detail in Pakistan, although it is an important phenomenon. In this study, the presence of this mechanism, which can be associated with the presence of MBLs, has been observed with the loss of oprD or loss of expression by a stop codon. The population structure of P. aeruginosa is very diverse, but international high-risk clones, including ST664, are well known.³⁵ The other clones belonging to ST815 and ST316 observed in our study were less predominant, but were associated with carbapenem-resistant isolates found in the Gulf countries and in Portugal.^36,37

In addition, the presence of multidrug-resistant opportunistic A. xylosoxidans, responsible for nosocomial infections,³⁸ should be monitored. To the best of our knowledge, we report here, for the first time, VIM variants (VIM-6 and VIM-11) in P. aeruginosa isolated from various clinical samples in Pakistan. Moreover, this is the only study in Pakistan describing the clonality of different Gram-negative bacteria by MLST, including E. coli, K. pneumoniae, E. cloacae, Acinetobacter spp., and P. aeruginosa isolates, thus allowing the description of the new ST1209 in A. baumannii.

Conclusion

This study highlights the diversity of Gram-negative bacteria resistant to carbapenems and the variety of resistance mechanisms that are associated with them in Pakistan.

The molecular structure of carbapenem resistance in Enterobacteriaceae and P. aeruginosa evolves with the emergence of different genes and gene variants encoding carbapenemases. Clone monitoring is essential to understand the evolution of a country's epidemiology and compare it with the regional and international epidemiology. In our case, although it is similar to the Indian subcontinent, some regional specificity in the clonal types was observed. The identification of high-risk clones with extensive multidrug resistance highlights the importance of a global surveillance of antimicrobial resistance.

Footnotes

Acknowledgment

The authors thank CookieTrad for proofreading the text.

Disclosure Statement

No competing financial interests exist.

Funding information

This work was supported by the French Government under the “Investissementsd'avenir” (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research) (reference: Méditerranée Infection 10-IAHU-03).

This work was supported by Région Provence Alpes Côte d'Azur and European funding FEDER PRIMI.

This work was also partially supported by Higher Education Commission (HEC), Pakistan, Project number 1813.

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