Carbapenem–Resistant Gram Negative Bacilli Are Predominantly Multidrug or Pan-Drug Resistant

Abstract

Carbapenems, although originally introduced against multidrug–resistant (MDR) Gram negative bacilli (GNB), are now advocated for initial empiric use resulting in increasing carbapenem-resistant (CR) GNB. In this study, we analyzed the frequencies of CR-GNB and compared their resistance patterns against other antibiotics. Overall, 42% (1,014/2,420) of CR-GNB were isolated (range: 29–59%), with similar frequencies among hospitalized and community-acquired infections. However, the CR frequencies in Acinetobacter baumannii were significantly higher in the hospitalized patients (>50%). In addition, the CR-GNB isolates showed significantly higher resistance to the other antibiotics—fluoroquinolones, aminoglycosides, sulfonamides, and ureidopenicillins compared to carbapenem-sensitive isolates, thereby limiting further treatment options. Majority of CR-GNB isolates were extended spectrum β-lactamase producers (38–72%) and MDR (19–61%). Pan-drug resistant (PDR) frequencies among these MDR isolates ranged from 21% (Proteus spp.) to 100% (A. baumannii). Overall, CR-GNB are predominantly MDR or PDR and so warrant continuous antibiotic surveillance to provide better management of the infectious diseases.

Introduction

Carbapenem is a recently introduced class of antibiotic mainly targeted against multidrug–resistant (MDR) Gram negative bacilli (GNB). A number of factors contribute to antibiotic resistance in GNB, including the lipopolysaccharide content that plays a role in intrinsic resistance, ability to acquire resistance genes, etc.¹ Cephalosporins (third and fourth generations) were the primary drugs of choice for treating GNB infections. This extended use of cephalosporins led to a rise in extended spectrum β-lactamase (ESBL) producing GNB.^2,3 With such emergence of ESBL producers, carbapenems became the empiric choice of drug.⁴ Although emergence of resistance to any antimicrobial agent is a natural survival phenomenon among microorganisms, it is augmented by its overuse. Soon after this increase in usage, carbapenem resistance emerged, posing a challenge to treat MDR GNB.

Carbapenems inhibit bacterial cell wall synthesis by interfering with the transpeptidation reaction of the peptidoglycan. Carbapenems, unlike β-lactams, are not affected by the β-lactamases. Carbapenem resistance is conferred by porin-mediated resistance, overproduction of efflux pumps, or carbapenemase enzyme–mediated resistance.⁵

Beyene et al. reported about 70% of carbapenem-resistant (CR) isolates from intensive care units (ICUs) and about 25% from general wards in Ethiopia.⁶ Veeraraghavan et al. reported 39% of CR-Klebsiella pneumoniae and 12% of CR-Escherichia coli in India.⁷ Mhondoro et al. reported a significant increase in CR-Acinetobacter baumannii and CR-Pseudomonas aeruginosa in Zimbabwe.⁸ In 2017, the World Health Organization (WHO) listed around 12 bacteria as global priority pathogens that need urgent new drug discoveries. In this list, WHO had classified the CR-GNB as critical priority pathogens.⁹ Lakoh et al. and Massart et al. reported that prior colonization, previous antibiotic use, existing comorbidities, and age greater than 65 years are relevant risk factors for the development of ESBL and CR bacterial infections.^10,11 A large study by Seifert et al. revealed very high frequencies of antibiotic resistance among GNB in the developing countries of Asia, Africa, and South America compared to the developed countries in North America and Europe.¹²

In this study, we analyzed the prevalence of CR over a period of 4 years in all the GNB isolates from various specimens received in the Microbiology laboratory of a tertiary care hospital that caters to the middle-to-lower socioeconomic population in South India. The findings of this study revealed high frequencies of CR among E. coli, Klebsiella spp., Proteus spp., P. aeruginosa, and A. baumannii in comparison to the other GNB among both the outpatients (OP) and inpatients (IP). The study also highlighted that the CR-GNB are predominantly MDR or pan-drug resistant (PDR).

Materials and Methods

The study was conducted in a tertiary care hospital-based laboratory that provides services to the middle-to-lower socioeconomic population of a metropolitan city in South India. The study was approved by the Institutional Ethics Committee. The Microbiology data from clinical specimens received between August 2016 and December 2019 were retrieved from the database retrospectively in an anonymous delinked manner and analyzed. Specimens (n = 9,057) processed for bacterial culture and sensitivity using conventional microbiological methods (such as standard biochemical tests for bacterial identification and Kirby Bauer disc diffusion method for antibiotic sensitivity) were included. Significant reportable growth was considered as culture positive in this study. Intermediate or resistant to Imipenem was considered as CR. Intermediate or resistant to at least one agent of three or more classes of antibiotics was defined as MDR isolates. Intermediate or resistant to at least one agent of all classes of antibiotics was defined as PDR isolates.¹³

Colistin resistance was further confirmed by minimum inhibitory concentration (MIC) broth dilution method. Serial two-fold dilutions from 40 to 0.078 μg/mL of Colistin Sulfate (HiMedia, India) were tested in duplicates with 10⁴ cfu/mL of the 4-hour bacterial inoculum using Brain heart infusion broth (HiMedia, India) in 96-well microtiter plates. MIC greater than or equal to 4 μg/mL was considered to be resistant, and an MIC less than or equal to 2 μg/mL was considered to be sensitive as per CLSI guidelines.¹⁴

Data analyses were done in Microsoft Excel, and statistical analyses were done using online calculators like VassarStats.

Results

High frequencies of carbapenem resistance

GNB were isolated from 2,420/9,057 (27%) specimens during a 4-year period from 2016 to 2019. Among these, members of Enterobacteriaceae (1,589/2,420; 66%) were the predominant isolates, followed by P. aeruginosa (333/2,420; 14%) and A. baumannii (131/2,420; 5%). Within the Enterobacteriaceae family, E. coli (668/1,589; 42%) was the most predominant followed by Klebsiella spp. (588/1,589; 37%) and Proteus spp. (199/1,589; 13%). Of the 2,420 GNB isolated, 1,014 (42%) were found to be CR (Table 1). CR rate was higher in these five predominantly isolated GNB (E. coli, Klebsiella spp., Proteus spp., P. aeruginosa, and A. baumannii—866/1,919; 45%) compared to the other GNB group (148/501; 30%), and this difference was statistically significant (p < 0.0001; chi square test). Although the isolation of CR-GNB isolates from various types of infections was almost similar, CR was highest among the isolates from wounds and abscesses (Table 2). The gender-wise and age-wise analyses among the OP and IP are shown in Table 3. Although there was a male preponderance in the CR-GNB frequencies, there was no significant difference between the genders. In the age-wise analyses, the pediatric age group showed the highest CR-GNB frequencies among OP, while the geriatric age group showed the highest CR-GNB frequencies among the IP. However, there was no significant difference between the age groups.

Table 1.

Carbapenem–Resistant Gram Negative Bacilli During 2016–2019

S. no.	Bacteria name	Total	CR (%)
1	Enterobacteriaceae	1,589	730 (46)
(a)	Escherichia coli	668	251 (37)
(b)	Klebsiella spp.	588	306 (52)
(c)	Proteus spp.	199	118 (59)
2	Acinetobacter baumannii	131	70 (53)
3	Pseudomonas aeruginosa	333	121 (36)
4	Other GNB	367	93 (25)
	Total	2,420	1,014 (42)

Number in parentheses indicates the percentage of CR bacteria over the total number of corresponding bacteria isolated.

CR, carbapenem-resistant; GNB, Gram negative bacilli; Other GNB included members of the Enterobacteriaceae family other than E. coli, Klebsiella spp., and Proteus spp., and nonfermenting GNB other than A. baumannii and P. aeruginosa.

Table 2.

Distribution of Carbapenem–Resistant Gram Negative Bacilli in Various Types of Infections During 2016–2019

S. no.	Infection type	Total	CR (%)
1	Septicemia	48	19 (40)
2	Urinary tract infection	680	279 (41)
3	Lower respiratory infection	548	255 (47)
4	Wound and abscess	543	275 (51)
	Total	1,819	828 (46)

Number in parentheses indicates the percentage of CR bacteria over the total number of corresponding specimens.

Table 3.

Gender-Wise and Age-Wise Distribution of Carbapenem–Resistant Gram Negative Bacilli During 2016–2019

	OP (N = 1,027)			IP (N = 1,393)
	GNB (%)	Pr-GNB (%)	CR-GNB (%)	GNB (%)	Pr-GNB (%)	CR-GNB (%)
Gender
Male	498 (48.5)	355 (71.3)	159 (44.8)	874 (62.7)	740 (84.7)	373 (50.4)
Female	529 (51.5)	396 (74.9)	154 (38.9)	519 (37.2)	424 (81.7)	179 (42.2)
Age
0–17	81 (7.9)	59 (72.8)	28 (47.0)	71 (5.1)	46 (64.8)	25 (54.0)
18–30	251 (24.4)	172 (68.5)	60 (34.9)	139 (10.0)	117 (84.2)	40 (34.2)
31–60	467 (45.5)	345 (73.9)	151 (43.8)	721 (51.8)	605 (83.9)	297 (49.1)
61–80	206 (20.1)	159 (77.2)	69 (43.4)	425 (30.5)	360 (84.7)	169 (46.9)
>81	22 (2.1)	16 (72.7)	5 (31)^a	37 (2.7)	31 (83.8)	21 (67.7)^a

Denotes a significant p-value of 0.04 by chi-square test.

The percentage is calculated over the total number of OP or IP numbers.

Pr-GNB—predominant five GNB, including Escherichia coli, Klebsiella spp., Proteus spp., Pseudomonas aeruginosa, and Acinetobacter baumannii; the percentage is calculated over the corresponding GNB columns.

CR-GNB—the percentage is calculated over the corresponding Pr-GNB columns.

IP, inpatients; OP, outpatients.

Correlation of CR with acquisition of infection and specimen type

Carbapenem resistance was further analyzed among the predominant five GNB (E. coli, Klebsiella spp., Proteus spp., P. aeruginosa, and A. baumannii) that were isolated from samples obtained from OP versus IP, as shown in Fig. 1A. CR-GNB were seen in 41% (322/796) of the OP isolates and in 47% (573/1,224) of the IP isolates. There was only a marginal difference in the CR frequencies among the OP and IP isolates, except A. baumannii. In A. baumannii, the CR frequencies were significantly higher in the IP isolates than the OP isolates (p = 0.007; chi square test).

FIG. 1.

Prevalence of carbapenem resistance among the five predominant GNB. Both the graphs have similar X and Y axes. The X-axes denote the five GNB—Escherichia coli (EC), Klebsiella spp. (K), Proteus spp. (P), Acinetobacter baumannii (AB), and Pseudomonas aeruginosa (PA). Y-axes denote the percentage of CR GNB. The percentages are depicted above the corresponding bars. (A) Distribution of CR-GNB in culture positive clinical samples collected from OP (white bars) and IP (black bars). (B) Specimen-wise analysis of the prevalence of CR isolates. White bars—blood samples; dark gray bars—urine samples; black bars—specimens from the LRT; light gray bars—pus aspirates/swabs. CR, carbapenem-resistant; GNB, Gram negative bacilli; IP, inpatients; LRT, lower respiratory tract; OP, outpatients.

The analysis based on type of infections showed that although E. coli (391/680; 58%) was predominantly isolated from urinary tract infections (UTI), a high percentage (44/72; 61%) of E. coli isolated from lower respiratory tract infections were found to be resistant to carbapenem (Fig. 1B). More than 50% of the Klebsiella spp. isolated from all the four different predominant specimen types was resistant to carbapenem. Among the Proteus isolates, those from the pus aspirates/swabs showed higher frequencies of carbapenem resistance. More than 50% of the A. baumannii isolates showed carbapenem resistance, except those from UTI. In contrast, Carbapenem Resistant Pseudomonas aeruginosa (CRPa) isolates were predominantly seen in UTI.

CR isolates were more frequently MDR or PDR

We compared the antibiotic resistance patterns, including MDR and PDR frequencies, among the isolates that were resistant or sensitive to carbapenem. Most carbapenem-resistant Enterobacteriaceae (CRE) isolates showed statistically higher resistance to other antibiotics like cephalosporins, fluoroquinolones, aminoglycosides, sulfonamides, and ureidopenicillins compared to their CS counterparts, except Proteus spp. (Table 4). Similarly, ESBL producers (Table 4) and MDR isolates (Table 5) were statistically higher among CR group compared to CS group. In addition, Table 5 shows that 21% (Proteus spp.) to 100% (A. baumannii) of the MDR GNB were also PDR. Overall, the data indicate that CR-GNB were most often MDR or PDR.

Table 4.

Comparison of Antibiotic Resistance Pattern Between Carbapenem-Resistant and Carbapenem–Sensitive Enterobacteriaceae Isolates from Various Clinical Samples Between 2016 and 2019

Bacteria name (N)	C3GR n (%)	FQR n (%)	AGR n (%)	SFR n (%)	UPR n (%)	ESBL n (%)
Escherichia coli
CR (251)	178 (71)	145 (58)	77 (31)	171 (68)	171 (68)	175 (70)
CS (410)	247 (60)	263 (64)	115 (28)	224 (55)	224 (55)	240 (59)
p value	0.007	0.1	0.5	0.0008	0.0008	0.005
Klebsiella spp.
CR (306)	219 (72)	150 (49)	109 (36)	152 (50)	152 (50)	219 (72)
CS (277)	104 (38)	77 (28)	52 (19)	101 (36)	101 (36)	94 (34)
p value	<0.0001	<0.0001	<0.0001	0.002	0.002	<0.0001
Proteus spp.
CR (118)	46 (39)	34 (29)	28 (24)	27 (23)	79 (67)	45 (38)
CS (79)	22 (28)	43 (54)	28 (35)	12 (15)	42 (53)	22 (28)
p value	0.1	0.0005	0.1	0.2	0.07	0.1
Other Enterobacteriaceae
CR (55)	31 (56)	29 (53)	19 (35)	31 (56)	21 (38)	29 (53)
CS (77)	24 (31)	21 (27)	11 (14)	27 (35)	18 (23)	23 (30)
p value	0.007	0.005	0.01	0.02	0.1	0.01

Numbers in parentheses denote the percentage of n/N for each column.

p Values were calculated using chi square test.

AGR, aminoglycoside resistant (resistant or intermediate to amikacin and gentamycin); C3GR, third generation cephalosporin resistant (resistant or intermediate to second generation cefuroxime and third generation cefixime); CS, carbapenem-sensitive; ESBL, extended spectrum β-lactamase (resistant or intermediate to cefuroxime, cefixime, and ampicillin); FQR, fluoroquinolone resistant (resistant or intermediate to ciprofloxacin and levofloxacin); SFR, sulfonamide resistant (resistant or intermediate to cotrimoxazole); UPR, ureidopenicillin resistant (resistant or intermediate to piperacillin/tazobactam).

Table 5.

Comparison of Multidrug and Pan-Drug Resistance Between Carbapenem-Resistant and Carbapenem–Sensitive Gram Negative Bacilli Isolates from Various Clinical Samples Between 2016 and 2019

Bacteria name	Total (N)	MDR n1 (%)	p	PDR n2 (%)
Escherichia coli	CR (251)	154 (61)		40 (26)
	CS (410)	95 (23)	<0.0001	—
Klebsiella spp.	CR (306)	153 (50)		86 (56)
	CS (277)	80 (29)	<0.0001	—
Proteus spp.	CR (118)	39 (33)		9 (21)
	CS (79)	21 (27)	0.4	—
Other Enterobacteriaceae	CR (55)	29 (53)		11 (38)
	CS (77)	19 (25)	0.002	—
Acinetobacter baumannii	CR (70)	38 (54)		38 (100)
	CS (60)	7 (12)	<0.0001	—
Pseudomonas aeruginosa	CR (121)	23 (19)		19 (83)
	CS (208)	8 (4)	<0.0001	—
Other GNB	CR (93)	2 (2)		2 (100)
	CS (239)	4 (2)	1.0	—

Numbers in parentheses denote the percentage of n1/N in the MDR column and n2/n1 in the PDR column.

p Values were calculated using chi square test.

MDR, multidrug-resistant (resistant or intermediate to cefixime, ampicillin, and carbapenem in case of CR isolates; resistant or intermediate to cefixime, ampicillin, and ciprofloxacin in case of CS isolates); PDR, pan-drug resistant (resistant or intermediate to at least one drug in every class of antibiotic).

PDR isolates were predominantly sensitive to colistin

Ten PDR GNB were tested for colistin sensitivity by MIC method. Only 1/10 (10%) isolate (K. pneumoniae) was resistant to colistin. All the other nine isolates (90%) were sensitive to colistin.

Discussion

Increasing frequencies of ESBL producers have curtailed the empiric use of cephalosporins and escalated the empiric use of carbapenems, which were previously used primarily to treat MDR bacteria. The subsequent surge in carbapenem resistance among GNB poses a serious threat to the treatment of bacterial infections. This warrants a continuous monitoring of the bacterial spectrum and their antibiotic resistance patterns to understand the constantly changing treatment options. In the current study, the predominantly isolated GNB were E. coli, Klebsiella spp., Proteus spp., A. baumannii, and P. aeruginosa. The carbapenem resistance of these five GNB was 45%, while the other GNB isolated exhibited significantly lower resistance rate (30%). The patients in this study predominantly belonged to the middle-to-lower socioeconomic population attending a tertiary care multispeciality hospital in South India. Studies have shown higher frequencies of antibiotic resistance in developing countries compared to developed countries.^12,14,15 Babu Rajendran et al. have reported 38% of CRE in European countries.¹⁶ A few other studies have reported a higher prevalence of these CR bacteria in patients admitted in the ICUs compared to those in the general wards, which consequentially lead to increasing mortality and cost of treatment.^6,17 In a multicentric study from India, the CR frequencies among E. coli and K. pneumoniae were 12% and 39%, respectively, between 2014 and 2016.⁷ In comparison to the previous study, we showed an increase in CR frequencies (ranging from 27% to 67%), indicating a continuing surge. Except carbapenem-resistant Acinetobacter baumannii (CRAb), which was significantly higher among hospitalized patients, all the other critical priority pathogens were equally prevalent among the OP and IP samples. This may imply a high prevalence of CR bacteria in the community. A number of factors like inappropriate antibiotic use, lack of access to clean water, inadequate sanitation measures, and lack of access to high quality health care and health care products have been attributed to the widespread resistance frequencies among communities in different countries.¹⁸

Majority of the CRE are ESBLs and so they are MDR. So, the medical community is left with very little treatment options in case of CR isolates. Therefore, we next analyzed the resistance patterns of the other commonly used antibiotics among the stratified CR and CS isolates to understand the available treatment options. Our data suggest that CR-E. coli, CR-Klebsiella spp., CRAb, and CRPa exhibited statistically higher resistance patterns to the other antibiotics compared to their CS counterparts. MDR frequencies were significantly higher among the CR isolates (more than 50% among most CRE and CRAb, and about 19% in CRPa) compared to the CS isolates. Our study also indicates that more than 50% of the MDR isolates of Klebsiella spp., A. baumannii, and P. aeruginosa are also PDR, which is quite alarming. In such cases, colistin is the last line antibiotic that is available for treatment. Colistin sensitivity was determined in 10 PDR GNB isolates. Majority of these isolates (90%) was sensitive to colistin, while only one isolate was resistant. Unfortunately, each dose of colistin costs about Rs. 4,000 ($57) in India, making it unaffordable to majority of the patients belonging to the middle-to-lower socioeconomic population.

Conclusions

Overall, our study highlights the GNB spectrum in the middle-to-lower socioeconomic population of South India, as well as their emerging MDR and PDR patterns in the context of their resistance or sensitivity to carbapenems. The study also indicates the limited treatment options for tackling CR isolates from the currently available antibiotic armamentarium. Continuous antibiotic surveillance policies and judicious use of antibiotics that can handle CR bacteria are imperative to medically manage the challenging infections.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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