Introduction

In this era of worsening antimicrobial resistance, gram negative bacteria resistant to carbapenem poses a special clinical challenge because they have been the most active and potent agents for a longtime against multidrug resistant gram negative bacteria. This is further supported by World Health Organization priority pathogens list for research and development of new antibiotics in 2017 which enlists carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Acinetobacter baumannii, and carbapenem-resistant Pseudomonas aeruginosa as critical priority [1].

The mechanisms responsible for carbapenem resistance including porin mutations, efflux pump upregulation and carbapenemase production are explained in detail by Nordmann and Poirel [2]. Of significance are chromosomally encoded porin gene mutation (such as OprD), overexpression of genes encoding for efflux pumps (such as MexAB-OprM, MexXY-OprM, or MexCD-OprJ) and the carbapenemases like KPC, VIM, IMP, NDM and OXA-48 types [2-4]. Resistance due to carbapenemase production is clinically most important as they not only hydrolyze almost all beta-lactams and confer high Minimum Inhibitory Concentration (MICs) but are also encoded by genes transferable horizontally by plasmids or transposons which also have genes encoding for other resistance determinants [5].

Reports of carbapenemase-mediated resistance continue to increase globally [6,7], leading clinicians to resort to other antibiotics with therapeutic success against carbapenemaseproducing Gram-negative bacteria. Currently, colistin (polymyxin E) and tigecycline have become the antibiotics of last resort for carbapenemase- producing Gram-negative bacteria [8,9]. Increasing colistin and tigecycline consumption has been concurrent with increasing reports of tigecycline and colistin resistance, especially during therapy. Already, clinical outbreaks involving colistin resistant KPC-producing Klebsiella pneumoniae have been reported in the United States and Italy with worrying recurrence leaving very limited treatment options [5,6]. Increasing nonsusceptibility to colistin and tigecycline during colistin and tigecycline monotherapy has engendered the use of colistin and/or tigecycline in double and triple combinations with a carbapenem, an aminoglycoside (amikacin, gentamycin and tobramycin), rifampicin, fosfomycin or fluoroquinolone [8,9].

In view of the above, this study was conducted to isolate and identify the carbapenemase producing Gram negative bacteria from patients admitted in various wards and determine their colistin and tigecycline susceptibility pattern.

Materials and Methods

Study Design

This cross-sectional study was carried out in the Institute of Microbiology Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai. The study was conducted for a period of 6 months from (April 2017 to September 2017).

Sample Size: 75 carbapenemase producing Gram negative bacteria

Inclusion Criteria

• Non-repetitive isolates of Carbapenemase producing Gram negative bacterial isolates.

• Only one isolate per sample in polymicrobial infection were included.

Bacterial Identification and Phenotypic Analysis of Carbapenems

Non-repetitive clinically relevant isolates of Gram negative bacteria from various clinical samples including blood, sputum, pus, wound swabs, bronchial wash, tracheal aspirates and urine were collected. The isolates were identified as per Standard Microbiological protocol. Antimicrobial susceptibility testing of the isolates were done by Kirby Bauer’s disc diffusion technique on Mueller-Hinton agar, as per Clinical Laboratory Standard Institute (CLSI) guidelines 2017. Isolates with intermediate susceptibility or resistance to meropenem and imipenem by disc diffusion method were screened for the production of carbapenemase.

Phenotypic Detection of Carbapenemases

A phenotypic detection of carbapenemases was done by Modified Hodge test.

Modified Hodge Test (MHT) [10]: 0.5 McFarland standard suspension of E.coli ATCC 25922 was prepared in saline and diluted 1:10 in saline. A lawn culture of 1:10 dilution E.coli ATCC 25922 was done on to a Mueller Hinton Agar plate and allowed to dry for 3-5 minutes. A 10µg meropenem disc is placed in the center of the test area. In a straight line, the test organism was streaked from the edge of the disc to the edge of the plate and incubated at 37°C for 16-20 hours. Positive and negative controls were included.

Interpretation

Enhanced growth (Clover-leaf indentation) = Positive

No enhanced growth = Negative

Colistin Suceptibility Test

Colistin MIC was determined by Epsilometer Test (E-test) using colistin E-strips of concentration gradient 0.016 to 256µg/ ml (HIMEDIA) as per the EUCAST guidelines 2017 [11]. E.coli ATCC 25922 was used for Quality control (QC range, 0.25-2µg/ml).

Four or five identical colonies were inoculated into 5ml of saline and incubated for 2 hours to match 0.5 McFarlands standards. Lawn culture of the suspension was made onto Mueller Hinton Agar plate. The E strip was placed on the Mueller Hinton Agar plate using a sterile applicator and incubated at 37°C for 20-24 hrs. After incubation whereby bacterial growth becomes visible, an elliptical zone of inhibition correlating with the gradient concentration of antibiotic. The MIC value is read from the scale in terms of µg/ml where the point of ellipse intersects the strip (Table 1).

Tigecycline Susceptibility Test

Pseudomonas species is intrinsically resistant to Tigecycline, hence Enterobacteriaceae and Acinetobacter spp were tested for Tigecycline MIC by using tigecycline E-strips of concentration gradient 0.016 to 256µg/ml (HIMEDIA) as per the EUCAST guidelines 2017. E.coli ATCC 25922 was used for Quality control (QC range, 0.03-0.25µg/ml) (Table 2).

Statistical Analysis

Data were entered in Microsoft Excel 2013. MIC₅₀ and MIC₉₀ were calculated for colistin and Tigecycline among various organisms. MIC₅₀ and MIC₉₀ were defined as lowest concentration of antibiotics(lowest MIC) at which 50% and 90% of the tested strains were inhibited.

Results

A total of 112 Carbapenem resistant gram negative isolates by disc diffusion were screened for Carbapenemase production by Modified Hodge Test, out of which 75 isolates tested positive. Clinical samples for the isolates included in the study and distribution of isolated bacteria are provided in (Table 3).

Among the 75 Modified Hodge Test positive(Carbapenemase producers) isolates, 50.6% isolates were from urine sample and the most frequently isolated organism was Acinetobacter spp (26.6%) followed by Klebsiella pneumoniae, E.coli, Klebsiella oxytoca and Pseudomonas spp.

Susceptibility pattern of the isolates to colistin and tigecycline is provided in (Table 4).

Among the 75 carbapenemase producing gram negative bacilli tested, one isolate of Acinetobacter spp. (Acinetobacter baumannii) from the pus sample of pyothorax was resistant to colistin with MIC of 8μg/ml. Two Klebsiella oxytoca showed intermediate susceptibility and six Acinetobacter spp. showed resistance to tigecycline. The Acinetobacter spp. which was resistant to colistin was also resistant to tigecycline with MIC of 0.75 μg/ml.

The distribution of colistin MIC among the carbapenemase producing gram negative bacteria is shown in (Table 5).

Maximum isolates (36/75) showed colistin MIC of 0.75 μg/ml, followed by 1 μg/ml(29/75).

The distribution of tigecycline MIC among the carbapenemase producing gram negative bacteria is shown in (Table 6).

Maximum isolates (14/63) showed tigecycline MIC of 0.064 μg/ml, followed by 0.5 μg/ml(13/63).

The colistin and tigecycline MIC₅₀ and MIC₉₀ was calculated for Enterobacteriaceae and non-fermenters separately are listed in (Table 7).

The Colistin MIC₅₀ was determined to be 1 μg/ml and 0.75 μg/ml for Enterobacteriacae and NFGNB respectively whereas colistin MIC₉₀ was determined to be 1.5 μg/ml for both Enterobacteriaceae and NFGNB. Both the colistin MIC₅₀ and MIC₉₀ for Enterobacteriaceae and NFGNB was found to be in the susceptible.

The Tigecycline MIC₅₀ and MIC₉₀ were determined to be 0.094 μg/ml and 0.5 μg/ml respectively for both Enterobacteriacae and NFGNB.

Discussion

The knowledge of antibiotics susceptibility pattern of the bacteria is necessary to overcome the problem of treating infections with resistant isolates. Available treatment options for infection caused by carbapenemase producing GNB are limited currently with colistin and tigecycline being the primary treatment. Increasing use of colistin and tigecycline for carbapenemase producing GNB infections has led to the emergence of colistin and tigecycline resistance in several countries worldwide. The prevalence of this emerging resistance varies between regions based on the availability and use of these antibiotics [13]. Hence this study was aimed to determine the colistin and tigecycline susceptibility pattern in Carbapenemase producing Gram negative bacteria isolated from clinical samples in a tertiary care hospital, Tamil Nadu.

A recent Global surveillance program study involving 39 countries, revealed 88% and 91.1% of carabapenemase producing Enterobacteriaceae were susceptible to colistin and tigecycline respectively [14] and a study conducted in Bangalore by Chandran, et al revealed 100% and 94.05% of carabapenemase producing Enterobacteriaceae were susceptible to colistin and tigecycline respectively [15]. While in the present study, the colistin and tigecycline susceptibility among Enterobacteriaceae were 100% and 94.43% respectively, which indicates lower prevalence of resistance comparatively.

The MIC₅₀ and MIC₉₀ for colistin and Tigecycline among Enterobacteriaceae in the present study were 1μg/ml & 1.5μg/ ml and 0.094 μg/ml & 0.5 μg/ml respectively while in the Global Surveillance Program were 0.12 μg/ml& 4 μg/ml and 1 μg/ml & 2 μg/ml; and in the study conducted in Bangalore by Chandran et al 0.094 μg/ml & 0.125 μg/ml and 1 μg/ml & 2 μg/ml respectively [14,15]. This breakdown helps us identify, the MIC₅₀ of colistin among Enterobacteriaceae is high in the present study compared to the other studies indicating a impeding rise of resistance in the near future, though the prevalence of colistin resistance is low currently. Thus, prompting implementation of appropriate approach in using these antibiotics.

In the present study, 100% and 95% of Pseudomonas spp and Acinetobacter spp. were susceptible to colistin with MIC₅₀ and MIC₉₀ being 0.75 μg/ml and 1.5 μg/ml respectively for both organism. While 70% of the Acinetobacter spp. were susceptible to Tigecycline with MIC₅₀ and MIC₉₀ being 0.094 μg/ml and 0.5μg/ml. In a study conducted in Egypt 2015 by Wesam Hatem Amer, Acinetobacter spp. revealed 100% and 43.3% susceptibility to Colistin and Tigecycline respectively with MIC₅₀ and MIC₉₀ 0.5 μg/ml & 1 μg/ml and 2 μg/ml & 4 μg/ml respectively [16]. In the study conducted by Sangeetha et al in 2012 at Vellore revealed Colistin and Tigecycline MIC₅₀ and MIC₉₀ of Pseudomonas spp. and Acinetobacter spp as 1 μg/ml & 2 μg/ml and 0.5 μg/ml & 64 μg/ml respectively [17]. These values strengthen the finding that the prevalence of resistance among NFGNB against colistin is much lower in the present study, though the same cannot be said for tigecycline but with proper guidelines the development of resistance can be delayed significantly.

There are few limitations in the study. There are chances for inaccuracies due to lack in use of genotypic methods. Polymyxins diffuses poorly in the media, hence the use of E-test tends to underestimate the resistance pattern.

Conclusion

With the findings in the current study, it is concluded that colistin and Tigecycline can be considered as an option in treating infections with carbapenemase producing Gram negative bacteria, however national and global surveillances studies are recommended to regulate the treatment protocol and dose adjustment. Emerging resistance against these drugs are also to be kept in mind, as it leaves us with very minimal options to treat them. Hence their judicious use and regular monitoring of their susceptibility pattern are also recommended.

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