Research Article | Open Access
Shivendra Mohan and Umar Farooq
Department of Microbiology, Teerthankar Mahaveer Medical College, Moradabad – 244 001, India.
J Pure Appl Microbiol, 2019, 13 (2): 1069-1078 | Article Number: 5580
Received: 10/03/2019 | Accepted: 20/05/2019 | Published: 29/06/2019
Abstract

Infections with MDR GNB are associated with mortality rates 21% higher than those of non resistant GNB and results in longer in patient stays and higher treatment costs. Several Indian studies have reported prevalence of carbapenemase producing Enterobacteriaceae, Pseudomonas and Acinetobacter species in a range of 11% to 81%, because of ample variation reported in prevalence and incidence of carbapenemases reported from different geographical region from time to time, we aimed to determine prevalence of carbapenemase producing organism and carbapenemase encoding genes among clinical MDR-GNB isolates from our area and also to assess the performance of the phenotypic tests. This was a cross sectional study. A total of 510 multi drug resistant isolates included were subjected to MHT and MBL E strip Test to detect carbapenamase production. In addition these isolates were subjected to PCR assay to confirm presence of carbapenamase genes encoding for these enzymes. The study found carbapenemase prevalence of 58.6% by phenotypic tests. blaNDM was the most common gene (24.7%) found by PCR assay followed by blaKPC (14.9%), blaVIM (9.6%) and blaOXA-48 (8.6%). Awareness of the prevalence and incidence of the carbapenem resistance and carbapenemase enzymes is crucial in the prevention of their spread and selection of appropriate treatment options. Study shows high prevalence rate of carbapenam resistant gram negative bacilli in this area, which indicates danger of limited treatment options and requirement of continuous detection of these cases to limit spread of resistant cases.

Keywords

Carbapenemase producing GNB, carbapenem resistant GNB, carbapenemase encoding genes.

Introduction

Bacterial resistance to anti-microbial treatment is emerging as one of the major public health problem. Carbapenamases may be defined as beta lactamases that significantly hydrolyze at least imipenem or meropenem. Resistant to carbapenams is mostly due to the production of carbapenemases, which are b- lactamase enzymes with a capacity to hydrolyze not only the carbapenams but also all the other beta lactam agents1,2. The most common carbapen-emases include verona integron metallo-beta–lactamases types (VIM), imipenemase (IMP) types, Klebsiella pneumoniea carbapenemase (KPC), oxacillinase-48 (OXA-48), and New Delhi metallo-beta-lactamase-1 (NDM-1), encoded by carbapenem resistance determining gene blaVIM, blaIMP, blaKPC, blaOXA-48 and blaNDM respectively1. Phenotypic assay are used to identify carbapen-emase activity while molecular assay have been developed to identify carbapenemase encoding genes2. Recently, increasing resistance to carbapenams in health care associated infections has been reported worldwide3-5. Thus, resistance to carbapenams becomes a real threat to the survival of patients with infections caused by MDR-GNB as mortality in such infections has been reported up to 50% who acquire blood stream infections and overall mortality rates are 21% higher than those of non resistant GNB and results in longer in patient stays and higher treatment costs1,6. Several Indian studies have reported prevalence of carbapenemase producing Enterobacteriaceae, Pseudomonas and Acinetobacter species in a range of 11% to 81%7-11. This study set out to determine the burden of carbapenam resistance, prevalence of carbapenemase producing organism and carbapenemase encoding gene among clinical MDR-GNB isolates obtained from patients. We also aimed to determine performance of Modified Hodge test (MHT) and Metallo b-lactamase (MBL) E Test by comparing them with results of Polymerase chain reaction (PCR) assay.

Materials and Methods

Study design and setting
This was a cross sectional laboratory based prospective study which was carried out in Microbiology department of Teerthankar Mahaveer Medical College and Research Centre, Moradabad, Uttar Pradesh, India, during the period of April 2016 to December 2018.

Sample collection
A total of 2562 non-duplicate samples from patients suspected of infection caused by Gram-negative bacteria like Urine, Pus, Blood, Body fluids, Tracheal secretion, Sputum, HVS, Foley’s Tip etc were collected as per standard sample collection technique reported earlier12.

Collected samples were subjected to conventional methods i.e. Gram staining, Culture, Biochemical tests. Out of 2562 samples 1507 were gram negative and out of 1507 gram negative bacterial isolates 510 were Multi Drug Resistant. Drug susceptibility test was done by Kirby Bauer disk diffusion method with following antibiotic disks Imipenem (IPM) 10µg, Meropenem (MRP) 10µg, Ertapenem (ETP) 30µg, Cefixime (CFM) 5mg, Cefepime (CPM) 30mg, Ceftazidime(CAZ) 30µg, Ceftazidime/Clavulanic Acid (CAC) 30µg, Ceftriaxone (CTX) 30µg, Cefoperzone/Sulbactum (CFS) 30µg, Ciprofloxacin (CIP) 5µg, Piperacillin/Tazobactam (PIT) 10µg, Amikacin (AK) 30µg, Tigecycline (TGC) 15µg and interpreted according to CLSI guidelines13. This was in order to find out MDR Strain and also to find relation between resistance to these drugs and carriage of carbapenemase gene. Multi Drug Resistant (MDR) strains were differentiated according to criteria given by Mattner et al.6 In brief, isolates that were resistant to three different classes of antibacterials but sensitive to carbapenems were included and isolates that were resistant to any one carbapenem but sensitive to other anti-bacterials were also included.

Detection of carbapenemase production
Phenotypic detection of carbapenemase production was done by MHT and MBL E Test. MHT was performed and interpreted according to guideline provided by CDC14. MBL E test strip were obtained by HiMedia Pvt. Ltd and test was performed and interpreted according to kit insert provided with kit15.

PCR amplification for carbapenemase genes
The entire molecular / PCR test (DNA extraction, amplification and gel electro-phoresis) were done in molecular laboratory of Subharti Medical College, Meerut.
DNA extraction was done using Qiagen DNeasy blood and tissue kit following manufacturer’s instructions16. for reaction mixture preparation, commercially available Genei® Master Mix kit was used. Manufacturer’s instruction manual was followed for using the kit. Primers of PrimeX targeting blaVIM, blaKPC, blaOXA-48 and blaNDM were obtained from Valine Life Sciences, India, as described in study by Asthana S et al.17 For reaction mix preparations following contents were added Molecular grade water 15µl, Master Mix. in kit 25µl, Primers 0.2µl and Template DNA 10µl. The amplification was done using Applied Biosystem Veriti 96 well thermal cycler. For blaKPC, blaOXA-48 and blaNDM the programme was initial denaturation at 94oC for 5 minutes followed by 30 cycles of 30 seconds denaturation at 94oC, annealing at 55oC for 30 seconds and extension at 72oC for 1 minute. An additional final extension step was performed at 72oC for 7 minute. For blaVIM the same programme was used except that annealing temperature was adjusted to 45oC for 60 seconds and had a final extension step of 72oC for 10 minutes.

5µl of PCR product were analyzed by electrophoresis in 1.0% agarose stained with ethidium bromide to detect the specific amplified product by comparing with 100 base-pair standard DNA ladder and visualized under gel doc. system. For quality control, of MHT well characterized strains were used. E. coli ATCC 25922 was used as a susceptible strain, K. pneumoniae ATCC BAA-1705 as a positive control while K. pneumoniae ATCC BAA-1706 was used as a negative control and for PCR tests, the following control strains were used; K. pneumoniae ATCC strain BAA-1705 for blaKPC, K. pneumoniae ATCC strain BAA-2146 for blaNDM, and E. coli ATCC BAA-2523 for blaOXA-48.

Ethical Approval
The study protocol was carefully reviewed and approved by the Institutional Ethics Committee of the Teerthankar Mahaveer Medical College and Research Centre.
Data Analysis
Data analysis was done using SPSS ver. 16.0. All categorical variables were represented by percentages and Comparison of categorical variables was done by Chi-square test. A p value of <0.05 was considered as evidence of significant statistical difference.

RESULTS

Distribution and Characteristics of Isolates Included in study
A total of 2562 samples were processed during study period of April 2016 to December 2018 in which 1507 were gram negative bacilli and out of 1507 gram negative bacterial isolates 510 were Multi Drug Resistant. MDR strains were differentiated according to criteria given by Mattner et al.6 most of which were resistant to three different classes of anti-bacterial. Overall 25.1% isolates were resistant to one or more carbapenem tested. Individually, 11%, 8.1%, 6% resistant rate was observed by Imipenem, Ertapenem and Meropenem respectively. Out of imipenem, ertapenem and meropenem resistant isolates genes were present in 59% 64%, and 68.8% isolates respectively.132 isolates were resistant to three different classes of anti-bacterial but sensitive to carbapenems tested. Distribution of antibiotic resistant isolates is shown in Table 1. Out of 510 MDR strains 267 were from male and 243 from female patients. The age of patients ranged from 1day to 82 years with a median of 35 years. Most common species isolated among MDR-GNB was E. coli (34.7%, 177/510) followed by K. pneumoniae (18.2%, 93/510) and P. aeruginosa (9.4%, 48/510). It was also observed that highest frequency of E. coli was from Gyne ward and that of K. pneumoniae was from Medicine ICU. Distribution of isolates from various sources is shown in Table 2. Highest numbers of MDR organisms were from MICU followed by SICU. Most common sample was Urine (140/510) followed by Pus (113/510) and Blood (85/510). Majority of Urine samples was received from FMW, Gyne and MMW. Majority of Pus samples were from MMW and Blood samples were from Medicine ICU and Surgical ICU as shown in Table 3.

Table (1):
Cross Tabulation showing  total drug resistant isolates and number of isolates with gene detected.

Drug Resistant
Gene Detected
No gene detected
Total Isolates
Imipenem
92
74
166
Ertapenem
75
47
122
Meropenem
57
33
90
Carbapenem resistant MDR-GNB
224
154
378
Carbapenem sensitive MDR-GNB
17
115
132
Total
241
269
510

Table (2):
Shows Ward wise Distribution of Species isolated.

Wards E.coli K.pneumoniae P.aeruginosa A.lwoffi C.freundi E.aerogenes Other Organism* Total
ICCU 02 01 01 04
MICU 14 14 04 09 09 13 63
NEU. ICU 03 02 03 06 02 01 09 26
NICU 05 05 01 02 05 18
PICU 01 01 02 01 02 06
SICU 14 07 07 06 05 03 07 49
ENT 01 02 03
FMW 18 08 01 01 01 05 34
FOW 01 01 02
FSW 06 04 01 01 05 17
GYNE 22 02 01 01 01 04 31
L/R 06 03 01 01 01 12
OBS 14 03 01 03 05 03 05 34
MMW 14 07 03 01 02 27
MOW 07 08 09 05 02 03 04 38
MSW 13 09 06 03 03 06 40
URO 01 01
OPD 09 05 01 01 04 09 29
PEDIA 04 02 01 01 08
PVT.     05 03 01 01 01 11
TBC     05 03 05 04 01 01 08 27
E/W     15 05 05 02 01 01 29
TOTAL 177 93 48 41 32 28 91

*Others Organism Include K. oxytoca (n=21), E. cloacae (n=17), C. koseri (n=13), P. mirabilis (n=4), P. vulgaris (n=3), Pseudomonas Species (n=29), A. baumanni Complex (n=4)

ICCU: Intensive Critical care Unit, MICU: Medicine Intensive care Unit, Neu ICU: Neuro Intensive care Unit, NICU: Neonatal Intensive care Unit, PICU: Paediatric Intensive care Unit, SICU: Surgical Intensive care Unit, FMW: Female Medicine Ward, FOW: Female Orthopaedic Ward, FSW: Female Surgery Ward, L/R Labour Room, MMW: Male Medicine Ward, MSW: Male Surgery ward, URO: Urology Ward, PVT.: Private ward, OPD: Out Patient Department, E/W: Emergency ward

Table (3):
Distribution of type of samples obtained from different wards

Wards
Urine
Pus
Blood
Sputum
ET. Secr.
Other Samples#
Total
ICCU
02
01
01
04
MICU
08
01
20
07
20
07
63
NEU. ICU
02
02
01
18
03
26
NICU
10
08
18
PICU
04
03
07
SICU
07
09
16
02
12
03
49
ENT
03
03
FMW
21
02
05
02
04
34
FOW
01
01
02
FSW
04
07
02
02
02
17
GYNE
22
05
04
31
L/R
03
09
12
OBS
16
05
13
34
MMW
21
02
02
02
27
MOW
35
02
01
38
MSW
05
18
07
03
03
04
40
URO
01
01
OPD
13
15
01
29
PEDIA
02
03
03
08
PVT.
03
03
02
01
01
01
11
TBC
01
03
13
10
27
E/W
09
03
09
08
29
TOTAL
140
113
85
42
56
74
510

#Other Samples Include: – BAL (n=12), Bronchial Biopsy specimen (n=2), Catheter Tip (n=1), CSF (n=2), D&E Specimen(n=1), Drain Fluid (n=3), Peritoneal Fluid (n=7), Rectal Swab (n=7), Vaginal Swab (n=3), Pleural Fluid (n=1), Foley’s Tip (n=18), HVS (n=17)

Prevalence of Carbapenemase producing organism based on MHT and MBL E-test
Carbapenemase activity was detected in 15.6% (80/510) isolates by MHT method, 23.5% (120/510) by MBL E test method. 19.4% (99/510) isolates were positive for both tests. Therefore, total number of isolates positive by MHT was 179/510 (35.0%) and by E test was 219/510 (42.9%) correlation of phenotypic results with that of number of gene detected is shown in Table 4. Among 510 MDR isolates total 299 were carbapenemase producers by phenotypic methods and 211 were non carbapenemase producers. Table 5.

Table (4):
Cross tabulation of Results of Phenotypic tests with Gene detection

MHT RESULT MBL E-TEST RESULT TOTAL NO. OF SAMPLES NO. OF SAMPLES IN WHICH GENE FOUND NO. OF SAMPLES IN WHICH GENE NOT FOUND
POSITIVE POSITIVE 99 86 13
POSITIVE NEGATIVE 80 51 29
NEGATIVE POSITIVE 120 104 16
NEGATIVE NEGATIVE 211 NIL 211
TOTAL NO OF SAMPLE  PROCESSED 510 241 269

Table (5):
Distribution of Number of Carbapenemase Producing and Non Carbapenemase producing isolates from different wards.

ICU’S
CPP
CPN
TOTAL ISOLATES
MICU
44
19
63
SICU
34
15
49
PICU
3
4
7
NICU
6
12
18
NEURO ICU
20
6
26
ICCU+ CCU
3
1
4
110
57
167
WARDS
CPP
CPN
TOTAL
MSW
26
14
40
MOW
19
19
38
MMW
12
15
27
FMW
20
14
34
FSW
8
9
17
FOW
1
1
2
GYNE
15
16
31
L/R
8
4
12
OBS
18
16
34
E/W
19
10
29
PEDIA
4
4
8
ENT
3
0
3
URO
1
0
1
TBC
14
13
27
PVT.
7
4
11
175
139
314

Prevalence of genes encoding for Carbapenemase enzymes
Based on PCR assays 47.2% (241/510) of the isolates were positive for one or more gene. E.coli was species with highest number of gene detected, whereas percentage wise highest rate of gene among MDR isolates were detected in E. cloacae (64.7%) followed by C. freundi (62.5%). 82 out of 177 MDR E. coli strains were positive for one or more gene followed by K. pneumoniae (40/93, 43%), A. lwoffi (25/41, 60.9%), P. aeruginosa (20/48, 37.9%) and C. freundi (20/32, 62.5%). 82 (16.07%) isolates had presence of one or more genes. Distribution of gene in various MDR isolates is shown in Table 6. All target genes were unevenly distributed among the isolated species with overall highest prevalence of blaNDM (24.7%) followed by blaKPC (14.9%), blaVIM (9.6%) and blaOXA (8.6%). Species wise distribution of genes detected is shown in Table 7.

Table (6):
Species wise distribution of Gene detected in number of MDR isolates.

ORGANISM ISOLATED
Total number of MDR organisms
GENES DETECTED IN
E.CLOACAE
17
11(64.7%)
C.FREUNDI
32
20(62.5%)
A.LWOFFI
41
25(60.9%)
K.OXYTOCA
21
12(57.1%)
P.MIRABILIS
4
2(50%)
E.COLI
177
82(46.3%)
C.KOSERI
13
6(46.1%)
K.PNEUMONIAE
93
40(43.0%)
P.AERUGINOSA
48
20(41.6%)
E.AEROGENES
28
11(39.2%)
PSEUDO.SPP
29
11(37.9%)
ACB COMPLEX
4
1(25%)
P.VULGARIS
3
0(0%)
TOTAL
510
241

Table (7):
Distribution of  different Carbapenemase genes detected in various Enterobacteriaceae, Pseudomonas and Acinetobacter species.

Group Organism NDM KPC VIM OXA NDM,KPC NDM,VIM NDM,OXA KPC,VIM KPC,OXA VIM,OXA NDMKPC

VIM

NDMKPCOXA NDMVIMOXA KPCOXAVIM NO. of isol.
 

 

 

 

 

Enterobacteriaceae

E.coli 28 14 10 11 06 04 02 05 01 01 82
K.pneumoniae 15 07 08 01 04 01 01 02 01 40
K.oxytoca 06 01 01 02 01 01 12
E.aerogenes 06 01 01 02 01 11
E.cloacae 04 00 01 02 01 02 01 11
C.freundi 10 01 01 01 02 02 01 01 01 20
C.koseri 02 01 01 01 01 06
P.mirabilis 01 01 02
P.vulgaris
Total 72 26 19 16 16 06 10 06 05 03 01 02 02 184
 

Pseudomonas

P.aeruginosa 08 05 04 01 01 01 20
Pseudo.Spp 05 02 03 01 11
Total 13 07 07 01 01 02 31
 

Acinetobacter

A.lwoffi 11 04 02 02 01 01 02 02 25
Acb Complex 01 01
Total 12 04 02 02 01 01 02 02 26
Grand Total 97 37 28 19 18 06 11 06 07 02 03 03 02 02 241

Correlation of phenotypic and genotypic tests
Out of 80 isolates positive by MHT 51 isolates showed presence of one or more gene and in 29 isolates no gene was detected. Out of 120 isolates positive by MBL E test genes were detected in 104 samples and in rest of 16 isolates which were phenotypically positive but no gene was detected in them. 99 isolates which were positive for both test MHT and MBL E test 86 were positive for gene detection and in 13 isolates no gene was detected. Table 4

Sensitivity and specificity of Modified Hodge test and MBL E test was calculated considering PCR as gold standard. MHT gave better performance for detection of Class A and Class D genes, sensitivity and specificity for blaKPC was 93.4% and 75.1% and sensitivity and specificity for blaOXA calculated was 84.7% and 69.8% whereas MBL E test is better for MBL detection, sensitivity and specificity for blaNDM was 99.2% and 78.3%. Overall sensitivity and specificity of MHT found was 56.8% and 78.8% and sensitivity and specificity of E-test was 82.4% and 86.7% respectively. Statistically it was also evident that MBL E Test had strong association with detection of blaNDM and blaVIM (p<0.05) and MHT showed a good association with detection of blaKPC and blaOXA-48 genes. (p<0.05).

DISCUSSION

Antibiotic resistance to reserve antibiotic class is on a continuous rise among gram negative bacteria especially in the family Enterobacteriaceae and among species of Acinetobacter and Pseudomonas (EPA Species)1,2. Recently, a newspaper article reported 13% of mortality rate in India is due to antibiotic resistant cases, which is more than double when compared to developed nations where mortality rate due to drug resistant cases is 2-7%18. Worldwide several studies had reported increased prevalence of carbapenemase producing organisms5,19,20. Our findings show out of 510 MDR strains 52.3% were from males and 47.6% were from females. Ratio of male to female patient in this study was 1:1.09 this shows almost equal distribution of Antibiotic resistant strains among both sexes. We found most common MDR organism was E.coli followed by K. pneumoniae and P. aeruginosa and the hotspot zone of these organism were medical and surgical ICU’S. Similar findings were reported by Mathias et al. from Ludhiana, Diwakar J. et al. from Etawah and Manohar et al. from Tamil Nadu region8,9,21. These findings may have vital role in making of hospital infection control policy. This study shows a prevalence rate of carbapenemase enzyme of 58.6% by phenotypic tests among EPA species resistant to three different classes of antibacterials or resistant to any one of the carbapenem tested. Our phenotypic prevalence is lower than then that reported by Diwakar et al. from Etawah, Mate et al. from Imphal and Saini et al. from Jaipur who reported phenotypic prevalence of 81.8%, 60% and 83% respectively9,10,22, whereas, higher as compared to that reported by Mathias et al. from Ludhiana, Gupta et al. from Varanasi and Singh et al from Navi Mumbai8,23,24. They reported phenotypic prevalence of 57%, 50% and 43.7% respectively. The difference in these findings could be because of variation in geographical regions which occur from time to time and also because of different inclusion criteria and test done. Our prevalence is also much higher than that reported in studies from western countries like United States, Canada, and Latin America19,25,26. These differences may be due to restricted use of antibiotics in these countries compared to India where many drugs are available over the counter without prescription of a clinician. In parallel, we found prevalence of genes encoding for carbapenemase was 47.2%. Variable rate of genotypic prevalence has been reported by various Indian studies ranging from 18% to 100%7-11,23. The difference might be due to different target genes as in some studies only single class of gene was targeted whereas in our study common genes of all classes of carbapenemase were included. The most prevalent gene among 510 MDR GNB isolates was blaNDM, (27.4%) This was in accordance to studies elsewhere in India viz., Delhi, Guwahati, Mumbai, Vellore and Puducherry reporting blaNDM as the commonest gene detected7,27-29. Although in western world most common gene encoding carbapenemase found is blaKPC19,30. Whereas a study from Africa reported highest prevalent gene were blaIMP types while another one reported blaVIM as the most common gene encoding for carbapenemase enzyme1,2. These findings are suggestive of inter-regional spread of the specific mechanism of carbapenem resistance. In our study we found 58(11.3%) samples were phenotypically positive but no gene was detected in them by PCR. This might be due to limited number of genes targeted in our study as well as to other mechanisms of resistance such as porin loss or mutations.
When we compared the performance of phenotypic tests to results obtained by PCR, it was found that Modified Hodge test was more sensitive and specific for Class A enzyme i.e. blaKPC (93% sensitive and 75.11% specific) and Class D enzyme i.e. blaOXA whereas MBL E test performed better for detection of Class B enzymes i.e. blaNDM and blaVIM. Similar results were reported by Girlich et al.31

CONCLUSION

Carbapenemases are globally distributed and their prevalence and incidence vary considerably across each continent, nation, region and even centre to centre, so awareness of the prevalence and incidence of the carbapenem resistance and carbapenemase enzymes is crucial in the prevention of their spread and selection of appropriate treatment options. Our study shows high prevalence rate of carbapenamase producing gram negative bacilli in this area, which indicates danger of limited treatment options and requirement of continuous detection of these cases to limit spread of resistant cases. We also found that combination of two phenotypic tests MHT and E strip Test can be done together to rule out false negative results whereas E Test should be done on regular basis to detect MBL as MBL encoding genes were more prevalent in our region as it is not feasible to do PCR on regular basis on every sample.

Declarations

Acknowledgments
Authors would like to acknowledge support of Dr. Sanjay Kumar and Dr. Seema Negi of Subharti Medical College Meerut for their guidance and support in processing of samples for gene detection. SM is thankful to Ms. Sana Nudrat for her valuable help in sample processing and data collection. Authors also acknowledges support of Dr. Umme Afifa for help in statistical analysis of collected data.

Conflicts Of Interest
The authors declare that there is no conflict of interest.

Author’s Contribution
SM performed the tests, collected data, did data analysis and wrote the manuscript. UF guided the study and reviewed the manuscript. SM and UF approved it for publication.

Funding
None.

Data Availability
All datasets generated or analysed during this study are included in the manuscript.

Ethics Statement
The study protocol was carefully reviewed and approved by the Institutional Ethics Committee of the Teerthankar Mahaveer Medical College and Research Centre, Moradabad U.P. India.

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