Research Article | Open Access
Ranjitha Shankare Gowda, Sowmya G.S. , Pavithra N., Raghavendra Rao M.,  Krishna Karthik M.  and Satya Sai B.
Department of Microbiology, JSS Medical College, JSSAHER, Mysore – 570 015, India.
J. Pure Appl. Microbiol., 2018, 12 (4): 2283-2287 | Article Number: 5308
https://doi.org/10.22207/JPAM.12.4.69 | © The Author(s). 2018
Received: 15/08/2018| Accepted: 09/10/2018 |Published: 29/12/2018
Abstract

Early onset Ventilator associated pneumonia (VAP) is usually less severe, associated with a better prognosis, and is more likely  caused by antibiotic- sensitive bacteria. Late-onset VAP, is usually caused by multidrug resistant (MDR) pathogens and is associated with increased morbidity and mortality. Therefore, the local microbial flora causing VAP needs to be studied and appropriate therapy based on the early endotracheal (ET) aspirate culture report can help managing this group of patients. The present study was carried out in the department of Microbiology, JSS Hospital, Mysore from January 2017 to December 2017. Bacterial pathogens and antibiotic resistance pattern of isolates from ET aspirates of patients on mechanical ventilation in Intensive care units were source of data. A total number of 1432 samples were received in the lab for ET aspirate culture. Among these 1432 samples, 1055 showed growth ranging from 10 2to 106 colony forming units/ml (CFU/ml), 124  had no growth at all and 253 had no significant growth (<102 CFU/ml) Out of 1055 isolates,1023 were Gram negative bacteria and  32 were Gram positive cocci . Acinetobacter, Pseudomonas and Klebsiella were the major pathogens in our study. Majority of them being MDR, maximum sensitivity was observed for Tigecycline and colistin in these isolates. Knowledge of causative microbial flora of VAP along with information on the susceptibility patterns will help in selection of the appropriate antibiotic for therapeutic use for better outcome.

Keywords

VAP, ET aspirates, Early onset, MDR, susceptibility pattern

Introduction

Ventilator associated pneumonia (VAP) is a globally  known notorious nosocomial infection that occurs more than 48 hours after a patient begins mechanical ventilation (MV)making recovery more difficult for patients who are already critically ill.1 Early onset VAP is usually less severe, associated with a better prognosis, and is more likely to be caused by antibiotic- sensitive bacteria. Late-onset VAP, is usually caused by multidrug resistant (MDR) pathogens and is associated with increased morbidity and mortality2,3. VAP results from microaspiration, which is primary route of bacterial entry into respiratory tract. The ET tube acts as an obstacle to host defences by inhibiting the action of cilia, swallowing, and spontaneous coughing by the patients. Risk factors that are associated with increased incidence of VAP are body position, state of consciousness, enteral nutrition, prior exposure to antibiotics, use of a nasotracheal tube and pre-existing conditions such as acute respiratory distress syndrome4.

Typically, diagnosis includes clinical, radiologic and microbiologic evidence of infection. However, the exact criteria for diagnosis varies widely among institutions and thus results in a wide range of reported VAP mortality rates, which is stated to be anywhere from 0-76% 5,6,7. Many studies from India have investigated the causative organisms of VAP. Pseudomonas species, Acinetobacter species, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus are the common pathogens with varying prevalence and quite often being MDR 6,8,9. Therefore, the local microbial flora causing VAP needs to be studied and an appropriate therapy based on the early ET aspirate report can reduce the mortality and cost in managing this group of patients

Aim
In view of increasing incidence of VAP and MDR nature of pathogens in our hospital, this study was taken up to detect the bacteriological profile and sensitivity pattern of isolates from Endotracheal aspirates Cyperus rotundus L. (family: Cyperaceae) is regarded as a traditionally medicinal herb that has been used for treating numerous clinical ailments such as diarrhea, diabetes, inflammation, malaria, and belly and bowel disorders. Phytochemical and pharmacological research printed that C. rotundus includes an excessive concentration of vital oils, ascorbic acids, phenolic acids, and flavonoids demonstrating its antibacterial, antioxidant, anti-inflammatory, anti-cancerous, and antimalarial4.

Nuclear magnetic resonance (NMR) considered the most advanced technique for the profiling and identifying the metabolites in complex matrices such as plant extracts. The technique is broadly used due to its ease in pattern instruction and the outcomes received are reproducible5. The NMR technique in combination with multivariate statistics evaluation (MVA) has been employed appreciably in recent studies for metabolites profiling for the discrimination of samples of same origin subjected to different processes6. Therefore, this study objective was to determine the antibacterial and antioxidant activities of C. rotundus L Fermented by lactic acid bacteria. Moreover, the metabolites in charge for the antibacterial and antioxidant activities of C. rotundus were identify by using 1H-NMR spectroscopy.

Materials and Methods

The present study was carried out in the Department of Microbiology, JSS Hospital, Mysore, Karnataka, for a period of 1 year from January 2017 to December 2017. This was a prospective study. ET aspirates sent from various intensive care units sent to Microbiology laboratory formed the material of our study

Bacterial identification and isolation
The ET samples received in the Microbiology lab were subjected to microscopy and culture.  Gram stain of Lower  respiratory tract secretions is an immediate procedure that can guide management and it has a reasonable correlation with culture results Semiquantitative culture was done on blood agar and MacConkey agar and incubated overnight at 37°C for 24 to 48 hrs  for bacterial growth and isolation. The isolated organisms were processed with Vitek 2 systems for identification as well as Antibiotic susceptibility testing. Colony count of  10 6 CFUml or more was considered significant for VAP . ET aspirates yielding growth between 10 2 to 10 CFU/ml, whose microscopy revealed  moderate to plenty of inflammatory cells were also processed.

RESULTS

A total number of 1432 samples were received in the lab for ET aspirate culture. Among these 1432 samples, 1055 showed growth ranging from 10 2to 106 CFU/ml or more, 124  had no growth at all and 253 had no significant growth (<102 CFU/ml) Among 1055 bacterial growth isolates, 377 isolates showed growth 10 6 CFUml or more which is considered significant for VAP and 731 isolates ranging 10 2to 106 CFU/ml.

Table (1):
Distribution of microbial isolates in colony-forming units per ml.

102
103
104
105
10or more
Acinetobacter
26
57
23
192
130
Klebsiella
19
32
20
136
77
Pseudomonas
10
27
9
117
62
E.coli
2
2
2
12
11
Proteus
0
0
0
0
3
Citrobacter
0
3
1
2
0
Enterobacter
0
0
2
3
1
Serratia
2
1
2
10
5
Stenotrophomonas
1
2
4
4
3
Burkholderia
0
0
0
3
3
Morganella
0
1
0
0
1
S.aureus
0
0
8
13
4
Coagulase negative Staphylococcus
2
5
0
0
0


Table (2):
Antibiotic Resistance pattern of Acinetobacter.

Antibiotics
Sensitive
Intermediate
Resistant
Piperacillin/Tazobactum
33(7%)
3(0.7%)
392(92%)
Ceftazidine
18(4%)
20(4.6%)
390(92%)
Cefoperazone/ Sulbactum
100(23%)
66(15%)
262(62%)
Cefepime
31(7.2% )
14(3.2%)
383(89.4%)
Aztreonam
10(2.0%)
5(1.16%)
413(95%)
Doripenem
37(8.64%)
391(92%)
Imipenem
34(7.94%)
8(1.86%)
386(90.1%)
Meropenem
39(9.11%)
8(1.86%)
381(89.01%)
Amikacin
17(3.97%)
1(0.23%)
410(95.79%)
Gentamicin
78(18.22%)
32(7.4%)
318(74%)
Ciprofloxacin
30(7.9%)
12(2.80%)
386(90%)
Levofloxacin
32(7.4%)
52(12.14%)
344(80.3%)
Minocycline
242(56.54%)
46(10.74%)
140(32.7%)
Tigecycline
346(80.84%)
75(17.52%)
7(1.63%)
Colistin
412(96.26%)
16(3.7%)
Cotrimoxazole
69(16.12%)
359(84%)


Table (3):
Antibiotic resistance pattern of Pseudomonas.

Antibiotics
Sensitive
Intermediate
Resistant
Ticarcillin/Clavulinic acid
72(32%)
59(26.22%)
94(42.22%)
Piperacillin/Tazobactum
110(48.88%)
25(11.11%)
90(40%)
Ceftazidine
125(55.55%)
20(8.88%)
80(35.55%)
Cefoperazone/ Sulbactum
127(56.44%)
49(21.77%)
49(21.77%)
Cefepime
119(52.8%)
20(8.88%)
86(38.22%)
Aztreonam
81(36%)
59(26.22%)
85(37.77%)
Doripenem
132(58.66%)
3(1.33%)
90(40%)
Imipenem
136(60.4%)
0(0%)
89(39.5%)
Meropenem
126(56%)
13(5.7%)
86(38.22%)
Amikacin
150(66.6%)
71(31.5%)
4(1.7%)
Gentamicin
119(52.88%)
3(1.33%)
32(14.22%)
Ciprofloxacin
118(52.44%)
13(5.77%)
94(41.77%)
Levofloxacin
121(53.77%)
9(4%)
95(42.22%)
Colistin
206(91.55%)
2(0.8%)
17(7.55%)

Out of 1055 isolates, 1023 were Gram negative bacteria and  32 were Gram positive cocci. Out of 1023 GNB, 428 were Acinetobacter species,284 were Klebsiella species,225 were Pseudomonas species, 29 were E.coli, 3 Proteus spp, 6 Citrobacter spp, 6 Enterobacter spp, 20 Serratia, 14 Stenotrophomonas, 6 Burkholderia spp and 2 Morganella spp. Acinetobacter baumanii, Klebsiella pneumoniae and Pseudomonas aeruginosa were the most common isolates in our study. Amongst the 32 gram positive isolates 25 were Staphylococcus aureus, 3 Streptococcus pneumoniae, 2 Beta hemolytic Streptococci and 2 coagulase negative Staphylococcus.

Table (4):
Antibiotic resistance pattern of Klebsiella.

Antibiotics
Sensitive
Intermediate
Resistant
Amoxyclav
41(14.43%)
44(15.49%)
199(70.07%)
Piperacillin/Tazobactum
48(16.9%)
10(3.5%)
226(79.5%)
Cefuroxime
43(15%)
6(2.11%)
235(82.7%)
Cefuroxime axetil
48(16.9%)
5(1.76%)
231(81.3%)
Cefoperazone/ Sulbactum
77(21%)
3(1%)
204(71.83%)
Cefipime
49(17%)
13(4.57%)
222(78.3%)
Ertapenem
98(34.5%)
13(4.57%)
173(60.91%)
Nalidixic acid
74(26.05%)
16(5.63%)
194(68%)
Imipenem
119(41.90%)
63(22.18%)
102(35.91%)
Meropenem
84(29.57%)
10(3.52%)
190(66.90%)
Amikacin
169(59.5%)
40(14.08%)
75(26.40%)
Gentamicin
120(42.2%)
6(2.11%)
158(55.63%)
Ciprofloxacin
83(29.22%)
24(8.45%)
177(62.32%)
Tigecycline
180(63.3%)
64(22.53%)
40(14%)
Colistin
270(95%)
1(0.35%)
13(4.57%)
Cotrimaxozole
102(35.9%)
2(0.70%)
180(63.38%)
Ceftriaxone
40(14.08%)
2(0.70%)
242(85.21%)


Table (5):
Range of Values of MIC of Tigecycline and Colistin for most frequent MDR isolates.

 

Tigecycline

Colistin

Organisms Sensitive
<0.5 – 2
Intermediate
>2 – 6
Resistant
>8 – 16
Sensitive
<0.5 – 2
Intermediate
>2 – 6
Resistant
>8 – 16
Acinetobacter 346 75 7 412 0 16
Klebsiella 180 64 40 270 1 13
Pseudomonas 206 2 17

Majority of Enterobacteriaceae apart from Klebsiella isolates were sensitive to Imipenem, Ertapenem, Meropenem, Gentamicin, Amikacin, Tigecycline, Colistin, Cotrimoxazole, Ampicillin. Among the 25 Staphylococcus aureus, 4 were Methicillin-resistant Staphylococcus aureus sensitive  Vancomycin and Linezolid.

DISCUSSION

Prompt identification of organisms from ET aspirates in ventilated patients and accurate selection of antimicrobial agents, based on MIC represent important clinical goals in prevention of VAP.  Sound knowledge of susceptibility pattern of the locally prevalent pathogens is necessary for choosing the appropriate antibiotics.

In our study, overall, Gram-negative bacilli predominated over Gram-positive cocci.  Acinetobacter spp, Klebsiella spp followed by Pseudomonas were the most common isolates identified. Other significant Gram negative isolates were E.coli, Serratia, Stenotrophomonas, Enterobacter, Citrobacter, Proteus and Burkholderia. Among Gram positive organisms Staphylococcus aureus was most common. The antibiogram pattern of isolates showed that Maximum resistance of Acinetobacter spp and Pseudomonas were seen in Aztreonam, Cefipime, Doripenem, Imipenem, Ciprofloxacin, Piperacillin/Tazobactum. Maximum sensitivity was observed with Colistin, Tigecycline and  Minocycline. Our findings were similar to the other studies6,7,8,9

In last two decades,  Gram negative bacteria have  become important nosocomial pathogens throughout the world and is a leading problem in treatment owing to its multidrug resistance. Colistin, which is an old antimicrobial agent, is very active against these. Susceptibility to Colistin was reported as 97.9–100% in various studies. There was least resistance to Colistin in our study. It appeared to be a good option apart from Tigecycline, in the treatment of VAP developed with MDR pathogens.

CONCLUSION

We conclude that VAP in mechanically ventilated patients is on rise and has been continually associated with indiscriminate and irrational use of antibiotics which contribute to emergence of drug resistant strains. Knowledge of their causative microbial flora in a local setting along with information on the susceptibility patterns will help in selection of the appropriate antibiotic for therapeutic use and a better outcome.  It is clear that Tigecycline and Colistin become the last resort antibiotics in most cases. Within no time in future, we can expect resistance to these antibiotics also making us reach the dead end. Hence, resistance rates should be pursued closely.  Newer alternative drugs revealing promising results to solve the problem is the need of the hour.

Declarations

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

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