To study Prevalence, Pre-disposing factors and Prevention of the following MDRO’s – Klebsiella pneumoniae Carbapenemase Producer, Methicillin Resistant Staphylococcus aureus, Multi Drug Resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli.

SEMINAR BY:NISHIKA BHAN

M.Sc. Microbial TechnologyEnrolment No. - A0928610015AIMT, AMITY UNIVERSITY

NOIDA, UP

Introduction

• Multidrug-resistant organisms are usually bacteria that have become resistant to the antibiotics used to treat them. Bacteria that resist treatment with more than one antibiotic are called multidrug-resistant organisms (MDRO’s).

• Multidrug-resistant organisms are found mainly in hospitals and long-term care facilities. They often affect people who are older or very ill and can cause bad infections.

MDRO’s in present study

• Klebsiella pneumoniae carbapenemases (KPCs) • MDR Pseudomonas aeruginosa • MDR Acinetobacter baumannii • MDR Escherichia coli• Methicillin Resistant Staphylococcus aureus

OBJECTIVES • To identify MDRO isolates from clinical samples of

patients admitted in the hospital. • To study pre-disposing factors for acquiring these MDRO‘s

from patients history. • To review the steps which can help in prevention of

MDRO‘s.

Materials & Methods• The study was conducted at a tertiary care cancer hospital,

Delhi from January 2012 to June 2012. The hospital serves as a major health care facility in the public sector.

• A total of 91 bacterial isolates of clinical importance including E.coli, P.aeruginosa, Klebsiella species, Acinetobacter species and S.aureus were selected for further study. The sensitivity pattern of clinically significant bacteria isolated from the samples brought to Microbiology Laboratory of a tertiary care cancer hospital was determined against commonly used antibiotics using disc diffusion method at the hospital laboratory. The pre-disposing factors were collected from the hospital records.

Sample Processing• Pus Samples Pus samples were collected and directly inoculated on

Blood agar and MacConkey agar and incubated for 24 to 48 hours at 37◦C.

• Urine Samples Urine samples were transferred to sterile centrifuged

tubes, centrifuged and streaked on CLED media. The plates were incubated for 24 to 48 hours at 37◦C. Growth on CLED medium shows that the urine contains pathogenic organisms. The bacterial growth was subcultured on Blood agar and MacConkey agar.

• Blood Samples Blood samples collected from admitted patients

were brought to the pathology laboratory. Blood was transferred to 3-5 ml of Brain Heart Infusion (BHI) broth, and incubated at 37 C for 24 hours. In case of no growth, incubation period was extended for another 24 hours. Growth was sub cultured on Blood agar and MacConkey agar plates, and incubated for ten days at 37 C. For broth with no growth even after 48 hours were still incubated upto ten days. The sample was considered as negative only if there no turbidity or growth on tenth day.

• Respiratory Samples

Sputum and swab samples were directly streaked/ swabbed on Blood agar and MacConkey agar and incubated for 24 to 48 hours at 37 ◦C.

Bacterial strains isolated from all type of samples were identified and characterized up to genus and species level, depending upon the requirement. After identification, routine susceptibility testing was done using disc diffusion method.

Identification and morphological characterization of bacterial strains

• Isolated colonies were initially gram stained. • The isolates were biochemically characterized and

identified. The following tests were carried out: Catalase test, Coagulase test, Citrate utilization test, Nitrate reduction test, Triple sugar iron test, for lactose/glucose fermentation, Manitol and Sucrose fermentation test, H2S production, Indole production test, Urease test and Motility test.

Antibiotic resistance patterns• Antibiotic resistance pattern of the isolated gram negative

bacilli and gram positive cocci were studied. The pattern among different groups of antibiotics, which are commonly in use, was determined by disc diffusion method of Bauer et al.

• Clinical Laboratory and Standard Testing Institute (CLSI, 2011) served to interpret diameter of inhibition zone, with specified potencies of the antibiotics, was used, and is outlined in Table 1 and Table 2.

Table 1 : ANTIBIOTIC PANEL (As per CLSI 2011 guidelines) GRAM POSITIVE COCCI

Antibiotic Abbrev. Disc content R I S S.aureusATCC25923

Amikacin Ak 30 mcg 14 15-16 17 20-26

Gentamicin Gen 10 mcg 12 13-14 15 19-27

Penicillin P 10 U 28 - 29 26-37

Ampicillin Amp 10 mcg 28 - 29 27-35

Cephalothin Cep 30 mcg 14 15-17 18 29-37

Augmentin Amc 20/10 mcg 19 - 20 28-36

Erythromycin E 15 mcg 13 14-22 23 22-30

Clindamycin Cd 2 mcg 14 15-20 21 24-30

Ofloxacin Of 5 mcg 12 13-15 16 21-28

Moxifloxacin Mo 5 mcg 20 21-23 24 28-35

Ciprofloxacin Cip 5 mcg 15 16-20 21 22-30

Tetracycline Te 30 mcg 18 19-22 23 24-30

Chloramphenicol C 30 mcg 12 13-17 18 19-26

Imipenem Ipm 10 mcg 13 14-15 16 -

Table 2: ANTIBIOTIC PANEL (As per CLSI 2011 guidelines)GRAM NEGATIVE BACILLI

Antibiotic Abbrev. Disc content R I S E.coliATCC25922

Amikacin Ak 30 mcg 14 15-16 17 19-26

Gentamicin Gen 10 mcg 12 13-14 15 19-26

Ampicillin Amp 10 mcg 13 14-16 17 16-22

Ciprofloxacin Cip 5 mcg 15 16-20 21 30-40

Moxifloxacin Mo 5 mcg 20 21-23 24 28-35

Imipenem Ipm 10 mcg 13 14-15 16 26-32

Aztreonam Atm 30 mcg 15 16-21 22 28-36

Mezlocillin Mz 75 mcg 17 18-20 21 23-29

Tigecycline Tgc 15 mcg - - - 20-27

Colistin Cl 25 mcg 10 - 11 11-17

Polymyxin - B PB 300 U 11 - 12 14-18

Cefuroxime Cxm 30 mcg 14 15-17 18 20-26

Cefotaxime Ctx 30 mcg 14 15-22 23 29-35

Identification of MDR Pseudomonas aeruginosa, MDR E.coli & MDR Acinetobacter baumannii

• Susceptibility Test Procedure: Mueller Hinton Agar plates were prepared for use in the Kirby-Bauer

Method.• Preparation of the Inoculum: Gram staining was done before starting the susceptibility test. 4-5

similar colonies were transferred with a wire, needle or loop to 5 ml Peptone Water and incubated at 35-37 ºC for 2-3 hours until light to moderate turbidity develops. The inoculum’s turbidity was compared with that of standard 0.5 McFarland (prepared by mixing 0.5 ml of 1.175% barium chloride and 99.5 ml of 0.36N sulphuric acid).

• A sterile cotton swab was dipped into the standardized inoculum and the soaked swab was firmly rotated against the upper inside wall of the tube to express excess fluid. The entire agar surface of the plate was streaked with the swab three times, turning the plate at 60º angle between each streaking. The inoculum was allowed to dry for 5-10 min. with lid in place.

• The discs were applied using aseptic techniques. The discs were deposited with centers at least 24 mm apart.

• The plates were incubated immediately at 35± 2 ◦C and examined after 16-18 hours or longer, if necessary.

• The zones showing complete inhibition were measured and the diameters of the zones were recorded to the nearest mm using a calibrated instrument.

Identification of Methicillin Resistant Staphylococcus aureus (MRSA) from clinical samples

• As recommended by CDC, the isolates of staphylococci were tested for oxacillin susceptibility by agar screen method using 1 mcg oxacillin disc. S.epidermidis ATCC-12228 was used as a negative control. M.H. agar plates were incubated at 35 degree Celsius and read after 24 hrs. of incubation. The bacterial growth on oxacillin screen agar was considered to be MRSA.

Picture a: MRSA showing resistance towards oxacillin (1 mcg) disc on MHA plate. PC stands for positive control (S.aureus ATCC-25923), NC for negative control (S.epidermidis ATCC-12228) and Test is the clinical isolate tested positive for MRSA.

Isolation and Identification of Klebsiella pneumoniae Carbapenemase producer (KPC) from clinical samples

• Modified Hodge Test (MHT)• A 0.5 McFarland dilution of the E.coli ATCC 25922 was prepared in 5 ml of

broth or saline. • Diluted 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of MHB or

saline. • Streaked a lawn of the 1:10 dilution of E.coli ATCC 25922 to a Mueller

Hinton agar plate and allowed to dry for 3–5 minutes. • A 10 µg meropenem susceptibility disk was placed in the center of the test

area. • In a straight line, test organism was streaked from the edge of the disk to

the edge of the plate. Up to four organisms can be tested on the same plate with one drug.

• Incubated overnight at 35ºC ± 2ºC in ambient air for 16–24 hours

Pre-disposing factors

• The pre-disposing factors which lead to MDRO infections were collected from the hospital records.

Table 4: Pre-disposing factors responsible for MDRO infections in patients

S.NO. RISK FACTORS

1. Existing Severe Illness

2. Diabetes

3. Chronic Kidney Disease

4. Skin Lesions

5. Previous use of Antibiotics

6. Dialysis

7. Urinary Catheter

8. Vascular Catheter

9. Regular Hospital visits

10. Regular Dialysis visits

11. Long stay

12. Elderly age Factor

13. Immuno-Compromised

RESULTS

• The present study was conducted on various clinical isolates from indoor (IPD) and outdoor (OPD) patients to determine the prevalence of clinically significant gram negative bacilli such as K. pneumoniae, P. aeruginosa and A. baumannii and gram positive cocci such as S. aureus at a tertiary care cancer hospital, Delhi from January 2012 to June 2012.

• The presence of carbapenemase was best

detected by MHT. The result on a MHA plate displayed characteristic cloverleaf-like indentation. High prevalence rate of carbapenemase producing strains have been reported in Klebsiella species.

• Of the 42 Klebsiella isolates, 29 (69%) were carbapenemase producers.

Picture b: The MHT performed on a 100 mm MHA plate. (1) K. pneumoniae ATCC 1705, positive control (2) K.pneumoniae ATCC 1706, negative control; and (3) a clinical isolate, positive result.

Picture c: MRSA showing resistance towards oxacillin (1 mcg) disc on MHA plate. PC stands for positive control (S.aureus ATCC-25923), NC for negative control (S.epidermidis ATCC-12228) and Test is the clinical isolate tested positive for MRSA.

Picture d: MRSA showing resistance towards oxacillin (1 mcg) disc on MHA plate. PC stands for positive control (S.aureus ATCC-25923), NC for negative control (S.epidermidis ATCC-12228).

Methicillin resistance was documented in 10 (22 %) of the S. aureus isolates. The rate of MRSA during six-month study was 0.05 %.

Prevalence of gram negative bacilli and gram positive cocci:

Prevalence of MDRO’s in pus samples during Jan’12 – June’12

Series11

Percentage of MDRO's in Pus Samples

Prevalence of MDRO’s in Urine samples during Jan’12 – June’12

Series11

Percentage of MDRO's in Urine Samples

Prevalence of MDRO’s in Respiratory samples during Jan’12 – June’12

Series11

Percentage of MDRO’s in Respiratory samples

Prevalence of MDRO’s in Blood samples during Jan’12 – June’12

Series11

Percentage of MDRO’s in Blood samples

Prevalence of MDRO’s in miscellaneous samples during Jan’12 – Jun’12

Series11

Percentage of MDRO’s in Miscellaneous samples

Month-wise prevalence of MDRO’s:

• No definite seasonal variation trend was observed.

• The occurrence of clinical isolates were scattered all through the six months.

Organism Total Isolates

No. of Isolated MDRO’s during 6 months period

Jan Feb Mar Apr May June

Klebsiella sp.

131 10 5 3 6 15 3

A.baumannii

27 1 5 3 2 6 1

P. aeruginosa

69 5 4 0 1 2 0

E.coli 53 2 0 0 1 4 2

S.aureus

45 1 1 4 2 2 0

Rate of MDRO’s during six month study

In the present study, of the 91 MDROs , 42 ( 46.15 %) isolates of Klebsiella species were found to be the most common MDRO’s followed by Acinetobacter baumannii ( 18 , 19.78 %), Pseudomonas aeruginosa ( 12 , 13.18 %), MRSA ( 10 , 10.98 % ) and E.coli ( 9 , 9.89 % ).

Organism Type Total Isolates No. of Isolated MDRO’s

Incidence Rate = No. of cases /

Total Patient Days X 100

(%)

Attack Rate = (No. of MDRO’s /

Total admissions in 6 months) X 100

(%)

% of antimicrobial

resistance within all strains = No. of MDRO’s / Total Isolates X

100 (%)

Klebsiella sp. 131 42 ( 42 / 17192 ) 100= 0.24 %

( 42 / 2738 ) 100= 10.53 %

( 42 / 131 ) 100= 32.06 %

Acinetobacter sp. 27 18 ( 18 / 17192 ) 100= 0.10 %

( 18 / 2738 ) 100= 0.65 %

( 18 / 27 ) 100= 66.66 %

Pseudomonas sp. 69 12 ( 12 / 17192 ) 100

= 0.06 %( 12 / 2738) 100

= 0.43 %( 12 / 69 ) 100

= 17.39 %

E.Coli 53 9 ( 9 / 17192 ) 100= 0.05 %

( 9 / 2738) 100= 0.32 %

( 9 / 53 ) 100

= 16.98 %

S. aureus 45 10 ( 10 / 17192 ) 100= 0.05 %

( 10 / 2738) 100= 0.36 %

( 10 / 45 ) 100

= 22.22 %

Klebsiella sp. Acinetobacter sp.

Pseudomonas sp.

E.coli S.aureus0

35

70

105

140MDROS from clinical samples studied during the period Jan’12 – June’12

Total Isolates Isolated MDRO's

Organisms

No. o

f Cas

es

Pre-disposing Factors

• In the present study, history of previous use of antibiotics was the major pre-disposing factor seen in 81.5 % of patients acquiring MDRO’s, followed by, regular hospital visits and immuno compromised status ( seen in 75 % of patients).

• Use of invasive procedures such as catheters (urinary/ vascular) was seen in 60 % of the patients infected with MDRO’s while 43.75 % was seen in elderly aged patients.

S.NO. RISK FACTORS No. of patients ( % )

1. Diabetes 37.5 %

2. Chronic Kidney Disease 25 %

3. Skin Lesions 25 %

4. Previous use of Antibiotics 81.5 %

5. Dialysis 12.5 %

6. Urinary Catheter 75 %

7. Vascular Catheter 62.5 %

8. Regular Hospital visits 75 %

9. Regular Dialysis visits 12.5 %

10. Elderly 43.75 %

11. Immuno-Compromised 75 %

Chronic

Kidn

ey Dise

ase

Skin

Lesio

ns

Diabete

s

Elderl

y age

facto

r

Vascula

r Cath

eter

Urinary

Cathete

r

Immun

o-Com

promise

d

Regula

r Hosp

ital vi

sits

Previo

us use

of Anti

biotics

0

0

0

1

1

0 0

0

0

1

1 1 1

1

Percentage of the risk factors contributing to the MDR infections No. of patients ( % )

Pre-disposing Factors

No.

of P

atie

nts

(%)

Discussion• The transmission and spread of isolates producing Klebsiella

pneumoniae carbapenemases (KPCs) has become a significant problem. The vast majority of Klebsiella infections are associated with hospitalization.

• As opportunistic pathogens, Klebsiella spp. primarily attack immunocompromised individuals who are hospitalized and suffer from severe underlying diseases such as diabetes mellitus or chronic pulmonary obstruction.

• Treatment of infection caused by these pathogens is thus a considerable challenge for clinicians. 69 % of isolates showed carbapenemase production, these isolates are ineffective in treatment because of production of carbapenemase. Tigecycline and Colistin may be used as treatment modality for such patients.

• Our study also revealed the incidence and pre-disposing factors of multiple MDRO’s in I.C.U. and surgical wards of a tertiary care cancer hospital.

• History of previous use of antibiotics, regular hospital visits and immuno compromised status was seen in 70 % of patients acquiring MDRO’s.

• The prevalence of MDR P. aeruginosa in this present study was found to be 17.39 % ( 12 / 69 ).

• Pseudomonas aeruginosa is the leading cause of nosocomial infections, including pneumonia, urinary tract infections, and bacteremia.

• S. aureus is one of the most frequently isolated nosocomial pathogens and in particular this organism is an important cause of surgical wound infections.

• According to this study, the overall incidence of S.aureus was found to be higher in Pus samples(20 %).

• The reason may be that S. aureus constitutes the normal flora of the skin, or the wound is much exposed and there is greater chance of dissemination, contamination and secondary infection.

Prevention• Adequate hand hygiene, antibiotic stewardship and MDRO

surveillance must be practiced to prevent the emergence and transmission of MDRO’s.

• Hand hygiene is a simple and effective infection control intervention. Dirty or contaminated hands can transmit microorganisms which may cause infection. Hand washing with soap and water is effective (Friedman et al. 2004).

• Contact precautions in addition to other infection prevention measures, e.g., hand hygiene, environmental cleaning, and restriction of antibiotics, have been shown to be effective in preventing transmission in outbreak situations, use of gloves and aprons/gowns.

Conclusions

• The most prevalent gram negative bacilli from clinical samples of urine, pus, respiratory, blood and other specimens ( miscellaneous ) were found to be Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumannii.

• The most prevalent gram positive organism from clinical

samples of urine, pus, respiratory, blood and other specimens ( miscellaneous ) were found to be Staphylococcus aureus.

• No definite seasonal variation trend was observed in month-

wise prevalence of the MDRO’s. The occurrence of clinical isolates were scattered all through the six months.

• Klebsiella species accounted for most of the MDR infections, K.pneumoniae being the most opportunistic pathogen of all.

• High prevalence rate of carbapenemase producing

strains have been reported in Klebsiella species. • The study points out limited use of

carbapenems in vivo in Klebsiella pneumoniae isolates because of production of carbapenemase (69 % of the cases).

• History of previous use of antibiotics was the major pre-disposing factor seen in patients acquiring MDRO’s, followed by, regular hospital visits and immuno compromised status. Use of invasive procedures such as catheters (urinary/ vascular) and elderly status of patients also contributed to MDR infections.

• Adequate hand hygiene, antibiotic stewardship and MDRO surveillance must be practiced to prevent the emergence and transmission of MDRO’s.

• Prevention of antimicrobial resistance depends on appropriate clinical practices that should be incorporated into all routine patient care. These include optimal management of vascular and urinary catheters, prevention of lower respiratory tract infection in intubated patients, accurate diagnosis of infectious etiologies, and judicious antimicrobial selection and utilization.

• The control measures include administrative support, judicious use of antimicrobials, surveillance (routine and enhanced), Standard and Contact Precautions, environmental measures, education and decolonization.

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