European Journal of Cardiovascular Medicine

Healthcare-associated infections (HAIs) are one of the leading causes of morbidity and mortality among hospitalized patients all over the world.1HAIs, also called ‘nosocomial infections’, are the infections acquired during hospital care occurring more than 48 h after admission, which are not present, or incubating at the time of admission.²

Approximately 1.4 million patients are affected with HAI worldwide, with global prevalence ranging anywhere between 7% and 12%, as per WHO estimation. Published literature from Indian subcontinent has indicated heterogeneity in reported prevalence with numbers ranging anywhere between 11-83%.¹

According to the National Health Safety Network (NHSN) and CDC criteria, HAIs have been classified into 4different types. HAI is diagnosed by using a combination of clinical, laboratory and imaging criteria.3 Clinical diagnostic characteristics for CAUTI are fever (>38°C), suprapubic tenderness, costovertebral angle pain or tenderness, urinary frequency, urinary urgency and dysuria etc,for CLABSI are some systemic characteristics such as fever, hypotension and bradycardia.4-5 The clinical diagnostic characteristics for VAP are fever (>38ºC), increased respiratory secretions, increased suctioning requirement, cough, dyspnea, tachypnea, bronchial breath sounds, increased oxygen requirements, and leukocytosis (>12000 WBC/mm3) or leukopenia (<4000 WBC/mm3) etc.³

HAIs, not only increase morbidity and mortality, but also impose a significant economic burden on the health care infrastructures.6 It is well known that inappropriate and irrational use of antibiotics to treat infections lead to the emergence of Multi Drug Resistant (MDR) strains among the common bacterial isolates so that occurrence of pathogens, along with the resistant genotypes of Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus, Extended-Spectrum Beta-Lactamase (ESBL)-generating Escherichiacoli and Klebsiella species& carbapenems-resistant E.coli, Klebsiella species, Proteus species, Pseudomonas aeruginosa and Acinetobacterbaumannii inflict HAIs especially inside the ICU setting.7 The frequency of occurrence of infection among patient to the ICU/ Ward may vary from one geographical region to another, from one hospital to another and even among the ICU within one hospital. The types of infection, the profile of pathogen causing these infections, their antimicrobial susceptibility pattern also vary according to the location.3Therefore, this study was conducted to determine the microbiological profile of HAIs and to identify potential microorganisms and their antimicrobial susceptibility pattern.

This observational prospective study was conducted on patients who were clinically suspected with HAIs and admitted in the Intensive Care Unit and wards of Govt. R. D. B. P. Jaipuria Hospital and Hospital of RUHS College of Medical Sciences, Jaipur fulfilling the inclusion and exclusion criteria during the study period from September 2022 to February 2023 after ethical committee approval.

Diagnostic criteria were based on CDC guidelines for HAIs. The samples such as blood, endotracheal tube aspirates, urine, wound swab, vascular catheter tips, sputum, etc. were collected according to standard procedure related to each sample, from the patients suspected of HAIs and immediately transported to the laboratory. In the microbiological laboratory, each sample was processed as per the Clinical and Laboratory Standard Institutes (CLSI) guidelines.⁸

Blood samples were collected in blood culture bottles and incubated at 37°C.If the blood culture was observed positive, it was sub-cultured on Nutrient agar, Blood agar, and MacConkey agar, while other types of samples were proceeded immediately after receiving, by inoculating on Blood agar, MacConkey agar, Nutrient agar (Gram’s Staining of all samples except urine, blood and wet mount of urine was done on the day of receiving), then incubated at 37°C for 18-24 hours. Microbiological identification was done on the basis on colony characteristics, microscopical characteristics, and biochemical tests as per standard guidelines/SOP. Antimicrobial susceptibility testing was performed as per standard guidelines. (Reference latest CLSI Guidelines).

During the study period of six months (from September 2022 to February 2023), a total of 861 samples from 749patients submitted to the microbiological laboratory of RUHS Medical College and RDBP Jaipuria Hospital, Jaipur was included in the study.

Out of 749, 97patients (12.95%, 105 samples) were confirmed having HAI. One patient had 3 HAI and six patients had 2 HAIs respectively, resulting in a total of 105 confirmed HAIs. Table 1 shows the distribution of observed HAI from different departments.

Table No.1: Distribution of observed HAIs from different departments

Although higher number i.e. 48/97 of HAIs were observed from the samples received from the ward as compared to ICU but the percentage of HAI observed in samples from ICU was more. Among the HAIs from the ward, majority of cases were CAUTI (24/48, 50%) followed by SSI (22/48, 48%). However, among HAI observed in patients admitted in ICU (43), maximum was VAP (26/43) followed by CAUTI (19/43).

Table 2 shows the microbiological profile of the various HAI. The Most common organism associated with patients of CAUTI(N-43) was E. coli 19/43(44.18%) followed by Candida spp. 11/43(25.58%), Pseudomonas spp. 5/43 (11.6%%), and Enterobacter spp. 2/43(4.65%) respectively while the microbiological profile of VAP showed Pseudomonas spp. Kleb. pneumoniae, Candida spp., Proteus mirabilus, Enterobacter spp., Enterococcus spp. Acinetobacter spp.3 and Citrobacter. The SSI showed the infection of S. aureus (10/29) followed by Pseudomonas (6/29), Klebsiellaoxytoca (3/29), E. coli. (3/29), CoNS (3/29), Acinetobacter spp. (2/29), Proteus mirabilus (1/29) and Kleb. pneumonae (1/29) respectively. Out of 4 cases of CLABSI, 3 showed infection of Acinetobacter spp. and1 showed Kleb. pneumonae.

Table No.2: Microbiological profile of various HAIs

Out of 105 isolates, 62 (59.04%) showed resistance to drugs while 43 didn’t show resistance to any drug. Table 3 shows the pattern of antimicrobial resistance in isolated organisms from various admission sites and Table 4 shows the resistance pattern of isolates in different types of HAIs. Resistance was seen in 30/51 (58.82%) isolates from the ICU. Among them 16/51(31.37%), were MBL producers, 7/51(13.7%) were AmpC positive, 5/51 (9.8%) were ESBL producers and1/5(12%) were HALR.

Bacterial isolates from the ward’s patients were 54. Among them ESBL, MBL producers were 10/54(20.42%) and 3/54 (10.20%) bacterial strains were MRSA. 4/54(8.16%) bacterial strains were AmpC Positive and the remaining 36/54(40.81%) bacterial isolates presented with no resistance pattern.

Table 3- Pattern of antimicrobial resistance in isolates

Table No.4: Antimicrobial Resistance Pattern in organisms associated with various HAIs (N-105)

All CLABSI isolates were found resistant, while 25/43 (58.18%), 16/29 (55.17%), and 17/29 (58.62%) isolates were found resistant in CAUTI, VAP and SSI cases respectively.

Table No.5: Antimicrobial Susceptibility Pattern of Gram-positive isolates

All Staphylococcus spp.(N-15) isolates showed sensitivity to Linezolid while highest resistance (10/15, 66.6%)was observed for Levofloxacin. Enterococcus (N-2) isolates showed 100% sensitivity to Linezolid, 100% resistance to Amikacin, Cefoxitin and Co-Trimoxazole, 50% resistance to Amoxyclav, Tetracycline and Gentamicin. Table5 shows the antimicrobial susceptibility pattern of Gram-positive isolates.

Table No. 6: Antimicrobial Susceptibility pattern of gram-negative (except pseudomonas spp.) isolates for common antibiotics

The total number of GNB isolated (except pseudomonas spp.) was 52. Among them, maximum isolates showed sensitivity to Meropenem (84%) followed by Gentamicin (75%), Ampicillin/Sulbactam (69.2%), and Imipenem (63.5%). Isolates showed high resistance to Cefotaxime, Cefoxitin (82%) followed by Ceftazidime (80%), Amoxyclav (69%) and Amikacin (53%). Table 6 shows the Antimicrobial Susceptibility pattern of gram-negative (except pseudomonas spp.) isolates for common antibiotics and Table 7 shows Antimicrobial Susceptibility pattern of Gram-negative isolates of Urine & Blood samples.

Table No. 7: Antimicrobial Susceptibility pattern of Gram-negative isolates of Urine & Blood samples.

Total GNB isolated from urine samples were 26. GNB isolates showed maximum resistance to Ceftriaxone and Cefoxitin (88.5%) followed by Cefotaxime, Ceftazidime and Nalidixic acid 80.8%. Co-trimoxazole 73.1%. Ciprofloxacin 57.7% and Amoxyclav 53.8%. Maximum sensitive antimicrobials were Meropenem 96.2%. Gentamicin 80.8% followed by Nitrofurantoin 76.9%, Ofloxacin 76.9%, Imipenem 69.2%, Amikacin69.2%, Ampicillin/Sulbactam 65.4%. Ceftazidime/ Clavulanate and Tetracycline both were 50% sensitive.

All Gram-negative isolates (n-4) of blood samples showed resistance (100%) to Amikacin, Amoxyclav, Cefotaxime, Ceftazidime/Clavulanate, Cefoxitin and Ciprofloxacin. Followed by Cefepime/Clavulanic acid 75% and Imipenem75%. Maximum sensitiveantimicrobialswerePolymyxin B and Ampicillin/Sulbactam 100%, followed by Tigecycline 100%, Gentamicin and Colistin were 50%, Meropenem, Co-trimoxazole sensitive were 50% sensitive.

Out of 749 patients, 97 patients were confirmed for 105 HAIs. with an infection rate of 12.95% percent in this study, which was less than the rates reported 24.3%,27.9% and 27.2% respectively.^9-11 This variation may be attributed due to the study site (ICU) and duration, sample size. Lack in following the standard (routine) precautions by HCWs, personal hygiene, and awareness status are responsible.

CAUTI was identified to be most common HAIs (35.2%) followed by SSI (27.6%), VAP (20.0%) and CLABSI (3.8%) which is similar to a study who also reported (only devise associated HAIs) CAUTI (55.55%) was most common HAIs, followed by VAP (38.27%) and CLABSI (5.55%).¹⁶ Other studies which are listed above show variations from our study may be due to different sites of study, populations and different criteria of patient selection.

Most common infectious agent of SSI was Staphylococcus spp. 44.82%. which is similar with the recent studies52.63% & 25.77% respectively.^12-13 Second most common microorganism is Pseudomonas spp. 20.7% in our study which is similar to above listed studies. The HCWs are the potential carriers of Staphylococcus aureus (multidrug resistance), it may be spread to patient either from HCWs, also from patients own endogenous flora so that Staphylococcus aureus is a most common agent of SSI in all above listed studies.

Most common infectious agent of CAUTI was Escherichia coli 45.9% similarly studies reported Escherichia coli as the most dominant organism isolated from CAUTI.^13,7 Second most common bacteria which is isolated from CAUTI in present study and Gemechu MM. et al is Pseudomonas spp.10.8%, 18.5% respectively.13 Some variation in microbiological profile of CAUTI in above listed studies may due to difference in study participants (present study involve all age group and wards) sample size and the standard personal hygiene and awareness status may be responsible.

The most common etiological agent of VAP was Pseudomonas spp. 33.3% followed by Acinetobacter spp. and Klebsiella spp. 14.3% which is well correlated with other study 27%, 22% and 15% respectively.¹⁰ They also showed the dominance of Acinetobacter spp. as an etiological agent of VAP and this variation in the microbiological profile of VAP may be due to the distribution of etiological agents varies from one ICU to another ICU and duration of hospitalization, antibiotic policy, patient’s status are other reasons.

A study reported the dominance of Gram-negative pathogens as a causing agent of VAP because Gram-negative pathogens colonize on healthy skin of ICU people, catheters, instruments, and environments that can be even transmitted through the air.

The most common etiological agent of CLABSI was Acinetobacter spp. 75% followed by Klebsiella spp. 25%, similar to the recent study14 25% and 12.5% respectively, another study7 reported Acinetobacter spp. 33.3% as the most dominant etiological agent of CLABSI. Some variation in etiological agents of CLABSI which is found in the above-listed studies may be due to deference in the environmental flora of every ICU/hospital, the status of patients, and the sample size of the study.⁷

A total of 17 strains of GPC were isolated from HAIs patients. MRSA accounted for 90% of the total number of Staphylococcus aureus. Which is closely similar to study who also reported 100% MRSA of a total number of Staphylococcus aureus.¹⁵

Only 2 strains of Enterococcus spp. were isolated from HAIs patients. Among them, one strain (50%) shows HLAR resistance, while another study which is conducted by AmanS.et al also reported 50% resistance to high-level gentamicin.⁷

The high rate of MRSA observed in this study might be attributed to the lack of molecular assay targeting the macA gene, the number of samples and use of antibiotics in that setup. The dominance of MBL producers from both Enterobacteriaceae and Non-fermenters. 26.08% and 35.29% respectively.¹⁶

Most of Enterobacteriaceae spp. like Citrobacter (100%), Enterobacter spp. (40%), Proteus mirabilus(40%), E. coli (27.2%), Klebsiella spp. (35.7%) were MBL producers. In Pseudomonas sppand Acinetobacter spp. MBL producer strains were 40% and 50% respectively. Due to the efficacy and low toxicity of β-lactam antibiotics, these antibiotics were mostly prescribed antibiotics in ICUs across the globe, in the last few decades, so most of the bacterial strains of hospitals and ICUs have become ESBL producers. For the treatment of ESBL-producing pathogens carbapenems group of antibiotics are the drug of choice at the present time, but above listed study shows that pathogens rapidly adapt and modify themselves to produce MBL and carbapenemase which have become a major issue for healthcare facilities.

Gram-positive isolates showed a resistance range from 26% to 100% which is closely comparable with 37% to 100%.19 All gram-positive isolates were 100% sensitive to linezolid, is similar to study who reported 100% sensitivity to linezolid.⁷

Resistance to levofloxacin (Fluoroquinolones), azithromycin, and cefoxitin was high in the present study which is similar to the study who reported high-level of resistance to these drugs.⁷

Enterococcus spp. were 100% sensitive to linezolid and 50% resistant to gentamicin, tetracycline and amoxiclav which is closely similar to the study who reported 100% sensitivity to linezolid and 50% resistance to gentamicin, erythromycin.¹⁷

Gram-negative isolates (except Pseudomonas spp.) showed maximum resistance to 2nd and 3rd generation of cephalosporin as Cefoxitin (82%), Cefotaxime (82%) and Ceftazidime (80.7%). Followed by Amoxyclav (69.2%) from the class of β-lactam+β- lactamase inhibitor antibiotics and Co-trimoxazole (67.3%) from the class of Sulfonamides and trimethoprim and showed notable sensitivity to carbapenems (meropenem 84% and imipenem 63.5%) and gentamicin 75%). Which was similar to the study which reported that the cephalosporin group of antibiotics (ceftriaxone, cefotaxime, ceftazidime, and cefepime) showed the least sensitivity to all gram-negative bacilli and notable sensitivity to gentamicin, imipenem and meropenem and tigecycline was the most effective antibiotic against all gram-negative isolates.⁷ In our study, gram-negative isolates of blood samples were also 100% sensitive to tigecycline.

Pseudomonas spp. showed 100% sensitivity to polymyxin B followed by Gentamicin (90%), Tobramycin (75%), Piperacillin +Tazobactam (75%), Amikacin (65%), Imipenem and Meropenem (60%) sensitive. Similarly, a study reported high sensitivity to Gentamicin (78.6%), Tobramycin (57.5%), Piperacillin +Tazobactam (78.6%), Amikacin (78%), Imipenem and Meropenem (92%).7 This suggested that the cephalosporin group of antibiotics was the most used drug of first-line treatment and the user of these antibiotics leads to resistance.

Some variation was found in antibiotic resistance patterns of isolated organisms from HAIs patients in different studies may be due to differences in prescribed drugs, the microbiological profile of HAIs and the resistance pattern of organisms.

The maximum number of isolated organisms from HAIs were gram- negative and showed antibiotic resistance patterns such as ESBL, MBL, and AmpC-β- lactamase hence it is concluded from the study that detection of antibiotic resistance patter should be reported in microbiology laboratory so that knowledge of the susceptibility pattern of causative microorganisms can guide the clinicians to choose the appropriate antibiotics. Most of the MBL producers were isolated from the ICU patients which indicates that antibiotics from the group of carbapenems were frequently used in the ICU because of the patient’s disease severity and for the treatment of ESBL-producing pathogens. Pathogens that are associated with HAIs are usually multidrug resistant thus making the treatment more complicated. Combination therapy should be used rather than monotherapy for HAIs strains. Polymyxin B, tigecycline, tobramycin, and Piperacillin +Tazobactam showed high sensitivity so these drugs should be used as reserved drugs.

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