SUMMARY
OBJECTIVE:
Beta-lactams resistance is a major clinical problem in treating pneumonia. This study aimed to detect the extended-spectrum beta-lactamases (ESBL) genes in Klebsiella pneumoniae among patients with community-acquired pneumonia (CAP) in Al-Najaf City, Iraq.
METHODS:
A total of 511 sputum samples were obtained from all suspected patients with CAP in Al-Najaf City, Iraq, from March 2020 to September 2020. Sputum samples were subjected to microbiological tests. The disk diffusion method was used to test antibiotic sensitivity. Production of ESBLs was identified using phenotypic and genotypic methods.
RESULTS:
The total prevalence of K. pneumoniae was 31.9% (163/511). Using CHROM agar, 41 (25.2%) isolates were ESBL producers. The imipenem 0.0% (n=0/41) and norfloxacin 0.0% (n=0/41) were the most effective antibiotics. The multiplex polymerase chain reaction showed that 46.3% (n=19/41) of isolates harbored ESBL genes. Out of 19 ESBL producers, 47.4% and 15.8% harbored bla CTX-M and bla SHV, respectively. While bla CTX-M and bla SHV genes were detected in 7 (36.8%) isolates, simultaneously.
CONCLUSIONS:
The imipenem and norfloxacin can be used in empirical treatment of K. pneumoniae isolates in Iraq. The emergence of K. pneumoniae strains harboring ESBL resistance genes necessitates the development of a regular surveillance program to prevent the spreading of these isolates more in Iraqi health care systems.
KEYWORDS: bla CTX-M , CAP, ESBL, Pneumonia, Klebsiella pneumoniae
INTRODUCTION
According to the British Thoracic Society, community-acquired pneumonia (CAP) is an acute symptomatic infection of the lung parenchyma that occurs outside a hospital or nursing home 1 . CAP is caused by various microorganisms including 2 . No exact information about the incidence of the CAP in Iraq has been found so far. Clinical burden of CAP in older adults has only been assessed by a few large databases, with incidence rates ranging from 7.6 to 13.4 per 1,000 individuals 3 . A previous study from Iraq revealed K. pneumoniae as the leading cause of pneumonia 4 .
Strains of K. pneumoniae that can produce extended-spectrum beta-lactamases (ESBLs) become seriously active against many types of beta-lactam antibiotics. In addition, these virulent strains are capable of becoming resistant to numerous classes of non-beta-lactams, making it difficult to treat infections, and are referred to as multidrug-resistant (MDR) strains 5 .
Nearly 450 forms of ESBLs enzymes have been documented worldwide, and among these types, bla SHV, bla TEM, and bla CTX-M were predominant. ESBLs are enzymes that contribute to resistance to a variety of beta-lactams 6 . ESBLs hydrolyze the beta-lactam ring of beta-lactam antibiotics, causing these antibiotics to lose their antimicrobial activity. These factors may contribute to the development of pneumonia complications 6 , 7 .
To date, there are no studies on the prevalence of ESBL-producing K. pneumoniae in patients with CAP in Iraq. Therefore, the present research attempted to identify the ESBL-producing K. pneumoniae in Iraqi patients with CAP by phenotypic and molecular genotypic methods.
METHODS
Sample collection and bacterial isolation
The sputum samples of suspected patients suffering from CAP referred to Al-Sader Teaching Hospital in Al-Najaf City, Iraq, from March 2020 to September 2020 were collected in sterile containers. All patients were selected and diagnosed by a respiratory infectious disease specialist based on clinical examination and radiological and laboratory findings. Sputums were processed within 1 to 2 h of collection, using standard microbiological procedures. Sputums were initially cultured on blood agar and MacConkey agar (Merck, Germany) and incubated at 37°C for 2 days. The suspected K. pneumoniae colonies were further tested and identified using a panel of appropriate biochemical tests, including citrate utilization, urease, methyl red/Voges Proskauer, and triple sugar iron agar 8 . The confirmed K. pneumoniae isolates were stocked in tryptic soy broth containing 20% glycerol and placed at -80°C for long preservation.
Phenotypic detection of ESBL-producing K. pneumoniae
CHROM agar
All K. pneumoniae isolates were streaked on plates of CHROMagarTM ESBL agar (Pioneer, France). Chrome agar plates were aerobically incubated at 35°C overnight. Colonies of ESBL producers were appeared greenish blue.
Antimicrobial susceptibility testing
The ESBL-producing K. pneumoniae isolates were tested for antimicrobial susceptibility testing (AST) using disk diffusion technique according to the Clinical and Laboratory Standards Institute (CLSI) instructions 9 . Antimicrobials were classified as follows: aztreonam (ATM, 30 μg), gentamicin (CN, 10 μg), ciprofloxacin (CIP, 5 μg), ceftazidime (CAZ, 30 μg), levofloxacin (LEV, 5 μg), amoxicillin/clavulanate (AMC, 30 μg), trimethoprim (TMP, 5 μg), norfloxacin (NOR, 10 μg), cefotaxime (CTX, 30 μg), nitrofurantoin (F, 300 μg), imipenem (IPM, 10 μg), chloramphenicol (C, 30 μg), tetracycline (TE, 30 μg), and ceftriaxone (CRO, 30 μg) (Bioanalyse, Turkey). MDR isolates were determined according to the previous definition (resistance to at least one member of three antibiotics classes) 10 . Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality control strains.
Molecular detection of ESBL genes among K. pneumoniae
The presence of ESBLs encoding genes (bla SHV and bla CTX-M) were investigated by multiplex polymerase chain reaction (M-PCR) using previously described primer pairs (Bioneer, Koria) 11 . The DNA was extracted using genomic DNA extraction kit (FavorPrep, USA), according to the supplier instructions. All the components of M-PCR were mixed in final volume of 20 μl as follows: 12.5 μl of Master Mix (iNtRON, Koria), 5 μl of DNA template, 1.5 μl of DNA/RNA free water, and 0.5 μl of each reverse and forward primer. M-PCR mixture was put in a thermocycler (Biosystems, USA) instrument with following program: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 50 s, annealing at 50°C for 40 s, elongation at 72°C for 60 s, and final extension at 72°C for 5 min. E. coli NCTC 13353 and K. pneumoniae ATCC 700603 were used as bla CTX-M- and bla SHV-positive controls, respectively.
Statistical analysis
The data for this research were analyzed statistically using the Statistical Package for Social Science (SPSS) version 20.0 (IBM Corp., Armonk, NY, USA).
RESULTS
Bacterial isolation
In total, 511 sputum samples were taken from 302 (59.1%) male and 209 (40.9%) female patients with CAP, from which 148 (29.0%) Gram-positive bacteria and 308 (60.3%) Gram-negative bacteria (GNB) were isolated. Also, 55 (10.7%) samples showed no bacterial growth. Out of 308 GNB, 163 (52.9%) isolates were identified as K. pneumoniae and were recorded as a major cause for pneumonia in this study. These isolates were obtained from 102 (62.6%) males and 61 (37.4%) females, respectively. The total prevalence of K. pneumoniae was 31.9% (163/511).
Phenotypic detection of ESBL producers
Using CHROM agar method, 41 (25.2%) and 122 (74.8%) K. pneumoniae isolates were found to be ESBL producers and non-ESBL producers, respectively.
Antibiotic susceptibility testing
This test was carried out on all ESBL-producing K. pneumoniae isolates against 14 antibiotics (Table 1). The ceftazidime (n=41/41; 100%), cefotaxime (n=40/41; 97.6%), ceftriaxone (n=38/41; 92.7%), and aztreonam (n=39/41; 95.1%) were among the less effective antibiotics, while imipenem (n=0/41; 0.0%) and norfloxacin (n=0/41; 0.0%) were the most effective antimicrobials. In total, 27 (65.9%) ESBL-producing K. pneumoniae isolates were MDR due to the resistance to at least one member of three antibiotics classes.
Table 1. An antimicrobial susceptibility testing of 41 extended-spectrum beta-lactamases producing isolates of Klebsiella pneumoniae.
| Antibiotic agent | Number (%) of isolates | ||
|---|---|---|---|
| Resistance | Intermediate | Susceptible | |
| Amoxicillin/clavulanate | 18 (43.9) | 0 (0.0) | 23 (56.1) |
| Aztreonam | 39 (95.1) | 0 (0.0) | 2 (4.9) |
| Cefotaxime | 40 (97.6) | 0 (0.0) | 1 (2.4) |
| Ceftazidime | 41 (100.0) | 0 (0.0) | 0 (0.0) |
| Ceftriaxone | 38 (92.7) | 0 (0.0) | 3 (7.3) |
| Chloramphenicol | 17 (41.5) | 3 (7.3) | 21 (51.2) |
| Ciprofloxacin | 11 (26.8) | 0 (0.0) | 30 (73.2) |
| Gentamicin | 19 (46.3) | 3 (7.4) | 19 (46.3) |
| Imipenem | 0 (0.0) | 0 (0.0) | 41 (100.0) |
| Levofloxacin | 9 (22.0) | 2 (4.9) | 30 (73.1) |
| Nitrofurantoin | 9 (22.0) | 0 (0.0) | 32 (78.0) |
| Norfloxacin | 0 (0.0) | 0 (0.0) | 41 (100.0) |
| Tetracycline | 29 (70.7) | 0 (0.0) | 12 (29.3) |
| Trimethoprim | 27 (65.9) | 0 (0.0) | 14 (34.1) |
Molecular detection of ESBLs encoding genes
The M-PCR showed that 46.3% (n=19/41) of isolates harbored ESBL genes, while 53.7% (n=22/41) of isolates were found to be negative for these genes. Out of total 19 ESBL-positive isolates, 47.4% (n=9) harbored bla CTX-M and 15.8% (n=3) harbored bla SHV genes. And, bla CTX-M and bla SHV genes were detected in 7 (36.8%) isolates simultaneously. The bla CTX-M was the most dominant gene and present either alone or in combination with bla SHV gene.
DISCUSSION
In this study, the bacterial isolates were obtained from 89.2% (n=456/511) of the CAP patients, which was significantly higher than those obtained in the studies by Kishimbo et al. 1 from Tanzania (20.4%) and Regassa 12 from South Ethiopia (42.9%). These discrepancies may be due to differences in the study population, sample size, and geographical variations. This study showed more prevalence of CAP in male patients than females, which was consistent with previous report from Australia 13 .
In this study, the total prevalence of K. pneumoniae was 31.9% among patients with CAP. This finding was lower than the previous study (54.0%) by Jaaffar et al. 4 from Iraq and higher (18.0%) than the former studies by Temesgen et al. 14 from Ethiopia.
In this study, the ESBL-producing K. pneumoniae showed high resistance rates against ceftazidime (100.0%), cefotaxime (97.6%), aztreonam (95.1%), ceftriaxone (92.7%), tetracycline (70.7%), and trimethoprim (65.9%), whereas all isolates were susceptible to imipenem and norfloxacin. These results were closely similar to those observed by Fils et al. 15 from France and Liu et al. 16 from China. In this research, K. pneumoniae isolates showed good susceptibility to fluoroquinolones and aminoglycosides. In line with our results, Zhang et al. 17 from China reported the good efficacy of ciprofloxacin and levofloxacin against K. pneumoniae causing community-onset infections.
Another finding of this study was the high frequency of 65.9% for MDR phenotype among ESBL-producing K. pneumoniae isolates. This finding was in parallel with the previous reports from Brazil (84.0%) 10 and Portugal (100%) 18 . Teklu et al. 19 concluded that the main explanation for these high resistance rates may be due to the widespread, excessive, irregular, unnecessary, and uncontrolled use of antibiotics to treat various infections. Carbapenems are still used as the best option to treat various infections, including pneumonia caused by ESBL-producing GNB. The results of this study were in agreement to the findings of most international studies, according to which imipenem has high efficacy against ESBL-producing K. pneumoniae 20 - 22 .
In this study, 25.2% (n=41/163) of K. pneumoniae isolates showed phenotypic positive result for ESBL production using CHROM agar. Ultimately, this research was unable to validate the existence of ESBL genes by M-PCR in all isolates that had phenotypic positive test. It was found that 19 of 41 ESBL producers harbored ESBL genes using M-PCR method. These findings may presumably be due to the involvement of additional resistance pathways such as Ambler class C beta-lactamases, the presence of other mechanisms of resistance to beta-lactams, and the presence of other ESBL genes such as bla TEM and bla PER leading to differences between the results of phenotypic and molecular methods 23 , 24 . According to the CLSI, the combination disk test (CDST) is recommended for confirmation of ESBL production in Enterobacteriaceae using CAZ (30 μg) and CTX (30 μg) alone and in combination with clavulanic acid 9 .
The results of this study showed that bla CTX-M was the most common ESBL gene among K. pneumoniae isolates. The worldwide spread of bla CTX-M producing K. pneumoniae is a major concern in most continents. In a meta-analysis by Eskandari-Nasab et al. 25 , the prevalence of bla CTX-M was documented in Bahrain, Turkey, Saudi Arabia, Iran, United Arab Emirates, Pakistan, and Kuwait as 10.0, 30.0, 35.3, 56.7, 64.4, 96.9, and 100.0%, respectively. While international studies recorded varying percentages for the presence of this gene among the isolates producing ESBLs, including North Africa, America, Russia, Latin America, Brazil, and European countries, the percentages were 7.4, 26.4, 34.9, 61.1, 62.1, and 84.5%, respectively 25 . Despite the fact that TEM and SHV variants are the most universal ESBLs, it seems that they have become less common over the past decade than CTX-M. The results of this study were consistent with previous studies that found the bla CTX-M gene as the most widespread ESBL type in K. pneumoniae isolates 16 , 17 , 25 . However, Ferreira et al. 10 from Brazil and Carvalho et al. 18 from Portugal showed a higher prevalence of bla SHV compared to bla CTX-M in K. pneumoniae, which was in contrast to our finding. Many factors, including the sample origin, sample size, studied population, and detection methods, can contribute to these differences.
Finally, our results showed the co-existence of ESBL genes in 36.8% of K. pneumoniae isolates. Previous studies from Brazil 10 , China 16 , 17 , and Portugal 18 reported the co-existence of various ESBL genes among clinical isolates of K. pneumoniae. This study had several limitations: the lack of screening of other ESBL genes such as bla TEM and bla PER, the lack of clinical data of patients to investigate the ESBL-related risk factors, and lack of sequencing for detected ESBL genes.
CONCLUSIONS
The emergence of MDR K. pneumoniae strains harboring ESBL resistance genes necessitates the development of a regular surveillance program to monitor, control, and prevent the more spread of these isolates in Iraqi health care systems.
Footnotes
Funding: none.
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