International Medical Journal Vol. 26, No. 5, pp. 376 - 380 , October 2019

IMMUNOLOGY

Phenotypic and Molecular Characterization of Extended-Spectrum β-Lactamase Producing Klebsiella spp. from

Nosocomial Infections in Egypt

Eman A. El-Masry1,2), Nahla A. Melake1,3), Ibrahim A.Taher2)

ABSTRACTObjectives: Identification of Extended spectrum beta-lactamase (ESβLs) and genes mediated resistance are important for

their survilance transmission and control. This study was designed to detect the prevalence of ESβL producing Klebsiella species among patients with nosocomial infections in Egypt.

Materials and Methods: Screening was performed using antimicrobial susceptibility methods. Phenotypic identification was confirmed by Double disk synergy test, confirmatory clavulinate combined disks test, ESβL-E-test and MicroScan panel identi-fication system. A uultiplex PCR was used to detect the blaSHV and blaTEM genes.

Results: Of 41 Klebsiella isolates tested, 25 (61%) were ESβL-producers. Out of these 25 ESβL-producing Klebsiella spp. 84% were resistant to amoxicillin/clavulanate, 80% to gentamicin, ampicillin/sulbactam, cefotaxime and trimethoprim/sulfamethox-azole. Multiplex PCR showed 18 (72%) carried one of the blaSHV and blaTEM genes. Both genes were present in 6 (24%) of isolates, 8 (32%) had blaSHV gene, while 4 (16%) were positive for blaTEM gene.

Conclusion: ESβL-producing Klebsiella spp. is an increasing problem in Egyptian hospitals. A national surveillance studies using molecular and epidemiological methods could be of help to identify the various ESβLs types and to help in their control and spread within hospitals.

KEY WORDSKlebsiella spp. Extended-spectrum β-lactamases (ESβLs), TEM, SHV, antibiotics resistance

Received on September 26, 2018 and accepted on January 22, 20191) Department of Medical Microbiology and Immunology, College of medicine, Menoufia University Egypt2) Department of Pathology, College of medicine, Jouf University KSA3) Department of Medical Microbiology and Immunology, College of medicine, Al-Imam Mohammad Ibn Saud Islamic University KSA Correspondence to: Ibrahim Taher(e-mail: itaher@ju.edu.sa)

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INTRODUCTION

The early 1980s saw the first intoduction of extended spectrum and third geneation cephalosporins (ceftazidime, cefotaxime and ceftriax-one) as very effective antimicrobial drugs against wide range of multi-resistant microrganisms causing nosocomial infections like Pseudomonas aeruginosa, Klebsiella spp., and Enterobacter spp.1).

The β-lactamases family is divided into four groups based on their substrate specificities; penicillinases, Extended-spectrum Beta-lactamases (ESβLs), carbapenemases, and AmpC-type cephalosporinas-es. B-lactamases-mediated antibiotic hydrolysis is considered to be the primary mechanism by which Enterobacteriaceae resist the action of β-lactams, while altered expression of efflux pumps and porins are said to play a minor role2).

ESβLs are made of a heterogeneous group of plasmid-mediated bac-terial enzymes that confer significant bacterial resistance to antimicrobi-al drugs such as penicillins, cephalosporins, and aztreonam, but not being affected by clavulanic acid3). In general, there has been a wide range of resistance towards cephalosporins and multiple types of ESβLs being detected in various organisms worldwide4).

Multiple antimicrobial resistant Enterobacteriaceae carrying ESβLs are considered to be one of the major causes of hospital acquired infec-

tions and infectious diseases agents in different parts of the world. Since these strains can confer resistance to multiple antimicrobial agents, making their treatment challenging and leaving only very few options to their therapeutics5). In particular, the increase incidence and spread of K. pneumoniae producing ESβLs in the community has led to surge in the community-onset and hospital-associated infections on a large scale worldwide6). So far, there have been over 200 types of ESβLs been described in various Enterobacteriaceae and other organisms. These organisms produce var iants of the TEM, SHV and CTX-M β-lactamases7). The TEM- and SHV- β-lactamases are mainly produced by E. coli and K. pneumoniae that have spread throughout hospital set-tings, while the CTX-M enzymes is predominantly prevalent in the community8).

In order to detect ESβLs, a number of different tests have been developed and suggested such as; the dilution tests or disk diffusion sus-ceptibility tests using ceftazidime, cefotaxime, ceftriaxone and cefpodx-ime9). Recently, there have been proposals to replace or complement the old phenotypic methods by using PCR and gene sequencing tech-niques10). Several studies in Egypt had reported high levels of ESβL pro-duction among Enterobacteriaceae. Of the isolates from bloodstream infections 80.6% of K. pneumoniae and 40.9% E. coli were identified as ESβLs producers11). In another study 50.8% resistance against carbapen-ems among other Gram-negative isolates was also reported12).

C 2019 Japan Health Sciences University & Japan International Cultural Exchange Foundation

El-Masry E. A. et al. 377

The present study was designed to investigate the spread of ESβLs, blaSHV and blaTEM genes among Klebisella spp. isolated from patients suf-fering from nosocomial infections in Giza chest hospital, Egypt.

MATERIALS AND METHODS

The study was conducted during the period from March 2017 to May 2018. An ethical approval from the Local Ethical Committee of Giza chest hospital was obtained for the study to be conducted, and all patients signed a written consnt for their specimens and other informa-tion to be included in this study. A total of 140 patients with various nosocomial infections who were admitted to the hospital were enrolled in this study. Seventy-four (55.7%) patients were females and 66 (47.1%) were males aging from 15 to 65 years old (m = 32.6, SD = ± 9.9 years). Patient's demographic data and clinical histories were record-ed including age, sex, residence, associated co-morbidities, history of drug administration, admission to word and/or intensive care units (ICUs), duration of hospitalization and exposure to invasive procedure.

Bacterial strains:

One hundred and fifty bacterial species were isolated from blood, urine, sputum, pus aspirates and endotracheal secretions and identified using a combination of conventional techniques, and API 20E system (Biomeriux SA, Montalien Vercica and France).

The disk diffusion method using Muller-Hinton agar plates (Oxoid, Basingstoke, UK) was used to test for the antimicrobial susceptibility testing according to the Clinical and Laboratory Standard Institute guidelines13).

Detection of ESβL producing strains:

Initial screening of ESβLs production was performed by the disk diffusion method using ceftazidime (CAZ; 30 μg), cefotaxime (CTX; 30 μg), ceftriaxone (CRO; 30 μg) and aztreonam (ATM; 30 μg). The CLSI criteria used for designation of ESβL production by Klebsiella spp. was followed (zone diameters of ≤ 22 mm for ceftazidime, ≤ 27 mm for cefotaxime or aztreonam or ≤ 25 mm for ceftriaxone)13). ESβL-producing Klebsiella spp. were confirmed by conventional phenotypic tests including double disk synergy test (DDST), confirmatory clavuli-nate combined disks test, ESβL-E-test and by MicroScan panel identifi-cation system (Dade Behring, Inc., West Sacramento, CA).

Double-disk synergy (DDS) test:

This was performed by placing an amoxicillin/clavulanic acid (AMC; 20/10 μg) and cefotaxime (CTX; 30 μg) disks at a distance of 30mm apart on an inoculated Muller Hinton agar plates (Oxoid LTD, Basingstoke, UK) incubated aerobically for 18-24h at 37℃. A clear expansion of cefotaxime inhibition zone towards the clavulanate disks was considered as synergy (ESβL-producer) (Figure 1). Antibiotic disks of ceftazidime (CAZ; 30 μg), ceftriaxone (CRO; 30 μg) and aztreonam (ATM; 30 μg) were also placed on the plate. In cases where there was suspicion or no clear evidence that Klebsiella spp. was not harboring ESβLs and was negative using the criterion of 30 mm between disks, the test was repeated using a closer distance of 20 mm14).

Confirmatory clavulinate combined disks test:

This phenotypic confirmatory test was performed using different combinations of antimicrobial drugs of ceftazidime (30 μg) versus cef-tazidime/clavulanic acid (30/10 μg), (Oxoid, UK), and cefotaxime (30 μg) disk versus cefotaxime/clavulanic acid (30/10 μg) disk (Figure 1). ESβL production (CLSI, 2015) was considered positive if ≥ 5 mm

increase in the zone diameter for the antimicrobial agent tested in com-bination with β-lactamase inhibitor versus its zone when tested alone was recorded13).

ESβL-E-test:

The E-test ESβL CT/CTL and TZ/TZL strips (AB Biodisk, Solna, Sweden) were used in this study. The strips are calibrated with reading scales in μg/ml (MIC) on one side, while two predefined exponential gradients are located on the other side. Cefotaxime is labelled as CT (0.25-16 μg/ml), and CTL codes cefotaxime (0.016-1 μg/ml) plus (4 μg/ml) clavulanic acid. Ceftazidime (0.5-32 μg/mL) is coded as TZ, and TZL for ceftazidime (0.064-4 μg/mL) plus (4 μg/mL) clavulanic acid. Bacterial isolatees were considered as ESβL-producers when the zone of inhibition was read from two halves of the strip that contained cefo-taxime/ceftazidime alone or cefotaxime/ceftazidime plus clavulanate. Detection of ESβL was confirmed by the appearance of deformation or a phantom zone (Figure 1) of the CT or TZ ellipse or when either the MIC of CT or TZ was reduced by ≥ 3 log2 dilutions in the presence of clavulanic acid14).

MicroScan analysis:

Following the manufacturer's recommendations, the MicroScan WalkAway 96 SI System (Dade Behring, Inc., West Sacramento, CA) a semiautomated system was used to screen for the ESβL-producing microorganisms. This screening depends on the growth of microrgan-isms in the presence of cefpodoxime, cefotaxime, ceftazidime and aztre-onam at concentrations recommended by the CLSI13) for ESβL screen-ing. Minimum inhibitory concentrations obtained for cefpodoxime, cefotaxime, ceftazidime and aztreonam were interpreted according to CLSI breakpoints (aztreonam, 0.5-16; cefpodoxime, 0.5-64; cefotaxime, 0.5-128 and ceftazidime, 0.5-128). The MicroScan test incoportae a clavulanate synergy test based on MIC for the confirmation of ESβLs production. Results are interpreted by MicroScan software program, fol-lowing CLSI13).

Table 1. PCR primer sequences used for ESβL genes detection.Target Primer Sequence (5'-3') Product size

TEM TEM- F ATA AAA TTC TTG AAG AC 1076 TEM- R TTA CCA ATG CTT AAT CA SHV SHV-F GGG TTA TTC TTA TTT GTC GC 930 SHV-R TTA GCG TTG CCA GTG CTC

Table 2. Demographic features of patients with ESβL-producer (n = 25) and Non-ESβL-producer (n = 16) Klebsiella spp. (n = 41 patients). Data shown are frequencies; n (%), and, mean ± SDM.

Variables P-value Non-ESβL-producer ESβL-producer

Age 34.2 ± 28 21 ± 31.8 < 0.005Gender: Male/Female 10 (62.5)/6 (537.5) 15 (60)/10 (40) < 0.005Residence: Urban/ Rural 8 (50)/8 (50) 16(64)/ 9(36) < 0.005Hospital admission word ICU 4 (25) 10 (40) < 0.005Medical word 8 (50) 7 (28) > 0.005Surgical word 4 (25) 8 (32) > 0.005Duration of admission (days) 9.5 ± 11.5 19.7 ± 31.3 > 0.005Clinical specimen Urine 7 (43.7) 10 (40) < 0.005Sputum 2(12.5) 6 (24) > 0.005Endotracheal aspirate 1(6.25) 1 (4) > 0.005Pus 3(18.7) 5 (20) > 0.005Blood 3(18.7) 3(12) > 0.005Intravascular or urinary 8 (50) 13 (52) > 0.005catheter Mechanical ventilation 4 (25) 6 (24) > 0.005Underlying disease 8 (50) 10 (40) > 0.005Previous use of third- 3 (18.8) 12 (48) < 0.0005generation cephalosporin

Extended-Spectrum β-Lactamase Producing Klebsiella spp.378

Molecular study

Detection of blaSHV and blaTEM genes

Plasmid extraction:

The Jemima and Verghese15) method was used for plasmid isolation (Mini Kit-12123, Quiagen, Germany), which were stored at -20℃ under sterile conditions.

Multiplex PCR:

A multiplex PCR using Taq PCR Master Mix (Qiagen, Germany) was used for the identification of β-lactams resistance genes (SHV and TEM). To amplify the 930 and 1076 pb fragments that spanned the entire blaSHV and blaTEM genes, respectively, two sets of primer as described in Table 1 were used15,16).

Each PCR mix was totaled 50 μL. The cycling conditions used included; one cycle at 94℃ for 5 min followed by 35 cycles at 94℃ for 45s, 56℃ for 45s, and 72 for 45s, and a final extension at 72℃ for 10 min, using MycyclerTM Thermal cycler (BioRad, USA). A ladder of 15.0-1000.0 bp was used to estimate allele sizes after the PCR products

were separated in 0.8% agarose gel, staining with ethidium bromide and visualization under UV light (Figure 2)17,18).

Statistical analysis

The "Statistical Package for the Social Sciences" (SPSS) program version 17 was used to analyse all the data. Results were exprssed as ranges, means ± S.D., Chi-square test and P-value of < 0.05 was cosid-ered as significant.

RESULTS

One hundred and forty patients were included in the present study; females were represented by 74 (55.7%) and males by 66 (47.1%). They

Table 3. Antibiotic resistance patterns of ESβL-producer (n = 25) and Non-ESβL-producer (n = 16) Klebsiella spp. (n = 41 patients). Data shown are frequencies; n (%).

Antimicrobial agents Non-ESβL-Producer ESβL-Producer R S R S No (%) No (%) No (%) No (%)

Amoxicillin (AMX) 10 (62.5) 6 (37.5) 16 (64) 9 (36)Amoxicillin/clavulanic acid (AMC) 8 (50) 8 (50) 21 (84) 4 (16)Amikacin (AK) 10 (62.5) 6 (37.5) 12 (48) 13 (52)Gentamicin (CN) 13 (81.2) 3 (18.7) 20 (80) 5 (20)Aztreonam (ATM) 9 (56.2) 6 (37.5) 15 (60) 10 (40)Ampicillin -sulbactam (SAM) 16 (100) 0 (0) 20 (80) 5 (20)Imipenem (IMP) 0 (0) 16 (100) 1 (4) 24 (96)Tobramycin (TOB) 8 (50) 8 (50) 10 (40) 15 (60)Ciprofloxacin (CIP) 12 (75) 4 (25) 18 (72) 7 (28)Cefotaxime (CTX) 10 (62.5) 6 (37.5) 20 (80) 5 (20)Ceftazidime (CAZ) 6 (37.5) 10 (62.5) 25 (100) 0 (0)Cefepime (FEP) 5 (31.2) 11 (68.7) 8 (32) 17 (68)Trimethoprim/sulfamethoxazole (SXT) 12 (75) 4 (25) 20 (80) 5 (20)Ceftriaxone (CRO) 9 (56.2) 7 (43.7) 18 (72) 7 (28)Ofloxacin (OFL) 3 (18.7) 13 (81.2) 3 (12) 22 (88)Cefoxitin (FOX) 12 (75) 4 (25) 5 (20) 20 (80)

R = resistant. S = Sensitive. CXT = Cefotaxime, CAZ = Ceftazidime, CFX = Cefotaxime, ATM = Aztreonam, CPD =

Cefpodoxime.

Table 4. Comparison between semiautomated MicroScan analy-sis and different conventional phenotypic methods for detection of ESβL-producing Klebsiella spp. isolates (n = 41). Data shown are frequencies; no (%).

Method Klebsiella spp. Sensitivity Specificity No. (%) % %

Semiautomated 20 (48.8) 80 76MicroScan analysisConventional Double-disk 23 (56.09) 92 88 synergy phenotypic (DDS)methods Confirmatory 22 (53.6) 88 84 clavulinate combined disks E-test 25 (60.9) 100 60.9

Figure 1. Phenotypic testing for ESβL-producing Klebsiella spp. (a) A representative result of Double Disk Synergy test (Cefotaxime/clavulanate). (b) Confirmatory Clavulinate Combined Disks test (cef-tazidime versus ceftazidime/clavulanate). (c) E-test ESβL; showing (ESβL-producer).

Figure 2. PCR detection of blaTEM β-lactamase gene. Ladder = DNA marker, lanes 1 and 2 = blaTEM negative control, and lanes 3-8 = posi-tive blaTEM Klebsiella spp. isolates, 9 = blaTEM positive control.

El-Masry E. A. et al. 379

aged from 15 to 65 years old and their mean age was 32.6 ± 9.9 years. A total of 150 bacterial isolates were recovered from these patients, 41 (27.3%) were identified as Klebsiella spp. [Table 2]. Twenty-five out of 41 (60.9%) of these Klebsiella spp. isolates were found to produce ES?L.

Table 2 summarizes and compares the demographic characters of the patients harboring ESβL-producers and those who did not harbor ESβL-producer Klebsiella spp. Although, the majority of the studied parameters were not found to be significantly associated with the colo-nization of Klebsiella spp. (p > 0.05), however, previous treatment with third-generation cephalosporin (p < 0.0005), age, sex, and ICU admis-sion were all found to be strongly correlated with the colonization of Klebsiella spp. (p < 0.005).

The antimicrobial susceptibility pattern of 25 ESβL-producing Klebsiella spp. revealed a resistance rates of 84% towards amoxicillin/clavulanate, 80% resistant to gentamicin, ampicillin/sulbactam, tri-methoprim/sulfamethoxazole, and cefotaxime, 72% resistant to cipro-floxacin and ceftriaxone, 64% resistant to amoxicillin, and 60% were resistant to aztreonam [Table 3]. The lowest resistance rate of 4% was recorded against imipenem.

Table 4 shows a comparison between semiautomated MicroScan analysis and different conventional phenotypic tests for detection of ESβL-producing Klebsiella spp. Of the 41 Klebsiella spp. tested; only 20 (48.8%) isolates were detected as ESβL-producers by semiautomated Microscan analysis, while 25 (60.9%) isolates by E-test, 23 (56.09%) by double-disk synergy (DDS) test and 22 (53.6%) by confirmatory clavi-nate combined test. The E-test was the most sensitive (100%) followed the DDS test (92%), confirmatory clavinate combined test (88%) and finally the semiautomated Microscan analysis (80%). Of these methods, DDS was the most specific (88%) followed by confirmatory clavinate combined test (84%), then semiautomated Microscan (76%) and E-test (60.9%). (Table 4, Figure 1)

Detection of blaSHV and blaTEM genes by multiplex PCR showed that among isolated organisms 18 (72%) carried one of the two genes,while, 7(28%) were negative for any gene. Both genes were present in 6(24%) of isolates, 8(32%) isolates possesed blaSHV gene, while 4(16%) out of 25 isolates were positive for blaTEM gene [Figure 2].

DISCUSSION

This study was conducted on 25 ESβL-producing Klebsiella spp. The incidence of ESβL was more common in urine samples 10 (40%), than in others such as; sputum 6 (24%), endotracheal aspirate 1 (4%), pus aspirates 5 (20%), and blood 3 (12%). In a similar study by Elsherif and Maamoun19), urine cultures provided the highest percentage of ESβL isolates (34%), followed by (21.5%) sputum, blood cultures (17%), wound specimens (13%), stool of healthy carrier (10%), and bronchoal-veolar lavages (4.5%).

Several studies have reported the spread of ESβL-producing organ-isms worldwide. For instance, Lal et al.20) in an Indian study reported one of the highest ESβLs rates of 97% produced by Klebsiella isolates. Lower rates of ESβL production were reported in other studies; as in a Japanese hospital, 4.9% of the total K. pneumoniae isolates were shown to produce ESβL21), while 19.8% of Enterobacteriaceae species isolates were ESβL-producers in Hyderabad, India22). In Egypt; the highest rate of ESβL production among Gram-negative bacteria was 70%23). In another study among neonatal intensive care patients in Cairo, Egypt, reported that 80% of the isolates were K. pneumoniae of which 58% were ESβL producers24). While, 21% rate among K. pneumoniae was reported by Ahmed et al.25).

These findings demonstrate that there is a wide spread of ESβL-producing bacteria in different parts of the world. Some of the factors that had been associated with the spread of ESβL included prolonged hospitalization, urinary catheters, and indiscriminate use of beta-lactam drugs. Other likely causes could be attributed to the spread of a single clone or a plasmid conferring resistance to ESβL within a hospital set-ting19).

In our study, we found that older patients were more likely to be affected by nosocomial infections caused by ESβL-producing Klebsiella spp. which coincide with the findings of some previous studies26). Similarly, patients admitted to ICUs are also prone to be infected by ESβL-producing Klebsiella spp., as they are at high risk of being sub-jected to invasive procedures and exposed to different antimicrobial drugs27). We also found that previous use of third-generation cephalospo-rins correlated with the production of ESβL in our study.

The overall antimicrobial susceptibility patterns of our 25 ESβL-

producing Klebsiella spp. were higher than those reported by Ahmed et al., 201325), but are in line with the findings by Chong et al.,29) who found a significant association between ESβL-production by different bacterial species and resistance to almost all cephalosporins genera-tions28).

In the present study, ESβL-producing isolates were resistant to the majority of the antimicrobial drugs used; this could be explained by co-transfer of resistant genes among these bacteria28), which in turn may limit the therapeutic choices against these pathogens. Of the antibiotics tested, imipenem was found to be the most effective with 96% of the ESβL-producing Klebsiella spp. being sensitive. Imipenem has been reported to be highly effective (100%) against ESβL-producing K. pneu-moniae isolates29), and with the addition of meropenem, both drugs were considered to be the drugs of choice in the treatment of life-threatening infections caused by ESβL-producing Enterobacteriaceae29). Among the fluroquinolones tested in the present study, ofloxacin was found to be the most active against ESβL-producing Klebsiella spp. isolates (88%), compared to 72% being resistant to ciprofloxacin which is higher than that reported in other studies30).

Of the 41 Klebsiella spp. tested; only 48.8% were identified as ESβL-producers by the semiautomated Microscan analysis compared with 60.9% were detected by E-test, 56.09% by DDS test and 53.6% by the confirmatory clavinate combined test. The E-test was the most sen-sitive followed by DDS test and the confirmatory clavinate combined test and finally semiautomated Microscan analysis. DDS test was the most specific followed by the confirmatory clavinate combined test then semiautomated MicroScan and E-test.

Changes in the structures of TEM and SHV genes are reported to lead to the spread of ESβL enzymes among Klebsiella spp.30). In the present study, PCR results showed that 18 (72%) of our isolates carried one of the two genes, and 7(28%) were negative for any gene. Both genes were found in 6 (24%) of the isolates, 8 (32%) had blaSHV gene, while blaTEM gene was seen in 4 (16%) of the isolates. Among 77 Klebsiella spp. tested by Elsherif and Maamoun19), SHV gene was pres-ent in 45.5% of their isolates. Likewise, 20% of ESβL-producing K. pneumoniae tested were found to harbor TEM/SHV23). 45% of Klebsiella spp. were found to be positive for blaCTX-M and blaSHV genes as demonstrated by Jemima and Verghese15), which is compatible with other several studies in other countries that have shown higher preva-lence of SHV compared to TEM genes22).

CONCLUSION

In view of the high prevalence rate of ESβL-producing K. pneumo-niae in our region, implementation of infection control measures and antibiotic control policies are of paramount importance. A national sur-veillance studies in Egypt using molecular and epidemiological means could be of help to identify the various ESβLs types, their incidence, and finally control.

ACKNOWLEDGEMENT

The authors would like to acknowledge the support of Dr. Hesham Ali El-Masry, ICU specialist, Giza Chest Hospital, Egypt for his assis-tance with the sample collection.

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