Iranian Journal of Medical Microbiology

Sample Collection

 All samples, which were referred to Payvand Clinical and Specialty Laboratory for culture during March 2020-September 2020 (6 months), were inclu-ded in the present study. The demographic data were submitted during sample collection. The information of all patients was kept private during data analysis and manuscript preparation. All the isolated microo-rganisms, including gram-positive and gram-negative bacteria, as well as isolated yeasts, were stored in a -70°C freezer in trypticase soy broth (TSB) with 15% glycerol. But based on the aims of this study only gram negative bacteria were included for further study and the remained were role out.

Bacterial Identification and AST

 Bacterial identification and AST were carried out using an automated VITEK 2 system (BioMerieux, France). In this system, pure cultures are needed for bacterial inoculation preparation. Consequently, different media were used for isolation based on the clinical samples. Sheep blood agar and MacConkey agar (QUELAB, UK) were utilized for urine samples, and blood agar, MacConkey agar, chocolate agar, and Sabouraud dextrose agar (QUELAB, UK) were applied for sputum specimens. In addition, all the four mentioned media and TSB were used for tracheal tube culture (11).
All inoculated plates were incubated at 37ºC for 24 h. Afterward, gram staining was performed, and using two or three pure colonies, a bacterial suspension was prepared by special PBS of the VITEK 2 system from each bacterial sample with turbidity equal to standard 0.5 McFarland (1.5×10⁸ CFU/mL). The OD of bacterial and fungal suspensions by VITEK 2 spectrophotometer must be in the ranges of 0.5-0.63 and 1.8-2.2, respectively. Further dilution was executed for AST based on the manufacturer's protocol. Moreover, especial GP, GN, N240, GN76, AST-STO3, and AST-GP75 cards were used for microbial isolation and AST. According to the protocol, all the bacterial suspen-sions must be used 20 min after preparation. For quality control, standard ATCC bacteria, namely E. coli 25922, K. pneumoniae ATCC700603, and P. aerugin-osa ATCC27853 were injected into the VITEK 2 system simultaneously with the clinical samples.

Confirmation of ESBls Production by Disk Synergy Test

 Flagged samples as extended-spectrum beta-lactamase (ESBLs) producers by VITEK 2 system were confirmed by manual double-disk synergy test (DDST). In order to complete the DDST, a bacterial suspension with 0.5 McFarland turbidity (1.5×10⁸ CFU/mL) was prepared and inoculated to Mueller-Hinton agar. A ceftazidime disk alone and a ceftazidime-clavulanic acid disk were placed by sterile forceps at a distance of 20 mm from the center. All plates were incubated for 24 h at 37ºC. A difference of ≥ 5 mm between the diameter of the zone of inhibition around ceftazidime-clavulanic acid disk versus ceftazidime disk alone was reported as ESBL producer (12-14).

Statistical Analysis

 The frequency of isolated XDR bacteria, as well as resistant and susceptible bacteria, was entered in an excel file and was reported after percentage calculation.

The length of hospitalization and the rising cost of care during infection with resistant organisms, especially MDR organisms, is a global challenge (1,15). The severity of gram-negative infections is usually higher than gram-positive infections, such as bacteremia (16-18). In such situations, an immediate antimicrobial prescription is needed. However, the chance of empirical therapy, which covers most cases, is decreasing because of antimicrobial limitations (19). In the present study, 4125 urine, 34 sputum, and 1 tracheal aspiration tube samples were submitted to Payvand Clinical and Specialty Laboratory for direct examination and microbial culture. Various gram-positive and gram-negative bacteria, as well as yeasts, were isolated. Exclusively the gram-negative bacteria were included for further study in the current investigation. The AST was performed simultaneously with isolation using VITEK 2 system and special cards as mentioned above.
Based on the AST results, 46% of E. coli isolates and 12.82% of K. pneumoniae isolates were ESBL-positive. Furthermore, 31 isolates of gram-negative bacteria were confirmed as XDR, while no PDR was detected. Zhou et al. in 2019 reported that E. coli and K. pneumoniae might be the main gram-negative XDR bacilli (20). Similarly, E. coli and K. pneumoniae were the most frequently isolated gram-negative bacteria in the present investigation. The MDR and XDR gram-negative prosthetic joint infections were evaluated by Papadoulous et al. (21, 22). In their study, the prevalent gram bacilli were E. coli, P. aeruginosa, K. pneumoniae, and Enterobacter spp. However, in the current research, the detected gram-negative bacteria were more variable than the latter study. E. coli, K. pneumoniae, P. aeruginosa, and Enterobacter spp. were similarly reported as the most frequent isolated bacteria from different clinical samples.
In the study performed by Mirzae et al., 3248 clinical samples were collected from the Burns Center of the Northeast of Iran. They observed that 309 cases were culture-positive, with 75 and 234 specimens being positive for P. aeruginosa and A. baumannii, respect-tively. Most samples were from the Burn Intensive Care Unit (ICU) (60.5%) and Burn Wards (20.4%). Moreover, they reported that 16.5% and 15.53% of P. aeruginosa isolates and 74.75% and 73.13% of A. baumannii isolates were MDR and XDR, respectively. Finally, they recommended improving the prevention criteria to inhibit the spreading of XDR bacteria.
It should be noted that sampling in both studies was performed only in one center. The frequency and variation of microbial isolates in the present study were higher than the mentioned research. According to the findings of AST, 52% and 100% of P. aeruginosa isolates were imipenem-resistant in the study cond-ucted by Mirzaie et al. and the current study, respectively. In addition, 62.7% and 100% of P. aeruginosa isolates, as well as 97.4% and 100% of A. baumannii isolates, were ciprofloxacin-resistant in the study by Mirzaei and our study, respectively. How-ever, the frequency of P. aeruginosa and A. baumannii isolates in the current study was lower than the results of Mirzaei et al. Both of these investigations revealed a high resistant rate to imipenem and ciprofloxacin as common antibiotics. Susceptibility to colistin was not assessed in their study, which is one of their limitations (23).
According to Magiorakos A-P et al. study to investigate MDR and PDR bacteria, all or almost all suggested antibiotics in the CLSI protocol should be tested. Similarly, in the current study, all CLSI sugges-ted antibiotics for each isolated bacteria were tested but no PDR (pan drug resistant)bacteria was detected (24).
Different studies showed that the rate of infections with gram-negative bacteria, including Enterobact-eriaceae, A. baumannii, P. aeruginosa, and S. malto-philia is increasing in China and other countries (24-27). Furthermore, gram-negative bacteria were the most frequent bacteria in the ICU, neonatal ICU (NICU), and Cardiac Care Unit of Saudi Arabia hospitals. It was shown that A. baumannii was the most prevalent isolated gram-negative bacteria in this region followed by K. pneumonia, E. coli, and S. maltophilia (25-28). The emergence of XDR bacteria is a global challenge because of limitations in the treatment of these pathogens (25-31). However, E. coli and K. pneumoniae were the most frequently isolated resistant bacteria from a teaching hospital in Sri Lanka and a tertiary care hospital in Nepal (29, 31).
Finally, to understand the accurate frequency of XDR and PDR organisms, multicenter sampling is recommended in future studies. Sampling from only one clinical laboratory was the main limitation of this study. Moreover, we investigated XDR bacteria only among gram-negative bacteria. As a result, a similar evaluation of gram-positive resistant bacteria is also recommended. Such local assessments may deter-mine whether any modifications to treatment guideli-nes are necessary.

 

The rate of XDR bacteria was high in the investigated laboratory in this study. Therefore, accurate screening based on a standard protocol, antimicrobial steward-ship, and surveillance is recommended in different medical centers of Iran. In addition, to decrease antimicrobial resistance, the monitoring of MDR and XDR organisms in all clinical laboratories is recom-mended.

 

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This study was done in Payvand clinical and Specialty laboratory (a private clinical laboratory), Tehran-Iran, under scientific supervision and management of Dr. Behzad Poopak. The idea and study design was done by Dr. Mojdeh Hakemi-Vala (Ph.D In medical bacter-iology), Dr. Hadi Rezaei (Ph.D In medical bacteriology) and Dr. Behzad Poopak (Ph.D in hematology, Doct-orate in Clinical Laboratory Sciences). Routine samp-ling was done based on the physician’s request and standard protocols. The practical parts including: bacterial isolation, identification and processing of Vitek 2 system was done by MS. Sepideh Ghasemshahi and Mr. Mohammad Ahmadpour under supervision of Dr. Hadi Rezaei, head of department of microbiology, Payvand Clinical and Specialty laboratory. All demo-graphic and practical data registration including age, gender, background diseases and bacterial reports was done by Mrs. Aazam Booskabadi. Data analysis and draft preparation of the recent paper was done by Dr. Mojdeh Hakemi-Vala.

 

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Conflicts of Interest

There is no any conflict to declare.