Molecular Characterization of Multidrug-Resistant Klebsiella pneumoniae Isolated from Some Hospitals in Benghazi, Libya

Klebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide, mainly in hospitalized or immune-compromised individuals. Also, this could be due to the emergence of Multidrug resistance (MDR) Extended Spectrum Beta Lactamase (ESBL) and carbapenemase-producing strains. This study's main goals were to evaluate the prevalence of resistance demonstrated by K. pneumoniae strains found in clinical samples from Benghazi Medical Center and AL-jalaa Hospital and to find evidence of ESBL strains and their resistance to certain antibiotic. During the study period, K. pneumoniae was isolated from 320 clinical samples (urine, sputum, blood and wound). The procedure for processing of samples, identification, susceptibility toward antimicrobials and evidence of ESBL, MBL strains were carried out according to the recommended standards. PCR was used to detect β-Lactamase and carbapenemase resistance genes. From a total K. pneumoniae isolates, 120 (37.5%) were isolated from hospital patients. The isolates exhibited high resistance to all used antibiotics. Forty-eight (40%) of the isolates were ESBL producers. MDR and XDR were identified in 89% and 56% of isolates respectively. ESBL-CTX-M-15 gene and OXA-48 were detected in all isolates. Moreover, SHV and NDM were identified in four isolates. In this study shows the high rate of MDR in clinical K. pneumoniae isolates in hospitals. There is an urgent need to implement an antibiotic resistance surveillance system to regulate and continuously monitor the emergence of antimicrobial resistance.


ABSTRACT
Klebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide, mainly in hospitalized or immune-compromised individuals.Also, this could be due to the emergence of Multidrug resistance (MDR) Extended Spectrum Beta Lactamase (ESBL) and carbapenemase-producing strains.This study's main goals were to evaluate the prevalence of resistance demonstrated by K. pneumoniae strains found in clinical samples from Benghazi Medical Center and AL-jalaa Hospital and to find evidence of ESBL strains and their resistance to certain antibiotic.During the study period, K. pneumoniae was isolated from 320 clinical samples (urine, sputum, blood and wound).The procedure for processing of samples, identification, susceptibility toward antimicrobials and evidence of ESBL, MBL strains were carried out according to the recommended standards.PCR was used to detect β-Lactamase and carbapenemase resistance genes.From a total K. pneumoniae isolates, 120 (37.5%) were isolated from hospital patients.The isolates exhibited high resistance to all used antibiotics.Forty-eight (40%) of the isolates were ESBL producers.MDR and XDR were identified in 89% and 56% of isolates respectively.ESBL-CTX-M-15 gene and OXA-48 were detected in all isolates.Moreover, SHV and NDM were identified in four isolates.In this study shows the high rate of MDR in clinical K. pneumoniae isolates in hospitals.There is an urgent need to implement an antibiotic resistance surveillance system to regulate and continuously monitor the emergence of antimicrobial resistance.

INTRODUCTION
Up to 10% of nosocomial infections are caused by the Gram-negative encapsulated bacteria Klebsiella pneumoniae, or KP.It is increasingly linked to invasive infections that have significant morbidity and mortality rates [1].The opportunistic pathogen; k. pneumoniae is a member of the Enterobacteriaceae family, that has emerged as major clinical problem causing both hospital-associated and community -acquired infections due to the rising prevalence of multidrugresistant strains(MDR) [2,3].MDR strains of K. pneumoniae can be difficult to treat, particularly in the elderly and in young children with immature immunity.K. pneumoniae causes a number of infections, including wound infections, bacteremia, pneumonia, and urinary tract infections.These infections are typically caused in hospitalized or immunecompromised people [4].Since the introduction and widespread use of new generation extended range antibiotics, there has been a significant increase in the prevalence of bacterial species resistant to drugs.By producing enzymes such as carbapenemase and extended spectrum lactamases (ESBLs) [5].Currently, K. pneumoniae strains producing (ESBL) and carbapenemases have spread globally.One pathway involves the expression of (ESBLs) that contributes to produce resistance in K. pneumoniae against cephalosporin and monobactam.Another extremely worse resistance mechanism is that the expression of carbapenemases by K. pneumoniae, which contribute to resistance of K. pneumoniae against most offered β-lactams as well as the carbapenems [1].In addition, K. pneumoniae has been reported to be the most common pathogenic bacteria to develop resistance to broad-spectrum beta-lactam antibiotics via (ESBL).The number of people who are more prone to infection has increased due to the emergence and spread of hyper-virulent strains.Before the gene became widespread in other pathogens, K. pneumoniae was the source of several novel antimicrobialresistance genes.Example of these genes are blaOXA-48-like, blaSHV-X, blaNDM-1, blaCTX-M-15 and blaTEM-X [6].The relevance of MDR bacteria has increased recently in Libya especially among Gram-negative bacteria including K. pneumoniae, which showed the highest resistance to most of antibiotics in a previous study on patients in intensive care unit at BMC in Benghazi, Libya [7].This might be due to the abuse of antimicrobials including Carbapenems, ESBL in hospitals and poorly applied infection control practices [8].Genotyping methods are important in finding the genetic affinity between bacterial isolates and also in the classification of bacteria, identification the sources of infection and characterization of the most pathogenic strain.Molecular epidemiology analysis gives us the information we need to create strategies to stop the spread of clinically dangerous strains by enabling us to ascertain the global spread of highrisk clones [6].A little information of antibiotic resistance in Libya is known and the genetic basis of Beta-lactam resistance was not available at a large scale in Libya, therefore the main goal of this study was to determine the antibiotic resistance profiles, incidence of MDR, XDR as well as Beta-lactams resistance genes among K. Pneumonia clinical isolates in Benghazi medical Centre (BMC) and Al-Jalaa Hospital at Benghazi, Libya.

Bacterial isolates
Three hundred and twenty specimens (blood, urine, sputum, wound), were obtained from November 2021 to February 2022, that were collected in sterilized containers from both genders, their ages ranged between 20-75, from BMC and AL-jalaa Hospital, Benghazi, Libya.All specimens were incubated onto blood agar as a rich media, and both differential media; MacConkey agar and CLED agar.The plates were incubated for 18-24 hr at 37°C. Isolation and identification of microorganisms were done recommended standard methods.Bacteria were identified by examination of colonial morphology, Gram staining and biochemical tests include (Catalase, Oxidase, Urease, Citrate, Indole, Dnase and Triple Sugar Iron), in addition the isolated bacteria were confirmed by Phoenix system.Molecular identification was carried out by storage of isolates in Nutrient broth media with glycerol and stored in -20°C in Alakeed laboratory then transported in ice bag to isolation of genomic DNA from bacterial cells by boiling method.Then the molecular identification was done by the amplification of 16S ribosomal region using the PCR.The amplified DNA were sent to sequencing in Laboratory of Molecular Biology in Science College, Tunis El-Manar University [9].Sequences were analyzed by BLAST and compared with those available at the National Centre for Biotechnology Information (NCBI) database) and Ribosomal Database Project (RDP) [9].

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by the method of disk diffusion according to guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).The culture of each isolate was diluted to have turbidity around 0.5 McFarland standard, then plated onto Muller-Hinton agar plate (HIMEDIA).Antibiotic disks (Bioanalyze) were applied to each plate.After incubation at 37°C for 18-24hrs, the zone of inhibition diameter was measured.The isolates which were resistant common antimicrobial drug examined by synergism experiment [9].

Detection of ESBL and MBL phenotyping
Antimicrobial susceptibility testing for ESBLs.Performed in Al-Akeed Laboratory, Benghazi by Routine anti-biograms was determined by the disk diffusion method on Mueller-Hinton (MH) agar by placing disks of ceftazidime, cefotaxime, and cefepime at a distance of 30 or 20 mm (center to center) from a disk containing AMC (amoxicillin-clavulanic acid).ESBL production was inferred when the cephalosporin zone was expanded by the clavulanate (Fig. 1A), using Imipenem and EDTA-Imipenem (IEH) disc as a simple method to detect MBL producing clinical isolates (Fig. 1.B).

Detection of antibiotic resistance genes
The presence of antimicrobial resistance genes was searched by PCR and sequencing in some isolates (10).Table 1 showed the primers sequences and PCR conditions of beta-lactams blaCTX-M-15, blaSHV, blaOXA-48 and blaNDM-1.

RESULTS
In this study, from 320 different clinical specimens obtained 120 (37.5%) of the K. pneumoniae isolates were obtained.The highest number of the K. pneumoniae isolates was acquired from the urine samples (n=43; 35.5%) and the lowest number was in blood samples (n=20; 17.4%).Analogous to gender, K. pneumoniae afflicted male patients more frequently than female patients (n = 62; 51.7.2%), with the former having a higher incidence rate.Furthermore, studies on the frequency of K. pneumoniae in urine samples showed that it was more common in females than in males, on the other hand, males were more likely to have the infection in samples of blood, sputum and wounds, as shown in table 2. Phenotypically, susceptibility testing revealed that each isolated strain was highly resistant to the majority of the antibiotics used in this research.Blood samples derived K. pneumoniae strains were resistant to the majority of antibiotics.(Figure 2).In contrast, Table 2 indicates that all K. pneumonia isolates from all samples showed low resistance to Doxycycline (urine=24%, blood=15%, wound=10% and sputum=7.5%).All K. pneumonia separates from all specimens showed high resistance (100%) toward amoxicillin / clavulanic acid, Azactam, Levofloxacin, and Tigecycline.Table 3 displayed the antibiotic resistance pattern of K. pneumoniae strains isolated from clinical samples.By conventional criteria, it is calculated to be designated statistically significant (p< 0.05).The P-value was 0.00001, comparatively, results were significant.Out of all the isolates, 71.2% produced MBL, while 48 (40%) of the strains generated ESBL.In addition, 89% and 56% of the organisms were found to be multidrug-resistant (MDR) and XDR, respectively.OXA-48 and NDM were found in six isolates and five isolates respectively.β-lactamase genes, ESBL-CTX-M-15 type and SHV were identified in four and three strains respectively.

DISCUSSION
Since K. pneumoniae is the cause of 14-20% of infections linked to the respiratory tract, lower biliary duct, surgical wounds, bacteremia, and urinary tract, it has gained significance in the healthcare industry [11].Antimicrobial resistance has been recognized as an important global health dilemma over the past few decades [12].The main causes of resistance are inadequate sanitation and hygiene, overuse of antibiotics in humans and animals, and ineffective infection prevention and control in healthcare settings.The effects of antibiotic resistance on health and the economy are profound.All clinical K. pneumoniae isolates detected showed high resistance to all antibiotics especially amoxicillin / clavulanic acid, Azactam, Levofloxacin and Tigecycline.In addition, the high percentage of resistance (>75%) to the tested antibiotics in this study could be caused by overuse or inappropriate use of these drugs in our country, particularly because they are easily accessible and Libya does not have an antibiotic policy in place.In a related study, high resistance to K. pneumoniae was reported [13,14].MDR-K.pneumoniae infections are highly lethal and frequently linked to a higher chance of receiving insufficient antibiotic treatment [15].High rates of MDR (89%) strains were recorded in this study besides 56% of isolates were XDR.These findings are in agreements with the results in Brazil, they found that 85% of K. pneumoniae isolated from intensive care unit were MDR [16].In China, most of K. pneumoniae strains isolated from neurosurgery patients were MDR and XDR [17].In this study, we found that 40% (n=48) of the K. pneumoniae isolates were ESBL producers.Information of ESBL production collected from different countries showed the diverse rates ESBL in the K. pneumoniae strains.In Syria, the frequency of ESBLs producers of K. pneumoniae were (67.5%) [14].In Palestine, 59.3% of clinical K. pneumoniae strains were ESBLs [13].In Arab Galf, the ESBL phenotypes among K. pneumoniae strains were 23.5% and 36% in Kuwait and United Arab Emirates respectively [18,19].The CTX-M family, which codes for extended-spectrum β-lactamases, is highly prevalent particularly CTX-M-15 variant and has emerged as a significant concern in global health settings [20].CTX-M-15 was detected in four strains in our study which has been identified in many countries [21,22].The CTX-M-15 enzyme is known to spread quickly over numerous nations.The easy dissemination of this gene is linked with the epidemic plasmid which can be horizontally mobilized [23].The present study identified the carbapenem resistance genes blaOXA-48, and blaNDM.These determinants were detected with other β-lactamases blaCTX-M and blaSHV in the same strain.There limited data about the carbapenem resistance genes in Libya.Mathlouthi et al., [24] reported blaNDM-1 and blaOXA-23 genes among Acinetobacter baumannii isolates from two Libyan hospitals.El Salabi et al., [25] detected blaNDM-1 and blaOXA-23 genes in clinical A. baumannii and P. aeruginosa in Libyan hospital.The most important mechanism of carbapenem resistance are capable of enabling bacteria to resist to different antimicrobial agents including third generation cephalosporins, and others [26,27].The detection of these elements reflects the situation in Libyan hospitals and will complicate the infection treatments.

Figure 2 .
Figure 2. Distribution of antibiotic resistance of K. pneumoniae strains according to samples source.