The incidence of Extended-spectrum β -lactamase enzymes and their connection to virulence genes in community-acquired urinary tract infection

: In community-acquired urinary tract infections, Klebsiella pneumoniae is considered as one of the most common etiological agents. In Klebsiella pneumoniae populations, multidrug resistance and virulence are common. In this research, fifty isolates of Klebsiella pneumoniae were from urine samples and diagnosed by a vitek 2 compact device. The Kirby–Bauer disk diffusion technique was used to perform antibiotic susceptibility tests. That showed about 92% of isolates were multidrug resistant (MDR). The Modified Double Disc Synergy Test revealed that 90% of the isolates produce extended spectrum-lactamases (ESBLs).Genotypic detection of antibiotic resistance and virulence genes was done via PCR assay. The ability of Klebsiella pneumoniae isolates to produce ESBL genes showed that the SHV gene was the most prevalence among ESBL genes (68%), followed by the CTX-M gene (66%). while none of the isolates possessed the TEM gene. The capacity of isolates to generate Virulence factor type 3 fimbriae (MrKD) genes and biofilm (BssS) genes found that the isolates contain the MrKD gene (82%). The BssS gene was also discovered to be present in (72%) of the isolates. Virulence genes within ESBL-producing Klebsiella pneumoniae isolates in this study show that only [n=3 (6%)] of isolates which are non-ESBL producing carry one or both virulence genes. While one or both virulence genes are present in [n=41(82%)] of ESBL-producing isolates, the prevalence of ESBL-producing Klebsiella pneumoniae in community patients was found to be high in this research. It's also possible that ESBL production and virulence factors are linked.


Introduction
Urinary tract infections have serious health and financial consequences for society. These infections considered the most prevalent type of bacterial infection, and they can strike at any moment during a person's life. People in hospitals and the general public are both susceptible to urinary tract infections. CAUTIs (community-associated urinary tract infections) are frequently identified in individuals with risk factors such as age, prior UTI history, sexual activity, and diabetes mellitus. Antibiotics are the most common therapy for bacterial UTIs 1 . After E.coli, K. pneumoniae is the most medically significant species in the Enterobacteriaceae family 2 . One of the frequent pathogens linked to both community and hospital-acquired urinary tract infections is K. pneumoniae 3 . Because bacterial etiology is common in UTIs, broad-spectrum antibiotics are frequently used, resulting in a rise of resistant uropathogens 1 . Antibiotic resistance among K. pneumoniae strains is a major public health concern, as well as a significant financial burden for UTI sufferers. Antibiotic resistance of these isolates is acquired by a variety of processes, including the creation of extended spectrum-lactamases (ESBLs).The formation of extended-spectrum beta-lactamase, an enzyme that attacks the beta-lactam ring in medicines and renders them useless, causes ESBL resistance 4 . Several ESBL families have been identified, however the big families such as SHV, TEM, and CTX-M account for the bulk of ESBLs 5 . The rapidly increasing resistance of ESBL producers to multiple antibiotic families is a major issue that limits the treatment options available against ESBL producers 6 . In addition, lipopolysaccharide (LPS), capsular polysaccharide, adhesions, and sidrophores are among the virulence components found in K. pneumoniae that contribute to its pathogenicity 7 . Adhesion is an important phase in the infection that must be strong enough to overcome the host's defensive mechanisms. Fimbriae, also known as pili, are proteinaceous structures that extend from the bacterial cell surface to a distance of 100 nm to several microns and are composed of adhesions that are thought to aid bacterial adhesion 8 . There are two kinds of fimbriae or pili in K. pneumoniae, types 1 and 3 9 . Type 3 fimbriae have a helix-like structure that gives them spring-like flexibility and stretch-ability. This fimbrial type is encoded by a chromosomally borne gene cluster that has previously been demonstrated to consist of five genes in various strains of K. pneumoniae. The mrkABCDF gene cluster encodes the primary structural component (mrkA) and the fimbrial adhesin (mrkD), whereas the genes mrkB and mrkC encode the chaperone and usher proteins, respectively 10 .Type 3 fimbriae have been shown to mediate adhesion to various structures in human kidney and lung tissue, as well as epithelial cells from human urine sediments and endothelial and bladder epithelial cell lines, in vitro investigations 11 . K. pneumoniae biofilm development on biotic and abiotic inert surfaces is commonly linked to Type 3 fimbriae 12 . Infections caused by K. pneumoniae producing biofilms are more resistant to therapy than other infections. Biofilms serve as shields for bacterial populations, allowing them to evade host defenses. The bac-teria are also protected from severe circumstances such as pH changes, forces of shear, and nutritional deficiencies 13 . As a result, the current study was designed to look into the distribution of extended spectrum-lactamase enzyme genes (TEM, SHV, CTX-M), virulence genes (type 3 fimbriae MrKD gene, biofilm BssS gene), and the relationship between them in K. pneumoniae isolates from urine samples.

Antibiotic Susceptibility test
The Kirby-Bauer disk diffusion technique was used to perform Antibiotic susceptibility testing for all of the isolates 14 . Antibiotics that listed below were utilized: Ampicillin (10 μg

Detection of Isolates that Produce ESBL
Detect ESBL enzymes production by using Modified Double Disc Synergy Test through the following steps 15 : After uniformly spread the inoculum onto sterile Mueller-Hinton agar (biolab/Hungary), the disc which contain amoxicillin-Clavulanate (20/10μg) was placed in the center of the plate and the discs of third generation cephalosporin [cefotaxime (30μg), ceftriaxone (30μg), and cefpopdoxime (10μg)] ,and fourth generation cephalosporin [cefepime (30μg)] ,were placed 15mm and 20 mm apart respectively, center to center to that the amoxicillin-Clavulanate disc. After Incubated for 18-24 h at 37 °C, any increase in the inhibition zone toward the disc of the amoxicillin-Clavulanate was considered as positive for ESBL production.

DNA extraction
For the isolation and purification of DNA from K. pneumoniae isolates, DNA was extracted using G-spin TM Total DNA Extraction Kit (Intron biotechnology/ Korea) according to the manufacturer's instructions. Concentration and purity estimated by using Nanovue plus™ spectrophotometer (GE Healthcare/UK).

Polymerase Chain reaction of Antibiotic Resistance and Virulence Genes
The genes coding for virulence factors (type 3 fimbriae MrKD, biofilm BssS) and extended spectrum β-lactamase genes (TEM, SHV, CTX-M) are detected by the PCR method. Primers used in this study were purchased from (Alpha DNA/ Canada) in lyophilized form as shown in Table 1. The PCR amplification reaction mixture components (25μl) which are used for the detection of each gene contain: DNA sample, primers, and 5x FIREPol® Master Mix (Solis Bio Dyne/Europe) 16 . After preparing the reaction volume in the PCR tube, the mixture was spun down via (Centrifuge/Vortex for PCR plates) and then PCR tubes were placed in the PCR thermal cycler (Bio-Rad/USA) to amplify the target DNA for (TEM, SHV, CTX-M, BssS, MrKD primers) using the following program 17 as shown in Table 2.

Results
In the current study, 50 K. pneumoniae isolates were collected from urine samples of community acquired urinary tract infection patients. All samples were identified via vitek 2compact device. The antibiotic susceptibility test was done via The Kirby-Bauer disk diffusion method, then interpreting the results of which was done using criteria published by the Clinical and Laboratory Standard Institute 18 . As seen in Table  3.The results showed that about 92% of K. pneumoniae isolates were multidrug resistance (MDR).
Modified Double Disc Synergy Test (MDDST) was used to detect the ESBL production. The results showed that approximately 90% of the isolates produce ESBLs, as shown in Figure 1.
The ability of K. pneumoniae isolates to produce ESBLs (TEM, SHV, and CTX-M) genes and the capacity of isolates to generate Virulence factor type 3 fimbriae (MrKD) genes and biofilm (BssS) genes are determined using PCR, which uses sets of primers that amplify the genes. The results showed that SHV gene was the most prevalence among ESBL genes (68%) then the CTX-M gene (66%). while none of the isolates possessed the TEM gene as showed in Figure 2. The results found that the isolates contain MrKD gene at (82%) as shown in the figure A (3). It also, found that (72%) of isolates contain the BssS gene as shown in the figure B (3).
The prevalence Virulence genes within ESBLs and non-ESBLs producing K. pneumoniae isolates were detected as seen in Table 4.

Discussion
In both community and hospital settings, K. pneumoniae is a leading cause of serious infections such as urinary tract infection, pneumonia, skin and soft tissue infection, intra-abdominal infection, bloodstream infection, meningitis, and pyogenic liver abscess. In humans, antimicrobials have long been used to treat K. pneumoniae infections 19 . The emergence of multidrug-resistant (MDR) K. pneumoniae strains across the world is a major source of worry 20 . In this study, the results showed that about 92% of K. pneumoniae isolates were multidrug resistance (MDR). MDR is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories 21 . These results were close to the results obtained by the research study of Mohamed et al. 22 Multidrug resistance was revealed in 86.66% of K. pneumoniae isolates. While MDR phenotypes were seen in 75% of K. pneumoniae isolates, according to the study done by23 MDR obstructs disease control by increasing the risk of resistant microorganisms spreading, lowering treatment efficacy and, as a result, causing patients to be infected for longer periods of time 24 . A variety of virulence factors contribute to K. pneumoniae's pathogenicity 25 . And the capacity to develop various antibiotic resistances quickly 26 . K. pneumoniae is, in fact, a significant ESBL host. ESBL development has substantially boosted bacterial resistance to β-lactams in human infections, causing major morbidity and death 27 . Extended-spectrum beta-lactamases (ESBLs) are enzymes that can give resistance towards β -lactam antibiotics such penicilins, scephalosporins, and aztreonam. This is achieved by hydrolyzing the antibiotics, and beta-lactamase inhibitors like clavulanic acid block these enzymes 28 .
In this research, Modified Double Disc Synergy Test (MDDST) was used to detect ESBL production. As a result, any increase in the inhibition zone (of cefeime, cefpodoxime, ceftriaxone, and cefotaxime) toward the amoxicillin-Clavulanate disc was interpreted as a sign of ESBl generation. The results showed that approximately 90% of the isolates produced ES-BLs. According to one study 29 , approximately 88.6% of K. pneumoniae isolates produced ESBLs.
The ability of K. pneumoniae isolates to produce ESBLs (TEM, SHV, and CTX-M) genes showed that the SHV gene was the most popular among ESBL genes at (68%) than the CTX-M gene at (66%). while none of the isolates possessed the TEM gene. On the other hand, a study done by (30) showed that SHV was the most prevalent type of ESBL genes (90.5%) followed by TEM (80.0%) and CTX-M (76.2%). Furthermore, research by (23) shows that the TEM gene has the highest prevalence in K. pneumoniae isolates at 64.7%, followed by the CTX-M gene at 41.1% and the SHV gene at 35.2%. According to a study conducted by (31) all isolates contained the CTX-M gene, whilst (97%) contained TEM, and (83%) harbored SHV genes. CTX-M was shown to be the most common ESBL in K. pneumoniae community isolates in the majority of investigations 32,33 . Antibiotic resistance has developed as a result of widespread over distribution and irresponsible usage. Because most antibiotics are accessible over the counter in underdeveloped nations and may be administered without a prescription, patients and the general public must be educated 34 . The capacity of K. pneumoniae isolates to generate Virulence factor type 3 fimbriae (MrKD) genes and biofilm (BssS) found that the isolates contain MrKD gene at (82%) while The results found that (72%) of isolates contain the BssS gene. The prevalence Virulence genes within ESBL-producing K. pneumoniae isolates in this research show that only [n=3 (6%)] of isolates which are non-ESBL producing carry one or both virulence genes. whereas [n=41(82%)] of ESBL-producing isolates contain one or both virulence genes. This is consistent with the study done by (35) that suggests a correlation between ESBL production and some virulence factors. Table 2. PCR thermocycler conditions for each gene.
The incidence of Extended-spectrum β-lactamase enzymes and their connection to virulence genes in community-acquired urinary tract infection Table 3. Antibiotic susceptibility of 50 K. pneumoniae isolates.

Conclusions
In conclusion, the prevalence of ESBL-producing K. pneumoniae in community patients was found to be high in this research. It's also possible that ESBL production and virulence factors are linked. Antibacterial resistance is a developing problem in clinical practice. As a result, improved clinical awareness and laboratory testing are required to decrease treatment failure and prevent the spread of ESBL-producing K. pneumoniae. Table 4. Distribution Virulence genes within ESBLs and non-ESBLs producing K. pneumoniae isolates.