Mutations in β-lactamases detected in multidrug resistant gram negative bacteria isolated from community acquired urinary tract infections in Assiut , Egypt

The aim of this study was to characterize the beta lactamases genes of bacteria isolated from urinary tract infection (UTI) in Assiut, Egypt. Results revealed that one hundred fifty nine [31.8%] out from 500 urine samples were culture-positive. Escherichia coli was the most common UTI pathogen [61%] followed by Klebsiella pneumoniae [23.3%], Proteus mirabilis [8.2%] and Pseudomonas aeruginosa [7.5%]. Sensitivity of isolates to ampicillin was [15%], amoxicillin/clavulanic acid [43.5%], ceftriaxone [24%], imipenem [95.6%], amikacin [75%], ciprofloxacin [21.4%] and trimethoprim /sulfamethoxazole [37%]. Confirmatory phenotypic detection of extended-spectrum β-lactamases [ESBLs] by ESBL E-test method resulted in [42.7%] isolates were ESBLs producers. Genotypic characterization of ESBLs genes in phenotypically positive isolates resulted in [91.2%] were ESBL producers. The presence of CTX-M type ESBL was [75%] followed by TEM [37%], OXA [24%] and SHV [21%]. Sequencing of ESBLs genes showed that CTX-M-15, OXA [1,116], TEM-1 and SHV [1, 11,111,115] as new ESBL types. Multiple sequence alignment of sequenced genes showed mutation in L31R in SHV-11[Novel SHV-115], E29Q in SHV-1[Novel SHV-111], and P65R in TEM-1 and I97M in OXA-1 [Novel OXA-116]. This study is one from first studies in Egypt that highlights the presence of multiple mutations in ESBLs.


INTRODUCTION
UTIs are ranked among the most common infectious diseases found in either the community or healthcare setting (Nicolle, 2005).UTIs have been described by the Egyptians as "sending forth heat from the bladder" since ancient times with the first documented description in the Ebers Papyrus 1550 BC (Al-Achi, 2008).Many studies reported that Escherichia coli and Klebsiella pneumoniae represented the most common pathogens that caused UTIs in various regions of the world, (Gupta et al., 2011) while Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter, Enterococcus species and Staphylococcus species represented the minority of the detected uropathogens (Thomson et al., 1994).
Emergence of antibiotic resistance in uropathogens increased sharply over the world.It varies according to geographical regions and is directly proportional to the excessive use and misuse of antibiotics (Gupta et al., 2001).Certain microorganisms produce defensive enzymes as ESBLs, which own hydrolytic activity enabling them to attack β-lactam ring of penicillins (Paterson and Bonomo, 2005) β-lactamases possess an active site serine, and generally inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam or tazobactam (Livermore, 1995).
TEM-1, SHV-1 and TEM-2 enzymes have limited hydrolytic activity, while mutations in these enzyme result in extended-spectrum phenotype [ESBLs] manifested in serious structural alterations within the active site of the protein which potentiate the β-lactamase activity towards the third-generation cephalosporins (Stürenburg and Mack, 2003).Other types of ESBLs including CTXM, OXA, BES, CME, VEB, PER, SFO and GES, which characterized by potent hydrolytic activity, have been emerged which reflecting the abundance of β-lactamase genes that are available in the bacterial gene pool (Ambler et al., 1991;Livermore, 1995;Philippon et al., 2002;Poirel et al., 2002;Stürenburg and Mack, 2003).
In many studies, a remarkable increase in the ESBL rate was reported from all regions of the world (Eisner et al., 2006;Gupta, 2007;Hosoglu et al., 2007).

Collection of urine samples
A total of 500 clinical samples were collected from Al Azhar university hospital, Assiut, during the period of 1 January 2014 to 1 July 2014, urine samples were collected in a sterile container according to the methods described by Cheesbrough from patients previously clinically diagnosed with UTIs (Cheesbrough, 2006).

Isolation and purification of uropathogens
Urine samples were centrifuged at 3.000 r.p.m for 5 min and the sediment was streaked on cysteine lactose electrolyte deficient agar [Oxoid, UK] for isolation of different uropathogens.

Morphological and Biochemical characterization of isolated bacteria
Purified isolates were examined macroscopically and microscopically.Catalase and oxidase tests performed for all isolates.API 20 E and API 20 NE [Biomerieux,France] were used for confirmatory identification of purified isolates from Enterobacteriaceae, and Pseudomonas spp.(Butler et al., 1975;Peladan and Monteil, 1988).
The assay was conducted in duplicate for each organism evaluated.The zone size around each antimicrobial disc was interpreted as susceptible, intermediate or resistant according to interpretative criteria recommended by CLSI (2013).Isolates with inhibition zone ≤ 25 mm to ceftriaxone 30 μg [Oxoid, UK] was considered as ESBLs producers (CLSI, 2013).

Genetical analysis
DNA was purified by using WIZARD Genomic DNA Purification Kit (Promega, Germany, catalog No. A1125), following instructions as directed by the manufacturer.
PCR was conducted for detection of specific genes according to the resistance phenotype using forward and reverse primers for the following genes bla-TEM, bla-SHV, bla-CTX-M, and bla-OXA (Oliver et al., 2002;Pagani et al., 2003).PCR reactions was carried out in 50 μl reactions with 2μl forward and reverse primers, 2 μl template DNA, and 10 ml of 5Xof Hot Master Mix [Solis BioDyne -Tartu Estonia].Thermal cycling consisted of different conditions for amplification of each gene (Table 1).

Agarose gel electrophoresis
The PCR products were visualized using agarose [1%] gel electrophoresis [Biometra-Agarose gel mini, Germany] (Brook, 2005).amplicons sizes were calculated by a comparison with 100 bp to 3kb molecular weight DNA ladder [Solis BioDyne -Tartu Estonia].PCR products were purified using the Agencourt XP Ampure Beads [Beckam Coulter, USA].The quality of the final products were assessed using a Bioanalyzer 2100 [Agilent Technologies, USA] and after quantification with a Qubit [Invitrogen, USA].

Sequencing of PCR products
β-lactamases were identified by sequencing the purified PCR amplicons using the dideoxynucleotide chain termination method with fluorescent cycle sequencing using dye-labelled terminators [BigDye Terminator version3.1cyclesequencing kit; Applied Biosystems, Grand Island, NY, USA] on an ABI prism 3730 automated DNA sequencer (Sanger et al., 1977).

Sequence assembly, analysis and alignment
The sequences obtained of ESBLs was assembled by [     The bacteria species showed varying susceptibility patterns to seven of the antimicrobial agents (Table 2).

DISCUSSION
In our results, the leading pathogen causing UTI was E. coli [61%] followed by K. pneumonia [23.3%],P. mirabilis [8.2%] and P. aeruginosa [7.5%] which nearly similar to that reported by Ibrahim et al. (2014) in Egypt.E. coli was the most common pathogen causing UTI in the world this in agreement with our study (Gupta et al., 2011).Blindly treatment of UTI leads to increase the resistance rate of these pathogens to antibiotics especially β-lactam antibiotics due to excessive and misuse of these antibiotics.Imipenem was the most effective antibiotic against UTI and activity more than 95% because of less used due to economic considerations.
In this study production of ESBLs varies from type of isolate to another.P. aeruginosa was the most powerful ESBLs producers [100%], followed by E. coli 97%, K. pneumoniae 82.6% and finally P. mirabilis 82%.In our study sequenced CTX-M showed that most CTX-M genes were CTX-M-15 as that detected in Egypt and middle east area (Amin et al., 2005;Thabit et al., 2011).In our study, multiple mutations detected among βlactamases; in SHV-11 gene there is mutation in position 31, amino acid Arginine instead of amino acid Leucine [L31R], results in novel SHV-115 K. pneumoniae strain MR1982 ESBL with accession number [KR780481].L31R mutation in Klebsiella was the first detected in middle east and the second detected in world after Mendonça et al. (2009)

Conclusion
Frequent consumption and misuse of antibiotics lead to mutations and the emergence of new genes more aggressive and more resistant to antibiotics, which leads to increased mortality and which calls for the search for new antibiotics and open new horizons in how to address these pathogens.

Table 1 .
McGinnis and Madden, 2004).The multiple sequence alignment was performed by the online software Clustal Omega Universal primers and PCR conditions for β-lactamases.

Table 2 .
Total antimicrobial susceptibility pattern of UTI isolates results.