Microbiological studies on resistance patterns of antimicrobial agents among Gram negative respiratory tract pathogens

Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia. Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. Department of Microbiology and Immunology, Faculty of Pharmacy, October University for Modern Sciences and Arts, 6 October City, Egypt.


INTRODUCTION
The Centers for Disease Control and Prevention (CDC) estimates that more than 100 million antibiotic prescriptions are written each year in the ambulatory care setting. With so many prescriptions written each year, inappropriate antibiotic use will promote resistance. In addition to antibiotics prescribed for upper respiratory tract infections with viral etiologies, broad-spectrum antibiotics are used too often when a narrow-spectrum antibiotic would have been just effective (Steinman et al., 2003).
Resistance to β-lactam antibiotics occurs primarily through the production of β-lactamases, enzymes that inactivate these antibiotics by splitting the amide bond of the β-lactam ring. β-Lactamases most likely coevolved with bacteria as mechanisms of resistance against natural antibiotics over time, and the selective pressure exerted by the widespread use of antimicrobial therapy in modern medicine may have accelerated their development and spread. β-Lactamases are encoded either by chromosomal genes or by transferable genes located on plasmids and transposons. In addition, βlactamase genes (bla) frequently reside on integrons, which often carry multiple-resistance determinants. If mobilized by transposable elements, integrons can faciletate further dissemination of multidrug resistance among different bacterial species (Weldhagen, 2004).
Four major groups of enzymes are defined by their substrate and inhibitor profiles: group 1 cephalosporinases that are not well inhibited by clavulanic acid; group 2 penicillinases, cephalosporinases and broadspectrum β-lactamases that are generally inhibited by active site-directed β-lactamase inhibitors; group 3 metallo β-lactamases that hydrolyze penicillins, cephalosporins and carbapenems and that are poorly inhibited by almost all β-lactam-containing molecules; and group 4 oxacillin-hydrolyzing enzymes that are not inhibited by clavulanic acid (Webb, 1984).
Another important mechanism of antibiotic resistance is efflux pumps. In general, multiple antibiotic resistance in Gram-negative bacteria often starts with the relatively limited outer membrane permeability to many antibiotic agents, coupled with the over expression of multi-drug resistance (MDR) efflux pumps, which can export multiple unrelated antibiotics. In addition, by reducing the intracellular concentration of the antimicrobial agent to less than the MIC required for bacterial killing, efflux mechanisms may allow bacterial survival for longer periods, facilitating the accumulation of new antibioticresistance mutations (e.g., those encoding topoisomerase IV or DNA gyrase targets, rendering fluoroquinolones ineffective) (Piddock, 2006).
Antimicrobial agents exert strong selective pressures on bacterial populations, favoring organisms that are capable of resisting them. Genetic variability occurs through a variety of mechanisms. Point mutations may occur in a nucleotide base pair, and this is referred to as microevolutionary change. These mutations may alter enzyme substrate specificity or the target site of an antimicrobial agent, interfering with its activity (Medeiros, 1997). This study focused on the genetic variability among Gram negative respiratory tract isolates and its relation to antimicrobial resistance including multi-drug resistant isolates.

Bacterial isolates
A total of 309 non replicate Gram negative respiratory tract isolates from 249 patients: 115 males, 134 females, between the ages of 3 and 50 from medical intensive care unit, MICU and surgical intensive care unit, SICU, with underlying upper and lower respiratory tract diseases with no history of antibiotic administration prior to sample acquisition for three months were collected from King Abdulaziz University Hospital, Jeddah, KSA. From September 2011 to June 2012 according to the generally accepted guidelines for specimen collection and transportation of common specimen types as illustrated in Table 1 (Murray, 2007), clinical specimens collected were isolated, identified using morphological, microscopy, biochemical tests and API kit method as well.

Characterization and molecular mechanisms of antimicrobial resistance pattern of Gram negative respiratory tract pathogens
Isolates that exhibited reduced susceptibility to one or more of ceftazidime, aztreonam, cefotaxime or ceftriaxone were considered as potential producers of ESβL. Double-disk synergy test (Figure 1) was done using ceftazidime and a ceftazidime + clavulanic acid (30 μg/10 μg) discs as confirmatory test for detection of ESβL production (Coudron et al., 1997). Isolates resistant to imipenem or meropenem were considered as suspicious for production of metallo-beta-lactamases (MβL), ethylene diamine tetraacetic acid (EDTA) disc synergy test (Figure 2) was done for detection of metallo-β-lactamases in the imipenem resistant isolates (Yong et al., 2002). Isolates resistant to one or more of cefoxitin, cefotetan, cefotaxime, ceftazidime and aztreonam were considered as suspicious for production of AmpC-beta-lactamases (AmpC-BL), combined disc test ( Figure 3) using cloxacillin as inhibitor of AmpC enzymes was done as confirmatory test for detection of AmpC producing isolates (Mirelis et al., 2006). Minimum inhibitory concentration (MIC) of ciprofloxacin against the clinical isolates was determined using the two-fold serial broth dilution method with an inoculum of 1 x 10 6 cells/ml. All experiments were done with and without 100 mg/L carbonyle cyanide-m-chlorophenylhydrazone (CCCP). The MIC was taken as the lowest concentration inhibiting visible growth after 18 h incubations at 37°C. CCCP inhibited multidrug resistant (MDR) efflux pump was inferred if the MIC with CCCP was four-fold or lower than the MIC without CCCP (Omoregie et al., 2007).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Table 1. Guidelines for specimen collection and transportation of common specimen types.

Specimen Collection methods Respiratory, Upper Nose
Premoistened swab was inserted 1-2 cm into nares and rotated against nasal mucosa. Nasopharynx Nasopharyngeal washings and swabs. Throat or pharynx The posterior pharynx was swabbed, avoiding saliva.

Respiratory, Lower
Bronchial alveolar lavage A large volume of fluid was collected; transported in sterile container.

Sputum (expectorated)
Patient was instructed to rinse or gargle with water to remove excess oral flora; then to cough deeply and expectorate secretions from lower airways; which were then collected and transported in a sterile container.

DNA sequencing
After initial screening for the amplification of β-lactamases and efflux pump genes on both chromosomal and plasmid, the plasmid and chromosomal borne genes were subjected to nucleic acid sequencing. The initial PCR amplified products were purified and treated with QIAquick PCR Purification Kit (QIAgen Inc.,Valencia CA, USA).
Direct sequencing of each amplicon was carried out using the Sanger dideoxynucleotide chain termination method with the ABI Prism Big Dye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Inc., Foster City, CA, USA) on an ABI Prism 3500 Automated Sequencer. Using data collection software version 2.0, and sequencing analysis software 5.1.1, for each sequencing reaction, 2 μl purified PCR product were added to a final reaction volume of 10 μl containing 1× of sequencing buffer; 4 μl BigDye reaction mix; and 3.2 pM of each of the Forward and Reverse primer. The sequencing cycle was composed of two stages; stage one is denaturing at 96°C for 1 min, while stage two is composed of 25 cycles of denaturing at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 4 min (Sabate et al., 2000).
Each cycle sequence product was purified by BigDye XTerminator Purification Kit. The purified PCR product was then placed in the DNA analyzer. The DNA sequences obtained were compared with those in the GenBank using the BLAST program (http://blast.ncbi.nlm.nih.gov/).  (34.6, 26.6, 13.9, 7.7, 6.4, 5.5, 1.4, 1.3, 1, 1, 0.3 and 0.3%. The distribution of organisms harboring β-lactamases and efflux pump among Gram negative respiratory tract isolates are illustrated in Table 2.

Detection and prevalence of beta-lactamases and efflux pump genes in Gram negative respiratory tract isolates
PCR and sequence analysis indicated the presence of bla SHV , bla CTX-M , bla TEM , bla IMP , bla VIM , ACC, DHA, AdeJ, MexX and MexE genes in the isolated respiratory tract isolates with distribution illustrated in Table 3.

DNA sequencing results
Nucleotide composition analysis of some A. baumannii isolates showed that, the RND family drug transporter (AdeJ) gene detected was of GCwith value of 41.5 and the detailed composition was: T (30.7), C (20.8), A (27.8) and G (20.8). Among the studied 659 nucleotide bases compromising for AdeJ gene, 655 bases were conserved while only 4 sites were variable. Surprisingly, 3 out of the four base substitutions were transitional changes, from T→C (356 and 389) and C→T (566). Only one base substitution was transversional change from G→T (449) (Figure 4). Nucleotide composition analysis of some P. aeruigenosa isolates showed that, the multidrug efflux membrane fusion protein encoding gene (MexE) detected was of high GC with value of 71.1 and the detailed composition was: T (10.7), C (38.7), A (18.2) and G (32.4). Among the studied 458 nucleotide bases compromising for MexE gene, 455 bases were conserved while only 3 sites were variable. Two out of the three base substitutions were transitional changes, from A→G (15 and 39). Only one base substitution was transversional change from C→A (77) ( Figure 5).     nucleotide bases compromising for MexX gene, 411bases were conserved while only 2 sites were transitional changes, from T→C (20) and C→T (404) (Figure 6).

DISCUSSION
The present study proposes a combined phenotypic and genotypic approach for the specific diagnosis of antibiotic resistance mediated by β-lactamases and efflux pump system harboring Gram negative respiratory tract isolates.
In the present study, bla CTX-M genes were predominant in A. baumannii and P. aeruginosa isolates with percentage of 39 and 31% respectively, followed by bla SHV genes in A. baumannii and E. cloacae isolates with percentage of 20 and 60% respectively. bla TEM genes were predominant in E. coli, K. pneumoniaea and S. maltophilia isolates with percentage of 50, 33 and 33%, respectively. bla SHV genes were predominant in E. cloacae with percentage of 60%. Similar findings were found in Indian study (Gupta, 2007), from a total of 94 isolates, 50 (n = 47), 14.89 (n = 14) and 11.70 (n = 11) ESβL rates for bla TEM , bla SHV and bla CTX-M type beta lactamases, respectively. bla TEM and bla CTX-M type ESβL were observed in 72.72 and 22.72% of E. coli isolates, respectively. Also, the present study revealed that bla IMP gene was predominant in A. baumannii isolates with percentage of 27%, followed by bla VIM gene 11%. bla VIM gene was predominant in P. aeruginosa isolates with the percentage of 44%, followed by bla IMP gene 18%. Both bla IMP and bla VIM genes were found together in E. coli isolates with the percentage of 33% followed by 11% of each alone. This was in accordance with Nordman and Poirel (2002), were a total of 8 pseudomonas isolates carried bla VIM -type gene, these data demonstrate that bla VIM -type gene are the most prevalent MβLs among clinical specimens of P. aeruginosa. ACC gene was predominant in A. baumannii and P. aeruginosa isolates with percentage of 52 and 36%, respectively, followed by DHA-1, DHA-2 genes with 13 and 6% respectively. This result differs significantly from the findings of several studies were the isolation numbers of ACC enzymes were still significantly lower than those of CIT (CMY), FOX and DHA (Philippon et al., 2002). AdeJ was detected in A. baumannii with percentage of 29.2%, while MexX gene was predominant in P. aeruginosa isolates with percentage of 46% followed by MexE, 3.8%. This differ from the findings of some biological observations made during a study where the basal expression level of MexX is much lower than that of MexA but that both efflux pumps are over-expressed 4 to 8 times in resistant strains, suggesting that a lower quantity of MexXY-OprM than MexAB-OprM protein may be needed for effective transport of the corresponding substrates (Llanes et al., 2004). Second, over-expression of MexX in clinical isolates is systematically associated with that of MexA. This may be related to the fact that MexXY uses OprM as a porin (Masuda et al., 2000).

Conclusion
The study figured out the most common genes responsible for the expression of β-lactamase enzymes and efflux pump system in Gram negative respiratory tract isolates. The study also revealed that, isolates harboring more than one gene from the same class have higher resistance pattern towards antimicrobial agents than those harboring only one; also, isolates having microevolutionary changes in their nucleotide composition of the detected genes have higher resistance pattern towards antimicrobial agents than those where all bases are conserved.