Ciprofloxacin and Azithromycin Resistance Among Bacterial Isolates from Different Infections.

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UNIVERSTITY OF KUFA                                    
Faculty of Science                                                
Department of investigation laboratory
    

               
Ciprofloxacin and Azithromycin Resistance Among Bacterial Isolates from Different Infections

A Research Submitted to the Faculty of Science, University of Kufa in Partial Fulfillment of  the Requirement for the Bachelor Degree in Science.


By
Hussein A. Khuttar
Zaid A. Imran
Qasem A. Najah


Supervisor
A. Professor Dr. Raed Ali Hussein  Aboshabaa

2018 A.D.                                                                   1439 A.H.






Ciprofloxacin and Azithromycin Resistance Among Bacterial Isolates from Different Infections
Abstract:
This study was aimed to presents the incidence of Ciprofloxacin and Azithromycin resistance among urinary tract infection (UTI), gastrointestinal infection, and burns, Azithromycin and Ciprofloxacin are broad spectrum antibiotics, bacteriostatic and bactericidal respectively, are used for this study because they are mostly used in Iraq and have less resistant from infectious bacteria. Eighty-four specimens were collected from patients admitting to Alforat al-Awsat hospital in An-Najaf province using transport swab, All the collected swabs were cultured on plates of nutrient agar, blood agar, MacConkey agar, and salmonella-shigella agar (SS agar), The microbial isolates were identified according to colony morphology and microscopical features and biochemical tests include Catalase, Oxidase, Mannitol Salt Agar, Coagulase test, IMViC tests and Novobiocin susceptibility. Antibiotic susceptibility tests were done to all the isolates. Out of eighty-four specimens collected during this study, 76 (90%) were positive for bacterial grow. The results of macroscopic and microscopic study showed that from all the 76 bacteria isolate a 20 (27%) were identified as staphylococcus aureus, 11(14%) were identified as Staphylococcus saprophyticus, 13 (17%) were identified as coagulase- negative Staphylococci, 10 (13%) were identified as Proteus mirabilis, 12 (16%) were identified as Klebsiella spp., 10 (13%) were identified as Shigella spp. The results of antibiotic sensitivity test study showed that from all the 76 bacteria isolates, A 20 isolates (100%) staphylococcus aureus were resistance for azithromycin and Ciprofloxacin, A 13 of Staphylococcus saprophyticus isolates, showed no resistance to Azithromycin and (100 %) resistance to ciprofloxacin, 13 isolates of coagulase- negative Staphylococci were (100%) resistance to Azithromycin and Ciprofloxacin, A 12 isolates of Klebsiella SPP showed no resistance to Azithromycin and (100%) resistance to Ciprofloxacin, A 10 isolates of Proteus mirabilis showed (100%) resistance Azithromycin and Ciprofloxacin, A 10 isolates of Shegilla spp. showed (100%) resistance to Azithromycin and no resistance to Ciprofloxacin.
1.Introduction:
Azithromycin and Ciprofloxacin are broad spectrum antibiotics, bacteriostatic and bactericidal respectively, are used for this study because they are mostly used in Iraq and have less resistant from infectious bacteria. 
Azithromycin is a broad-spectrum macrolide antibiotic with bacteriostatic activity against many Gram-positive and Gram-negative bacteria including Bordetella pertussis and Legionella species. It also has activity against Mycoplasma pneumoniae, Treponema pallidum, Chlamydia species and Mycobacterium avium complex. Staphylococcus aureus, Shigella spp, Klebsiella spp and others .1  Azithromycin reversibly binds to the 50S ribosomal subunit of the 70S ribosome of sensitive microorganisms, thereby inhibiting the translocation step of protein synthesis, wherein a newly synthesized peptidyl tRNA molecule moves from the acceptor site on the ribosome to the peptidyl (donor) site, and consequently inhibiting RNA-dependent protein synthesis leading to cell growth inhibition and cell death.1
Ciprofloxacin is bactericidal antibiotic, the most frequently prescribed fluoroquinolone for urinary tract infections, Lower respiratory tract infections, Skin infections, Bone and joint infections, Intra-abdominal infections, Kidney infections in children Acute sinus infections, Infectious diarrhea, Typhoid fever, Gonorrhea, Plague and Anthrax because of its availability in oral and intravenous formulations. It is well absorbed from oral doses and is rapidly excreted from the body under normal conditions Genetic and biochemical studies have identified the A subunit of the essential bacterial enzyme DNA gyrase as a target of ciprofloxacin and other quinolones. For a series of quinolones, inhibition of purified DNA gyrase correlated with antibacterial activity [2.3.4.].
Antibiotic Resistance:
Antimicrobial resistance (AMR) is defined as the resistance of microorganisms to an antimicrobial agent to which they were at first sensitive.5 This natural evolutionary phenomenon, enhanced by the misapplication of antimicrobial medicines and the global spread of AMR, mainly affects unhealthy and debilitated patients, giving rise to super microbes. AMR inflicts high costs in the public. There are many mechanisms of resistance in bacteria. Of these, five are the most frequently observed, showing high prevalence in clinical isolates. They are enzymatic inhibition, penicillin binding protein (PBP) modifications, porin mutations, efflux pumps, and target changes5-10.
Efflux Pumps
             A highly efficient mechanism of resistance is the production of an efflux pump, a proton-dependent system that effects an active removal of the antibiotic from inside the cell.8 There are five families of membrane-spanning efflux proteins, including major facilitators (MFs), small multidrug resistance (SMR), resistance nodulation cell division (RND), ATP-binding cassette (ABC), and multidrug and toxic compound extrusion (MATE).11 On the one hand, drug efflux from Gram-positive bacteria is commonly mediated by a single cytoplasmic membrane-located transporter of the MF, SMR, or ABC families. On the other hand, Gram-negative bacteria are more complex due to the presence of an outer membrane.12 The MF family consists of membrane transport proteins, with 1214 transmembrane domains (TMDs),13 implicated in the antiport, symport, or uniport of many substances.14 In MF and SMR family transporters, the propulsion force for drug efflux appear to be an electrochemical potential of H1 over the cell membrane.13 All members of this family have three conserved motifs: motif A, which acts as a cytoplasmic gate controlling the passage of the substrate to and from the cytoplasm; motif B, which is involved in energy coupling; and motif C, which determines the orientation of the unoccupied substrate-binding site and thus commands the direction of transport. The best characterized protein in this family is the tetracycline transporter (TetB), from E. coli, which has been shown to function as an electroneutral antiport system, catalyzing the exchange of a tetracycline-divalent-metal-cation complex for a proton.14

Enzymatic inhibition:
            The most common mechanism of resistance in bacteria is enzymatic inhibition. This mechanism is based on several strategies for modifying the structure of antibacterial compounds: with hydrolysis, a type of reaction that occurs mainly with β-lactam agents; transference of functional groups (acyl, phosphoryl, thiol, nucleotidil, ADP-ribosyl, glycosyl), which occurs with a lot of antibacterials, such as aminoglycoside, chloramphenicol, rifamycin, and lincosamide; and other chemical modifications (redox, lyase), which occur with tetracycline, rifamycin, and streptogramin.5,15

Penicillin Binding Protein (PBP) modification:
               PBPs are important proteins involved in the construction of peptidoglycan, which is the major constituent of bacterial cell walls.6 These enzymes catalyze the glycan strand (transglycosylation) and the cross-linking between glycan chains (transpeptidation).6,16 However, some PBP classes did not have transglycosylation activity, such as B PBPs and low-molecular-mass PBPs.16,17 The transpeptidase active site is the target of β-lactam agents.18 These compounds mimic the D-Ala-D-Ala dipeptide in peptidoglycan and form a very stable acyl-enzyme complex, leading to enzyme inactivation.18,19 Among the different modified PBPs, some of them have high prevalence, including PBP4 and PBP5, which confer resistance to penicillins; and PBP2x and PBP1a, which are responsible for conferring variable resistance to penicillins and other β-lactams, both of chromosomal origin.20 However, the most alarming is PBP2a (also called PBP20), a modified protein that confers resistance to penicillins and cephalosporins.21 This protein is the product of the gene mecA and the homologous genes mecB and mecC, all of plasmid origin.21 These modified PBPs change the active site, causing the β-lactam agents to lose or diminish their affinity with the target protein, promoting resistance.21,22.

Porin modifications:
               Gram-negative bacteria have a membrane outside the cell wall, the outer membrane, which consists of a lipid bilayer. The main constituent of this bilayer is the lipopolysaccharide, and due to its hydrophobicity, the passage of hydrophilic compounds is very difficult; thus, porins or outer membrane porins (Omps), which are proteins that aid in the passage of hydrophilic solutes across lipid bilayer membranes, are required.7,23 Many factors affect the ability of the drug to pass through porins, such as charge, shape, and size.23 There are some typical porins, such as OmpF, OmpC, and OmpE.24 Each bacterial species produces specific porins, and the loss or impairment of one or more Omps is a common contributing factor in establishing resistance (eg, loss of OprD in P. aeruginosa confers resistance to imipenem and meropenem; in other species, loss of OmpF can lead to multidrug-resistant (MDR) organisms).24,25 This phenomenon results in an increase in minimum inhibitory concentrations to hydrophilic antimicrobials and reduces the choices of antibacterial therapeutics in clinical practice24,25,26.

Molecular Modification of Antibiotic Targets:               
            Most antibiotics affect the protein synthesis process targeting the ribosome,9 and differences between the structure of this account for the selective action of antibiotics in bacterial, archaeal, and eukaryotic cells. Even among species, slight variations in the ribosomal structure may lead to idiosyncratic, species-specific interactions among the drugs and their targets.27 Antibiotics that target the translational machinery of the bacterial cell are potent inhibitors of prokaryotic pathogens.28 Nevertheless, over decades of clinical use, these pathogens have become resistant to antibiotics that inhibit protein synthesis.27 A notable mechanism of resistance is the modification of the molecular target of antibiotics. Commonly, this can arise through point mutations in selected genes, resulting in relatively rapid and easy resistance with a minimal impact on microbe fitness. Relatively small changes in an amino acid sequence alter the protein structure sufficiently to impede antibiotic binding and action. For example, single mutations in target genes such as gyrA provide high-level resistance, while interactive mutations in the same gene can increase the level of resistance. Furthermore, target modification can arise from catalytic resistance strategies. An example is the ribosome methyltransferase, where Erm enzymes modify the 23S ribosomal RNA (rRNA) of the large subunit of the ribosome at position A2058 (in E. coli). This confers resistance to macrolides, lincosamides, and type B streptogramins.8,28 .

1.1. Aim of study                                                                               
     This study was aimed to isolate and identify bacterial pathogens from different infections and to demonstrate their susceptibility to ciprofloxacin and azithromycin antibiotics among them.

2.Materials and Methods:

2.1 Spacemen collection:
           This study was conducted in the University of Kufa, Faculty of science, department of laboratory investigation during the period from October 2017 to march 2018. Eighty-four specimens were collected from patients admitting to Alforat al-Awsat hospital in An-Najaf province using transport swab.

2.2. Isolation and Identification:
           All the collected swabs were cultured on plates of nutrient agar, blood agar, MacConkey agar, and salmonella-shigella agar (SS agar), All the inoculated plates were incubated at 37 c for 24- 48 hours.

2.3. Macroscopic and microscopic identification:
         The colony features were observed including color, texture, size, blood hemolysis and lactose fermenting. Gram stained slides were observed under the microscope to demonstrate Gram features and bacterial shape.
All gram-negative bacteria were identified by using IMViC tests. Staphylococci were identified by catalase, coagulase, novobiocin.

2.4. Biochemical testes:
There following biochemical testes were used to identify the bacterial isolates in this study:

2.4.1. Oxidase:
            Oxidase test was done by putting a small piece of the bacterial colony using sterile loop on an oxidase disk (MASTDISKS, UK) (Figure 3), the developing of blue color after 10 seconds considered a positive result. 29-32.

2.4.2. Catalase:
The catalase test was done by putting a bacterial suspension on clean slide and adding a few drops of 3-6% hydrogen peroxide, the developing of bubbles within 10 seconds considered a positive result 33.     

2.4.3. Mannitol Salt Agar:                                                                                      
           Mannitol Salt Agar is used for the selective isolation of pathogenic    Staphylococci. This medium is recommended for the detection and enumeration of coagulase-positive    Staphylococci in milk food and other specimens34-35.
The medium contains beef extract and protease peptone which makes it very nutritious as they provide essential growth factors and trace nutrients. Many other bacteria except    Staphylococci are inhibited by 7.5% sodium chloride. Mannitol is the fermentable carbohydrate source. The differential action of the medium is attributed to D-Mannitol.    Staphylococcus aureus ferments mannitol to produce yellow colonies with yellow cones. Most coagulase-negative species of    Staphylococci and    Micrococci do not ferment mannitol and therefore the medium remains red in color. The color of the medium is due to the reactivity of phenol red to the pH of the medium; phenol red is red at pH 8.4 and yellow at 6.8. Presumptive    Staphylococcus showing yellow colored medium should be further tested for production of coagulase36.           
Mannitol Salt Agar plates were inoculated with the bacterial specimens and incubated for 24-48 hours at 37°C , the yellow colonies were identified as coagulase-positive    Staphylococcus aureus.

2.4.4. Coagulase test:
Members of staphylococcus are differentiated by the ability to clot plasma by the action of the enzyme coagulase. The mechanism of coagulase action is not known33, was done by putting a few drops of plasma on clean slide and adding a 1-2 bacterial colony and emulsify it in the plasma, There will a visible clumping of cells within 10-15 seconds.

2.4.5. IMViC tests:
·       The indole test determines the ability of an organism to produce indole from the degradation of the amino acid tryptophan. The tryptophan is hydrolyzed by tryptophanase to produce three possible end products – one of which is indole33. A colored product is produced when the indole is complained with certain aldehydes37, this test was done by inoculate the tryptophan or (peptone water) with test organism and incubate at 37 c° for 24-48 hours, add 0.5 ml of the Kovac's reagent, and examine the upper layer of liquid ,the positive result = red color , negative result = yellow color.
·       Methyl red and Voges proskauer: The methyl red (MR) test is a quantitative test based on the use of the pH indicator, methyl red, to determine the amount of acid produced by an organism from glucose fermentation in MR-VP broth.  A positive test reaction is indicted by a red color reaction when the methyl red reagent is added38.
The VP test is credited to Voges and Proskauer since they were the first bacteriologists to observe a red color change on culture media after treatment with potassium hydroxide.  Barritt was the first individual to use both potassium hydroxide and α-naphthol for the VP test; an alternate, rapid VP test was reported by Barry and Feeney using an additional reagent, creatine. Tow tube of MR-VP broth was inoculated with bacterial colony, and incubated at 37° C for 4 days, 5 drops of the methyl red indicator solution was added to the first tube (for Voges-Proskauer test, Barrit’s reagent is added to another tube). A positive reaction is indicated, if the colour of the medium changes to red within a few minutes. Voges -Proskauer Test:  VP Positive (+):  Pink or red color at the surface of the medium. VP Negative (–): No change; yellow or copper color at the surface of the medium
·       Simmons Citrate Agar: This test is based on the work of Koser who first developed a liquid medium for differentiating coliforms from fecal coliforms based on the utilization of citrate as the sole source of carbon.  The Koser medium required additional testing, as the uninoculated medium appeared turbid.  Simmons improved upon the Koser medium be adding agar and the pH indicator bromothymol blue, direct inoculum streak the slant of the medium from the bottom up in a fish-tail motion by used sterile loop.  Ensure that the inoculum is not too heavy. the slant tube was incubated at 35°C , and examined after 24 and 48 hours41-43.

2.4.6. Novobiocin:
             The mechanisms of novobiocin-resistance include inhibition of cell wall synthesis as well as inhibition of protein and nucleic acid synthesis. Novobiocin resistance is intrinsic in S. saprophyticus. The Novobiocin Disk can be used to differentiate S. saprophyticus from other coagulase-negative staphylococci by the overnight disk test method or the 5-hour broth disk procedure44. The novobiocin test was done by inoculating Mueller Hinton Agar plates with a suspension prepared from a pure 18-24-hour culture, using sterile swabs to obtain confluent growth, one novobiocin disk was aseptically applied onto the inoculated agar surface and lightly pressed down to ensure full contact with the medium, and was incubated aerobically for 18-24 hours at 35°-37°C.It was measured (in millimeters) the diameter of the zone of inhibition around the novobiocin disk, and record as susceptible or resistant.
Sensitive - A zone of inhibition greater than 16mm.
Resistant - A zone of inhibition less than or equal to 16mm45.

2.5. Antibiotic susceptibility tests:
           Disk diffusion method was used in this study according to CLSI, Muller Hinton agar by spread method and antibiotic disk diffusion was placed on the plate with Azithromycin, Chloramphenicol, Ciprofloxacin, Doxycycline, Penicillin, Tobramycin, (table 1).


Table (1): antibiotic of inhibition zone standards according to CLSI( 2017)46,47


 3. Results:
3.1. Bacterial isolates:
  Out of eighty-four specimens collected during this study, 76 (90%) were positive for bacterial grow.
3.2 Identification results:
The results of macroscopic and microscopic study showed that from all the 76 bacteria isolate a 20 (27%) were identified as staphylococcus aureus, 11(14%) were identified as Staphylococcus saprophyticus, 13 (17%) were identified as coagulase- negative Staphylococci, 10 (13%) were identified as Proteus mirabilis, 12 (16%) were identified as Klebsiella spp., 10 (13%) were identified as Shigella spp. 
3.3. Biochemical tests results:
Catalase:
this test was used to differentiate between gram positive bacteria streptococcus and staphylococcus. A 44 (100%) of gram positive bacteria was catalase positive.
Coagulase test:                                                                                                            
was used to differentiate between staphylococcus spp. Bacteria. Gram positive Bacteria with coagulase positive was identified as staphylococcus aureus bacteria. A 20 (45%) of gram positive bacteria was identified coagulase positive.
Oxidase:
staphylococcus with coagulase positive were identified with oxidase test for differentiate between Micrococcus spp. and coagulase-negative Staphylococcus spp. A 24 (54% of gram positive bacteria) were negative oxidase test, and identified as coagulase-negative Staphylococcus spp.
IMViC tests results:
These tests were used to diagnose Gram negative bacteria, and include: Indole test, Methyl red test, Voges Proskauer test, and Citrate test. 
Indole test:
The indole test determines the ability of an organism to produce indole from the degradation of the amino acid tryptophan. This test was proceed on gram negative bacteria (A 32 isolates 42%), all isolates were have a negative indole test.
Methyl red test:
test is a quantitative test based on the use of the pH indicator, methyl red, to determine the amount of acid produced by an organism from glucose fermentation in MR-VP broth.
The results was : A 20 isolates (62% of gram negative bacteria) with a positive result, and A 12 isolates (38% of gram negative bacteria ) with a negative result.

Voges Proskauer test:
The results of this test were: 32 isolates (100% of gram negative bacteria) with a negative result.
Citrate test:
This test was used to diagnose bacteria that utilization of citrate as the sole source of carbon.
The results were: 10 isolates (32% of gram negative bacteria ) with Citrate negative result, A 22 isolates ( 68% of gram negative bacteria ) with citrate positive result. 
3.5. Novobiocin:
Novobiocin antibiotic was used in this study to differentiate between coagulase-negative Staphylococcus spp. the sensitive bacteria were identified as Staphylococcus saprophyticus. 13 (54%) of coagulase-negative Staphylococcus spp. were identified as Staphylococcus saprophyticus

3.6 Antibiotic susceptibility test:
The results of antibiotic sensitivity test study showed that from all the 76 bacteria isolates, 20 isolates (100%) staphylococcus aureus were resistance for azithromycin and Ciprofloxacin,13 of Staphylococcus saprophyticus isolates, showed no resistance to Azithromycin and (100 %) resistance to ciprofloxacin, 13 isolates of coagulase- negative Staphylococci were (100%) resistance to Azithromycin and Ciprofloxacin,12 isolates of Klebsiella SPP showed no resistance to Azithromycin and (100%) resistance to Ciprofloxacin,10 isolates of Proteus mirabilis showed (100%) resistance Azithromycin and Ciprofloxacin,10 isolates of Shegilla spp. showed (100%) resistance to Azithromycin and no resistance to Ciprofloxacin (Table 2).

Table 2: Bacterial isolates resistant to Ciprofloxacin and Azithromycin


Discussion:
Azithromycin resistance increased, the increase was greater in those on azithromycin. Regression analyses showed that long-term use of azithromycin was associated with lower recovery rates of S. aureus. Still, macrolide-resistant S. aureus were significantly more often isolated from patients using azithromycin. Three mechanisms may be responsible for the increase in macrolide resistance. First, isolates with an intrinsic resistance for macrolides may surface as susceptible ones are eradicated. Second, resistance may be acquired through one- or multi-step mutation. And third, resistant isolates may be acquired through cross-infection from other patients under antibiotic pressure. The increase in resistance among those not using azithromycin could be due to cross-infection, or due to increased prescriptions of macrolides by family practitioners of patients48.
High-level resistance to Ciprofloxacin in several bacterial isolates was recently reported. Development of ciprofloxacin-resistant occurred within 3 months of clinical use of ciprofloxacin49.
Conclusion:
From our  study we concluded that from all of  staphylococcus aureus isolates and coagulase- negative Staphylococci isolates were resistance for azithromycin and Ciprofloxacin, 13 of Staphylococcus saprophyticus isolates, were resistance to ciprofloxacin and sensitive  to Azithromycin (100 %), 13 were (100%) resistance to Azithromycin and Ciprofloxacin,12 isolates of Klebsiella SPP showed no resistance to Azithromycin and (100%) resistance to Ciprofloxacin,10 isolates of Proteus mirabilis showed (100%) resistance Azithromycin and Ciprofloxacin,10 isolates of Shegilla spp. showed (100%) resistance to Azithromycin and no resistance to Ciprofloxacin.

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