Quinolones are synthetic, broad-spectrum antibacterial drugs with bactericidal activities against a wide range of bacteria. The mechanism of action is through the arrest of deoxyribonucleic acid (DNA) replication fork progression.¹ Quinolones are often the first line in the treatment of several forms of bacterial infections especially in developing countries, and they are also extensively used in livestock and agriculture.^1,2

Quinolones are synthetic antibiotics, thus it was thought that mutations in target genes would be the only mechanisms for acquired resistance.² Truly, the common mechanisms of resistance to quinolones reported after the discovery of nalidixic acid were chromosomal, involving alterations in target enzymes.² However, the first plasmid-mediated resistance gene in quinolones, qnrA1,was reported in 1998^3,4 and since then, more plasmid-mediated quinolone resistance (PMQR) determinants have been identified: qnrA, qnrS, qnrB, qnrC, qnrD, qnrE, and qnrVC.^2-5 Also, some other elements like efflux pumps genes: oqxAB and qepA, with a quinolone modifying enzyme gene aac(6’)-Ib-cr have also been shown to contribute to quinolone resistance.^2-8 The PMQR genes confers low-level resistance to fluoroquinolones, they enable the survival of pathogens under quinolone exposure and facilitate selection of chromosomal mutations.^7,8

Studies done earlier in Nigeria reported uncommon resistance to the quinolones among clinical isolates because the drugs were not widely used before the expiration of patents for these drugs in the early 2000s.⁹ More recently, there are reports of widespread resistance to quinolones in Nigeria as well as neighboring countries in West Africa.^9-14 A systematic review of published articles on fluoroquinolone-resistant enteric bacteria in sub-Saharan Africa not only found a high prevalence of PMQR genes but also an up-regulation in efflux pumps and the drug-modifying mechanism across several species of bacteria¹⁴ even though the qnr-mediated target protection mechanism was the first to be discovered. As part of a continuous antimicrobial resistance mechanisms surveillance in the global efforts to contain the emergence and dissemination of antimicrobial resistance, we determined the distribution of PMQR genes in gram-negative bacilli from a tertiary healthcare institution in South-West Nigeria.

Study site

This study was conducted at the Department of Medical Microbiology and Parasitology of Obafemi Awolowo Teaching Hospitals Complex (OAUTHC) and the Central Science Laboratory, Obafemi Awolowo University (OAU), Ile-Ife, Osun State. Approval was obtained from the Research and Ethics Committee of the OAUTHC, Ile-Ife for the study (protocol number ERC/2016/01/09). OAUTHC is a first-generation teaching hospital with about 600-bed complement and a large capacity for outpatients, averaging about 220,000 annually.

This was a cross-sectional study with a sample size of 390 clinical isolates. All gram-negative bacilli isolated from clinical infections at different body sites such as blood, urine, wound and aspirates during the period of study were included in the study while all isolates suspected to be contaminants and isolates from patients without any focus of infection were excluded from the study.

Processing of specimens

Blood collected in BACTEC (Becton Dickinson, Belgium) culture bottles were incubated between 24 hours to 5 days in a semi-automated BACTEC 9050 blood culture machine (Becton Dickinson). The blood culture bottles which signaled to be positive were sub-cultured on 5% sheep blood agar, chocolate agar and MacConkey agar plates. Cerebrospinal fluid (CSF) samples were examined macroscopically and microscopically, gram staining was done on the specimen and then plated immediately on blood agar, MacConkey and chocolate agar. Semi-quantitative urine culture was done with a calibrated loop, a loopful (0.001 mL) of well-mixed un-centrifuged urine inoculated onto the surface of cysteine-lactose-electrolyte-deficient medium (CLED). All other samples including wound biopsy and swabs (ear, eye and wound swabs), sputum, tracheal aspirates, and stool collected in sterile screw-top container were processed according to standard laboratory practices. Incubation for MacConkey plates, CLED, deoxycholate citrate agar (DCA), Selenite-F or thiosulfate-citrate-bile-salts-sucrose agar (TCBS) were under atmospheric aerobic condition while blood and chocolate agar were under 5% CO₂ enriched atmosphere at 37°C for 18-24 hours.

Identification of bacterial isolates

Identification of the isolates was initially done by colonial morphology on the agar plate, gram staining, and standard biochemical tests. Microbact^{TM 2}GNB 24E and GNB 12E were also used in the identification of the isolates. Interpretation of the result was by use of Microbact^TM (Oxoid, England) identification software package, version 2009. All tests were done according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI).¹⁵

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was done by the modified Kirby-Bauer disc diffusion method.¹⁶ All isolated gram-negative bacilli were tested against nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), ofloxacin (5 μg), ampicillin (10 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), co-amoxiclav (20/10 μg), gentamicin (10 μg), imipenem (10 μg), and nitrofurantoin (300 μg) which was used for urinary isolates only. The results were interpreted according to CLSI guidelines and WHONET software version 5.6.

Determination of presence of PMQR

Plasmid DNA was extracted from pure colonies of isolates that were resistant to quinolones using the boiling method. The DNA suspension from the supernatant was used as a template DNA in the polymerase chain reaction (PCR) based detection. All the primers used were commercially synthesized by Inqaba biotechnical Industries (Pty) Ltd, South Africa.

The multiplex PCR protocol developed for PMQR by Cesiekzuk et. al.¹⁷ was used in this study. All isolates were screened twice for the PMQR genes in two sets using specific primers for qnrA, qnrB, qnrS, qnrC, qnrD and also for aac(6’)-Ib-cr, qepA, and oqxAB. The amplification conditions for the first set made up of qnrA, qnrC, qnrB, qepA, were as follows: an initial denaturation at 94°C for 4 mins; 30 cycles of 94°C for 30 s, optimized annealing temperature 55°C for 30 s and 72°C for 1 min; followed by a final extension at 72°C for 5 min.

The second set included qnrD, qnrS, aac(6’)-lb-cr, oqxAB, and the amplification condition was initial denaturation at 94°C for 4 mins, followed by 30 cycles of denaturation at 94°C for 45s, annealing at 55°C for 45 s, extension at 72°C for 1 min then a final extension at 72°C for 3 min.

Each amplicon (10 μL) was electrophoresed on a 1.5% agarose gel pre-stained with 0.5 μg/mL ethidium bromide in 1X tris-borate-EDTA (TBE) buffer and viewed with a UVitec transilluminator (Avebury, Cambridge UK). The positions of the amplified product were estimated by the position of 100 base pair molecular weight marker (Bio Lab).

Sequencing

Amplimers resulting from PCR reactions for two isolates found to have all the targets were sequenced at Inqaba biotechnical Industries (Pty) Ltd. South Africa, to confirm their identities. The resulting forward and reverse sequences were aligned with the Blast multiple alignment tool at http://www.ncbi.nlm.nih.gov. Sequences were accepted when they corresponded with known PMQR sequences in the Blast database. A repeat PCR was then done and isolates with the sequenced genes were used as controls.

Data analysis and statistical techniques

The data was analysed using the IBM Statistical Package for Social Sciences (SPSS) version 20 (IBM Corp., USA) and WHONET software version 5.6. Categorical variables were summarized and presented in frequency tables with simple proportions and charts as appropriate. Inferential bivariate analyses were performed using Chi-square and Fisher’s exact test. The level of statistical significance was determined at p-values less than 0.05.

A total of 390 gram-negative bacilli isolates were isolated from clinical specimens of 382 patients between December 2016 and August 2017; 256 patients were inpatients while 126 others were outpatients. There were 211 males and 171 females, with a wide age distribution between 1 and 90 years. The average age distribution was between 31-40 years. More than half of the isolates (n=201; 51.5%) were from patients with urinary tract infections, 131 (34.3%) isolates were from wound infections, and 47 (12.1%) isolates were obtained from the blood of patients with sepsis while 8 (2.1%) isolates were from patients with other infections (Table 1).

 Table 1. Distribution of the isolates from clinical infections

 

 

The isolates were commonly resistant to nalidixic acid (n=244; 62.6%), norfloxacin (n=204; 52.3%), ofloxacin (n=203; 52.1%), ciprofloxacin (n=199; 51.0%), ampicillin (n=137; 35.1%), cefotaxime (n=181; 46.4%) and co-amoxiclav (n=168; 50.8%) but were least resistant to imipenem (n=36; 9.2%) – Table 2.

 

Table 2. Antimicrobial resistance pattern in the isolates

 

 

A total of 244 isolates were resistant to at least one quinolone, 180 (73.8%) of these harbored at least one PMQR gene: most were efflux pump determinants qepA (22.5%) and oqxAB (21.1%), and others were aac(6’)-Ib-cr (19.7%), qnrB (13.2%), qnrS (8.7%), qnrA (5.9%), qnrD (4.5%), and qnrC (4.2%) – Table 3. The PMQR genes were distributed across isolates occurring either singly or in combinations in all quinolone-resistant isolates.

Our study showed E. coli, Klebsiella spp. and P. aeruginosa as the most predominant causes of gram-negative infections in our environment. Some of the infections could be healthcare-associated as about two-thirds (n=256; 67%) of the patients were on hospital admission. A high level of resistance to common antibiotics in routine use was observed among the isolates and most importantly, there was widespread quinolone resistance with at least one in every two gram-negative bacilli isolated being quinolone-resistant. Resistance to the first-generation quinolone nalidixic acid was found to be higher than to the fluoroquinolones.

Quinolone-resistant isolates were also found to be resistant to antibiotics in other classes used in this study: aminoglycoside, penicillins and cephalosporins except for imipenem, a carbapenem. This is in agreement with studies that showed fluoroquinolone-resistant strains are usually resistant to multiple antimicrobials, as quinolone-resistance specific mechanisms evolved on a background of existing mechanisms conferring resistance to multiple classes of antibiotics.²

Quinolone resistance was found more commonly in E. coli, Klebsiella spp. and P. aeruginosa isolates, while only an isolate of Salmonella spp. was resistant to the four quinolones used in this study; this is in keeping with studies done on resistance to quinolones in Salmonella spp. in Africa, which found ciprofloxacin-resistant salmonellae to be uncommon in humans.¹⁴

There was a wide distribution of PMQR genes among the quinolone-resistant isolates, which is likely contributing to the high level of quinolone resistance found among these isolates. Up to 180 (73.8%) of these isolates harbored one PMQR gene or more of these genes. Of all the E. coli isolates which accounted for 31% of the total isolates, 84 were resistant to quinolones and harbor one or more PMQR genes, more prominently aac(6’)-lb-cr, which was found alone or in addition to other PMQR genes in 33 of the E. coli isolates representing 39.3% of the total E. coli found to be resistant to quinolones. Almost half (34, 47.2%) of Klebsiella spp. isolated also had qepA gene alone or in addition to other PMQR especially oqxAB, which was found to be more prevalent in Klebsiella spp., this was not surprising as oqxAB genes are naturally present on the chromosome of K. pneumoniae with different levels of expression.⁶

Aac(6’)-lb-cr was also predominant in P. aeruginosa, found in 12% of the whole P. aeruginosa isolates. The only spp. of Salmonella found to be resistant to quinolone had the oqxAB gene. The most prevalent PMQR gene overall was qepA found in 32.8% of the whole quinolone-resistant isolates. Although target protection mechanism (qnr proteins) was the first to be discovered, this study found more recent determinants appearing to be more successful with efflux mechanisms (qepA and oqxAB) and drug-modifying (acetylating; aac(6’)-lb-cr) mechanism being the most common determinants of resistance than the qnr proteins. This is similar to findings from studies done across regions in sub-Sahara Africa which also found a high prevalence of PMQR determinants among quinolone-resistant organisms with efflux pumps and drug-modifying mechanisms being more common than qnr-mediated target protection mechanism.¹⁴

This study showed there is a high level of quinolone resistance, with at least one in every two gram-negative bacilli isolated being quinolone-resistant and up to 73.8% of these quinolone-resistant isolates harboring one or more PMQR genes. The wide distribution of these genes is ultimately contributing to the more established and successful chromosomal mutations to enhance more rapid widespread resistance to quinolones, as well as resistance to other broad-spectrum antibiotics.

We recommend a review of the use of quinolones as first-line drugs in the management of patients, a continuous antimicrobial resistance surveillance and antimicrobial stewardship to guide appropriate use of antibiotics.

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