Molecular screening of antibiotic-resistant determinants among multidrug-resistant clinical isolates of Proteus mirabilis from SouthWest Nigeria.

Background: Globally, and particularly in developing countries, the menace of anti-microbial resistance is an accelerating prob-lem. In Nigeria, increase in bacterial resistance has been phenotypically established but due to high cost, few molecular studies have been reported. Objectives: This study screened for presence of transferable resistance genes and mobile genetic elements (MGEs) such as integron among multi-drug resistant (MDR) P. mirabilis . Methods: A total of 108 P. mirabilis strains collected from five tertiary hospitals in SouthWest Nigeria were subjected to antibi otic susceptibility study using disc-diffusion method. Transferable resistance genes and MGEs were amplified using Polymerase chain reaction (PCR) analysis and amplicons sequenced. Results: Varied resistance was observed against all the antibiotics tested. About 56% of the isolates were MDR including those from 0-12 years old children. PCR analysis revealed the presence of aac(6’)-Ib (33.3%), plasmid mediated quinolone resistance (PMQR) genes [qnrA (36.7%), acc(6’)-Ib-cr (5%)], TEM (48.3%), CTX-M (6.7%) and integrons class 1 (58.3%) and class 2 (26.7%). Sequencing analysis revealed bla TEM-1 , bla CTX-M-15 associated with IS Ecp1 and eight different arrays of gene cassettes: aadA1 , aadA1-qacH, aadB-aadA2, aadA5, dfrA7, dfrA15, dfrA17, dfrA17-aadA5 . Conclusion: Transferable resistance genes in association with MGEs are present in Nigerian P. mirabilis thus their potential in disseminating resistance.


Introduction
Anti-microbial drug resistance has become a global concern recognized by the World Health Organization 1 . Proteus species, common inhabitants of the soil and part of the normal flora of the enteric region of man and animals, have been found to cause opportunistic infections in several anatomical sites. 2,3 Among the various species of these genera, Proteus mirabilis is of medical importance and usually responsible for most of the common nosocomial opportunistic infections such as urinary tract infections, wounds, ear and other infections. 3 Their possession of a number of virulence factors including antibiotic resistance genes, have placed the organism on the list of medically important nosocomial agents. 4,5,6,7,8 The micro-organism has been implicated in several nosocomial infection outbreaks and community acquired infections in different parts of the world, including Nigeria. 9,10,11,12 Among other antibiotics used in the treatment of nosocomial infections in Nigeria, the commonest classes of antibiotics usually employed for life threatening cases are the third generation cephalosporins, fluoroquinolones and aminoglycosides. However, several authors have reported resistance of Enterobacteriaceae including Proteus mirabilis to these classes of antibiotics. 13,14,15,16 Resistance to third generation cephalosporins has been attributed to the presence of certain resistance determinants called extended-spectrum beta-lactamase enzymes, ESBLs. 17 Several variants of these enzymes which were believed to have evolved from the wild-type beta-lactamases (TEM-1 and SHV-1) via mutation at one or more point in the gene coding for their amino acid sequence exist and are spread among bacterial isolates worldwide. 17 Another widely spread ESBL group are the CTX-M-type discovered in the 1980s in Munich, Germany from clinical isolate of Escherichia coli and currently five phylogroups exist. 18 They are known to have high hydrolytic property against cefotaxime. 18 Resistance to fluoroquinolones was initially thought to be majorly due to chromosomal mutation involving gyrA and topoisomerase genes until plasmid mediated quinolone resistance genes (PMQR) such as the qnr, qepA and aac(6')-Ib-cr was later reported in 1998. 19 Since then PMQR genes have been described in several bacterial isolates particularly members of the Enterobacteriaceae family worldwide. 13,15,20,21,22 The qnr genes are known to produce proteins (QNR proteins) that protect the quinolone targets from inhibition. Studies have shown that they produce a low level resistance, but facilitate a higher level resistance in association with chromosomal mutations in bacteria harbouring them. 23,24,25 The aac(6')-Ib-cr, which encode a variant of aminoglycoside transferase that confers reduced susceptibility to ciprofloxacin and norfloxacin by N-acetylation of their piperazinyl amine, causes an increase in their MIC two to four fold 26 and qepA gene, which encodes quinolone-specific efflux pump 27 initiate decreased susceptibility to quinolones by up to 64-fold. 28 Resistance to aminoglycosides such as gentamicin, amikacin and tobramycin, have been attributed to the aminoglycoside-modifying enzymes. Among the different classes of these aminoglycoside-modifying enzyme that have been reported, the one encoded by the aac(6')-Ib gene is the most commonly isolated in many clinical isolates particularly among the Enterobacteriaceae worldwide. 13,15 Common resistance determinant genes that have been detected among isolates belonging to the Enterobacteriaceae family in Nigeria are ESBLs (TEM, CTX-M, SHV and OXA), AmpC Beta-Lactamase (CMY, DHA and ACT), plasmid-mediated quinolone resistance (qnrA, qnrB, qnrD, aac(6')-Ib-cr and qepA) and aminoglycoside resistance (aac(6')-Ib) genes. 15,29 Most of these resistant genes are acquired from other bacterial isolates through mobile genetic elements such as integrons.
Integrons are composed of three key elements: a gene encoding an integrase, a primary recombination site, and a promoter that directs the transcription of captured genes. 30 Integrase is involved in capturing of resistance genes and incorporating them by site-specific recombination within the integrons conserved segments. 30 The aim of this study was to screen for the presence of common transferable resistance genes and mobile genetic elements such as integron and their associated gene cassettes among MDR clinical isolates of P. mirabilis from SouthWest Nigeria.

Clinical bacterial isolates
A total of 108 non-duplicated clinical isolates of P. mirabilis were randomly collected during a 12 months period (January to December, 2011) from Microbiology units of five selected tertiary hospitals in SouthWest Nigeria, namely University Teaching Hospital (UCH) Ibadan (n = 43), Olabisi Onabanjo University Teaching Hospital (OOUTH) Sagamu (n = 10), Federal Medical Centre (FMC) Abeokuta (n = 13), Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife (n = 28) and Lagos state University Teaching Hospital (LUTH) Lagos (n = 14). The isolates were obtained from different clinical samples (wound, ear, eye, vaginal swabs, urine, sputum, peritoneal effluents, finger abscesses, pus and stool). They were identified by using Gram stain, MacConkey and Chocolate agar, swarming activity and confirmed with Microbact 12E Identification kit (Oxoid Ltd. Basingstoke, Hants, UK). Information on the patient's data such as age, sex and specimen types was also recorded.

Amplification of common resistance genes
Clinical isolates that showed resistance to at least three classes of antibiotics were regarded as MDR isolates 32 and were selected for amplification of resistance genes using polymerase chain reaction (PCR) technique.

Beta-Lactamase gene detection
MDR strains that showed resistance to third generation cephalosporins (cefotaxime and ceftazidime), aztreonam but were susceptible to cefoxitin were screened by simplex PCR for bla TEM , bla SHV and bla CTX-M gene. 33,34,35 The CTX-M-positive strains were screened by simplex PCR for commonly associated insertion sequences (ISEcp1, IS26 and IS903) using them and bla CTX-M primer as forward and reverse respectively. 36 Those MDR isolates that showed resistance to the third generation cephalosporins, aztreonam and cefoxitin but were susceptible to imipenem were screened for common variants of AmpC (bla CMY , bla DHA-1 bla FOX ) and bla OXA that are usually found in Enterobacteriaceae using a multiplex PCR. 37 Detection of genes encoding plasmid mediated quinolone resistance (PMQR) and aminoglycoside-modifying enzyme (AAC). MDR isolates with reduced susceptibility or resistance to quinolones and aminoglycosides were screened for the presence of qnrA, qnrB, qnrS, qepA and aac(6')-Ib genes using multiplex PCR. 21 The aac(6')-Ib amplicons were further digested with BstF5I (New England Biolabs, Ipswich, MA) and electrophoresed on 1% agarose gel to identify the aac(6')-Ib-cr which lacks the BstF5I restriction site present in the wild-type aac(6')-Ib gene. 38

Detection of classes 1, 2 and 3 Integrons and associated gene cassettes
All the MDR isolates were screened for the presence of classes 1, 2 and 3 integron-associated integrase genes using specific primers. 39 The variable region of the integrase positive strains were then amplified by PCR using specific primers to know if they carry gene cassettes. 39 Purification and sequencing of amplified PCR products The resulting PCR amplification products of the detected beta-lactamase genes and the amplified variable region of the integrons were purified using GF-1 PCR cleanup kit according to the manufacturer's guidelines (Qiagen, Izasa, Portugal) and then sequenced in both forward and reverse nucleotide chains (Stabvida, Portugal) to identity the genes.

Conjugation assay for ESBL positive strains
The conjugation assay was performed using a modified method described by Kim et al. (2004). 12 The ESBL positive donor strains were conjugated with the standard strain E. coli J53 (resistant to sodium azide) as a recipient cell. MacConkey agar containing 4 µg/mL of cefotaxime and 200 µg/mL of sodium azide was used as the selection medium. The growth of the sodium azide resistant E. coli J53 in the selection plate was interpreted as an indication of an ESBL embedded plasmid transfer. The transconjugants were confirmed by determining the antimicrobial susceptibility profile and PCR amplification.

Mutagenic treatment of the isolates harbouring ESBL genes
Isolates harbouring ESBL genes were subjected to R-plasmid curing experiment using the modified method described by Adeleke et al. (2002). 40 Briefly, the overnight culture of each ESBL-positive P. mirabilis strains was exposed to 200 and 100 μg/ml of ethidium bromide and incubated for 24 hours at 37 0 C. Pure colonies of the ethidium bromide treated and untreated isolates were first selected on MacConkey agar plates and then on Nutrient agar, after which they were then diluted to 0.5 MacFarland standard for susceptibility testing against selected antibiotics (cefotaxime, ceftazidime, aztreonam, ciprofloxacin, gentamicin and trimethoprim-sulphamethoxazole) by disc-diffusion method, and zones of inhibition interpreted by comparing with the CLSI guideline. 31
Sixty (55.6%) of the isolates were classified as MDR since they were resistant to at least three classes of antibiotics. Twelve (20%) of these MDR isolates were from children within 0 -12 years of age suffering from various infections such as otitis media (50%), conjunctivitis (8.3%), burn wounds (25%), abscess (8.3%) and UTI (8.3%). The MICs of the selected antibiotics against some of the MDR isolates are presented in Table 1

Antimicrobial resistance genes
Amplification and sequencing of the beta-lactamase genes from the MDR strains resistant to cefotaxime or/and ceftazidime revealed that 29 (48.3%) harbored bla TEM-1 and 4 (6.7%) harboured bla CTX-M-15 . The bla SHV, bla C-MY , bla OXA , bla DHA-1 and bla FOX genes were not found. Two of the bla CTX-M-15 genes were associated with ISEcp1. One of the bla CTX-M-15 genes was detected from an MDR isolate collected from a six years old male child with an infected burn wound (Table 1). Twenty (33.3%) of the MDR isolates carried the aac(6')-Ib gene, including two isolated from a 2½ and 10 year old female and male patients, respectively, both suffering from ear infections ( Table 1). The aac(6')-Ib-cr and qnrA genes, associated with quinolone resistance, were present in 3 (5%) and 22 (36.7%) of the MDR isolates, respectively, while none was positive for qepA. All (100%) MDR isolates harboured integrase genes, either IntI1, IntI2 or both, among which 96.7% harboured IntI1, 53.3% IntI2, 51.7% carried both IntI1 and IntI2 while none harboured IntI3. Amplification of the inte-grons variable region produced positive amplicons in 76.7%, which included 58.3% and 26.7% of the IntI1 and IntI2 positive strains respectively. Nine of the children within the ages 0 and 12 years were infected with integron-positive MDR strains. Sequencing analysis revealed eight different gene cassette arrays (aadA1, aadA1-qacH, aadB-aadA2, aadA5, dfrA7, dfrA15, dfrA17 and dfrA17-aa-dA5) spread among the integron positive MDR isolates. The gene cassette array dfrA17-aadA5 (47.1%) was the most common among the MDR isolates.

Conjugation and curing assays
The conjugation experiment performed with four CTX-M-15 strains did not yield any noticeable growth of transconjugants in the selection plates. Moreover, the curing experiment did not alter the susceptibility profile of the CTX-M-15 positive isolates to the cephalosporins and aztreonam significantly but rather increased the susceptibility of the isolates to the effect of the sulphamethoxazole-trimethoprim, gentamicin and to ciprofloxacin, as shown in Table 2. All the primers used in the amplification of the genes detected are presented in table 3.   GAT GCC ATC GCA AGT ACG AG Discussion P. mirabilis is becoming a common opportunistic pathogen particularly within the Nigerian hospital setting. 41 P. mirabilis is often associated with urinary tract infections especially among patients with long-term indwelling catheters or under frequent antibiotic therapy. 42,43 However, in this study, high occurrence in wound infections (43.5%) was found compared to urinary tract infections (21.3%) but a similar percentage occurred in ear infections (22%).
Other studies performed in Nigeria, though with a lower number of isolates, also reported a higher isolation of P. mirabilis in wounds than in urine. 3,15 The reason for the observed increased cases of wounds infected with P. mirabilis in previous and current studies cannot be explained but calls for serious caution in infection control program in these hospitals. Though the majority of the patients (62%) were between the age of 18 and 59 years, about 30% of the isolates were recovered from infected children less than 17 years (including newborn and children that were a few months old). This should be a worrisome development to healthcare professionals, highlighting that control of nosocomial infection must be drastically improved in Nigerian tertiary hospitals.
The high frequency of MDR phenotype observed among the P. mirabilis isolates in this study (55.6%) confirms earlier reports that MDR is increasing in Nigeria among members of the Enterobacteriaceae family. 15 It is a concern to see that 20% of the MDR isolates detected in this study are from children between the ages 0 and 12 years. This may not be surprising since various reports mention that self-medication of children is rampant among nursing mothers, who usually take antibiotics irrationally even during breastfeeding. 44,45,46,47 A low prevalence of bla CTX-M-15 was found, (6.7%) contrary to the 27.3% prevalence of bla SHV and bla CTX-M-15 reported by Ogbolu et al. 15 among 11 clinical P. mirabilis isolates from SouthWest Nigeria. Although bla CTX-M-15 has been previously described in clinical P. mirabilis isolates in Southwest Nigeria,1 5 this study reported ISEcp1 associated bla CTX-M-15 usually involved in the transposition of the gene and its dissemination. 48 The association of bla CTX-M-15 with mobile genetic elements such as insertion sequences and conjugative plasmids usually explain its worldwide dissemination. However, the conjugation assays performed in this study with all the CTX-M-15 positive strains did not produce any transconjugants suggest-ing that it is not likely to be associated with conjugative plasmid. Furthermore, the R-plasmid curing experiments with ethidium bromide against these strains retained the resistance profile conferred by the bla CTX-M-15 , this still suggest a non-plasmid based gene. Although bla CTX-M-15 was already found within a chromosomal location associated with ISEcp1 in P. mirabilis strains isolated in Tunisia 49 and Korea, 50 the two phenotypic experiment done in this study are inadequate to conclude a chromosomally based bla CTX-M-15 . A molecular analysis, which was not the objective of this study, is required to finally confirm the actual genetic location of the bla CTX-M-15 detected in this study.
Plasmid mediated quinolone resistance (PMQR) genes are known to confer resistance to nalidixic acid and to increase the minimum inhibitory concentrations of fluoroquinolones up to 32-fold or more. 26 In this study, the detection of qnrA and aac(6')-Ib-cr associated with the reduced susceptibility to fluoroquinolones showed that these genes are spreading among clinical isolates in Nigeria. Previously, these genes have been reported in some Enterobacteriaceae in Nigeria, together with the qnrD gene variant which was not detected in this study. 15 The aac(6')-Ib gene is the most prevalent aminoglycoside modifying enzyme, conferring resistance to tobramycin, kanamycin and amikacin. 51 The gene was first identified in Klebsiella pneumoniae isolates in 1986 and since then several variants of this enzyme have been described. 26,51 However, in this study, 3 (5%) of the MDR isolates harboured aac(6')-Ib-cr, a variant of the aac(6')-Ib gene.
Of relevance is the high prevalence of MDR P. mirabilis strains with class 1 and 2 integrase genes, Intl1 and Intl2, and the occurrence of eight different gene cassette arrays (aadA1, aadA1-qacH, aadB-aadA2, aadA5, dfrA7, dfrA15, dfrA17 and dfrA17-aadA5) within the variable region of the integrons in some of the isolates. The size of the amplicons (variable region) ranged from 300bp to 600bp depending on the type and number of gene cassettes present. This result shows the potential of these opportunistic micro-organisms to acquire resistance genes, which is a threat for under-resourced countries, where more potent antibiotics against MDR strains are usually expensive or inexistent.
Overall, accumulation of resistant determinants through the activities of mobile genetic elements is associated with the observed multidrug phenotype of P. mirabilis clinical isolates from SouthWest Nigeria. Our findings showed that these strains have the tools to acquire resistance genes as much as possible. Therefore, strict surveillance of anti-microbial resistance, rational prescription of antibiotics and strict laws to be enforced against overthe-counter sales of antibiotics must be implemented in Nigeria, to reduce to a minimum antibiotic resistance cases in the country. The frequent isolation of P. mirabilis in infected wounds in tertiary hospitals in Nigeria suggests the urgent need to review the existing infection control measures in hospital settings and to educate the clinical staff on this scenario.