Following emergence of resistance to inexpensive broad-spectrum antimicrobials across much of Africa, quinolone antibacterials have recently been introduced and are widely used. West African studies that sought quinolone resistance in commensal or diarrhoeagenic Escherichia coli before 2004 reported no or very low incidences of resistance to nalidixic acid and the fluoroquinolones 1 2 3 4 . Thus, available data suggests that resistance to the quinolones was rare in West Africa until the first decade of the 21^st century. More recent anecdotal reports and surveillance studies point to emergence of quinolone resistance among enteric pathogens and faecal enteric bacteria in Ghana and elsewhere in West Africa 5 6 7 8 . In a study by Nys et al. (2004) faecal isolates of adult volunteers in eight different countries were assessed for susceptibility to antimicrobials in the same laboratory 8 . Resistance to broad spectrum first-generation antibiotics was common and ciprofloxacin resistance was found to be slowly emerging in Asian, South American and African countries, including Ghana 8 . Newman et al. (2004) collected 5099 clinical bacterial isolates (1105 of which were E. coli) from nine of the ten regions in Ghana and tested them for antimicrobial susceptibility. They found that over 70% of the isolates were resistant to tetracycline, trimethoprim-sulphamethoxazole, ampicillin and chloramphenicol and reported that 11% of the isolates were ciprofloxacin-resistant 7 .

Quinolones inhibit the activity of bacterial DNA gyrase and DNA topoisomerase enzymes, which are essential for replication. Single nucleotide polymorphisms (SNPs) in the quinolone resistance determining regions (QRDR) of gyrA and parC, the two genes that encode DNA gyrase and topoisomerase IV respectively, can lead to conformational changes in these enzymes that cause them to block quinolones from binding to the DNA- substrate complex, yet still preserve their enzymatic function 9 . In Escherichia coli and related Gram-negative bacteria, DNA gyrase is the first target for fluoroquinolones. If gyrA has resistance-conferring mutations, the primary target of fluoroquinolone switches from DNA gyrase to topoisomerase IV 10 11 . Studies from other parts of the world have found that resistance-conferring mutations are typically selected in gyrA first, and then parC.

Although mutations in the QRDR of gyrA and parC are the most commonly documented resistance mechanisms, resistance has also been known to be conferred by mutations in the second topoisomerase gene, parE. Another mechanism of quinolone resistance relies on upregulation of efflux pumps, which export quinolones and other antimicrobials out of the bacterial cell. For example, mutations in the gene encoding a repressor of the acrAB pump genes, acrR, are associated with quinolone resistance 12 . Quinolone resistance can also be acquired horizontally through transferable quinolone resistance (qnr) or other DNA. The Qnr gene product inhibits quinolones binding to target proteins 13 . Other horizontally acquired quinolone resistance genes include aac(6')-Ib , encoding a fluroquinolone acetylating enzyme, as well as qepA and oqxAB, which encode horizontally transmitted efflux pumps 14 15 16 .

Resistance to the quinolones often emerges at low-levels by acquisition of an initial resistance-conferring mutation or gene. Acquisition of subsequent mutations leads to higher levels of resistance to the first-generation quinolone, nalidixic acid and a broadening of the resistance spectrum to include second-generation quinolones (first-generation fluoroquinolones) such as ciprofloxacin, followed by newer second- and third-generation fluoroquinolones 17 .

Although multiple mechanisms of quinolone resistance have been reported from other continents, there are few data from sub-Saharan Africa on the molecular basis for quinolone resistance. We performed antimicrobial susceptibility testing on fecal E. coli isolates from Accra, Ghana in 2006, 2007 and 2008. We identified isolates that were resistant to nalidixic acid and screened these strains for mutations in the QRDR of gyrA and parC as well as horizontally-acquired quinolone-resistance genes. In order to gain some insight into resistance dissemination, we also studied inter-strain relatedness among quinolone-resistant E. coli isolates by multilocus sequence typing.

Evolution of reduced susceptibility to the quinolones is causing concern following rapidly rising rates of fluoroquinolone-resistant E. coli in many parts of the world 20 . In African countries with a high infectious disease burden, formal and informal health systems depend heavily on broad spectrum orally-administrable antibacterials. In this study, we found that most commensal E. coli isolates are resistant to ampicillin, sulphonamides, tetracycline and trimethoprim, as well as streptomycin, which have been used to treat actual and supposed bacterial infections in Ghana for over four decades, and that resistance to these agents is increasing with time. We also found that about a third of isolates were resistant to chloramphenicol. Fluoroquinolone antimicrobials have been recently introduced as an effective alternative to older antibacterials that have been compromised by resistance. However, although resistance rates were markedly lower for this class of drugs, we also found that quinolone resistance was increasingly common among fecal E. coli in this study.

We determined that 12-18% of fecal E. coli isolated from healthy individuals in Accra in 2006, 2007 and 2008 are quinolone resistant. Twenty-three of the 40 QREC isolated were resistant to the fluoroquinolone ciprofloxacin. Ciprofloxacin-resistant QREC, showing high-level nalidixic acid resistance, were more commonly isolated in 2008 than in 2006 and 2007. Strains with one or no mutations in gyrA were typically ciprofloxacin sensitive. However most isolates had accumulated a second gyrA mutation and/or mutations in parC and were fluoroquinolone resistant. The QRDR polymorphisms most commonly detected in this study are those most frequently reported in the literature 10 . As has been validated experimentally in isogenic strains, high-level nalidixic acid resistance and fluoroquinolone resistance in isolates in this study was associated with parC substitutions in strains also harbouring substitutions in gyrA 17 . However, gyrA and parC mutations did not absolutely correlate with nalidixic acid MICs, partly due to horizontally-acquired quinolone-resistance genes. We sought qnrA, qnrB, qnrS and qepA genes by PCR and confirmed all amplicons by sequencing. We found that two isolates without mutations in the QRDRs of gyrA and parC, as well as ten isolates with QRDR mutations carried a qnrS1, a qnrB or a qepA allele. The presence of horizontally-acquired genes accounted in part for elevated nalidixic acid MICs in strains that harboured these genes, but not completely. It is therefore possible that other resistance mechanisms, such as ParE polymorphisms, other horizontally acquired resistance genes (such as oqxAB and aac(6')-Ib for example), over-active efflux, or even novel mechanisms are present in some of the isolates.

Resistance patterns in pathogens often mirror those in commensals. This is borne out by our recent documentation of quinolone resistance in Vibrio cholerae isolates recovered in the same time frame as the E. coli strains presented in this report 21 . Fifteen of the 40 QREC isolates identified in this study belonged to ST10, or were single- or double-locus variants of this ST, pointing to the possibility of clonal expansion. ST10-complex strains were isolated in all three years and therefore over-representation of these STs in our sample cannot be explained by short-term, localized clustering. There are four major E. coli phylogenetic clades: ECOR A, B1, B2 and D. Few studies have looked at the geographical variance in the distribution of these groups but overall, QREC from Ghana were predominantly drawn from ECOR group A. Of the STs identified in this study that are classified into ECOR clades at the E. coli MLST database, ST10 complex (14 isolates) belong to ECOR group A, ST131 (1 isolate) to ECOR B2, STs101 and 410 (3 isolates) to ECOR B1 and STs 156, 206 and 210 (4 isolates) are hybrids of ECOR A and B1, that is AxB1. Available data appear to suggest that ECOR A strains are highly prevalent in Africa, compared to some other world regions 22 . However, when we compared the sequence types of quinolone-resistant and -susceptible strains from Ghana only, we still found that resistant strains were over-represented in the ST10 complex. Pandemic clonal expansion of some QREC lineages has been reported in the literature 23 24 25 26 27 28 . For example, ST131 is a globally disseminated multi-resistant clone and was detected once among the QREC in this study. Recent reports suggest that isolates from Europe and North America that belong to ST10- or ST131- clonal complexes may be less likely to carry virulence factors for invasive disease, but more likely to be fluoroquinolone resistant 24 25 26 27 28 . However it is equally likely that mutations to fluoroquinolone resistance are more likely to be stably inherited in a specific genetic background. Our own data also appear to suggest that, although horizontally acquired, qnrS1 is associated with ST10 complex.

A recent paper by Davidson et al suggests that the antimalarial chloroquine may select for fluoroquinolone-resistant fecal bacteria in malaria endemic areas and proposes that chloroquine-mediated selection accounts for high levels of QREC in fecal flora in villages in South America 29 . However, data from within Africa (where chloroquine has seen heaviest use) to support this hypothesis are currently lacking and evidence from elsewhere supports a link between quinolone resistance rates in E. coli and fluoroquinolone consumption at population levels 30 31 . Our own data suggest that even if resistance was present at low levels prior to the introduction of the quinolones, an upsurge in resistance may reflect a selective advantage that came with quinolone introduction. This is particularly likely for Ghana were artemisinin combination therapies have recently been introduced to replace chloroquine. Furthermore, because almost all quinolone resistant-E. coli were multiply resistant, selective pressure from other, even more commonly applied, antimicrobials will help to maintain the quinolone-resistant clonal groups we identified in this study.

Fluoroquinolones, largely ciprofloxacin, were introduced very recently to Ghana with high expectations. This study demonstrates that resistance to these drugs is already common and occurs through multiple mechanisms, suggesting that heavy use of these valuable drugs may rapidly obliterate their usefulness. In addition to the impact that the emergence and dissemination of quinolone resistant bacteria may have on the use of fluoroquinolone antibacterials, we found that QREC were almost invariably resistant to multiple antimicrobials. This is worrisome because it means that, if the commensal flora is reflective of resistance profiles in pathogens, there may be few low-cost alternatives for managing infections due to Gram-negative enteric organisms. Additionally, horizontally-acquired resistance to the quinolones, and presumably other agents may be present on mobile elements that could be transmitted to pathogens. Recent calls for antimicrobial development have spotlighted hospital pathogens and Gram-positive community-acquired pathogens such as Staphylococci 32 . Our data suggest that there is also a pressing need for orally administrable drugs with activity against Gram-negative organisms, which can be used to manage community enteric infections in Ghana and other parts of Africa. Additionally, known strategies for containing antimicrobial resistance need to be more rigorously applied 33 34 35 .

SSN performed molecular experiments, analysed and interpreted data, and contributed to writing the paper. JAO collected isolates and performed microbiology experiments. RSL designed and performed molecular experiments. MJN co-conceived the study and collected isolates. INO co-conceived the study, performed microbiology and molecular experiments, analysed and interpreted data and wrote the manuscript. All authors read and approved the final manuscript.

This work was supported by a Branco Weiss Fellowship from the Society in Science, ETHZ, Zürich to INO. SSN and RSL were HHMI-supported undergraduate researchers, and RSL was also an Arnold and Mabel Beckman Scholar, at Haverford College.