Prevalence and multidrug resistance of Escherichia coli from community-acquired infections in Lagos , Nigeria

Introduction: The emergence of multidrug resistance (MDR; resistance to ≥ 2 more antimicrobials) in Escherichia coli is of concern due to complications encountered in treatment. Methodology: In this study, prevalence, antimicrobial resistance, and genetic characteristics of MDR community isolates of E. coli from Lagos, Nigeria were determined. Urine and stool samples were obtained from outpatients attending Lagos State hospitals and from animal handlers in abattoirs, poultries, and open markets, from December 2012 to July 2013. Results: Approximately 50% of urine (200/394) and 88% of stool samples (120/136) were positive for E. coli. Based upon β-lactamase production, a subset of those isolates was selected for further study. Of the 22 antimicrobials tested, E. coli exhibited resistance to all antimicrobials except amikacin and piperacillin/tazobactam. The highest levels of resistance were to tetracycline (182/247; 73.7%), trimethoprim/sulfamethoxazole (152/247; 61.5%), and ampicillin (147/247; 59.1%). Resistance to the cephalosporins ranged from 1.6%–15% including the thirdand fourth-generation cephalosporins, cefpodoxime (20/247; 8.1%) and cefepime (4/247; 1.6%), respectively. MDR was observed in 69.6% (172/247) of the isolates. Forty-eight E. coli resistant to at least five antimicrobials were selected for further analysis using pulsed-field gel electrophoresis; seven distinct clusters were observed among the diverse patterns. Of the 48 MDR E. coli, 30 different sequence types (ST) were detected using multilocus sequence typing, including four ST131. Conclusions: This study demonstrated circulating MDR E. coli in the Nigerian community. Monitoring of antimicrobial resistance in developing countries is necessary to optimize empiric treatment and the prudent use of antimicrobials.


Introduction
Antimicrobial drugs are important in the treatment of infections to reduce mortality and morbidity [1].However, the emergence of antimicrobial resistance has posed a worldwide challenge to public health by hampering effective chemotherapy [2,3].The use of antimicrobials in both human and veterinary medicine has led to the selection of resistant organisms, resulting in dissemination of drug-resistant bacteria in hospitals and the community [4,5].The development of multidrug resistance (MDR; resistance to ≥ 2 antimicrobials) to major classes of antimicrobials within a short period of time after approval for medical use is cause for concern, especially due to the time required to produce new antimicrobial agents [6,7].
Escherichia coli, a normal inhabitant of the intestinal tract of mammals, is a common cause of intestinal infections, urinary tract infections (UTIs), and bacteremia in humans of all ages [8][9][10][11].Resistance to a number of antimicrobials including aminoglycosides, β-lactams, cephalosporins, fluoroquinolones, sulfonamides, tetracycline, and trimethoprim have all been described in E. coli from clinical settings and food-producing animals [12][13][14].MDR E. coli from the community have been described worldwide [15].In the United States, prevalence of MDR E. coli among community isolates increased more than 56% from the 1950s to the 2000s [1].In Latin America, the widespread dissemination of phylogenetically related strains of uropathogenic E. coli in community and hospital settings has been observed and attributed to a common source in the community such as food and water or person-to-person transmission [16,17].
Many of the resistances in MDR E. coli are located on plasmids, which increases the possibility of clonal dissemination of these resistance classes in the community [18][19][20][21].Spread of MDR E. coli globally can also be attributed to clones, such as E. coli sequence type 131 (ST131), known for its resistance to fluoroquinolones, aminoglycosides and trimethoprimsulfamethoxazole, as well as its virulence and propensity to exchange genetic material, characteristics which further complicate therapy.A number of reports exist on the emergence of this clonal group in E. coli from the community and hospital infections in developed and developing countries [10,22,23].In Nigeria, the emergence of E. coli ST131 and ST617 among clinical isolates of E. coli was reported in 2012 [21].However, there is a paucity of data from community infections in Nigeria on the MDR profile of E. coli and their genetic lineages or reports on the major clonal complex circulating in the community.The potential spread of MDR E. coli is significant, especially for developing countries, which may have low financial resources for healthcare systems and poor infection control management.The antimicrobial resistance issue is exacerbated in these areas due to large numbers of the populace who tend to live in unhygienic conditions and have little access to healthcare and may be able to easily obtain antimicrobials, including counterfeit antimicrobials.
To provide more information on the extent of MDR E. coli in the human population in Nigeria, the prevalence and antimicrobial resistance of E. coli among outpatients and animal handlers in the community was determined.E. coli isolates in this study were tested against a wide range of broadspectrum antimicrobials primarily used for treating human infections, including some of the most recently approved antimicrobials for human use.In addition, phylogenetic grouping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) were used to compare a subset of MDR E. coli.

Bacterial isolates
Between December 2012 and July 2013, nonduplicate urine and stool samples were collected from asymptomatic patients at outpatient clinics in selected government hospitals in Lagos State and from animal handlers working in food production in the market and abattoirs in the State.Patients and animal handlers who were on antimicrobial therapy or had been on admission in any hospital six months prior to sampling were excluded from the study.Other requirements for participation in the study included urinary bacterial load of ≤ 10 5 CFU/mL, no history of diarrhea in the preceding three months prior to the study, and willingness to respond to the questionnaire.Ethical approval for sampling was obtained from the Lagos State Health Service Commissions Board and Institutional Review Board of the hospitals.Informed consent was obtained from all human participants in this study.
All samples were collected in sterile universal bottles and kept on ice before processing; all were processed within 4 hours of collection.Stool samples were collected in modified Cary-Blair transport medium (Oxoid, Basingstoke, UK) and directly inoculated onto the agar plates.Urine and stool samples were cultivated on MacConkey agar and 5% sheep's blood agar (Oxoid, Basingstoke, UK) for the presence of members of the Enterobacteriaceae and incubated aerobically at 37°C for 18-24 hours.One to two colonies were selected from each sample.Isolates were Gram-stained and identified using API 20E according to the manufacturer's directions (bioMérieux, Basingstoke, UK).Colonies representative of E. coli were confirmed with the VITEK ® 2 System using the VITEK 2GN cards (bioMérieux, Durham, USA) according to the manufacturer's directions.A subset of β-lactamase-positive E. coli using the β-lactamase test strips (Oxoid, Basingstoke, UK) were used for the study.All bacterial strains were stored at -80°C in brain-heart infusion (BHI) broth containing 30% glycerol.

Phylogenetic analysis
Phylogenetic grouping of the eight phylo-groups for Escherichia (i.e., A, B1, B2, C, D, E, F, and cryptic clade I) was performed using the extended quadruplex polymerase chain reaction (PCR) as previously described [25].

Pulsed-field gel electrophoresis
A 24-hour E. coli PFGE procedure was performed as previously described [27].Briefly, cells from an overnight culture were embedded in 1.0% Seakem Gold agarose (BioWhittaker Molecular Applications, Rockland, USA) and digested with 10 U of XbaI (Roche Molecular Biochemicals, Indianapolis, USA).DNA standards were prepared from Salmonella enterica serotype Braenderup H9812.Digested DNA was separated using the CHEF-DRII PFGE system per the manufacturer's instructions (Bio-Rad, Hercules, USA).Electrophoresis was performed at 6 V for 19 hours with a ramped pulse time of 2.16-54.17seconds in 0.5X Tris-borate-EDTA (TBE) at 14°C.Cluster analysis was determined with BioNumerics software using Dice coefficient and the unweighted pair group method (UPGMA).Optimization settings for dendrograms were 1.5% with a position tolerance of 1.5%.Isolates with ≥ 65% similar profiles were considered to represent the same clone [28].

Statistical analysis
Probability values of statistical significance were generated using Chi-square analysis (SAS version 9.1.3,SAS Institute Inc., Cary, USA).Statistical significance was defined as a probability value of less than or equal to 0.05 (p ≤ 0.05).

Results
Prevalence and antimicrobial resistance of E. coli A total of 530 samples of human urine (n = 394) and stool (n = 136) were obtained from outpatients and from food animal handlers in the community.Of those, 50.8% (200/394) urine samples and approximately 88% of stool samples (120/136) were positive for E. coli (Table 1).Although the Gram-negative susceptibility plate used in this study contained 22 antimicrobials, the majority of those were β-lactams.Therefore, the E. coli selected for further study (n = 247) were those positive for β-lactamase production.
Fifty-three resistance patterns were observed among all E. coli isolates (Table 3).Almost 70% of the isolates (172/247) were MDR, exhibiting resistance ranging from two to sixteen antimicrobials and two to five antimicrobial classes.Fifty-two isolates were resistant to five or more antimicrobials; two isolates that were resistant to sixteen antimicrobials collectively were resistant to the entire antimicrobial drug classes used in the study (Table 3).The largest groups of MDR, based upon different patterns of antimicrobials, belonged to those composed of three antimicrobials; fifteen combinational patterns were observed for this group.Surprisingly, nine different antimicrobial resistance combinations were observed for the groups composed of two antimicrobial classes and also five antimicrobial classes, suggesting wide-ranging MDR in the isolates.The three antimicrobials for which the E. coli isolates exhibited the highest level of resistance (ampicillin, tetracycline, and trimethoprim/sulfamethoxazole) also resulted in the most common resistance pattern, AmpTetSxt (n = 43) (Table 3).

Molecular characteristics
A subset of MDR E. coli (n = 48) resistant to five or more antimicrobials were selected for further analysis.Using the extended quadraplex E. coli phylo-typing method, five phylogenetic groups (A, B1, B2, C, and E) were identified; two isolates could not be assigned a phylo-group (unassigned; U) (Figure 1).Most of the isolates were identified as group B1 (n = 16), B2 (n = 11), or C (n = 11), while slightly fewer were group A (n = 7).Interestingly, the majority of MDR E. coli belonging to groups A, B1, and C were isolated from stool samples (n = 22) compared to urine samples (n = 12), while the majority of isolates from the virulent phylogenetic groups were isolated from urine samples (n = 10) rather than stool samples (n = 1).However, these results were not significantly different.
Among the 48 MDR E. coli, 30 STs were identified and frequently represented only once among the isolates (Figure 1).Two STs, ST410 (n = 5) and ST131 (n = 4), contained the greatest number of isolates; isolates belonging to ST410 were all related to commensal phylogenetic groups, while isolates identified as ST131 were also virulence associated.

Genetic relatedness
Using PFGE analysis, MDR E. coli isolates grouped into seven clusters (A-F) having ≥ 65% similarity (Figure 1).Clusters were examined to determine if they grouped according to PFGE pattern, antimicrobial resistance phenotype, source, phylogenetic group, or sequence type.Overall, the clusters appeared to contain isolates with a variety of different phenotypic and genotypic characteristics; however, source appeared to be a dominant character for a few clusters.Cluster A2 contained three isolates all from urine, and clusters E and F were composed of isolates all from stool samples (Figure 1).One of the larger clusters, cluster C, contained eleven isolates, mostly from urine samples.The other large clusters, A1 and B, were composed of almost even numbers of isolates from both urine and stool samples.STs represented by more than one isolate also appeared to cluster together, such as ST120 and ST155; however, that was not always consistently observed, as other STs (e.g., ST1284, ST73) were located in different clusters (Figure 1).
Although not many isolates were identical, the three sets of isolates that had the same PFGE pattern (H232ii and H233; H204ii and H204iii; and H322i and H322ii) each belonged to the same phylogenetic group and the same ST.However, only isolates H322i and H322ii had identical antimicrobial resistance patterns (Figure 1).The four isolates identified as ST131 all grouped in cluster B and were resistant to twelve to thirteen antimicrobials, including antimicrobials in the aminoglycoside, fluoroquinolone, and sulfonamide classes.The two isolates resistant to sixteen antimicrobials (H124b.iand H98) were located in different clusters and had different STs, but were associated with the commensal E. coli phylogenetic group.

Discussion
E. coli is a leading cause of community and opportunistic infections in humans [29,30].The increasing prevalence of MDR E. coli among community isolates is challenging because those isolates can occupy multiple niches, including human and animal hosts, thereby acquiring or transmitting antimicrobial resistance genes horizontally and vertically [31].Earlier studies from clinical samples on antimicrobial resistance profiles of E. coli from Nigeria recorded a high prevalence to commonly administered antibiotics such as amoxicillin, amoxicillin-clavulanic acid, tetracycline, erythromycin, and trimethoprim/sulfamethoxazole [32][33][34][35].However, few studies have assessed the antimicrobial and MDR profile of E. coli from community isolates in West Africa [36,37].Most available data are specific to pathogenic E. coli isolates [13,38].In this study, surveillance data was obtained on prevalence and MDR profiles of E. coli from outpatients and food-producing animal handlers in the community.The genetic lineages and the major clonal complexes of MDR E. coli circulating in the community were also investigated, which may be responsible for observed therapeutic failures [10].This information is important for making informed decisions in the therapy of primary healthcare patients and forming strategies in the event of an outbreak of infection that could arise from these circulating clones in the community.
In this study, resistance to tetracycline was the most prevalent.This was not unexpected, as other studies from Nigeria have reported > 80% resistance to tetracycline [39,40].Tetracycline is a commonly used antimicrobial in the Nigerian community, which may have given rise to the observed high resistance in this study.For MDR isolates, resistance to tetracycline, trimethoprim/sulfamethoxazole, and ampicillin was the predominant resistance phenotype.Similar findings on the resistance pattern of MDR of E. coli strains have been reported from Bangladesh and other parts of the world [41][42][43][44][45].This may be attributed to the common use of these broad-spectrum antimicrobials for therapeutic purposes by clinicians in treating infections [35,38].The sale of these drugs by medicine hawkers (non-professionals) in the Nigerian community has encouraged the practice of self-medication, leading to under-or over-dosage, which may also contribute to these observed resistance patterns [46].More significantly, this study also indicated that most antibiotics used as first-choice oral empiric treatment are no longer appropriate since the prevalence of resistance exceeds 10% [47].
Cephalosporins are frequently prescribed by clinicians in Nigerian hospitals for treatment of bacterial infections [12,13], and studies on clinical isolates have shown high levels of resistance to cephalosporins [13,35].In contrast to those studies, resistance to cephalosporins was low among E. coli isolates in this study.The non-phenotypic expression of resistance among isolates may be due to the study design, which excluded patients with previous intake of antimicrobial drugs within the previous six months of the study or admission into any healthcare facility.
The increasing rise in resistance to β-lactams, tetracyclines, and sulfa antimicrobials has led to the use of quinolones in difficult-to-treat infections and also in the agricultural and veterinary sector [12,[48][49][50].This may have resulted in the recorded emergence of quinolone resistance in recent years.Nevertheless, this study tested only resistance to ciprofloxacin in the E. coli isolates, and it was observed that resistance to ciprofloxacin was reduced (less than 15%), which is similar to the conclusion of another study that reported that most of the uropathogens causing uncomplicated UTIs in outpatients are susceptible to fluoroquinolones [51,52].This reduced resistance leading to low-level quinolone resistance can be attributed to various mechanisms, including reduced target expression or altered porins [53,54].
Carbapenem resistance exhibited by some MDR isolates in this study was unexpected because these antimicrobials are rarely used in Nigerian hospitals except in cases of emergencies or life-threatening conditions, primarily due to the high cost to purchase the drugs.These drugs are the last-line drugs in therapy of infections; resistance to carbapenems is an emerging concern in human medicine.In a study by Ogbolu et al. [55], carbapenem resistance with novel mechanisms of resistance was detected among Gram-negative bacterial isolates from hospital infections.No data exists on community isolates exhibiting resistance to carbapenems in Nigeria; however, studies have documented resistance to carbapenems in Enterobacteriaceae in other parts of the world among community isolates [56][57][58][59].The presence of resistance to carbapenems in Lagos may be due to its geographical location, as Lagos is a border state with a high influx of people from surrounding African countries.In addition, easy access to imported frozen foods, which are cheaper than other meat sources, has been documented to be a major source of MDR bacteria in the country [60].
Resistance to broad-spectrum and easily available antibiotics (i.e., ampicillin and trimethoprim/sulfamethoxazole) was high, similar to a report by Sahm et al. [61] that demonstrated dissemination of E. coli exhibiting resistance to broadspectrum antibiotics among community isolates in the United States.Other drugs, such as tigecycline, are not easily available in Nigeria, which may account for the low level of resistance to tigecycline in E. coli isolates in this study.Nonetheless, the observed resistance, although low, may be due to the overexpression of efflux pumps or other mechanisms of resistance to new antibiotics existing before the drug was introduced into medical use [62,63].The indiscriminate consumption of medicinal herbs by the community, which may contain a measure of natural antimicrobials, may also have contributed to the observed MDR among these isolates from the community [64].Poor sanitation and overcrowding in the community may also be a significant risk factor in the dissemination of drug resistance among the isolates.
Genotyping of isolates has become important due to the possibility of an outbreak or horizontal transmission of certain bacterial strains.In the absence of whole genome sequencing, the discriminatory power and reproducibility has qualified PFGE as the gold standard in epidemiological studies for determining related strains from the same source [27].PFGE analysis of the MDR E. coli revealed a genetically diverse population among human isolates.The majority of isolates belonging to the same cluster groups had at least 65% similarity, which is similar to a previous report by Nsofor et al. [65].However, four pairs of isolates had 100% similarity, indicating that the isolates belonged to a single clonal group.One pair of identical isolates within the four groups was also resistant to the same antimicrobials.It is of note that some of the isolates that were in the same cluster had different antimicrobial phenotypes, suggesting that isolates with similar PFGE types do not necessarily have the same antimicrobial phenotypes because they may have acquired different antibiotic resistance genes and unique DNA rearrangements [21,66,67].
Genetic analysis of the isolates also determined common genetic lineages among isolates, and diverse STs were identified.This study confirmed the presence of 30 STs for E. coli belonging to phylogenetic groups A, B1, B2, C, and E circulating in the community.Commensal E. coli typically belong to group A, B1, or C, while isolates from phylogenetic group B are associated with virulence and account for the majority of extra-intestinal infections [68,69].Phylogenetic group B isolates have also been associated with avian phylogenetic groups, which pose a potential zoonotic risk [70][71][72].Dissemination of these clones to humans through the food chain is a possibility since Lagosians depend on food-producing animals, especially poultry, as a main source of protein [14,72].Phylogenetic group A has been reported to be more prevalent within the United States than abroad and infects urinary tract and non-urinary tract sites [73].These strains have also been associated with trimethoprim-sulfamethoxazole resistance, which was also observed in this study.Phylogenetic group C has only been recognized in the last few years and is closely related to phylogenetic group B1 [25].A recent study has shown that isolates belonging to this group possess traits such as colonization and ability to disseminate in the intestinal tract, which may increase the pathogenicity of these isolates [74].
Little data exists on the presence of ST131 and other clonal groups in Africa.E. coli ST131 virulenceassociated clonal group was detected in the present study with similar MDR patterns, suggesting the possibility of clonal spread of these MDR E. coli.The presence of E. coli ST131 isolates in West Africa was reported by Aibinu et al. [21] from hospital infections in Lagos, while one other E. coli ST131 was identified from a study of inpatients from Oyo State, Nigeria [36].In a report from another African country, approximately 45% of extended-spectrum β-lactamase (ESBL)producing E. coli isolates were ST131, which suggested a high rate of virulence-associated E. coli in circulation in Cape Town [75].Travel to Africa has also been indicated as a factor in E. coli ST131 travel-related ESBL-producing E. coli infections [76].These clones threaten public health due to their virulence factors, rapid dissemination, and the lack of new drugs to combat the spread [23].

Conclusions
An urgent need for antimicrobial surveillance in the public health sector is of utmost importance; this study provides needed data from the community to assist in this effort.The declining rate of production of new antimicrobials and an increase in MDR pathogens in the clinic and community is precipitating a global health crisis [77].Continuous migration and international travel have led to increased antimicrobial resistance in this group of bacteria due to mobile genetic elements that are easily spread through direct contact and ingestion of contaminated water or food [29,78,79].It is therefore necessary to screen outpatients for asymptomatic bacterial infections and also food animal handlers before they handle animal food products sold to the community to prevent transmission or circulation of clonal strains of MDR E. coli.

Table 1 .
Prevalence of Escherichia coli among humans from the community.
*Isolates selected for inclusion in this study were positive for β-lactamase production.

Table 2 .
Antimicrobial resistance of Escherichia coli isolated from humans.
*No isolates were resistant to amikacin or piperacillin-tazobactam.

Table 3 .
Single and multidrug resistance patterns in Escherichia coli from humans.