Predictors of blaCTX-M-15 in varieties of Escherichia coli genotypes from humans in community settings in Mwanza, Tanzania

Background Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae commonly cause infections worldwide. BlaCTX-M-15 has been commonly detected in hospital isolates in Mwanza, Tanzania. Little is known regarding the faecal carriage of ESBL isolates and blaCTX-M-15 allele among humans in the community in developing countries. Methods A cross-sectional study involving 334 humans from the community settings in Mwanza City was conducted between June and September 2014. Stool specimens were collected and processed to detect ESBL producing enterobacteriaceae. ESBL isolates were confirmed using disc approximation method, commercial ESBL plates and VITEK-2 system. A polymerase chain reaction and sequencing based allele typing for CTX-M ESBL genes was performed to 42 confirmed ESBL isolates followed by whole genome sequence of 25 randomly selected isolates to detect phylogenetic groups, sequence types plasmid replicon types. Results Of 334 humans investigated, 55 (16.5 %) were found to carry ESBL-producing bacteria. Age, history of antibiotic use and history of admission were independent factors found to predict ESBL-carriage. The carriage rate of ESBL-producing Escherichia coli was significantly higher than that of Klebsiella pneumoniae (15.1 % vs. 3.8 %, p = 0.026). Of 42 ESBL isolates, 37 (88.1 %) were found to carry the blaCTX-M-15 allele. Other transferrable resistance genes were aac(6’)Ib-cr, aac(3)-IIa, aac(3)-IId, aadA1, aadA5, strA, strB and qnrS1. Eight multi-locus sequence types (ST) were detected in 25 E. coli isolates subjected to genome sequencing. ST-131 was detected in 6 (24 %), ST-38 in 5 (20 %) and 5 (20 %) clonal complex − 10(ST-617, ST-44) of isolates. The pathogenic phylogenetic groups D and B2 were detected in 8/25 (32 %) and 6/25 (24 %) of isolates respectively. BlaCTX-M-15 was found to be located in multiple IncY and IncF plasmids while in 13/25(52 %) of cases it was chromosomally located. Conclusion The overlap of multi-drug resistant bacteria and diversity of the genotypes carrying CTX-M-15 in the community and hospitals requires an overall approach that addresses social behaviour and activity, rationalization of the antibiotic stewardship policy and a deeper understanding of the ecological factors that lead to persistence and spread of such alleles.


Background
Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are currently a major problem in hospitalized patients worldwide [1][2][3]. The prevalence of ESBLs among clinical isolates varies between countries and from institution to institution [2,4]. Tanzania is one of the sub-Saharan African countries facing increasing numbers of health care associated infections due to multi-drug resistant Gram-negative bacteria [5][6][7][8]. Data regarding ESBL isolates in Tanzania are limited to tertiary hospital based studies only.
Several studies performed in developed countries have demonstrated ESBL carriage in the community [9][10][11][12][13]. In addition, in many studies from developed and middle-income countries it could be demonstrated that ESBL-producing bacteria are common in domestic and companion animals in the community [14,15]. Human to animal contact seems to play a role in the transfer of ESBL producing bacteria between both populations [14,16,17]. In Thailand, different factors such as better education, history of hospitalization and the use of antibiotics have been found to be independently predictors for ESBL carriage [18]. Despite the potential risk of ESBL acquisition in the community and transfer between humans and animals there is no study which has documented ESBL carriage and associated risk factors in the human community in Tanzania. Therefore this study was done to investigate the magnitude of ESBL carriage and diversity of ESBL genotypes, and to identify factors associated with it among humans in the community in Mwanza.

Sample size and sampling
A cross-sectional study was conducted between June and September 2014 in three rural districts (Igogo, Mbugani and Kirumba) with squatters in Mwanza city. The characteristics of the rural districts are described in Table 1. Sample size was calculated using the formula by Cochran [19]. A prevalence of 22.1 % from a study performed in Madagascar was used for calculation [20]. The minimum sample size obtained was 297.
Streets within these rural districts with characteristics of squatter settlements were purposively selected. A total of 3144 households were obtained from these streets. The number of households in each street was obtained from the household registers at the street Executive Officer's office. Simple random sampling was used to select 334 households which were included in the study. Using a random number generator, the households to be included in the study were determined. A total of 334 stool samples (one per participant) were collected. From every participant enrolled, additional information (with the use of an interview questionnaire) such as age, gender, size of the family, history of antibiotic use in the past one month and history of admission in the past one year, were collected.

Laboratory procedures
A total of 334 non-repetitive stool specimens were obtained from humans. All specimens were cultured on MacConkey agar supplemented with 2 μg/mL cefotaxime (Oxoid, Basingstoke, UK) and plain MacConkey agar plates to isolate lactose fermenting colonies to investigate these for the antimicrobial susceptibility pattern.

Strain selection
One colony from predominant morphologically similar colonies was selected on the MacConkey agar plate with 2 μg/mL cefotaxime for subsequent characterization while a representative of predominant morphologically similar lactose fermenter colonies was also selected from a plain MacConkey agar plate.

Whole genome sequencing
Twenty five CTX-M-15-producing Escherichia coli strains were randomly selected for whole genome sequencing (WGS). Genomic DNA was isolated using Purelink Genome DNA Mini kit (Invitrogen, Darmstadt, Germany) according to the manufacturer's instruction. WGS was carried out on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) with an Illumina Nextera XT library with 2x300bp paired-end reads. The reads were assembled using SPAdes (version 3.0) [23]. The assembled contigs were analysed and examined for the presence of transferrable resistance genes, virulence genes, multi-locus sequence types, and plasmid replicon types using ResFinder, VirulenceFinder, MLST 2.0, and PlasmidFinder software, [24][25][26][27], respectively.

Data analysis
Data were double-entered into Microsoft Excel and analysed using STATA version 11. Results were summarized using proportions (%) for categorical data and medians (IQR) for continuous variables. Categorical variables were compared using either Pearson's Chi-squared or Fisher's exact test, where appropriate. To determine the predictors of ESBL carriage, univariate followed by multivariate logistic regressions analysis was performed. The predictors tested included age, sex, number of residents in a household, antibiotic use in the last month, admission history and presence of animals at home. Odd ratios with respective 95 % confidence interval (CI) were reported. Predictors with a p-value of less than 0.05 were considered statistically significant.

Limitations of the study
In this study the primers pair used are not for amplification of all CTX-M groups, and might have introduced a great bias towards group 1 to which CTX-M-15 belongs. However, the sequence covered aligned clearly align to the product with CTX-M-15 standard and this was further confirmed for the 25 isolates which underwent WGS. The WGS of 25 randomly selected isolates also confirmed the presence CTX-M-15 in all 25 isolates. Finally, 13 ESBL isolates could not be recovered for PCR and sequencing.

ESBL carriage and rates of ESBL by isolates
Of 334 individuals from the Mwanza city community, 55 (16.5 %) were found to be colonized by ESBL-producing Enterobacteriaceae. A total of 323 lactose fermenting isolates (270 E. coli and 53 Klebsiella pneumoniae) were obtained from plain MacConkey agar plates. Out of E. coli and Klebsiella pneumoniae isolates, 42/270 (15.5 %) and 2/53 (3.8 %) were ESBL producers, respectively (p = 0.026). E. coli (42/55; 76.3 %) formed the majority of ESBL isolates in this population. A total of eleven ESBL isolates were other Enterobacteriaceae species (Enterobacter spp: n = 4, Proteus mirabilis: n = 5 and Proteus vulgaris: n = 2). Their ESBL rates could not be calculated because these isolates were not targeted for in using plain MacConkey agar plates.
Of 208 individuals who had a history of using antibiotics in the past one month, 44 (21.1 %) were found to be colonized with ESBL isolates as compared to 11 (8.7 %) of those with no history of antibiotic use (OR = 2.88, 95 % CI 1.3-5.66, p = 0.004). In addition, significantly higher rate of colonization was observed among individuals with history of admission in the past one year compared to those with no history (66.6 % vs. 15.1 %; p = 0.001).
On multivariate logistic regression analysis only history of admission, history of antibiotic use and increasing age were independent predictors of ESBL carriage (Table 3).  Table 4). All sequenced isolates carried trimethoprim and sulphamethoxazole resistance genes.  All but two isolates harboured tetracycline resistance genes.

Multi locus sequence types (MLST), phylogenetic groups and plasmid MLST
Eight different sequence types were observed in the genome-sequenced strains ( Table 4) (Table 4).
In most cases, isolates exhibiting an identical sequence type were found to carry different plasmid types.

Virulence genes
Isolates belonging to the phylogenetic group B2 harboured the most virulence genes followed by phylogroup D isolates. Glutamate decarboxylase (gad) that confers resistance to bile salts and the increased serum survival genes (iss) were detected in 13 (52 %) of the isolates. All phylogroup B2 isolates harboured the sat and Iha genes encoding for a serine-protease autotransporter and an iron-dependent adhesion protein, respectively (Table 5).

Discussion
The presented study identifies a high proportion of faecal carriage of ESBL-producing E. coli in the Mwanza community. The prevalence of 21 % in Igogo rural district is almost the same as that of 25 % obtained in E. coli clinical isolates in the same town [5]. Compared to a previous study [28] which investigated ESBL carriage in women and neonates admitted at Bugando Medical Centre, similar findings are observed with the magnitude of carriage observed in women. However, the carriage is significantly lower than that obtained in neonates. Our findings are within the range of the ESBL carriage reported in Africa which has been found to range from 10 % in Senegal to 31 % in Niger [20,[29][30][31]. The high carriage in Niger could be attributed to the fact that the population studied suffered from malnutrition with majority of individuals having previous antibiotic exposure. As observed previously [18,28], antibiotic exposure, history of admission, or increase in age were found to predict ESBL carriage on multivariate logistic regression analysis in our setting as well.
As in previous studies [16,29], the bla CTX-M-15 was the commonest allele observed in the community in Mwanza, Tanzania. This allele has also been found to be the commonest allele in E. coli and Klebsiella pneumoniae isolates from Bugando Medical Centre [6,32]. This could possibly be explained by contacts between individuals admitted to the hospital, and due to the fact that the Bugando Medical Centre is the only tertiary hospital in the region. Also environmental contamination by hospital sewages and transfer through the food-chain in the city such as the fish-consumption could play a significant role [33]. This is further supported by the fact that, as in hospital E. coli isolates from a previous study [32], ST-131 was commonly found in the community. However, more studies are needed to establish the transmission pathways especially the role of food-chain and environment in the persistence and spread of ESBL isolates in the city.
Though marginally significant on univariate analysis, people from Igogo were 1.9 times more likely to carry ESBL isolates than those from Kirumba. The population of these two rural districts are almost equal. However, Igogo rural district is nearer to the Bugando Medical Centre and has more industrial and garage activities than Kirumba. These could contribute to more environmental contamination that might lead to bacteria being resistant to various metals and chemicals. In addition, the population per pharmacy/ medicine shops is low at Igogo rural district as compared to that in Kirumba rural district; this might contribute to an easy accessibility to antibiotics and more environmental contamination. More research to explore human's activities, environmental contamination and the role of antibiotic usage are needed. A geographical factor worth studying regarding the influence on ESBL carriage is the fact that in all rural communities studied a majority of the population lives in the hills, and use latrines that are not connected to city sewage. As compared to hospital isolates [5,32], E. coli isolated in the community were more resistant to gentamicin, sulphamethoxazole/trimethoprim and tetracycline. These results necessitate an urgent review whether these antibiotics are of value for empirical treatment of infections caused by E. coli such as urinary tract infections.
This study further confirms the role of IncF plasmids with multiple resistance genes to be responsible in transmitting resistance genes among Enterobacteriaceae isolates [32,34]. An important finding in this study is the detection of IncY plasmids in 20 % of isolates carrying quinolone resistance gene (qnrS1) and aminoglycosides resistance genes (aadA5, strA and strB) in addition to bla CTX-M-15 . Similar plasmids have been recently detected in Nigeria among healthy pregnant women [35]. In addition, 57 % (13/25) of the isolates displayed a chromosomally located CTX-M-15, thereby enabling a vertical transfer of the resistance. The findings underscore the importance to investigate the epidemiology of ESBL-producing bacteria on the African continent to ascertain transmission pathways and factors associated to the persistence.

Conclusion
High ESBL carriage, especially of bla CTX-M-15 , is observed in the community among Escherichia coli in Mwanza city. Predictors for ESBL carriage are the use of antibiotic, history of hospital admission and increase in age. This information is useful for introducing pre-admission screening, planning empirical treatment by identifying high risk ESBL patients and adapting empirical treatment of infections towards covering ESBL isolates in patients with urosepsis. The findings that hospital clones and plasmids are also in the community isolates require more studies using a One Health approach to determine the role of human's activities in relation to the persistence, circulation, spread and transmission of CTX-M-15-producing E. coli strains in Mwanza city in order to provide appropriate recommendations to control this public health threat.

Ethical considerations
The protocol to perform this study was approved by Catholic University of Health and Allied Sciences and Bugando Medical Centre (CUHAS/BMC) ethics review board (CREC/043/2014). In addition, all participants signed written informed consent for participation in the study. Where participants were children, a parent or guardian signed the consent.