Studies have shown the presence of extended-spectrum β-lactamase-producing Enterobacteriaceae (E-ESBLs) in Cameroon, not only in hospitals but also in the community [1-4]. However, although E-ESBLs are present and increasingly prevalent in the country, little is known about their dissemination. To find out the prevalence of faecal carriage of E-ESBLs and how they are spreading, an epidemiological study of E-ESBLs involving outpatients, inpatients, inpatient carers, hospital workers and members of their households was performed in hospitals in Ngaoundere, Cameroon. Written informed consent was obtained from all participants. Participants' characteristics are shown in Table 1.
|Characteristic||Outpatients (n = 232)||Inpatients (n = 208)||Inpatient carers (n = 63)||Hospital workers (n = 48)||Household members (n = 39)|
|E-ESBL+ n = 104 (45%)||E-ESBL– n = 128 (55%)||E-ESBL+ n = 140 (67%)||E-ESBL– n = 68 (33%)||E-ESBL+ n = 36 (57%)||E-ESBL– n = 27(43%)||E-ESBL+ n = 21 (44%)||E-ESBL– n = 27 (56%)||E-ESBL+ n = 18 (46%)||E-ESBL– n = 21 (54%)|
|Median age, years (±SD)||35 ± 15||36 ± 15.5||37 ± 17.4||41 ± 18||42 ± 14.8||39 ± 13.5||35 ± 6.72||37 ± 10.52||17 ± 13||14 ± 9.24|
|Male gender (n)||40||60||71||39||6||8||9||16||8||14|
|Previous hospital admission|
|Previous antimicrobial treatment|
A total of 334 Enterobacteriaceae were isolated on two selective media, Drigalski and MacConkey agars, supplemented, respectively with cefotaxime and ceftazidime. Detection of ESBL producers was carried out by the double-disc synergy test . Susceptibility of the isolates to antibiotics was determined using the Vitek system (bioMérieux, Marcy l'Etoile, France). All the presumptive ESBL producers were further analysed by PCR aimed at detecting ESBL genes (one isolate of each morphotype from each patient). PCR amplification of bla genes (blaTEM, blaSHV, blaOXA and blaCTX-M) was performed using primers and methods described previously [6, 7] (Table 2). All PCR products were sequenced using a 3100 ABI Prism Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequence alignment and analyses were performed online using the BLAST program available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
|PCR name||β-Lactamase(s) targeted||Primer name||Sequence (5′-3′)||Amplicon size (bp)||Primer concentration (pmol/μL)||Reference|
|Multiplex TEM,||TEM variants including, TEM-1 and TEM-2||MultiTSO-T_for||CATTTCCGTGTCGCCCTTATTC||800||0.4||[  ]|
|SHV||SHV variants, including SHV-1||MultiTSO-S_for||AGCCGCTTGAGCAAATTAAAC||713||0.4|
|and OXA-1- like||OXA-1, OXA-4 and OXA-30||MultiTSO-O_for||GGCACCAGATTCAACTTTCAAG||564||0.4|
|CTX-M group 1||Variants of CTX-M group 1, including CTX-M-1, CTX-M-3 and CTX-M-15||CTX-M3G_for||GTTACAATGTGTGAGAAGCAG||1050||0.5||[  ]|
To determine the relatedness of bacterial strains, 44 isolates (22 Escherichia coli and 22 Klebsiella pneumoniae) were recovered from 44 participants and fingerprinted using the automated repetitive-sequence-based PCR DiversiLab system (bioMérieux). The strains were selected based on their β-lactamase pattern, their origin and the relationship between participants. The relationships between repetitive-sequence-based PCR profiles were designated as recommended by the manufacturer: different, 3+ band differences (similarity <95%); similar, 1–2 band differences (similarity 95–97%); and indistinguishable, no band differences (similarity >97%).
Subsequently, the 22 E. coli isolates that had undergone DiversiLab analysis were investigated by a series of PCRs [8-10] to determine the phylogenetic groups (A, B1, B2 and D), the presence of ISEcp1 elements and the aac(6′)-Ib-cr variant (the gene that can induce resistance to aminoglycoside and fluoroquinolone simultaneously).
All 22 E. coli ESBL-producing isolates were screened for sequence type 131 (ST131) using a PCR for the pabB allele, described by Clermont et al. . Multilocus sequence typing was performed on positive pabB isolates using the Achtman typing scheme (http://www.mlst.ucc.ie/mlst/dbs/Ecoli).
Statistical analysis was performed with Epi Info version 3.5.3. Fisher's exact test, when appropriate, was used for the univariate comparison of all variables. A p value of p <0.05 was considered to be statistically significant.
The overall prevalence of faecal carriage of E-ESBLs in this study was 54.06%. Regarding participant groups and their relatives, the prevalence was also high (see Table 2). However, the prevalence of faecal carriage among inpatients was not significantly different from that of their carers (p >0.05). Similarly, the prevalence of faecal carriage among hospital workers was not significantly different from that of their household members (p >0.05).
Of the 334 bacteria isolated, 216 (64.67%) were E. coli, 74 (22.15%) were Klebsiella spp., 23 (6.88%) were Enterobacter spp. and 21 (6.28%) were Citrobacter freundii. Regardless of group, participants presented similar ESBLs and CTX-M-15 was the most widespread ESBL found in all strains (Table 3).
|ESBL types (number of isolates)|
|Strain (n = 334)||Outpatients||Inpatients||Inpatient carers||Hospital workers||Household members|
|Citrobacter freundii (n = 21)||CTX-M-15, OXA-1 (8)||CTX-M-15 (2)||CTX-M-15, TEM-1 (1)|
|CTX-M-15, TEM-1 (2)||CTX-M-15, OXA-1, TEM-1 (2)||CTX-M-15, OXA-1, TEM-1 (1)|
|SHV-12, TEM-1 (2)||CTX-M-15, TEM-1 (1)|
|SHV-12, TEM-1 (1)|
|Enterobacter spp. (n = 23)||CTX-M-15, OXA-1, TEM-1 (14)||CTX-M-15, OXA-1, TEM-1 (5)||CTX-M-15, OXA-1, TEM-1 (2)|
|SHV-12, TEM-1, OXA-1 (1)||SHV-12, TEM-1 (1)|
|Escherichia coli (n = 216)||CTX-M-15, OXA-1 (26)||CTX-M-15, OXA-1, TEM-1(42)||CTX-M-15, OXA-1, TEM-1 (11)||CTX-M-15, OXA-1 (8)||CTX-M-15, TEM-1 (8)|
|CTX-M-15, TEM-1 (15)||CTX-M-15, OXA-1 (30)||CTX-M-15, OXA-1 (9)||CTX-M-15, OXA-1, TEM-1 (4)||CTX-M-15, OXA-1, TEM-1 (5)|
|CTX-M-15, OXA-1, TEM-1 (14)||CTX-M-15, TEM-1 (24)||CTX-M-15, TEM-1 (8)||CTX-M-15, TEM-1 (2)|
|CTX-M-15 (3)||CTX-M-15 (3)||CTX-M-15, SHV-12, OXA-1, TEM-1(1)|
|SHV-12, TEM-1 (1)||CTX-M-15 (1)|
|Klebsiella spp. (n = 74)||CTX-M-15, SHV-1, TEM-1 (8)||CTX-M-15, SHV-1, TEM-1 (13)||CTX-M-15, SHV-1, TEM-1 (7)||CTX-M-15, SHV-1, TEM-1 (2)||CTX-M-15, SHV-1, TEM-1 (1)|
|CTX-M-15, TEM-1 (8)||CTX-M-15, TEM-1 (6)||CTX-M-15, SHV-1, OXA-1, TEM-1(1)||CTX-M-15, OXA-1, TEM-1 (2)||CTX-M-15 (1)|
|CTX-M-15, OXA-1, TEM-1 (5)||CTX-M-15, SHV-1, OXA-1, TEM-1(5)||CTX-M-15, TEM-1 (1)||CTX-M-15, OXA-1 (1)|
|CTX-M-15, OXA-1 (2)||CTX-M-15, OXA-1, TEM-1 (4)||CTX-M-15, OXA-1 (1)||CTX-M-15, TEM-1 (1)|
|CTX-M-15, SHV-1, OXA-1, TEM-1 (1)||CTX-M-15, SHV-12, TEM-1 (1)||SHV-12 (1)|
|SHV-12 (1)||CTX-M-15 (1)|
As demonstrated by the DiversiLab results (see Supplementary material, Fig. S1 and Fig. S2), multiple clones were seen among ESBL-producing E. coli and using a cut-off of 95% similarity (vertical dashed red line, Fig. S1) it was possible to establish six distinct clusters (types I, III, V, VI, VII and X), indicating the overall close relationship of the isolates, and five singleton patterns (types II, IV, VIII, IX and XI) (Table 4). These six outbreaks occurred between related or non-related individuals. This finding confirms that in some cases the acquisition of E-ESBLs has occurred via a common source or a common environmental reservoir (hands of hospital workers, through contaminated surfaces or via food) as described previously [12-17].
|Alternative identification||Type of participants (department in which the participant was at the time of sampling)||Type of relationship/gender (age)||Phylogenetic group||Cluster type||ST131 PCR||PMQR||ISEcp1 element||ESBL types||Resistance to antibiotics (other than β-lactam)|
|f32||Household member of p3||Daughter (3)||D||I||−||–||−||CTX-M-15, TEM-1||GEN, SXT|
|20||Maintenance staff (medicine)||M (42)||D||I||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1, TEM-1||GEN, SXT|
|hn50||Inpatient (medicine)||F (29)||D||II||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||CIP, GEN, SXT|
|hn89||Inpatient (medicine)||M (31)||B2||III||+||aac(6′)Ib-cr||+||CTX-M-15, OXA-1||CIP, SXT|
|p13||Nurse (medicine)||F (28)||B2||IIIa||+||aac(6′)Ib-cr||+||CTX-M-15, OXA-1||CIP, SXT|
|p3||Nurse (surgery)||F (41)||B2||IIIb||+||–||+||CTX-M-15, TEM-1||CIP, SXT|
|p25||Nurse (intensive care unit)||F (35)||B2||III||+||–||−||CTX-M-15, TEM-1||CIP, SXT|
|hn69||Inpatient (surgery)||M (40)||B2||IV||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||CIP, GEN, SXT|
|p5||Nurse (medicine)||F (30)||A||V||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, GEN, NIT, SXT|
|16||Maintenance staff (medicine)||M (38)||A||V||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, GEN, NIT, SXT|
|hn82||Inpatient (medicine)||M (39)||A||Va||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, GEN, NIT, SXT|
|12||Maintenance staff (operating room)||F (25)||A||Vb||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, GEN, NIT, SXT|
|p4||Nurse (medicine)||M (38)||A||Vc||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, NIT, SXT|
|p17||Nurse (surgery)||F (27)||A||VI||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, SXT|
|3||Maintenance staff (operating room)||M (41)||A||VIa||−||aac(6′)Ib-cr||−||CTX-M-15, OXA-1||CIP, SXT|
|f2||Household member of 11||Sister (18)||A||VII||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||GEN, SXT|
|11||Maintenance staff (maternity)||F (35)||A||VII||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||GEN, SXT|
|ng33||Carer of hn57||Husband (53)||A||VIII||−||–||−||CTX-M-15, OXA-1, TEM-1||CIP, SXT|
|ng41||Carer of hn69||Wife (35)||B1||IX||−||–||−||CTX-M-15, OXA-1, TEM-1||CIP, SXT|
|ng32||Carer of hn57||Sister (55)||B1||X||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||CIP, GEN, SXT|
|p16||Nurse (medicine)||F (33)||B1||X||−||aac(6′)Ib-cr||+||CTX-M-15, OXA-1, TEM-1||GEN, SXT|
|hn57||Inpatient (medicine)||F (43)||D||XI||−||–||−||CTX-M-15, OXA-1, TEM-1||CIP, GEN, SXT|
Molecular typing of K. pneumoniae isolates showed them to be more genotypically diverse than the E. coli isolates. The repetitive-sequence-based PCR analysis allowed the identification of two clusters and 17 unique profiles (see Supplementary material, Fig. S3 and Fig. S4). Our results emphasize the high endemicity of CTX-M-15 producers in the study setting (demonstrated by the very high prevalence in all participant categories). In addition, these findings could also explain the dramatic increase in the prevalence of faecal carriage of E-ESBLs previously observed in the same area during two non-outbreak periods separated by 1 year (in 2009 and 2010) [3, 4] (and data of this study). Moreover, examination of the different clusters (III, V and VI) showed that different strains of E. coli produced the same type of β-lactamase, indicating that the spread does not occur by strain but by another mode of dissemination (e.g. dissemination by plasmids: further studies are ongoing to clarify this).
The PCR for the pabB allele of ST131 status identified cluster III with four related isolates as belonging to ST131. The ST131 status was confirmed by multilocus sequence typing. In addition, isolates from this cluster could be assigned to phylogenetic group B2. The remaining ESBL-producing E. coli isolates (negative by PCR for pabB) belonged to phylogenetic groups A, B2, D and B1 (ten, one, four and three isolates, respectively). Interestingly, strains that belonged to the same phylogenetic group and clustered together showed a similar resistance profile to the antibiotics tested (Table 4). Many of the E. coli carrying blaCTX-M-15 from different countries in Europe and North America are homogeneously grouped as E. coli O25:H4-ST131 [11, 18-20]. In addition, in all blaCTX-M-15 tested, the aac(6′)-Ib-cr gene was carried by 16 strains (16/22) and the ISEcp1 element was found in nine of the strains analysed (9/22). One study has reported that CTX-M ESBL genes are often associated with an ISEcp1 element, which may provide a higher level of expression of the plasmid-located blaCTX-M genes and facilitate the spread of resistance . Our study provides the first evidence for the presence of an E. coli ST131 clone in Cameroon. Strict hygiene measures, staff training to improve hand-washing procedures and changes to the antibiotic policy are essential to limit the spread of these E-ESBLs in hospitals and the community.