Sequence analysis of genes mediating extended-spectrum beta-lactamase (ESBL) production in isolates of Enterobacteriaceae in a Lagos Teaching Hospital, Nigeria

Background Extended-spectrum β-lactamases (ESBLs) in Gram-negative organisms is now a major concern in Enterobacteriaceae worldwide. This study determined a point-prevalence and genetic profiles of ESBL-producing isolates among members of the family Enterobacteriaceae in Lagos State University Teaching Hospital Ikeja, Nigeria. Methods Consecutive non-repetitive invasive multidrug-resistant isolates of the family Enterobacteriaceae obtained over a period of 1 month (October 2011) were studied. The isolates were identified using VITEK-2/VITEK MS Systems. Susceptibility testing was performed using E test technique; results were interpreted according to the criteria recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012). ESBL production was detected by E test ESBL method and confirmed by polymerase chain reaction (PCR). Results During the one-month study period, 38 isolates with ESBL phenotypic characteristics were identified and confirmed by PCR. Of these, 21 (55.3 %) were E. coli, 12 (31.6 %) K. pneumoniae, 3 (7.9 %) Proteus spp., 1 (2.6 %) each M. morganii and C. freundii. Thirty (79 %) harbored blaCTX-M genes. Sequence analysis revealed that they were all blaCTX-M-15 genes. Twenty-nine (96.7 %) of these, also harbored blaTEM genes simultaneously. All the CTX-M-15-producing isolates carried insertion sequence blaISEcP1 upstream of blaCTX-M-15 genes. The E. coli isolates were genetically heterogeneous, while the K. pneumoniae had 98 % homology. Conclusions Our point-prevalence surveillance study revealed a high prevalence of Enterobacteriaceae isolates harboring blaCTX-M-15 in the Hospital. Urgent implementation of antibiotic stewardship and other preventive strategies are necessary at this time in our hospital.


Background
Gram-negative organisms belonging to the family Enterobacteriacae commonly produce beta-lactamases, which confer resistance to most penicillins but not to expanded-spectrum cephalosporins. The genes encoding these beta-lactamases are plasmid-borne, and belong to the TEM-1, TEM-2, and SHV-1 types [1]. Infections caused by members of the family Enterobacteriaceae are treated with cephalosporins, particularly the third-and fourthgeneration cephalosporins. However, resistance to these drugs has emerged throughout the world with widespread of resistant strains. By the middle of the 80s, resistance to the expanded-spectrum cephalosporin became evident and various studies have shown that the resistance was mediated by structural mutation in the older enzymes [1,2].
The literature is awash with evidence of global dissemination of CTX-M type ESBL of pandemic proportion [9,13,14]. The production of this enzyme is mediated by the bla CTX-M gene, which confers resistance to the thirdgeneration cephalosporins particularly in Escherichia coli and Klebsiella spp. [15]. Several phenotypic and genotypic studies have documented the emergence of CTX-M-type extended-spectrum β-lactamases as well as the genes encoding their production in Enterobacteriaceae, in Nigeria. However, most of these studies have been on randomly selected E. coli and K. pneumoniae [16][17][18][19] and Salmonella enterica serovar Typhi [20] and thus the true prevalence of CTX-M in most parts of the country is unknown. In our hospital, cephalosporins are first line antibiotics used in the treatment of Gram-negative sepsis and other infective conditions. A previous study conducted earlier in the same hospital demonstrated high resistance rates among clinically significant species of the family Enterobacteriaceae against the cephalosporins and other β-lactam antibiotics [21]. With such high resistance rates, it is conceivable that CTX-M would also be the dominant ESBL type responsible for this high level of resistance in our hospital.
This study was undertaken to investigate a pointprevalence and genetic profiles of ESBL-producing isolates among members of the family Enterobacteriaceae causing infections in patients on admission in a tertiary hospital in Lagos.

Bacterial isolates and setting
Thirty-eight consecutive isolates of multidrug-resistant invasive species of the family Enterobacteriaceae were obtained over a period of one month (October 2011), during routine laboratory investigation, from in-patients at the Lagos State University Teaching Hospital (LASUTH) located in Ikeja, a suburban part of Lagos. The hospital serves as a referral center for about 6 million Lagosians. It has one adult Intensive Care Unit (ICU), 1 Critical Care Unit (CCU), a dialysis unit and an oncology unit. The age, sex, nationality, previous hospital admissions, and documented travel history were all carefully noted. Duplicate isolates were omitted from the study.

Susceptibility testing
Susceptibility testing of the isolates was performed by determining the minimum inhibitory concentrations (MICs) of amikacin, amoxicillin-clavulanic acid, cefepime, cefotaxime, cefoxitin, ceftazidime, ciprofloxacin, colistin, ertapenem, imipenem, gentamicin, meropenem, piperacillin-tazobactam, and tigecycline on Mueller-Hinton agar plates using E test (bioMerieux) technique. A quality control strain, Escherichia coli ATCC 25922 was included in each run. The results were interpreted according to the break-points and criteria recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012) [22].

Confirmation of ESBL
All the ESBL-producing isolates were phenotypically detected by E test ESBL method using cefotaxime (CT)/cefotaxime combined with clavulanic acid (CTL) and ceftazidime (TZ)/ceftazidime combined with clavulanic acid (TZL) (bioMerieux) and were confirmed by polymerase chain reaction (PCR). In-house ESBL-producing E. coli strain K31 [23] and ESBL-negative strain were included in the test runs as positive and negative controls, respectively.

PCR amplification and sequencing
PCR assays were carried out with a series of primers to detect the following genes mediating bla SHV , bla TEM , bla CMY-6 , bla CMY-4 , and bla CTX-M [24]. PCR products were sequenced with a 3130xl Genetic Analyzer (Applied Biosystems, Hitachi High-Technologies Corporation, Tokyo, Japan). Sequences were compared and aligned with reference sequences available in the GenBank.

Genotyping of isolates
Fifteen randomly selected strains of the bla CTX-M-15positive isolates (9 E. coli and 6 K. pneumoniae) were investigated for genetic relatedness using pulsed-field gel electrophoresis (PFGE) with Xba1 digestion of the genomic DNA separated by electrophoresis in 1.2 % agarose gel [26] and the strains compared by differences in number and mobility of the bands.

Prevalence of bla CTX-M ESBL-positive Enterobacteriaceae
The ESBL-producing E. coli and K. pneumoniae were multidrug-resistant isolates (MDR) showing resistance to five or more antibiotics ( Table 1). The number of MDR P. mirabilis, M. morganii and C. freundii isolates was too small for any analysis.
As shown in Table 2, of the 38 ESBL-producing isolates, 30 (79 %) harbored bla CTX-M genes. Sequence analysis of these genes revealed that they were all bla CTX-M-15 . Twenty-nine (96.7 %) of these, also harbored bla TEM genes simultaneously. A combination of bla CTX-M-15 , bla TEM and bla SHV were found in 6 isolates; the distribution of the bla SHV was bla SHV-11 (2), bla SHV-12 (2) and bla SHV-112 (2). All 30 isolates positive for bla CTX-M carried insertion sequence bla ISEcP1 upstream of the bla CTX-M-15 genes.
The ESBL E-test for ESBL production detected all the bla CTX-M , bla TEM and bla SHV -positive isolates confirmed by PCR. Testing both cefotaxime and ceftazidime was necessary for detection of CTX-M-positive isolates. As demonstrated in Table 1, the MIC 90 s of the third-generation cephalosporins were all >256 μg/ml. Our bla CTX-M -positive isolates, with or without SHV and TEM, were susceptible to amikacin, colistin, imipenem, meropenem and tigecycline. Three isolates (2 E. coli and 1 K. pneumoniae) were not inhibited at the cut-off breakpoint (0.5 μg/ml) of ertapenem but were susceptible to imipenem (MIC = 0.125 and 0.5 μg/ml) and meropenem (0.5 and 0.75 μg/ml). There was no specific distribution of the CTX-M-15-positive isolates among ethnic groups of the Yoruba race. Two E. coli and a Proteus spp. isolates were positive for ESBL but the genes encoding their production were not detected.

Clonal relatedness of the bla CTX-M-15 harboring isolates
The fingerprinting of the genomic DNA of the bla CTX-M-15 -positive randomly selected isolates of E. coli and K. pneumoniae showed that the E. coli isolates were genetically heterogeneous, as the isolates did not fall within a particular cluster. On the other hand, there was about 98 % similarity with the K. pneumoniae isolates.

Discussion
The predominant genotype of the ESBL found in the clinical isolates of Enterobacteriaceae in this study was bla CTX-M -type genes with all being bla CTX-M-15 ; over 96 % of these 38 isolates also harbored a bla TEM β-lactamase gene. Five isolates co-harbored narrowspectrum bla SHV-11 and other bla SHV genes. Our data demonstrate a very high prevalence of bla CTX-M type ESBL genes among the multidrug-resistant (MDR) isolates studied. This lends credence to the worldwide pandemic spread of the CTX-M β-lactamase enzyme, a phenomenon that has reached epidemic proportion among members of the family Enterobacteriaceae. The ESBL mediated by bla CTX-M type β-lactamase genes are  undoubtedly the most widespread enzymes produced among members of this family. This assertion is predicated on the fact that over 79 % of our ESBL-producing isolates harbored bla CTX-M , the gene that mediates CTX-M enzyme production. Remarkably, all the CTX-M enzymes were CTX-M-15, making this type of β-lactamase the most common type detected in Enterobacteriaceae in this Lagos hospital. The dominance of bla CTX-M-15 β-lactamase genes in our snap surveillance study confirms what other workers had earlier reported in North America [15,27,28], Europe [29,30], South America [31] the Middle East [23,32] and Nigeria [16][17][18][19][20]. This ESBL gene has also been incriminated in community outbreaks of multidrug resistant (MDR) E. coli infections in some parts of the UK [30] and elsewhere [15]. The literature on the genetic characteristics of ESBLs in Nigeria and, indeed, Africa is sparse. The data emanating so far from Nigeria does not include the prevalence of the CTX-M ESBL in an in-patient setting.
With this study we have demonstrated that the prevalence of Enterobacteriaceae isolates carrying genes that encode CTX-M-15 ESBL enzymes is at an unacceptable level with potential clinical and financial implications for the hospital.
The clinical implication of this finding is that many patients infected by MDR Gram-negative bacteria stand the risk of treatment failure and cases of fatalities may increase. Thus, treatment of such infections assumes a great challenge to the clinician and the clinical microbiologists as treatment options are limited to very expensive and sometimes toxic drugs. Added to this burden is the fact that the location of the mobile genetic element, ISEcp1, a single copy insertion sequence responsible for mobilization of bla genes, was found upstream of the bla CTX-M genes. This has grave consequences as it might conceivably facilitate the spread of these genes among the Enterobacteriaceae within the hospital. Transfer of the bla CTX-M-15 genes to recipient E. coli J53 has been shown to be quite readily achievable. This suggests that resistance genes can easily move from one species to another with the possibility of easy interspecies transfer. One of the limitations of this study was that plasmids were not studied to determine if these genes were the same.

Conclusion
In conclusion, our study demonstrated an explosive emergence of isolates harboring bla CTX-M-15 gene mediating CTX-M-type ESBL production in invasive members of family Enterobacteriaceae. Immediate implementation of antibiotic stewardship and other preventive strategies are necessary to stem the tide of dangerous spread of MDR Enterobacteriaceae in this Lagos hospital.