Dissemination of Incf Plasmids Carrying Beta-lactamase Genes in Gram-negative Bacteria from Nigerian Hospitals

Introduction: Production of beta-lactamases is the predominant cause of resistance to beta-lactam antibiotics in Gram-negative bacteria. We investigated the diversity of plasmid-borne beta-lactamase genes and replicon type of the plasmids carrying the respective genes in Gram-negative bacteria recovered from clinical infection in Nigerian hospitals. Methodology: A total of 134 Gram-negative bacteria of 13 species were analyzed for antimicrobial susceptibility, phenotypic and genotypic detection of various beta-lactamases, and plasmid analysis, including replicon typing. Multiplex PCR showed that 6 isolates carried plasmidic AmpCs (ACT-1, DHA-1 and CMY-2); these enzymes were detected only in isolates possessing CTX-M beta-lactamases. Of 13 (76.9%) representative plasmids investigated in detail, 9 (69.2%) were self-transferable when selected by a beta-lactam and the plasmids once transferred coded for beta-lactam resistance. Replicon typing indicated IncF as the common vector encoding for beta-lactamases. Conclusions: The study showed a diversity of beta-lactamase genes disseminated by conjugative IncF plasmids in Gram-negative bacteria; TEM-1, SHV-1, OXA-1, CTX-M-15, CTX-M-3and plasmidic AmpC enzymes are in common circulation in Nigeria.


Introduction
The presence of multidrug-resistant bacterial strains harbouring several expanded-spectrum betalactamases simultaneously (e.g., extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases) that are also resistant to fluoroquinolones and aminoglycosides, has been reported worldwide but there is scarcity of information in Nigeria.Indeed, only a few studies have reported isolation and frequency of beta-lactamases from Nigerian hospitals [1][2][3].The CTX-M beta-lactamases are the most widespread ESBL enzymes globally [4].They were initially reported in the second half of the 1980s, and their rate of dissemination among bacteria and in most parts of the world has increased dramatically since 1995 [5].
Enterobacteriaceae and Pseudomonadaceae are the most important Gram-negative causes of nosocomial and community acquired infections, and they are a major cause of nosocomial infections associated with high mortality.In Nigeria, extendedspectrum cephalosporins, particularly third-generation and fluoroquinolones, remain the drugs of choice to treat infections caused by these microorganisms.Carbapenems have recently been introduced in Nigeria and are rarely used by physicians.ESBLs have been described in Africa [6,7] and have considerable implications for the developing world, where use of antibacterial agents is largely indiscriminate and uncontrolled.
Recent studies have demonstrated the clonal expansion of certain enterobacterial clones that are able to acquire plasmids harbouring different ESBLs and plasmidic AmpCs [8].The high prevalence of bla ESBL genes in different parts of the world is caused by the horizontal transfer of plasmids among unrelated clones and also within local or international epidemic clones.Plasmid transmission has played a significant role in the global spread of beta-lactamases.IncF plasmids have been associated with the abrupt worldwide emergence of clinically relevant ESBLs, such as CTX-M-15 [9][10][11], but also with the spread of plasmid-mediated AmpC genes (bla CMY and bla DHA ) [12,13].Characterization of plasmids encoding bla CTX- M-15 from Canada, France, Spain, and the United Kingdom has classified most of them as members of incompatibility group FII [14,15].Similarly, Coque et al. reported IncF plasmids harbouring multiple antimicrobial drug resistance determinants, including the aac(6')-Ib-cr gene, in several countries from America, Asia, Middle East and Europe [9].The present study investigated the diversity of plasmidborne beta-lactamases and replicon type of the plasmids carrying the respective genes in Gramnegative bacteria isolated from clinical specimens of patients attending tertiary care hospitals in Nigeria.

Bacterial strains
One hundred and thirty-four clinical Gramnegative bacterial isolates of 13 species were obtained from 585 non-duplicate clinical specimens including aspirates, ear swab, wound swab, throat swab, high vaginal swab, eye swab, sputum, urine, cerebrospinal fluid and blood culture for the period between 2005 and 2007.Single isolates from each specimen were retained.The isolates were from four teaching hospitals in south-western Nigeria: University College Hospital, Ibadan; Obafemi Awolowo University Teaching Hospital, Ile-Ife; Ladoke Akintola University of Technology Teaching Hospital; and Osogbo and Olabisi Onabanjo University Teaching Hospital, Sagamu.All isolates were speciated using API 20E strips (BioMerieux, Marcy l'Etoile, France).

Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of beta-lactam antibiotics were determined.MIC testing was performed using the agar dilution method according to the recommendation of the British Society for Antimicrobial Chemotherapy (BSAC) (http://www.bsac.org.uk/susceptibility_testing/guide_to_antimicrobial_susceptibility_testing.cfm).All runs included the control organisms Escherichia coli (NCTC 10418) and Pseudomonas aeruginosa (NCTC 10662).A start and finish plate without antibiotic was also included as a growth control.

Detection of beta-lactamases by nitrocefin hydrolysis
Broth cultures of each test strain as well as an ampicillin-resistant strain (E. coli NCTC 10418, carrying pUC18) were incubated overnight in 5 ml of LB broth.Two hundred microlitres of the resulting overnight cultures were transferred into wells of a microlitre tray; sterile broth was included to serve as a negative control.Ten microlitres of nitrocefin solution prepared according to the manufacturer's instructions (Fisher Scientific, Loughborough, UK), were added to each well.Beta-lactamase production was inferred when the broth turned red within 30 minutes of nitrocefin addition, as directed by the manufacturer.

Detection of extended-spectrum beta-lactamases (ESBLs) by the double disc method
Suspensions of broth culture of each test strain were prepared in sterile water to give an inoculum equivalent to a 0.5 McFarland standard before being used to inoculate the surface of iso-sensitest agar plates [16].ESBL positive (K.pneumoniae ATCC 700603) and negative (E. coli ATCC 25922) control strains were used in these experiments.The double disc method described by Jalier et al. was followed [17].

Detection of AmpC by simple disc diffusion
AmpC beta-lactamase production was detected for strains producing ESBL using cefepime and cefpodoxime disks in combination with clavulanate as previously described [18].The plate and the inoculum were essentially the same as for ESBL detection described above.Following overnight incubation at 37ºC, AmpC beta-lactamases were detected using a difference of ≥14 mm between cefepime/cloxacillin and cefpodoxime/cloxacillin discs.

Amplification of beta-lactam genes
PCR was used to detect genes encoding resistance to beta-lactams (bla OXA , bla SHV , bla TEM , bla CTX-M ).Multiplex PCR was used for bla AmpC genes [19].Amplimers resulting from these PCR reactions were sequenced to confirm the identity and specific variant of each gene identified, and sequences were aligned to known reference sequences using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Gene transfer
Transformation experiments were performed by electroporation (Gene pulser; Bio-rad, Hemel Hempstead, UK) of purified plasmid DNA into competent Escherichia coli α-select (Bioline, London, UK, efficiency of > 10 9 ).The protocol of the manufacturer was followed to determine the properties of plasmids.The mixtures (200 µl) containing transformants were plated onto selective agar (ampicillin, 16µg/ml), allowed to stand for a few minutes, and incubated at 37ºC overnight.To control for antibiotic activity, the wild type strain was also inoculated onto the selective plates.Minimum inhibitory concentrations of the transformants and recipient strain were measured.Plasmid DNA from the transformants were isolated and restricted with EcoRV and BamHI.
Conjugation experiments were performed by broth and filter mating assay using E. coli DH5α with a chromosomal mutation conferring rifampicin resistance as recipient cells.Suspensions of 200 µl were plated out onto selective plates containing rifampicin (100 µg/ml) and ampicillin (16 µg/ml).For controls, test strains were plated on rifampicin plates, while recipient cells were plated on separate plates of rifampicin and ampicillin.

Estimation of plasmid size
Plasmid size was estimated as previously described [20].

Identification of plasmids by PCR-based replicon typing
Incompatibility/replicon PCR-based typing was used to trace plasmids conferring drug resistance.In this method, 18 pairs of primers were designed for five multiplex-and three simplex-PCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Iy, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons, representing the major plasmid incompatibility groups circulating among the Enterobacteriaceae [21].Because of the high level of homology between the K and B/O replicons, the same forward primer was used in both these simplex PCRs.Plasmid DNA of transformants and transconjugants were generated as a DNA template for PCR [22].

Identification of bacterial species
A total of 13 Gram-negative enteric bacteria were identified and they consisted of Klebsiella pneumoniae, Escherichia

Susceptibility testing
Determination of precise MIC values confirmed the numbers of isolates resistant to clinical breakpoint concentrations for all antibiotics using BSAC guidelines.The MIC results also showed that the level of resistance to beta-lactam antibiotics was extremely high, MIC 50 greater than clinical breakpoints.The mean percentage of strains resistant to each of the beta-lactam drugs was 85% (Table 1).

Phenotypic detection of beta-lactamase
Beta-lactamase production was inferred when diluted broth turned red within 30 minutes of adding nitrocefin.Of the 134 isolates, 111 (82.8%) were found to be beta-lactamase positive by this method: 85.7% of K. pneumoniaes (54 out of 63), 71.4% of E. coli (20 out of 28), 69.2% of P. aeruginosa (9 out of 13), and 81.8% of P. mirabilis (9 out of 11).The rest of the strains were uniformly positive in the nitrocefin reactions.

Phenotypic detection of ESBLs
ESBL production was inferred when the zone of inhibition around the ceftazidime and cefotaxime discs was expanded by the clavulate by ≥ 5 mm.Among the 134 isolates, 28 (20.9%) were found to be ESBL positive by the combined disc test and all 20.9% ESBL-positive isolates were also beta-lactamase positive by nitrocefin.

Phenotypic detection of AmpC
Phenotypic detection of AmpC by disc diffusion using cefepime and cefpodoxime revealed that AmpC enzymes were present in 29 of 44 ESBL (ESBL by DDDT and CTX-M by PCR) producing isolates (65.9%).Twenty-five AmpC positive isolates were detected and interpreted as positive using an increase of ≥ 14 mm in zone diameter around the antibiotic disc with added inhibitor compound between cefepime and cefpodoxime.

Genotypic detection of plasmidic AmpC enzymes
Multiplex PCR was used to screen for isolates with plasmidic AmpCs.The results showed that six isolates were positive by PCR for AmpC genes and these genes were only found in isolates carrying CTX-M genes.The AmpC-positive strains cut across most of the clinical sites of the specimens, ranging from wound, ear, aspirate, blood culture, and catheter tip.Out of the six AmpC-positive genes, one was amplified using EBC primers, four by DHA group primers and one by CIT primers.Sequencing identified these genes as ACT-1, DHA-1 and CMY-2, respectively.DHA-like enzymes were present predominantly in the strains.No enzymes belonging to the ACC, FOX, or MOX family were detected.Only four of the six AmpC-positive strains produced an inducible AmpC.AmpC betalactamase-positive strains appeared to have low-level resistance to the third-generation cephalosporins with an MIC range of 0.25 to 256 µg/ml comparatively.Resistance to quinolones among AmpC-positive strains was high with MICs ranging between 8 µg/ml and 256 µg/ml (Table 3).

Plasmid DNA analysis
The strains were found to harbour plasmids ranging from < 20 to > 200 kb as observed by plasmid extraction and electrophoresis.A number of plasmids were common to multiple isolates.Restriction digestion to further type plasmids used EcoRV endonuclease to select representative restriction patterns from the diverse profiles for further studies.Digestion products were found to yield several bands with sizes ranging from 6kb to > 200 kb (data not shown).

Gene transfer
Transformation experiments revealed that the plasmids once transferred coded for beta-lactam resistance.Beta-lactamase transformants showed significantly higher resistance to amoxicillin and ceftazidime (Table 4).The transformant strains carried a common plasmid approximately of 108-kb.

Replicon type of plasmids
To identify the replicon type of the common 108kb plasmid observed, 18 specific primer pairs were used to amplify replicon specific sequences, on the basis of the multiple comparative analysis of nucleotide sequence deposited in the Gene Databank.A positive result was seen for IncF, indicating this is the replicon type of the common plasmids encoding beta-lactamases.

Discussion
This study indicates that bla genes were located on epidemic plasmids encoding different resistance phenotypes from strains from different hospitals with an estimated size of 108-kb.This result implies the spread of a common plasmid among different strains and essentially in different parts of Nigeria.Some of these plasmids encoded more than one type of ESBLs or AmpC.Plasmids with the ability to spread efficiently, or epidemic plasmids, are believed to be responsible for disseminating CTX-M ESBLs [23] by conjugative plasmids rather than by clonal expansion of a bacterial host strain [24].The replicon type of the common plasmids for the beta-lactamases was IncF; this suggests the circulation of a prevalent IncF plasmid carrying different bla genes.To our knowledge, this study is the first report of plasmid identification by incompatibility group based on PCR in Nigeria.This observation suggests interesting clues on the evolution of related plasmids that can gain additional resistance determinants and/or lose the transferability gene-associated functions, evolving in different plasmid variants.Our study corroborates the current worldwide spread of clinically relevant bla genes such as CTX-M-15 [9][10][11], also with the spread of plasmid-mediated AmpC genes (bla CMY and bla DHA ) [12,13].
IncF plasmids have been reported to be of low copy number with a size of > 100 kb having more than one replicon promoting the initiation of replication.The multi-replicon status has been described to be one means by which plasmids with a narrow host range can accomplish broad host range replication [11], which explains why they are widely diffused in clinically relevant Enterobacteriaceae, representing one of the most frequently encountered plasmid types.The IncF plasmids carried various ESBLs including AmpC-type DHA-1 and CMY-2 genes.IncF plasmids harbouring multiple antimicrobial drug-resistance determinants including aac(6')-Ib-cr gene have been described [9,25].The phenotypic detection of betalactamases indicated a high prevalence in clinical isolates in this country; these strains prove a clinical treatment challenge and are likely to be associated with high levels of mortality and morbidity.TEM-and SHV-derived ESBLs are most often found in E. coli and K. pneumoniae [26,27].TEM-1 was the most commonly identified beta-lactamase in this study.In previous African studies, this enzyme has previously been reported only from South Africa, where it was identified initially in ESBL-producing Klebsiella species, Proteus species, Enterobacter species, and E. coli strains [28,29] and in strains of Salmonella [30].

AmpC
There has been an increase in the isolation of CTX-Mproducing bacteria globally, with reports from Europe, North and South America, Africa and Asia [4].In Africa, clonal CTX-M-12-producing K. pneumoniae strains were isolated in Kenya [31].There are few reports of bla genes, especially bla CTX-M , in Nigeria.Twenty-five isolates were CTX-M-positive (18.7%), although among 28 ESBL-producing isolates, only 8 were CTX-M-positive by PCR.Plasmids coding for ESBLs that express a low level of resistance to betalactams [32] or contain multiple silenced antibiotic resistance genes [33] are of particular concern, as they may serve as reservoirs of antibiotic resistance determinants in bacteria that cannot be detected phenotypically.A previous report of CTX-M in Nigeria identified CTX-M-15 [2].In this study, sequencing identified most of the CTX-M enzymes to be CTX-M-15 and only one was CTX-M-3.CTX-M-14, CTX-M-15, CTX-M-9, CTX-M-3 and CTX-M-2 are the most commonly isolated enzymes worldwide.The prevalence of plasmidic AmpC-mediated resistance in Nigeria is not known due largely to the few surveillance studies seeking clinical strains producing AmpC beta-lactamases and to the technical difficulties for laboratories to detect this resistance mechanism.Plasmid-mediated AmpC was detected in 6 isolates by PCR and these genes were found in isolates co-producing other ESBLs such as CTX-M-15, TEM-1, SHV-1and OXA-1.The AmpC-positive strains cut across most of the clinical sites of the specimens, ranging from wound, ear, aspirate, blood culture and catheter tip.The majority of these patients had actually been treated with beta-lactam antibiotics.Sequencing identified these genes as ACT-1, DHA-1 and CMY-2.Only four of the six AmpC-positive strains produced an inducible AmpC with the disc diffusion method, while two of the AmpC positive strains did not express inducible AmpC by the phenotypic method.

Conclusion
In conclusion, we describe the dissemination of IncF plasmids in K. pneumoniae, E. coli, P. mirabilis, and P. aeruginosa carrying various types of ESBLs including CTX-M-15 and CTX-M-3 as well as AmpCs including DHA-1, CMY-2 and ACT-1 in hospitals in Nigeria.

Table 1 .
MICs of selected strains to beta-lactam antibiotics

Table 2 .
Beta-lactamase production by the isolates n: frequency, a: positive by nitrocefin test b: positive for ESBL production by DDDT ( ): number in parenthesis is %

Table 4 .
Resistance profiles of donor strains and transformants