Ibukun E. Aibinu

Extended-Spectrum β-Lactamase Enzymes in Clinical Isolates of Enterobacter Species from Lagos, Nigeria

ABSTRACT Over a 9-month period, 8 of 40 nonduplicate isolates of Enterobacter spp. producing extended-spectrum β-lactamase (ESBL) were detected for the first time from two hospitals in Lagos, Nigeria. Microbiologic and molecular analysis confirmed the presence of ESBL. Only four isolates transferred ESBL resistance as determined by the conjugation test, and pulsed-field gel electrophoresis showed genetically unrelated isolates.

Emergence of bla CTX-M-15, qnrB1 and aac(6′)-ib-cr resistance genes in Pantoea agglomerans and enterobacter cloacae from Nigeria (sub-Saharan Africa)

Resistance of Enterobacter species to extended-spectrum cephalosporins is known to be mediated by hyperproduction of chromosomal AmpC b-lactamases. However, the additional expression of plasmid-encoded extended-spectrum b-lactamases (ESBLs) has become more prevalent worldwide in recent years (Ko et al., 2008). In Nigeria, ESBL production in Enterobacter species has been associated with TEMand SHV-type ESBLs (Aibinu et al., 2003; Kasap et al., 2010). Other b-lactamase resistance determinants, conferring resistance to extendedspectrum cephalosporins, such as blaVEB, blaOXA and blaCMY, have recently been reported in Nigerian Providencia species strains (Aibinu et al., 2011). In addition, the worldwide spread of CTX-M-15 (Cantón & Coque, 2006) has reached Nigeria, having being identified in Klebsiella species and Escherichia coli (Soge et al., 2006; Olowe et al., 2010). There is no documented report yet on ESBL production mediated by blaCTX-M-15 or the association of the spread of PMQR determinants in Enterobacter species from Nigeria. This study reports the phenotypic and genotypic characteristics of ten clinical isolates of Enterobacter species and one isolate of Pantoea agglomerans with respect to the occurrence of blaCTX-M and other resistance genes. The Enterobacter species, which consisted of Enterobacter asburiae (n51), Enterobacter aerogenes (n51) and Enterobacter cloacae (n58), and the Pantoea agglomerans isolate represented 9.5 % of all members of the Enterobacteriaceae isolated within a period of 6 months from October 2008 to March 2009 at Lagos University Teaching Hospital (LUTH), a tertiary hospital, in Nigeria. Enterobacter agglomerans was previously renamed Pantoea agglomerans to reflect its genetic distance from the genus Enterobacter (Sanders & Sanders, 1997).

Emergence of β-lactamases OXA-10, VEB-1 and CMY in Providencia spp. from Nigeria

Sir, Resistance to cephalosporins of the third and fourth generations in Enterobacteriaceae is an increasing problem worldwide. This resistance is mainly attributed to the production of extended-spectrum b-lactamases (ESBLs). In Nigeria SHV-, TEMand CTX-M-type ESBLs have been reported in Enterobacter spp., Klebsiella spp. and Escherichia coli. Besides these classical ESBLs, there are various other plasmid-mediated b-lactamases that are less common but which are regarded as emerging and increasing in frequency among the b-lactamase family. Here we report the emergence of OXA-10, VEB-1 and CMYb-lactamases and mobile genetic elements in three clinical isolates of Providencia spp. isolated between October 2008 and April 2009 in two tertiary hospitals in Nigeria. Three strains of Providencia spp. were identified using the VITEK 2 GN card system following cultivation of clinical samples on MacConkey agar and blood agar base with 5% sheep blood (Oxoid, UK). Isolate Providencia rettgeri 58K was recovered in late 2008 from the catheter tip of a patient hospitalized at Lagos University Teaching Hospital (LUTH) in Lagos, Nigeria. The patient was notably hypertensive and was diagnosed with an exacerbation of chronic kidney disease, secondary to adult polycystic kidney disease. He had been catheterized for 19 days, with insertion of the catheter from the private hospital where he was hospitalized before his referral to LUTH. The patient was not febrile at any time and there was no infection documented. Isolate Providencia stuartii V1 was recovered from the gunshot wound of a patient admitted to the National Orthopaedic Hospital Igbobi (NOHI), Lagos, in early 2009, while isolate P. stuartii V2 was also recovered at NOHI from the wound swab of a patient who had had a motorcycle accident. The three patients had not received antibiotics prior to the isolation of the strains. The three clinical strains of Providencia spp. were multiply resistant to different b-lactams, fluoroquinolones and aminoglycosides but remained susceptible to carbapenems using the VITEK 2 GN card system (Table 1). ESBL and AmpC production was confirmed using the double disc synergy tests (ESbL/AmpC test D68C; Mast Diagnostica GmbH, Reinfeld, Germany) and ESBL-Etest strips (bioMerieux, Nurtingen, Germany). By PCR and sequence analysis, b-lactamase genes blaCMY-4, blaTEM-1, blaVEB-1 and blaOXA-10 were identified in isolate P. rettgeri 58K. Isolate P. stuartii V2 harboured blaCMY-41 and blaTEM-52, while ESBL genes blaVEB-1 and blaOXA-10 were found in isolate P. stuartii V1. The blaVEB-1 genes were located on sul-type class 1 integrons and ISCR2 mobile elements. The blaOXA-10 genes were also encoded on class 1 integrons. Furthermore, the plasmid-mediated quinolone resistance gene qnrA1 was found in all three clinical isolates (Table 1). Transfer of all b-lactamase genes and gene qnrA1 was successfully performed for the clinical isolates P. rettgeri 58K and P. stuartii V1 using broth mating assays with a sodium azide-resistant E. coli J53 recipient. In order to ascertain the potential transfer and acquisition of b-lactamase genes or resistant strains via the food chain, we investigated resistance determinants of Providencia spp. from faecal samples of apparently healthy farm animals. Farm animals serve as major sources of meat products for the population of Lagos. Between October 2008 and April 2009 we recovered 97 Enterobacteriaceae isolates from 115 faecal samples of different animals from three local farms. Using the VITEK 2 GN card system we identified seven Providencia spp. recovered from chickens (n1⁄46) and pigs (n1⁄41). Typing of human and animal Providencia spp. isolates by enterobacterial repetitive intergenic consensus PCR revealed different patterns, indicating no clonal relationship of these isolates. In contrast to the clinical strains, all animal isolates were susceptible to most of the antibiotics and none produced b-lactamases (Table 1). The variable regions of class 1 integrons in clinical and animal strains harboured the classic qacED-sul1 region and dfrA genes (dfrA1, dfrA14 and dfrA15). The class 2 integron was additionally found in two isolates from chickens. Furthermore, the sequence of one P. rettgeri isolate from a chicken (156K) showed 99% identity with the integrating conjugative element ICEPalban1 described in a Providencia alcalifaciens isolate from the USA (GenBank accession no. GQ463139). The absence of b-lactamase genes and quinolone resistance genes in the animal isolates suggested no correlation of horizontal transfer of these resistance genes between animal and human Providencia spp. strains. However, simultaneous occurrence of class 1 and 2 integrons in two isolates from chickens highlighted a possible variety of recombinatorial events among these genetic platforms according to Machado et al. This study presents the first report of Providencia spp. producing CMY-type and OXA-10 b-lactamases. To our knowledge, the only known documentation of a blaCMY gene in Providencia spp. is the submission of a blaCMY-16 gene sequence from a P. stuartii isolate from Tunisia (GenBank accession no. FJ855437.1). Horizontal gene transfer has played a major role in the global spread of b-lactamases and Qnr determinants into various Gram-negative species. Furthermore, this is the first report of ICEPalban1 in a P. rettgeri animal isolate. Though we only found a small number of Providencia spp. isolates, our data suggest that further population-based prevalence studies are needed in order to monitor the ability of clinical Providencia spp. to be a reservoir of different resistance genes. The nucleotide sequences of some of the resistance genes in this study have been deposited in the GenBank nucleotide sequence database under accession numbers GU056840, GU056841, GU056843 and GU056844. Research letters