CO-PRODUCTION OF EXTENDED-SPECTRUM BETA-LACTAMASES AND METALLO BETA-LACTAMASES AMONG MULTI-DRUG RESISTANT GRAM-NEGATIVE BACTERIA ISOLATES COLLECTED FROM TERTIARY HOSPITALS IN OYO STATE, NIGERIA

Extended-spectrum beta-lactamases (ESBLs) and metallo beta-lactamases (MBLs) are compromising the chemotherapeutic use of cephalosporins and carbapenems respectively. This study investigated the burden of ESBLs and MBLs co-production among multi-drug resistant (MDR) Gram-negative bacteria collected from two tertiary hospitals in Oyo State. A total of 240 non-duplicated clinical isolates of Escherichia coli, Klebsiella spp. and Pseudomonas spp. were collected from the Microbiology units of two tertiary hospitals in Oyo State and their identities authenticated using standard identification techniques. Antimicrobial susceptibility testing was carried out by disc-diffusion method and isolates exhibiting resistance to ≥3 classes of antibiotics selected as MDR strains. ESBL and MBL production was detected by double-disc synergy test (DDST) and combined-disc-diffusion test (CDDT) respectively. Selected beta-lactamase genes were detected by PCR, amplicons sent out for sequencing and phylogenetic tree of the sequences constructed using Mega X software. MDR was exhibited by 43.8% of the isolates. ESBLs and MBLs were produced by 32.4% and 7.6% of the MDR isolates respectively. Co-production of ESBL and MBL was observed in 6.7% of the MDR isolates. BlaCTX-M-15 (67.7%), blaTEM-1 (55.9%), blaSHV-1 (47.1%), co-existing blaTEM + blaSHV, blaTEM + blaCTXM, blaCTX-M + blaSHV (each in 5.9%) and blaCTX-M +blaTEM + blaSHV (26.5%) were detected among the ESBLproducers. MBL genes were not detected among the MBL-producers. Only blaTEM-1 sequences showed two different claudes on the phylogenetic tree. The occurrence of MDR isolates co-harbouring different classes of beta-lactamse genes observed in this study is of public health concern and hence, requires stricter control of antibiotic use.


INTRODUCTION
Antibiotics resistance is a major health problem worldwide with serious impact on cost of care, morbidity, mortality and the length of hospital stay of patients (Carlet et al., 2012, Prestinaci et al., 2015Founou et al., 2017;Shrestha et al., 2018). The development of antibiotic resistance has been attributed to various factors including selective pressure imposed by use and overuse of antibiotics in human medicine, agriculture and veterinary medicine among others (Hao et al., 2014;Chang et al., 2015;Blaser, 2016). The overuse and misuse of antibiotics in both clinical and community settings has led to the emergence of multi-drug resistance among microorganisms which are occasionally implicated in many infectious outbreaks worldwide (Alabi et al., 2017;Chaw et al., 2018;Chokshi et al., 2019). The lack of frequent monitoring of the use of antibiotics and lack of routine assessments of the potencies and efficacies of the various brands of antimicrobial agents available in the pharmaceutical markets, particularly in developing countries, have led to increase in antibiotic resistance (Lucet et al., 1999;Ayukekbong et al., 2017;Chokshi et al., 2019). In most developing countries like Nigeria, drugs could be brought into the country illegally through the back door without evaluation by relevant regulatory bodies. Some unpatriotic citizens out of the quest to get rich overnight engaged in adulteration and counterfeiting of drugs, particularly antibiotics, and thus exerting a huge impact on the development of antibiotic resistance (Glass, 2014;Kelesidis and Falagas, 2015).
The beta-lactam antibiotics are group of antibiotics widely used in the treatment of infections in both community and clinical settings, and they are divided into narrow, broad and extendedspectrum beta-lactams. While it has been well established that the narrow and some of the broad-spectrum classes of these group of antibiotics are now mostly ineffective clinically due to the emergence of the conventional beta-lactamases (i.e. the class of enzymes that inactivates the narrow spectrum antibiotics), the extended-spectrum beta-lactam antibiotics, such as the third generation cephalosporins (cefotaxime, ceftazidime, ceftriaxone) and the monobactams (aztreonam), developed to overcome the resistance conferred by the conventional beta-lactamases are gradually becoming less effective against most bacterial pathogens due to the advent of superior beta-lactamases known as extended-spectrum betalactamases (ESBLs) that hydrolyses the beta-lactam ring structure of the antibiotics (Alabi et al., 2016). The carbapenems, which were beta-lactam antibiotics of last resort commonly used in the treatment of the extended-spectrum beta-lactamase (ESBL) producing bacteria pathogens are also gradually becoming ineffective against some of these pathogens due to the development of a more superior betalactamases generally named carbapenemases part of which are the metallo beta-lactamases (MBLs) (Gaude and Hattiholli, 2013;Worthington and Melander, 2013;Levashov, 2014, Balsalobre et al., 2019, Mirza et al., 2019. Microbial resistance as a result of ESBL production was first reported in Europe and subsequently in the United States in a single isolate of Klebsiella ozaenae (Bradford, 2001) while MBL was initially reported in a clinical isolate of Serratia marcescens conferring resistance to imipenem (Jiang et al., 2018). Since then, they have emerged in other countries of the world including Nigeria among Gram-negative bacteria particularly the Enterobacteriaceae (Mendonca et al., 2007;Olowe et al., 2012) and Pseudomonas spp. (Oberoi et al., 2013;Kaur and Singh, 2018). Although, the genes coding for these enzymes are mostly borne on resistance plasmids and thus are transferable to other genera or species (Bradford, 2001;Kaur and Singh, 2018;Teklu et al., 2019), some have been reported to be on the chromosome (Mahrouki et al., 2011;Song et al., 2011).
The ESBL enzymes are known to be ineffective against the cephamycin (cefoxitin, cefotetan) and carbapenem (imipenem, meropenem, ertapenem) classes of the beta-lactam antibiotics and hence, the use of carbapenems as the antibiotic of last resort against the ESBL producing bacteria (Teklu et al., 2019). The emergence of the carbapenemases or metallo betalactamases (MBLs) known to hydrolyse the carbapenems and all other beta-lactams, thereby conferring resistance against the antibiotics, is of serious clinical and therapeutic problem (Pitout and Laupland, 2008;Bora et al., 2014;Nepal et al., 2017;Kaur and Singh, 2018).
The scenario of having a microorganism producing different variants of ESBL or MBL and even co-producing both will definitely be of significant public health concern because of their effects on the therapeutic options and treatment outcomes. This study, therefore, screened MDR Gram-negative bacteria genera from two selected tertiary hospitals in Oyo state, for the production of ESBLs and MBLs and further determined the occurrence of ESBL and MBL genes among these bacteria isolates.

MATERIALS AND METHODS Ethical Approval
There was no direct contact or an invasive procedure involved in this study and thus ethical approval is not required. Clinical isolates that are already isolated and characterized from patients that visited the out-patient sections of the teaching hospitals were collected from the microbiology laboratories of the two institutions involved in the study.

Study design and setting
This was a laboratory-based cross-sectional study involving Microbiology laboratory units of the two tertiary hospitals in Oyo State: University College Hospital (UCH), Ibadan and Ladoke Akintola University of Technology Teaching Hospital (LTH), Ogbomoso, Oyo State, Nigeria. The Gram-negative clinical isolates used in this study were collected using convenient sampling technique from the Microbiology laboratory units of the two tertiary hospitals and the research bench work conducted at the Molecular and Microbiology laboratory of the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Nigeria.

Collection of isolates
A total of 240 non-duplicated Gram-negative bacteria were collected between September and November, 2018 by convenience sampling on Nutrient agar (Oxoid Ltd, UK) slants at the Microbiology laboratories of University College Hospital (UCH), Ibadan and Ladoke Akintola University of Technology Teaching Hospital (LTH), Ogbomoso, Oyo state, Nigeria and transported to the main laboratory of the Department of Pharmaceutical Microbiology, University of Ibadan within 12 hours of collection for incubation at 37 o C for 24 hours. From the individual laboratory records, the entire isolates included in this study were from patients who visited the out-patient clinic of the hospitals and the isolates were recovered previously from different specimens such as urine, wound, ear swabs, sputum, catheter tips, pus, semen, aspirate, blood, high vagina swabs, stool, eye swabs and pleural fluid.

Confirmation of the identities of isolates
After 24 hours of incubation of the isolates collected on slants, they were sub-cultured on different selective media to observe their cultural characteristics and purity. Presumptive E coli collected were sub-cultured on MacConkey and Eosin Methylene Blue Agar (Oxoid Ltd, UK) to observe for the pink and green metallic sheen colonies respectively, characteristic of E coli. Presumptive Pseudomonas spp. were sub-cultured on cetrimide agar which permits the growth of the bacteria only. Presumptive Klebsiella spp. were sub-cultured on MacConkey agar to observe for the pink, mucoid colonies, characteristic of the bacteria. All the streaked plates were incubated in an inverted position for 24 hours at 37 o C.The emergent pure colonies were further subjected to standard biochemical test to confirm their identities. Confirmed isolates were streaked on fresh agar slants, incubated at 37 o C for 24 hours and kept at -4 o C for further studies.

Antibiotics susceptibility test
The confirmed isolates were subjected to antimicrobial susceptibility testing against eleven antibiotics using the discdiffusion method on Mueller-Hinton agar. The selected antibiotics are amoxicillin-clavulanic acid (20/10µg), cefotaxime (30µg), ceftazidime (30µg), cefoxitin (30µg), cefepime (30µg), aztreonam (30µg), gentamicin (10µg), ciprofloxacin (5µg), imipenem (10µg), meropenem (10µg) and ertapenem (10µg). Briefly, an overnight culture of each isolate was suspended in sterile normal saline and the turbidity adjusted to 0.5 McFarland standard by dilution. With the aid of sterile cotton swab, each bacterial suspension was inoculated on the dried-surface of Mueller-Hinton agar plate to form a single cell layer lawn. With the aid of a sterile forcep, each of the antibiotic disc was transferred on the inoculated agar plates at equidistance, allowed to stand on the bench for preincubation diffusion for 1 hour and then incubated in an inverted position for 24 hours at 37 o C. The Clinical Laboratory Standard Institute (CLSI, 2017) breakpoints for interpreting antibiotic susceptibility test results as resistant, intermediate or sensitive was used and isolates that exhibited resistance to three or more classes of antibiotics were selected as multidrug resistant (MDR) isolates (Magiorakos et al., 2011).

Phenotypic detection of ESBL production
All isolates that exhibited resistance to all the third generation cephalosporins based on the CLSI breakpoint guideline were screened for ESBL production by double-disc synergy test (DDST). Briefly, bacterial suspension of overnight culture diluted to 0.5McFarland standard was inoculated on to the dried-surface of Mueller-Hinton agar plate by surface spreading as described previously above. With the aid of sterile forcep, discs (Oxoid Ltd, UK) of cefotaxime (30µg), ceftazidime (30µg) and cefepime (30µg) were placed at 20mm distance centre-to-centre around amoxicillin-clavulanic acid (20/10µg) disc, allow for pre-incubation diffusion for 1hour and then incubated in an inverted position for 24 hours at 37 o C. Isolates that exhibited enhanced susceptibility to one or more of the cephalosporin antibiotics (i.e. enhanced zone of inhibition) were selected as potential producers of ESBL.

Phenotypic detection of MBL production
Bacterial isolates that exhibited resistance to all or any of the three carbapenem antibiotics used in this study were screened for the production of MBL using the combined-disc-diffusion test (CDDT) method as described by Lee et al.(2003) and Pitout et al. (2005). The CDDT was performed using discs of imipenem (10µg), meropenem (10µg) and ertapenem (10µg) alone and a second set of the discs to which 750 µg EDTA has been added to make carbapenem+EDTA combined discs. An increase in the zone diameter of ≥7 mm around any or all of the carbapenem+EDTA discs when compared to that of the carbapenem discs alone was considered positive for MBL production.

Template DNA extraction
The DNA of the ESBL-and MBL-producers was extracted by boiling method as described by Mendonca et al (2007). Briefly, pure colonies of the ESBL-and MBL-producer were dispersed each into 500µL of sterile distilled deionized water in an eppendorf tube. The inoculum was vortexed to give homogenous bacterial suspension. The suspension was then centrifuge at 10 RCF for 3mins and the supernatant was carefully discarded leaving the bacterial suspension. A 100 µL of fresh sterile deionized water was added into the bacterial cell pellet and re-suspended by vortexing. A small hole was made on the cap of the eppendorf tube and then boiled at 100 O C for 15mins. The eppendorf tube was then centrifuged for 5mins at 10 RCF and 50 µL of the supernatant was picked with a sterile micropipette into a new sterile eppendorf tube. The DNA was stored at -20 O C for PCR analysis.

PCR detection of ESBL and MBL genes
For amplification of all the beta-lactamase genes, a reaction volume of 20 µL containing 10 µL of pre-mixed master mix (Inqaba Biotech, SA), 8 or 7µL of sterile distilled deionized water (for simplex and multiplex reactions respectively), 0.5µL of each of the forward and reverse primers and 1µL of the template DNA was prepared. For blaCTX-M gene (Forward: TTT GCG ATG TGC AGT ACC AGT AA and Reverse: CGA TAT CGT TGG TGG TGC CAT A), conditions for the simplex PCR protocol was 5 minutes at 94 0 C followed by 31 cycles of 1_minute at 94 0 C, 1_minute at 56 0 C, 1 minute at 72 0 C and concluded by 10 minutes at 72 0 C. For blaTEM (Forward: GAGTATTCAACATTTTCGT and Reverse: ACCAATGCTTAATCAGTGA) and blaSHV (Forward: TCGCCTGTGTATTATCTCCC and Reverse: CGCAGATAAATCACCACAATG), conditions for the multiplex PCR protocol include 5 min at 94°C, followed by 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 1.5 min.
For blaNDM-1 gene (Forward: GGTTTGGCGATCTGGTTTTC, Reverse: CGGAATGGCTCATCACGATC), the PCR conditions was 10 min at 94°C; 36 cycles of amplification consisting of 30 sec at 94°C, 40 sec at 52°C, and 50 sec at 72°C; and 5 min at 72°C for the final extension.
For blaIMP (Forward: TTTCATATGGCAGAGTCTTTGCCAGATT, Reverse: ATCCTAGAAATTTAGTTGCTTGGTT) and blaVIM (Forward: ATGTTCAAACTTTTGAGTAAG, Reverse: CTACTCAACGACTGAGCG), multiplex PCR conditions include 95°C for 5 min, followed by 30 cycles at 95°C for 1 min, annealing at 45°C for 1 min, an extension at 68°C for 1 min, ending with incubation for 5 min at 68°C. For blaKPC gene (Forward: ATGTCACTGTATCGCCGTC, Reverse: CTCAGTGCTCTACAGAAAACC), the PCR conditions include 5 min at 95°C, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 30sec, extension at 72°C for 1 min 30 sec and final extension at 72°C for 10 min. The PCR products were visualized on 1.5% Agarose gel stained with EZ vision and visualized with UVP® GelMax® UV Gel Imager. The expected amplicon sizes of blaTEM, blaSHV, blaCTX-

DISCUSSION
Gram-negative bacteria exhibiting multiple resistance to several classes of antibiotic are on the increase and cut across almost all genera and species, most especially among the Enterobacteriaceae and Pseudomonadaceae (Leverstein-van Hall et al., 2003;Lynch III et al., 2013;Yadav et al., 2015;Cilloniz et al., 2019). The ability of the individual microorganism toacquire resistance determinants through mobile genetic elements and transfer the resistance traits among themselves coupled with the principle of selective pressure as a result of misuse or overuse of broad and extended-spectrum antibiotics have encouraged the spread of multiple antibiotic resistance among these microorganisms (Thanner et al., 2016;Manyi-Loh et al., 2018). The increase in the use of beta-lactam antibiotics, particularly the broad and extendedspectrum beta-lactams in both community and clinical infectious cases have partly contributed to the high prevalence of ESBL and MBL producing bacteria causing infections worldwide (Rawat and Nair, 2010;Teklu et al., 2019). In this study, three most commonly encountered bacteria genera in most infectious cases were selected for collection and the highest percentage were recovered from urine (32.9%), most especially E coli (55.6%). This is not surprising as Enterobacteriaceae in particular, are part of the leading causes of urinary tract infection (UTI), particularly among female subjects due to the proximity of their anus to the urinary tract compared to the male subjects (Behzadi et al., 2010).
In this study, higher percentage of the isolates exhibited resistance to amoxicillin-clavulanic acid (73.8%) and gentamicin (58.8%) while lower resistance was observed against imipenem (14.2%), meropenem (8.3%) and ertapenem (7.9). This result was relatively similar to that described by Teklu et al. (2019) (2019) reported higher (38.0%) occurrence of MBL producers among the carbapenem resistance clinical Gram-negative bacteria from Northwest Nigeria with Klebsiella spp. (36.8%) being the highest producers compared to the other genera. Co-production of both ESBL and MBL was recorded in 6.7% of the isolates in this study which was highest among the Pseudomonas spp. (8.7%), followed by the E coli (7.7%) and then the Klebsiella spp. (4.7%). Coexistence of two beta-lactamase genes was recorded only in 5.9% of the Klebsiella spp. in this study with 31.6% of the Klebsiella spp. and 23.1% of the E coli isolates harbouring the three selected ESBL genes (blaTEM+blaSHV+blaCTX-M). Similar scenario was also reported by Ugwu et al. (2020) among clinical isolates of Klebsiella pneumoniae and E coli from Southeastern Nigeria. Coexistence of blaTEM+blaSHV+blaOXA-Like genes was recorded among the Klebsiella spp. (21.4%) but only blaTEM+blaSHV was recorded among the E coli (5.2%) by Ugwu et al. (2020). In this study, isolates producing MBL was detected but PCR amplification of the target MBL genes did not produced amplicons. This suggests that the isolates are probably resisting the carbapenems via different mechanisms, or possibly, the types of MBL genes harboured by the isolates are different from the specific MBL genes targeted in this study. This however, requires further screening of the isolates for more of the MBL genes by PCR technique or to carry out whole genome sequencing and analysis of the MBL-producers so as to elucidate the exert mechanism(s) of resistance to the carbapenems. The evolutionary relationships expressed by the phylogenetic tree in this study revealed close relationship among all the blaSHV-1 genes and among the blaCTX-M-15 genes sequenced with their respective downloaded sequences. However, some level of evolutionary differences was observed among the blaTEM-1 sequences. While 5 (26.3%) of the blaTEM-1 sequences were similar to the three downloaded sequences, 14 (73.7%) had relatively different gene sequences compared with the downloaded and the five sequenced genes.

CONCLUSION
This study reveals the presence of MDR bacteria isolates from community acquired infections co-harbouring multiple types of ESBL genes and co-producing ESBLs and MBLs. This signified a serious public health threat, especially for the carbapenem resistance and thus required continuous surveillance and strict restrictions in the use of extended-spectrum beta-lactams and carbapenems for community based infections to prevent further increases and spread within the community.