René Magritte (Lessines, Bégica, 1898 – Bruselas 1967)

This study provides genetic epidemiological data on ESBL-carrying enterobacteriaceae in the clinical setting of a University Hospital in Germany and BMC Mwanza, Tanzania. In both Institutions ESBL producers were commonly found in Escherichia coli, Klebsiella pneumoniae and Enterobacter spp with most of the strains recovered from urine, blood and wound swab samples. As described previously a predominance of certain specie producing ESBL in specific institution was observed in this study, whereby in Giessen University Hospital Escherichia coli was predominant while Klebsiella pneumoniae was predominant at BMC. Among Klebsiella pneumoniae isolates from these two institutions, the prevalence of ESBL was higher than in other enteric gram negative bacteria. The predilection for ESBL production by Klebsiella pneumoniae has never been clearly explained. Almost all non- ESBL producing Klebsiella pneumoniae isolates have chromosomally mediated SHV-1 β-lactamase [73]. This could also explain why 100% of our Klebsiella pneumoniae were resistant to ampicillin. Also Klebsiella pneumoniae was the commonest isolate from ICU and a significant number of them were ESBL producers. This finding agrees with those described in more than 75% of previous studies, where the majority of Klebsiella pneumoniae isolates were found to produce ESBL [73-76].

At BMC approximately three quarters of the isolates were from inpatients and of these significant proportions were found to produce ESBL (p=0.00001). Some other studies have demonstrated a statistically significant increase in antibiotic resistance in those organisms isolated after 72 hours of admission [77, 78]. This suggests that nosocomial acquired organisms are more likely to become ESBL producer and this may result in treatment failure with empirical use of cephalosporins.

Almost all phenotypic confirmed ESBLs harboured ESBL genes. In a few cases (<2%) the PCR for the ESBL genes tested were negative. The study has demonstrated that CTX-M ESBLs are the most common ESBL types in Giessen and Mwanza. CTX-M- types were found in 76% of all ESBLs isolates in Giessen; in all Escherichia coli in Mwanza and in 76% of Klebsiella pneumoniae in Mwanza. The blaCTX-M-15 allele was the commonest allele among all ESBLs alleles and was more common in Klebsiella pneumoniae than in Escherichia coli in Giessen and it was vice versa in Mwanza. The predominance of blaCTX-M-15 indicates that this allele might be as common in Germany and Tanzania as in other European countries (such as UK, Poland, Greek, France etc) [7, 44]. The blaCTX-M genes are associated with an ISEcp1 element which facilitates their transfer and may explain why they are becoming the most common ESBL types in the world. In this study the ISEcpl element was found in all Escherichia coli strains tested habouring blaCTX-M-15 alleles. The blaTEM-1 was commonly associated with the blaCTX –M-15 allele in the present study. Karisik et al. reported that 6 out of 11 Escherichia coli harboured both blaCTX –M-15 and TEM-1[11].

Extensive studies investigating the association of the MLST clonal complex ST131 and blaCTX-M-15 have been reported for Canada, India, Kuwait, France, Switzerland,

Portugal, Spain, Korea and Japan; and worldwide dissemination of blaCTX-M-15

seems to be linked to this clone [12, 13]. In the present study ST131 was detected in 38% of Escherichia coli and this is the first report of ST131 in Tanzania. Also other multiple ST clones were detected to harbour blaCTX-M-15 allele, an observation that indicates a high diversity of Escherichia coli carrying ESBL genes in Tanzania. Both Escherichia coli and Klebsiella pneumoniae, isolates carrying blaCTX-M-15 were significantly more resistant to ciprofloxacin, gentamicin and co-trimoxazole as compared to other ESBL alleles. Other studies have reported cross-resistance to tetracycline, aminoglycosides, quinolones and co-trimoxazole in ESBL-producing organisms [6, 7, 12, 13, 15]. Other studies have shown that the plasmids harbouring the blaCTX-M-15 gene also carry other genes of resistance such as tet(A) and aac(6’)- ib-cr [6, 11, 44]. In this study gentamicin, tetracycline and SXT resistance were transferable by conjugation in 50% of cases. In non conjugative isolates like Klebsiella pneumoniae isolates from Giessen University Hospital, the mechanisms of co-resistance could not be explained, which suggests the possibility of other coexisting mechanisms of resistance in these isolates or location in non conjugative plasmids [6, 7, 44].

This study found that all isolates habouring blaCTX-M-15 were resistant to cefepime with most of them having a MIC greater than 32µg/ml (p=0.001) when compared to other CTX-M alleles. Mutation in (Asp-240-Gly) in blaCTX-M-15 which is conferring

cefepime in strains harboring this allele [5, 19, 61]. Furthermore, this study demonstrated that most of the isolates with TEM- ESBL alleles were significantly sensitive to cefepime (p=0.02).

All ESBL isolates used in our study were sensitive to carbapenems as it has been found in most studies; these groups of drugs have been recommended as the treatment of choice for ESBL isolates [59, 60]. Of the tested, ESBL isolates habouring CTX-M types 97% were found to be sensitive to tigecycline with a MIC < 2µg/ml. Thus, this could be a good alternative to carbepenems in case of systemic infection due to ESBL producing organisms [62] and also in avoiding overuse of carbepenems with attendant resistance problems. The only limitation could be cost because tigecycline is more expensive than carbapenems so its use in developing countries is questionable. The clinical impact of ESBL infection was assessed among neonates (Article III). As in other studies high mortality rate was observed among neonates infected with ESBL producing organisms in the present study [79, 80]. The majority of neonates with multi-resistant organisms died within 72hrs of initiation of antimicrobials. Good survival was demonstrated in neonates with sensitive organisms, 80.8% of them improved after 72hrs of treatment [81].