Literature reviews show that most studies on the presence of clinical isolates of ESBL-producing Enterobacterales in Africa have been conducted in Northern and Eastern Africa, with a relative lack of data in Sub-Saharan Africa [13, 24-26]. In general, and particularly in Mozambique there is very limited data from surveillance or clinical studies documenting the susceptibility pattern of common pathogens. Previous studies have revealed a great variety in proportions of ESBL-producing Enterobacterales, underlining the importance of surveillance studies and local data in order to guide antimicrobial therapy and infection control [24, 25].
In this study, the overall prevalence of plasmid-mediated AmpC- and/or ESBL-production in clinical isolates of E. coli was 10.8% (25/230) and 32.6% (75/230), respectively. Both phenotypes were present in urinary tract and blood culture isolates and with a significant higher prevalence of ESBL in urinary isolates obtained from inpatients (8%) compared to outpatients (47%). CTX-Ms were the most dominant ESBL-type, with CTX-M group 1 and blaCTX-M-15 as the major subtype and allele, respectively. Most of the blaCTX-M negative, ESBL-positive isolates were negative for blaTEM, but positive for blaSHV, indicating an SHV-ESBL-type. This is in accordance with the international situation [5, 7] and the recent meta-analysis of ESBL-producing Enterobacterales in East Africa hospitals .
Moreover, a study of ESBL-producing Enterobacterales in stool samples in Mali, Niger and Cameroon showed that CTX-M was the dominant ESBL-type . Finally, our results are also in line with the recent detection of a relatively high prevalence of ESBL CTX-M-type producing Enterobacterales in stool samples from Mozambique university students .
A total of 11% (25/230) of the isolates expressed an AmpC phenotype and all those were pAmpC-PCR positive. Surprisingly, all isolates contained two to three pAmpC genes of which blaFOX was the most prevalent. These observations are in contrast with the worldwide observations of blaCMY as the most prevalent pAmpC gene in E. coli populations [8, 9]. CMY-2 has the broadest geographic spread among pAmpCs and is an important cause of extended-spectrum beta-lactam resistance in E. coli as well as in non-typhoid Salmonella strains in many countries . The finding of multiple pAmpC-bla genes in single strains has recently been reported in a Tunisian study. Briefly, a total of 11 out of 75 pAmpC positive clinical strains of Enterobacterales were shown to contain up to three different pAmpCs . In contrast to our study, CMY-2 was the most common pAmpC-type in their study. Moreover, the combination of MOX-, FOX- and CMY-2 type enzymes was dominant in their isolates, in contrast to ours that mostly contained MOX- and FOX-types in combination with DHA.
ERIC-PCR has been a useful rapid method in various molecular epidemiological studies to describe the genetic relatedness in Enterobacterales strain collections . Our ERIC-PCR results revealed an overall genetic diversity of pAmpC - and/or ESBL -positive E. coli strains at the Maputo Central Hospital. The results indicate that there is not a dominant clone of ESBL-/pAmpC positive E. coli. However, there are several clusters with clonal relatedness indicating minor outbreaks between patients at specific departments. This notion is supported by the isolation of CTX-M-15 producing strains with similar resistance patterns from the Pediatric department linked in time within cluster B.
The observation of multidrug-resistant pAmpC- and/or ESBL-producing E. coli in a high proportion of clinical isolates during a period of three months is a major concern. E. coli is the most prevalent cause of urinary tract infections and Gram-negative bacteremia in most countries [29, 30]. A large proportion of the ESBL-producing strains also expressed resistance to fluoroquinolones, aminoglycosides and trimethoprim-sulfamethoxazole, limiting treatment options to last resort antibiotics such as carbapenems, piperacillin-tazobactam, colistin or tigecycline. Those drugs are not easily available at Maputo Central Hospital and in developing countries in general. Recent 2015-data from the Pharmacy Department at Maputo Central Hospital showed that betalactams represented 75% of the total in-house antibiotic use, in which ceftriaxone (a third generation cephalosporin) covered 21% of the betalactams (Zimba TF et al. unpublished). Carbapenems were not in use in 2015. Thus, a significant proportion of clinical E. coli strains at the Maputo Central Hospital is in fact not treatable with the locally available antibiotics. This is in line with the recently reported antimicrobial surveillance data of blood culture Gram-negative pathogens from Blantyre, Malawi .
In conclusion, our study has shown: (i) a high proportion of pAmpC- and/or ESBL-producing clinical isolates of E. coli from the urinary tract and blood cultures at the Central Hospital.  CTX-Ms and FOX/MOX were the dominant ESBL- and pAmpC-types, respectively. (iii) All ESBL- and pAmpC-producing isolates were multidrug-resistant, only susceptible to antibiotics that are not easily available at the hospital. (iv) Studies of the genetic relatedness between ESBL- and / or pAmpC-producing isolates demonstrate genetic diversity and some clusters indicating within-hospital spread of isolates. The overall findings strongly support the urgent need for accurate and rapid diagnostic services to guide antibiotic treatment of life-threatening infections and improved infection control measures. The findings have a probable transfer value to other hospitals in Mozambique as the Central Hospital has reference functions with transfer of patients to and from other hospitals.