INTRODUCTION

Nursing homes are long-term care institutions with a high prevalence of infectious diseases ¹; urinary tract infection (UTI), whose main etiological agent is Escherichia coli, is the most frequent infection in this type of institution ¹. Likewise, between 20 to 40% of bacteremias in the elderly population are caused by bacteria that ascend through the urinary tract; E. coli and Proteus mirabilis are the most common microbial agents ².

Nursing home residents have a high probability of colonization and infections by multidrug-resistant E. coli ³. In addition, the high recurrence of UTI and inappropriate treatment of asymptomatic bacteriuria increases the use of antibiotics, which leads to the selection of resistant bacteria ¹, with an increase in the mortality rate of residents ².

ß-lactam antibiotics represent approximately 50% of the antibiotics prescribed worldwide ⁴; however, their therapeutic efficiency has decreased due to the rapid selection of resistance mechanisms. Extended-spectrum ß-lactamases (ESBLs) represent the most important mechanism for resistance to third-generation cephalosporins in E. coli. CTX-M enzymes, due to their high propagation efficiency, have displaced SHV and TEM type ß-lactamases as the most prevalent worldwide ⁴. CTX-M has a heterogeneous lineage that includes six groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25 and KLUC) that are different from each other by 10% or more amino acid residues ⁴. ESBL-producing E. coli. are highly frequent in Peru; in 2011, a multinational surveillance program that evaluated eleven Latin American countries found that 54.0% of E. coli. were ESBL-producing ⁵.

On the other hand, although the dissemination of CTX-M type ESBL in uropathogenic E. coli (UPEC) is a global problem that decreases therapeutic options in patients, the severity of infection depends on the pathogenic capacity of UPEC ⁶. UPEC have adherence factors (e.g. Pap G II) that allow them to successfully initiate infection and migrate to the upper regions of the urinary tract, aggravating the patient’s condition ⁶. Likewise, UPEC produce siderophore systems (e.g. ChuA, Aer) and exotoxins (e.g. α-Hly, TcpC, Cnf-1) that together constitute a protein system that allow them to evade and/or affect the immune system to the detriment of the patient’s health ⁶. In bacteremias caused by an UTI, UPEC require the expression of proteins that allow them to adapt to the environmental conditions of the blood tissue (e.g. nanA) ⁷.

To our knowledge, no studies that describe the frequency of CTX-M type ESBL-producing UPEC in nursing homes have been published in Peru. The population of these nursing homes is susceptible to UTI and systemic complications; therefore, the aim of this study was to determine the frequency of virulence genes and the presence of the bla _CTX-M gene in UPEC isolated from residents of nursing homes located in Metropolitan Lima.

THE STUDY

FINDINGS

Antimicrobial Susceptibility

Thirty-five ESBL-producing UPEC from nursing homes were analyzed. Most isolates were resistant to cefotaxime (25/35), there were no carbapenem-resistant isolates, and only two (2/35) isolates were resistant to cefoxitin. In addition to resistance to ß-lactams, we observed that UPEC strains were highly resistant to ciprofloxacin, 82.9% (29/35) of the cases. UPEC resistant to PTZ, MEM and IMP were not found. Figure 1 shows the frequencies of resistance by antibiotic.

Figure 1 Antimicrobial resistance profile of extended-spectrum ß-lactamase-producing Escherichia coli isolates (n=35). 

Genotyping of UPEC carrying bla _CTX-M

Of the 35 isolates, 57.1% (20/35) carried the bla _CTX-M gene, of which 70% (14/20) corresponded to bla _{CTX-M-group 2}, 40% (8/20) to bla _{CTX-M-group 1}, 30% (6/20) to bla _{CTX-M-group 9} and 20% (4/20) to bla _{CTX-M-group 8}. On the other hand, 60% (12/20) of the UPEC carrying the bla _CTX-M gene had two or more allelic variants of the bla _CTX-M gene in the same bacterium. In addition, there was a higher level of confluence between the genes from bla _{CTX-M-group 1} and bla _{CTX-M-group 2} (Table 2). The presence of other ß-lactamase-producing genes (bla _SHV and bla _TEM) was also evaluated in bla _CTX-M producers and non-producers. Of a substratum of the analyzed UPEC, 48.1% (13/27) did not carry the bla _CTX-M gene, but did carry the bla _SHV and/or bla _TEM genes. Likewise, 51.9% (14/27) of the UPEC presented the bla _CTX-M gene, in addition to the bla _SHV and/or bla _TEM genes. The nucleotide sequences of the allelic variants that would confirm belonging to the ESBL group were not analyzed.

Table 2 Distribution of blaCTX-M gene clusters in uropathogenic Escherichia coli producing extended-spectrum ß-lactamases 

Genotyping of virulence factors in ESBL-producing UPEC

After analyzing genes related to exotoxins in the 35 ESBL-producing UPEC, we obtained a frequency of 46% (15/35) and 37% (13/35) of hly-alpha and cnf-1, respectively. aer (94%, 33/35) and chuA (94%, 33/35) were the most frequent genes associated with iron metabolism. Furthermore, adhesin pap GII was found in 46% (16/34) of the isolates and the nanA gene in 91% (32/35) (Figure 2).

Figure 2 Frequency of virulence genes in uropathogenic Escherichia coli. 

DISCUSSION

The results of this study show that 57.1% of the ESBL-producing Escherichia coli carried the bla _CTX-M gene, 70% of which belonged to bla _{CTX-M-group 2}. On the other hand, the genes: hly-alpha and cnf-1, that code for exotoxins, were detected with a frequency of 46 and 37%, respectively.

Nursing homes are long-stay institutions where the prevalence of UTI caused by multidrug-resistant bacteria is high ³. In this study, 57.1% of the UPEC carried the bla _CTX-M gene. This result is similar to those described by Galván et al.¹³ and Arce-Gil et al.¹¹, who reported a frequency of 54.7% and 51.4%, respectively. However, they differ from those described by Ramirez et al. ¹⁴ and Chavez ¹⁵ in various hospitals in Lima (70% and 91.7%). The latter demonstrates the great variability in the presence of this gene in health institutions.

Results regarding the bla _CTX-M subgroups showed that bla _{CTX-M-group 2} (14/20) followed by bla _{CTX-M-group 1} (8/20) were the most frequent. This differs from what was reported by Chavez ¹⁵, where bla _{CTX-M-group 2} was not detected and bla _{CTX-M-group 1} (26/33) had the highest frequency; and by Palma et al. ¹⁶, where bla _{CTX-M-group 1} predominated (13/27). However, recent studies indicate that bla _{CTX-M-group 2} is still significant in South America ¹⁷ and a recent review in Brazil identifies bla _{CTX-M-group 2} and bla _{CTX-M-group 1} as the most prevalent variants in the region ¹⁸.

We also detected bla _TEM (54.3%) and bla _SHV (51.4%) genes, which were found among UPEC carrying the bla _CTX-M gene. It is important to note that in 37% of the isolates, only the genes bla _TEM and/or bla _SHV were identified, despite the fact that its allelic variant and its belonging to the ESBL group was not determined, this is a strong indicator of its membership. None of the evaluated resistance genes were detected in 5.7% of the isolates, which indicates the presence of other ESBL-type resistance genes that were not analyzed in this study.

Regarding virulence factors, the pap GII gene was present in 47.1% of UPEC. These findings differ from the results obtained by Paniagua-Contreras et al. ¹⁹, who reported a 21.1% frequency of the pap GII gene in community UPEC in Mexico. We also found a high frequency of genes associated with iron transport, similar to what was reported by Dadi et al. ²⁰, with 54.5% for the iucC gene.

Regarding exotoxins, we obtained a frequency of 44.2% and 38.2% of hly-alpha and cnf-1, respectively, these results are comparable to those reported by Dadi et al. ²⁰ who found 50.4% and 29% of hly-alpha and cnf-1, respectively. On the other hand, in this study we did not find UPEC carrying the tcpC gene, in contrast to other studies that have reported prevalence of up to 25% ¹⁰.

We also found a frequency of 88.2% for the nanA gene, which is important for energy production. It has been proposed that the presence of the nanA gene creates high competitiveness in UPEC to cause bacteremias, although its role in the pathogenesis of UTI is less important ⁶, the results point to the existence of high risk in residents with bacteremia that develops into sepsis.

This study has some limitations. Although it was carried out in several nursing homes, no clinical information was obtained from the patients, in addition to having a limited number of samples. Moreover, we did not analyze further virulence and resistance genes (other types of ESBL) relevant to the epidemiology of UPEC.

In conclusion, this is the first report of ESBL-producing UPEC in nursing homes in Peru, which shows predominance of the bla _CTX-M gene, 70% of which belong to bla _{CTX-M-group 2}. Also, we can point out that ESBL-producing UPEC in nursing homes present a high frequency of exotoxins and the nanA gene, which gives them an advantage to disseminate into the bloodstream. We hope that these findings will allow strengthening the epidemiological surveillance of multidrug-resistant bacteria and prevent their dissemination.