Food Protection Trends - September/October 2020 - 316

ethanol solution before taking the water sample. The pH and
chlorine residual concentrations of all water samples were
determined by using a pH meter (model pH 209, HANNA
Instruments, Sarmeola di Rubano-PD, Italy) and the N,Ndiethyl-p-phenylene-diamine method (25), respectively. Each
sample was tested for the presence of aerobic mesophilic
bacteria (AMB), total coliforms (TC), and fecal coliforms
(FC), following methods approved by the Mexican official
guidelines NOM-201-SSA1-2015 (25) and NOM-210SSA1-2014 (26). For the isolation of Escherichia coli, for
each E. coli tube positive for FC, a loopful was transferred to
tubes containing E. coli broth with 4-methylumbelliferyl-β-Dglucuronide and incubated for 24 h at 35 ± 0.5°C. Each tube
was examined for growth (turbidity, gas) and determined for
fluorescence production. From the positive tubes, a loopful
of broth was removed and streaked on an eosin methylene
blue agar plate that was incubated for 24 h at 35 ± 0.5°C. Two
to three E. coli-like colonies that grew were then selected and
biochemically characterized with the IMViC test (indole
test, methyl red test, Voges-Proskauer test, and citrate test).
All E. coli strains isolated from eosin methylene blue plates
were analyzed using two multiplex PCR assays to identify
different diarrheagenic E. coli pathotypes (6, 22). All data
were analyzed according to guideline NOM-041-SSA1-1993
(041 Guideline) (24). This guideline establishes that purified
water must have a pH between 6.5 and 8.5, a free of residual
chlorine concentration of up to 0.1 ppm, must not exceed
the limit of 2 log CFU/mL (100 CFU/mL) of aerobic
mesophilic bacteria (AMB), and the presence of TC must not
be detectable in any 100 mL (<1.1 most probable number
[MPN]/100 mL) of sample.
Isolation and identification of mycobacteria
Five hundred milliliters of water from each jug was filtered
using the CORNING sterile filtration system with a 0.22-µm
membrane. Subsequently, the membrane was placed onto
Middlebrook 7H10 agar plates (Difco, BD) supplemented
with albumin dextrose catalase (BD BBL prepared media),
cycloheximide (500 μg/mL), and the PANTA cocktail (BD
BBL: 40 U/mL polymyxin B, 4 μg/mL amphotericin B, 16
μg/mL nalidixic acid, 4 μg/mL trimethoprim, and 4 μg/mL
azlocillin). Plates were incubated at 35°C and were examined
daily for the first 8 days and thereafter once a week for 2
months. Once bacterial growth had been observed on the
Middlebrook 7H10 agar, the number of CFU/500 mL was
directly determined for each plate, and the identification
of acid-fast bacilli was carried out by Ziehl-Neelsen stain.
Isolates belonging to the genus Mycobacterium and to the
M. tuberculosis complex were identified by two PCR assays
described previously (8). NTM species were identified by
three methods: (i) PCR restriction enzyme pattern analysis
of the 65-kDa heat shock protein gene hsp65, as described
by Telenti et al. (37); (ii) sequencing of the hypervariable
region 2 of the 16S rRNA gene (20); and (iii) sequencing

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Food Protection Trends September/October

of a fragment (723 bp) of the rpoB gene (2). Nucleotide
sequences were compared with known sequences in the
GenBank database by using the Blastn algorithm. Species
identifications were based on the 100% similarity cut-off for
the 16S rRNA gene and ≥97% for the rpoB gene.
RESULTS
Chemical and microbiological quality of purified water
The pH range of the water samples was found to be
between 6.8 and 7.8, and the chlorine concentration was
<0.1 ppm. All samples were thus found to be within the
chemical standards range for pH recommended by Mexico's
official guidelines for purified bottled water. Regarding the
microbiological quality, all 20 samples analyzed were positive
for AMB (Table 1). Concentrations of AMB ranged from
1.12 to 6.45 log CFU/mL. In total, 11 (55%), 6 (30%), and
2 (10%) water samples were positive for TC, FC, and E. coli,
respectively (Table 1). In the positive samples, TC and FC
concentration ranged from 1.1 to >8 MPN/100 mL and
from 1.1 to 4.6 MPN/100 mL for E. coli. None of the six
E. coli strains isolated from two water samples belonged to
diarrheagenic pathotypes. In total, 18 (90%) of the water
samples were found to fall outside Mexico's accepted official
guidelines: 7 (35%) water samples exceeded the maximum
allowed limit for AMB, 5 (25%) for TC, and 6 (30%) samples
exceeded both indicators (AMB plus TC; Table 2).
Mycobacteria isolation and identification
NTM were isolated from 5 (25%) of the 20 water samples,
recovering a total of five isolates. Concentrations of mycobacteria ranged from 3 to 25 CFU/500 mL. According to three
molecular methods (PCR restriction enzyme pattern analysis
of the gene hsp65 gene and the sequencing of the 16S rRNA
and rpoB genes), all NTM identified in this study belonged to
the Mycobacterium fortuitum complex: Mycobacterium porcinum (n = 2), M. fortuitum (n = 1), Mycobacterium conceptionense (n = 1), and Mycobacterium sp. (n = 1).
DISCUSSION
In this work, the microbiological quality of purified bottled
water obtained from small water purification plants was
evaluated, and 90% of purified bottled water samples did not
comply with the 041 Guideline. These results are similar to
those published by Cerna-Cortes et al. (7) in Mexico City
and Pant et al. (29) in Dharan, Nepal, who found that 72.9
and 87.5%, respectively, of bottled water samples analyzed
did not comply with the World Health Organization
microbiological criteria for drinking water (<100 CFU/
mL of AMB and absence of coliforms) (39, 40). However,
our results were higher than those reported by Abd ElSalam et al. (1) in Egypt and Halage et al. (13) in Kampala,
Uganda, who reported that 54.8 and 15%, respectively, of
bottled water samples evaluated exceeded the World Health
Organization guidelines.
Food Protection Trends - September/October 2020

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